• 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.

• Molecules and Cells
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• 제40권6호
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• pp.426-433
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• 2017

Ephrin-A5 has been implicated in the regulation of brain morphogenesis and axon pathfinding. In this study, we used bacterial homologous recombination to express a LacZ reporter in various ephrin-A5 BAC clones to identify elements that regulate ephrin-A5 gene expression during mesencephalon development. We found that there is mesencephalon-specific enhancer activity localized to a specific +25.0 kb to +30.5 kb genomic region in the first intron of ephrin-A5. Further comparative genomic analysis indicated that two evolutionary conserved regions, ECR1 and ECR2, were present within this 5.5 kb region. Deletion of ECR1 from the enhancer resulted in disrupted mesencephalon-specific enhancer activity in transgenic embryos. We also found a consensus binding site for basic helix-loop-helix (bHLH) transcription factors (TFs) in a highly conserved region at the 3'-end of ECR1. We further demonstrated that specific deletion of the bHLH TF binding site abrogated the mesencephalon-specific enhancer activity in transgenic embryos. Finally, both electrophoretic mobility shift assay and luciferase-based transactivation assay revealed that the transcription factor Ascl1 bound the bHLH consensus binding site in the mesencephalon-specific ephrin-A5 enhancer in vitro. Together, these results suggest that the bHLH TF binding site in ECR1 is involved in the positive regulation of ephrin-A5 gene expression during the development of the mesencephalon.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.303-307
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• 2006

We have screened phage display peptide libraries to select for peptides binding to various sized $TiO_2$ nanoparticles. Phage libraries displaying random 7mer, 12mer, and C-7-Cmer peptides were used for screening. The size of target $TiO_2$ particles used were 7 nm, 15 nm, and 25 nm in diameter. We could select peptides binding each nanoparticles from all 3 libraries. Their binding was confirmed by transmission electron microscopy (TEM). Each peptide investigated was also shown to bind the other sized particles, meaning that the binding was specific for the nature of the particle rather than for the size of it. One of the 7mer peptides (PEP9, SVSPISH) was chosen for further analysis. The binding was shown to be in a dose-dependent manner, suggesting a specific interaction.

• Animal cells and systems
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• 제6권3호
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• pp.253-261
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• 2002

Although cardiac distribution of specific receptors for atrial natriuretic peptide (ANP) was mainly observed in the ventricular endocardium, the modulation of ANP receptors in relation to cardiac development is not defined. The present study was undertaken to investigate ANP receptor modulation in rat during development. In the developmental stages examined (fetus, after postnatal 3-days, 1-, 2-, 3-, 4-, and 8-week-old Sprague Dawley rats) specific ANP binding sites were localized in the right and left ventricular endo-cardia by quantitative in vitro receptor autoradiography using (equation omitted)-rat ANP as labeled ligand. The specific bindings to endocardium were much higher in the right than the left ventricle. The binding affinities of ANP were much higher in the right than the left ventricular endocardium. The difference of these binding affinities among various developmental stages was not observed in the right ventricle, whereas the binding affinity in left ventricle was gradually increased with aging and reached the peak value at 8 weeks. No significant difference in maximal binding capacities of endocardial bindings was observed in the right and left ventricular endocardia during developmental stages. Also, cGMP production via activation of particulate guanylyl cyclase-coupled receptor subtypes in the ventricular membranes was gradually decreased with close relationship to aging. Therefore, the present study show that the endocardial ANP receptor is modulated with close relationship to cardiac development in the left ventricle rather than the right ventricle, and may be involved in regulating myocardial contractility in left heart.

• 한국발생생물학회지:발생과생식
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• 제20권3호
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• pp.179-185
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• 2016

The insulin-like growth factor (IGF) pathway is a key signal transduction pathway involved in cell proliferation, migration, and apoptosis. In dairy cows, IGF family proteins and binding receptors, including their intracellular binding partners, regulate mammary gland development. IGFs and IGF receptor interactions in mammary glands influence the early stages of mammogenesis, i.e., mammary ductal genesis until puberty. The IGF pathway includes three major components, IGFs (such as IGF-I, IGF-II, and insulin), their specific receptors, and their high-affinity binding partners (IGF binding proteins [IGFBPs]; i.e., IGFBP1-6), including specific proteases for each IGFBP. Additionally, IGFs and IGFBP interactions are critical for the bioactivities of various intracellular mechanisms, including cell proliferation, migration, and apoptosis. Notably, the interactions between IGFs and IGFBPs in the IGF pathway have been difficult to characterize during specific stages of bovine mammary gland development. In this review, we aim to describe the role of the interaction between IGFs and IGFBPs in overall mammary gland development in dairy cows.

• Archives of Pharmacal Research
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• 제14권3호
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• pp.231-236
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• 1991

The high affinity binding sites for angiotensin II were solubilized from rat liver membranes by treatment with CHAPS. The binding protein was also partially purified by angiotensin III inhibitor-coupled Affi-gel affinity chromatography. Binding to the intact membrances as well as to the solubilized preparation was specific and saturable. According to the Scatchard plot, the membrane preparations exhibited a single class of high affinity binding sites with a Kd OF 0.71 nM. The solubilized preparation also showed the presence of a single class of bindings sites with less affinity (Kd of 14 nM). Meanwhile the competition studies using angiotensin II analogues represented two separate binding sites for angiotensin II and single binding site for antagonist. These latter findings were correlated to the results provided by Garrison's research group. More works are needed to clarify this discrepancy.

• KSBB Journal
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• 제14권3호
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• pp.273-278
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• 1999

The molecularly imprinted polymers(MIPs) synthesized at various polymerization conditions were examined as ibuprofen receptors in terms of binding characteristics. The 4-vinylpyridine polymers had 1.2 times higher adsorption capability for (S)-(+)-ibuprofen than the methacrylic acid polymers. The methacrylic acid polymers synthesized by UV radiation had 1.9 times higher selectivity for (S)-(+)-ibuprofen compared to those by thermal initiation. Effects of various solvents for binding were also examined in this research. According to the Scatchard analysis, the (S)-(+)-ibuprofen artificial receptors had two different kinds of binding sites for (S)-(+)-ibuprofen while having only single kind of binding site for ketoprofen. The binding sites of (S)-(+)-ibuprofen, n were calculated as 4.3~4.9 $\mu$mol/g and the dissociation constants, $K_D$ were 0.68 mM for the specific binding.

• Bulletin of the Korean Chemical Society
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• 제25권4호
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• pp.539-544
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• 2004

The ability of protoberberine alkaloids, berberine and berberrubine, to act as topoisomerase II poisons is linked to the anti-cancer activity. Minor alterations in structure have a significant effect on their relative activity. Berberine, which has methoxy group at the 19-position, is significantly less potent than berberrubine. Several observations support non-specific binding to HP14 by the berberine: (i) nonspecific upfield changes in $^1H$ chemical shift for protons of the berberine; (ii) the broadening of imino protons of HP14 upon binding of the berberine; (iii) very small increases in duplex melting temperature in the presence of the berberine. Our results reveal that substitution of a hydroxyl group to a methoxy group on the 19-position, thereby converting the berberrubine to the berberine is associated with a non-specific DNA binding affinity and a reduced topoisomerase II poisoning. The presence of a bulky 19-methoxy substituent decreases intercalating properties of berberine and makes it inactive as topoisomerase II poison.

• 대한핵의학회지
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• 제12권2호
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• pp.1-5
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• 1978

For clinical application of radioreceptor assay, we studied preliminarily the distribution of estrogen receptor in various organs of rabbit by a dextran-charcoal method using $6,7-^3H-estradiol$. The results were expressed as binding index, which is the ratio of specific estradiol receptor binding radioactivity to total radioactivity. The materials consist of 5 female rabbits and 3 male rabbits. The results were as follows: 1) Female rabbits The binding index was highest in the uterine tissue. This binding index of the uterine tissue was 9.4 times that of the liver, 21.9 times that of the kidney, 24.6 times that of the brain, 28.1 times that of the lung and 65.7 times that of the muscle. 2) Male rabbits The binding index was highest in the liver and decreased in the order of the kidney, the testis, the lung, the brain and the muscle. It is suggested that the estrogen receptor is not confined to any specific target organ but is widely distributed in the various organs, to a different degree.

• 대한약리학회지
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• 제24권2호
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• pp.165-178
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• 1988

백서에서 전기충격 (electroconvulsive shock; ECS)이 뇌내 및 혈중 opiate system에 미치는 효과와 이에 대한 수종의 psychoactive drugs의 영향을 검토코저 1일 1회씩, 1, 3, 7및 14일간 ECS를 가하거나, 14일간 상기 약물과 ECS를 병행처리한 백서의 뇌내 specific $[^3H]$-morphine binding, Met-enkephalin 함량, ${\beta}-endorphin$ 함량 또는 혈중 ${\beta}-endorphin$ 농도를 측정하여 다음과 같은 결과를 얻었다. 1. 뇌내 Met-enkephalin의 함량은 1회의 ECS에 의해서 증가되는 경향을 보였으며 장기간의ECS를 가한 군에서는 최종 ECS 1시간 후부터 유의하게 증가되어 7일후까지 지속되었다. 2. 뇌내 ${\beta}-endorphin$의 함량은 ECS처리 횟수에 관계없이 최종 ECS 1시간후에는 유의하게 감소되었으나 24시간, 3일, 7일 및 14일후의 측정치는 대조군과 차이가 없었다. 3. 혈중 ${\beta}-endorphin$의 농도는 ECS처리 친수에 관계없이 최종 ECS 5분후에 유의하게 증가되었으나 1시간, 24시간, 7일 및 14일후의 측정치는 대조군과 차이가 없었다. 4. 뇌내 specific $[^3H]$-morphine binding의 Bmax는 1회의 ECS에 의해 변동되지 않았으나 장기간의 ECS를 가한 군에서는 ECS 1시간후부터 유의하게 감소되어 7일후까지 지속되었다. 한편 Kd치는 모든 실험군에서 변동되지 않았다. 5. ECS장기처리군에서 ECS 30분전 phenobarbital(100 mg/kg) 전처리는 ECS에 의한 뇌내Met-enkephalin함량증가를 현저히 억제 하였으며, ECS에 의한 뇌내 specific $[^3H]$-morphine binding의 Bmax감소, 뇌내 ${\beta}-endorphin$ 함량감소와 혈중 ${\beta}-endorphin$ 농도증가에 대해서는 영향을 주지 못하였다. 6. Imipramine 또는 pargyline 장기처리는 자체로써 뇌내 ${\beta}-endorphin$함량증가, 혈중 ${\beta}-endorphin$ 농도증가, 뇌내 specific $[^3H]$-morphine binding의 Bmax감소를 일으켰으나 뇌내 Met-enkephalin의 함량과 ECS작용에 영향을 미치지 못했다. 7. Reserpine, chlorpromazine 또는 haloperidol 장기처리는 자체로써 뇌내 Met-enkephalin의 함량증가, 뇌내 ${\beta}-endorphin$함량증가, 혈중 ${\beta}-endorphin$ 농도증가, 뇌내 specific $[^3H]$-morphine binding의 Bmax감소를 일으켰고, ECS효과를 강화시켰다. 8. 장기간 ECS를 가한 백서의 뇌내 specific $[^3H]$-morphine binding의 Bmax는 뇌내 Met-enkephalin 함량과는 유의한 역상관 관계를 보이 나 뇌내 ${\beta}-endorphin$ 함량과는 관계 가 없었다. 이상의 실험성적은 전기충격요법이 생체내에서의 작웅기전에 중추 또는 말초 opiate계가 개입되어 있음을 시사하며 또한 ECT의 효과가 수종의 중추신경계에 작용하는 약물에 의해 변동될수 있음을 보여준다.