This study is to stabilize insoluble and unstable active ingredient which is Idebenone (INCI name: hydroxydecyl ubiquinone) in a multi-lamellar vesicle (MLV) and to stabilize it in the skin care cosmetics. Idebenone is good effective raw material in the treatment of Alzheimer's disease in the medical field and a powerful antioxidant in dermatology. It is well known as a substance that inhibits the formation of melanin and cleans the skin pigment. However, it did not dissolve in any solvent and it was difficult to apply in cosmetic applications. Niosome vesicle was able to develop a nano-particle by making a multi-layer of idebenone encapsulated with a nonionic surfactant, hydrogenated lecithin and glycine soja (soybean) sterols and passing it through a high pressure microfluidizer. Idebenone niosome vesicle (INV) has been developed to have the ability to dissolve transparently in water and to promote transdermal penetration. The appearance of the INV was a yellowish liquid having specific odor, and the particle size distribution of INV was about 10~80 nm. The pH was 5~8 (mean=6.8). This capsulation with idebenone was stored in a 45°C incubator for 3 months and its stability was observed and quantitatively measured by HPLC. As a result, the stability of the sample encapsulated in the niosome vesicle (97.5%) was about 66.3% higher than that of the non-capsule sample of 32.5%. Idebenone 1% INV was used for the efficacy test and clinical trial evaluation as follows. The anti-oxidative activity of INV was 38.2%, which was superior to that of 12.8% tocopherol (control). The melanin-reducing effect of B16 melanoma cells was better than INV (17.4%) and Albutin (control) (9.6%). Pro-collagen synthesis rate was 128.2% for INV and 89.3% for tocopherol (control). The skin moisturizing effect was 15.5% better than the placebo sample. The elasticity effect was 9.7% better than the placebo sample. As an application field, INV containing 1% of idebenone is expected to be able to develop various functional cosmetic formulations such as skin toner, ampoule essence, cream, eye cream and sunblock cream. In addition, it is expected that this encapsulated material will be widely applicable to emulsifying agents for skin use in the pharmaceutical industry as well as the cosmetics industry.

Dipalmitoyl phosphatidyl choline(DPPC), Polyphenol 유도체, 그리고 Hematoxylin-Eosin은 5분 동안 산성 상태에서 직접 초음파 처리하여 명확한 표준용액을 제공했다. 25 ℃에서 DPPC 계의 레시틴소포에서 폴리페놀유도체의 흡수특성은 흡수분광학에 의해 분석하였다. 레시틴소포에서 단량체와 이량체 사이의 폴리페놀유도체의 평형은 폴리페놀유도체의 저농도에서 존재했지만 올리고머는 레시틴소포 의 고농도에서 소포안에 형성되었다. 폴리페놀유도체의 일정한 농도에 Bacteriorhodopsin(BR)을 첨가함으로써 DPPC의 상전이동안 폴리페놀유도체의 흡수비율(α/β)이 감소되었다. 기둥이 있는 곳에 라멜라 소포와 uni- and multilamella 응집체의 혼합물이 존재한다. 용출된 기둥과 혼합물사이의 속도 차이가 관찰되었으므로 기둥 용출된 라멜라반응 보다 촉매 효과를 나타냈다. DPPC 및 폴리페놀유도체에 대한 가수분해의 상전이 온도는 DPPC 및 폴리페놀유도체 보다 높게 측정되었다.

This study was performed to establish a new parameter for estimating gestational age and predicting parturition day by ultrasonographic measurement of deep portion of telencephalic vesicle (DPTV) diameter in small dogs. Fetal head diameter (HD) and DPTV diameter were measured in 15 pregnant Pekingese bitches, from Day 15 to the parturition day, and evaluated the correlation between gestational age. HD was measured from day 29 of pregnancy to parturition day and increased from 4.9 ± 2 mm to 25.5 ± 0.7 mm. Especially, from day 38 of pregnancy to parturition day, HD uniformly increased about 0.6 mm per day and was significantly and linearly relative to gestational age during this period (r2>0.99). DPTV diameter was measured from day 35 to day 60 of pregnancy and increased from 3.2 ± 0.9 mm to 11.5 ± 0.7 mm. Especially from day 38 to day 60 of pregnancy, DPTV diameter uniformly increased about 1 mm per 3 days and was significantly and linearly relative to gestational age during this period (r2>0.99). In conclusion, DPTV diameter could to be a useful parameter for the estimation of gestational age and the prediction of parturition day when used alone or in combination with HD during the second half of pregnancy.

피부 노화를 방지하고 지속적으로 보습을 유지하기 위해 다양한 베시클들이 연구되고 있다. 최근에 활성물질의 흡수, 투과 및 보습의 유지와 관련하여 리포좀, 액정 및 다중층 라멜라 에멀젼 같은 많은 제조 방법들이 소개되고 있다. 본 연구에서는 인지질과 유사한 cetearyl alcohol/ceteth-20 phosphate/dicetyl phosphate 계면활성제를 이용하여 전단세기 및 pH 변화에 따른 다중층 라멜라 베시클을 개발하였으며 편광현미경을 통해 확인하였다. 결과로서 낮은 전단세기 및 pH에서는 라멜라 베시클 입자의 형태가 불균일하게 형성됨을 확인하였다. 42℃에서 2개월 간의 라멜라 베시클 내의 레티놀 함량의 변화를 측정한 결과 낮은 pH에서 레티놀의 함량이 감소하였다. 또한, 이 라멜라 베시클은 일반 O/W 에멀젼에 비해 피부 수분손실량이 14% 감소됨을 확인하였으며, O/W 썬 크림과 내수성 in vitro SPF를 측정하여 비교한 결과 UVB와 UVA 영역 모두에서 자외선을 잘 차단하여 유사한 내수성을 보이고 있음을 확인하였다.

In all the studies of mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configurations). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configurations) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. As not all the species show the SN configuration and gene transcription always stops at the late stage of oocyte growth, it is suggested that a thorough condensation of GV chromatin is essential for transcriptional repression. Because the GV chromatin status is highly correlated with oocyte competence, oocytes must end the NSN configuration before they gain the full meiotic competence and they must take on the SN/corresponding configurations and stop gene transcription before they acquire the competence for early embryonic development. In this study, we firstly investigated whether the layer of cumulus cells and size of oocytes could determine chromatin configurations in porcine oocytes. Using Hoechst3342 staining, the GV nucleolus and chromatin of porcine oocytes was classified into SN and NSN configurations. Next, we examined the changes in GV chromatin configurations during growth and maturation of porcine oocytes. In addition, the maturation and parthenogenetic development abilities in vitro were significant different between the SN and NSN configurations oocytes. These results indicated that chromatin changes in GV oocytes affect the development potential of parthenogenetic embryos.

본 연구에서는 M16 A계통 '부지화'를 강세대목인 대목 '스윙글 시트루멜로' 또는 보통대목인 '탱자'에 접목하여 무가온 하우스에서 재배한 잎과 과실 사양의 수분상태 변화 및 과실 성숙기의 과실 가용성 고형물과 산 함량변화를 조사했다. 강세대목인 '스윙글 시트 루멜로'와 보통대목인 '탱자'에 접목한 M16 A계통 '부지화'의 대목에 따라 과실의 품질은 영향을 받는 것으로 나타났는데, '스윙글 시트루멜로' 대목은 '탱자' 대목보다 과실 가용성 고형물과 산함량이 낮았다. 동트기 직전에 측정된 과실사양의 수분포텐셜 및 팽압은 '스윙글 시트루멜로' 대목이 '탱자' 대목보다 조금 높은 경향을 보였으나, 삼투 포텐셜의 값은 '탱자' 대목이 '스윙글 시트루멜로' 대목보다 낮았다. 과실사양과 같은 시간대에 측정된 엽수분포텐셜 값의 변화도 과실 사양의 삼투포텐셜 변화와 유사한 경향을 보여주었다. 이상의 결과로 보아, M16 A계통 '부지화'에 있어 두개의 대목에 따라 잎의 수분 상태와 과실사양의 삼투포텐셜 차이가 있었으며, 이러한 차이로 인하여 과실 가용성 고형물과 산 함량에 차이가 나타나는 것으로 판단되며, 대목에 따른 수분 상태와 과실 품질의 차이는 대목의 뿌리분포가 영향을 미쳤기 때문인 것으로 사료된다.

Correlations between cumulus cells and germinal vesicle (GV) chromatin configuration were examined in porcine oocytes. Cumulus-oocyte complexes (COCs) were collected from 2～6 mm follicles and divided into three categories according to cumulus cell morphology. "A" group was compacted COCs with more than three cumulus cell layers. "B" group was COCs with less cumulus cell layers than "A" group. "C" group was COCs with one or less layer of cumulus cells. Cumulus cells were removed 0.1% hyaluronidase, and denuded oocytes were stained with Hoechst 33342. GV chromatin configuration was classified into GV-Con and GV-Dis. GV-Con meant that a nucleus was surrounded by condensed chromatin in a ring. GV-Dis meant that filamentous chromatin clumps were distributed in nucleus. The proportion (80.2%) of GV-Con in "A" group was significantly higher than "B" (62.0%) or "C" (44.9%). The proportion (55.1%) of GV-Dis in "C" group was significantly higher than "A" (19.8%) or "B" (38.0%). The meiotic competence of COCs was examined after 44 h culture. The proportion (90.0%) of oocytes reaching to metaphase II (M-II) in "A" group was significantly higher than "B" (76.5%) or "C" (45.5%). In conclusion, oocytes with good quality cumulus cell layers are synchronized early GV stage, and early GV stage is important for meiotic competence in pigs.

The aim of this study was to investigate what components of porcine epididymal fluid (pEF) influences the nuclear maturation of porcine germinal vesicle oocytes. Porcine cumulus-oocytes complexes from follicles were cultured in TCM 199 containing pEF. After 48 h cultures, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. Maturation rate of oocytes was significantly increased in media supplemented with 10% pEF during in vitro maturation (IVM) than in those without pEF. When lipid component of pEF was removed by treating n-heptane, no significant difference was observed in maturation of oocytes between n-heptane treatrment and intact pEF group. However, the proportion of oocytes reaching at metaphase II (M II) was significantly (p<0.05) decreased in the oocytes cultured in media containing trypsin-treated pEF compared to those in media with intact pEF. When porcine GV oocytes were matured in the medium supplemented with intact pEF or pEF heated at 56'C and 97'C, rates of oocytes remained at GV stage were 11.7%, 29.4% and 42.0%, respectively. However, there were no difference in proportion of oocytes reaching at MII stage among intact pEF group and 56'C group. Present study suggests that 1) pEF contains an enhancing component(s) for nuclear maturation in vitro of oocytes, 2) protein(s) of pEF may be capable to promote nuclear maturation in vitro, and 3) enhancing component for nuclear maturation may consist of two factors, which are responsible for germinal vesicle breakdown (GVBD) and promotion of MII stage.

In vitro maturation of porcine immature cumulus-enclosed oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. The aim of this study was to determine whether the addition of spermatozoa into the culture medium can stimulate the meiosis resumption of porcine cumulus-enclosed oocytes arrested at germinal vesicle (GV). Cumulus-enclosed oocytes (CEOs) were collected from follicles of 3 to 5mm diameter. Porcine CEOs were cultured in tissue culture medium containing various meiosis inhibitors and spermatozoa. Oocytes were examined for evidence of GV and GV breakdown after 24 h culture. After 24 h culture 43.8% of oocytes cultured in only TCM 199 remained at GV stage whereas 56.2% of oocytes were able to resume meiosis. When porcine CEOs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 90% of oocytes were not able to resume meiosis. However, co-culture of porcine CEOs with spermatozoa was able to overcome the inhibitory effect of dbcAMP and Fo. Irrespective of the presence of 3-isobutyl-1-methylxanthine (IBMX), no difference was observed in the proportion of oocyte reached germinal vesicle breakdown (GVBD). The present study suggests that dbcAMP and Fo prevent the spontaneous maturation of competent oocyte in culture after isolation from follicles and that mammalian spermatozoa contain a substance(s) that improves meiosis resumption in vitro of porcine cumulus-enclosed oocytes.

Maturation of oocytes is maintained by complex procedures along with follicular genesis and is a critical step for embryonic development. Purine known as an oocyte maturation regulator is present in follicular fluid. In this study, the roles of guanosine as a strong inhibitor of GVBD and a modulator of cyclic GMP concentration in ooyctes were revealed. Denuded immature oocytes were treated with guanosine, and the maturation rates and cGMP concentration of oocytes were measured. GVBD was blocked in a concentration dependent manner by guanosine, but this effect was reversible. However, GVBD was lagged yet not significant by adenosine. Both guanosine and adenosine modified cGMP concentration in oocytes. The characteristic of the guanosine-treated oocyte was significantly higher cGMP compared with the adenosine-treated oocyes at initial time of the maturation. Based these results, guanosine may be a strong and reversible GVBD inhibitor. Although the precise mechanism of guanosine presently is unclear, the results suggest that guanosine may lead the accumulation of cGMP in oocyte cytoplasm, which in turn suppresses GVBD.

In vitro maturation of denuded porcine immature oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. This study was to determine whether the addition of spermatozoa into the culture medium could influence the nuclear maturation of porcine cumulus-enclosed germinal vesicle (GV) oocytes. Cumulus-oocyte complexes (COCs) were collected from follicles of 3- to 5-mm diameter. Porcine COCs were cultured in tissue culture medium containing spermatozoa. After 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II was significantly (P < 0.05) increased in the oocytes cultured in media containing spermatozoa compared to those in media without spermatozoa (52.3% vs 12.5%). No difference in the percentage of metaphase II was observed among the different periods of spermatozoa exposure and among the spermatozoa from different species. The proportion of oocytes reaching metaphase II was significantly different between high and low concentrations of spermatozoa. The present study suggests that manunalian spermatozoa contain a substance(s) that improves nuclear in vitro maturation of porcine cumulus-enclosed GV oocytes. Enhancing effect of spermatozoa for in vitro maturation of oocytes is a highly dose-dependent.

Thermograms of methylene blue(MB) in L-α-lecithin vesicle and incorporated purple membrane vesicle(InPM) systems have been studied by photochemical reaction differential scanning calorimetry at 25~55℃. Phase transition temperatures of lecithin vesicle, purple membrane(PM), and InPM were found to be independent of illumination of light(436nm) at 39~40℃, but endothermic phase transition was found in InPM vesicle. In MB-InPM system, endothermic phase transition was found on unillumination of light at 40~42℃, but exothermic phase transition was found on steady illumination of light at 48~52℃. It was estimated that the light energy absorbed from MB on vesicular surface was transferred to PM, and the transferred energy was redistributed to hydrophobic site of membrane. Therefore, the exothermic phase transition was measured at high temperature because of the increased hydrophobicity of acyl chain.