• Applied Biological Chemistry
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• v.51 no.1
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• pp.84-87
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• 2008

In this research, we screened for glucosyltransferase (GTase) inhibitors that effectively prevent the dental caries from 420 kinds of boiled water extracts of herbs and wild plants and searched for GTase inhibitory activities. Among them, 13 kinds of hot water extracts had high GTase inhibitory activities and especially, we focused on Foeniculum vulgare which showed the highest inhibitory activity on GTase. The boiled water extract of F. vulgare was stable at high temperature and showed as a mixed type of competitive and uncompetitive inhibition kinetic behavior. It did not have antibacterial effect on Streptococcus mutans and had inhibitory activity on GTase. Specially, in the clinical trial, the group treated by boiled water extract of F. vulgare showed more decrease of plague index at 4.8 point than untreated group. These results suggested that boiled water extract of F. vulgare can effectively suppress the plague formation as it inhibits the GTase activity.

• Molecules and Cells
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• v.46 no.9
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• pp.545-557
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• 2023

Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg²⁺-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg²⁺, Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Fe²⁺, and Fe³⁺ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg²⁺-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

• Korean Journal of Microbiology
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• v.48 no.3
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• pp.180-186
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• 2012

Pseudomonas aeruginosa, a Gram-negative bacterium, is an ubiquitous and opportunistic human pathogen, which expresses many virulence factors through quorum sensing (QS) regulation. QscR, one of the QS signal receptors of P. aeruginosa, has unique features that make it possible to distinguish QscR from other QS receptors. In the present study, we focused on amino acid residues responsible for such a broad signal specificity of QscR. Thus we constructed mutant QscRs: $QscR_{T72I}$, $QscR_{R132M}$, and $QscR_{T140I}$ by substituting $72^{nd}$ threonine, $132^{nd}$ arginine, and $140^{th}$ threonine residues with isoleucine, methionine, and isoleucine, respectively by site-directed mutagenesis. When we examined the activity of these mutant QscRs, $QscR_{R132M}$ failed to respond to N-3-oxododecanoyl homoserine lactone (3OC12-HSL), but $QscR_{T72I}$ and $QscR_{T140I}$ remained the ability to respond to 3OC12-HSL despite much reduction of the sensitivity. When we treated a variety of acyl-HSLs with different structure, $QscR_{T72I}$ and $QscR_{T140I}$ showed better responsiveness to N-decanoyl HSL (C10-HSL) or N-dodecanoyl HSL (C12-HSL) that has no oxo-moiety at $3^{rd}$ carbon of acyl group than to 3OC12-HSL, and $QscR_{R132M}$ showed no responsiveness to any acyl-HSLs tested here. In addition, $QscR_{T72I}$ and $QscR_{T140I}$ were inhibited by 5f, a QscR inhibitor as similarly as wild type QscR was. These results suggest that while the $130^{th}$ arginine is crucial in both activity and acyl-HSL binding of QscR, the $72^{nd}$ and $140^{th}$ threonines are important in the activity, but they are little responsible for the discrimination of acyl-HSLs or competitive inhibitor.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.26 no.1
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• pp.163-186
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• 2000

We have previously screened 150 medicinal plants on the inhibition of elastase and found a significant inhibitory effects of the extracts of Areca catechu L. on the aging and inflammation against the skin tissues. To isolate and identify the compounds having biological activity, we was further purified by each of the solvent fractions, silica gel column chromatography, preparative TLC and reversed-Phase HPLC. Peak in HPLC, which coincided with the inhibitory activity against elastase, was identified as Phenolic substance using various colorimetric methods, UV, and IR. $IC_{50}$/ values of phenolic substance purified from Areca catechu were 26.9 $\mu\textrm{g}$/$m\ell$ for porcine pancreatic elastase (PPE) and 60.8 $\mu\textrm{g}$/$m\ell$ for human neutrophil elastase (HNE). This Phenolic substance showed more potent activity than those of reference compounds, oleanolic acid (76.5 $\mu\textrm{g}$/$m\ell$ for PPE, 219.2 $\mu\textrm{g}$/$m\ell$ for HNE) and ursolic acid (31.0 $\mu\textrm{g}$/$m\ell$ for PPE, 118.6 $\mu\textrm{g}$/$m\ell$ for HNE). According to the Lineweaver-Burk Plots, the inhibition against both PPE and HNE by this phenolic substance was competitive with substrate. Phenolic substance from Areca catechu exhibited high free radical scavenging effect ($SC_{50}$/ : 6 $\mu\textrm{g}$/$m\ell$) and inhibited effectively hyaluronidase activity ($IC_{50}$/: 210 $\mu\textrm{g}$/$m\ell$). These results suggest that the Phenolic substance Purified from Areca catechu showed anti-aging effect by protecting connective tissue proteins.

• Development and Reproduction
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• v.2 no.1
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• pp.9-20
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• 1998

It has been wel known that phospholipase C(PLC) plays an important role in the intracellular signaling in a variety of cell types. However, involvement of PLC in mouse oocyte maturation and preimplantation embryo development remains unknown. The present study examined the expression patterns of the mouse PLC \beta 1 and \gamma 1 during oocyte maturatio and preimplantation embryo development study examined the expression patterns of the mouse PLC \beta 1 and \gamma 1 during oocyte maturation and preimplantation embryo development by the competitive reverse transcription-polymerase chain reaction (RT-PCR method). PLC \gamma 1 mRNA (0.1 fg) was readily detected in germinal vesicle (GV)-stage oocyte and its level was reduced as meiotic resumption proceeded. PLC-\beta 1 mRNA (<0.1 fg) as detected at low level at GV-stage oocytes and scarcely detected at germinal vescle breakdown (GVBD)-stage oocytes. After fertilization, both PLC \beta 1 and \gamma 1 mRNA levels began to increase at morula-stage embryos (0.2 fg) and were more prominent in blastocyst-stage embryos(1 fg). to elucidate the possible involvement of PLC via protein kinase C(PKC) pathway during oocyte maturation and preimplantation embryo development , the effects of sphingosine (PKC inhibitor), sn-$diC_{8}$(PKC activator) anc U73122 (PLC ingibitor) were examined. Treatment of GV-stage oocytes with sphingosine (20 \mu M) facilitated the meiotic resuption by 10-20 over the control within 1 h as judged by GVBD, whereas U73122 failed to show any significant effect. U73122 (10 \mu M) effectively blocked the compaction of morula, while sn-$diC_{8}$(50 \mu M). In summary, the present study shows that the mouse PLC \beta 1 and \gamma 1 are expressed in a developmental stage-specific manner and PLC-PKC pathway may be involved in early preimplantation embryo development.

• Korean Journal of Biological Psychiatry
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• v.1 no.1
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• pp.88-97
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• 1994

Many evidences are compatible with the correlation between the inhibition of [$^3H$] imipramine([$^3H$]IMI) and [$^3H$]paroxetine([$^3H$]PAT) binding to the 5-hydroxytryptamine(5-HT) transporter complex and the 5-HT uptake of 5-HT neurons and platelets, and most antidepressants have been shown to inhibit the [$^3H$]IMI and [$^3H$]PAT binding and the neuronal 5-HT uptake. However, several paradoxical research findings led to doubt about the pharmacological significance of the [$^3H$]IMI and [$^3H$]PAT binding sites. This study was carried to clarify the correlation between the [$^3H$]IMI and [$^3H$]PAT binding parameters and the tissue 5-HT content or/and [$^3H$]5-HT uptake in the rabbit platelet, which contains 40 times ad much 5-HT as that of human platelet and shows the 10 fold higher $B_{max}$ of the 5-HT transporter binding to a 5-HT uptake inhibitor. The rabbits were treated for 28 days with amitriptyline(4mg/kg/day : AP), fluoxetine(0.5mg/kg/day : FO), and sertraline(0.5mg/kg/day : SA) via an Alzet osmotic pump implanted for constant infusion. The [$^3H$]IMI binding $B_{max}$ and $K_d$ of the rabbit platelets were $6.4{\pm}1.2$pmol/mg protein and $10.9{\pm}2.1$nM and those in the [$^3H$]PAT binding were $8.6{\pm}1.1$pmol/mg protein and $1.6{\pm}0.3$nM, respectively. AP slightly increased $B_{max}$ of [$^3H$]IMI binding and both [$^3H$]IMI binding and [$^3H$]PAT binding $K_d$, and i contrast, it slightly decreased $B_{max}$ of [$^3H$]PAT binding. FO Slightly increased $K_d$ of both and [$^3H$]IMI and [$^3H$]PAT binding and slightly decreased $B_{max}$ of [$^3H$]IMI and [$^3H$]PAT binding. SA produced the significant increase of [$^3H$]PAT binding $B_{max}$ and the slight increase of both [$^3H$]IMI and [$^3H$]PAT binding $K_d$ and in contrast, it slightly decreased $B_{max}$ and of [$^3H$]IMI binding. And, the $V_{max}$ and $K_m$ of platelet [$^3H$]5-HT uptake were $24.2{\pm}2.4$pmol/$10^8$ platelets/min and $3.3{\pm}0.3$nM, respectively. The $V_{max}$ was little affected by AP, FO, or SA, but the [$^3H$]5-HT uptake $K_m$ value was moderately increased by FO. However, the platelet 5-HT content was moderately decreased by all of the 5-HT uptake inhibitors used in this study. These results seem to be consistent with the allosterical and competitive interaction of 5-HT uptake inhibiting antidepressants with each other as well as 5-HT in the 5-HT transporter binding, and provide no support for the view that the potencies of 5-HT uptake inhibitors to inhibit the [$^3H$]IMI or [$^3H$]PAT binding with 5-HT transporter complex correlate with their antidepressant potencies.