• Title/Summary/Keyword: Affinity binding

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Detection of Bacillus Cereus Using Bioluminescence Assay with Cell Wall-binding Domain Conjugated Magnetic Nanoparticles

  • Park, Chanyong;Kong, Minsuk;Lee, Ju-Hoon;Ryu, Sangryeol;Park, Sungsu
    • BioChip Journal
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    • v.12 no.4
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    • pp.287-293
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    • 2018
  • Bacillus cereus can cause blood infections (i.e., sepsis). Its early detection is very important for treating patients. However, an antibody with high binding affinity to B. cereus is not currently available. Bacteriophage cell wall-binding domain (CBD) has strong and specific binding affinity to B. cereus. Here, we report the improvement in the sensitivity of an ATP bioluminescence assay for B. cereus detection using CBD-conjugated magnetic nanoparticles (CBD-MNPs). The assay was able to detect as few as 10 colony forming units (CFU) per mL and $10^3CFU\;per\;mL$ in buffer and blood. CBD-MNPs did not show any cross-reactivity with other microorganisms. These results demonstrate the feasibility of the ATP assay for the detection of B. cereus.

An in silico Appraisal to Identify High Affinity Anti-Apoptotic Synthetic Tetrapeptide Inhibitors Targeting the Mammalian Caspase 3 Enzyme

  • Kelotra, Seema;Jain, Meeta;Kelotra, Ankit;Jain, Ish;Bandaru, Srinivas;Nayarisseri, Anuraj;Bidwai, Anil
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.23
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    • pp.10137-10142
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    • 2015
  • Apoptosis is a general phenomenon of all multicellular organisms and caspases form a group of important proteins central to suicide of cells. Pathologies like cancer, Myocardial infarction, Stroke, Sepsis, Alzheimer's, Psoriasis, Parkinson and Huntington diseases are often associated with change in caspase 3 mediated apoptosis and therefore, caspases may serve as potential inhibitory targets for drug development. In the present study, two series of synthetic acetylated tetrapeptides containing aldehyde and fluromethyl keto groups respectively at the C terminus were proposed. All these compounds were evaluated for binding affinity against caspase 3 structure. In series 1 compound Ac-DEHD-CHO demonstrated appreciable and high binding affinity (Rerank Score: -138.899) against caspase 3. While in series 2 it was Ac-WEVD-FMK which showed higher binding affinity (Rerank Score: -139.317). Further these two compounds met ADMET properties and demonstrated to be non-toxic.

Identification and Classification of the Muscarinic Receptors in the Uterus (자궁 무스카린수용체의 확인 및 분류)

  • Lee, Shin-Woong;Lee, Jeung-Soo;Park, Young-Joo
    • YAKHAK HOEJI
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    • v.36 no.3
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    • pp.220-229
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    • 1992
  • The muscarinic acetylcholine receptors of the dog unpregant uterus were characterized using $[^3H]quinuclidinyl$ benzilate(QNB) as a radioligand and the binding of muscarinic receptor agonists and antagonists in the uterus was compared to that in the urinary bladder which contains almost exclusively the M2 receptors in order to determine the receptor subtypes in the uterus. $[^3H]QNB$ binding to uterus and bladder was rapid, saturable and reversible. Scatchard analysis of the saturation data gave linear plots and the Hill coefficients were close to unit, which indicated that each preparation contained a single population of specific binding sites for $[^3H]QNB$. The KD values(120 pM) for QNB were almost identical in both organs, whereas the $B_{max}$ value of 256 fmol/mg protein in the uterus was significantly different from that of 563 fmol/mg protein in the bladder. Muscarinic agonists and antagonists inhibited in a competitive manner the $[^3H]QNB$ binding to the same extent in both organs. The competition binding studies using antagonists(atropine and pirenzepine) exhibited a single binding site and this site had a low affinity for pirenzepine with the Ki value of about 330 nM. However, high and low affinity binding sites were observed with carbachol, methacholine and oxotremorine. These binding studies with agonists and antagonists did not show any differences in drug affinities between uterus and bladder. These results indicate that the muscarinic receptors in the uterus are M2 receptors which have a low affinity for pirenzepine.

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Characterization of binding specificity using GST-conjugated mutant huntingtin epitopes in surface plasmon resonance (SPR)

  • Cho, Hang-Hee;Kim, Tae Hoon;Kim, Hong-Duck;Cho, Jae-Hyeon
    • Korean Journal of Veterinary Service
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    • v.44 no.4
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    • pp.185-194
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    • 2021
  • Polyglutamine extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyglutamine aggregates in Huntington's disease (HD). Mutant huntingtin can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. To better understand the mechanism by which an elongated polyglutamine causes aggregates, we have developed an in vitro binding assay system of polyglutamine tract from truncated huntingtin. We made GST-HD exon1 fusion proteins which have expanded polyglutamine epitopes (e.g., 17, 23, 32, 46, 60, 78, 81, and 94 CAG repeats). In the present emergence of new study adjusted nanotechnology on protein chip such as surface plasmon resonance strategy which used to determine the substance which protein binds in drug discovery platform is worth to understand better neurodegenerative diseases (i.e., Alzheimer disease, Parkinson disease and Huntington disease) and its pathogenesis along with development of therapeutic measures. Hence, we used strengths of surface plasmon resonance (SPR) technology which is enabled to examine binding specificity and explore targeted molecular epitope using its electron charged wave pattern in HD pathogenesis utilize conjugated mutant epitope of HD protein and its interaction whether wild type GST-HD interacts with mutant GST-HD with maximum binding affinity at pH 6.85. We found that the maximum binding affinity of GST-HD17 with GST-HD81 was higher than the binding affinities of GST-HD17 with other mutant GST-HD constructs. Furthermore, our finding illustrated that the mutant form of GST-HD60 showed a stronger binding to GST-HD23 or GST-HD17 than GST-HD60 or GST-HD81. These results indicate that the binding affinity of mutant huntingtin does not correlate with the length of polyglutamine. It suggests that the aggregation of an expanded polyglutamine might have easily occurred in the presence of wild type form of huntingtin.

Contribution of Second Metal Binding Site for Metal Specificity of D-Xylose Isomerase

  • Cha, Jae-Ho
    • Journal of Microbiology and Biotechnology
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    • v.9 no.6
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    • pp.757-763
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    • 1999
  • The metal specificity of D-xylose isomerase from Streptomyces rubiginosus was examined by site-directed mutagenesis. The activation constants for metal ion ($Mg^{2+},{\;}Mn^{2+},{\;}or{\;}Co^{2+}$) of wild-type and mutant enzymes were determined by titrating the metal ion-free enzyme with $Mg^{2+},{\;}Mn^{2+},{\;}and{\;}Co^{2+}$, respectively. Substitutions of amino acids either on coordinated or around the M2 site (His-22O, Asn-185, Glu-186, and Glu-221) dramatically affected the activation constants as well as activity. A decrease of metal binding affinity was most significant in the presence of $Mg^{2+}$. When compared with the wild-type enzymes, the binding affinity of H220S and Nl85K for Mg^{2+} was decreased by 10-15-fold, while the affinity for $Mn^{2+}{\;}or{\;}Co^{2+}$ only decreased by 3-5-fold. All the mutations close to the M2 site changed their metal preference from $Mg^{2+}{\;}to{\;}Mn^{2+}{\;}or{\;}Co^{2+}$. These altered metal preferences may be caused by a relatively weak binding affinity of $Mg^{2+}$ to the enzyme. Thermal inactivation studies of mutants at the M2 site also support the importance of the M2 site geometry for metal specificity as well as the thermostability of the enzyme. Mutations of other important groups hardly affected the metal preference, although pronounced effects on the kinetic parameters were sometimes observed. This study proposes that the metal specificity of D-xylose isomerase can be altered by the perturbation of the M2 site geometry, and that the different metal preference of Group I and GroupII D-xylose isomerases may be caused by nonconserved amino acid residues around the M2 site.

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Effect of Avidin and Biotin in Attachment of Human Adipose Stem Cells to Micronized Acellular Dermal Matrix (지방줄기세포가 무세포 분쇄진피기질(Acellular micronized dermal matrix) 부착에 있어 Avidin과 Biotin의 효과)

  • You, Gyeol;Rhie, Jong Won;Lim, Jin Soo
    • Archives of Plastic Surgery
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    • v.35 no.1
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    • pp.1-6
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    • 2008
  • Purpose: In tissue engineering, it is important that the scaffolds have high affinity with cells for making efficient use of cells. The authors studied the binding affinity of human adipose stem cells(ASCs) to micronized acellular dermal matrix(alloderm) using biotin and avidin linkages.Methods: Human ASCs were harvested from adipose tissue obtained by abdominoplasty. ASCs($1{\times}10^4$, $5{\times}10^4$, $1{\times}10^5$, $5{\times}10^5$, $1{\times}10^6$, $5{\times}10^6$ cells) were attached to micronized alloderm(1mg) in three groups; 1) control group in which no ASCs and alloderm was treated; 2) serum group in which alloderm was exposed to fetal bovine serum; and 3) biotin group in which biotinylated cells were attached to biotinylated alloderm. The binding affinities were determined 1 day after making ASC-alloderm complexes. The proliferation rates were determined by XTT assays in 4, 7, 14, and 21 days and scanning electron microscopic examination was performed in 7 and 21 days after culture of ASC-alloderm complexes.Results: The binding affinities of the biotin group were significantly increased in all cell concentrations. Maximum binding affinity was observed at $5{\times}10^4/mg$ of micronized dermal matrix in biotin group. The viabilities were lowest in biotin group in contrast to binding affinity, but the difference was not significant. SEM showed well attachment of cells to micronized dermal matrix in all groups. Conclusion: The use of avidin/biotin facilitated human ASCs attaching to micronized acellular dermal matrix. This attachment would not disturb adipose stem cells viabilities. The present study suggests that avidin/ biotin can be used as making efficient use of cells in adipose tissue engineering.

The Search of Pig Pheromonal Odorants for Biostimulation Control System Technologies: III. Comparative Molecular Field Analysis (CoMFA) on Binding Affinities between Ligands of 2-(Cyclohexyloxy) Tetrahydrofurane Derivatives and Porcine Odorant Binding Protein (생물학적 자극 통제 수단으로 활용하기 위한 돼지 페로몬성 냄새 물질의 탐색: III. 2-(Cyclohexyloxy) Tetrahydrofurane 유도체와 Porcine Odorant Binding Protein 사이의 결합 친화력에 관한 비교 분자장 분석)

  • Sung Nack-Do;Park Chang-Sik;Jung Hoon-Sung;Seong Min-Kyu
    • Reproductive and Developmental Biology
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    • v.30 no.1
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    • pp.13-19
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    • 2006
  • To search of new porcine pheromonal odorants for biostimulation control system technologies to improve reproductive efficiency in livestock species, the comparative molecular field analysis (CoMFA) for binding affinity constant $(p(Od)_{50})$ between porcine odorant binding protein (pOBP) and ligands of odorant 2-(cyclohexyloxy) tetrahydrofurane derivatives as substrate molecule was conducted and discussed. In the optimized CoMFA model AIV with chirality $(C_1'(R),\;C_2(S))$ in substrate molecule and atom based fit alignment (A) of odorants, the statistical results showed the best predictability of the binding affinities $(p(Od)_{50})$ based on the LOO cross-validated value $r^2_{cv}.\;(q^2=0.886)$ and non-cross-validated conventional coefficient $(r^2_{ncv}.=0.984)$. the binding affinity constants exhibited a good correlation with steric (40.8%), electrostatic (14.6%) and hydrophobic (44.6%) factors of the substrate molecules. from the analytical results of the contour maps, which may give us some valuable informations to the modification of odorants for effective binding affinity.

Binding affinity of some herbal extracts on the muscarinic acetylcholine receptor subtype 1 $(mAChR-M_1)$ (수종 생약추출물의 muscarin성$(M_1\;type)$ acetylcholine 수용체$(mAChR-M_1)$에 대한 친화력 검색)

  • Kim, Young-Sup;Kim, Jeoung-Seob;Kim, Seong-Kie;Heor, Jung-Hee;Lee, Byung-Eui;Ryu, Shi-Yong
    • Korean Journal of Pharmacognosy
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    • v.32 no.3 s.126
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    • pp.219-225
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    • 2001
  • The water extracts of 82 Korean medicinal herbs were examined for the binding affinity on the recombinant human muscarinic acetylcholine receptor subtype 1 $(mAChR-M_1)$ produced from the CHO (Chinese Hamster Ovary) cell line. Of those tested, the extracts of Coptidis Rhizoma, Phellodendri Cortex, Hedyotis Herba and of Terminariae Fructus were found to exhibit a significant competition with $[^3H]$ N-methyl-scopolamine for the specific binding to $mAChR-M_1$ in a dose dependent manner, respectively.

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Isolation of cDNA Encoding Double-Stranded RNA Binding Protein (RBFII) (이중선RNA결합담백질(RBFII)의 cDNA분리)

  • 박희성
    • Journal of Life Science
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    • v.7 no.3
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    • pp.167-171
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    • 1997
  • As an initial effort to elucidate RNA: protein binding in a way to regulate translation initiation and phosphorylation, a cDNA encoding a double-stranded RNA binding factor (RBFII)was isolated from Hela ZAPII cDNA library by affinity screening using [$\alpha$$^{-32}$P] UMP-labeled HIV Rev-responsive element(RRE) RNA. The nucleotide sequence of RBF (or TRBP) cDNA except the 5’end. At the 5’end, This common ORF was fused in-frame to N-terminal residues of Lac-Z through a unique 138 nt sequence encoding 46residues in the case of RBFII and a 63 nt sequence encoding 21 residuces in the case of RBFI. The context of ATG appearing first in the sequences suggests that both these cDNA inserts are incomplete at the 5’end.

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Importance of Accurate Charges in Binding Affinity Calculations: A Case of Neuraminidase Series

  • Park, Kichul;Sung, Nack Kyun;Cho, Art E.
    • Bulletin of the Korean Chemical Society
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    • v.34 no.2
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    • pp.545-548
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    • 2013
  • It has been shown that calculating atomic charges using quantum mechanical level theory greatly improves the accuracy of docking. A protocol was developed and shown to be effective. That this protocol works is just a manifestation of the fact that electrostatic interactions are important in protein-ligand binding. In order to investigate how the same protocol helps in prediction of binding affinities, we took a series of known cocrystal structures of influenza neuraminidase inhibitors with the receptor and performed docking with Glide SP, Glide XP, and QPLD, the last being a workflow that incorporates QM/MM calculations to replace the fixed atomic charges of force fields with quantum mechanically recalculated ones at a given docking pose, and predicted the binding affinities of each cocrystal. The correlation with experimental binding affinities considerably improved with QPLD compared to Glide SP/XP yielding $r^2$ = 0.83. The results suggest that for binding sites, such as that of neuraminidase, which are laden with hydrophilic residues, protocols such as QPLD which utilizes QM-based atomic charges can better predict the binding affinities.