Dong-Mok Lee

Identification of Genes Differentially Expressed in Myogenin Knock-Down Bovine Muscle Satellite Cells during Differentiation through RNA Sequencing Analysis

Background The expression of myogenic regulatory factors (MRFs) consisting of MyoD, Myf5, myogenin (MyoG) and MRF4 characterizes various phases of skeletal muscle development including myoblast proliferation, cell-cycle exit, cell fusion and the maturation of myotubes to form myofibers. Although it is well known that the function of MyoG cannot be compensated for other MRFs, the molecular mechanism by which MyoG controls muscle cell differentiation is still unclear. Therefore, in this study, RNA-Seq technology was applied to profile changes in gene expression in response to MyoG knock-down (MyoGkd) in primary bovine muscle satellite cells (MSCs). Results About 61–64% of the reads of over 42 million total reads were mapped to more than 13,000 genes in the reference bovine genome. RNA-Seq analysis identified 8,469 unique genes that were differentially expressed in MyoGkd. Among these genes, 230 were up-regulated and 224 were down-regulated by at least four-fold. DAVID Functional Annotation Cluster (FAC) and pathway analysis of all up- and down-regulated genes identified overrepresentation for cell cycle and division, DNA replication, mitosis, organelle lumen, nucleoplasm and cytosol, phosphate metabolic process, phosphoprotein phosphatase activity, cytoskeleton and cell morphogenesis, signifying the functional implication of these processes and pathways during skeletal muscle development. The RNA-Seq data was validated by real time RT-PCR analysis for eight out of ten genes as well as five marker genes investigated. Conclusions This study is the first RNA-Seq based gene expression analysis of MyoGkd undertaken in primary bovine MSCs. Computational analysis of the differentially expressed genes has identified the significance of genes such as SAP30-like (SAP30L), Protein lyl-1 (LYL1), various matrix metalloproteinases, and several glycogenes in myogenesis. The results of the present study widen our knowledge of the molecular basis of skeletal muscle development and reveal the vital regulatory role of MyoG in retaining muscle cell differentiation.

Gene expression profiles during differentiation and transdifferentiation of bovine myogenic satellite cells

The molecular mechanisms underlying myogenic satellite cells (MSCs) differentiation into myotube-formed cells (MFCs) and transdifferentiation into adipocyte-like cells (ALCs) are unclear. As a step towards understanding the molecular mechanisms underlying MSC differentiation and transdifferentiation, we attempted to identify the genes differentially expressed during differentiation and transdifferentiation using gene microarray analysis (GMA). Thirty oligonucleotide arrays were used with two technical replicates and nine and six biological replicates for MFCs vs. MSCs and ALCs vs. MSCs, respectively, to contrast expression profile differences. GMA identified 1,224 differentially expressed genes by at least 2-fold during differentiation and transdifferentiation of MSCs. To select the highly expressed genes for future functional study, genes with a 4-fold expression difference were selected for validation by real time RT-PCR and approximately 96.9% of the genes were validated. The up-regulation of marker genes for myogenesis (MYL2, MYH3) and adipogenesis (PPARγ, and FABP4) was observed during the differentiation and transdifferentiation of MSCs into MFCs and ALCs, respectively. KOG analysis revealed that the most of the genes up-regulated during differentiation and transdifferentiation of MSCs were related to signal transduction. Again the exact location of 109 differentially expressed genes by 4-fold were analyzed by chromosome mapping. Among those, co-localization of 29 genes up-regulated during transdifferentiation with QTL for marbling score and intramuscular fat percentage supports the involvement of these genes in cellular transdifferentiation. Interestingly, some genes with unknown function were also identified during the process. Functional studies on these genes may unfold the molecular mechanisms controlling MSC differentiation and transdifferentiation.

Characterization of gender-specific bovine serum

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 17β-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

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 TTRwt (TTR wild type) and TTRkd (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.

Effects of dietary Macsumsuk® supplementation on growth performance, haematological parameters, disease resistance and body composition of juvenile Nile tilapia, Oreochromis niloticus L.

An eight-week feeding trial was conducted to evaluate the effects of dietary Macsumsuk® supplementation on growth performance, haematological parameters, disease resistance and body composition of juvenile Nile tilapia, Oreochromis niloticus. A total of 375 fish averaging 1.0 ± 0.05 g (mean ± SD) were randomly distributed into 15 tanks in groups of 25, and each tank was then randomly assigned to one of three replicates of five diets containing 0(M0), 0.3(M0.3), 0.6(M0.6), 1.2(M1.2) and 2.4(M2.4)% dietary Macsumsuk®. At the end of eight weeks of feeding trial, average weight gain (WG), specific growth rate (SGR), feed efficiency (FE) and protein efficiency ratio (PER) of fish fed M1.2 diet were significantly higher than those fed M0 diet (P < 0.05). Significantly higher values for haematological parameters, such as red blood cell (RBC), haemoglobin (Hb) and haematocrit (HCT), were obtained in fish fed M0.6 diet compared to the fish offered M2.4 diet. Although an analysis of variance (ANOVA) test suggested that the optimum level of dietary Macsumsuk® in juvenile Nile tilapia, O. niloticus, could be 1.2%, broken-line analysis of WG indicated a level of 0.803% when Macsumsuk® is used as the dietary feed additive.

Gene expression profiles analyzed by DNA sequencing of cDNA clones constructed from porcine preadipocytes and adipocytes

As a step towards understanding the molecular mechanism of adipogenesis in pigs, preadipocytes purified from the back fat of 1 day-old female piglets were used for in vitro culture. Normalized cDNA libraries were constructed with 1.6×107 and 1.1×107 independent clones from preadipocyte and mature adipocyte mRNAs, respectively. Polymerase chain reaction (PCR) result using primers T3 and T7 (universal primer) confirmed the presence of the insert in the vector. Sequencing of 2,112 randomly selected clones from each cDNA library identified 217 clusters, 1,169 singletons, and 216 contigs in preadipocytes and 231 clusters, 1,100 singletons, and 233 contigs in mature adipocytes. Expressed sequence tag (EST) identified 24 genes with known annotation highly expressed in adipocytes and 21 in preadipocytes by at least four EST number. Among those 45 genes, when analyzed by real time RT-PCR, 76% of the gene showed significant difference between preadipocytes and mature adipocytes. Highly expressed genes in mature adipocytes were related to adipogenesis, extracellular matrix control and oncogenes, whereas cytoskeleton-related genes were down-regulated. An interesting similarity found during gene profile studies indicated a correlation between cancer and adipogenesis.

Effect of different ingredients in traditional Korean medicine for human uterine leiomyoma on normal myometrial and leiomyomal smooth muscle cell proliferation

Crude water extracts of 13 traditional Korean medicinal ingredients used for leiomyomal treatment were prepared and used to treat human uterine normal myometrial and leiomyomal cell cultures. All the ingredients inhibited proliferation and altered the morphology of both myometrial and leiomyomal cells. Among the 13 ingredients, n-hexane-, chloroform-, and ethylacetate-soluble fractions were extracted from seven ingredients that potently inhibited cell proliferation in their water extract form. Among these, the ethylacetate-fraction of Phlomis umbrosa and Spatholobus suberectus, and the chloroform-fraction of Curcuma zedoaria and S. suberectus inhibited leiomyomal cell proliferation significantly compared to myometrial cell proliferation. Similarly, immunohistochemical analysis showed the inhibition of transforming growth factor-beta receptor 2 in leiomyomal tissue after treatment with the fractions of the ingredients. Moreover, the chloroform-fraction of C. zedoaria was subfractionated by open column chromatography. Two of the eight subfractions (fractions 6 and 7) potently inhibited cell proliferation in leiomyoma compared to myometrium. Further study will be performed with the goal of isolating specific compounds from two effective subfractions of C. zedoaria, ethylacetate-fraction of P. umbrosa, and the ethylacetate and chloroform-fractions of S. suberectus. The present study may be helpful in developing an alternative remedy to leiomyoma with minimal side-effects compared to the current treatments.

Development of a Novel Electrostatic Precipitator System Using a Wet-Porous Electrode Array

The authors introduce a novel electrostatic precipitator using a wet-porous electrode (WPE) array, for application to various work processes (e.g., molding and extrusion), to improve the removal performance of ultrafine particles and water-soluble gaseous pollutants generated during manufacturing. The WPE array electrostatic precipitator (WPE-ESP) consists of an ionization component for particle charging and a collection component equipped with the WPE array to maintain a high humidity environment. The performance of the WPE-ESP was evaluated in terms of the removal efficiency of airborne particles and water-soluble gases under ESP operating conditions in which the ionization charge current, the applied electric field strength of the collection component, and the relative humidity (RH) were varied. The collection efficiency of the WPE-ESP was enhanced by increasing the RH, due to water adsorption on the particle surface and the enhanced electric field strength near the collecting plate. The maximum total collection efficiency of the ESP system was ˜99.2% for a 73% RH collection environment, which was approximately 3.3% higher than that of conventional dry-type ESP (C-ESP). The proposed system also removed sulfur dioxide (SO2), a representative source (90.7%) of undesirable emissions during manufacturing processes, and mitigated ozone (O3, <10 ppb), a by-product of the corona discharge in the ESP, regardless of the field strength. The proposed WPE-ESP included a simple mounting system; additionally, the system generated a more uniform water film than that of wet-ESPs. Thus, the WPE-ESP shows great potential for application to workplaces and machining devices that require the simultaneous removal of ultrafine particles and water-soluble gaseous pollutants. Copyright 2015 American Association for Aerosol Research