• Journal of Photoscience
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• 제9권2호
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• pp.448-450
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• 2002

DNA samples extracted from Japanese cypress leaf tissues contain isopropyl alcohol-precipitable, high molecular weight compounds, which interfere ELISA for cyclobutane pyrimidine dimers (CPD). Removal of the compounds is achieved by DEAE ion-exchange column chromatography and improves the ELISA responses of the DNA. When extracting DNA repeatedly from the same leaf tissues, the DNA samples show CPD responses which increase with the order in sequential extraction, and hence for a reliable detennination of DNA lesion a thorough extraction of DNA is required. Clearing these two problems it was demonstrated that CPD level was slightly higher in the leaves of trees growing under full sunlight than in those growing under UBV -cut sunlight.

• BMB Reports
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• 제34권6호
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• pp.537-543
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• 2001

In the present study, an in vitro ELISA system to assess the interaction between Src homology (SH)2 domains and phosphotyrosine that contain peptides was established using purified GST-conjugated SH2 proteins and synthetic biotinylated phosphotyrosine that contain oligopeptides. The SH2 domains bound the relevant phosphopeptides that were immobilized in the streptavidin-coated microtiter plate in a highly specific and dose-dependent manner. The epidermal growth factor receptor (EGFR)-, T antigen (T Ag)-, and platelet-derived growth factor receptor (PDGFR)-derived phosphopeptides interacted with the growth factor receptor binding protein (Grb)2/SH2, Lck/SH2, and phosphatidyl inositol 3-kinase (PI3K) p85/SH2, respectively. No cross-reactions were observed. Competitive inhibition experiments showed that a short phosphopeptide of only four amino acids was long enough to determine the binding specificity. Optimal concentrations of the GST-SH2 fusion protein and phosphopeptide in this new ELISA system for screening the binding blockers were chosen at 2nM and 500nM, respectively. When two candidate compounds were tested in our ELISA system, they specifically inhibited the Lck/SH2 and/or p85/SH2 binding to the relevant phosphopeptides. Our results indicate that this ELISA system could be used as an easy screening method for the discovery of specific binding blockers of protein-protein interactions via SH2 domains.

• Asian-Australasian Journal of Animal Sciences
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• 제26권3호
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• pp.334-342
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• 2013

Proteases and protease inhibitors play key roles in most physiological processes, including cell migration, cell signaling, and cell surface and tissue remodeling. Among these, the matrix metalloproteinase (MMPs) pathway is one of the most efficient biosynthetic pathways for controlling the activation of enzymes responsible for protein degradation. This also indicates the association of MMPs with the maturation of spermatozoa. In an attempt to investigate the effect of MMP activation and inhibitors in cultures with various hormones during sperm capacitation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3), as well as their expression profiles. Matured spermatozoa were collected from cultures with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and Lutalyse at 1 h, 6 h, 18 h, and 24 h. ELISA detected the expression of MMP-2, MMP-9, TIMP-2, and TIMP-3 in all culture media, regardless of medium type (FSH-supplemented fertilization Brackett-Oliphant medium (FFBO), LH-supplemented FBO (LFBO), or Lutalyse-supplemented FBO (LuFBO)). TIMP-2 and TIMP-3 expression patterns decreased in LFBO and LuFBO. MMP-2 and MMP-9 activity in FBO and FFBO progressively increased from 1 h to 24 h but was not detected in LFBO and LuFBO. The localization and expression of TIMP-2 and TIMP-3 in sperm heads was also measured by immunofluorescence analysis. However, MMPs were not detected in the sperm heads. MMP and TIMP expression patterns differed according to the effect of various hormones. These findings suggest that MMPs have a role in sperm viability during capacitation. In conjunction with hormones, MMPs play a role in maintaining capacitation and fertilization by controlling extracellular matrix inhibitors of sperm.

• The Korean Journal of Physiology and Pharmacology
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• 제21권4호
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• pp.429-437
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• 2017

The aim of this study was to evaluate the relaxant and anti-inflammatory effects of two thalidomide analogs as phosphodiesterase-4 (PDE-4) inhibitors in pregnant rat uterus. Uteri from Wistar female rats were isolated at 19 day of pregnancy. Uterine samples were used in functional studies to evaluate the inhibitory effects of the thalidomide analogs, methyl 3-(4-nitrophthalimido)-3-(3,4- dimethoxyphenyl)-propanoate (4NO2PDPMe) and methyl 3-(4-aminophthalimido)- 3-(3,4-dimethoxyphenyl)-propanoate (4APDPMe), on prostaglandin-$F2{\alpha}$ ($PGF2{\alpha}$)-induced phasic, $K^+$-induced tonic, and $Ca^{2+}$-induced contractions. Accumulation of cAMP was quantified in uterine homogenates by ELISA. Anti-inflammatory effect was assessed by using ELISA for determination of the pro-inflammatory cytokines tumor necrosis factor-${\alpha}$ ($TNF{\alpha}$) and interleukin (IL)-$1{\beta}$, and anti-inflammatory IL-10, from uterine explants stimulated with lipopolysaccharide (LPS). Nifedipine, forskolin and rolipram were used as positive controls where required. Both thalidomide analogs induced a significant inhibition of the uterine contractions induced by the pharmaco- and electro-mechanic stimuli. Nifedipine and forskolin were more potent than the analogs to inhibit the uterine contractility, but these were more potent than rolipram, and 4APDPMe was equieffective to nifedipine. Thalidomide analogs increased uterine cAMP-levels in a concentration-dependent manner. The LPS-induced $TNF{\alpha}$ and $IL-1{\beta}$ uterine secretion was diminished in a concentration-dependent fashion by both analogs, whereas IL-10 secretion was increased significantly. The thalidomide analogs induced utero-relaxant and anti-inflammatory effects, which were associated with the increased cAMP levels as PDE-4 inhibitors in the pregnant rat uterus. Such properties place these thalidomide analogs as potentially safe and effective tocolytic agents in a field that urgently needs improved pharmacological treatments, as in cases of preterm labor.

• Journal of Acupuncture Research
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• 제24권6호
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• pp.137-148
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• 2007

Objectives : Following central nervous system (CNS) injury, inhibitory influences at the site of axonal damage occur. Glial cells become reactive and form a glial scar, know as gliosis. As well,myelin debris such as MAG inhibits axonal regeneration. Astrocyte-rich gliosis relates to up-regulation of GFAP and CD81, and eventually becomes a physical and mechanical barrier to axonal regeneration. It is postulated that when the astrocytic reaction is absent, regeneration of axons can occur. It was reported that treatment with anti CD81 antibodies enhanced functional recovery in rats with spinal cord injury. Methods : MAG is one of several endogenous axon regeneration inhibitors that limit recovery from central nervous system injury and disease. It was reported that molecules which block such inhibitors enhanced axon regeneration and functional recovery. Results : In this current study, the author investigated the effect of the water extract of Ginseng Radix on the regulation of CD81, GFAP and MAG which increases when gliosis occurs. MTT analysis was performed to examine cell viability, and cell based ELISA, Western Blot and PCR were used to detect the expression of CD81, GFAP and MAG. Immunohistochemistry was also performed to confirm in vivo. Conclusions : We observed that Ginseng Radix significantly down-regulates the expression of CD81, GFAP and MAG by means of cell based ELISA, Western Blot and PCR. In immunohistochemistry, expression of CD81, GFAP and MAG also decreased. Taken together, these results suggest that Ginseng Radix can be a candidate for regenerating CNS injury.

• Bulletin of the Korean Chemical Society
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• 제27권6호
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• pp.909-917
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• 2006

2,2-Dimethyl-3,4-dialkoxy-substituted 6-aminobenzopyran analogues (eg., 7 and 8) were identified as prolyl 4-hydroxylase inhibitors via a screening process using HSC-T6 and LI 90 cells that express an immortalized rat hepatic stellate cell line and as part of a test of the type I collagen contents employing the ELISA method. A subsequent lead optimization effort based on solid-phase parallel synthesis led to the identification of 2,2-dimethyl-3,4-dialkoxy-substituted 6-aminobenzopyrans as potent inhibitors of prolyl 4-hydroxylase.

• IMMUNE NETWORK
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• 제3권1호
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• pp.61-68
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• 2003

Background: Fibroblast functions both as a structural element and as a vital immunoregulatory cell. Fibroblasts regulate inflammation through governing of chemokine expression. In order to elucidate the mechanisms by which the expressions of chemokines were regulated, the co-stimulatory effects of Th1 and proinflammatory cytokines were compared using nasal mucosal fibroblasts. Methods: Human nasal mucosa was obtained from surgery for septal deviation and the growth of fibroblasts was established. Fibroblasts from 4th to 6th passage were stimulated with various combinations of cytokines. To inhibit selected signaling pathways, fibroblasts were pretreated with cyclosporin A, wortmannin, staurosporine, and dexamethasone prior to the stimulation with cytokines. The supernatants were collected and chemokines were detected with a sandwich enzyme-linked immunosorbent assay. Results: $TNF-{\alpha}/IFN-{\gamma}$-induced production of RANTES was inhibited by all inhibitors used. MCP-1 was produced constitutively and $TNF-{\alpha}$-induced or $TNF-{\alpha}/IFN-{\gamma}$-induced production of MCP-1 was not inhibited by cyclosporin A or wortmannin, but by stauroporine or dexamethasone. All inhibitors used in this experiment inhibited $TNF-{\alpha}/IFN-{\gamma}$-induced or $IL-1{\beta}/IFN-{\gamma}$-induced production of MCP-2 in nasal mucosal fibroblasts. Although staurosporine or dexamethasone showed strong inhibitory effects, cyclosporin A or wortmannin did not inhibit the production of MCP-3 by $IL-1{\beta}/IFN-{\gamma}$ treatment. Conclusion: Chemokines were strongly induced by stimulation of cytokines in combination and showed different pattern of inhibition by the inhibitors. Therefore, it was assumed that cytokines acted on multiple pathways or on unknown pathways which converged to gene-specific transcription factors.

• Applied Biological Chemistry
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• 제42권4호
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• pp.310-316
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• 1999

대두가공제품 중에 존재하는 Bowman-Birk protease inhibitor(BBPI) 함량을 protease 저해활성 측정 및 경합 Enzyme-Linked Immunosorbent Assay(ELISA)로 살펴보았다. 항체제조를 위한 BBPI는 ion exchange chromatography와 전기영동 후 gel slicing 방법으로 시판 soybean trypsin-chymotrypsin inhibitor로부터 순수, 분리하였다. 순수분리한 BBPI를 면역원으로 rabbit anti-BBPI antibody를 조제하였으며 단백질 농도별 titration방법으로 BBP에 비교적 선택적으로 결합하는 항체임을 확인하였다. 이를 이용한 경합 ELISA 방법으로 BBPI를 정량하기 위한 표준 정량곡선을 작성하였으며 시료용액중의 BBPI 함량이 $0.03{\sim}30\;{\mu}g/ml$ 범위일 경우에 정량적인 분석이 가능하였다. 대두품종별 chymotrypsin 저해활성은 $8,462{\sim}12,428\;U/g$이었으며 BBPI 함량은 $482{\sim}692\;mg%$ 이었다. 시판 대두 가공제품 중에서 5종의 콩나물은 건물량 기준으로 $10,695{\sim}13,249\;U/g$의 chymotrypsin 저해활성과 $529{\sim}803\;mg%$의 BBPI 함량을 나타내었으며 일부 두부제품에서도 68.9 mg%정도의 BBPI가 검출되었다. 그 밖의 두유, 된장, 고추장 및 간장 등의 대두발효식품, 탈지대두박 등에서는 chymotrypsin 저해활성 및 BBPI가 거의 검출되지 않았다.

• Journal of Periodontal and Implant Science
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• 제35권2호
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• pp.427-436
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• 2005

Objective: MMP-8 is a neutrophil enzyme and its level increases in some inflammatory diseases, including periodontal disease. We knew that the lipopolysaccharide of E.coli(E-LPS) induced MMP-8 release from human neutrophils. E-LPS is known to induce the production and release of inflammatory cytokines through CD14, Toll-like receptor(TLR). In the present study, we investigated whether MMP-8 release by E-LPS is induced via CD14-TLR pathway and the cellular mechanism of MMP-8 release in human neutrophils. Material and methods: Human neutrophils were isolated from the peripheral blood of healthy donors and pre-incubated in medium containing antibodies against CD14, anti-TLR2 and anti-TLR4 or several inhibitors of microtubules and microfilaments and then incubated with E-LPS. The cells were treated TPCK and E-LPS simultaneously. The MMP-8amount in the culture medium was determined using ELISA. Results: E-LPS increased MMP-8release from neutrophils and its induction was inhibited by anti-CD14 and anti-TLR4 but not by anti-TLR2 antibodies. The inhibitors of microtubule and microfilament polymerization significantly decreased E-LPS-induced MMP-8release. TPCK inhibited E-LPS-induced MMP-8 release. Conclusion: These results suggest that MMP-8 release is induced by E-LPS via the CD14-TLR4 signal pathway in human neutrophils and may be depedent on microtubule and microfilament systems and $NF-{\kappa}B$ pathway.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.743-746
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• 2003

The major pathological lesion in Parkinson's disease(PD) is selective degeneration and loss of pigmented dopaminergic neurons in substantia nigra (SN). Although the initial cause and subsequent molecular signaling mechanisms leading to the dopaminergic cell death underlying the PD process is elusive, the potent neurotrophic factors (NTFs), brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF), are known to exert dopaminergic neuroprotection both in vivo and in vitro models of PD employing the neurotoxin, MPTP. BDNF and its receptor, trkB are expressed in SN dopaminergic neurons and their innervation target. Thus, neurotrophins may have autocrine, paracrine and retrograde transport effects on the SN dopaminergic neurons. This study determined the BDNF secretion from SN dopaminergic neurons by ELISA. Regulation of BDNF synthesis/release and changes in signaling pathways are monitored in the presence of free radical donor, NO donor and mitochondrial inhibitors. Also, this study shows that BDNF is able to promote survival and phenotypic differentiation of SN dopaminergic neurons in culture and protect them against MPTP-induced neurotoxicity via MAP kinase pathway.