• Korean Journal of Microbiology
• /
• v.47 no.3
• /
• pp.200-208
• /
• 2011

Most problematic antibiotic resistance mechanism for MLS (macrolide-lincosamide-streptogramn B) antibiotics encountered in clinical practice is mono- or dimethylation of specific adenine residue at 2058 (E. coli coordinate) of 23S rRNA which is performed by Erm (erythromycin ribosome resistance) protein through which bacterial ribosomes reduce the affinity to the antibiotics and become resistant to them. ErmSF is one of the four gene products produced by Streptomyces fradiae to be resistant to its own antibiotic, tylosin. Unlike other Erm proteins, ErmSF harbors idiosyncratic long N-terminal end region (NTER) 25% of which is comprised of arginine well known to interact with RNA. Furthermore, NTER was found to be important because when it was truncated, most of the enzyme activity was lost. Based on these facts, capability of NTER peptide to inhibit the enzymatic activity of ErmSF was sought. For this, expression system for two different proteins to be expressed in one cell was developed. In this system, two plasmids, pET23b and pACYC184 have unique replication origins to be compatible with each other in a cell. And expression system harboring promoter, ribosome binding site and transcription termination signal is identical but disparate amount of protein could be expressed according to the copy number of each vector, 15 for pACYC and 40 for pET23b. Expression of NTER peptide in pET23b together with ErmSF in pACYC 184 in E. coli successfully gave more amounts of NTER than ErmSF but no inhibitory effects were observed suggesting that there should be dynamicity in interaction between ErmSF and rRNA rather than simple and fixed binding to each other in methylation of 23S rRNA by ErmSF.

• The Korean Journal of Pharmacology
• /
• v.30 no.1
• /
• pp.87-100
• /
• 1994

It has been known for some time that dopamine-containing cells are existed in sympathetic ganglia, i.e., small, intensely fluorescent cells. However, its role and mechanism of action as a peripheral neurotransmitter are poorly understood so far. In the present study, an attempt was made to examine the effect of apomorphine, which is known to be a selective agonist of dopaminergic $D_2$. receptor on secretion of catecholamines (CA) from the isolated perfused rat adrenal gland. The perfusion of a low concentration of 10uM apomorphine into an adrenal vein for 20 min produced significant reduction in CA secretion induced by 5.32 mM ACh, 56 mM KCl, 100 uM DMPP and 100 uM McN-A-343. Increasing apomorphine concentration to 30 uM led to more markedly decreased CA secretion as compared to the case of 10 uM apomorphine and also did inhibit clearly CA release by $10^{-5}M$ Bay-K-8644. Furthermore, in adrenal glands preloaded with a higher dose of 100 uM apomorphine, CA releases evoked by ACh, excess $K^+$, DMPP and McN-A-343 were almost abolished by the drug. The perfusion of $3.3{\pm}10^{-5}M$ metoclopramide, which is well-known as a selective dopaminergic $D_2$ antagonist, produced significantly inhibitory effect of CA release by ACh, DMPP and McN-A-343 but did not affect that by excess $K^+$. However, preloading of 30uM apomorphine in the presence of metoclopramide did not modify the CA secretory effect of excess $K+$ and DMPP. These experimental results demonstrate that apomorphine causes dose-dependent inhibition of CA secretion by cholinergic receptor stimulation and also by membrane depolarization from the isolated perfused rat adrenal gland, suggesting that these effects appear to be exerted by inhibiting influx of extracellular calcium into the rat adrenal medullary chromaffin cells through activation of inhibitory dopaminergic receptors.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.12
• /
• pp.4773-4780
• /
• 2014

Background: To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment. Materials and Methods: The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment. Results: The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5nmol/L~50nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations ($IC_{50}$) of siRNA1384, siRNA1276 and siRNA1786 for 24hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50nmol/L siRNA transfection. Conclusions: Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.

• The Korean Journal of Pharmacology
• /
• v.28 no.2
• /
• pp.147-162
• /
• 1992

The objectives of the present experiments were to characterize the effects of the peptides belonging to the pancreatic polypeptide family on the contractility of cerebral arteries and to observe the interactions of these peptides with the cyclic nucleotide activators and the potassium channel openers. Dogs of either sex, $20{\sim}30\;Kg$ in weight, were sacrificed. Basilar and middle cerebral arteries from brain were isolated and prepared for myography in the PSS equilibrated with 95% $O_2$ and 5% $CO_2$ at $37^{\circ}C$. The endothelial cells of the spiral strips were removed by CHAPS solution (0.3% w/v, 15 seconds). 1. PP, PYY and NPY contracted the arterial strips concentration-dependently with a rank order of potency of PYY>NPY>PP. These peptides were 20 to 200 times more potent than norepinephrine, and only PYY showed a greater potency than 5-HT. 2. Cyclic nucleotide activators, forskolin (for cAMP) and sodium nitroprusside (for cGMP) reduced the basal tone and inhibited the PP-, PYY- and NPY- induced contractions by concentration-dependent manners. Forskolin was more potent in reducing basal tone than sodium nitroprusside. 3. Potassium channel openers, RP 49356, P 1060 and BRL 38227 reduced the basal tone concentration-dependently and tended to inhibit the PP-, PYY- and NPY- induced contractions. Notably, BRL 38227 with low concentration $(0.1\;{\mu}M)$ enhanced the contractions induced by those peptides while P 1060 inhibited the contractions concentration-dependently. 4. The combinations of the cyclic nucleotide activators and the potassium channel openers were slightly additive in reducing the basal tone. P 1060 and BRL 38227 enhanced the relaxant effect of sodium nitroprusside significantly. On the PYY-induced contration $(0.1\;{\mu}M)$, $K^+$ channel openers tended to inhibit the inhibitory actions of forskolin and sodium nitroprusside. P 1060 and BRL 38227 antagonized the inhibitory action of sodium nitroprusside significantly. The results of the present study may be summarized that in canine cerebral arteries, not only NPY but also PYY may play a role in a cerebrovascular spasm, and intracellular concentration of either cAMP or cGMP may be involved in the mechanism of vasoconstrictive actions of these peptides, which may be affected either positively or negatively by a $K^+$ channel opener.

• The Korean Journal of Food And Nutrition
• /
• v.26 no.4
• /
• pp.806-813
• /
• 2013

Agrimonia pilosa Ledeb. is a medicinal plant with anti-tumor, anti-oxidant, anti-inflammatory and anti-hyperglycemic activities. However, few studies of the anti-diabetic effect of A. pilosa on insulin resistance status have been performed. In the present study, the anti-diabetic effect of A. pilosa water extract (AP) was determined by investigating its ${\alpha}$-glucosidase inhibitory property, glucose utilization, and uptake, as well as insulin resistance mechanism of action in C2C12 skeletal muscle cells. Compared to positive control (acarbose), AP ($10mg/m{\ell}$) showed a similar ${\alpha}$-glucosidase inhibitory capacity. Glucose uptake was significantly increased by $1{\mu}m$ insulin treatment (p<0.05). However, palmitic acid (FFA, 1 mM) induced muscle insulin resistance and glucose uptake dysfunction. On the other hand, AP ($10{\mu}g/m{\ell}$) was capable of reversing the FFA-induced insulin resistance in C2C12 myotubes. Compared to control, AP ($100{\mu}g/m{\ell}$ without insulin) significantly increased the utilization of glucose (p<0.05) in C2Cl2 myotubes cultured in normal glucose (7 mM). AP treatment significantly increased the relative mRNA and protein expression levels of Akt. In particular, the effect of A. pilosa on the insulin signaling system is associated with the up-regulation of Akt genes and glucose uptake in C2Cl2 myotubes. These results suggest that A. pilosa is useful in the prevention of diabetes and the treatment of hyperglycemic disorders.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.17 no.2
• /
• pp.529-541
• /
• 2003

Yukmijihwang-tang has been widely used as an and-aging herbal medicine for hundred years in Asian countries. Numerous studies show that Yukmijihwangtang has anti-oxidative effect both in vivo and in vitro. It has been reported that Moutan Cortex Radicis extract (MCR) was the most effective herb in Yukmijihwang-tang on undifferentiated PC12 cells upon oxidative-stressed with hydrogen peroxide. The purpose of this study is to; 1) evaluate the recovery of neuronal damage by assessing the anti-oxidant effect of MCR on PC12 cells differentiated with nerve growth factor (NGF), 2) identify candidate genes responsible for anti-oxidative effect on differentiated PC12 cells by oligonucleotide chip microarray. PC12 cells, which were differentiated by treating with NGF, were treated without or with hydrogen peroxide in the presence or absence of various concentration of MCR. Cell survival was determined by using MTS assay. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H2DCFDA assay The viability of cells treated with MCR was significantly recovered from stressed PC12 cell. In addition, wide rage of concentrations of MCR shows dose-dependent inhibitory effect on ROS production in oxidative-stressed cells. Total RNAs of cells without treatment(Control group), only treated with H₂O₂ (stressed group) and treated with both H₂O₂ and of MCR (MCR group) were isolated, and cDNAs was synthesized using oligoT7(dT) primer. The fragmented cRNAs, synthesized from cDNAs, were applied to Affymetrix GeneChip Rat Neurobiology U34 Array. mRNA of Calcium/calmodulin-dependent protein kinase II delta subunit(CaMKII), neuron glucose transporter (GLUT3) and myelin/oligodendrocyte glycoprotein(MOG) were downregulated in Stressed group comparing to Control group. P2X2-5 receptor (P2X2R-5), P2X2-4 receptor (P2X2R-4), c-fos, 25 kDa synaptosomal attachment protein(SNAP-25a) and GLUT3 were downregulated, whereas A2 adenosine receptor (A2AR), cathechol-O-methyltransferase(COMT), glucose transporter 1 (GLUT1), EST223333, heme oxygenase (HO), VGF, UI-R-CO-ja-a-07-0-Ul.s1 and macrophage migration inhibitory factor (MIF) were upregulated in MCA group comparing to Control group. Expression of Putative potassium channel subunit protein (ACK4), P2X2A-5, P2X2A-4, Interferon-gamma inducing factor isoform alpha precursor (IL-18α), EST199031, P2XR, P2X2 purinoceptor isoform e (P2X2R-e), Precursor interleukin 18 (IL-18) were downregulated, whereas MOO, EST223333, GLUT-1, MIF, Neuronatin alpha, UI-R-C0-ja-a-07-0-Ul.s1, A2. adenosine receptor, COMT, neuron-specific enolase (NSE), HO, VGF, A rat novel protein which is expressed with nerve injury (E12625) were upregulated in MCR group comparing to Stressed group. The results suggest that decreased viability and AOS production of PC12 cell by H₂O₂ may be, at lease, mediated by impaired glucose transporter expression. It is implicated that the MCR treatment protect PC12 cell from oxidative stress via following mechanisms; improving glucose transport into the cell, enhancing expression of anti-oxidative genes and protecting from dopamine cytotoxicity by increment of COMT and MIF expression. The list of differentially expressed genes may implicate further insight on the action and mechanism behind the anti-oxidative effects of herbal extract Moutan Cortex Radicis.

• Radiation Oncology Journal
• /
• v.20 no.4
• /
• pp.367-374
• /
• 2002

Purpose : To investigate the growth inhibitory effects, and the underlying mechanism of human colon cancer cell (HT-29) death, induced by a new synthetic bile acid derivative (HS-1200). Materials and Methods : Human colon cancer cells (HT-29), in exponential growth phase, were treated with various concentrations of a new synthetic bile acid derivative (HS-1200). The growth inhibitory effects on HT-29 cells were examined using a frypan blue exclusion assay. The extent of apoptosis was determined using agarose gel electrophoresis, TUNEL assays and Hoechst staining. The apoptotic cell death was also confirmed by Western blotting of PARP, caspase-3 and DNA fragmentation factor (DFF) analysis. To investigate the involvement of mitochondria, we employed immunofluorescent staining of cytochrome c and mitochondrial membrane potential analyses. Results : The dose required for the half maximal inhibition $(IC_{50})$ of the HT-29 cell growth was $100\~150\;{\mu}M$ of HS-1200. Several changes, associated with the apoptosis of the HT-29 cells, were reveal by the agarose gel eletrophoresis, TUNEL assays and Hoechst staining, following their treatment with $100\;{\mu}M$ of HS-1200. HS-1200 treatment also induced caspase-3, PARP and DFF degradations, and the western blotting showed the processed caspase-3 p20, PARP p85 and DFF p30 and p11 cleaved products. Mitochondrial events were also demonstrated. The cytochrome c staining indicated that cytochrome c had been released from the mitochondria in the HS-1200 treated cells. The mitochondrial membrane potential $(\Delta\Psi_m)$ was also prominently decreased in the HS-1200 treated cells. Conclusion : These findings suggest that the HS-1200 - induced apoptosis of human colon cancer cells (HT-29) is mediated via caspase and mitochondrial pathways.

• Journal of Applied Biological Chemistry
• /
• v.61 no.3
• /
• pp.267-273
• /
• 2018

Matricaria chamomilla L. has been used as a bath agent in Europe because of its sterilization effect on the skin. Flowers contain terpenes, flavonoids are effective in relieving inflammation. Matricaria chamomilla L. has been reported to have various drug efficacies such as sedation, anti-diabetic effect and anti-arthritic effect, but there is little research on the scientific efficacy of whitening effect. The purpose of this study was to examine the whitening effect of Matricaria chamomilla L. extract and to investigate the mechanism of inhibition of melanogenesis. The extracts were used to determine tyrosinase inhibitory activity. The tyrosinase inhibitory activity of extracts was ineffective for water extract but in 60% ethanol extract was shown in a concentration-dependent manner. B16F10 melanoma cell was measured using a powder obtained by lyophilization of 60% ethanol extract. The toxicity was observed at a concentration of $75{\mu}g/mL$. And concentration range was selected to be at most $50{\mu}g/mL$. The effect of tyrosinase, MITF, TRP-1 and TRP-2 on the expression of melanin protein was investigated in melanoma cells of B16F10 melanoma cells. As a result, it was confirmed that as the concentration of the extract increased, the melanogenesis level decreased and the protein expression level also decreased in a concentration dependent manner. Therefore, it was concluded that Matricaria chamomilla L. extract inhibited melanogenesis in cells. Based on the above results, it is expected that it will be used as a useful basic data for industrialization of whitening functional food of Matricaria chamomilla L.

• Journal of the korean academy of Pediatric Dentistry
• /
• v.31 no.4
• /
• pp.605-616
• /
• 2004

Subinhibitory concentrations (sub-MICs) refer to concentrations below minimum inhibitory concentrations (MICs). The antimicrobial agents may be present at relatively high concentration, at least higher than bacterial MIC and thereafter be deserted off a surface and function at sub-MICs, perhaps by interfering with bacterial metabolism. Consequently, the aim of this study was to determine the effects of growth, in the presence of sub-MICs of antimicrobial agents, on the cell surface properties and virulence factors of mutans streptococci and to investigate the efficacy of a chemical approach in vitro. Streptococcus mutans Ingbritt and Streptococcus sobrinus 6715-7 were used. Eight antimicrobial agents (Sanguinaria extract;SG, Chlorhexidine digluconate;CHX, Fluoride;F, Propolis;PP, Hydrogen peroxide;HP, Triclosan;TC, Sodium dodecyl sulfate;SDS Cetylpyridinium chloride; CC) were diluted serially in broth to determine MICs and to compare the growth rate, acid production, hydrophobicity, adhesion activity to saliva coated hydroxyapatite, glucan synthesis and cellular aggregation of experiment groups (in the presence of sub-MICs) with those of control (in the absence of antimicrobial agents). Sub-MICs of antimicrobial agents affected the growth of cells, hydrophobicity, and adhesion of bacteria to saliva coated hydroxyapatite and glucan synthesis. They also resulted in a significant reduction in pH after 12 hours (p<0.05). By cells pretreated with proteinase K, either the aggregation induced by antimicrobial agents was completely inhibited or the aggregation titers were markedly increased. According to the results of the present study, each antimicrobial agent at sub-MICs could affect similar as its known action mechanism and could continually inhibit cariogenic bacteria at such concentrations. Thus, the use of these antimicrobial agents would be one of the effective methods to prevent dental caries.

• Food Science and Preservation
• /
• v.22 no.1
• /
• pp.27-35
• /
• 2015

Anti-obese effects of mulberry (Morus alba L.) root bark was investigated in vitro by measuring its inhibitory effect against 3T3-L1 preadipocyte differentiation and digestive enzymes such as ${\alpha}$-amylase, ${\alpha}$-glucosidase and pancreatic lipase. Ethanol extract of mulberry root bark (MRE) showed the potent inhibitory activities on ${\alpha}$-amylase, ${\alpha}$-glucosidase and pancreatic lipase with $IC_{50}$ values of $7.86{\pm}0.36$, $0.12{\pm}0.03$ and $7.93{\pm}0.11mg/mL$, respectively. Furthermore, MRE significantly suppressed cellular lipid accumulation in 3T3-L1 cells in a dose-dependent manner. To elucidate the mechanism of MRE, we performed qRT-PCR and Western blotting for the expression of genes related with adipogenesis and lipogenesis. Treatment of MRE markedly suppressed the protein expression of $PPAR{\gamma}$, $C/EBP{\alpha}$ and SREBP-1c, as well as FAS and ACC, which are the key transcription factors and metabolic enzymes in adipogenesis and lipogenesis. In addition, qRT-PCR analysis indicated that the anti-adipogenesis effect of MRE might be due to its inhibition at transcription levels. These results demonstrate that MRE can effectively suppress adipocyte differentiation and inhibit key enzymes related to obesity. Our findings suggest that mulberry root bark may have a potential benefit in preventing obesity.