• KSBB Journal
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• v.18 no.4
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• pp.312-317
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• 2003

The addition of glycine up to 0.5％ (w/v) to Luria broth (LB) media on the secretion of levansucrase in a recombinant strain Escherichia coli JM109/pUPLK1 was observed to enhance the release of periplasmic proteins from the cell to the broth, without significantly affecting the cell growth rate and protein productivity. However, the glycine concentration at 1 ％ (w/v), the cell density attainable at the stationary phase fell to about 50％ and the extracellular activity of levansucrase corresponded to about 80％ of the total (extracellular plus intracellular) activity and increased by 2.6-fold, comparing to the cells grown in the absence of glycine. The increased pH at stationary phase accelerated the degradation of levansucrase. Maximal extracellular activity was attained when 1 ％ glycine was supplemented at the onset of strain growth.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.9
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• pp.1245-1252
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• 2017

Objective: Phellodendron amurense (P. amurense) and Humulus japonicus (H. japonicus) are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos. Methods: After in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense ($0.01{\mu}g/mL$) and H. japonicus ($0.01{\mu}g/mL$). The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated. Results: We observed that the blastocysts rate was significantly increased (p<0.05) in P. amurense ($28.9%{\pm}2.9%$), H. japonicus ($30.9%{\pm}1.5%$), and a mixture of P. amurense and H. japonicus ($34.8%{\pm}2.1%$) treated groups compared with the control group ($25.4%{\pm}1.6%$). We next confirmed that the intracellular levels of reactive oxygen species (ROS) were significantly decreased (p<0.01) in P. amurense and/or H. japonicus extract treated groups when compared with the control group. Our results also showed that expression of cleaved caspase-3 and apoptotic cells of blastocysts were significantly decreased (p<0.05) in bovine blastocysts derived from both P. amurense and H. japonicus extract treated embryos. Conclusion: These results suggest that proper treatment with P. amurense and H. japonicus extracts in the development of preimplantation bovine embryos improves the quality of blastocysts, which may be related to the reduction of ROS level and apoptosis.

• Journal of Microbiology and Biotechnology
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• v.31 no.6
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• pp.775-783
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• 2021

Sea cucumber, Holothuria scabra, is a well-known traditional Asian medicine that has been used for suppressing inflammation, promoting wound healing, and improving immunity. Moreover, previous studies demonstrated that the extract from H. scabra contains many bioactive compounds with potent inhibitory effect on tumor cell survival and progression. However, the effect of the methanolic extract from the body wall of H. scabra (BWMT) on human prostate cancer cells has not yet been investigated. In this study, we aimed to investigate the effects and underlying mechanism of BWMT on prostate cancer cell viability and metastasis. BWMT was obtained by maceration with methanol. The effect of BWMT on cell viability was assessed by MTT and colony formation assays. The intracellular ROS accumulation was evaluated using a DCFH-DA fluorescence probe. Hoechst 33342 staining and Annexin V-FITC/PI staining were used to examine the apoptotic-inducing effect of the extract. A transwell migration assay was performed to determine the anti-metastasis effect. BWMT significantly reduced cell viability and triggered cellular apoptosis by accumulating intracellular ROS resulting in the upregulation of JNK and p38 signaling pathways. In addition, BWMT also inhibited the invasion of PC3 cells by downregulating MMP-2/-9 expression via the ERK pathway. Consequently, our study provides BWMT from H. scabra as a putative therapeutic agent that could be applicable against prostate cancer progression.

• Toxicological Research
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• v.11 no.2
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• pp.199-203
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• 1995

The focus of this study was to investigate that cellular parameters and glucose uptake might be altered by extracellular calcium and starvation. Addition of 1 mM $Ca^{++}$ to hepatocytes (equalling to the free calcium concentration of blood) significantly increased intracellular $Na^+$ and decreased $Na^+$ & LDH leakage. This pertains to the hepatocytes of control rats as well as those of rats fasted for 24 and 48. hr. These effects might be come from the membrane-stabilizing effects of calcium. But calcium had no effects on cell volumes, superoxide-formation and glucose uptake. Actually hepatocytes of starved rats showed changes in several cellular parameters. Starvation increased LDH leakage, glucose uptake and the total concentration of $Na^+$ and $Na^+$ whereas it markedly decreased cell volumes. Since total tonicity remained unchanged, intracellular $Na^+$ and $Na^+$ could contribute to a higher share of total osmolarity in starvation. Starvation increased the cytoplasmic pH because $R-NH^{3+}$ions and their corresponding counterions disappeared. This increase may be related to suppress the protonization of amino groups in proteins. Starvation decreased hepatic glycogen, a major compound that affects cytosolic volume of hepatocytes. The data indicate that starvation increases the glucose transport activity. The possible molecular basis will be discussed.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.3
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• pp.304-310
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• 2012

The present study showed that Transforming growth factor beta 1 (TGF-${\beta}_1$) can induce apoptosis of bovine mammary epithelial cells. This apoptosis was also observed with phosphorylation of Smad2/3 within 0.5-2 h. Afterwards the signal transferred into the nucleus. Moreover, intracellular free $Ca^{2+}$ concentration was significantly elevated as well as Caspase-3 activated and DNA lysised, thereby inducing the programmed cell death. This signaling pathway of TGF-${\beta}_1$ was blocked by SB-431542 ($10^{-2}{\mu}M$) via inhibiting ALK-5 kinase activity, which thus reversed the anti-proliferation and apoptosis effect of TGF-${\beta}_1$ in mammary epithelial cells. These results indicated that TGF-${\beta}_1$ induced apoptosis of bovine mammary epithelial cells through the ALK-5-Smad2/3 pathway, which plays an important role in inhibiting survival of mammary epithelial cells. Moreover, intracellular $Ca^{2+}$ also played a critical role in TGF-${\beta}_1$-induced cell apoptosis.

• BMB Reports
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• v.38 no.3
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• pp.281-289
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• 2005

Tumor necrosis factor (TNF) signaling is mediated via two distinct receptors, TNFR2 and TNFR1, which shows partially overlapping signaling mechanisms and biological roles. In the present study, TNFR2 and TNFR1 signal transduction mechanisms involved in activation of $NF{\kappa}B$ and CMV promoter-enhancer were compared with respect to their susceptibility towards inhibitors of intracellular signaling. For this, we used SW480 cells, where we have shown that TNF-signaling can occur independently through each of the two receptors. The TNFR1 response was inhibited by D609, bromophenacyl bromide (BPB), nordihydroguararetic acid (NDGA), and by sodium salicylate, while TNFR2-mediated activation of $NF{\kappa}B$ and CMV promoter-enhancer was resistant to these compounds. The signaling mechanisms known to be affected by these inhibitors include phospholipases as well as redox- and pH-sensitive intracellular components. Our results imply that TNFR2 signaling involved in $NF{\kappa}B$ activation proceeds independently of these inhibitor-sensitive signaling components, indicating distinct signaling pathways not shared with TNFR1.

• Journal of Microbiology
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• v.35 no.3
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• pp.213-221
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• 1997

The secretion of the alkaliphilic Bacillus sp. S-1 extracellular pullulanase involves translocation across the cytoplasmic membrane of the Gram-positive bacterial cell envelope. Translocation of the intracellular pullulanase PUL-I, was traced to elucidate the mechanism and pathway of protein secretion from an alkaliphilic Bacillus sp. S-1. Pullulanase could be slowly bue quantitatively released into the medium during growth of the cells in medium contianing proteinase K. The released pullulanase lacked the N-terminal domain. The N-terminus is the sole membrane anchor in the pullulanase protein and was not affected by proteases, confirming that it is not exposed on the cell surface. Processing of a 180,000M$\_$r/ pullulanase to a 140,000M$\_$r/ polypeptide has been demonstrated in cell extracts using antibodies raised against 140,000M$\_$r/ extracellular form. Processing of the 180,000 M$\_$r/ protein occured during the preparation of extracts in an alkaline pH condition. A modified rapid extraction procedure suggested that the processing event also occured in vivo. Processing apparently increased the activity of pullulanase. The western blotting analysis with mouse anti-serum against 140-kDa extracellular pullulanase PUL-E showed that PUL-I is processed into PUL-X via intermediate form of PUL-E. Possible explanationa for the translocation are discussed.

• Journal of Embryo Transfer
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• v.33 no.1
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• pp.1-11
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• 2018

Cellular cyclic adenosine-3' 5'-monophosphate (cAMP) modulator is known as meiotic inhibitor and can delays spontaneous maturation in IVM experiment. Among many cAMP modulators, the role of Pituitary adenylate cyclase activating polypeptide (PACAP) on IVM isn't known. The purpose of this study is to improve the maturation of oocytes derived from follicles ${\leq}3mm$ in diameter through PACAP as meiotic inhibitor during pre-in vitro maturation (pre-IVM). First, we checked PACAP and its receptors in cumulus cells and, to establish the optimal phase and concentration of PACAP for pre-IVM, we conducted chromatin configuration assessments. As a result, the rate of GV (Germinal Vesicle) according to duration of pre-IVM was significantly decreased 12 h and 18 h after IVM (87.1 and 84.1%, respectively) compared to 0 h (99.4%). When COC was cultured for 18 h, the GV rate in the $1{\mu}M$ of PACAP treatment group (82.1%) was significantly higher than any other PACAP treatment groups (60.5, 64.1, 74.4 and 69.9 %, respectively). So, we divided into four groups as follows; MF (the conventional IVM group, obtained from follicle from 3 to 6 mm in diameter), SF (the conventional IVM group, obtained from follicle ${\leq}3mm$ in diameter), Pre-SF(-)PACAP (IVM group including 18 h pre-IVM without $1{\mu}M$ of PACAP, obtained from follicle ${\leq}3mm$ in diameter) and Pre-SF(+)PACAP (IVM group including 18 h pre-IVM with $1{\mu}M$ of PACAP, obtained from follicle ${\leq}3mm$ in diameter). To examine the effect of PACAP during pre-IVM, we investigated analysis of nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. In cumulus cells, PACAP receptors, ADCYAP1R1 and VIPR1 were detected but were not detected in oocytes. After IVM, the Pre-SF(+)PACAP had the highest Metaphase II rate (91.7%) among all groups (P<0.05). The GSH levels in the MF and Pre-SF(+)PACAP were significantly higher than in the other groups (P<0.05) and ROS levels was no significant difference among all groups. In conclusion, these results indicated that even though the oocytes were derived from SF, pre-IVM application of PACAP improved meiotic and cytoplasmic maturation by regulating intracellular oxidative stress.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.2
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• pp.83-89
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• 2010

In this study, we studied whether hydrogen sulfide ($H_2S$) has an effect on the pacemaker activity of interstitial cells of Cajal (ICC), in the small intestine of mice. The actions of $H_2S$ on pacemaker activity were investigated using whole-cell patch-clamp technique, intracellular $Ca^{2+}$ analysis at $30^{\circ}C$ and RT-PCR in cultured mouse intestinal ICC. Exogenously applied sodium hydrogen sulfide (NaHS), a donor of hydrogen sulfide, caused a slight tonic inward current on pacemaker activity in ICC at low concentrations (50 and $100{\mu}m$), but at high concentration ($500{\mu}m$ and 1 mM) it seemed to cause light tonic inward currents and then inhibited pacemaker amplitude and pacemaker frequency, and also an increase in the resting currents in the outward direction. Glibenclamide or other potassium channel blockers (TEA, $BaCl_2$, apamin or 4-aminopydirine) did not have an effect on NaHS-induced action in ICC. The exogenous application of carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and thapsigargin also inhibited the pacemaker activity of ICC as NaHS. Also, we found NaHS inhibited the spontaneous intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) oscillations in cultured ICC. In doing an RT-PCR experiment, we found that ICC enriched population lacked mRNA for both CSE and CBS, but was prominently detected in unsorted muscle. In conclusion, $H_2S$ inhibited the pacemaker activity of ICC by modulating intracellular $Ca^{2+}$. These results can serve as evidence of the physiological action of $H_2S$ as acting on the ICC in gastrointestinal (GI) motility.

• Korean Journal of Animal Reproduction
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• v.17 no.1
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• pp.49-56
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• 1993

Mouse mammary epithelial cells(NMuMG) were plated onto 24 well phates(100,000 cells/well), in DMEM supplemented with 10% fetal calf serum. After serum starvation for 24 hours, EGF)0~100ng/ml) was added simultaneously with IGF-I(10ng/ml), 1$\mu$M photoreactive cAMP(4,5-dimethoxy-2-nitrobenzyl adenosine-3',5' cyclic monophosphate, DMNB) or IGF-I plus DMNB. After 2 hours, the cells were expposed to UV light(300nm, 3 second pulse0 in order to activate DMNB which induces a rapid transient increase in intracellular cAMP upon UV irradiation. DNA synthesis was estimated as incorporation of 3H-thymidine into DNA(1 hour pulse with 1$\mu$Ci/ml, 18~19 hours after UV exposure). Without IGF-I or DMNB, EGF(10 or 100ng/ml) increased DNA synthesis from 8,362 dpm/well in control to 16,345 or 18,684 dpm/well with EGF(pooled SE=1,239 dpm/well, P<0.05). IGF-I or IGF-I plus DMNB alone increased DNA synthesis from 8,362 dpm/well in control to 17,307 or 20,427 dpm/well, respectively(P<0.05). Addition of IGF-I, DMNB or IGF-I plus DMNB into 0~100ng/ml EGF did not significantly change the shape of dose response curve of EGF alone. In other experiment, EGF or IGF-I plus DMNB into 10ng/ml EGF group exhibited interaction effect in DNAsynthesis [EGF(10ng/ml)=18,497; IGF-I＋EGF=22,837; DMNB＋EGF=20,658 ; IGF-I＋DMNB＋EGF=29,658, pooled SE=1,055, P<0.05]. These results indicate that simultaneous activation of EGF, IGF-I and intracellular cAMP interact in DNA synthesis of mouse mammary epithelial cells.