• Molecules and Cells
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• v.41 no.9
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• pp.868-880
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• 2018

Pancreatic cancer (PC) is one of the most aggressive cancers presenting with high rates of invasion and metastasis, and unfavorable prognoses. The current study aims to investigate whether EZH2/miR-139-5p axis affects epithelial-mesenchymal transition (EMT) and lymph node metastasis (LNM) in PC, and the mechanism how EZH2 regulates miR-139-5p. Human PC and adjacent normal tissues were collected to determine expression of EZH2 and miR-139-5p, and their relationship with clinicopathological features of PC. Human PC cell line was selected, and treated with miR-139-5p mimics/inhibitors, EZH2 vector or shEZH2 in order to validate the regulation of EZH2-mediated miR-139-5p in PC cells. Dual-luciferase report gene assay and chromatin immunoprecipitation assay were employed to identify the relationship between miR-139-5p and EZH2. RT-qPCR and Western blot analysis were conducted to determine the expression of miR-139-5p, EZH2 and EMT-related markers and ZEB1/2. Tumor formation ability and in vitro cell activity were also analyzed. Highly-expressed EZH2 and poorly-expressed miR-139-5p were detected in PC tissues, and miR-139-5p and EZH2 expressions were associated with patients at Stage III/IV, with LNM and highly-differentiated tumors. EZH2 suppressed the expression of miR-139-5p through up-regulating Histone 3 Lysine 27 Trimethylation (H3K27me3). EMT, cell proliferation, migration and invasion were impeded, and tumor formation and LNM were reduced in PC cells transfected with miR-139-5p mimics and shEZH2. MiR-139-5p transcription is inhibited by EZH2 through up-regulating H3K27me3, thereby down-regulation of EZH2 and up-regulation of miR-139-5p impede EMT and LNM in PC. In addition, the EZH2/miR-139-5p axis presents as a promising therapeutic strategy for the treatment of PC.

• Journal of Microbiology
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• v.44 no.2
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• pp.192-199
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• 2006

Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene $(C_1-C_4)$, biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99 %) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.

• Herbal Formula Science
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• v.23 no.2
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• pp.199-208
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• 2015

Objectives : In the present study, we investigated the effects of ethanol extract of Scutellaria baicalensis (EESB) on the progression of cell cycle in human renal cell carcinoma Caki-1 cells. Methods : The effects of EESB on cell growth and apoptosis induction were evaluated by trypan blue dye exclusion assay and flow cytometry, respectively. The mRNA and protein levels were determined by Western blot analysis and reverse transcription-polymerase chain reaction, respectively. Results : It was found that EESB treatment on Caki-1 cells resulted in a dose-dependent inhibition of cell growth and induced apoptotic cell death as detected by Annexin V-FITC staining. The flow cytometric analysis indicated that EESB resulted in G2/M arrest in cell cycle progression which was associated with the down-regulation of cyclin A expression. Our results also revealed that treatment with EESB increased the mRNA and proteins expression of tumor suppressor p53 and cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/CIP1), without any noticeable changes in cyclin B1, Cdk2 and Cdc2. In addition, the incubation of cells with EESB resulted in a significant increase in the binding of p21 and Cdk2 and Cdc2. These findings suggest that EESB-induced G2/M arrest and apoptosis in Caki-1 cells is mediated through the p53-mediated upregulation of Cdk inhibitor p21. Conclusions : Taken together, these findings suggest that EESB may be a potential chemotherapeutic agent and further studies will be needed to identify the biological active compounds that confer the anti-cancer activity of S. baicalensis.

• The Journal of Korean Medicine
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• v.31 no.3
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• pp.79-97
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• 2010

Background: Peripheral nerve injuries are commonly encountered clinical problems and often result in severe functional deficit. Bee venom acupuncture has traditionally been used to treat several inflammatory diseases and chronic pain conditions. Objectives: The aims of this study were to compare the effects of bee venom (general bee venom, BV) and sweet bee venom (allergen-removed bee venom, SBV) acupuncture on the recovery rate of locomotor function, the expression of brain-derived neurotrophic factor (BDNF) in the sciatic nerve, and the expression of c-Fos in the brain following sciatic crushed nerve injury in rats, and to evaluate differences due to administration areas. Method: Walking track analysis, Western blot for BDNF and tyrosine receptor kinase B (TrkB), and immunohistochemistry for c-Fos were performed. In this study, comparative analyses of the effects of BV and SBV acupuncture in relation to administration sites, contralateral side or ipsilateral side, were conducted. Results: In the present result, sciatic function index (SFI) in walking track analysis significantly decreased following sciatic crushed nerve injury. The expressions of BDNF and TrkB in the sciatic nerve increased after induction of sciatic crushed nerve injury. C-Fos expression in the ventrolateral periaqueductal gray (vlPAG) and paraventricular nucleus (PVN) also increased. BV and SBV acupuncture treatment improved the SFI in walking track analysis. Treatment with SBV at 1mg/kg showed more potent enhancing effect on SFI compared to BV. Treatment with 1mg/kg BV or 1mg/kg SBV acupuncture suppressed the BDNF and TrkB expression in the sciatic nerve. BV and SBV acupuncture treatment also suppressed c-Fos expression in the PVN and vlPAG regions. Treatment with SBV at 1mg/kg showed more potent suppressing effect on c-Fos expression compared to BV when injected into the contralateral side of the injured nerve. Generally we could not find significant difference in the effects between contralateral side and ipsilateral side of the injured nerve. Conclusion: We have shown that BV and SBV acupuncture treatment can be used as the effective therapeutic modality to ameliorate the symptoms of sciatic crushed nerve injury.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.6
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• pp.695-702
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• 2017

The sustained tonic currents ($I_{tonic}$) generated by ${\gamma}$-aminobutyric acid A receptors ($GABA_{A}Rs$) are implicated in diverse age-dependent brain functions. While various mechanisms regulating $I_{tonic}$ in the hippocampus are known, their combined role in $I_{tonic}$ regulation is not well understood in different age groups. In this study, we demonstrated that a developmental increase in GABA transporter (GAT) expression, combined with gradual decrease in $GABA_AR{\alpha}_5$ subunit, resulted in various $I_{tonic}$ in the dentate gyrus granule cells (DGGCs) of preadolescent rats. Both GAT-1 and GAT-3 expression gradually increased at infantile ($P_{6-8}$ and $P_{13-15}$) and juvenile ($P_{20-22}$ and $P_{27-29}$) stages, with stabilization observed thereafter in adolescents ($P_{34-36}$) and young adults ($P_{41-43}$). $I_{tonic}$ facilitation of a selective GAT-1 blocker (NO-711) was significantly less at $P_{6-8}$ than after $P_{13-15}$. The facilitation of $I_{tonic}$ by SNAP-5114, a GAT-3 inhibitor, was negligible in the absence of exogenous GABA at all tested ages. In contrast, $I_{tonic}$ in the presence of a nonselective GAT blocker (nipecotic acid, NPA) gradually decreased with age during the preadolescent period, which was mimicked by $I_{tonic}$ changes in the presence of exogenous GABA. $I_{tonic}$ sensitivity to L-655,708, a $GABA_AR{\alpha}_5$ subunit inverse agonist, gradually decreased during the preadolescent period in the presence of NPA or exogenous GABA. Finally, Western blot analysis showed that the expression of the $GABA_AR{\alpha}_5$ subunit in the dentate gyrus gradually decreased with age. Collectively, our results suggested that the $I_{tonic}$ regulation of altered GATs is under the final tune of $GABA_AR{\alpha}_5$ subunit activation in DGGCs at different ages.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.421-431
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• 2016

Recently, poly-γ-glutamic acid synthetase A (pgsA) has been applied to display exogenous proteins on the surface of Lactobacillus casei or Lactococcus lactis, which results in a surface-displayed component of bacteria. However, the ability of carrying genes encoded by plasmids and the expression efficiency of recombinant bacteria can be somewhat affected by the longer gene length of pgsA (1,143 bp); therefore, a truncated gene, pgsA, was generated based on the characteristics of pgsA by computational analysis. Using murine IL-10 as an exogenous gene, recombinant Lactobacillus plantarum was constructed and the capacity of the surface-displayed protein and functional differences between exogenous proteins expressed by these strains were evaluated. Surface expression of IL-10 on both recombinant bacteria with anchorins and the higher expression levels in L. plantarum-pgsA'-IL-10 were confirmed by western blot assay. Most importantly, up-regulation of IL-1β, IL-6, TNF-α, IFN-γ, and the nuclear transcription factor NF-κB p65 in RAW264.7 cells after stimulation with Poly(I:C) or LPS was exacerbated after co-culture with L. plantarum-pgsA. By contrast, IL-10 expressed by these recombinant strains could reduce these factors, and the expression of these factors was associated with recombinant strains that expressed anchorin (especially in L. plantarum-pgsA'-IL-10) and was significantly lower compared with the anchorin-free strains. These findings indicated that exogenous proteins could be successfully displayed on the surface of L. plantarum by pgsA or pgsA', and the expression of recombinant bacteria with pgsA' was superior compared with bacteria with pgsA.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1720-1728
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• 2020

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40℃, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.23-23
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• 2003

It has been suggested that the impairment of smooth muscle cell (SMC) function by alterations in the $Ca^{2+}$-activated $K^{+}$ ( $K_{Ca}$ ) channels accounts for the reduction in coronary reserve during left ventricular hypertrophy (LVH). However, this hypothesis has not been fully investigated. The main goal of this study was to assess whether the properties of $K_{Ca}$ channels in coronary SMCs were altered during LVH. New Zealand white rabbits (0.8-1.0 kg) and Sprague-Dawley rats (300-400 g) were randomly selected to receive either an injection of isoproterenol (300 $\mu\textrm{g}$/kg body weight) or an equal volume of 0.9％ saline (1 mL/kg body weight). The animals developed LVH 10 days after injection. In patch-clamp experiments, the unitary current amplitude and open probability for the $K_{Ca}$ channels were significantly reduced in LVH patches compared with control patches. The concentration-response curve of the $K_{Ca}$ channel to [C $a^{2+}$]$_{i}$ was shifted to the right. Inhibition of the $K_{Ca}$ channels with TEA was more pronounced in LVH cells than in the control cells. The whole-cell currents of $K_{Ca}$ channels were reduced during LVH. Western blot analysis indicated no differences in $K_{Ca}$ channel expression between the control and LVH coronary SM membranes. In contraction experiments, the effect of a high $K^{+}$concentration on the resting tension of the LVH coronary artery was greater than on that of the control. The effect of TEA on the resting tension of the LVH coronary artery was reduced as compared with the effect on the control. Our findings imply a novel mechanism for reduced coronary reserve during LVH.ing LVH.

• IMMUNE NETWORK
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• v.2 no.1
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• pp.60-64
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• 2002

Background: Chemotherapy with 5-fluorouracil (5-FU) has been one of the mainstay in breast cancer treatment. The effects of high dose 5-FU on cell cycle regulation were studied in breast caner cells. Methods: A breast cancer cell line MCF-7 was used. Protein expressions of G1/S cyclins, $p21^{Waf1/Cip1}$, cdk2, E2F1 and retinoblastoma were tested by western blot analysis. Immunoprecipitation and immune complex kinase assay were done for the assessment of E2F1/RB interacton and the activity of cdk2 respectively. Results: $p21^{Waf1/Cip1}$ expression was barely detectable in control cells. With addition of 5-FU level of $p21^{Waf1/Cip1}$ were induced and cyclin D3 level was decreased as cell growth decreases. In accordance with increased expression of $p21^{Waf1/Cip1}$, cyclin E-associated cdk2 kinase activity was reduced. Retinoblastoma protein (RB) became dephosphorylated and E2F-1 binding activity with RB was increased. Conclusion: In this situation of high concentration of 5-FU breast cancer cells tend to be G1/S cell cycle arrested. Overexpression of $p21^{Waf1/Cip1}$ and dephosphorylation of RB may mediate the effectss of 5-FU by inhibiting E2F-1 activity, which contributes to G1/S cell cycle arrest. These results could be an indicating landmark for further study of high dose chemotherapy with 5-FU.

• IMMUNE NETWORK
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• v.11 no.2
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• pp.107-113
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• 2011

Background: Metabolic disorders, including type II diabetes and obesity, present major health risks in industrialized countries. AMP-activated protein kinase (AMPK) has become the focus of a great deal of attention as a novel therapeutic target for the treatment of metabolic syndromes. In this study, we evaluated whether dietary aloe could reduce obesity-induced inflammation and adipogenesis. Methods: Male C57BL/6 obese mice fed a high-fat diet for 54 days received a supplement of aloe formula (PAG, ALS, Aloe QDM, and Aloe QDM complex) or pioglitazone (PGZ) and were compared with unsupplemented controls (high-fat diet; HFD) or mice fed a regular diet (RD). RT-PCR and western blot analysis were used to quantify the expression of obesity-induced inflammation. Results: Aloe QDM complex downregulated fat size through suppressed expression of scavenger receptors on adipose tissue macrophages (ATMs) compared with HFD. Both white adipose tissue (WATs) and muscle exhibited increased AMPK activation through aloe supplementation, and in particular, the Aloe QDM complex. Obesity-induced inflammatory cytokines (IL-$1{\beta}$ and -6) and $HIF1{\alpha}$ mRNA and protein were decreased markedly, as was macrophage infiltration by the Aloe QDM complex. Further, the Aloe QDM complex decreased the translocation of NF-${\kappa}B$ p65 from the cytosol in the WAT. Conclusion: Dietary aloe formula reduced obesity-induced inflammatory responses by activation of AMPK in muscle and suppression of proinflammatory cytokines in the WAT. Additionally, the expression of scavenger receptors in the ATM and activation of AMPK in WAT led to reduction in the percent of body fat. Thus, we suggest that the effect of the Aloe QDM complex in the WAT and muscle are related to activation of AMPK and its use as a nutritional intervention against T2D and obesity-related inflammation.