• Animal cells and systems
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• 제1권1호
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• pp.93-98
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• 1997

The present study investigated the cellular localization of a-subunit of Gq (Gaq) protein in developing L8E63, rat skeletal muscle cell line. The colocalization of Gaq with actin cytoskeleton was demonstrated by double-labeling experiments. In mononucleated myoblasts, the immuno-fluorescence staining pattern of Gaq was almost identical with that of F-actin visualized with rhodamine-conjugated phalloidin. However, this colocalization of Gaq with cytoskeleton was not maintained in multinucleated myotubes. The staining pattern of Gaq in myotubes did not match with any specific subcellular structure, but appeared as a uniformly distributed diffuse staining throughout the whole cell surface. Interestingly, change in the expression level of Gaq was not detected during myoblast differentiation, suggesting that actin-associated Gaq protein might dissociate from the cytoskeleton as cells differentiate. Immunocytochemical experiments using specific antibodies directed against several G proteins indicated that the subcellular localizations of Gai1, Gai2, Gai3, and Gao were different from those obtained with Gaq.

• 한국동물생명공학회지
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• 제34권3호
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• pp.212-221
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• 2019

Sestrin-2 (SESN2) as a stress-metabolic protein is known for its anti-oxidative effects as a downstream factor of PERK pathways in mammalian cells. However, the expression patterns of SESN2 in conjunction with the UPR signaling against to ER stress on porcine oocyte maturation in vitro, have not been reported. Therefore, we confirmed the expression pattern of SESN2 protein, for which to examine the relationship between PERK signaling and SESN2 in porcine oocyte during IVM. We investigated the SESN2 expression patterns using Western blot analysis in denuded oocytes (DOs), cumulus cells (CCs), and cumulus-oocyte complexes (COCs) at 22 and 44 h of IVM. As expected, the SESN2 protein level significantly increased (p < 0.01) in porcine COCs during 44 h of IVM. We investigated the meiotic maturation after applying ER stress inhibitor in various concentration (50, 100 and 200 μM) of tauroursodeoxycholic acid (TUDCA). We confirmed significant increase (p < 0.05) of meiotic maturation rate in TUDCA 200 μM treated COCs for 44 h of IVM. Finally, we confirmed the protein level of SESN2 and meiotic maturation via regulating ER-stress by only tunicamycin (Tm), only TUDCA, and Tm + TUDCA treatment in porcine COCs. As a result, treatment of the TUDCA following Tm pre-treatment reduced SESN2 protein level in porcine COCs. In addition, SESN2 protein level significantly reduced in only TUDCA treated porcine COCs. Our results suggest that the SESN2 expression is related to the stress mediator response to ER stress through the PERK signaling pathways in porcine oocyte maturation.

• 한국미생물·생명공학회지
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• 제23권6호
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• pp.730-736
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• 1995

The solubilization and proteolytic process of $\delta $-endotoxin was analvsed to compare the biochemical property of the toxin isolated from B. thuringiensis subsp. sotto. The purified crystals were dissolved in 50 mM carbonate buffer containing 10 mM dithiothreitol at pH 10 for various times. The electrophoretic pattern showed that a rapid disappearance of 138 kDa protein band. This disappearance of protein with high molecular weight was accompanied by the appearance of new protein fragment with 104 kDa, 60 kDa, and 25 kDa. For proteolvtic processing, the soluble crystals were digested with trypsin for various times. The soluble crystal protein of 104 kDa was completely disappeared. However, the protein fragment of 60 kDa and 25 kDa still remained after complete proteolysis. The comparative immunoblot analysis showed that the antiserum against intact crystals showed strong immunoreactivity to the homologous inclusion protein of 138 kDa, 104 kDa, and 25 kDa, and to the intact spores of 221 kDa and 138 kDa, but not to the vegetative cell homogenate. The sera against crystals and spores had no immunoreactivity to the vegetative cell homogenate.

• Journal of Animal Science and Technology
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• 제56권4호
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• pp.12.1-12.7
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• 2014

This study investigated changes in gene expression by dietary fat source, i.e., beef tallow, soybean oil, olive oil, and coconut oil (each 3% in feed), in both male and female growing-finishing pigs. Real-time PCR was conducted on seven genes (insulin receptor; INSR, insulin receptor substrate; IRS, phosphatidylinositol (3,4,5)-triphosphate; PIP3, 3-phosphoinositide-dependent protein kinase-1; PDK1, protein kinase B; Akt, forkhead box protein O1; FOXO1 and cGMP-inhibited 3', 5'-cyclic phosphodiesterase; PDE3) located upstream of the insulin signaling pathway in the longissimus dorsi muscle (LM) of pigs. The INSR, IRS, PIP3, and PDE3 genes showed significantly differential expression in barrow pigs. Expression of the PIP3 and FOXO1 genes was significantly different among the four dietary groups in gilt pigs. In particular, the PIP3 gene showed the opposite expression pattern between barrow and gilt pigs. These results show that dietary fat source affected patterns of gene expression according to animal gender. Further, the results indicate that the type of dietary fat affects insulin signaling-related gene expression in the LM of pigs. These results can be applied to livestock production by promoting the use of discriminatory feed supplies.

• 한국발생생물학회지:발생과생식
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• 제18권4호
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• pp.233-240
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• 2014

The early growth response protein 1 (Egr-1) is a widely reported zinc finger protein and a well known transcription factor encoded by the Egr-1 gene, which plays key roles in many aspects of vertebrate embryogenesis and in adult vertebrates. The Egr-1 expression is important in the formation of the gill vascular system in flounders, which develops during the post-hatching phase and is essential for survival during the juvenile period. However, the complete details of Egr-1 expression during embryo development in olive flounder are not available. We assessed the expression patterns of Egr-1 during the early development of olive flounders by using reverse transcription polymerase chain reaction (RT-PCR) analysis. Microscopic observations showed that gill filament formation corresponded with the Egr-1 expression. Thus, we showed that Egr-1 plays a vital role in angiogenesis in the gill filaments during embryogenesis. Further, Egr-1 expression was found to be strong at 5 days after hatching (DAH), in the development of the gill vascular system, and this strong expression level was maintained throughout all the development stages. Our findings have important implications with respect to the biological role of Egr-1 and evolution of the first respiratory blood vessels in the gills of olive flounder. Further studies are required to elucidate the Egr-1-mediated stress response and to decipher the functional role of Egr-1 in developmental stages.

• Fisheries and Aquatic Sciences
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• 제18권1호
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• pp.73-80
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• 2015

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring ${\beta}$-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

• 대한의용생체공학회:의공학회지
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• 제28권5호
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• pp.601-608
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• 2007

2 Dimensional Gel Electrophoresis(2DGE) is an essentialmethodology for analysis on the expression of various proteins. For example, information for the location, mass, expression, size and shape of the proteins obtained by 2DGE can be used for diagnosis, prognosis and biological progress by comparison of patients with the normal persons. Protein spot matching for this purpose is comparative analysis of protein expression pattern for the 2DGE images generated under different conditions. However, visual analysis of protein spots which are more than several hundreds included in a 2DGE image requires long time and heavy effort. Furthermore, geometrical distortion makes the spot matching for the same protein harder. In this paper, an iterative algorithm is introduced for more efficient spot matching. Proposed method is first performing global matching step, which reduces the geometrical difference between the landmarks and the spot to be matched. Thus, movement for a spot is defined by a weighted sum of the movement of the landmark spots. Weight for the summation is defined by the inverse of the distance from the spots to the landmarks. This movement is iteratively performed until the total sum of the difference between the corresponding landmarks is larger than a pre-selected value. Due to local distortion generally occurred in 2DGE images, there are many regions in whichmany spot pairs are miss-matched. In the second stage, the same spot matching algorithm is applied to such local regions with the additional landmarks for those regions. In other words, the same method is applied with the expanded landmark set to which additional landmarks are added. Our proposed algorithm for spot matching empirically proved reliable analysis of protein separation image by producing higher accuracy.

• Asian-Australasian Journal of Animal Sciences
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• 제20권6호
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• pp.811-816
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• 2007

Adipocyte fatty acid-binding protein (A-FABP), which belongs to the FABP family, plays an essential role in long-chain fatty acid uptake and metabolic homeostasis, especially in adipose tissue. The pattern of A-FABP gene mRNA expression in different growth stages and its relation to intramuscular fat (IMF) accretion in pigs was studied. Fifteen female $Duroc{\times}Landrace{\times}Yorkshire$ pigs in five groups of three pigs each, weighing 1, 30, 50, 70 and 90 kg were used to study developmental gene mRNA expression of A-FABP in various adipose tissues by means of semi-quantitative RT-PCR. Results showed that A-FABP mRNA levels in subcutaneous and ventral adipose tissues first increased from 1 to 50 kg, then gradually declined from 50 to 90 kg. Moreover, the rank order of A-FABP mRNA levels determined in three adipose tissues was as follows: subcutaneous adipose>ventral adipose>mesenteric adipose. A-FABP mRNA expression in mesenteric adipose tissue was constant during development. In addition, a positive correlation from 1 to 50 kg BW pigs and a negative correlation from 50 to 90 kg BW between A-FABP mRNA levels in subcutaneous and ventral adipose and IMF content were found.

• Asian-Australasian Journal of Animal Sciences
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• 제30권3호
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• pp.328-337
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• 2017

Objective: An experiment was conducted to determine the relationship between the KAP11.1 and the regulation wool fineness. Methods: In previous work, we constructed a skin cDNA library and isolated a full-length cDNA clone termed KAP11.1. On this basis, we conducted a series of bioinformatics analysis. Tissue distribution of KAP11.1 mRNA was performed using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. The expression of KAP11.1 mRNA in primary and secondary hair follicles was performed using real-time PCR (real-time polymerase chain reaction) analysis. The expression location of KAP11.1 mRNA in primary and secondary hair follicles was performed using in situ hybridization. Results: Bioinformatics analysis showed that KAP11.1 gene encodes a putative 158 amino acid protein that exhibited a high content of cysteine, serine, threonine, and valine and has a pubertal mammary gland) structural domain. Secondary structure prediction revealed a high proportion of random coils (76.73%). Semi-quantitative RT-PCR showed that KAP11.1 gene was expressed in heart, skin, and liver, but not expressed in spleen, lung and kidney. Real time PCR results showed that the expression of KAP11.1 has a higher expression in catagen than in anagen in the primary hair follicles. However, in the secondary hair follicles, KAP11.1 has a significantly higher expression in anagen than in catagen. Moreover, KAP11.1 gene has a strong expression in inner root sheath, hair matrix, and a lower expression in hair bulb. Conclusion: We conclude that KAP11.1 gene may play an important role in regulating the fiber diameter.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.70-70
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• 2003

The uterus undergoes dynamic changes during the cycle and displays many features typical of developmental process. In order to be prepared for implantation, endometrium undergoes predictable, sequential phases of proliferation and secretory changes. The uterus during estrus cycle synthesize a complex of signaling molecules with specific spatial and temporal modes of expression and which are critical for cell proliferation and differentiation. The purpose of this investigation was to use cDNA microarrays to evaluate the expression of genes of rat uterus in estrus cycle. Animals were sacrificed on proestrus, estrus, metestrus, diestrus. Differential gene expression profiles were revealed(growth-related c-myc reponsive protein RCL, heat shock 47-kDa protein (HSP47), cytochrome c oxidase polypeptide Vlc2 (COX6C2), calreticulin (CALR)). Reverse transcription polymerase chain reaction (RT-PCR) was used to validate the relative expression pattern. Using this approach, we found several genes whose expression in rat uterus was altered with estrus cycle. Our long-term goal is to determine the role of these differentially expressed genes during estrus cycle. This study was supported by through the Biohealth Products Research Center(BPRC), Inje University.