• Journal of Microbiology and Biotechnology
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• v.23 no.5
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• pp.637-643
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• 2013

We have demonstrated that 1-deoxynojirimycin (DNJ) isolated from Bacillus subtilis MORI could enhance the levels of adiponectin and its receptors in differentiated 3T3-L1 adipocytes, which has been shown to be effective in lowering blood glucose levels and enhancing insulin sensitivity. DNJ was not toxic to differentiated 3T3-L1 adipocytes for up to a concentration of $5{\mu}M$. In terms of expression levels of adiponectin and its receptors (AdipoR1 and AdipoR2), DNJ in concentrations as low as $0.5{\mu}M$ elevated both mRNA and protein levels of adiponectin and transcript levels of AdipoR1 and AdipoR2. In addition, DNJ increased phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK) in a statistically significant manner. Finally, treatment with DNJ resulted in increased mRNA expression of glucose transporter 4 (GLUT4), which encodes for a glucose transporter, along with a significant increase in glucose uptake into the adipocytes based on results of a 2-deoxy-D-[$^3H$] glucose uptake assay. Our findings indicate that DNJ may greatly facilitate glucose uptake into adipose tissues by increasing the action of adiponectin via its up-regulated expression as well as its receptor genes. In addition, the glucose-lowering effects of DNJ may be achieved by an increased abundance of GLUT4 protein in the plasma membrane, as a consequence of the increased transcript levels of the GLUT4 gene and the activation of AMPK.

• Journal of Pharmaceutical Investigation
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• v.31 no.3
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• pp.151-160
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• 2001

The purpose of this study is to investigate the interaction of progesterone with various cyclodextrins (CDs) in the aqueous solution and in solid state, and finally to formulate a parenteral aqueous formulation. CDs used were ${\alpha}-$, ${\beta}-$, and ${\gamma}-CD$, $2-hydroxypropyl-{\beta}-CD$ (HPCD), sulfobutyl $ether-{\beta}-CD$ (SBCD), $dimethyl-{\beta}-CD$ (DMCD) and $trimethyl-{\beta}-CD$ (TMCD). The solubility studies of progesterone were performed in the presence of various CDs as a function of concentration or temperature. The solubility of progesterone increased in the rank order of ${\alpha}-CD$ < ${\beta}-CD$ < ${\gamma}-CD$ < TMCD$ < HPCD < DMCD < SBCD. Addition of SBCD (200 mg/ml) in water increased the aqueous solubility $(9.36\;{\mu}g/ml)$ about 3,200 times, and lowering the temperature facilitated the solubilization of progesterone. However, the addition of HPCD and SBCD in 20:80 (v/v) polyethylene glycol 300-water and propylene glycol-water cosolvents markedly decreased the solubility of progesterone, compared with solubilizing effects in water. Physical mixtures and solid dispersions of progesterone with HPCD or SBCD were prepared, and evaluated by differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FT-IR), near IR spectroscopy and dissolution studies. By DSC and IR studies, it was found that progesterone was dispersed in HPCD in monotectic state and dissolved rapidly from both solid dispersions. Based on solubility studies, new aqueous progesterone fonnulations (5 mg/ml) containing SBCD (200 mg/ml) could be prepared and did not form precipitates even after 2 months at $4^{\circ}C$. The solution was transparent when mixed with normal saline and 5% dextrose injection at 1: 1, 1:10 and 1:20 (v/v) even after 7 days. Permeation rates of progesterone through a cellulose membrane from 20% PEG 300 solution $(50\;{\mu}g/ml)$ containing HPCD or SBCD were compared with oily formulation. Permeation of progesterone from oily formulation did not occur up to 8 hr, but aqueous formulations showed fast permeation rates from early stage of permeation study. The addition of HPCD or SBCD retarded the permeation rates of progesterone with the increase of CD concentrations, suggesting the possibility of a controlled absorption from the site administered intramuscularly. These results demonstrate that it is feasible to develop a new progesterone parenteral aqueous injection (5 mg/ml) using SBCD.

• The Korean Journal of Pharmacology
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• v.26 no.1
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• pp.55-66
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• 1990

In both resting and opsonized zymosan activated neutrophils, ATP stimulated superoxide generation, whereas adenosine inhibited it slightly. The superoxide generation in activated neutrophils to ATP was greater than that of resting neutrophils. In $Ca^{++}$ free medium, inhibitory effect of adenosine on superoxide generation was detectable, whereas ATP did not have any effect. The stimulatory effect of ATP on superoxide generation was inhibited by adenosine in a dose dependent manner. Neither ATP nor adenosine had any effect on NADPH oxidase acitivity. Effects of ATP or adenosine on superoxide generation were more prominent than that by other triphosphate nucleotides or nucleosides. ATP and ADP further stimulated $Ca^{++}$ uptake and increased cytosolic free $Ca^{++}$ level in neutrophils activated by opsonized zymosan, but adenosine inhibited a $Ca^{++}$ mobilization. Verapamil effectively and tetrodotoxin slightly inhibited an increase of cytosolic free $Ca^{++}$ level induced by ATP. Inhibitory effect of either verapamil or tetrodotoxin on superoxide generation in the ATP plus opsonized zymosan-activated neutrophils was greater than in the cells activated by opsonized zymosan alone. Tetraethylammonium chloride had no apparent effect on superoxide generation. CCCP, 2,4-dinitrophenol, diphenylhydantoin and procaine all inhibited superoxide generation in neutrophils activated by opsonized zymosan. Among these, CCCP only inhibited a stimulatory effect of ATP. ATP further stimulated a loss of sulfhydryl groups in activated neutrophils, whereas adenosine had no effect on it. These results suggest that functional responses of neutrophils may be regulated at least partly by purines. ATP and adenosine may further after functional responses of activated neutrophils through their effect on $Ca^{++}$ uptake, membrane phosphorylation and oxidation of soluble sulfhydryl groups.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.3
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• pp.119-125
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• 2010

We investigated the effects of a hot-water extract of Artemisia iwayomogi, a plant belonging to family Compositae, on cardiac ventricular delayed rectifier $K^+$ current ($I_K$) using the patch clamp technique. The carbohydrate fraction AIP1 dose-dependently increased the heart rate with an apparent $EC_{50}$ value of $56.1{\pm}5.5\;{\mu}g/ml$. Application of AIP1 reduced the action potential duration (APD) in concentration-dependent fashion by activating $I_K$ without significantly altering the resting membrane potential ($IC_{50}$ value of $APD_{50}$: $54.80{\pm}2.24$, $IC_{50}$ value of $APD_{90}$: $57.45{\pm}3.47\;{\mu}g/ml$). Based on the results, all experiments were performed with $50\;{\mu}g/ml$ of AIP1. Pre-treatment with the rapidly activating delayed rectifier $K^+$ current ($I_{Kr}$) inhibitor, E-4031 prolonged APD. However, additional application of AIP1 did not reduce APD. The inhibition of slowly activating delayed rectifier $K^+$ current ($I_{Ks}$) by chromanol 293B did not change the effect of AIP1. AIP1 did not significantly affect coronary arterial tone or ion channels, even at the highest concentration of AIP1. In summary, AIP1 reduces APD by activating $I_{Kr}$ but not $I_{Ks}$. These results suggest that the natural product AIP1 may provide an adjunctive therapy of long QT syndrome.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.6
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• pp.341-346
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• 2005

The mechanism underlying oxidant-induced intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) increase was studied in cultured bovine aortic endothelial cells (BAECs) using fura-2 AM. In the presence of 2 mM extracellular $Ca^{2+}$, the application of DTBNP ($20{\mu}M$), a membrane-permeable oxidant, caused an increase in $[Ca^{2+}]_i$, and DTT (2 mM) as a reductant completely reversed the effect of DTBNP. The $[Ca^{2+}]_i$ increase induced by DTBNP was also observed in an extracellular $Ca^{2+}$-free/2 mM EGTA solution, indicating the release of $Ca^{2+}$ from intracellular store(s). After endoplasmic reticulum was depleted by an $IP_3$-generating agonist, ATP ($30{\mu}M$) or an ER $Ca^{2+}$ pump inhibitor, thapsigargin ($1{\mu}M$), DTBNP-stressed BAECs showed an increase of $[Ca^{2+}]_i$ in $Ca^{2+}$-free/2 mM EGTA solution. Ratio-differences before and after the application of DTBNP after pretreatment with ATP or thapsigargin were $0.42{\pm}0.15$ and $0.49{\pm}0.07$, respectively (n=7), which are significantly reduced, compared to the control value of $0.72{\pm}0.07$ in a $Ca^{2+}$-free/2 mM EGTA solution. After the protonophore CCCP ($10{\mu}M$) challenge to release mitochondrial $Ca^{2+}$, the similar result was obtained. Ratio-difference before and after the application of DTBNP after pretreatment with CCCP was $0.46{\pm}0.09$ (n=7). Simultaneous application of thapsigargin and CCCP completely abolished the DTBNP-induced $[Ca^{2+}]_i$ increase. The above results together indicate that the increase of $[Ca^{2+}]_i$ by DTBNP resulted from the release of $Ca^{2+}$ from both endoplasmic reticulum and mitochondria.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.4
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• pp.177-183
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• 2008

The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and $[Ca^{2+}]_c$ of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH ($pH_e{\leq}5.5$) activated outwardly rectifying $Cl^-$ current ($I_{Cl,pH}$) with slow kinetics of voltage-dependent activation. $I_{Cl,pH}$ was potently inhibited by an anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 73.5% inhibition at 1${\mu}$M). $I_{Cl,pH}$ became more sensitive to $pH_e$ by raising temperature from $24^{circ}C$ to $37^{circ}C$. HaCaT cells also expressed $Ca^{2+}$-activated $Cl^-$ current ($I_{Cl,Ca}$), and the amplitude of $I_{Cl,Ca}$ was increased by relatively weak acidic $pH_e$ (7.0 and 6.8). Interestingly, the acidic $pH_e$ (5.0) also induced a sharp increase in the intracellular [$Ca^{2+}$] (${\triangle}[Ca^{2+}]_{acid}$) of HaCaT cells. The ${\triangle}[Ca^{2+}]_{acid}$ was independent of extracellular $Ca^{2+}$, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed ${\triangle}[Ca^{2+}]_{acid}$. In summary, we found $I_{Cl,pH}$ and ${\triangle}[Ca^{2+}]_{acid}$ in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.301-310
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• 2010

Bacillus subtilis strains produce a broad spectrum of bioactive peptides. The lipopeptide surfactin belongs to one well-known class, which includes amphiphilic membrane-active biosurfactants and peptide antibiotics. Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Bs-M49, obtained by means of low-energy ion implantation in wild-type Bs-916, produced significantly lower levels of surfactin, and had no obvious effects against R. solani. Occasionally, we found strain Bs-M49 decreased spore formation and the development of competence. Blast comparison of the sequences from Bs-916 and M49 indicate that there is no difference in the srfA operon promoter PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild-type Bs-916 and mutant M49 strains were significantly different. When we integrated the comA ORF into the chromosome of M49 at the amyE locus, M49 restored hemolytic activity and antifungal activity. Then, HPLC analyses results also showed the comA-complemented strain had a similar ability to produce surf actin with wild-type strain Bs-916. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis. ComA protein 3D structure prediction and motif search prediction indicated that ComA has two obvious motifs common to response regulator proteins, which are the N-terminal response regulator receiver motif and the C-terminal helix-turn-helix motif. The three residues in the ComA N-terminal portion may be involved in phosphorylation activation mechanism. These structural prediction results implicate that three mutated residues in the ComA protein may play an important role in the formation of a salt-bridge to the phosphoryl group keeping active conformation to subsequent regulation of the expression of downstream genes.

• The Korean Journal of Pharmacology
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• v.29 no.2
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• pp.165-174
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• 1993

$Ca^{2+}$ is an important regulator of neurite elongation and growth cone movements but the mechanism(s) mediating these $Ca^{2+}-dependent$ effects is unclear. Since cytoskeletal proteins are rapidly degraded by $Ca^{2+}-dependent $ proteinases (calpains) in vitro and in vivo, we investigated whether $Ca^{2+}-induced$ pruning or regression of neuronal processes is mediated by calpains. Isolated hippocampal pyramidal-like neurons were cultured and the ability of the membrane-permeable calpain inhibitors EST (etyl (+)-(2S,3S)-3-[(S)-methyl-1-(3-methlbutylcarbamoyl)-butylcarbamoyl]2-oxiranecarboxylate) and MDL28170 (carbobenzoxyl-Val-Phe-H) to block the $Ca^{2+}$ ionophore A23187-induced suppression in neurite outgrowth was investigated. Addition of 100 nM A23187 to the culture medium resulted in a retraction of dendrites without altering axonal elongation. The addition of 300 nM A23187 to the culture medium resulted in a signiciant decrease in the rate of axonal elongation as well as a retraction of dendritic processes. Administration of EST $(5\;or\;20{\mu}M)$ to the culture medium completely blocked the pruning effect of 100 nM A23187 on dendrites and of 300 nM A23187 on axons, while EST alone did not significantly affect neurite outgrowth rate. MDL 28170 $(20\;{\mu}M)$ showed the same effect as EST in preventing ionophore-induced pruning of dendrites and axons at 100 nM and 300 nM concentrations, respectively, of A23187. EST $(20\;{\mu}M)$ did not block the A23187-induced rise of $[Ca^{2+}]_{i}$ as measured with fura-2. These results show that $Ca^{2+}-induced$ pruning of neurites in isolated hippocampal pyramidal neurons is mediated by calpains.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.1
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• pp.61-70
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• 2009

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on $Ba^{2+}$ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary(CHO) cells and used the whole cell patch-clamp technique to record $K^+$ currents in the absence and presence of externally applied $Ba^{2+}$. Wild type Kir2.1 was blocked by externally applied $Ba^{2+}$ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of $Ba^{2+}$ blockage. The estimated $K_d(0)$ was 108 ${\mu}M$ for Kir2.1 wild type, 124 ${\mu}M$ for a concatameric WT-Y145V dimer, 109 ${\mu}M$ for a WT-Y145L dimer, and 267 ${\mu}M$ for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for $Ba^{2+}$, also showing two components with very different time courses. Mutants D172K and D172R(lining the central, aqueous cavity of the channel) showed both a decreased affinity to $Ba^{2+}$ and a decrease in the on transition rate constant(${\kappa}_{on}$). These results imply that residues stabilising the cytoplasmic end of the selectivity filter(T141, S165) and in the central cavity(D172) are major determinants of high affinity $Ba^{2+}$ blockage in Kir 2.1.

• Resources Recycling
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• v.24 no.5
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• pp.33-39
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• 2015

A basic study was conducted to effectively recover nitric acid from a waste solder stripper by diffusion dialysis using anion exchange membranes. The effects of flow rate, flux ratio, nitrate concentration, and metallic ion types and concentration on the recovery percentage of nitric acid were investigated. The recovery percentage of nitric acid was decreased with the increase of flow velocity. But the recovery percentage of nitric acid was increased as the increase of flux ratio(W/F) and showing a recovery percentage of nitric acid of about 99% at a flux ratio of 1.5 or more. As the increase of nitric acid concentration in feed solution, the recovery percentage of nitric acid was increased up to 3.0M, but in case of greater than 3.0M, the recovery percentage gradually was decreased. Leakage percentage of metallic ions through the membrane were in the order of Pb, Na and Cu but Fe and Sn did not leakaged. As a result of diffusion dialysis using real waste solder stripper at a flow rate of $0.9L/hr-m^2$, W/F = 1.3, a recovery percentage of nitric acid of approximately 94% was gained.