• Animal Bioscience
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• v.35 no.9
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• pp.1327-1339
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• 2022

Objective: Fat deposition in poultry is an important factor in production performance and meat quality research. miRNAs also play important roles in regulating adipocyte differentiation process. This study was to investigate the expression patterns of miRNAs in duck adipocytes after differentiation and explore the role of miR-214 in regulating carnitine palmitoyltransferases 2 (CPT2) gene expression during duck adipocyte differentiation. Methods: Successful systems for the isolation, culture, and induction of duck primary fat cells was developed in the experiment. Using Illumina next-generation sequencing, the miRNAs libraries of duck adipocytes were established. miRanda was used to predict differentially expressed (DE) miRNAs and their target genes. The expression patterns of miR-214 and CPT2 during the differentiation were verified by quantitative real-time polymerase chain reaction and western blot. Luciferase reporter assays were used to explore the specific regions of CPT2 targeted by miR-214. We used a miR-214 over-expression strategy in vitro to further investigate its effect on differentiation process and CPT2 gene transcription. Results: There were 481 miRNAs identified in duck adipocytes, included 57 DE miRNA candidates. And the 1,046 targets genes of DE miRNAs were mainly involved in p53 signaling, FoxO signaling, and fatty acid metabolism pathways. miR-214 and CPT2 showed contrasting expression patterns before and after differentiation, and they were selected for further research. The expression of miR-214 was decreased during the first 3 days of duck adipocytes differentiation, and then increased, while the expression of CPT2 increased both in the transcriptional and protein level. The luciferase assay suggested that miR-214 targets the 3'untranslated region of CPT2. Overexpression of miR-214 not only promoted the formation of lipid droplets but also decreased the protein abundance of CPT2. Conclusion: Current study reports the expression profile of miRNAs in duck adipocytes differentiated for 4 days. And miR-214 has been proved to have the regulator potential for fat deposition in duck.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.11
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• pp.1562-1570
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• 2014

The effects of Lactobacillus mucosae (L. mucosae), a potential direct fed microbial previously isolated from the rumen of Korean native goat, on the rumen fermentation profile of brewers grain were evaluated. Fermentation was conducted in serum bottles each containing 1% dry matter (DM) of the test substrate and either no L. mucosae (control), 1% 24 h broth culture of L. mucosae (T1), or 1% inoculation with the cell-free culture supernatant (T2). Each serum bottle was filled anaerobically with 100 mL of buffered rumen fluid and sealed prior to incubation for 0, 6, 12, 24, and 48 h from which fermentation parameters were monitored and the microbial diversity was evaluated. The results revealed that T1 had higher total gas production (65.00 mL) than the control (61.33 mL) and T2 (62.00 mL) (p<0.05) at 48 h. Consequently, T1 had significantly lower pH values (p<0.05) than the other groups at 48 h. Ammonia nitrogen ($NH_3$-N), individual and total volatile fatty acids (VFA) concentration and acetate:propionate ratio were higher in T1 and T2 than the control, but T1 and T2 were comparable for these parameters. Total methane ($CH_4$) production and carbon dioxide ($CO_2$) were highest in T1. The percent DM and organic matter digestibilities were comparable between all groups at all times of incubation. The total bacterial population was significantly higher in T1 (p<0.05) at 24 h, but then decreased to levels comparable to the control and T2 at 48 h. The denaturing gradient gel electrophoresis profile of the total bacterial 16s rRNA showed higher similarity between T1 and T2 at 24 h and between the control and T1 at 48 h. Overall, these results suggest that addition of L. mucosae and cell-free supernatant during the in vitro fermentation of dried brewers grain increases the VFA production, but has no effect on digestibility. The addition of L. mucosae can also increase the total bacterial population, but has no significant effect on the total microbial diversity. However, inoculation of the bacterium may increase $CH_4$ and $CO_2$ in vitro.

• BMB Reports
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• v.38 no.6
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• pp.668-675
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• 2005

A full-length cDNA encoding taxadiene synthase (designated as TmTXS), which catalyzes the first committed step in the Taxol biosynthetic pathway, was isolated from young leaves of Taxus media by rapid amplification of cDNA ends (RACE). The full-length cDNA of TmTXS had a 2586 bp open reading frame (ORF) encoding a protein of 862 amino acid residues. The deduced protein had isoelectric point (pI) of 5.32 and a calculated molecular weight of about 98 kDa, similar to previously cloned diterpene cyclases from other Taxus species such as T. brevifolia and T. chinenisis. Sequence comparison analysis showed that TmTXS had high similarity with other members of terpene synthase family of plant origin. Tissue expression pattern analysis revealed that TmTXS expressed strongly in leaves, weak in stems and no expression could be detected in fruits. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in Taxol biosynthetic pathway in different tissues of Taxus plants. Phylogenetic tree analysis showed that TmTXS had closest relationship with taxadiene synthase from T. baccata followed by those from T. chinenisis and T. brevifolia. Expression profiles revealed by RT-PCR under different chemical elicitor treatments such as methyl jasmonate (MJ), silver nitrate (SN) and ammonium ceric sulphate (ACS) were also compared for the first time, and the results revealed that expression of TmTXS was all induced by the tested three treatments and the induction effect by MJ was the strongest, implying that TmTXS was high elicitor responsive.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.23 no.4
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• pp.306-323
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• 2001

종양발생에서 유전자 발현을 확인하고 profile 변화를 monitor하는 것은 병리학적 변화의 원인뿐 아니라 질병탐지와 진료의 새로운 목표를 확인하기 위한 새로운 기회를 제공해준다. cDNA microarray는 수천개의 유전자 발현을 동시에 연구할 수 있는 최신의 방법으로 피부, 유방, 간을 비롯한 다른 인체장기에서는 일부 이루어졌으나 array를 이용해 타액선 종양 연구에서는 전혀 이루어지지 않았다. 인간의 타액선 세포의 악성형질전환을 조절하는 분자적 상태를 연구하기 위해 본 연구는 약 2,000개의 유전자가 print된 cDNA microarray를 이용하여 인간 타액선 도관상피세포주(HSG)와 악하선에서 기원한 미분화 선암종(SGT)간에 비교연구를 하였다. Cy3와 Cy5 dye로 각각의 세포주에서 얻은 RNA와 reciprocal hybridize시키고 GenePix 4000 scanner로 스캔하고 GenePix Pro로 분석한 후 log2로 평균발현비율을 전환시켜 최소 2배이상의 발현을 보이는 유전자를 분석대상으로 하였다. 90%이상의 유전자가 비슷한 발현을 보였으며 2배이상의 발현을 보이는 경우 HSG가 SGT에 비해 72개 유전자가, SGT가 HSG에 비해 111개의 유전자 발현이 up-regulation되어 총 10%미만의 발현차이를 보였고 반복된 hybridization 으로부터 얻은 선택된 spot의 Pearson 상관계수는 -0.85이였다. HSG에서는 6번 p 염색체에서 과발현되는 유전자가 가장 많았고, SGT에서는 11번 q 염색체에서 가장 많았는데 HSG에서는 SGT에 비해 9, 13, 17, 18, 20, 21, 22염색체에서 과발현 되는 유전자 수가 많았고, SGT에서는 HSG에 비해 2, 7, 10, 15 염색체에서 유전자 발현 증가가 관찰되었다. HSG와 SGT간의 유전 발현을 기능별로 분석한 결과 몇 가지 주요 경로가 세포악성에 관련됨을 발견하였고, 타액선 도관상피세포에서 선암종을 구별하는데 기여하는 관련된 몇종의 과다 발현된 유전자를 찾았는데 전사인자, 성장인자 및 수용기, 세포골격 및 세포외기질 단백, 세포내 신호전달조절자 및 인자, 세포표면 항원등의 그룹으로 분류할 수 있었다. 따라서 이러한 microarray를 이용한 분자학적 표지자 연구가 악성 타액선 종양 발생과정에서 큰 도움을 줄 수 있을 뿐 아니라 유전자 조절에 의한 진단, 예후, 치료에서의 정확성을 개선시킬 수 있으리라 여겨진다.

• Journal of Ecology and Environment
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• v.41 no.9
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• pp.271-280
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• 2017

Background: Soil microorganisms play key roles in nutrient cycling and are distributed throughout the soil profile. Currently, there is little information about the characteristics of the microbial communities along the soil depth because most studies focus on microorganisms inhabiting the soil surface. To better understand the functions and composition of microbial communities and the biogeochemical factors that shape them at different soil depths, we analyzed microbial activities and bacterial and fungal community composition in soils up to a 120 cm depth at a fallow field located in central Korea. To examine the vertical difference of microbial activities and community composition, ${\beta}$-1,4-glucosidase, cellobiohydrolase, ${\beta}$-1,4-xylosidase, ${\beta}$-1,4-N-acetylglucosaminidase, and acid phosphatase activities were analyzed and barcoded pyrosequencing of 16S rRNA genes (bacteria) and internal transcribed spacer region (fungi) was conducted. Results: The activity of all the soil enzymes analyzed, along with soil C concentration, declined with soil depth. For example, acid phosphatase activity was $125.9({\pm}5.7({\pm}1SE))$, $30.9({\pm}0.9)$, $15.7({\pm}0.6)$, $6.7({\pm}0.9)$, and $3.3({\pm}0.3)nmol\;g^{-1}\;h^{-1}$ at 0-15, 15-30, 30-60, 60-90, and 90-120 cm soil depths, respectively. Among the bacterial groups, the abundance of Proteobacteria (38.5, 23.2, 23.3, 26.1, and 17.5% at 0-15, 15-30, 30-60, 60-90, and 90-120 cm soil depths, respectively) and Firmicutes (12.8, 11.3, 8.6, 4.3, and 0.4% at 0-15, 15-30, 30-60, 60-90, and 90-120 cm soil depths, respectively) decreased with soil depth. On the other hand, the abundance of Ascomycota (51.2, 48.6, 65.7, 46.1, and 45.7% at 15, 30, 60, 90, and 120 cm depths, respectively), a dominant fungal group at this site, showed no clear trend along the soil profile. Conclusions: Our results show that soil C availability can determine soil enzyme activity at different soil depths and that bacterial communities have a clear trend along the soil depth at this study site. These metagenomics studies, along with other studies on microbial functions, are expected to enhance our understanding on the complexity of soil microbial communities and their relationship with biogeochemical factors.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.1
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• pp.19-31
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• 2007

Objective: Pathogenesis of the endometriosis is very complex and the etiology is still unclear. Our hypothesis is that there may be some difference in gene expression patterns between eutopic endometriums with or without endometriosis. In this study, we analyzed the difference of gene expression profile with cDNA microarray. Methods: Endometrial tissues were gathered from patients with endometriosis or other benign gynecologic diseases. cDNA microarray technique was applied to screen the different gene expression profiles from early and late secretory phase endometria of those two groups. Each three mRNA samples isolated from early and late secretory phase of endometrial tissues of control were pooled and used as master controls and labeled with Cy3-dUTP. Then the differences of gene expression pattern were screened by comparing eutopic endometria with endometriosis, which were labeled with Cy5-dUTP. Fluorescent labeled probes were hybridized on a microarray of 4,800 human genes. Results: Twelve genes were consistently over-expressed in the endometrium of endometriosis such as ATP synthase H transporting F1 (ATP5B), eukaryotic translation elongation factor 1, isocitrate dehydrogenase 1 (NADP+), mitochondrial ribosomal protein L3, ATP synthase H+ transporting (ATP5C1) and TNF alpha factor. Eleven genes were consistently down-regulated in the endometriosis samples. Many extracellular matrix protein genes (decorin, lumican, EGF-containing fibulin-like extracellular matrix protein 1, fibulin 5, and matrix Gla protein) and protease/protease inhibitors (serine proteinase inhibitor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 1), and insulin like growth factor II associated protein were included. Expression patterns of selected eight genes from the cDNA microarray were confirmed by quantitative RT-PCR or real time RT-PCR. Conclusion: The result of this analysis supports the hypothesis that the endometrium from patients with endometriosis has distinct gene expression profile from control endometrium without endometriosis.

• Parasites, Hosts and Diseases
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• v.54 no.1
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• pp.1-8
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• 2016

Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, $RIDA^{(R)}$ Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

• Molecules and Cells
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• v.38 no.9
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• pp.765-772
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• 2015

Saussurea lappa has been reported to possess anti-atopic properties. In this study, we have confirmed the S. lappa's anti-atopic properties in Nc/Nga mice and investigated the candidate gene related with its properties using microarray. We determined the target gene using real time PCR in in vitro experiment. S. lappa showed the significant reduction in atoptic dermatitis (AD) score and immunoglobulin E compared with the AD induced Nc/Nga mice. In the results of microarray using back skin obtained from animals, we found that S. lappa's properties are closely associated with cytokine-cytokine receptor interaction and the JAK-STAT signaling pathway. Consistent with the microarray data, real-time RT-PCR confirmed these modulation at the mRNA level in skin tissues from S. lappa-treated mice. Among these genes, PI3Kca and $IL20R{\beta}$ were significantly downregulated by S. lappa treatment in Nc/Nga mouse model. In in vitro experiment using HaCaT cells, we found that the S. lappa components, including alantolactone, caryophyllene, costic acid, costunolide and dehydrocostus lactone significantly decreased the expression of PI3Kca but not $IL20R{\beta}$ in vitro. Therefore, our study suggests that PI3Kca-related signaling is closely related with the protective effects of S. lappa against the development of atopic-dermatitis.

• BMB Reports
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• v.43 no.5
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• pp.349-354
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• 2010

Previously, we reported that overexpression of Opa (Neisseria gonorrhoeae opacity-associated)-interacting protein 5 (OIP5) caused multi-septa formation and growth defects, both of which are considered cancer-related phenotypes. To evaluate OIP5 as a possible cancer therapeutic target, we examined its expression level in 66 colorectal cancer patients. OIP5 was upregulated about 3.7-fold in tumors and over 2-fold in 58 out of 66 colorectal cancer patients. Knockdown of OIP5 expression by small interfering RNA specific to OIP5 (siOIP5) resulted in growth inhibition of colorectal and gastric cancer cell lines. Growth inhibition of SNU638 by siOIP5 caused an increase in sub-G1 DNA content, as measured by flow cytometry, as well as an apoptotic gene expression profile. These results indicate that knockdown of OIP5 may induce apoptosis in cancer cells. Therefore, we suggest that OIP5 might be a potential cancer therapeutic target, although the mechanisms of OIP5-induced carcinogenesis should be elucidated.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.75-75
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• 2003

Transition of the resting primordial follicle to the growing primary follicle is a critical process for female reproduction, but its mechanism is poorly understood. The present study was conducted to investigate gene expression profile at the primordial-primary follicle transition process. We isolated total RNA of female mouse ovary at day1 (contains only primordial follicles) and day5 (contains primordial and primary follicles) and synthesized cDNA using annealing control primers (ACP; Seegene, Inc., Seoul, Korea). ACP provides annealing specificity and sensitivity to the template and allows to identify only authentic differentially expressed genes (DEGs). We used total 80 ACPs for PCR, observed PCR products on 2% agarose gel, cloned 42 DEGs using TOPO TA cloning vector, sequenced, and analyzed by BLAST search. Sequences of 34 clones significantly matched database entries while 4 clones were novel and 4 clones were EST. Two of 34 genes were specifically expressed only in day 5 ovaries (Sui1-rs1, Apg3p/Aut1p-like), and rest of 32 genes were expressed in both stages but were differential in amount. Differential expression was confirmed using semiquantitative RT-PCR, and there was no false positive. Anx11 and Pepp2-pending were highly expressed genes in day1-, while BPOZ, Ches1, Kcmf1, NHE3, Nid2, Ninj1, SENP3 and Survivin were highly expressed genes in day5-ovary. List of genes would provide insight for further study of mechanism regulating primordial-primary follicle transition.