Expression of Transthyretin during bovine myogenic satellite cell differentiation.

Pokharel, Smritee,Kamli, Majid Rasool,Mir, Bilal Ahmad,Malik, Adeel,Lee, Eun Ju,Choi, Inho Springer 2014 In vitro cellular & developmental biology Animal Vol.50 No.8

<P>Adult myogenesis responsible for the maintenance and repair of muscle tissue is mainly under the control of myogenic regulatory factors (MRFs) and a few other genes. Transthyretin gene (TTR), codes for a carrier protein for thyroxin (T4) and retinol binding protein bound with retinol in blood plasma, plays a critical role during the early stages of myogenesis. Herein, we investigated the relationship of TTR with other muscle-specific genes and report their expression in muscle satellite cells (MSCs), and increased messenger RNA (mRNA) and protein expression of TTR during MSCs differentiation. Silencing of TTR resulted in decreased myotube formation and decreased expression of myosin light chain (MYL2), myosin heavy chain 3 (MYH3), matrix gla protein (MGP), and voltage-dependent L type calcium channel (Cav1.1) genes. Increased mRNA expression observed in TTR and other myogenic genes with the addition of T4 decreased significantly following TTR knockdown, indicating the critical role of TTR in T4 transportation. Similarly, decreased expression of MGP and Cav1.1 following TTR knockdown signifies the dual role of TTR in controlling muscle myogenesis via regulation of T4 and calcium channel. Our computational and experimental evidences indicate that TTR has a relationship with MRFs and may act on calcium channel and related genes.</P>

Potential use of adult bovine serum obtained during the slaughtering process as a biological reagent

유지은,김민수,Smritee Pokharel,김지회,최인호,최농훈,남상섭 한국통합생물학회 2013 Animal cells and systems Vol.17 No.2

Starting from 2013, the Korean ‘Marine Environment Management Act,’ in response to international regulation on marine pollution, will prohibit the marine disposal of land wastes such as animal husbandry by-products and waste produced during the slaughtering process. Currently, most bovine blood is wasted during the slaughtering process and causes environmental problems in Korea. Therefore, long-term measures are required to process blood waste produced during the slaughtering process. The aim of this research is to investigate the potential use of adult bovine serum obtained during the slaughtering process for use in cell cultures. Primary mouse astrocytes, primary human fibroblasts, and L6 cells were cultured with adult male, adult female, and castrated male bovine sera for comparison with fetal bovine serum (FBS). The performance of adult bovine serum was comparable to that of FBS in culture of astrocytes and fibroblasts. Compared to FBS, adult bovine serum supported enhanced proliferation and differentiation of L6 cells. These results suggest that adult bovine serum obtained from the slaughtering process has a potential use as a biological reagent. Moreover, recycling adult bovine serum will contribute to solving the environmental issues arising from the disposal of waste produced during the slaughtering process.

Characterization of gender-specific bovine serum

김지회,김민수,남상섭,이동목,Smritee Pokharel,최인호 한국통합생물학회 2011 Animal cells and systems Vol.15 No.2

Animal cell cultures generally require a nutrient-rich medium supplemented with animal serum. Adult bovine serum contains a variety of nutrients including inorganic minerals, vitamins, salts, proteins and lipids as well as growth factors that promote animal cell growth. To evaluate the potential use of gender-specific bovine serum (GSBS) for cell culture, the biochemical properties of male serum (MS), female serum (FS) and castrated-male serum (CMS)were investigated. Overall, the chemical profile of GSBS was similar to that of bovine references except for glucose,creatine kinase, lactate dehydrogenase and potassium. FS showed elevated total protein and sodium concentrations compared to MS and CMS. Proteins present in MS, FS and CMS but absent in fetal bovine serum (FBS) were selected by two-dimensional gel electrophoresis and identified by peptide mass fingerprinting. Some of the identified proteins are known to be involved in immune responses and the others have unknown physiological roles. Moreover,it was found that some proteins such as alpha-2-macroglobulin appeared to be gender-specific with higher contents in FS. Insulin and testosterone was significantly higher in MS, and 17b-estradiol and estrone were higher in FS, as compared to the other sera. Taken together, the results indicate that each GSBS has a different ratio of components. Differences in serum constituents may affect cell cultures in a different manner and could be beneficial, depending on the specific aim of cell cultures.

Transthyretin: A Transporter Protein Essential for Proliferation of Myoblast in the Myogenic Program

Lee, Eun Ju,Pokharel, Smritee,Jan, Arif Tasleem,Huh, Soyeon,Galope, Richelle,Lim, Jeong Ho,Lee, Dong-Mok,Choi, Sung Wook,Nahm, Sang-Soep,Kim, Yong-Woon,Park, So-Young,Choi, Inho MDPI 2017 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.18 No.1

<P>Irregularities in the cellular uptake of thyroid hormones significantly affect muscle development and regeneration. Herein, we report indispensable role of transthyretin (TTR) in maintaining cellular thyroxine level. TTR was found to enhance recruitment of muscle satellite cells to the site of injury, thereby regulating muscle regeneration. Fluorescence-activated cell sorting (FACS) and immunofluorescence analysis of TTR<SUB>wt</SUB> (TTR wild type) and TTR<SUB>kd</SUB> (TTR knock-down) cells revealed that TTR controlled cell cycle progression by affecting the expression of Cyclin A2. Deiodinase 2 (D2) mediated increases in triiodothyronine levels were found to regulate the expression of myogenic marker, myogenin (MYOG). Moreover, use of a coumarin derivative (CD) revealed a significant reduction in cellular thyroxine, thereby indicating that TTR play a role in the transport of thyroxine. Taken together, these findings suggest that TTR mediated transport of thyroxine represents a survival mechanism necessary for the myogenic program. The results of this study will be highly useful to the strategic development of novel therapeutics to combat muscular dystrophies.</P>

Animal Biotechnology : Effect of Gender-Specific Adult Bovine Serum on Gene Expression During Myogenesis

( Eun Ju Lee ),( Smritee Pokharel ),( Jie Hoe Kim ),( Sang Sup Nam ),( In Ho Choi ) 한국동물자원과학회 ( 구 한국축산학회 ) 2012 한국축산학회지 Vol.54 No.3

Gender specificity in muscle growth and development is well known. Genesis of muscle is dependent on proliferation and differentiation potential of resident myogenic satellite cells(MSCs) present in muscle fibers. Multipotential capacity of forming myocyte, osteocyte, and adipocyte like cell makes MSCs a unique stem cell. To understand the molecular mechanism involved in determination of muscle quality due to difference in hormone concentration of different gender of animals, MSCs were isolated from bovine skeletal muscle and cultured in male, female, and castrated serum supplemented media. DNA microarray used consisted of 24,000 spots with 70 mer oligo in each spot. A total of 88 genes were up-regulated and 551 genes were down-regulated by more than two fold. Among up-regulated gene, 33, 34, and 21 genes were found up-regulated in cells grown in male, female, and castrated serum, respectively. Interestingly, male serum showed 4, female 11 and castrated male showed 4 genes expressed highly in each gender. Further study on the highly up-regulated gene may unfold the mystery of gender specificity found in muscle development. Also, the identification of differentially expressed genes in gender-specific serum will add information on infrastructure of bovine genome research.

Characterization of gender-specific bovine serum

Kim, Ji-Hoe,Kim, Min-Soo,Nahm, Sang-Soep,Lee, Dong-Mok,Pokharel, Smritee,Choi, In-Ho The Korean Society for Integrative Biology 2011 Animal cells and systems Vol.15 No.2

Animal cell cultures generally require a nutrient-rich medium supplemented with animal serum. Adult bovine serum contains a variety of nutrients including inorganic minerals, vitamins, salts, proteins and lipids as well as growth factors that promote animal cell growth. To evaluate the potential use of gender-specific bovine serum (GSBS) for cell culture, the biochemical properties of male serum (MS), female serum (FS) and castrated-male serum (CMS) were investigated. Overall, the chemical profile of GSBS was similar to that of bovine references except for glucose, creatine kinase, lactate dehydrogenase and potassium. FS showed elevated total protein and sodium concentrations compared to MS and CMS. Proteins present in MS, FS and CMS but absent in fetal bovine serum (FBS) were selected by two-dimensional gel electrophoresis and identified by peptide mass fingerprinting. Some of the identified proteins are known to be involved in immune responses and the others have unknown physiological roles. Moreover, it was found that some proteins such as alpha-2-macroglobulin appeared to be gender-specific with higher contents in FS. Insulin and testosterone was significantly higher in MS, and $17{\beta}$-estradiol and estrone were higher in FS, as compared to the other sera. Taken together, the results indicate that each GSBS has a different ratio of components. Differences in serum constituents may affect cell cultures in a different manner and could be beneficial, depending on the specific aim of cell cultures.