• The Korean Journal of Physiology and Pharmacology
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• v.7 no.6
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• pp.349-355
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• 2003

We previously shown that LES contraction depends on $M_3$ receptors linked to PTX insensitive $G_q$ protein and activation of PLC. This results in production of $IP_3$, which mediates calcium release, and contraction through a CaM dependent pathway. In the esophagus ACh activates $M_2$ receptors linked to PTX sensitive $G_{i3}$ protein, resulting in activation of PLD, presumably, production of DAG. We investigated the role of PLC isozymes which can be activated by $G_q$ or $G{\beta}$ protein on ACh-induced contraction in LES and esophagus. Immunoblot analysis showed the presence of 3 types of PLC isozymes, $PLC-{\beta}1$, $PLC-{\beta}3$, and $PLC-{\gamma}1$, but not $PLC-{\beta}2$, $PLC-{\beta}4$, $PLC-{\gamma}2$, $PLC-{\delta}1$, and $PLC-{\delta}2$ from both LES and esophageal muscle. ACh produced contraction in a dose dependent manner in LES and esophageal muscle cells obtained by enzymatic digestion with collagenase. $PLC-{\beta}1$ or $PLC-{\beta}3$ antibody incubation reduced contraction in response to ACh in LES but not in esophageal permeabilized cells, but $PLC-{\gamma}1$ antibody incubation did not have an inhibitory effect. The inhibition by $PLC-{\beta}1$ or $PLC-{\beta}3$ antibody on Ach-induced contraction was antibody concentration dependent. The combination with $PLC-{\beta}_1$ and $PLC-{\beta}_3$ antibody completely abolished the contraction, suggesting that $PLC-{\beta}1$ and $PLC-{\beta}3$ have a synergism to inhibit the contraction in LES. $PLC-{\beta}1$, -${\beta}3$ or -${\gamma}1$ antibody did not reduce the contraction of LES cells in response to DAG ($10^{-6}$ M), suggesting that this isozyme of PLC may not activate PKC. When $G_{q/11}$ antibody was incubated, the inhibitory effect of the incubation of PLC ${\beta}3$, but not of PLC ${\beta}_1$ was additive (Fig. 6). In contrast, when $G_{\beta}$ antibody was incubated, the inhibitory effect of the incubation of PLC ${\beta}_1$, but not of PLC ${\beta}_3$ was additive. This data suggest that $G_{q/11}$/11 or $G{\beta}$ may activate cooperatively different PLC isozyme, $PLC{\beta}_1$ or $PLC{\beta}_3$ respectively.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.23 no.2
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• pp.13-26
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• 2010

Objectives : The present study was conducted to evaluate the anti-inflammatory effects of the Gamroeum water extracts (GRE) in vivo and in vitro. Methods : The effects of GRE on anti-inflammation were measured by production of NO, $PGE_2$ (Prostaglandin $E_2$), iNOS (inducible Nitric Oxide Synthase), COX-2, $NF{\kappa}B$ (Nuclear Factor kappa B), TNF-$\alpha$ (Tumor Necrosis Factor-alpha) and IL-$1{\beta}$ (Interleukin-$1{\beta}$), IL-6 in Raw 264.7 macrophage cells stimulated with LPS. Results : 1. In machrophage cells, LPS displayed significant stimulatory effects on the production of NO and $PGE_2$. However, GRE showed significant inhibitory effects on NO and $PGE_2$ release. The level of NO and $PGE_2$ was decreased by GRE in a concentration dependent manner as compared with LPS only group. 2. Immunoblot analysis verified that LPS stimulation significantly increased the iNOS and COX-2 protein level, but GRE suppressed the induction of iNOS and COX-2 protein at a concentration dependent manner. 3. GRE reduced the elevated production of TNF-$\alpha$, IL-$1{\beta}$ and IL-6 by LPS. Moreover, the inhibitory effects of GRE was occurred in a dose-dependent manner. 4. GRE significantly reduced the expression of NF-${\kappa}B$ protein in nuclear fraction. 5. GRE effectively inhibited the increases of hind paw skin thicknesses and inflammatory cell infiltrations induced by carrageenan treatment. It, therefore, considered that GRE will be favorably inhibited the acute edematous inflammations. Conclusions : These results indicated that GRE could have anti-inflammatory capacity by inhibiting the production of NO, $PGE_2$ and cytokines in vitro and by reducing the formation of carrageenan-induced paw edema in vivo. Moreover, inhibitory effects of GRE on the macrophage activation were attributable to the reduction of some of inflammatory factors by inhibiting iNOS and COX-2 through the suppression of NF-${\kappa}B$.

• Korean Journal of Soil Science and Fertilizer
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• v.29 no.1
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• pp.60-66
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• 1996

Bradyrhizobium japonicum with different colony morphology populated in five Yeongnam soils of Korea was examined for intrinsic antibiotic resistance to eight antibiotics, serological property by immunoblot and immunodiffusion, and protein profile differentiation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Colony morphological distribution of one hundred and twenty B. japonicum isolates was 47% for "dry". 41% for "wet", and 12% for "dry/wet" type. The total isolates showed such a strong correlation between the morphology and antibiotic resistance. Colony morphology, which though was dominantly consisted of the same type within a serogroup, wasn't absolutely linked to serological property of B. japonicum. Based on these data, colony morphology was too simple to identify variations with B. japonicum isolates : antibiotic resistance such complicated compared with serological analyses.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.34 no.4
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• pp.370-377
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• 2001

Flounder brain cytosol contains protein inhibitors that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) which is dependent on phosphatidylinositol 4,5-bisphosphate ($PIP_2$) but insensitive to ADP-ribosylation factor (ARF), The PLD inhibitors have been enriched through several chromatographic steps and characterized with respect to size and mechanism of inhibition. Sequential chromatography of the brain cytosol yielded six inhibitor fractions, Two (IIA and IIB) of six inhibitor fractions showed the $PIP_2$-phosphatase activities. IIA was identified as synaptojanin, a nerve terminal protein that has known to be a member of the inositolpolyphosphate 5-phosphatase family, by immunoblot analysis. IIB showed an apparent molecular mass of 158 kDa by Superose 12 gel filtration chromatography and was immunologically distinct from synaptojanin. IIB hydrolyzed $PIP_2$, yielding only phosphatidylinositol phosphate (PIP) as product, suggesting that IIB hydrolyzes only one phosphate from either the 4- or 5-position of PI (4,5)$P_2$. These studies demonstrate that the existence of multiple $PIP_2$-phosphatases have been implicated in the negative regulation of $PIP_2$-dependent PLD activity within flounder brain.

• Journal of agricultural medicine and community health
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• v.26 no.2
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• pp.181-191
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• 2001

Toxocariasis is produced by the migration of Toxocara canis larvae into the extra- intestinal tissue of unnatural hosts or natural hosts under unsuitable conditions. Soil contaminated with T. canis embryonated eggs in the main source of infection of man. In the present study, ELISA with T. canis adult crude antigen was used for determination the seroprevalence of T. canis infection in two areas of Korea. It was found that antibody positive rate was 15.7% in Keoje- Island. In the analysis according to sex, female group presented significantly higher positive rate than male group (23.8% in female and 7.4% in male, p<0.05). In Inchon city, the positive rate was 13.1%, and there was no significant difference between female and male group. Immunoblot analysis was performed to some positive patient sera. As the results, 9 cases of 15 cases were positive in Keoje-Island, and 22 cases of 27cases were positive in Inchon city by immunoblotting.

• Tuberculosis and Respiratory Diseases
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• v.71 no.2
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• pp.88-96
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• 2011

Background: It is known that cigarette smoke (CS) causes cell death. Apoptotic cell death is involved in the pathogenesis of CS-related lung diseases. Some members of the protein kinase C (PKC) family have roles in cigarette smoke extract (CSE)-induced apoptosis. This study was conducted to investigate the role of PKC epsilon in CSE-induced apoptosis in human lung fibroblast cell line, MRC-5. Methods: Lactate dehydrogenase release was measured using a cytotoxicity detection kit. The MTT assay was used to measure cell viability. Western immunoblot, Hoechst 33342 staining and flow cytometry were used to demonstrate the effect of $PKC{\varepsilon}$. Caspase-3 and caspase-8 activities were determined using a colorimetric assay. To examine $PKC{\varepsilon}$ activation, Western blotting was performed using both fractions of membrane and cytosol. Results: We showed that CSE activated $PKC{\varepsilon}$ by demonstrating increased expression of $PKC{\varepsilon}$ in the plasma membrane fraction. Pre-treatment of $PKC{\varepsilon}$ peptide inhibitor attenuated CSE-induced apoptotic cell death, as demonstrated by the MTT assay (13.03% of control, 85.66% of CSE-treatment, and 53.73% of $PKC{\varepsilon}$ peptide inhibitor-pre-treatment, respectively), Hoechst 33342 staining, and flow cytometry (85.64% of CSE-treatment, 53.73% of $PKC{\varepsilon}$ peptide inhibitor-pre-treatment). Pre-treatment of $PKC{\varepsilon}$ peptide inhibitor reduced caspase-3 expression and attenuated caspase-3, caspase-8 activity compared with CSE treatment alone. Conclusion: $PKC{\varepsilon}$ seem to have pro-apoptotic function and exerts its function through the extrinsic apoptotic pathway in CSE-exposed MRC-5 cells. This study suggests that $PKC{\varepsilon}$ inhibition may be a therapeutic strategy in CS-related lung disease such as chronic obstructive pulmonary disease.

• The Journal of Korean Academy of Prosthodontics
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• v.37 no.5
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• pp.698-713
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• 1999

Adherence of Candida albicans(C. albicans) to the surface of a denture is believed to be an initial and essential step in the formation of denture-induced stematitis. Previous studies have provided enormous infomation on the relationship between composition of palatine gland/parotid saliva and upper denture stomatitis. Relatively little information is available on the correlation between lower denture stomatitis and sublingual-submandibular ( SLSM ) saliva. The plaque samples were collected from the two sites($100mm^2$) on the inner surface of lower partial denture corresponding to the stematitis and healthy region of the lower partial dentures of 12 denture stomatitis patients and 6 nor-mal persons who wore lower partial dentures. The samples were plated to isolate C. albicans on a selective Saboraud's dextrose agar plate and the isolates were identified by germ tube test and gram staining. The subjects were divided into group I (stomatitis with C. albican), group II (lesion without C. albicans), group III (no lesion but C. albicans), and group IV (normal and healthy denture wearer). Individual SLSM saliva($20{\mu}g$ of protein) was analyzed by SDS-PAGE (SDS -poly-acrylamide gel electrophoresis) with Coomassie brilliant blue and PAS(Periodic Acid Schinff) stain-ing. The salivary proteins separated in the polyacryamide gels were subjected to immunoblot anaysis using anti-lactoferrin, anti-sIgA, and anti-secretory component of sIgA. In this study using custom made acrylic denture resin beads(5mm in diameter) coated with stimulated individual SLSM saliva, the binding ability of individual C. albicans strains to the beads was observed. Levels of C, albicans adhered to the acrylic resin beads were determined by measuring the optical density of the bound C. albicans to the beads at 580nm. The results showed that a higher number of C. albicans was observed in the lesion site than healthy site. The saliva of group I contained more high molecular weight glycoprotein(mucin, MGI) as compared to group II, III and IV. And lactoferrin and sIgA affected to the binding ability of C. albicans to acylic resin beads. Binding ability of individual C. albicans to the acrylic resin coated with respective individual saliva was found to be greater in group I than the other 3 groups. And when bound cells of C. albicans isolated from individual subject #2 to the saliva coated beads were used binding ability of subject #2 saliva coated beads was founed to be greater than the other sutjects. These results suggested that denture induced stomatitis is related to individual patient's salivary protein composition, especially MG-1. Future studies will be directed toward saliva exam-ination of patients who have general disease and analysis of pellicles formed on prosthesis with respect to oral disease.

• Journal of Periodontal and Implant Science
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• v.46 no.5
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• pp.320-328
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• 2016

Purpose: Human saliva, as a vital part of the immune defense system, contains a number of distinct proteins and peptides. Recently human common salivary protein 1 (CSP1) has been identified as an abundant salivary protein and may play a role in promoting the binding of cariogenic bacteria to salivary pellicles. However, nothing else is known regarding the role of CSP1 in periodontology. The aim of this study was to quantify and compare CSP1 levels between healthy subjects and periodontal patients. Methods: This controlled clinical study was conducted in periodontally healthy individuals and patients with chronic periodontitis Chonbuk National University Hospital, with Institutional Review Board approval. Whole saliva samples were collected from 36 healthy subjects and 33 chronic periodontitis patients and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immune blotting were conducted to ensure that anti-CSP1 monoclonal antibody (mAb) binds to CSP1 in human saliva. A sandwich enzyme-linked immunosorbent assay (ELISA) system was house-fabricated using mAb-hCSP1#14 and mAb-hCSP1#4 as a capture and a detector mAb, respectively. The CSP1 concentrations in saliva from 36 healthy subjects and 33 periodontal patients were quantified using the CSP1 sandwich ELISA system, and the results were analyzed using the Student's t-test. Results: Immunoblot analysis using mAb-hCSP1 as a probe confirmed that CSP1 in human saliva existed as a single band with a molecular weight of approximately 27-kDa. The quantification of CSP1 concentrations by CSP1 ELISA showed that the median values (25th to 75th percentiles) of periodontal patients and healthy subjects were 9,474 ng/mL (range, 8,434.10,139 ng/mL) and 8,598 ng/mL (range, 7,421.9,877 ng/mL), respectively. The Student's t-test indicated the presence of a statistically significant difference between the 2 groups (P=0.024). Conclusions: The presence of a significant difference in CSP1 levels between healthy subjects and periodontal patients suggests that CSP1 may be a potential biomarker for the detection or screening of periodontitis patients.

• Biomedical Science Letters
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• v.2 no.2
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• pp.175-185
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• 1996

The mitogen-activated protein(MAP) kinase signal transduction pathway represents an important mechanism by which mitogen, such as serum and PMA, regulate cell proliferation and differentiation. Target substrates of the MAP kinase are located within several compartments containing plasma membranes and nucleus. We now report that serum addition induces proliferation of the P388 murine leukemia cell, but PMA does not, while both serum and PMA treatment cause translocation of the MAP kinase, mainly p42$^{mapk}$ isoform, from cytosol into the nucleus, which was monitored by immunoblot analysis using polyclonal anti-ERK1 antibodies. We investigated whether the MAP kinase was capable of phosphorylating c-Jun protein and GST-fusion proteins, the P562$^{kk}$N-terminal peptides (1-77 or 1-123 domain) of the T cell tyrosine kinase, using the partially purified MAP kinase by SP-sephadex C-50, phenyl superose and Mono Q column chromatography. We found that the partially purified MAP kinase was able to phosphorylate c-Jun protein and the GST-fusion protein expressed using E.coli DH5$\alpha$ which is transformed with pGEX-3Xb plasmid vector carrying of p562$^{kk}$N-terminal peptide-encoding DNA. These results imply that tyrosine kinase receptor/Ras/Raf/MAP kinase pathway is a major mechanism for mitogen-induced cell proliferation in P388 murine leukemia cell and that the various MAP kinase isoforms may have their own target substrates located in distinct subcellular compartments.

• Journal of Physiology & Pathology in Korean Medicine
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• v.28 no.2
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• pp.178-185
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• 2014

Leonuri Fructus, a semen of Leonuri Herba, has been used for the treatment of menstrual disorders such as amenorrhea, dysmenorrhea and leukorrhea and for the remedy of hyperemia. The present study was conducted to evaluate the anti-inflammatory effects of the Leonuri Fructus extract (Leonurus japonicus Houtt. EtOH extract; LJE) in vivo and in vitro. In vitro study, the MTT assay for cell viability was conducted to determine the non-cytotoxic concentration of LJE treatment in media. The levels of NO were measured with Griess reagent. Pro-inflammatory cytokines were detected by ELISA method. The inflammation-related proteins of this study were detected by immunoblot anlaysis. The increases of NO production and iNOS expression were detected in LPS-treated cells compared with control, but LJE attenuated the increases of NO and iNOS by LPS. LJE reduced the production of TNF-${\alpha}$ and IL-$1{\beta}$ induced by LPS stimulation. LJE suppresses the signaling pathways of NF-${\kappa}B$ and MAPKs in LPS-induced macrophage cells. In vivo study, carrageenan-induced hind paw acute edematous inflammation rat model was used for evaluation of anti-inflammatory activity of LJE. LJE significantly inhibited the increases of hind paw swelling, skin thicknesses and inflammatory cell infiltrations, and decreased the numbers of mast cell induced by carrageenan injection. These results suggest that LJE has an anti-inflammatory therapeutic potential, which is mediated through modulating NF-${\kappa}B$ activation and MAPK phosphorylation. Inhibition of the rat paw edema induced by carrageenan is considered as direct evidence that LJE may be a useful source to treat inflammation.