• Asian-Australasian Journal of Animal Sciences
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• v.33 no.2
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• pp.219-229
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• 2020

Objective: Considering the physiological and clinical importance of leptin receptor (LEPR) in regulating obesity and the fact that porcine LEPR expression is not known to be controlled by lncRNAs and miRNAs, we aim to characterize this gene as a potential target of SSC-miR-323 and the lncRNA TCONS_00010987. Methods: Bioinformatics analyses revealed that lncRNA TCONS_00010987 and LEPR have SSC-miR-323-binding sites and that LEPR might be a target of lncRNA TCONS_00010987 based on cis prediction. Wild-type and mutant TCONS_00010987-target sequence fragments and wild-type and mutant LEPR 3'-UTR fragments were generated and cloned into pmiRRB-REPORT^TM-Control vectors to construct respective recombinant plasmids. HEK293T cells were co-transfected with the SSC-miR-323 mimics or a negative control with constructs harboring the corresponding binding sites and relative luciferase activities were determined. Tissue expression patterns of lncRNA TCONS_00010987, SSC-miR-323, and LEPR in Anqing six-end-white (AQ, the obese breed) and Large White (LW, the lean breed) pigs were detected by real-time quantitative polymerase chain reaction; backfat expression of LEPR protein was detected by western blotting. Results: Target gene fragments were successfully cloned, and the four recombinant vectors were constructed. Compared to the negative control, SSC-miR-323 mimics significantly inhibited luciferase activity from the wild-type TCONS_00010987-target sequence and wild-type LEPR-3'-UTR (p<0.01 for both) but not from the mutant TCONS_00010987-target sequence and mutant LEPR-3'-UTR (p>0.05 for both). Backfat expression levels of TCONS_00010987 and LEPR in AQ pigs were significantly higher than those in LW pigs (p<0.01), whereas levels of SSC-miR-323 in AQ pigs were significantly lower than those in LW pigs (p<0.05). LEPR protein levels in the backfat tissues of AQ pigs were markedly higher than those in LW pigs (p<0.01). Conclusion: LEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.245-255
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• 2004

In recent years, an increasing number of studies on pig in vitro maturation(IVM) and in vitro fertilization(IVF) have been separated. the wide range of new technologies, including that in applied molecular genetics, has increased this interest. the production of viable porcine embryos in vitro is a prerequisites for the successful production of transgenic pigs to date. The efficiency of IVM/IVF techniques in the porcine is lower than that obtained in other species such as cattle and mouse. The several problems are generally thought to be the cause of poor results: the low rate of MPN formation derived from inadequate IVM of oocytes, the high incidence of polyspermy after IVF and cell blocking at 4 cell during embryos culture. For there reasons overcoming, many studies have been conducted to improve in vitro embryo-genic competence of oocytes. In the last several years, many maturation culture media have been evaluated and various exogenous factors such as hormones and grows factors have been tested to improve the efficiency of porcine in vitro system. In the study several antioxidants have been examined to improve in vitro fertilization and development of porcine oocytes. In this study, several antioxidants were examined to determine the effects on the development of oocytes to the cleavage, morula and blastocyst stage when added at the maturation(IVM) or in vitro fertilization(IVF) or in vitro culture(IVC) of porcine embryos. Porcine oocytes were matured, fertilized and embryos were cultured in defind conditioned medium in vitro with or without supplementation with the antioxidents of cysteine, catalase and glutathione. 1. Significant improvement of blastocyst rate (27.2％ versus 15.4％, p<0.05) were achieved when catalase(500U/$m\ell$) were added to TCM-199 medium and morula rate(72.0％ versus 53.9％, p<0.05) were significantly higher when glutathione(1.0mM/$m\ell$) were added to TCM-199 medium than those of control. 2. In mTBM medium for oocytes fertilization, the addition of cysteine, catalase and glutathione had no positive effect on embryonic development. glutathione had no positive effect on embryonic development. In conclusion, this study shows that addition of catalase, gluththione during IVM improved the rate of porcine embryo development.

• Korean Journal of Poultry Science
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• v.30 no.4
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• pp.245-251
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• 2003

Three experiments were conducted to investigate the effect of feeding yeast accumulated transgenic ferritin(FRT, Saccharomyces cerevisiae) as a probiotic on the performance, iron contents in the liver, spleen, bone and yolk of laying hens and broiler chicks. Effects of feeding FRT were compared with that of feeding wild-type yeast(W0) and yeast grown on 20 mM ferric citrate-added medium (W20). In Expt 1, to investigate the effect of feeding yeast (control, W0 FRT) on performance and iron content of organs of broiler chicks which were fed basal diet supplemented with 75mg/kg iron(Fe75) or not (Fe0), three hundred sixty one-day-old male broiler chicks were fed a corn-sov based diet for five weeks. Weight gain, feed intake and feed conversion were measured weekly. In Expt 2, fifteen 33-week-old ISA Brown laying hens were placed in individual cages and were fed control, W0 and FRT diets for Four weeks. In Expt 3, twenty four 45-week-old ISA Brown laying hens were placed in individual cages and were fed a basal diet for a week. Then, experimental diets (control, W0, W20, FRT) were fed for three weeks. Iron contents in the liver, heart, spleen and tibia were determined at the end of all experiments. Iron content in yolk was measured weekly (expt 2, 3). The level of yeast added and iron concentration of FRT were $1{\times}10^8$cfu/kg diet and 500 mg/kg cell (DM) respectively in Expt 3, yeast was supplemented at $2{\times}10^{10}$cfu/kg diet and the iron content of FRT was 1000mg/kg cell (DM). In Expt 1. birds fed Fe75 showed significantly higher weight gain compared with Fe0 (P<0.05). However, weight gain and feed intake of birds fed FRT was significantly lower than control (P<0.05). In Expt 2, the iron content of the liver was decreased in the FRT treatment (P<0.05). In Expt 3, iron concentration of the liver and spleen tended to be increased by feeding FRt. However, the iron content of the tibia tended to be decreased in the FRT treatment. These results suggest that feeding FRT as a probiotic cannot improve performance and iron content in organs of broiler chicks and laying hens.

• The Korean Journal of Pharmacology
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• v.29 no.1
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• pp.139-148
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• 1993

Both SCC 12 and SCC 13 cell lines were derived from squamous cell carcinoma (SCC) of the skin (Wu and Rheinwald, 1981). In the present study, we compared the inherent invasive activity in their raft cultures where most in vivo characteristics of epidermis can be reproduced by cell culture method. The raft culture of SCC 12 cell line produced many invading colonies within the collagen lattice and basal-like cells in the middle of differentiating cell layers, but no invasive activity was observed in the SCC 13 raft culture. We investigated which factors are implicated in inherent invasive activity of SCC 12 cell line by examining basal levels of type I collagenase, EGF receptor, fibronectin, and its receptor in two cell lines. Among them, only type I collagenase was significantly higher in invasive SCC 12 cells than in non-invasive SCC 13 cells. Furthermore, we tried to investigate mechanisms underlying between SCC 12 cell's inherent invasive activity and its high basal level of type I collagenase. As one of them, discrepancy in TGF alpha mediated responses between two cell lines was observed. In SCC 13 cells, TGF alpha initially stimulated type I collagenase at 12 h after TGF alpha treatment and then its down regulation was followed from 24 h even though TGF alpha was continuously present in the medium. However in SCC 12 cells, TGF alpha continuously stimulated type I collegenase up to 48 h. We propose that defect in EGF receptor's down-regulation may be involved in lack of type I collagenase's down-regulation and its possible connection to invasive activity of SCC 12 cell line.

• Tuberculosis and Respiratory Diseases
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• v.47 no.3
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• pp.347-355
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• 1999

Background: Phospholipase C(PLC) plays a central role in cellular signal transduction and is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC reported to this date. Hydrolysis of phosphatidylinositol 4,5-bisphosphate($PIP_2$) by PLC produces two important second messengers, inositol 1,4,5-trisphosphate($IP_3$) and diacylglycerol. PLC-${\gamma}1$, previously, was known to be activated mainly through growth factor receptor tyrosine kinase. Other mechanisms of activating PLC-yl have been reported such as activation through tau protein in the presence of arachidonic acid in bovine brain and activation by $IP_3$, phosphatidic acid, etc. Very recently, another PLC-${\gamma}1$ activator protein such as tau has been found in bovine lung tissue, which now is considered to be AHNAK protein. But there has been no report concerning AHNAK and its associated disease to this date. In this study, we examined the expression of the PLC-${\gamma}1$ activator, AHNAK, in lung cancer specimens and their paired normal. Methods: From surgically resected human lung cancer tissues taken from twenty-eight patients and their paired normal counterparts, we evaluated expression level of AHNAK protein using immunoblot analysis of total tissue extract Immunohistochemical stain was performed with primary antibody against AHNAK protein. Results: Twenty-two among twenty-eight lung cancer tissues showed overexpression of AHNAK protein (eight of fourteen squamous cell lung cancers, all of fourteen adenocarcinomas). The resulting bands were multiple ranging from 70 to 200 kDa in molecular weight and each band was indistinct and formed a smear, reflecting mobility shift mainly due to proteolysis during extraction process. On immunohistochemistry, lung cancer tissues showed a very heavy, dense staining with anti-AHNAK protein antibody as compared to the surrounding normal lung tissue, coresponding well with the results of the western blot Conclusion: The overexpression of PLC-${\gamma}1$ activator protein, AHNAK in lung cancer may provide evidence that the AHNAK protein and PLC-${\gamma}1$ act in concerted manner in carcinogenesis.

• Korean Journal of Agricultural Science
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• v.26 no.2
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• pp.39-48
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• 1999

This study was carried out to investigate the difference and genetic similarity at the level of molecular genetics. Genomic DNA was extracted from blood of Holstein, Korean cattle, Charolais, and hybrid between Korean cattle and charolais and RAPD(random amplified polymorphic DNAs) was analyzed by PCR(polymerase chain reaction). After genetic similarity value from different breeds are analyzed, genetic similarity was estimated by UPGMA(unweighted pair-group method using average). The results obtained from this study can be summarized as follows: 1. When genomic DNA which was extracted from different breeds was subjected to electrophoresis on 1.5% agarose gel, bigger than 12.2kb was appeared. Ratio by absorbance of $A_{260}/A_{280}$ was 1.75~2.10, indicating that genomic DNA was quite pure for RAPD analysis. 2. Different band patterns by RAPD were appeared according to the breeds in cattle. The best primer used to distinguish Holstein from other breeds was 5'-GAC CGC TTG T-3'. 3. A 340bp fragment was amplified in $33.0^{\circ}C$ of annealing temperature for the Holstein and Charolais breeds, but any amplification was not occurred in this annealing temperature for Korean cattle and hybrid. In addition, a 340bp fragment was amplified in $37.5^{\circ}C$ of annealing temperature for the Holstein and Korean cattle, but any amplification was not occurred in this annealing temperature for Charolais and hybrid. For the reaction of PCR. $37.5^{\circ}C$ and $33.0^{\circ}C$ of annealing temperature was shown to be best for genetic marker identification from Holstein, Charolais, and hybrid between Korean cattle and Charolais. 4. When genetic similarity from different breeds are analyzed at the both temperature of $33.0^{\circ}C$ and $37.5^{\circ}C$, the genetic similarity value of Holstein and Korean cattle, Holstein and Charolais, Korean cattle and Charolais, and Korean cattle and hybrid were 0.666~0.777, 0.615~0.666, 0.400~0.461 and 0.857~0.888, respectively. 5. It could be concluded that different breeds are capable of distinguishing by RAPD used random primer 5'-GAC CGC TTG T-3', genetic similarity from different breeds was appeared the higher genetic similarity value of Korean cattle and Charolais than that of Holstein between Korean cattle and Charolais by UPGMA.

• Tuberculosis and Respiratory Diseases
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• v.40 no.6
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• pp.653-658
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• 1993

Background: Recent advancement of molecular genetics has revealed that malignant transformation of a cell may be a complex multistep process and this process is grouped, in general, into two distinct categories, activation of protooncogenes and inactivation of tumor suppressor genes. This study was focused on the mutation of p53 tumor suppressor gene, because p53 gene mutation is now generally accepted to be one of the most frequent genetic changes in a variety of human cancers. Although lung cancer is one of the common cancers in Korea, the genetic change in the carcinogenesis process is not yet known clearly. To investigate the role of p53 gene mutation in lung cancer, we examined the mutations of exon 4-8 of the p53 gene in humna lung cancer cell lines, because most of the mutations of p53 gene have been reported to develop in exon 4-8. Method: Genomic DNA was obtained by the digestion of proteinase K and the extraction by phenol-chloroform-ethanol method from two human pulmonary adenocarcinoma cell lines, PC-9 and PC-14, and one human small cell lung cancer cell line, H69. To detect the mutations of exon 4-8 of the p53 gene, polymerase chain reaction single-strand conformation polymorphism(PCR-SSCP) analysis was performed with the DNA extracted from the cells. Results: The mutation of p53 gene was present in all three cell lines tested. In PC-9, PC-14 and H69, the altered mobility was detected in exon 7, 7 and 5, respectively. Conclusion: These results suggest that p53 gene mutation plays an important role in certain steps of the carcinogenesis of human non-small cell and small cell lung cancer.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.18 no.3
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• pp.99-106
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• 2018

Mucolipidosis type III (pseudo-Hurler polydystrophy) is a mucolipids degrading disorder caused by a mutation in the GNPTAB gene and is inherited by autosomal recessive. It is diagnosed by examining highly concentrated mucolipids in blood and the diagnosis can be confirmed by genetic testing. Mucolipidosis type III is a rare and progressive metabolic disorder. Its initial signs and symptoms usually occur around 3 years of age. Clinical manifestations of the disease include slow growth, joint stiffness, arthralgia, skeletal abnormalities, heart valve abnormalities, recurrent respiratory infection, distinctive facial features, and mild intellectual disability. Here, we are presenting two siblings of mucolipidosis type III, a 4-year-old female and a 2 years and 7 months old male with features of delayed growth and coarse face. The diagnosis was confirmed by [c.2715+1G>A(p.Glu906Leufs*4), c.2544del(p.Glu849Lysfs*22)] mutation in targeted gene panel sequencing. In this case, c.2544del is a heterozygote newly identified mutation in mucolipidosis type III and was not found in the control group including the genome aggregation database. And it is interpreted as a pathogenic variant considering the association with phenotype. Here, we report a Korean mucolipidosis type III patients with novel mutations in GNPTAB gene who have been treated since early childhood. Owing to recent development of molecular genetic techniques, it was possible to make early diagnosis and treatment with pamidronate was initiated appropriately in case 1. In addition to these supportive therapies, efforts must be made to develop fundamental treatment for patients with early diagnosis of mucolipidosis.

• Journal of Animal Environmental Science
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• v.10 no.1
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• pp.11-22
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• 2004

This work was carried out to measure volatile fatty acids emissions from different manure (poultry, swine, cattle) incubated at $10^{\circ}C$, $25^{\circ}C$, and $37^{\circ}C$ for 6 days under anaerobic condition. Following are summary of these tests results. 1. Amounts of Acetic acid generated were 1,128.05mg/kg, 628.21mg/kg and 592.50mg/kg for swine, poultry, and cattle manure, respectively, during the period of incubation. In the case of swine and cattle manure, 83.87%(946.10mg/kg) and 57.49%(340.63mg/kg) from all the temperature treatments were produced in the $25^{\circ}C$, respectively. 83.57% in swine and 78.79% in cattle manure were intensively emerged from 3 day, 4 day and 5 day of the $25^{\circ}C$ treatment. In the case of poultry manure, 45.36%(284.93mg/kg) and 45.36%(284.93mg/kg) in the $25^{\circ}C$ and in the $37^{\circ}C$, respectively, were produced. Accordingly, acetic acid generated from poultry manure was characteristic of being mainly produced in more than $25^{\circ}C$. 2. Amounts of propionic acid generated were 238.56mg/kg, 162.14mg/kg and 155.49mg/kg for swine, poultry, and cattle manure, respectively, during the period of incubation. In the case of swine manure, 78.52%(187.32mg/kg) of propionate emitted from all the temperature treatments was produced in the $25^{\circ}C$ and 79.1% of them was intensively emerged from 3day, 4day and 5day of the $25^{\circ}C$ treatment. In the case of poultry manure, 35.12%(56.95mg/kg) and 45.89%(74.40mg/kg) of the propionate amounts were produced in the $25^{\circ}C$ and in the $37^{\circ}C$, respectively. In the case of cattle manure, 28.21% (43.86mg/kg) and 49.30% (76.66mg/kg) of the propionate amounts were produced in the $10^{\circ}C$ and in the $25^{\circ}C$, respectively. Accordingly, propionate evolved from poultry manure was characteristic of being mainly produced in more than $25^{\circ}C$ and from cattle manure, in less than $25^{\circ}C$, respectively. 3. Amount of butyric acid generated were 1,463.87mg/kg, 96.72mg/kg and 129.18mg/kg for swine, poultry, and cattle manure, respectively, during the period of incubation. The time intensively emerged from the period of incubation was differently generated from the incubation temperature and animal feces. 4. Amounts of iso-valeric acid generated were 6,885.99mg/kg, 399.28mg/kg and 307.47mg/kg for swine, cattle and poultry manure, respectively, during the period of incubation. In the case of swine and cattle manure, 28.22%(1,943.52mg/kg) and 48.56%(193.90mg/kg) in the $25^{\circ}C$, 68.76%(4,734.90mg/kg) and 46.93%(187.40mg/kg) in the $37^{\circ}C$, respectively, were occupied. Accordingly, iso-valeric acid evolved from swine and cattle manure was characteristic of being mainly produced in more than $25^{\circ}C$. In the case of poultry manure, 59.89%(184.13mg/kg) of iso-valeric acid generated from all the temperature treatments was produced in the $37^{\circ}C$ and 100% of them was intensively emerged from 2 day and 3 day of the $37^{\circ}C$ treatment.