Kisspeptin, a neuropeptide and the master controller of reproductive axis upstream to GnRH neurons, and its receptor are also expressed in extrahypothalamic tissues, such as ovaries. As systemic kisspeptin has been shown to modulate follicular dynamics in cattle, we hypothesized that kisspeptin has direct actions on the ovarian follicular development. We also hypothesized that kisspeptin regulation of primordial follicle development is via modulation of VEGF expression. In order to test these hypotheses, we cultured caprine ovarian cortical strips in vitro for 7 days with supplementation of kisspeptin at 1, 10 and 100 μM concentration and observed the development of primordial follicles into intermediate, primary and secondary follicles. We also studied the alteration in the expression profile of VEGF and VEGF transcript variant 2 mRNA during follicular development in the presence of kisspeptin. We confirmed the presence of GPR54 in goat ovaries in our preliminary studies. Supplementation of kisspeptin at 1 and 10 μM concentration facilitated the development of primordial follicles into intermediate, primary and secondary follicles with less number of degenerated follicles while the same at 100 μM resulted in degeneration of follicles. We observed a drastic increase in the expression profile of VEGF and VEGF transcript variant 2 mRNA upon culture which was independent of kisspeptin treatment. In conclusion, our studies show that kisspeptin facilitates ovarian primordial development in vitro.

The aim of present study was to investigate regulatory mechanism of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation of porcine oocytes. Basically, immature cumulus-oocyte complexes (COCs) were incubated for 22 h in IVM-I to which hormone was added, and then further incubated for 22 h in IVM-II without hormone. As a result, relative cumulus expansion was increased at 22 h after IVM and it was enhanced by treatment of ALA compared with control group (p < 0.05). During IVM process within 22 h, cAMP level in oocytes was decreased at 6 h (p < 0.05) and it was recovered at 12 h in ALA-treated group, while oocytes in control group recovered cAMP level at 22 h. In cumulus cells, it was reduced in all time point (p < 0.05) and ALA did not affect. Treatment of ALA enhanced metaphase-I (MI) and MII population of oocytes compared with oocytes in control group at 22 and 44 h, respectively (p < 0.05). Intracellular GSH levels in ALA group was increased at 22 and 44 h after IVM (p < 0.05), whereas it was increased in control group at 44 h after IVM (p < 0.05). In particular, the GSH in ALA-treated oocytes during 22 h of IVM was higher than control group at 22 h (p < 0.05). Lipid amount in oocytes from ALA group was higher than control group (p < 0.05). Treatment of ALA did not influence to absorption of glucose from medium. Cleavage and blastocyst formation of ALA-treated oocytes were enhanced compared with control group (p < 0.05). These findings suggest that supplementation of ALA could improve oocyte maturation and development competence through increasing GSH synthesis, lipid storage, and regulation of cAMP accumulation during early 22 h of IVM, and these might be mediated by cumulus expansion.

본 연구는 스프레이 국화의 기내 대량 증식을 위한 적정 배지를 선발하고, 발근시 절편체의 부위와 순화시기가 조직배양 묘의 품질에 미치는 영향을 조사하기 위해 수행하였다. 먼저 대량증식을 위한 적정 배지를 선발하기 위해 서로 다른 조성의 식물생장조절물질을 첨가한 MS배지에 ‘Field Green’, ‘Green Diamond’, ‘Snow Pangpang’ 등 스프레이 국화 3품 종을 기내배양 하였다. 배양 6주 후의 배양체의 생육을 조사한 결과, MS+NAA 0.1 mg·L-1의 배양체의 초장이 무처리에 비해 1.5~2.0배 더 길었고, 줄기의 두께도 더 두꺼웠다. 그러나 BA 0.1 mg·L-1 이상이 첨가된 모든 배지에서는 캘러스와 다신초가 유도되었고 뿌리가 발생하지 않았으며, 신초와 마디의 길이가 짧아졌다. 따라서, MS+NAA 0.1 mg·L-1 배지가 스프레이 국화의 기내 대량증식 단계에 가장 효율적인 것으로 선발되었다. 두번째로 스프레이 국화 우량묘를 양성하기 위하 여 ‘Field Green’ 품종에서 생장점을 포함하고 있는 줄기의 선단부와 액아를 포함하는 줄기의 중단부를 각각 기내배양 한 후, 배양 2주 후부터 일주일 간격으로 순화하여 순화된 묘의 생육특성을 조사하였다. 그 결과, 선단부 배양체의 뿌리출현일수가 11.2일로 중단부 배양체보다 1.7일 더 빨랐으며, 순화율 과 생장의 균일성도 순화시기에 관계없이 중단부 배양체보다 더 좋았다. 또한 초장, 엽수, 줄기의 굵기, 생체중과 같은 조직 배양묘의 소질과 관련된 특성도 선단부 배양체에서 더 좋게 나타났다. 순화시기에 따른 묘 소질 조사에서는 기내배양 2주 후에 순화한 선단부 배양체의 경우 최종 조사일에 초장이 3.8±0.5 cm로 가장 짧았지만, 줄기의 굵기와 생체중은 1.5±0.2 mm와 1.3±0.3g으로 기내배양 3주와 4주 후에 순화한 묘에 비해 1.5~2.0배 더 두껍고 무거워 품질이 우수하였다. 이러한 결과를 바탕으로 스프레이 국화의 조직배양 우량묘를 양성하기 위해서는 기내 발근시 줄기의 선단부를 절편체로 사용 하고, 기내배양 2주 후에 짧고 굵은 뿌리가 발생하기 시작했을 때 순화하는 것이 적절하다.

이 연구의 목적은 근육 세포의 증식 배양을 위해 필요한 핵심 인자인 혈청이 혈청 대체물의 첨가에 의해 대체될 수 있는지를 분자 생물학적 측면에서 검증하는 것이다. 혈청 대체물이 첨가된 low serum (5% FBS) 기반의 promo cell 배지는 성장 배양액으로 사용되었고, 마우스 하지 골격근의 근육세포는 pre-plating (pp) 방법에 의해 분리되었다. Pre-plating 4의 세포는 작고 둥근 형태의 굴절성을 가진 Myoblast/satellite like cells의 형태가 관찰되었으며, 근육 줄기세포 전사인자들(Pax3/7, Myf5, Myod1)과 골격근 발달 전사인자(Myog)의 발현량이 섬유아세포와 비교하여 높게 나타났다. 따라서 그들을 Myoblast derived cells로 명명하고, 기본 세포주로 사용하였다. 분리된 MDCs는 5%, 10% 혹은 20% 혈청이 첨가된 배양액에서 2주간 배양되었다. 배양 6일째부터 대조군(5% 혈청)과 비교하여 20% 혈청은 세포 수가 증가하였으며, 양적 혈청 농도 의존성이 확인되었다. 증식 및 세포사멸 관련 유전자들은 대조군과 비교하였다. 배양 1주 차에 20% FBS군은 세포증식 촉진 유전자인 Myc 발현이 증가한 반면 pro-apoptosis 유전자인 Bax의 발현량이 감소했고, 2주 차에는 세포주기정지 인자인Cdkn1a의 감소와 Myc의 지속적인 증가와 Bax/Bcl의 감소가 나타났다. 각기 다른 FBS 처리농도에서 배양된 Myoblast derived cells을 동일한 Myotube유도 배양액에서 2주간 분화하였다. 대조군과 비교하여 20% FBS군은 Myod1, Myog, Myf6, Myh1의 유의적 증가가 1주부터 확인되었다. 결론적으로 혈청 대체 물질은 근육세포 배양액에서 혈청의 효과를 완벽히 모사할 수 없음이 증명되었고, 따라서 체외에서 상업적 목적의 근육 세포 대량 증식 및 분화를 위해서는 적절한 혈청 대체물 개발이 선행돼야 할 것으로 사료된다.

The treatment of microbial infections requires a large use of antibiotics which is, partially, responsible for the appearance of resistant strains to antibiotics in dairy cows. However, many photochemical compounds, including the essential oils, are becoming interesting as potential source of natural bioactive molecules. The antibacterial activity of the studied essential oils was evaluated by aromatogram and microdilution in medium liquid. The results show that the essential oil of Thymus fontanesii has the biggest antibacterial action against all the bacterial strains comparing with the Eucalyptus oil. However, the aqueous extracts of Thymus fontanesii and Eucalyptus indicate a moderate antibacterial activity against the strains of Staphylococcus aureus. On the other hand there is no effect on Escherichia coli strains. The most strong activity inhibitory was get with the essential oil of Eucalyptus against Staphylococcus aureus strains with CMI of 0.39 μl/ml for Staphylococcus aureus ATCC and 1.562 μl/ml for Staphylococcus aureus pathogen in comparison with the essential oil of Thymus fontanesii which is more active against the Escherichia coli strains. The aqueous extract of Eucalyptus showed the best CMI and CMB against the Staphylococcus aureus strains in comparison with the aqueous extract of Thymus fontanesii. Comparative tests to the activity have been made with cefoxitin and gentamicin discs.

본 연구의 목적은 포졸란의 항생제 내성균과 장내 유해 미생물 억제에 관한 체외배양 항균 활성을 조사하는 것이었다. 대조군 (CON, 포졸란 무첨가 대조군)과 DP0.3 (증류수와 포졸란 분말 0.3%를 혼합하여 제조한 배지군), DP0.5 (증류수와 포졸란 분말 0.5%를 혼합하여 제조한 배지군), PE (포졸란 분말 추출물을 이용하여 제조한 배지군)으로 구분하였다. Lacctobacillus casei 균수는 CON과 비교할 때 DP0.3 처리군에서 유의하게 높았으나 (P<0.05) 기타 처리구 사이의 차이는 없었다. Clostridium butyricum, E. coli, Salmonella typhimurium 균수는 CON과 비교할 때 포졸란 처리구가 유의하게 낮았다 (P<0.05). Clostridium butyricum, Salmonella typhimurium 균수는 DP0.3, DP0.5와 PE 사이, E. coli 균수는 DP0.5와 PE 사이의 유의차가 있었다 (P<0.05). MRSA와 VRE의 균수는 CON과 비교할 때 포졸란 처리구가 유의하게 낮았다 (P<0.05). MRSA 균수는 DP0.5와 DP0.3, PE 사이의 차이가 있었고, VRE 균수는 PE> DP0.3> DP0.5> CON 처리구 순서로 유의하게 높았다 (P<0.05). 이와 같은 결과를 통해 포졸란의 항생제 내성균 및 장내 유해 미생물에 대한 항균활성이 확인됨에 따라 가축용 천연항균제, 보조사료 규산염제로써의 활용이 가능할 것으로 보인다.

This study was conducted to evaluate the effect of lactic acid bacteria (LAB) inoculation to domestically-cultivated Italian ryegrass (IRG) on silage fermentation and in vitro ruminal fermentation. There were six treatments based on the LAB inoculants: 1) no addition of LAB (negative control: NC), additions of 2) commercially-available LAB (positive control: PC), 3) Lactobacillus plantarum (LPL), 4) L. paracasei (LPA), 5) L. acidophilus (LA), and 6) L. pentosus (LPT). All treatments were inoculated at a concentration of 106 CFU/g and ensiled for 3, 7, 21, and 42 days in triplicate and analyzed for nutritive values when ensiling was terminated. Day 42 silage from all treatments were also examined for in vitro ruminal fermentation. After 42 days, LAB-inoculated silages had higher (P<0.05) lactic acid concentration compared to the NC. In terms of nutritive values, the silages treated with LPA, LA, and LPT showed higher (P<0.05) crude protein and lower (P<0.05) neutral detergent fiber and acid detergent fiber content compared to the rest of the treatment. In vitro ruminal dry matter degradability was not affected by LAB addition. However, LAB-treated IRG had shown higher (P<0.05) ammonia-N compared with that of the NC. LPA had shown the highest (P<0.05) volatile fatty acid concentration among the LAB examined. In conclusion, the addition of a single strain of LAB appeared to produce a quality IRG silage compared with the NC and the PC. Among the strains examined, LPA seemed to be superior to the others.

Melia azedarach is commonly used in traditional and folk medicine in Korea and China to treat a variety of diseases including diarrheal, diabetic, rheumatic, and hypertensive disease. The aim of this study was to determine the potential prophylactic and therapeutic effects of Melia azedarach against a broad spectrum of viruses in in vitro cell culture model and the protective effect against different influenza A subtypes in BALB/c mice model. An effective dose of pre-treatment, co-treatment, and post-treatment of Melia azedarach significantly reduced the replication of coxsackievirus, herpes simplex virus, influenza A virus, enterovirus, and bovine rhinovirus in both epithelial and macrophage cell lines. Melia azedarach treatment remarkably promoted the phosphorylation of the key molecules associated with the type-1 interferon and NF-κB signaling pathways. Furthermore, it induced the secretion of type-1 interferon and pro-inflammatory cytokines and the subsequent stimulation of the antiviral state in both epithelial and macrophage cells. Interestingly, oral inoculation of an effective dose of herb extract significantly improved viral clearance in the lungs of BALB/c mice, thus exhibiting protection against several subtypes of influenza A virus. Together with our results indicate that an extracts of Melia azedarach and its components could exhibit a potential natural source of an antiviral drug candidate for a broad spectrum of viruses in animal and humans.

Our objective of this study is to design and develop a polyethylene glycol ( PEG2000)-modified multiwall carbon nanotube (PEGylated MWCNT) formulation for oral controlled metronomic chemotherapeutic drug delivery. Multiwall carbon nanotubes undergo various chemical modifications including oxidation with strong acids, conjugation of polyethylene glycol, and coating with cellulose acetate phthalate which resulted in the formation of aqueous dispersion and prevention of drug degradation in acidic environment. Advanced analytical procedure such as Fourier transform infra-red, X-ray diffraction, differential scanning calorimetry, thermal gravimetric analysis, transmission electron microscopy, and dynamic light scattering techniques were used to evaluate physicochemical characterization. We also performed in vitro cytotoxic study by MTT assay and results revealed that carboplatin-loaded PEGylated MWCNTs did not show significant detrimental effect on the viability of MDA-MB-231 (human breast cancer) cells. The maximum encapsulation and drug-loading capacity were determined to be 71.58 ± 0.04 and 39.62 ± 0.07%, respectively. The release of carboplatin from PEGylated MWCNTs was investigated at simulated intestinal fluid (SIF), pH 6.8, after optimizing at simulated gastric fluid (SGF), pH 1.2, by enteric coating. Enteric-coated PEGylated MWCNTs exhibit pH-responsive drug activity in a sustained manner especially at pH 6.8. This surface modification strongly suggests that PEGylated MWCNTs could be a potential carrier for metronomic chemotherapeutic agent for high drug resistance, drug with maximum adverse effect and poorly oral bioavailable drugs.

The evergreen oak tree, Quercus myrsinaefolia Blume, is not only economically important for wood, medicine, landscape trees, etc., but also becoming more important in terms of ecology due to climate change. However, asexual reproduction was difficult, so this study was conducted to establish the optimum conditions for micropropagation by shoot multiplication. The surface sterilized seeds of Q. myrsinaefolia were successfully germinated in WPM basal medium. BAP (1.0 mg/L) treatment was most effective for inducing multiple shoots. The highest induction rates of adventitious roots from the multiple shoots was shown in the treatment of 1.0 mg/L NAA. Both MS and WPM medium were most effective for growth of multiplied plantlets. For ex vitro acclimatization, the survival rates of multiplied plantlets were 100% in vermiculite and commercial soil. The results of this study can be used for proliferation and supply, and establishment of ex situ conservation of Q. myrsinaefolia elite trees.