• Asian-Australasian Journal of Animal Sciences
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• v.19 no.10
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• pp.1508-1513
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• 2006

Myostatin (MSTN) and insulin-like growth factors (IGFs) are a known inhibitor and stimulators of proliferation and differentiation of muscle cells, respectively. The present study was performed to investigate the relationship of MSTN-induced growth inhibition to expression of the IGF system components and myogenin, a muscle cell-specific transcription factor, in rat L6 myoblasts. The L6 cells transfected with a green fluorescent protein-MSTN plasmid expression construct had a 47% less cell number than mock-transfected cells after 3-d serum-free culture, accompanied by delayed differentiation which was suggested by inhibited aggregation of cells. Moreover, cells transfected with the expression construct had decreased expression of IGF-II and myogenin genes, but not IGF-I or its receptor genes, as examined by reverse transcription-polymerase chain reaction. The reduced mitosis of the L6 cells transfected with the MSTN-expression construct increased following an addition of either IGF-I or IGF-II to the culture medium, but not to the level of mock-transfected cells. By contrast, myogenin gene expression in these cells increased after the addition of either IGF to the level of mock-transfected cells. Collectively, these results suggest that the inhibitory effect of MSTN on L6 cell proliferation and differentiation is likely to be partly mediated by serially suppressed expression of IGF-II and myogenin genes, not IGF-I gene.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.4
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• pp.500-506
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• 2009

A modified Tris-buffered medium (mTBM) has been widely used as an insemination medium for porcine in vitro fertilization (IVF). We examined whether mTBM could be used for bovine IVF. Bovine cumulus-oocyte complexes (COCs) were cultured in a serum-free medium containing 30 ng/ml EGF for 22 h. After culture, COCs were inseminated with spermatozoa for 12 h in mTBM containing 5 mM caffeine and 10 g/ml heparin. The penetration of oocytes increased significantly (p<0.05) as the sperm concentration increased from 0.1 (30%) to 1-10 $(87-100%){\times}10^6$ cells/ml. This was significantly different from values obtained at 1 (87%) and 10 $(100%){\times}10^6$ cells/ml. However, when COCs were inseminated with spermatozoa from different bulls, the proportions (62-100%) of oocytes penetrated varied according to the bull. The proportion (18%) of oocytes penetrated was significantly (p<0.05) lower in a fertilization medium without caffeine and heparin but increased with the addition of caffeine and/or heparin to the medium, and the proportion (93-96%) of oocytes penetrated increased significantly (p<0.05) when the medium was supplemented with heparin and caffeine. In this medium, sperm penetration was first observed at 3 h after insemination. Irrespective of the presence of glucose in the fertilization medium, the proportion (93-97%) of oocytes penetrated and the proportion (83-84%) of embryos at the ${\geq}2$-cell stage cultured in a chemically defined medium were not significantly different. However, the proportion of embryos developing to the blastocyst stage was significantly (p<0.05) higher in the presence (11%) of glucose in the fertilization medium than in its absence (2%). In conclusion, the present study demonstrated that bovine oocytes penetrated in vitro in mTBM can develop to the blastocyst stage and mTBM may be used for the in vitro production of bovine embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.3
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• pp.414-421
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• 2014

This study was conducted to determine the effects of saturated long-chain fatty acids (LCFA) on cell proliferation and triacylglycerol (TAG) content, as well as mRNA expression of ${\alpha}s1$-casein (CSN1S1) and genes associated with lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows, and were passaged twice. Then cells were cultured with different levels of palmitate or stearate (0, 200, 300, 400, 500, and 600 ${\mu}M$) for 48 h and fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L). The results showed that cell proliferation tended to be increased quadratically with increasing addition of stearate. Treatments with palmitate or stearate induced an increase in TAG contents at 0 to 600 ${\mu}M$ in a concentration-dependent manner, and the addition of 600 ${\mu}M$ was less effective in improving TAG accumulation. The expression of acetyl-coenzyme A carboxylase alpha, fatty acid synthase and fatty acid-binding protein 3 was inhibited when palmitate or stearate were added in culture medium, whereas cluster of differentiation 36 and CSN1S1 mRNA abundance was increased in a concentration-dependent manner. The mRNA expressions of peroxisome proliferator-activated receptor gamma, mammalian target of rapamycin and signal transducer and activator of transcription 5 with palmitate or stearate had no significant differences relative to the control. These results implied that certain concentrations of saturated LCFA could stimulate cell proliferation and the accumulation of TAG, whereas a reduction may occur with the addition of an overdose of saturated LCFA. Saturated LCFA could up-regulate CSN1S1 mRNA abundance, but further studies are necessary to elucidate the mechanism for regulating milk fat and protein synthesis.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.3
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• pp.761-766
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• 2012

Objective: Although various human cancer stem cells (CSCs) have been defined, their applications are restricted to immunocompromised models. Developing a novel CSC model which could be used in immunocompetent or transgenic mice is essential for further understanding of the biomolecular characteristics of tumor stem cells. Therefore, in this study, we analyzed murine lung cancer cells for the presence of CSCs. Methods: Side population (SP) cells were isolated by fluorescence activated cell sorting, followed by serum-free medium (SFM) culture, using Lewis lung carcinoma cell (LLC) line. The self-renewal, differentiated progeny, chemosensitivity, and tumorigenic properties in SP and non-SP cells were investigated through in vitro culture and in vivo serial transplantation. Differential expression profiles of stem cell markers were examined by RT-PCR. Results: The SP cell fraction comprised 1.1% of the total LLC population. SP cells were available to grow in SFM, and had significantly enhanced capacity for cell proliferation and colony formation. They were also more resistant to cisplatin in comparison to non-SP cells, and displayed increased tumorigenic ability. Moreover, SP cells showed higher mRNA expression of Oct-4, ABCG2, and CD44. Conclusion: We identified SP cells from a murine lung carcinoma, which possess well-known characteristics of CSCs. Our study established a useful model that should allow investigation of the biological features and pharmacosensitivity of lung CSCs, both in vitro and in syngeneic immunocompetent or transgenic/knockout mice.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.183-187
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• 2008

Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. $VEGF_{165}$ is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human $VEGF_{165}$ $(rhVEGF_{165})$ protein. The production rate of the established CHO cells was over 80mg/l of $rhVEGF_{165}$ protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The $rhVEGF_{165}$ protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified $rhVEGF_{165}$ protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the $rhVEGF_{165}$ protein in CHO cells differed from that in insect cells. The purified $rhVEGF_{165}$ protein in this study was functionally active with a half-maximal effective concentration of 3.8ng/ml and specific activity of $2.5{\times}10^5U/mg$.

• Journal of Microbiology and Biotechnology
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• v.23 no.12
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• pp.1747-1756
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• 2013

The aim of this paper was to study the effect of spent medium recycle on Spodoptera exigua Se301 cell line proliferation, metabolism, and baculovirus production when grown in batch suspension cultures in Ex-Cell 420 serum-free medium. The results showed that the recycle of 20% of spent medium from a culture in mid-exponential growth phase improved growth relative to a control culture grown in fresh medium. Although both glucose and glutamine were still present at the end of the growth phase, glutamate was always completely exhausted. The pattern of the specific glucose and lactate consumption and production rates, as well as the specific glutamine and glutamate consumption rates, suggests a metabolic shift at spent medium recycle values of over 60%, with a decrease in the efficiency of glucose utilization and an increase in glutamate consumption to fuel energy metabolism. Baculovirus infection provoked a change in the metabolic pattern of Se301 cells, although a beneficial effect of spent medium recycle was also observed. Both growth rate and maximum viable cell density decreased relative to uninfected cultures. The efficiency of glucose utilization was dramatically reduced in those cultures containing the lowest percentages of spent medium, whereas glutamine and glutamate consumption was modulated, thereby suggesting that infected cells were devoted to virus replication, retaining their ability to incorporate the nutrients required to support viral replication. Recycle of 20% of spent medium increased baculovirus production by around 90%, thus showing the link between cell growth and baculovirus production.

• Journal of Life Science
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• v.28 no.10
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• pp.1147-1155
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• 2018

Agaricus blazei mycelial liquid culture extract (ABMLCE) promoted the production of testosterone (TS) in TM-3 mouse Leydig testis cells. Now, we report that ABMLCE containing eritadenine (EA) as a minor constituent (15.3 mg/100 g) reduced $5{\alpha}-reductase$ 2 ($5{\alpha}-R2$) enzyme activity and dihydrotestosterone (DHT) content which are key constituents for the benign prostatic hyperplasia (BPH) inductions. RWPE-1 prostate cells were grown in a Keratinocyte serum-free medium (K-SFM) containing ABMLCE (0~50 ppm), EA (0~10 ppm,), and finasteride (FS $10{\mu}M$: a positive control) in a 24-well plate for 24 hr. Supernatants collected from cell-cultured media were used for the assay of $5{\alpha}-R2$, superoxide dismutase (SOD), catalase (CAT) and cyclooxygenase-2 (COX-2) enzyme activities, and for TS, DHT, tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$) and $interleukin-1{\beta}$ ($IL-1{\beta}$) contents by their assay kits. The $5{\alpha}-R2$ activity and DHT content were proportionally reduced (p<0.05) to concentrations of ABMLCE. The SOD and CAT enzyme activities were significantly (p<0.05) elevated concomitant with ABMLCE concentrations, while COX-2, $TNF-{\alpha}$ and $IL-1{\beta}$ showed reverse results (p<0.05). Similarly, the effects of EA were similar to those of ABMLCE. Efficacies of ABMLCE 50 ppm and EA 10 ppm in $5{\alpha}-R2$ and DHT reduction were similar to those of $10{\mu}M$ FS. These results suggest that ABMLCE and EA reduced $5{\alpha}-R2$ and DHT through their anti-inflammatory and anti-oxidative actions. This implies that ABMLCE containing EA could be a beneficial material in the cure of BPH in humans.

• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.3
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• pp.512-517
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• 2004

The effects of liquid culture of Coriolus versicolor on the body weight gain, lipid metabolism, protein levels and enzyme activities were studied in growing male rats. Sprague-Dawley rats were divided into four groups; control group (C. versicolor free water), 10%, 20% or 30% C. versicolor groups (10%, 20% or 30% C. versicolor in water) according to the levels of C. versicolor supplementation. The rats were fed ad libitum each of the experimental beverage for 5 weeks. The body weight gain, food intake, food efficiency ratio, and organ weight of the all C. versicolor groups were similar to those of the control group. There were no differences in the concentrations of triglyceride, total cholesterol, HDL-cholesterol, total protein and albumin, and the activities of GOT, GPT, and alkaline phosphatase of the serum among the experimental groups. However, the atherogenic index of the 30% C. versicolor groups were significantly lower than those of the control group, and the serum HDL-cholesterol/total cholesterol ratio increased.

• Journal of Life Science
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• v.31 no.6
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• pp.587-594
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• 2021

By 2050, 70% more food will be needed to fulfill the demands of a growing population. Among the solutions, cultured meat or clean meat is presented as a sustainable alternative for consumers. Scientists have begun to leverage knowledge and tools accumulated in the fields of stem cell and tissue engineering in efforts aimed at the development of cell-based meat. Cultured meat has to recreate the complex structure of livestock muscles with a few cells. Cells start to divide after they are cultured in a culture medium, which provides nutrients, hormones, and growth factors. An initial problem with this type of culture is the serum used, as in vitro meat aims to be slaughter free. Thus, it is contradictory to use a medium made from the blood of dead calves. The serum is expensive and affects to a large extent the production cost of the meat. A positive aspect related to the safety of cultured meat is that it is not produced from animals raised in confined spaces and slaughtered in inhumane conditions. Thus, the risk of an outbreak is eliminated, and there is no need for vaccinations and animal welfare issues. The production of cultured meat is presented as environmentally friendly, as it is supposed to produce less greenhouse gas, consume less water, and use less land in comparison to conventional meat production.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.63-69
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• 2001

This study was carried out to examine the effects of nitric oxide compounds (hemoglobin and L-NAME) on the development of porcine in vitro maturation (IVM) and in vitro fertilization (IVF) oocytes. Cumulus cell free embryos derived from porcine IVM/IVF oocytes were cultured in NCSU23 medium containing 1~5 $\mu\textrm{g}$/$m\ell$ hemoglobin added to 44 and 96hrs in culture times, and in NCSU23 medium containing 0, 10, 50 or 100mM L-NAME. The developmental rates beyond morulae stage in 0, 1 and 5 $\mu\textrm{g}$/$m\ell$ hemoglobin groups add to 44hrs in vitro culture times were 52.4%, 57.6% and 57.4%, respectively. The addition of hemoglobin groups made it slightly higher than the control group. The proportion of embryos developed to morulae and blastocysts in 1 $\mu\textrm{g}$/$m\ell$ hemoglobin add to 96hrs after in vitro culture (70.8%) was a little higher than those of 0 and 5 $\mu\textrm{g}$/$m\ell$ hemoglobin (66.2% and 62.8%). There was no significant difference in all groups (P〉0.05). The developmental rates beyond morulae stage in 0, 10, 50 and 100mM of L-NAME groups add to 96hrs after in vitro culture were 65.2%, 73.5%, 70.1% and 53.3%, respectively 10mM and 50mM L-NAME groups were significantly higher than in 0 and 100mM of L-NAME groups (P＜0.05). In conclusions, these results indicate that L-NAME (10mM, 50mM) can increase the proportion of embryos that develop into morulae and blastocysts but hemoglobin did not affect.