• Applied Microscopy
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• 제20권1호
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• pp.90-104
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• 1990

This study was carried out to investigate the ultrastructural changes of the proximal epiphyseal plate of the femur in young chickens that had been treated with monosodium glutamate(MSG). Eighty 1-day old broiler chickens(Hubbard strain) were divided into control and experimental groups. The experimental group received daily administration of MSG(3mg/g of body weight in 0.75% saline) per orally for 1, 3, 6, 9, 12, 15, 18 and 21 days, and were sacrificed with exanguination. The control group received an equal volume of 0.75% saline. For the transmission electron microscopy, the prehypertrophic cartilage zone of epiphyseal plate was cleaved, fixed with 2% glutaraldehyde(containing 0.2% ruthenium red), postfixed with 1 % osmium tetroxide, embedded in Epon 812, and stained with uranyl acetate and lead citrate. For the scanning electron microscopy, the calcified zone of epiphyseal plate was cleaved and coated with gold palladium. The results obtained were as follows; 1. On transmission electron microscopic examination, the sacculation decreased from 12 day to 21 day MSG administrated groups, and the vesiculation decreased in 18 and 21 day MSG administrated groups in rough endoplasmic reticulum of chondrocytes in prehypertrophic cartilage zone. The ruthenium red binding particles in pericellular rim, territorial matrix and interterritorial matrix increased from 9 day to 21 day MSG administrated groups, but the crystalloid materials decreased. 2. On scanning electron microscopic examination, the trabecular formation and calcospherites of calcification zone decreased in 18 and 21 day MSG administrated groups. The resorption cavities widened from 15 day to 21 day MSG administrated groups.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.252-252
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• 1996

We Investigated the peripheral excitatory effect of capsaicin and KR-25018, a newly synthesized capsaicin derivative which was demonstrated to have a potent analgesic activity. KR-25018 and capsaicin were found to be both potent efficacious contractors of isolated guinea pig bronchial smooth muscle. KR-25018 was equipotent with capsaicin and [Sar$\^$9/,Met(O$_2$)$\^$11/]-substance P, 10-fold more potent than histamine and 10-fold less potent than (${\beta}$ -Ala$\^$8/)-neurokinin A(4-10), and their -log(M)EC$\_$50/ values were 6.94${\pm}$0.08, 6.86${\pm}$0.05, 6.96${\pm}$0.07, 5.64${\pm}$0.04, 7.96${\pm}$0.02, respectively. Contractile responses to KR-25018 and capsaicin were potentiated by phosphoramidon (1 ${\mu}$M), an inhibitor of neuropeptide-inactivating endopeptidase, but completely abolished in a calcium-free medium. These responses to KR-25018 and capsaicin were unaffected by the NK-1 antagonist CP96345 (1${\mu}$M), partially inhibited by the NK-2 antagonist SR48968 (1 ${\mu}$M) but almost completely abolished by a combination of the antagonists. A vanilloid receptor antagonist capsazepine competitively antagonized the responses to both KR-25018 and capsaicin (pA$_2$: aganst KR-25018, 5.98${\pm}$0.47; against capsaicin, 5.80${\pm}$0.31), and a capsaicin-sensitive cation channel antagonist ruthenium red caused significant reduction in the maximum responses to KR-25018 and capsaicin (pD'$_2$: against KR-25018, 4.61${\pm}$0.33; against capsaicin 4.96${\pm}$0.21). In conclusion, the present results suggest that KR-25018 and cpasaicin act on the same vanilloid receptor inducing the influx of calcium through ruthenium red-sensitive cation channel and produce contractile responses via the release of tachykinins that act on both NK-1 and NK-2 receptor subtypes.

• The Korean Journal of Physiology and Pharmacology
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• 제1권4호
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• pp.377-384
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• 1997

To investigate the properties of ryanodine binding sites of the bird skeletal SR vesicles, SDS PAGE, purification of RyR, and $[^3H]ryanodine$ binding study were carried out in the SR vesicles prepared from the chicken pectoral muscle. The chicken SR vesicles have two high molecular weight (HMW) protein bands as in eel SR vesicles on SDS PAGE. The HMW bands on SDS PAGE were found in the $[^3H]ryanodine$ peak fraction $(Fr_{3-5})$ obtained from the purification step of the ryanodine receptor protein. Bmax and KD of the chicken $[^3H]ryanodine$ binding sites were 12.52 pmol/mg protein and 14.53 nM, respectively. Specific $[^3H]ryanodine$ binding was almost maximal at $50{\sim}100$ ${\mu}M$ $Ca^{2+}$, but was not increased by 5 mM AMP and not inhibited by high $Ca^{2+}$. Binding was significantly inhibited by $20{\sim}100$ ${\mu}M$ ruthenium red and 1 mM tetracaine, but slightly inhibited by $Mg^{2+}$. From the above results, it is suggested that chicken SR vesicles have the ryanodine binding sites to which the binding of ryanodine is almost maximal at $50{\sim}10$ ${\mu}M$ $Ca^{2+}$, is significantly inhibited by ruthenium red and tetracaine, slightly inhibited by $Mg^{2+}$, but not affected by AMP and not inhibited by high $Ca^{2+}$.

• Biomolecules & Therapeutics
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• 제20권6호
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• pp.550-555
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• 2012

TRPA1 and TRPV1 are members of the TRP superfamily of structurally related, nonselective cation channels. TRPA1 and TRPV1 are often co-expressed in sensory neurons and play an important role in somatosense such as cold, pain, and irritants. The first leaves of Kalopanax pictus Nakai (Araliaceae) have long been used as a culinary ingredient in Korea because of their unique chemesthetic flavor. In this study, we observed the intracellular $Ca^{2+}$ response to cultured cells expressing human TRPA1 (hTRPA1) and human TRPV1 (hTRPV1) by $Ca^{2+}$ imaging analysis to investigate the ability of the first leaves of K. pictus to activate the hTRPA1 and hTRPV1. An 80% ethanol extract of K. pictus (KPEx) increased intracellular $Ca^{2+}$ influx in a response time- and concentration-dependent manner via either hTRPA1 or hTRPV1. KPEx-induced response to hTRPA1 was markedly attenuated by ruthenium red, a general blocker of TRP channels, and HC-030031, a specific antagonist of TRPA1. In addition, the intracellular $Ca^{2+}$ influx attained with KPEx to hTRPV1 was mostly blocked by ruthenium red, and capsazepine, a specific antagonist of TRPV1. These results indicate that KPEx selectively activates both hTRPA1 and hTRPV1, which may provide evidence that the first leaves of K. pictus primarily activate TRPA1 and TRPV1 to induce their unique chemesthetic sense.

• The Korean Journal of Physiology and Pharmacology
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• 제20권5호
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• pp.449-457
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• 2016

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though $Ca^{2+}$ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ($[Ca^{2+}]_i$) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on $[Ca^{2+}]_i$ in human neutrophils. We observed that NAC ($1{\mu}M{\sim}1mM$) and cysteine ($10{\mu}M{\sim}1mM$) increased $[Ca^{2+}]_i$ in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in $[Ca^{2+}]_i$ in human neutrophils was observed. In $Ca^{2+}$-free buffer, NAC- and cysteine-induced $[Ca^{2+}]_i$ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in $[Ca^{2+}]_i$ in human neutrophils occur through $Ca^{2+}$ influx. NAC- and cysteine-induced $[Ca^{2+}]_i$ increase was effectively inhibited by calcium channel inhibitors SKF96365 ($10{\mu}m$) and ruthenium red ($20{\mu}m$). In $Na^+$-free HEPES, both NAC and cysteine induced a marked increase in $[Ca^{2+}]_i$ in human neutrophils, arguing against the possibility that $Na^+$-dependent intracellular uptake of NAC and cysteine is necessary for their $[Ca^{2+}]_i$ increasing activity. Our results show that NAC and cysteine induce $[Ca^{2+}]_i$ increase through $Ca^{2+}$ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

• 대한약리학회지
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• 제30권1호
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• pp.125-136
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• 1994

갑각류 골격근의 SR에서 칼슘유리 channel protein complex의 성격을 규명하기 위해 민물가재 및/또는 바다가재의 SR vesicles을 분리하여 $^{45}Ca$ 유리, $[^3H]ryanodine$결합, 및 immunoblot 실험을 실시하여 다음과 같은 결과를 얻었다. 1.민물가재 SR의 $[^3H]ryanodine$결합 실험에서 민물가재 SR의 maximal binding site및 affinity모두 바다가재에서 보다 낮았으나, high affinity binding site이었다. Extravesicles 칼슘농도를 증가시켰을 때 $[^3H]ryanodine$결합은 약간 증가되었으나, AMP나 AMP와 caffeine을 동시에 첨가하였을 때는 현저히 증가되었다(p<0.05). 이런 증가 현상은 $MgCl_2$나 tetracaine으로 유의성 있게 억제되었으나(p<0.001), ruthenium red에 의해서는 약간 억제되었다. 2.민물가재 SR을 전기영동하였을 때 바다가재의 ryanodine receptor band (HMWBr)와 비슷하나 포유류의 것(HMWBS) 보다는 약간 빠른 mobility를 나타낸다. 3.바다가재 HMWBr에 대한 polyclonal Ab를 이용한 민물가재, 바다가재 및 토끼 골격근의 칼슘유리 channel간의 면역학적 교차반응에서 민물가재와 바다가재의 칼슘유리 channel 간에는 교차반응이 있었으나, 포유류의 것과는 아무런 반응이 없었다. 4.민물가재 SR에서 $^{45}Ca$유리는 extravesicles의 칼슘농도 증가에 따라서 증가되었고, 낮은 외부 칼슘 농도에서 바다가재 보다 빠르게 일어났으나, AMP와 caffeine에 의해 영향을 받지 않았고, $MgCl_2$와 tetracaine으로 약간($3{\sim}8%$) 그리고 고농도의 ruthenium red로 중등도(23%) 억제되었다. 이상의 실험성적으로 갑각류 칼슘유리 channel protein은 포유류의 것과는 기능적으로나 면역학적으로 매우 다른 특징을 가지고 있고, 민물가재와 바다가재 칼슘유리 channel은 서로 유사한 특징을 갖지만, 민물가재의 칼슘유리 channel이 바다가재의 것보다 외부칼슘에 예민한 기능을 갖는 것으로 사료된다.

• Bulletin of the Korean Chemical Society
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• 제9권5호
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• pp.317-322
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• 1988

Premicellar precipitation, resolubilization and luminescing behaviors of $RuL_3^{2+}$ (L = bpy, phen, $Me_2bpy$) in aqueous alkylsulfate and sulfonate solutions were studied. Addition of the anionic surfactants to $RuL_3\;^{2+}$ solutions caused initial precipitation which was redissolved by further addition of the surfactants. The apparent solubility products $K_{sp}$'s of the precipitates were evaluated assuming 1:2 salt formation. The values were smaller as the ligand is more hydrophobic and the length of hydrocarbon chain of the surfactant is longer. The $K_{sp}$ values for L = bpy were constant over wide surfactant concentration range. However, those for L = $Me_2bpy$ and also for phen, but to less extent, increased with the surfactant concentration. The resolubilization of 1:2 salts was followed by red-shift of emission band and extensive emission quenching above critical concentration of the surfactants. The critical concentration was lower for more hydrophobic surfactant. For L = $Me_2bpy$, the blue-shifted emission band with enhanced emission intensity was observed in intermediate surfactant concentration region. The high ionic strength of media prevented the precipitate formation, but facilitated the red-shift of the emission bands. The results support that the precipitate is dissolved by accretion of surfactant anions to the salts to form water-soluble surfactant-rich $RuL_3$-surfactant anionic species. These species appeared to aggregate cooperatively to produce large clusters which exhibited the red-shifted emission.

• Journal of Microbiology and Biotechnology
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• 제7권6호
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• pp.435-437
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• 1997

The role of $Ca^{2+}$ in making functional malate synthase in Corynebacterium glutamicum was investigated using the cloned DNA coding for the enzyme. Introduction of cloned aceB into C. glutamicum overexpressed malate synthase as judged by SDS-PAGE. However, the increase in enzyme activity of the expressed malate synthase did not match the level of overexpression observed in SDS-PAGE. Addition of $Ca^{2+}$ to the growth medium specifically increased the activity. The malate synthase could be stained with ruthenium red in a $Ca^{2+}$-specific manner. This agrees with the previous observation which reported a potential $Ca^{2+}$-binding domain in the N-terminal region of the protein.

• Bulletin of the Korean Chemical Society
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• 제21권10호
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• pp.1052-1054
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• 2000

Binding geometries of $[Ru(II)(110-phenanthroline)_2L]^2+$, complexes (where L = dipyrido [3,2-a:2',3'-c]phena-zine (DPPZ) or benzodipyrido[3,2-a:2',3'-c] phenazine (BDPPZ)) to poly(dG)${\cdot}$poly(dC)${\cdot}$poly(dC) + triplex DNA (CGC + triplex) has been investigated by linear dichroism and normal absorption spectroscopy. Analysis of the linear dichroism for the CGC+ triplex and $[Ru(II)(phen)_2BDPPZ]^2+$ complex indicates that the extended ligand of the metal complex lie perpendicular to the polynucleotide helix axis. Together with strong hypochromism and red shift in the interligand absorption region, we concluded that the extended BDPPZ or DPPZ ligand in-tercalated between the bases of polynucleotide. The spectral properties of the metal complexes bound to CGC+ triplex are similar to those bound to $poly(dA)[poly(dT)]^2$ triplex (Choi et al., Biochemistry 1997, 36, 214), sug-gesting that the metal complex is located in the minor groove of the CGC+ triplex.

• International Journal of Oral Biology
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• 제35권3호
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• pp.129-135
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• 2010

Recent studies have implicated reactive oxygen species (ROS) as determinants of the pathological pain caused by the activation of peripheral neurons. It has not been elucidated, however, how ROS activate the primary sensory neurons in the pain pathway. In this study, calcium imaging was performed to investigate the effects of NaOCl, a ROS donor, on the intracellular calcium concentration ($[Ca^{2+}]i$) in acutely dissociated dorsal root ganglion (DRG) neurons. DRG was sequentially treated with 0.2 mg/ml of both protease and thermolysin, and single neurons were then obtained by mechanical dissociation. The administration of NaOCl then caused a reversible increase in the $[Ca^{2+}]i$, which was inhibited by pretreatment with phenyl-N-tertbuthylnitrone (PBN) and isoascorbate, both ROS scavengers. The NaOCl-induced $[Ca^{2+}]i$ increase was suppressed both in a calcium free solution and after depletion of the intracellular $Ca^{2+}$ pool by thapsigargin. Additionally, this increase was predominantly blocked by pretreatment with the transient receptor potential (TRP) antagonists, ruthenium red ($50\;{\mu}M$) and capsazepine ($10\;{\mu}M$). Collectively, these results suggest that an increase in the intracellular calcium concentration is produced from both extracellular fluid and the intracellular calcium store, and that TRP might be involved in the sensation of pain induced by ROS.