• 한국발생생물학회지:발생과생식
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• 제21권2호
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• pp.157-165
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• 2017

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were $127{\pm}18.9$. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as ${\alpha}-1,3-galactosyltransferase$ knock-out /hCD46 for xenotransplantation.

• Applied Microscopy
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• 제17권1호
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• pp.83-97
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• 1987

In order to discriminate the enteroendocrine cell types in the mucosal epithelium of the normal duodenum of the Korean hedgehog (Erinaceus koreanus). The tissues were fixed in the mixture of 1% paraformaldehyde and 1% glutaraldehyde in phosphate buffer (pH 7.2), and postfixed in 2% osmium tetroxide (phosphate buffer, pH 7.2). They were embedded in Araldite, and the ultrathin sections were made by LKB-V ultratome following the inspection of semithin sections stained with toluidine blue-borax solutions. Ultrathin sections contrasted with uranyl acetate and lead citrate were observed with JEM 100B electron microscope. At least six types of enteroendocrine cells distributed in the mucosal epithelium of the duodenum were identified according to their morphological characteristics mainly based on the size, shape, number and electron density of the secretory granules. Type I cells had moderately developed organelles. The secretory granules were pleomorphic ($370X510nm$), and the granule cores with high electron density were enveloped in limiting membrane and characterized by a narrow halo. Type II cells contained an indented nucleus and well-developed organelles. The secretory granules were round (350 nm) and classified in two kinds by electron density, moderate and high. Both granules were surrounded by limiting membrane and those with high electron density showed often a wide halo. Type III cells had an indented nucleus. The secretory granules with various electron density were round (220 nm) in shape. The granules with high electron density were enveloped in limiting membrane and characterized by a narrow halo, but those with low or moderate electron density had not been observed the limiting membrane. Type IV cells contained an indented nucleus and moderately developed organelles. The secretory granules were round (180 nm) in shape, and the granule cores with high electron density were enveloped in limiting membrane and showed often a wide halo. Type V cells had a large amount of rough endoplasmic reticulum. Secretory granules with low or moderate electron density were round (230 nm) in shape, and surrounded by limiting membrane and showed a narrow halo. Type VI cells contained an oval nucleus and well-developed organelles, especially Golgi complex. The secretory granules with high electron density were round (210 nm) in shape. The granules were enveloped in limiting membrane and showed often a wide halo.

• Applied Microscopy
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• 제38권3호
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• pp.259-264
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• 2008

소취 및 소독제로서 사용되어온 안정화 이산화염소($S-ClO_2$)의 조직에 대한 부패억제 효과를 확인하고자 본 연구를 수행하였다. 실험을 위해 8주령 SD계 흰쥐 콩팥(kidney)을 사용하였고, 안정화 이산화염소를 처리하지 않은 대조군과, 안정화 이산화염소의 분말과 수용액을 처리한 실험군으로 구분하여 광학 및 전자현미경으로 관찰하였다. 광학현미경 관찰 결과, 부패양상은 시간경과에 따라 토리, 보우만주머니 및 세뇨관을 구성하는 세포의 핵과 세포사이 경계가 불분명해지며, 세뇨관의 경우 신장되었다가 결국에 수축되었다. 대조군의 1일군부터 괴사(necrosis)가 시작되어 3일군 이후는 전체적인 조직 괴사로 형태를 구별하기 어려웠다. 실험군에서 3일군의 경우, 조직의 전반적 형태와 괴사정도가 대조군의 1일군과 유사하였다. 전자현미경 관찰 결과, 시간경과에 따라 세포소기관 및 미토콘드리아의 부분적 붕괴로 시작되어 결과적으로 모든 세포내소기관이 붕괴되었다. 대조군의 1일군에서 세포소기관의 부분적 붕괴가 관찰되었으며, 실험군의 3일군에서 세포소기관 및 미토콘드리아의 부분적 붕괴 현상이 관찰되었다. 대조군의 3일군 이후에서는 세포소기관을 구별할 수 없었다. 이상의 연구 결과에서 $37^{\circ}C$, 습도 $80{\pm}5%$에서 안정화 이산화염소($S-ClO_2$)가 부패와 변성을 억제하는 부패억제제로서 효과가 있고, 억제 정도는 실험군의 3일군이 대조군의 1일군에 해당하는 것으로 보아 최소 2일 정도의 부패억제 효과가 있는 것으로 확인되었다.

• 한국축산식품학회지
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• 제31권6호
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• pp.907-913
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• 2011

본 연구는 한외여과기법이 모짜렐라 치즈의 갈변화, 지방분리, 신전성 그리고 용융성에 미치는 영향을 모색하는데 그 목적이 있다. 한외여과로 농축한 우유로 제조한 UF-모짜렐라 치즈의 갈변화, 지방분리, 그리고 신전성은 모두 원료유의 지방함량, 모짜렐라 치즈 제조 시의 스타터와 렌넷 첨가량, 완성된 모짜렐라 치즈의 굽는(baking) 온도에 따라서 많은 영향을 받았다(p<0.05). 갈변화와 지방분리는 굽는 온도가 높고 저장기간이 길수록 증가하여 바람직하지 않았고, 신전성은 온도가 올라갈수록 향상되었지만 저장기간이 길어짐에 따라서 감소하였다(p<0.05). 용융성은 지방함량, 농축계수, 저장기간에 많은 영향을 받는 것으로 나타났다(p<0.05). 본 연구에서 한외여과기술로 처리된 UF-치즈유를 모짜렐라 치즈 제조에 사용하여도 기호성에 적절한 기능성 특성을 가지고 있다는 결과를 보여주었다.

• Asian-Australasian Journal of Animal Sciences
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• 제23권12호
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• pp.1566-1577
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• 2010

Nandrolone, 19-nortestosterone, is a synthetic androgenic-anabolic steroid promoting muscle growth. Nandrolone is also present in pig meat and sera at non-negligible levels. A number of scientific reports have suggested a positive relationship between incidence of infertility and increased meat consumption in humans. The present study was designed to determine out the effect of feeding nandrolone on the testis of the male reproductive tract. Mixtures of food and nandrolone at different concentrations (0.005 ppm and 0.5 ppm) were supplied to pubertal male rats for 6 weeks. Body weight was recorded every week during the entire experimental period. At the end of the treatment, the testis, epididymis, and epididymal fat were collected and weighted. Sperm numbers in the caudal epididymis were counted. Differential gene or protein expression of steroidogenic enzymes in the testes among experimental groups was determined by semi-quantitative real-time PCR or western blotting analysis, respectively. Histological changes of the testis induced by nandrolone treatment were examined by hematoxylin and eosin staining. Immunohistochemical analysis was employed to detect changes in the localization of steroidogenic enzymes in the testes among experimental animals. There were no significant changes on body, testis, epididymis, and epididymal fat weights among experimental groups. A significant increase of caudal sperm number was found in the 0.5 ppm nandrolone-treated group. Histological examination of the testes noted a high frequency of germ cell sloughing in seminiferous tubules of 0.5 ppm nandrolone-treated rats. Even though transcript levels of $3{\beta}$-hydroxysteroid dehydrogenase (HSD) I, $17{\beta}$-HSD4, and $17{\alpha}$-hydroxylase were influenced by nandrolone treatments, protein levels of all molecules examined in the present study were not significantly affected. Immunohistochemical analysis showed no visible changes in the localization of steroidogenic enzymes in the testes among experimental groups. The current study showed that oral intake of nandrolone in male rats for 6 weeks did not cause significant damage to the testis. It is considered that a feeding effect of nandrolone on male fertility would not be remarkable.

• Interdisciplinary Bio Central
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• 제1권3호
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• pp.9.1-9.6
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• 2009

Introduction: In the mass spectrometry-based proteomics, biological samples are analyzed to identify proteins by mass spectrometer and database search. Database search is the process to select the best matches to the experimental mass spectra among the amino acid sequence database and we identify the protein as the matched sequence. The match score is defined to find the matches from the database and declare the highest scored hit as the most probable protein. According to the score definition, search result varies. In this study, the difference among search results of different search engines or different databases was investigated, in order to suggest a better way to identify more proteins with higher reliability. Materials and Methods: The protein extract of human mesenchymal stem cell was separated by several bands by one-dimensional electrophorysis. One-dimensional gel was excised one by one, digested by trypsin and analyzed by a mass spectrometer, FT LTQ. The tandem mass (MS/MS) spectra of peptide ions were applied to the database search of X!Tandem, Mascot and Sequest search engines with IPI human database and SwissProt database. The search result was filtered by several threshold probability values of the Trans-Proteomic Pipeline (TPP) of the Institute for Systems Biology. The analysis of the output which was generated from TPP was performed. Results and Discussion: For each MS/MS spectrum, the peptide sequences which were identified from different conditions such as search engines, threshold probability, and sequence database were compared. The main difference of peptide identification at high threshold probability was caused by not the difference of sequence database but the difference of the score. As the threshold probability decreases, the missed peptides appeared. Conversely, in the extremely high threshold level, we missed many true assignments. Conclusion and Prospects: The different identification result of the search engines was mainly caused by the different scoring algorithms. Usually in proteomics high-scored peptides are selected and low-scored peptides are discarded. Many of them are true negatives. By integrating the search results from different parameter and different search engines, the protein identification process can be improved.

• BMB Reports
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• 제44권5호
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• pp.329-334
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• 2011

Many proteins with poor transduction efficiency were reported to be delivered to cells by fusion with protein transduction domains (PTDs). In this study, we investigated the effect of levosulpiride on the transduction of PEP-1 ribosomal protein S3 (PEP-1-rpS3), and examined its influence on the stimulation of the therapeutic properties of PEP-1-rpS3. PEP-1-rpS3 transduction into HaCaT human keratinocytes and mouse skin was stimulated by levosulpiride in a manner that did not directly affect the cell viability. Following 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice, levosulpiride alone was ineffective in reducing TPA-induced edema and in inhibiting the elevated productions of inflammatory mediators and cytokines, such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1${\beta}$, and tumor necrosis factor-${\alpha}$. Anti-inflammatory activity by PEP-1-rpS3 + levosulpiride was significantly more potent than by PEP-1-rpS3 alone. These results suggest that levosulpiride may be useful for enhancing the therapeutic effect of PEP-1-rpS3 against various inflammatory diseases.

• Journal of Yeungnam Medical Science
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• 제24권2호
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• pp.262-274
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• 2007

AdBMP-2와 AdBMP-7을 형질도입 시킨 사람 섬유아세포와 사람 골수기질세포를 면역결핍 생쥐의 척추 옆 근육으로 주입하여 척추 유합을 유도한 결과, AdBMP-7/BMSC가 AdBMP-2/BMSC 또는 AdBMP-7/HuFb와 AdBMP-2/HuFb 보다 골 형성능이 우수하였으며, 척추 유합을 잘 유도하였음을 확인하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권14호
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• pp.5815-5818
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• 2014

For an exact comparison of mRNA transcription in different samples or tissues with real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), it is crucial to select a suitable internal reference gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) have been frequently considered as house-keeping genes to normalize for changes in specific gene expression. However, it has been reported that these genes are unsuitable references in some cases, because their transcription is significantly variable under particular experimental conditions and among tissues. The present study was aimed to investigate which reference genes are most suitable for the study of gastric cancer tissues using qRT-PCR. 50 pairs of gastric cancer and corresponding peritumoral tissues were obtained from patients with gastric cancer. Absolute qRT-PCR was employed to detect the expression of GAPDH, ACTB, RPII and 18sRNA in the gastric cancer samples. Comparing gastric cancer with corresponding peritumoral tissues, GAPDH, ACTB and RPII were obviously upregulated 6.49, 5.0 and 3.68 fold, respectively. Yet 18sRNA had no obvious expression change in gastric cancer tissues and the corresponding peritumoral tissues. The expression of GAPDH, ${\beta}$-actin, RPII and 18sRNA showed no obvious changes in normal gastric epithelial cells compared with gastric cancer cell lines. The carcinoembryonic antigen (CEA), a widely used clinical tumor marker, was used as a validation gene. Only when 18sRNA was used as the normalizing gene was CEA obviously elevated in gastric cancer tissues compared with peritumoral tissues. Our data show that 18sRNA is stably expressed in gastric cancer samples and corresponding peritumoral tissues. These observations confirm that there is no universal reference gene and underline the importance of specific optimization of potential reference genes for any experimental condition.

• 대한의생명과학회지
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• 제11권2호
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• pp.165-173
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• 2005

A potent demethylating agent, 5-Azacytidine (5-AzaC) has been widely used as in many studies on DNA methylation, regulation of gene expression, and cancer biology. The mechanisms of the demethylating activity were known to be formation of complex between DNA and DNA methyltransferase (MTase), which depletes cellular MTase activity. However, 5-AzaC can also induce hypermethylation of a transgene in a transgenic cell line, G12 cells and it was explained as a result of defense mechanisms to inactivate foreign gene(s) somehow. This finding evoked the question that whether the phenomenon of hypermethylation induced by 5-AzaC is limited to the transgene or it can be occurred in endogenous gene(s). In order to answer the question, mutagenicity test of 5-AzaC and molecular characterization of mutants obtained from the test were performed using an endogenous gene, thymidine kinase (tk) in Chinese hamster V79 cells. When V79 and V79-J3 subclone cells were treated with 1, 2.5 ,5, $10{\mu}M$ of 5-AzaC for 48 hours, their maximum mutant frequencies were revealed as $6\times10^{-3}\;at\;5{\mu}M$(350-fold induction over background) and $8\times10^{-3}\;at\;2.5{\mu}M$ (l,800-fold induction over background) respectively. Since the induction rates were too high to be induced by true mutations, many trifluorothymidine (TFT)-resistant $(TFT^R)$ cells were subjected to Northern blot analysis to check the presence of tk transcripts. Surprisingly, all clones tested possessed the transcripts in a similar level, that implicates the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the gene in spite of unusually high mutation frequency. In addition, it has shown that the TK activity in the pool of 5-AzaC-induced $TFT^R$ cells has about a half of that in spontaneously-induced $TFT^R$ cells or in non-selected parental V79-J3 cells. This result suggests that the mechanism(s) underlying the TFT-resistance between spontaneously occurred and 5-AzaC-induced cells may be different. These findings have shown that the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the tk gene, and 5-AzaC may be induced by one or combined pathways among many drug resistance mechanisms. The exact mechanisms for the 5-AzaC-induced $TFT^R$ phenotype remain to elucidate.