• Experimental and Molecular Medicine
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• 제50권11호
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• pp.6.1-6.12
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• 2018

Morphine tolerance remains a challenge in the management of chronic pain in the clinic. As shown in our previous study, the dopamine D2 receptor (D2DR) expressed in spinal cord neurons might be involved in morphine tolerance, but the underlying mechanisms remain to be elucidated. In the present study, selective spinal D2DR blockade attenuated morphine tolerance in mice by inhibiting phosphatidylinositol 3 kinase (PI3K)/serine-threonine kinase (Akt)-mitogen activated protein kinase (MAPK) signaling in a ${\mu}$ opioid receptor (MOR)-dependent manner. Levo-corydalmine (l-CDL), which exhibited micromolar affinity for D2DR in D2/CHO-K1 cell lines in this report and effectively alleviated bone cancer pain in our previous study, attenuated morphine tolerance in rats with chronic bone cancer pain at nonanalgesic doses. Furthermore, the intrathecal administration of l-CDL obviously attenuated morphine tolerance, and the effect was reversed by a D2DR agonist in mice. Spinal D2DR inhibition and l-CDL also inhibited tolerance induced by the MOR agonist DAMGO. l-CDL and a D2DR small interfering RNA (siRNA) decreased the increase in levels of phosphorylated Akt and MAPK in the spinal cord; these changes were abolished by a PI3K inhibitor. In addition, the activated Akt and MAPK proteins in mice exhibiting morphine tolerance were inhibited by a MOR antagonist. Intrathecal administration of a PI3K inhibitor also attenuated DAMGO-induced tolerance. Based on these results, l-CDL antagonized spinal D2DR to attenuate morphine tolerance by inhibiting PI3K/Akt-dependent MAPK phosphorylation through MOR. These findings provide insights into a more versatile treatment for morphine tolerance.

• Toxicological Research
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• 제24권3호
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• pp.175-182
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• 2008

DNA-dependent protein kinase(DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination and is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PKcs. It has been suggested that DNA-PK might be $2^{nd}$ upstream kinase for protein kinase B(PKB). In this report, we showed that Ser473 phosphorylation in the hydrophobic-motif of PKB is blocked in DNA-PK knockout mouse embryonic fibroblast cells(MEFs) following insulin stimulation, while there is no effect on Ser473 phosphorylation in DNA-PK wild type MEF cells. The observation is further confirmed in human glioblastoma cells expressing a mutant form of DNA-PK(M059J) and a wild-type of DNA-PK(M059K), indicating that DNA-PK is indeed important for PKB activation. Furthermore, the treatment of cells with doxorubicin, DNA-damage inducing agent, leads to PKB phosphorylation on Ser473 in control MEF cells while there is no response in DNA-PK knockout MEF cells. Together, these results proposed that DNA-PK has a potential role in insulin signaling as well as DNA-repair signaling pathway.

• Molecules and Cells
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• 제38권10호
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• pp.876-885
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• 2015

N-acetyl-D-glucosamine kinase (NAGK) plays an enzyme activity-independent, non-canonical role in the dendritogenesis of hippocampal neurons in culture. In this study, we investigated its role in axonal development. We found NAGK was distributed throughout neurons until developmental stage 3 (axonal outgrowth), and that its axonal expression remarkably decreased during stage 4 (dendritic outgrowth) and became negligible in stage 5 (mature). Immunocytochemistry (ICC) showed colocalization of NAGK with tubulin in hippocampal neurons and with Golgi in somata, dendrites, and nascent axons. A proximity ligation assay (PLA) for NAGK and Golgi marker protein followed by ICC for tubulin or dynein light chain roadblock type 1 (DYNLRB1) in stage 3 neurons showed NAGK-Golgi complex colocalized with DYNLRB1 at the tips of microtubule (MT) fibers in axonal growth cones and in somatodendritic areas. PLAs for NAGK-dynein combined with tubulin or Golgi ICC showed similar signal patterns, indicating a three way interaction between NAGK, dynein, and Golgi in growing axons. In addition, overexpression of the NAGK gene and of kinase mutant NAGK genes increased axonal lengths, and knockdown of NAGK by small hairpin (sh) RNA reduced axonal lengths; suggesting a structural role for NAGK in axonal growth. Finally, transfection of 'DYNLRB1 (74-96)', a small peptide derived from DYNLRB1's C-terminal, which binds with NAGK, resulted in neurons with shorter axons in culture. The authors suggest a NAGK-dynein-Golgi tripartite interaction in growing axons is instrumental during early axonal development.

• The Korean Journal of Physiology and Pharmacology
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• 제2권2호
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• pp.251-260
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• 1998

Cromakalim (BRL 34915), known as an airway smooth muscle relaxant, inhibited the releases of mediators in the antigen-induced mast cell activation. It has been suggested that cromakalim, in part, inhibited mediator releases by inhibiting the initial increase of 1,2-diacylglycerol (DAG) produced by the activation of the other phospholipase system which is different from phosphatidylcholine-phospholipase D pathway. The aim of this study is to further examine the inhibitory mechanism of cromakalim on the mediator release in the mast cell activation. Guinea pig lung mast cells were purified by using enzyme digestion and percoll density gradient. In purified mast cells prelabeled with $[^3H]PIP_2$, phospholipase C (PLC) activity was assessed by the production of $[^3H]$insitol phosphates. Protein kinase C (PKC) activity was assessed by measuring the protein phosphorylated from mast cells prelabeled with $[{\gamma}-32P]ATP$, and Phospholipase $A_2\;(PLA_2)$ activity by measuring the lyso-phosphatidylcholine produced from mast cell prelabeled with 1-palmitoyl-2-arachidonyl $phosphatidyl-[^{14}C]choline$. Histamine was assayed by fluorometric analyzer, and leukotrienes by radioimmunoassay. The PLC activity was increased by activation of the passively sensitized mast cells. This increased PLC activity was decreased by cromakalim pretreatment. The PKC activity increased by the activation of the passively sensitized mast cells was decreased by calphostin C, staurosporine and cromakalim, respectively. The $PLA_2$ activity was increased in the activated mast cells. The pretreatment of cromakalim did not significantly decrease $PLA_2$ activity. These data show that cromakalim inhibits histamine release by continuously inhibiting signal transduction processes which is mediated via PLC pathway during mast cell activation, but that cromakalim does not affect $PLA_2$ activity related to leukotriene release.

• Nutrition Research and Practice
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• 제9권6호
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• pp.606-612
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• 2015

BACKGROUND/OBJECTIVES: Several medicinal properties of Smilax china L. have been studied including antioxidant, anti-inflammatory, and anti-cancer effects. However, the antiobesity activity and mechanism by which the water-soluble fraction of this plant mediates its effects are not clear. In the present study, we investigated the lipolytic actions of the water-soluble fraction of Smilax china L. leaf ethanol extract (wsSCLE) in 3T3-L1 adipocytes. MATERIALS/METHODS: The wsSCLE was identified by measuring the total polyphenol and flavonoid content. The wsSCLE was evaluated for its effects on cell viability, lipid accumulation, glycerol, and cyclic adenosine monophosphate (cAMP) contents. In addition, western blot analysis was used to evaluate the effects on protein kinase A (PKA), PKA substrates (PKAs), and hormone-sensitive lipase (HSL). For the lipid accumulation assay, 3T3-L1 adipocytes were treated with different doses of wsSCLE for 9 days starting 2 days post-confluence. In other cell experiments, mature 3T3-L1 adipocytes were treated for 24 h with wsSCLE. RESULTS: Results showed that treatment with wsSCLE at 0.05, 0.1, and 0.25 mg/mL had no effect on cell morphology and viability. Without evidence of toxicity, wsSCLE treatment decreased lipid accumulation compared with the untreated adipocyte controls as shown by the lower absorbance of Oil Red O stain. The wsSCLE significantly induced glycerol release and cAMP production in mature 3T3-L1 cells. Furthermore, protein levels of phosphorylated PKA, PKAs, and HSL significantly increased following wsSCLE treatment. CONCLUSION: These results demonstrate that the potential antiobesity activity of wsSCLE is at least in part due to the stimulation of cAMP-PKA-HSL signaling. In addition, the wsSCLE-stimulated lipolysis induced by the signaling is mediated via activation of the ${\beta}$-adrenergic receptor.

• 대한의생명과학회지
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• 제10권4호
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• pp.375-383
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• 2004

It has been reported that osteoporosis induced by the deficiency of estrogens in menopause is associated with the unbalance of Ca/sup 2+/ and Pi levels. Proximal tubule is very important organ to regualte Ca/sup 2+/ and Pi level in the body. However, the effect of estrogens on Ca/sup 2+/ and Pi regulation was not elucidated. Thus, we examined the effect of 17-β estradiol (E₂) on Ca/sup 2+/ and Pi uptake in the primary cultured rabbit renal proxiaml tubule cells. In the present study, E₂(> 10/sup -9/M) decreases Ca/sup 2+/uptake and stimulates Pi uptake over 3 days. E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by actinomycin D (a gene transcription inhibitor), cycloheximide (a protein synthesis inhibitor). tamoxifen, and progesterone (estrogen receptor antagonists). E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by SQ22536 (an adenylate cyclase inhibitor), Rp-cAMP (a cAMP antagonist), and PKI (a protein kinase A inhibitor). Indeed, E₂ increased cAMP formation. In addition, E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by staurosporine, H-7, and bisindolylmaleimide I (protein kinase C inhibitors) and E₂ translocated PKC from cytoslic fraction to membrane fraction. In conclusion, E₂ decreased Ca/sup 2+/ uptake and stimulated Pi uptake via cAMP and PKC pathway in the PTCs.

• Molecules and Cells
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• 제38권7호
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• pp.604-609
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• 2015

The active metabolite of vitamin D such as $1{\alpha}$,25-dihydroxyvitamin ($D_3(1{\alpha},25(OH)_2D_3)$ is a well-known key regulatory factor in bone metabolism. However, little is known about the potential of vitamin D as an odontogenic inducer in human dental pulp cells (HDPCs) in vitro. The purpose of this study was to evaluate the effect of vitamin $D_3$ metabolite, $1{\alpha},25(OH)_2D_3$, on odontoblastic differentiation in HDPCs. HDPCs extracted from maxillary supernumerary incisors and third molars were directly cultured with $1{\alpha},25(OH)_2D_3$ in the absence of differentiation-inducing factors. Treatment of HDPCs with $1{\alpha},25(OH)_2D_3$ at a concentration of 10 nM or 100 nM significantly upregulated the expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein1 (DMP1), the odontogenesis-related genes. Also, $1{\alpha},25(OH)_2D_3$ enhanced the alkaline phosphatase (ALP) activity and mineralization in HDPCs. In addition, $1{\alpha},25(OH)_2D_3$ induced activation of extracellular signal-regulated kinases (ERKs), whereas the ERK inhibitor U0126 ameliorated the upregulation of DSPP and DMP1 and reduced the mineralization enhanced by $1{\alpha},25(OH)_2D_3$. These results demonstrated that $1{\alpha},25(OH)_2D_3$ promoted odontoblastic differentiation of HDPCs via modulating ERK activation.

• 한국동물학회지
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• 제30권2호
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• pp.177-192
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• 1987

온열처리가 근세포의 분화에 어떤 영향을 미치는 지를 알아보기 위하여 배양한 근세포에 여러가지 온열처리를 한 다음, 단백질 합성, 세포증식, 융합지수, creatine kinase(CK)의 활성 및 cholesterol 함량의 변화를 조사하였다. 배양한지 24시간지나 45$^{\circ}C$에서 1 hr의 온열처리를 하면 근세포의 융합과 CK 활성이 지연되었으며, 55시간지나 같은 온열처리를 하면 세포막내(세포내 양의 95% 이상)의 chloesterol 양이 일시적으로 증가함과 아울러 세포융합이 지연되었다. 그러나 세포증식은 대조군과 뚜렷한 차이가 없었다. 이상과 같은 실험 결과로부터 온열처리가 분화에 미치는 영향은 온열처리를 받게되는 근원세포의 분화정도에 따라 차이가 있으며 온열처리에 따른 chloesterol 양의 일시적인 증가가 근세포 융합에 영향을 미칠 수 있다는 가능성이 제시되었다. 한편, 근세포는 온열처리를 받으면 평소의 단백질 합성 수준이 떨어짐과 더불어 heat shock protein(HSP)합성이 증대 내지는 유도 되었으며 HSP 합성의 유도는 actinomycin D의 처리로 억제되었다. 또한 온열처리로 근세포는 열내성을 얻어 세포융하과 CK 활성은 동일한 온열처리를 4시간 간격으로 두번 주어도 한번 주었을 경우와 차이가 없었으며 두번째 온열처리에 의해서는 새로운 HSP 합성이 유도되지도 않았다.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1170-1178
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• 2008

The cyclopentenone prostaglandins (cyPGs) prostaglandin $A_1$ ($PGA_1$) and 15-deoxy-${\Delta}^{12,14}$-prostaglandin $J_2$ (15d-$PGJ_2$) have been reported to exhibit antiinflammatory activity in activated monocytes/macrophages. However, the effects of these two cyPGs on the expression of cytokine genes may differ. In this study, we investigated the mechanism of action of $PGA_1$ in lipopolysaccharide (LPS)-induced expression of inter leu kin (IL)-10 mRNA in mouse peritoneal macrophages. 15d-$PGJ_2$ inhibited expression of LPS-induced IL-10, whereas $PGA_1$ increased LPS-induced IL-10 expression. This synergistic effect of $PGA_1$ on LPS-induced IL-10 expression reached a maximum as early as 2 h after simultaneous $PGA_1$ and LPS treatment ($PGA_1$/LPS), and did not require new protein synthesis. The synergistic effect of $PGA_1$ was inhibited by GW9662, a specific peroxisome proliferator-activated receptor ${\gamma}(PPAR{\gamma})$ antagonist, and Bay-11-7082, a NF-${\kappa}B$ inhibitor. The extracellular signal-regulated kinases (ERK) inhibitor PD98059 increased the expression of $PGA_1$/LPS-induced IL-10 mRNA, rather than inhibiting the IL-10 expression. Moreover, $PGA_1$ inhibited LPS-induced ERK phosphorylation. The synergistic effect of $PGA_1$ on LPS-induced IL-10 mRNA and protein production was inhibited by p38 inhibitor PD169316, and $PGA_1$ increased LPS-induced p38 phosphorylation. In the case of stress-activated protein kinase/c-Jun $NH_2$-terminal kinase (SAPK/JNK), the SAPK/JNK inhibitor SP600125 did not inhibit IL-10 mRNA synthesis but inhibited the production of IL-10 protein remarkably. These results suggest that the synergistic effect of $PGA_1$ on LPS-induced IL-10 expression is NF-${\kappa}B$-dependent and mediated by mitogen-activated protein (MAP) kinases, p38, and SAPK/JNK signaling pathways, and also associated with the $PPAR{\gamma}$ pathway. Our data may provide more insight into the diverse mechanisms of $PGA_1$ effects on the expression of cytokine genes.

• International Journal of Oral Biology
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• 제45권4호
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• pp.152-161
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• 2020

D-pinitol is an analog of 3-methoxy-D-chiro-inositol found in beans and plants. D-pinitol has anti-inflammatory, antidiabetic, and anticancer effects. Additionally, D-pinitol induces apoptosis and inhibits metastasis in breast and prostate cancers. However, to date, no study has investigated the anticancer effects of D-pinitol in oral cancer. Therefore, in this study, whether the anticancer effects of D-pinitol induce apoptosis, inhibit the epithelial-to-mesenchymal transition (EMT), and arrest cell cycle was investigated in squamous epithelial cells. D-pinitol decreased the survival and cell proliferation rates of CAL-27 and Ca9-22 oral squamous carcinoma cells in a concentration- and time-dependent manner. Evidence of apoptosis, including nuclear condensation, poly (ADP-ribose) polymerase, and caspase-3 fragmentation, was also observed. D-pinitol inhibited the migration and invasion of both cell lines. In terms of EMT-related proteins, E-cadherin was increased, whereas N-cadherin, Snail, and Slug were decreased. D-pinitol also decreased the expression of cyclin D1, a protein involved in the cell cycle, but increased the expression of p21, a cyclin-dependent kinase inhibitor. Hence, D-pinitol induces apoptosis and cell cycle arrest in CAL-27 and Ca9-22 cells, demonstrating an anticancer effect by decreasing the EMT.