• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.499-509
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• 2008

Foxo1 plays an important role in the integration of hormone-activated signaling pathways with the complex transcriptional cascade that promotes preadipocyte differentiation of clonal cell lines from rodents. We isolated the full-length cDNA of porcine FoxO1 gene using RACE, confirmed by visual Northern blotting. The deduced amino acids indicated 94% and 90% identities with the corresponding human and mice aa. Analysis of the aa sequence, showed that it included a Forkhead domain (aa 167-247), a transmembrane structure domain (aa 90-113), a LXXLL motif (aa 469-473), and 51 Ser, 8 Thr, and 4 Tyr phosphorylation sites, indicating a potential important role for FoxO1 transcriptional activity in vivo. Using the IMpRH panel, we mapped FoxO1 gene to chromosome 11p13. Our data provide basic molecular information useful for the further investigation on the function of FoxO1 gene. Time-course analysis of FoxO1 expressions indicated that levels of mRNA and protein gradually increased from day 0 to 3, and it reached almost maximal level at day 3, then decreased from day 5 to 7 in porcine primary preadipocyte differentiation. After induction by IGF-1, GPDH activity and accumulation of lipid increased, however, expressions of FoxO1 mRNA and protein were inhibited in a dose dependent manner. These results suggest that FoxO1 takes part in porcine preadipocyte differentiation and expressions of FoxO1 were regulated by IGF-1.

• BMB Reports
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• v.31 no.4
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• pp.370-376
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• 1998

Src-Homology 2 (SH2) domains have a capacity to bind phosphotyrosine-containing sequence context and play essential roles in various cellular signaling pathways. Due to the specific nature of the binding between SH2 domains and their counterpart proteins, inhibitors of SID domain binding have drawn extensive attention as a potential candidate for therapeutic agents. Here, we describe the binding assay system to screen for the ligands or blockers of the SH2 domains with an emphasis on the $p56^{lck}$ SH2 domain. In our assay system, SID domains expressed and purified as fusion proteins to Glutathione-S-transferase (GST) were covalently attached to 96-well microtitre plates through amide bond formation, which were subsequently allowed to bind the biotinylated phosphotyrosine (pY)containing synthetic pep tides. The binding of biotinylated pY peptides was detected by the horseradish peroxidase (HRP)-conjugated streptavidin. Using the various combinations of SH2 domain-pY peptides, we observed that: (1) The binding of pY-peptides to its counterpart SH2 domain is concentration-dependent and saturable; (2) The binding is highly specific for a particular combination of SH2 domain-pY peptide pair; and (3) The binding of Lck SH2-cognate pY-peptides is specifically competed by the nonbiotinylated peptides with expected relative affinity. These results indicate that the established assay system detects the SH2-pY peptide interaction with reproducible sensitivity and specificity and is suitable for screening the specific inhibitors of $p56^{lck}$ SH2 function.

• International Journal of Industrial Entomology and Biomaterials
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• v.37 no.1
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• pp.21-28
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• 2018

CRISPR/Cas9 gene editing system is an efficient method to mutation in a sequence specific manner. Here we report the direct transfection of the Cas9 nuclease and gene specific guide RNA can be used in BM-N cell line derived from Bombyx mori ovarian tissue to enfeeble function of endogenous gene in vitro. We have used gene editing system to negative regulation components of major signaling cascade, the Toll pathway, which controls B. mori resistance to microbe infections, such as fungi and gram positive bacteria. We demonstrate that the $I{\kappa}B-like$ protein Cactus may controls the activation of transcription factors such as Rel A and Rel B. The direct transfection of Cas9 nuclease and Cactus-specific guide-RNA complex may be used in BM-N cells to disrupt the function of endogenous genes in vitro. A mutation frequency of 30-40% was observed in the transfected cells, and various mutations caused the target region. Moreover, RT-PCR analysis revealed that Cactus gene was down regulated after these mutations. More importantly, mutation of BmCactus stimulated expression of lysozyme, moricin, and lebocin genes. These results suggest that the CRISPR/Cas9 systems are expected to efficiently induce site-specific mutations and it was possible to produce antimicrobial peptide through the gene editing.

• Molecules and Cells
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• v.27 no.3
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• pp.383-390
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• 2009

To investigate the regulatory mechanism underlying the contractile response in the intestinal smooth muscle of the nile tilapia (Orechromis niloticus), we used pharmacologic and molecular approaches to identify the muscarinic subreceptors and the intracellular signaling pathways involved in this motility. Myography assays revealed that an M1- and M3-subtype selective antagonist, but not a M2-subtype selective antagonist, inhibited carbachol HCl (CCH)-induced intestinal smooth muscle contraction. In addition, a phospholipase C inhibitor, but not an adenylate cyclase inhibitor, blocked the contractile response to CCH. We also cloned five muscarinic genes (OnM2A, OnM2B, OnM3, OnM5A, and OnM5B) from the nile tilapia. In the phylogenetic analysis and sequence comparison to compare our putative gene products (OnMs) with the sequences obtained from the near complete teleost genomes, we unexpectedly found that the teleost fish have respectively two paralogous genes corresponding to each muscarinic subreceptor, and other teleost fish, except zebrafish, do not possess muscarinic subreceptor M1. In addition, the expression pattern of the nile tilapia muscarinic subreceptor transcripts during CCH-induced intestinal smooth muscle contraction in the proximal intestinal tissue was analyzed by real-time PCR surveys and it was demonstrated that CCH increased the OnMs mRNA expression rapidly and transiently.

• BMB Reports
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• v.48 no.11
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• pp.595-596
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• 2015

Skeletal muscle exhibits a loss of muscle mass and function with age. Decreased regenerative potential of muscle stem/progenitor cells is a major underlying cause of sarcopenia. We analyzed microRNAs (miRNA) that are differentially expressed in young and old myoblasts, to identify novel intrinsic factors that play a degenerative role in aged skeletal muscle. miR-431, one of decreasing miRNAs in old myoblasts, improved the myogenic differentiation when overexpressed in old myoblast, but suppressed their myogenic capability in knockdowned young myoblasts. We found that miR-431 directly binds to 3` untranslated regions (UTR) of Smad4 mRNA, and decreases its expression. Given that SMAD4 is one of the downstream effectors of TGF-β, a well-known degenerative signaling pathway in myogenesis, the decreased miR-431 in old myoblast causes SMAD4 elevation, thus resulting in defective myogenesis. Exogenous expression of miR-431 greatly improved the muscle regeneration in the cardiotoxin-injured hindlimb muscle of old mice by reducing SMAD4 levels. Since the miR-431 seed sequence is conserved in human SMAD4 3'UTR, miR-431 regulates the myogenic capacity of human skeletal myoblasts in the same manner. Our results suggest that age-associated miR-431 is required for the maintenance of the myogenic capability in myoblasts, thus underscoring its potential as a therapeutic target to slow down muscle aging.

• Molecules and Cells
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• v.26 no.6
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• pp.595-605
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• 2008

Genome wide transcription analysis in response to stresses is essential to provide the basis of effective engineering strategies to improve stress tolerance in crop plants. In order to perform transcriptome analysis in Brassica rapa, we constructed a B. rapa oligo microarray, KBGP-24K, using sequence information from approximately 24,000 unigenes and analyzed cold ($4^{\circ}C$), salt (250 mM NaCl), and drought (air-dry) treated B. rapa plants. Among the B. rapa unigenes represented on the microarray, 417 (1.7%), 202 (0.8%), and 738 (3.1%) were identified as responsive genes that were differently expressed 5-fold or more at least once during a 48-h treatment with cold, salt, and drought, respectively. These results were confirmed by RT-PCR analysis. In the abiotic stress responsive genes identified, we found 56 transcription factor genes and 60 commonly responsive genes. It suggests that various transcriptional regulatory mechanisms and common signaling pathway are working together under the abiotic stresses in B. rapa. In conclusion, our new developed 24K oligo microarray will be a useful tool for transcriptome profiling and this work will provide valuable insight in the response to abiotic stress in B. rapa.

• The Plant Pathology Journal
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• v.21 no.1
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• pp.47-54
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• 2005

Identification of host genes involved in disease progresses and/or defense responses is one of the most critical steps leading to the elucidation of disease resistance mechanisms in plants. Soybean mosaic virus (SMV) is one of the most prevalent pathogen of soybean (Glycine max). Although the soybeans are placed one of many important crops, relatively little is known about defense mechanism. In order to obtain host genes involved in SMV disease progress and host defense especially for virus resistance, two different cloning strategies (DD RT-PCR and Subtractive hybridization) were employed to identify pathogenesis- and defenserelated genes (PRs and DRs) from susceptible (Geumjeong 1) and resistant (Geumjeong 2) cultivars against SMV strain G7H. Using these approaches, we obtained 570 genes that expressed differentially during SMV infection processes. Based upon sequence analyses, differentially expressed host genes were classified into five groups, i.e. metabolism, genetic information processing, environmental information processing, cellular processes and unclassified group. A total of 11 differentially expressed genes including protein kinase, transcription factor, other potential signaling components and resistant-like gene involved in host defense response were selected to further characterize and determine expression profiles of each selected gene. Functional characterization of these genes will likely facilitate the elucidation of defense signal transduction and biological function in SMV-infected soybean plants.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3627-3629
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• 2016

Background: Activation and inactivation of nuclear factor of kappa light chain gene enhancer in B cells (NFKB) is tightly regulated to ensure effective onset and cessation of defensive inflammatory signaling. However, mutations within NFKB, or change in activation and inactivation molecules have been reported in a few cancers. Although oral squamous cell carcinoma is one of the most prevalent forms of cancer in India, with a development associated with malignant transformation of precancerous lesions, the genetic status of NFKB and relative rates of change in oral precancerous lesions remain unknown. Hence in the present study we investigated all twenty four exons of NFKB gene in two precancerous lesions, namely oral submucous fibrosis (OSMF) and oral leukoplakia (OL) to understand its occurrence, incidence and assess its possible contribution to malignant transformation. Materials and Methods: Chromosomal DNA isolated from twenty five each of OSMF and OL tissue biopsy samples were subjected to PCR amplification with intronic primers flanking twenty four exons of the NFKB gene. The PCR amplicons were subsequently subjected to direct sequencing to elucidate the mutation status. Results: Sequence analysis identified a novel heterozygous mutation, c.419T>A causing substitution of leucine with glutamine at codon 140 (L140Q) in an OL sample. Conclusions: The identification of a substitution mutation L140Q within the DNA binding domain of NFKB in OL suggests that NFKB mutation may be relatively an early event during transformation. To the best of our knowledge, this study is the first to have identified a missense mutation in NFKB in OL.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.1
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• pp.3-12
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• 2018

Objective: DNA methylation plays a major role in regulating the expression of genes related to traits of economic interest (e.g., weight gain) in livestock animals. This study characterized and investigated the functional inferences of genome-wide DNA methylome in the loin (longissimus dorsi) muscle (LDM) of swine. Methods: A total of 8.99 Gb methylated DNA immunoprecipitation sequence data were obtained from LDM samples of eight Duroc pigs (four pairs of littermates). The reference pig genome was annotated with 78.5% of the raw reads. A total of 33,506 putative methylated regions (PMR) were identified from methylated regions that overlapped at least two samples. Results: Of these, only 3.1% were commonly observed in all eight samples. DNA methylation patterns between two littermates were as diverse as between unrelated individuals (p = 0.47), indicating that maternal genetic effects have little influence on the variation in DNA methylation of porcine LDM. The highest density of PMR was observed on chromosome 10. A major proportion (47.7%) of PMR was present in the repeat regions, followed by introns (21.5%). The highest conservation of PMR was found in CpG islands (12.1%). These results show an important role for DNA methylation in species- and tissue-specific regulation of gene expression. PMR were also significantly related to muscular cell development, cell-cell communication, cellular integrity and transport, and nutrient metabolism. Conclusion: This study indicated the biased distribution and functional role of DNA methylation in gene expression of porcine LDM. DNA methylation was related to cell development, cell-cell communication, cellular integrity and transport, and nutrient metabolism (e.g., insulin signaling pathways). Nutritional and environmental management may have a significant impact on the variation in DNA methylation of porcine LDM.

• Korean Journal of Veterinary Service
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• v.28 no.3
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• pp.259-265
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• 2005

The melanocortin 1 receptor (MC1R) encoded by the coat color extension gene (E) plays a key role in the signaling pathway of melanin synthesis. The primers for the amplification of bovine MC1R gene were designed based on a bovine MC1R gene sequence (GenBank accession no. Y19103). A size of 483bp (482bp for Hanwoo) was amplified by PCR, digested with Hpa II restriction enzyme and electrophoresed in $1.5\%$ agarose gel. When the amplified DNA product (483 bp) was digested with Hpa II restriction enzyme, Hanwoo meat showed a single band of 482bp, whereas two fragments of 325bp and 158bp were detected in Holstein, Angus and meat of Hanwoo / Holstein cross cow having back coat color phenotype, respectively. The results of this experiment Indicate that new designed primers of bovine MCIR gene may be useful for identification of Hanwoo meat from Holstein, Black Angus and Hanwoo / Holstein cross cow meat.