• Clinical and Experimental Reproductive Medicine
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• v.38 no.2
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• pp.68-74
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• 2011

Objective: Previously, we found that oocyte specific homeobox (Obox) 4 plays significant role in completion of meiosis specifically at meiosis I-meiosis II (MI-MII) transition. The purpose of this study was to determine the mechanism of action of $Obox4$ in oocyte maturation by evaluating downstream signal networking. Methods: The $Obox4$ dsRNA was prepared by $in$ $vitro$ transcription and microinjected into the cytoplasm of germinal vesicle oocytes followed by $in$ $vitro$ maturation in the presence or absence of 0.2 mM 3-isobutyl-1-metyl-xanthine. Total RNA was extracted from 200 oocytes of each group using a PicoPure RNA isolation kit then amplified two-rounds. The probe hybridization and data analysis were used by Affymetrix Gene-Chip$^{(R)}$ Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. Results: Total 424 genes were up (n=80) and down (n=344) regulated after $Obox4$ RNA interference (RNAi). Genes mainly related to metabolic pathways and mitogen-activated protein kinase (MAPK) signaling pathway was changed. Among the protein kinase C (PKC) isoforms, PKC-alpha, beta, gamma were down-regulated and especially the MAPK signaling pathway PKC-gamma was dramatically decreased by $Obox4$ RNAi. In the cell cycle pathway, we evaluated the expression of genes involved in regulation of chromosome separation, and found that these genes were down-regulated. It may cause the aberrant chromosome segregation during MI-MII transition. Conclusion: From the results of this study, it is concluded that $Obox4$ is important upstream regulator of the PKC and anaphase-promoting complex action for maintaining intact germinal vesicle.

• Animal Bioscience
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• v.34 no.11
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• pp.1766-1775
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• 2021

Objective: The oxidative stress status and changes of chicken ovary tissue after shading were studied, to determine the mechanism of the effect of shading on follicular development. Methods: Twenty healthy laying hens (40 weeks old) with uniform body weight and the same laying rate were randomly divided into two groups (the shading group and normal light group). In the shading group, the cage was covered to reduce the light intensity inside the cage to 0 without affecting ventilation or food intake. The normal lighting group received no additional treatment. After 7 days of shading, oxidative stress related indicators and gene expression were detected. Results: Analysis of paraffin and ultrathin sections showed that apoptosis of ovarian granulosa cells (GCs) increased significantly after light shading. Enzyme linked immunosorbent assay results revealed that the levels of total antioxidant capacity, malondialdehyde, superoxide dismutase (SOD), glutathione, catalase (CAT), and other substances in the sera, livers, ovaries, and follicular GCs of laying hens increased significantly after shading for 7 days; and reactive oxygen species (ROS) levels in the livers of laying hens also increased significantly. ROS in the serum, ovarian and GCs also increased. After shading for 7 days, the levels of 8-hydroxy-2 deoxyguanosine in the sera and ovarian tissues of laying hens increased significantly. Cell counting kit-8 detection showed that the proliferation activity of GCs in layer follicles decreased after shading for 7 days; the expression level of the anti-apoptotic gene B-cell lymphoma-2 in ovarian tissue and follicular GCs was significantly reduced, and the expression levels of pro-apoptotic caspase 3 (casp3), and SOD, glutathione peroxidase 2 (GPX2), and CAT were all significantly increased. Conclusion: Oxidative stress induced by shading light has a serious inhibitory effect on follicular development during reproduction in laying hens.

• Genomics & Informatics
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• v.20 no.3
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• pp.30.1-30.9
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• 2022

Hereditary spastic paraplegia is a not common inherited neurological disorder with heterogeneous clinical expressions. ALDH18A1 (located on 10q24.1) gene-related spastic paraplegias (SPG9A and SPG9B) are rare metabolic disorders caused by dominant and recessive mutations that have been found recently. Autosomal recessive hereditary spastic paraplegia is a common and clinical type of familial spastic paraplegia linked to the SPG11 locus (locates on 15q21.1). There are different symptoms of spastic paraplegia, such as muscle atrophy, moderate mental retardation, short stature, balance problem, and lower limb weakness. Our first proband involves a 45 years old man and our second proband involves a 20 years old woman both are affected by spastic paraplegia disease. Genomic DNA was extracted from the peripheral blood of the patients, their parents, and their siblings using a filter-based methodology and quantified and used for molecular analysis and sequencing. Sequencing libraries were generated using Agilent SureSelect Human All ExonV7 kit, and the qualified libraries are fed into NovaSeq 6000 Illumina sequencers. Sanger sequencing was performed by an ABI prism 3730 sequencer. Here, for the first time, we report two cases, the first one which contains likely pathogenic NM_002860: c.475C>T: p.R159X mutation of the ALDH18A1 and the second one has likely pathogenic NM_001160227.2: c.5454dupA: p.Glu1819Argfs Ter11 mutation of the SPG11 gene and also was identified by the whole-exome sequencing and confirmed by Sanger sequencing. Our aim with this study was to confirm that these two novel variants are direct causes of spastic paraplegia.

• Journal of Animal Science and Technology
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• v.64 no.4
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• pp.792-799
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• 2022

Dopamine (DA) is known to be a key modulator of animal behaviors. Thus, the plasma concentration of DA might be used as a biomarker for the behavioral characteristics of horses. The behavioral characteristics of horses vary depending on the breed, age, and sex. Moreover, the DA receptor genotypes are also related to horse behaviors. Thus, the aim of this study was to investigate the DA concentration variations of horse plasma by breed, age, sex, or genotype of its receptor. The horses were divided by breed into Thoroughbred (n = 13), Pony (n = 9), Warmblood (n = 4), and Haflinger (n = 5). The age variable was divided into three different groups: post-pubertal (2-5 years, n = 6), adult (6-13 years, n = 19), and aged horses (15-24 years, n = 6). The sex variable was divided into geldings (n = 8) and mares (n = 23). Approximately 10 mL of blood was collected, and an ELISA kit was used to measure the plasma concentration of DA. Polymerase chain reaction analysis was performed to identify the genetic variation in the DA D4 receptor gene (DRD4). SPSS statistical software was used for statistical analysis. The DA concentrations in geldings were significantly lower than those in mares. There was no significant difference in DA concentrations among breed and age groups. Horses with the GG and GA genotypes had significantly higher plasma concentrations of DA compared to horses with the AA genotype for the G292A gene. Briefly, the plasma concentration of DA varied depending on the sex and genotype of G292A. These factors should be considered when the concentration of DA is used as a biomarker for the behavioral characteristics of horses. In conclusion, the DA concentration or DRD4 genotype of horse plasma has the potential to be used as a biomarker that can predict the behavioral characteristics of horses.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.85-85
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• 2003

Human lactoferrin (hLF) is an 80 kDa iron-binding glycoprotein that is expressed in high concentration in milk and in lesser amount in the secondary or specific granules of neutrophils and in plasma, LF is classically considered to be related to the binding, transport, and storage of iron. The transgenic mice carrying the human hLF gene in conjunction with the bovine $\beta$-casein promoter produced the human hLF in their milk during lactation. To screen transgenic mice, PCR was carried out using chromosomal DNA extracted from tail or toe tissues. In this study, stability of germ line transmission and expression of hLF were monitored up to generation Fl7 of a transgenic line. When female mouse of generation F9 was crossbred with normal male, generation F9 to Fl7 mice showed similar transmission rates ($66.0 \pm 12.57%, 42.0 \pm 14.98%, 72.2 \pm 25.45%, 50.0 \pm 16.70%, 65.7 \pm 6.45%, 48.6 \pm 14.65%, 54 1 \pm 18 11%, 57.8 \pm 16.16% and 48.6 \pm 20.66$, respectively), implying that the hLF gene can be transmitted stably up to long term generation in the transgenic mice For ELISA analysis, hLF expression levels were determined with an hLF ELISA kit in accordance with the supplier's protocol. Expression levels of human hLF from milk of generation F9 to Fl3 mice were $ 3.2 \pm 0.69 mg/ml, 3.1 \pm 0.81 mg/ml, 4.6 \pm 1.38 mg/ml, 3.1 \pm 0.42 mg/ml, and 4.5 \pm 1,48 mg/ml$, respectively. These expression levels were lower than that of founder (6.6 mg/$m\ell$) mouse. We concluded that transgenic mice faithfully passed the transgene on their progeny and successively secreted target proteins into their milk through several generations.

• Journal of Genetic Medicine
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• v.5 no.2
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• pp.105-110
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• 2008

Purpose: Preecalmpsia is a pregnancy-specific disorder that reflects widespread endothelial dysfunction resulting from increases of adhesion molecule expression. Intercellular adhesion molecule-1 (ICAM-1) is involved in the pathogenetic mechanisms responsible for preeclampsia, and ICAM-1 plasma levels and/or function is genetically influenced. Therefore, we evaluated the distribution of ICAM-1 gene K469E polymorphism in pregnant Korean women with preeclampsia and evaluated the association between this polymorphism and preeclampsia. Methods: The K469E polymorphism was analyzed in peripheral blood samples from 197 preeclamptic pregnancies and 193 normotensive pregnancies by a SNapShot kit and an ABI Prism 3100 Genetic analyzer. Results: Genotypic and allelic frequencies of ICAM-1 gene polymorphism (K469E) did not differ between preeclamptic and normotensive pregnancies. The distributions of the KK, KE, and EE genotypes were 40.6%, 43.7%, and 15.7%, respectively, in preeclamptic pregnancies and 38.9%, 45.1%, and 16.1%, respectively, in normotensive pregnancies. The frequencies of K and E alleles were 0.62 and 0.38, respectively, in preeclamptic pregnancies and 0.61 and 0.39, respectively, in normotensive pregnancies. By multiple logistic regression analysis, there was no increased risk of preeclampsia in subjects with ICAM-1 KE (OR, 1.08; P=0.74) or EE (OR, 1.07; P=0.88) genotypes. Conclusion: This study suggests that the ICAM-1 gene K469E polymorphism does not associate with an increased risk of preeclampsia in pregnant Korean women.

• Tuberculosis and Respiratory Diseases
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• v.55 no.4
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• pp.344-352
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• 2003

Background : The prevalence of multidrug resistant tuberculosis (MDR-TB), resistant to isoniazid (INH) and rifampin (RFP), was 5.3% worldwide in 1995 and its increment has raised important public health problems. Resistance to RFP, one of the key drugs in the treatment of tuberculosis, results in grim clinical outcome. Recently rapid detection of RFP-resistant mutations in rpoB gene based on PCR method has become available. This study evaluated the prevalence of RFP resistance in first diagnosed, treatment failure, and recurred patients using INNO-LiPA test, and compared the results of INNO-LiPA with those of conventional mycobacterial drug susceptibility test. Methods : Forty-six patients, who were diagnosed of pulmonary tuberculosis and had revealed positive sputum AFB smear, were enrolled in this study from 1998 to 2002. The cases were classified as one three groups; first diagnosed, treatment failure, or recurred. RFP resistance was studied using an INNO-LiPA Rif. TB kit and compared with that obtained from drug susceptibility based on M. tuberculosis culture study. Results : Twenty-one out of 46 patients were enrolled under first diagnosis of pulmonary tuberculosis, 17 under treatment failure with first line drugs, and 8 under recurrence. The positive and negative predictive values of INNO-LiPA test in diagnosis in RFP resistant tuberculosis compared with conventional mycobacterial drug susceptibility test were 85.7% and 76.0%, respectively. INNO-LiPA result revealed rpoB gene mutation in 20 (80.0%) out of 25 patients who were diagnosed as treatment failure or recurrence, but in only 4 (19.0%) out of 21 patients who were first diagnosed as pulmonary tuberculosis. Conclusion : This study showed that RFP resistance could be diagnosed rapidly and accurately using INNO-LiPA test and that this test might be helpful for choosing second line anti-mycobacterial drugs. It might be of great help in clinical diagnosis and decision when used in complimentarily with drug susceptibility test based on M. tuberculosis culture.

• Animal Bioscience
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• v.36 no.9
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• pp.1336-1349
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• 2023

Objective: The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation. Methods: The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8). Results: A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC]=0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes. Conclusion: Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.

• Journal of Microbiology and Biotechnology
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• v.21 no.1
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• pp.100-108
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• 2011

A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 ($TCID_{50}$) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.

• Journal of Industrial Technology
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• v.28 no.A
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• pp.141-147
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• 2008

Several contaminated bacteria such as Lactobacillus brevis and Pediococcus damnosus in beer production cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. Recently, two contaminated strains were isolated from vessel of beer production and identified as Lactobacillus species by API kit identificaton as well as 16S-23S ITS sequencing analyses. Two isolated strains were named as Lactobacillus sp. HLA1 and Lactobacillus HLB2, respectively. A polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Lactobacillus sp.. Two sets of primer pairs (HLA1-F/HLA1-R and HLB2-F/HLB2-R) were designed for the amplification of a 1576 base pair (bp) fragment of the HLA1 16S-23S rRNA gene and 1888 bp fragement of the HLB2 16S-23S rRNA. Amplified PCR products were highly specific to detect corresponding bacteria when other contaminated strains were used as PCR templates. However, detection of both strains were limited when $100{\mu}{\ell}$ of cultured samples were mixed with $100m{\ell}$ of beer sample in arbitrary manner. The sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.