• Journal of Life Science
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• v.25 no.1
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• pp.1-7
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• 2015

Cyclin D is a member of the cyclin protein family, which plays a critical role as a core member of the mammalian cell cycle machinery. D-type cyclins (D1, D2, and D3) bind to and activate the cyclin-dependent kinases 4 and 6, which can then phosphorylate the retinoblastoma tumor suppressor gene products. This phosphorylation in turn leads to release or derepression of E2F transcription factors that promote progression from the G1 to S phase of the cell cycle. Among the D-type cyclins, cyclin D3 encoded by the CCND3 gene is one of the least well studied. In the present study, we have investigated the biochemistry of the proteolytic mechanism that leads to loss of cyclin D3 protein. Treatment of human prostate carcinoma PC-3-M cells with lovastatin and actinomycin D resulted in a loss of cyclin D3 protein that was completely reversible by the peptide aldehyde calpain inhibitor, LLnL. Additionally, using inhibitors for various proteolytic systems, we show that degradation of cyclin D3 protein involves the $Ca^{2+}$-activated neutral protease calpain. Moreover, the half-life of cyclin D3 protein half-life increased by at least 10-fold in PC-3M cells in response to the calpain inhibitor. We have also demonstrated that the transient expression of the calpain inhibitor calpastatin increased cyclin D3 protein in serum-starved NIH 3T3 cells. These data suggested that the function of cyclin D3 is regulated by $Ca^{2+}$-dependent protease calpain.

• Development and Reproduction
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• v.6 no.2
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• pp.123-129
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• 2002

Leptin, a product of the obese gene, is associated not only with obesity but also with female reproductive function, but it has not yet been ascertained whether leptin acts directly on the ovary or indirectly via the hypothalamus-pituitary pathway. Therefore, the object of this study was to investigate the expession of leptin and its receptor in the rat ovary by immunohistochemistry and RT-PCR during the estrous cycle. Immunohistochemistry results showed that leptin was stained in the theca cells and in part of granulosa cells in atretic follicles, whereas leptin receptor was localized in the interstitial cells and ova in preantral follicies. In particular, leptin and its receptor in atretic follicles displayed more intensive staining compared to those in normal follicles. During the estrous cycle, the mRNA expression of leptin and its receptor in the ovary was detected by RT-PCR and estradiol, progesterone, and leptin levels in the serum was measured by ELISA. The leptin level in the serum on metestrous phase was significantly higher than that on estrous phase. Similar to leptin level, progesterone level increased on metestrous phase. Leptin mRNA was not detected throughout the estrous cycle, whereas leptin receptor mRNA was expressed on all phases of estrous cycle excepting the diestrous phase. These results suggest that leptin might be directly involved in the regulation of ovarian function in rat.

• Journal of Ginseng Research
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• v.41 no.3
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• pp.257-267
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• 2017

Background: Heat-processed ginseng, sun ginseng (SG), has been reported to have improved therapeutic properties compared with raw forms, such as increased antidiabetic, anti-inflammatory, and antihyperglycemic effects. The aim of this study was to investigate the antiobesity effects of SG through the suppression of cell differentiation and proliferation of mouse 3T3-L1 preadipocyte cells and the lipid accumulation in Caenorhabditis elegans. Methods: To investigate the effect of SG on adipocyte differentiation, levels of stained intracellular lipid droplets were quantified by measuring the oil red O signal in the lipid extracts of cells on differentiation Day 7. To study the effect of SG on fat accumulation in C. elegans, L4 stage worms were cultured on an Escherichia coli OP50 diet supplemented with $10{\mu}g/mL$ of SG, followed by Nile red staining. To determine the effect of SG on gene expression of lipid and glucose metabolism-regulation molecules, messenger RNA (mRNA) levels of genes were analyzed by real-time reverse transcription-polymerase chain reaction analysis. In addition, the phosphorylation of Akt was examined by Western blotting. Results: SG suppressed the differentiation of 3T3-L1 cells stimulated by a mixture of 3-isobutyl-1-methylxanthine, dexamethasone, and insulin (MDI), and inhibited the proliferation of adipocytes during differentiation. Treatment of C. elegans with SG showed reductions in lipid accumulation by Nile red staining, thus directly demonstrating an antiobesity effect for SG. Furthermore, SG treatment down-regulated mRNA and protein expression levels of peroxisome proliferator-activated receptor subtype ${\gamma}$ ($PPAR{\gamma}$) and CCAAT/enhancer-binding protein-alpha ($C/EBP{\alpha}$) and decreased the mRNA level of sterol regulatory element-binding protein 1c in MDI-treated adipocytes in a dose-dependent manner. In differentiated 3T3-L1 cells, mRNA expression levels of lipid metabolism-regulating factors, such as amplifying mouse fatty acid-binding protein 2, leptin, lipoprotein lipase, fatty acid transporter protein 1, fatty acid synthase, and 3-hydroxy-3-methylglutaryl coenzyme A reductase, were increased, whereas that of the lipolytic enzyme carnitine palmitoyltransferase-1 was decreased. Our data demonstrate that SG inversely regulated the expression of these genes in differentiated adipocytes. SG induced increases in the mRNA expression of glycolytic enzymes such as glucokinase and pyruvate kinase, and a decrease in the mRNA level of the glycogenic enzyme phosphoenol pyruvate carboxylase. In addition, mRNA levels of the glucose transporters GLUT1, GLUT4, and insulin receptor substrate-1 were elevated by MDI stimulation, whereas SG dose-dependently inhibited the expression of these genes in differentiated adipocytes. SG also inhibited the phosphorylation of Akt (Ser473) at an early phase of MDI stimulation. Intracellular nitric oxide (NO) production and endothelial nitric oxide synthase mRNA levels were markedly decreased by MDI stimulation and recovered by SG treatment of adipocytes. Conclusion: Our results suggest that SG effectively inhibits adipocyte proliferation and differentiation through the downregulation of $PPAR{\gamma}$ and $C/EBP{\alpha}$, by suppressing Akt (Ser473) phosphorylation and enhancing NO production. These results provide strong evidence to support the development of SG for antiobesity treatment.

• Journal of Plant Biotechnology
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• v.35 no.4
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• pp.317-327
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• 2008

Phytocystatins, which are inhibitors of plant cysteine peptidases, are involved in the regulation of protein turnover and in the defense against insect pests and pathogens. Extensive searches in the Brassica rapa genome allowed the prediction of at least eight different phytocystatin genes on seven chromosomes in the B. rapa genome. Structure comparisons based on alignments of the all BrCYS ($\underline{B}$. $\underline{r}apa$ $phyto{\underline{cys}}tatin$) proteins using the CLUSTALW program revealed conservation of the three consensus motifs known to interact with the active site of cysteine peptidases. According to the phylogenetic analysis based on the deduced amino acid sequences, the eight BrCYS proteins were divided into several clusters related to the orthologous phytocystatin. The predicted three-dimensional structure models of the eight BrCYS proteins demonstrate that all of these proteins are similar to the reported crystal structure of oryzacystatin-I (OC-I). Digital northern and RT-PCR analyses indicated that the eight BrCYS genes exhibit different expression patterns in B. rapa tissues and respond differently to abiotic stimuli. The differences in gene structure and expression between the eight BrCYS genes suggest that these proteins may play diverse physiological roles in B. rapa and may interact with cysteine peptidases through different mechanisms.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.5
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• pp.664-670
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• 2016

Cheonggukjang (CKJ) is a Korean traditional food made of fermented soybeans. In comparison to normal intake of soybeans, Cheonggukjang has high digestibility with bioactive, antioxidant substances, and thrombolytic enzymes. Recent studies have reported anti-oxidant, anti-cancer, anti-inflammatory, anti-obesity activities as well as inhibitory activities against osteoporosis for CKJ. In this study, we identified the effects of CKJ on osteoblast differentiation by increasing the polyglutamic acid (PGA) content of CKJ. Alkaline phosphatase (ALP) activity and mineralization significantly increased in response to treatment with both natural CKJ (CKJ A) and PGA-increased CKJ (CKJ B). However, CKJ B exhibited higher ALP activity and mineralization than CKJ A. Real-time reverse transcription PCR demonstrated that mRNA expression of osteoblastic-associated genes such as type I collagen, alkaline phosphatase, osteocalcin, and osteopontin in C2C12 cells was significantly up-regulated by CKJ A or B treatment. These results indicate that treatment with CKJ has an anabolic effect on bone by increasing osteoblastic differentiation and ALP activity. Increasing PGA content in CKJ had a greater effect than CKJ A on up-regulation of osteoblastic gene expression in osteoblast cells.

• Molecules and Cells
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• v.38 no.7
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• pp.643-650
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• 2015

The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects.

• Korean Journal of Microbiology
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• v.55 no.2
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• pp.103-111
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• 2019

DNA methylation is involved in diverse processes in bacteria, including maintenance of genome integrity and regulation of gene expression. CcrM, the DNA methyltransferase conserved in Alphaproteobacterial species, carries out $N^6$-adenine or $N^4$-cytosine methyltransferase activities using S-adenosyl methionine as a co-substrate. Celeribacter marinus IMCC12053 from the Alphaproteobacterial group was isolated from a marine environment. Single molecule real-time sequencing method (SMRT) was used to detect the methylation patterns of C. marinus IMCC12053. Gibbs motif sampler program was used to observe the conversion of adenosine of 5'-GANTC-3' to $N^6$-methyladenosine and conversion of $N^4$-cytosine of 5'-GpC-3' to $N^4$-methylcytosine. Exocyclic DNA methyltransferase from the genome of strain IMCC12053 was chosen using phylogenetic analysis and $N^4$-cytosine methyltransferase was cloned. IPTG inducer was used to confirm the methylation activity of DNA methylase, and cloned into a pQE30 vector using dam-/dcm- E. coli as the expression host. The genomic DNA and the plasmid carrying methylase-encoding sequences were extracted and cleaved with restriction enzymes that were sensitive to methylation, to confirm the methylation activity. These methylases protected the restriction enzyme site once IPTG-induced methylases methylated the chromosome and plasmid, harboring the DNA methylase. In this study, cloned exocyclic DNA methylases were investigated for potential use as a novel type of GpC methylase for molecular biology and epigenetics.

• Journal of Life Science
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• v.13 no.2
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• pp.184-190
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• 2003

Myxococcus xanthus is a Gram negative, rod-shaped, soil bacterium that displays a social behaviors, and multicellular development upon nutrient deprivation. The csgA gene encoding a cell surface protein is essential for developmental behaviors including rippling, aggregation, fruiting body formation and sporulation. csgA mutants show normal vegetative growth, but lack all these developmental phenotypes. Expression of the CsgA (C-signal) specific genes are eliminated or dramatically reduced in csgA mutants. In order to identify components of C-signal transduction pathway, second site mutations were introduced into csgA mutants and were identified which can fully or partially restore development of csgA mutants (Rhie, H. G. et. al. 1989. J. Bacteriol. 171, 3268-3276). One of such csgA suppressor mutations, socD500 restores only sporulation to csgA mutants at 15$^{\circ}C$. The socD500 mutaion however eliminates the three basic developmental requirements, starvation, high cell density and a solid surface. Only sporulation, not accompanied with fruiting body formation is induced simply by shifting the temperature of vegetatively growing cells from $32^{\circ}C$ to $15^{\circ}C$. Spores induced by socD500 mutation is not as thick as that of wild-type fruiting body. In socD500 genetic background, two of ten C-signal dependent genes, $\Omega$DK4506 and $\Omega$DK4406 are more highly expressed in growing cells at $15^{\circ}C$. These results indicate that the socD500 mutation may be partly involved in the regulation of expression of two C-signal dependent genes and genes for sporulation in this transduction pathway.

• Journal of Life Science
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• v.20 no.4
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• pp.503-510
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• 2010

Most allergens have protease activities, suggesting that proteases may be a key link between Th2-type immune reactions in allergic responses. Protease activated receptor (PAR) 2 is activated via the proteolytic cleavage of its N-terminal domain by proteinases. To know the role of PAR2 in Aspergillus protease allergen activated Th2 immune responses in airway epithelial cells, we investigated and compared immune cell recruitment and level of chemokines and cytokines between PAR2 knock out (KO) mice and wild type (WT) mice. There were evident immune cell infiltrations into the bronchial alveolar lavage fluid (BALF) of WT mice, but the infiltrations in PAR2 KO mice were significantly lowered than those of WT mice. The IL-25, TSLP, and eotaxin gene expressions were profoundly increased after Aspergillus protease, but their expression was significantly lowered in PAR2 KO mice in this study. Compared to PAR2 KO mice, OVA specific IgE concentrations in serum of WT mice were quite increased; moreover, the IgE level of PAR2 KO mice was lower than in WT mice. The IL-25 expression by Aspergillus protease stimulation was significantly reduced by p38 specific inhibitor treatment. In this study, we determined that Th2 response was initiated with IL-25 and TSLP mRNA up-regulation in lung epithelial cells via PAR2 after Aspergillus protease allergen treatment.

• Journal of Life Science
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• v.21 no.11
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• pp.1607-1611
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• 2011

To analyze the role of Bombyx mori transferrin (BmTf) in response to microbes or oxidative stress, we investigated the level of BmTf transcripts in B. mori treated with various microbes and oxidative stress inducers. BmTf mRNA was mainly expressed in the epidermis and fat in the bodies of B. mori injected with Escherichia coli, and up regulated in response to microbes such as bacteria, fungi, or viruses, but was hardly altered in response to oxidative stress inducers such as $H_2O_2$, Cu, or $FeCl_3$. We also confirmed that BmTf mRNA expression was increased in Bm5 cells treated with ERK, PLC, PKA, PI3K, MAPK, or JNK inhibitors, respectively. To identify the major inducer of BmTf expression, we analyzed the amount of serum iron in the hemolymph of B. mori after injection or feeding with E. coli or $FeCl_3$. The results showed that the amount of serum iron was not changed by injection and feeding with E. coli, although BmTf mRNA was increased by injection with E. coli. On the contrary, injection and feeding with $FeCl_3$ significantly increased the amount of serum iron, although they did not alter the BmTf mRNA level. On the basis of these results, we assume that up-regulation of BmTf in B. mori is closely related to the defense of microorganism, and BmTf may be expressed at the basal constitutive level when it plays a role in iron metabolism by maintaining iron homeostasis and in the insect defense mechanism against oxidative stress.