벼 흰잎마름병균의 정확한 진단을 위하여 PCR용 진단 kit를 개발하였다. 본 PCR kit를 개발하기 위하여 벼 흰잎마름 병균 유전체 정보 중 phage-related integrase and transposase gene의 염기서열을 이용하여 프라이머를 각각 제작하였다. 프라이머 염기서열은 XOP-F (5-CGG TCT GCT CAA TGA GGA AGA-3)와 XOP-R2 (5-TGC AAT TGG TGT TCTCCA GG-3), XOT-F (5-GTC ATA GGT GAG GCT TCCC-3)와 XOT-R2 (5-AGT GCG ATC TTT CAG CAG G-3)로 벼 흰잎마름병균의 DNA를 401bp와 492bp를 증폭하게 제작하였다. PCR 증폭은 벼 흰잎마름병균만 증폭하였으며 다른 세균인 Escherichia coli, Agrobacterim, Pectobacterium caratovora subsp. cartovorum, P. atrosepticum, Pseudomonasputida, P. syringae, P. savastanoi pv. phaeolicola, P. savastanoipv. savastanoi and P. marginalis pv. Marginalis 등은증폭되지 않아 특이성이 인정 되었다. 본 프라이머로 병이 의심되는 벼잎과 논물에서 병원균을 3시간 이내에 검출할 수 있었다.

본 연구는 반추동물의 조사료 자원으로서 거대억새를 개발하기 위한 목적으로 수행되었다. 우리나라에서 새롭게 개발된 품종인 거대억새 1호를 완숙기 이후에 채취하여 in vitro 반추위 발효를 이용해반추위내 pH, 암모니아태 질소, 가스발생량, 휘발성 지방산 생성량 및 건물소화율을 조사하였으며,볏짚과 비교하여 평가하였다. 거대억새는 볏짚에 비하여 유의적으로 높은 반추위내 pH를 나타내었다(p<0.01). 암모니아태 질소의 경우 배양 12시간 이후에는 두 처리구간의 유의적인 차이를 나타내지 않았다 (p>0.05). 배양 6시간 이후 부터는 거대억새의 가스발생량이 볏짚에 비하여 유의적으로 낮게 나타났다 (p<0.05). 휘발성 지방산 생성량에 있어 acetate, propionate, butyrate, valerate 및 총생상량에서 볏짚이 거대억새보다 높게 나타났다. 그러나 iso-butyrate와 iso-valerate에서는 두 조사료원별 차이는 발견되지 않았다. 건물소화율에 있어 배양 12~24시간 사이의 거대억새 소화율이 볏짚에 비하여 유의적으로 나타났다. 결론적으로 거대억새의 이용성은 볏짚의 약 80% 수준인 것으로 나타났다.

To identify subspecies and stocks of minke whale meats purchased from Korean markets during 2005-2007, we first obtained their complete sequences of mitochondrial DNA cytochrome b and control region sequences, and compared these sequences to the corresponding sequences of the common minke whale (Balaenoptera acutorostrata), obtained from GenBank. From analyses with partial cytochrome b sequences (383 bp) and non-coding, partial control region sequences (463 bp), Korean mink whale meats are identified as products from the North Pacific minke whale (B. a. scammoni). In addition, the sequences of the partial control region from these meats showed G at site no. 298 and G or A at site no. 463, and the meats appeared to originate from the J stock within this subspecies. Thus, because the J stock has been protected since 1986, implementation of strict regulation measures to reduce their accidental fisheries by catch seems urgent. In addition, B. a. scammoni is distinct from B. a. acutorostrata, with an average Jukes-Cantor distance of 2.21% in the complete control region sequence analysis (935 bp) and 1.31% in the complete cytochrome b gene sequence analysis; the current results support the current subspecies classification, although further sequencing analyses with nuclear genes are necessary.

Anestrus is one of the most important production limiting disorders in dairy buffaloes and its underlying causes have been a current topic of studies. The objectives of this study were to explore the causes of anestrus in buffaloes with the application of ultrasonography. Two examinations were performed by transrectal ultrasonography at 12 days apart in buffalo cows that were not seen in oestrus at 60 or more days postpartum. As high as 54.5% buffaloes had silent ovulation and 45.5% suffered from the true anestrus with ovarian dysfunction. The duration of anestrus after calving was 60～90, 91～120, 121～180 and 181～365 days in 27%, 32%, 18% and 23% buffalo cows, respectively. Treatment with prostaglandin of cyclic buffalo cows with a corpus luteum (72.7%) resulted in higher estrous rate as compared with close observation of estrus (23.1%) by the farmer (p=0.021). Acyclic buffalo cows without any corpus luteum on ovaries were successfully treated with gonadotropin releasing hormone (70%), resulting in higher estrous detection rate than those treated with a vitamin-mineral mixture (20%) (p=0.035). In conclusion, poor heat detection due to silent ovulation is the most important cause of apparent anoestrus in buffaloes; however the percentage of the true anestrous is also quite high in postpartum buffaloes.

Porcine has been known to have a great impact on the studies of organ transplantation, biomaterial production and specific biomodel development such as transgenic animals. To achieve such therapeutic purposes, establishment of porcine embryonic stem cells (pESCs) will be needed. Especially, in vitro differentiation toward neural cells from pESCs can be a useful tool for the study of early neural development and neurodegenerative disorders. In addition, these cells can also be used in cell replacement therapies and drug development for neuroprotective and/or neurotoxic reagents. Although several studies reported the successful isolation of pES-like cells, it has been a big challenge to determine optimal conditions to generate pESCs without loss of pluripotency for a long time. The present study was performed for generation and characterization of putative pESCs, and differentiation into neurons and astrocytes. In this study, porcine blastocysts were produced by parthenogenetically activated oocytes. The putative pESCs were cultured in pESC growth media supplemented with a growth factor and cytokines (bFGF, LIF and SCF). Subculture of pESCs was conducted by mechanical dissociation using syringe needles after 4-5 days of incubation. As results, six putative pESC lines were maintained over thirty passages. The putative pESCs were compact, round, flat, and single layered, which were similar to human embryonic stem cell morphologically. Six pES-like cells were positive for alkaline phosphatase activity at every three passages. Furthermore, Oct-3/4, Sox-2, Nanog and SSEA-4 were shown to be expressed in those cells. Also, normal karyotypes of pESCs were observed by Giemsa-staining. Differentiation potential into the three germ layers of the putative pESCs was demonstrated by the formation of embryoid bodies (EB). Besides, the study of ESC is very important in aspect of its application to not only the cell-based replacement therapies but also cellular differentiation research. Our results also showed that RA and N2 supplements activated the neural differentiation in pESC5. Neurofilament-l60 were expressed in neural precursor cells. The expression of markers for specific neural lineages, such as Microtubule-associated protein-2 expressed in matured neuron, was also induced from embryonic neural progenitors. In summary, the pESCs were generated from the parthenogenetically activated blastocysts and the typical characteristics of the cells were maintained for the long term culture. Furthermore, it was successful to differentiate the pESCs into various neural lineages through in vitro neurogenesis system. Eventually, pESCs will be excellent biomedicine in incurable and/or zoonotic diseases by regenerating the damaged tissue.

Secondary air pollution can be caused by aerosol formation through reactions of ozone and volatile organic compounds (VOCs) emitted from household products used in the indoor environment. In this study, we investigated the potential for aerosol production during the reactions of ozone and VOCs emitted from a home insecticide, a popular commercial product extracted from natural ingredients, in a 1-m³ reaction chamber. The major chemical component of the test product was prallethrin, which has very high efficacy of mosquito and housefly elimination. Toluene, α-pinene, cymene, d-limonene, α-terpinene, and α-thujone were also identified as constituents of the insecticide. Injected ozone concentrations of 50, 100, and 200 ppb generated particle mass concentrations, corrected for wall loss and air exchange loss, of 7.3, 33.1, and 40.0 μg/m³, respectively, after a 4-h reaction time. These concentrations are lower than those generated by an air freshener in a previous study under the same experimental conditions. It was concluded that the home insecticide tested had the potential to initiate secondary aerosol formation under ozone exposure due to the biogenic VOCs it contained.

The sensitivities of PrP Sc detection methods, western blotting (WB), immunohistochemistry (IHC) and protein mis-folding cyclic amplification (PMCA) techniques were compared from brains, spleens and blood of mice challenged with PrP Sc of murine-adapted BSE strain 301C. PrP Sc was detected in the spleen from 30 dpi by IHC and at 60 dpi by WB. At 30 dpi, disease-specific signals of PrP Sc was observed in only two follicles of a single spleen. PrP Sc was detected in spleen at 10 dpi with PMCA after 5 rounds of amplification. Clinical signs were obviously shown from 240 dpi, and coincided with first detection of PrP Sc in brains by WB, IHC and PMCA after one round amplification. In addition, PrP Sc was also detected in blood at 60, 180 and 240 dpi with PMCA after 5 rounds of amplification. The FDC-M1 epitope, which appears in immature FDCs, and PrP Sc were detected in follicles first at 30 dpi, whilst the FDC-M2 epitope of mature FDCs was detected at 60 dpi. More FDC-M2 epitope and PrP Sc were detected in follicles as disease progressed. The CD21/35 epitope is expressed on both FDCs and germinal center B cells. The pattern of CD21/35 expressing cells was similar to but less dominant than that of FDCs.

Tooth development involves bud, cap, bell and hard tissue formation stages, each of which is tightly controlled by regulatory molecules. The aim of this study was to identify genes that are differentially expressed during dental hard tissue differentiation. Sprague-Dawley rats at postnatal days 3, 6 and 9 were used in the analysis. Differential display RT-PCR (DD-PCR) was used to screen differentially expressed genes between the 2nd (root formation stage, during mineralization) and 3rd (cap stage, before minerali-zation) molar germs at postnatal day 9. The DNA detected in the 2nd molar germs showed homology to osteonectin only (GenBank accession no. NM_012656.1). The level of osteonectin mRNA expression was much higher in the 2nd molar germs than in the 3rd molar germs and was found to increase in a time-dependent manner from the early bell stage to the root formation stage in the 2nd molar germs. The pattern of osteonectin protein expression was consistent with these RT-PCR results. Osteonectin protein was found by immunofluorescent analysis to localize in odontoblasts and preodontoblasts rather than the dentin matrix itself. Further studies are needed to validate the involvement of osteonectin in mineralization and root formation.

The use of high throughput screening (HTS) in drug development is principally for the selection new drug candidates or screening of chemical toxicants. This system minimizes the experimental environment and allows for the screening of candidates at the same time. Umbilical cord-derived stem cells have some of the characteristics of fetal stem cell and have several advantages such as the ease with which they can be obtained and lack of ethical issues. To establish a HTS system, optimized conditions that mimic typical cell culture conditions in a minimal space such as 96 well plates are needed for stem cell growth. We have thus established a novel HTS system using human umbilical cord derived-mesenchymal stem cells (hUC-MSCs). To determine the optimal cell number, hUC-MSCs were serially diluted and seeded at 750, 500, 200 and 100 cells per well on 96 well plates. The maintenance efficiencies of these dilutions were compared for 3, 7, 9, and 14 days. The fetal bovine serum (FBS) concentration (20, 10, 5 and 1%) and the cell numbers (750, 500 and 200 cells/well) were compared for 3, 5 and 7 days. In addition, we evaluated the optimal conditions for cell cycle block. These four independent optimization experiments were conducted using an MTT assay. In the results, the optimal conditions for a HTS system using hUC-MSCs were determined to be 300 cell/well cultured for 8 days with 1 or 5% FBS. In addition, we demonstrated that the optimal conditions for a cell cycle block in this culture system are 48 hours in the absence of FBS. In addition, we candidates using our HTS system which demonstrates the feasibility if using hUC-MSCs for this type of screen. Moreover, the four candidate compounds can be tested for stem cell research application.

Great positive has big lungs and weak liver, great negative has strong liver and small lungs, small positive has large spleen and small kidney and small negative has big kidney and small spleen. The formation of DNA at initial conception of human cell is equivalent to atomic bonding while the formation of proteins of the cell growth in a human body is equivalent to molecular bonding. Rotational electro-magnetic wave photo-synthetic starch has no molecular bonding but only gluco pyranoses. The atomic bonding are equivalent to right constitutions while the molecular bondings are equivalent to left constitutions. Five Uns and six Gis are rotational electro-magnetic waves from heaven and land. Jang (lung, liver, spleen, kidney, heart) becomes strong or weak by Uns while Bu(colon, gall, stomach, urinary bladder, small intestine, samcho) becomes strong or weak by Gis. In view of modern physics of life energies four phase constitutions are generated due to more four kinds of life energies than the others. Korean four phase constitutions considered apriority and postreriority at the same time while Chinese Un-Gi constitutions considered only apriority and are the same as the four phase constitutions in apriority. The energy-synthesis from land and heaven is an abnormal phenomenon, that is to say, a kind of energy diseases. The four phase constitutions is very effective for medical curing therefore. The theory of meridian flow in a body is the same as the electric-battery. The constitution are the results from chaotic process.

We developed the “Nest Finder System” to detect the breeding ecology of cavity nesters. Nest Finder System is composed of three parts, camera, transfer and recorder parts. In camera part, we utilized three types like pin camera, CCTV camera and endoscope with CMOS lens. Electricity and information was transferred with cable lines, and the information were directly recorded into sony digital recorder (GV-HD 700) or hard disk. To survey the breeding status of cavity nester, we disassembled the cone lens (TVC-MN4428C) applicable to enter the natural tree hole ranging no less than 30 mm. To support the camera and transfer part reaching at the hole located about 15m height, we used a couples of carbon poles in size of 1.8m, and its slender ending part was designed to fit each other. Nest Finder System can be applied to monitor breeding status of forest wildlife including cavity nesters, canopy nesters and aquatics. We discussed the potential problem in applying the equipment and analyzing the obtained data. Nest Finder System enable us to monitor the inner part of nest located at the upper part of trees not only coniferous and deciduous forests, and it can be applied to monitor the breeding ecology of aquatic organism.

Major and reckless development which have been continued during the last half century, have caused decrease and damage of urban green spaces in the point of qualitative and quantitative view. Particularly, it brought about reduction to urban neighborhood park which has taken the main role for landscape ecological value. Recently, neighborhood park planning based on the landscape ecological results has been increasing gradually. Most of all, diverse attempts such as the application of shape character analysis and the step of landscape ecological planning in urban park planning have been proposed. Today, we recognize the importance of comprehensive approach in urban green planning and neighborhood park planning, but landscape ecological approach which is analyzing character and making proposal with isolation, connection and circulation is still insufficient. Most of neighborhood parks in Korea are surrounded by buildings and isolated from adjacent green spaces. Besides, these parks have landscape ecological problems such as reduction of size, isolation from adjacent green spaces, decline of nature, and excessive pavement which we ignored during urban development process. We have sympathy for understanding landscape ecological characters and considering improvement proposals for neighborhood parks Therefore, the purpose of this study was to 1) select five neighborhood parks in Daegu, 2) analyze landscape ecological characters with isolation, connection and circulation, and 3) compare data. It is certain that these results should be the main data for the arrangement of improvement proposals which landscape ecological characters were appled to.

We identified cdf based on screening of the Arabidopsis cDNA library for functional suppressors of the AtBI-1 (a gene described to suppress the cell death induced by Bax gene expression in yeast). The cdf was located on Chr. V and was composed of 5 exons and 4 introns. It encodes a protein of 258 amino acid residues with a molecular weight of 28.8 kDa. The protein has 3 transmembrane domains in the C-terminal region. The cdf has one homologue, named cdf2, which was found in Arabidopsis. Like cdf, cdf2 also induced growth defect in yeast. The effect of the cell growth defect factor was somewhat lower than Bax. cdf could arrest the growth of yeast. Its localization to the nucleus was essential for the suppression of yeast cell proliferation. Morphological abnormality of intracellular network, which is a hallmark of AtBI-1, was attenuated by expression of cdf.

In this study, we examined whether Hanganutziu‐Deicher (H‐D) antigens are important as an immunogenic non‐a1,3‐galactose (Gal) epitope in pigs with a disrupted a1,3‐ galactosyltransferase gene. The targeting efficiency of the AO blood genotype was achieved (2.2%) in pig fibroblast cells. A total of 1800 somatic cell nuclear transfer (SCNT) embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. The a1,3‐galactosyltransferase activity in lung, liver, spleen, and testis of heterozygote a1,3‐galactosyltransferase gene knockout (GalT‐KO) pigs was significantly decreased, whereas brain and heart showed very low decreasing levels of a1,3‐ galactosyltransferase activity when compared to those of control. Enzyme‐linked lectinosorbent assay showed that the heterozygote GalT‐KO pig had more sialyla2,6‐ and sialyla2,3‐ linked glycan than the control. Furthermore, the heart, liver, and kidney of the heterozygote GalT‐KO pig had a higher N‐glycolylneuraminic acid (Neu5Gc) content than the control, whereas the lung of the heterozygote GalT‐KO pig had Neu5Gc content similar to the control. Collectively, the data strongly indicated that Neu5Gc is a more critical xenoantigen to overcoming the next acute immune rejection in pig to human xenotransplantation.

Genetic modification of the pig of which the gene is relevant to human diseases allows the pig to be used as a source of biomedical animal model. The promoter which could drive efficient expression constitutively or specifically of the interest gene in porcine organs is essentially required to increase versatility of a biomedical porcine model. In this study, we compared different promoters of activities driving efficient expression in different types of porcine cells including primary fibroblasts, kidney-derived PK-15, and primary endothelial cells (EC). To this end, we inserted CMV, EF1-α, CMV/EF1-α, CAG, human and porcine membrane cofactor protein gene promoters(MCP and Mcp), and porcine intercellular adhesion molecule-2 (Icam-2) promoter into pGL3 basic vector. Luciferase assay revealed that CAG promoter led to highest promoter activity in fibroblasts and PK-15 cells. CMV, EF1-α, CMV/EF1-α promoters showed moderate activities for luciferase expression in fibroblasts and PK-15 cells. Interestingly, CMV/EF1-α promoter, in which CMV promoter was linked to the front of EF1-α promoter as an enhancer led to highest luciferase expression in EC. The MCP, Mcp and Icam-2 promoters showed very low level of luciferase expression in three types of cells. Taken together, this study demonstrated that promoter activity in different porcine cells is differently expressed.

Hyperacture rejection (HAR) of pig organs, upon xenotransplantation into primates, could partly be overcome by knocking out the alpha-Gal gene. However, xenotransplanted organs may still undergo immunological acture rejection (AR) or acute vascular rejection (AVR). Among several genes involved in AR and AVR, the hCD47 evades the monocyte/ macrophage mediated phagocytosis by identifying the self/non-self signal (CD47-SIRPa) whereas hTFPI participates in the regulation of coagulation pathway by acting upstream of the thrombin. In this study, we investigated hCD47 and hTFPI as two possible candidates for avoiding AR and AVR, respectively upon pig-to-human xenotransplantation. A co-expression vector for hCD47 and hTFPI was constructed using 2A peptides system (F2A) and transfected into the porcine kidney cell line (PK-15). The transfected cells stably expressed both hCD47 and hTFPI mRNA and proteins. Co-culture of non-transfected, hCD47-transfected, hTFPI-transfected or hCD47+hTPFI-transfected PK15 cells with natural killer (NK) cells, monocytes and macrophages confirmed the cytotoxic effect of hCD47 and revealed a synergistic effect of hCD47 and hTFPI co-transfection. There was an attractive survivability of 25～30% on each type of innate immune cell, NK cell and macrophage. These results suggest that transgenic pigs, genetically modified for hCD47 and hTFPI may be useful for overcoming xenograft rejection. Furthermore, cotransfection with hTFPI may enhance the cytotoxic effect of hCD47, possibly by assisting the hCD47-SIRPa binding by an unknown mechanism.

Genomic reprogramming factors in the GV cytoplasm improved cloning efficiency in mice through the pre‐exposure of somatic cell nuclei to a GV cytoplasmic extract prior to nuclear transfer. In this study, a pig GV oocyte extract (pGV extract) was developed. Treatment of pig fibroblasts with the pGV extract promoted colony formation after 2–3 weeks in culture, concomitant with the expression of stem cell markers (Oct‐4, Rex1, Nanog, Sox2) and repression of differentiated cell markers (CKAP2, NPR3 ). Using fibroblasts transfected with human Oct‐4 promoter‐driven enhanced green fluorescent protein (Oct4‐EGFP), pGV extract treatment induced the reactivation of the Oct‐4 promoter in Oct4 ‐ EGFP cells by 10 days post‐treatment. These transgenic donor cells were injected into 8‐cell embryos. Oct‐4 promoter activity was subsequently detected in most ICM cells of the host blastocyst. Interestingly, reconstructed embryos with pGV extract‐treated Oct4‐ EGFP fibroblast nuclei showed prolonged expression of Oct4 in the ICM of embryos. Additionally, the pGV extract promoted somatic cell reprogramming and cloned embryo development when assessed by measuring histone H3‐K9 hypomethylation, the expression of Oct4 and Nanog in blastocysts, and the production of increased numbers of high‐ quality blastocysts. Under specific culture conditions, pGV extract‐treated fibroblast cells differentiated into neuronal, pancreas, cardiac, and endothelial lineages that were confirmed by antibodies against specific marker proteins. These data provide evidence for the generation of stem‐like cells from differentiated somatic cells by treatment with GV oocyte extracts in pig.

Hematopoietic stem cells (HSCs) are the self‐renewing, multipotent progenitors that give rise to all types of mature blood cells. The hallmark properties of HSCs are the ability to balance self‐renewal versus differentiation cell fate decisions to provide sufficient primitive cells to sustain haematopoiesis, while generating more mature cells with specialized capacities. In the present experiment, we optimized the techniques for isolation and identification of hematopoietic stem cells from cow peripheral blood. The objective of this study was to optimize the more accurate methodology for separation of mononuclear cells (MNCs) from peripheral blood and identification of HSCs by using a specific cell surface marker i.e. CD34. A total 10 peripheral blood samples were collected from Holstein dairy cows from jugular vein. We used Ficoll 400 in different concentrations from 1 to 12% and Ficollpaque Plus (1.077 g/ml) at different centrifugation speed and time. After Giemsa staining, we found more than 98% recovery of monocytes with Ficollpaque Plus (1.077 g/ml). It was demonstrated that Ficollpaque Plus (1.077 g/ml) and centrifugation at 400xg for 30 min is the best method for separation of MNCs from bovine peripheral blood. Separated MNCs were immediately subjected for magnetic activated cell sorting (MACS) by using CD34 microbead kit. HSCs (CD34+ cells) recovery was 0.307% of peripheral blood. Peripheral blood MNCs and CD34+ cells were morphologically characterized by Giemsa staining. CD34+ cells were also confirmed by immunochemistry using FITC conjugated CD34 antibodies. HSCs were also confirmed by progenitor assay including burst forming unit‐erythroid (BFU‐E), colony forming cells‐ granulocyte (CFC‐G), colony forming cells‐ macrophage (CFC‐M), colony forming cells‐ granulocyte macrophage (CFU‐GM) and colony forming cells‐ granulocyte erythroid macrophage monocyte (CFCGEMM) on Methocult 4435.

Oct4 and Nanog are transcription factors involved in pluripotency of stem cells. In general, Oct4 is up-regulated by Nanog. In previous results, however, Oct4 was differentially regulated by various doses of Nanog in P19 cells. High dose Nanog down-regulated the Oct4 expression. This negative feedback event was performed by DNMT and HDAC through the inhibitor assays (5-AZA-cytidine and trichostatin A). To identify the precise recruited sites for DNMT and HDAC, ChIP assay was performed in differential doses of Nanog. As a result, HDAC1, HDAC2, DNMT3A and Nanog were recruited to CR2, CR3, CR1, and CR4 upon high dose Nanog, respectively. Next, to investigate the differentiation potency of the cells upon high dose Nanog, RT-PCR with specific markers for three germ layers was performed. However, there was no increase for three germ layers in high dose Nanog treated cells except E-cadherin expression. E-cadherin is a specific marker for epithelial cells. Taken together, high dose Nanog induces Oct4 down-regulation and results in differentiating embryonic carcinoma cells to epithelial cell type. These results will be helpful for study on regulation of pluripotency-related genes in embryonic stem cells. * This study was supported by 2012 Post Doctoral Fellowship Program of National Institute of Animal Science, Rural Development Administration, Republic of Korea. This work received grant support from the Agenda Program (No.PJ007577), Rural Development Administration, Republic of Korea.