Mechanistic models enable the rational use of in vitro drug-target binding kinetics for better drug effects in patients.

PubMed

de Witte, Wilhelmus E A; Wong, Yin Cheong; Nederpelt, Indira; Heitman, Laura H; Danhof, Meindert; van der Graaf, Piet H; Gilissen, Ron A H J; de Lange, Elizabeth C M

2016-01-01

Drug-target binding kinetics are major determinants of the time course of drug action for several drugs, as clearly described for the irreversible binders omeprazole and aspirin. This supports the increasing interest to incorporate newly developed high-throughput assays for drug-target binding kinetics in drug discovery. A meaningful application of in vitro drug-target binding kinetics in drug discovery requires insight into the relation between in vivo drug effect and in vitro measured drug-target binding kinetics. In this review, the authors discuss both the relation between in vitro and in vivo measured binding kinetics and the relation between in vivo binding kinetics, target occupancy and effect profiles. More scientific evidence is required for the rational selection and development of drug-candidates on the basis of in vitro estimates of drug-target binding kinetics. To elucidate the value of in vitro binding kinetics measurements, it is necessary to obtain information on system-specific properties which influence the kinetics of target occupancy and drug effect. Mathematical integration of this information enables the identification of drug-specific properties which lead to optimal target occupancy and drug effect in patients.

Soy lecithin replaces egg yolk for cryopreservation of human sperm without adversely affecting postthaw motility, morphology, sperm DNA integrity, or sperm binding to hyaluronate.

PubMed

Reed, Michael L; Ezeh, Peace C; Hamic, Amanda; Thompson, Douglas J; Caperton, Charles L

2009-11-01

Semen specimens (one ejaculate from each of 20 consenting study participants) were subjected to routine semen analysis, an in vitro sperm binding assay (HBA), and a sperm chromatin dispersion assay (HaloSperm), both before and after cryopreservation using cryoprotectant media supplemented with either egg yolk or soy lecithin. Comparing the equivalency of the two phospholipid cryopreservation supplements with regard to postthaw functional parameters demonstrated that there were no statistically significant differences between the two supplements for [1] recovery of motile sperm, [2] maintenance of sperm cell morphology, [3] maintenance of the ability of sperm to bind to hyaluronate in vitro, or [4] maintenance of sperm DNA integrity.

Estrogenicity of halogenated bisphenol A: in vitro and in silico investigations.

PubMed

Zhang, Jie; Li, Tiezhu; Wang, Tuoyi; Yuan, Cuiping; Zhong, Shuning; Guan, Tianzhu; Li, Zhuolin; Wang, Yongzhi; Yu, Hansong; Luo, Quan; Wang, Yongjun; Zhang, Tiehua

2018-03-01

The binding interactions of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs) to human estrogen receptor α ligand binding domain (hERα-LBD) was investigated using a combined in vitro and in silico approach. First, the recombinant hERα-LBD was prepared as a soluble protein in Escherichia coli BL21(DE3)pLysS. A native fluorescent phytoestrogen, coumestrol, was employed as tracer for the fluorescence polarization assay. The results of the in vitro binding assay showed that bisphenol compounds could bind to hERα-LBD as the affinity ligands. All the tested halogenated BPAs exhibited weaker receptor binding than BPA, which might be explained by the steric effect of substituents. Molecular docking studies elucidated that the halogenated BPAs adopted different conformations in the flexible hydrophobic ligand binding pocket (LBP), which is mainly dependent on their distinct halogenation patterns. The compounds with halogen substituents on the phenolic rings and on the bridging alkyl moiety acted as agonists and antagonists for hERα, respectively. Interestingly, all the compounds in the agonist conformation of hERα formed a hydrogen bond with His524, while the compounds in the antagonist conformation formed a hydrogen bond with Thr347. These docking results suggested a pivotal role of His524/Thr347 in maintaining the hERα structure in the biologically active agonist/antagonist conformation. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation, which might be applicable for the structure-based design of novel bisphenol compounds with reduced toxicities and for environmental risk assessment. In addition, based on hERα-LBD as a recognition element, the proposed fluorescence polarization assay may offer an alternative to chromatographic techniques for the multi-residue determination of bisphenol compounds.

Endocrine disrupters--testing strategies to assess human hazard.

PubMed

Baker, V A

2001-01-01

During the last decade an hypothesis has been developed linking certain chemicals (natural and synthetic) to observed and suspected adverse effects on reproduction in both wildlife and humans. The issue of 'endocrine disruption' originally focused on chemicals that mimic the action of the natural hormone oestrogen. However, the concern is now encompassing effects on the whole endocrine system. In response to public awareness, regulatory agencies (including the US EPA) and the OECD are formulating potential testing strategies and have begun the process of validating defined tests to systematically assess chemicals for their endocrine-disrupting activities. In order to investigate chemicals that have the potential to cause endocrine disruption, a large number of in vitro and in vivo assays have been identified. In vitro test systems (particularly when used in combination) offer the possibility of providing an early screen for large numbers of chemicals and can be useful in characterising the mechanism of action and potency. In vitro assays in widespread use for the screening/characterisation of endocrine disrupting potential include hormone receptor ligand binding assays (determination of the ability of a chemical to bind to the hormone receptor), cell proliferation assays (analysis of the ability of a chemical to stimulate growth of oestrogen sensitive cells), reporter gene assays in yeast or mammalian cells (analysis of the ability of a chemical to stimulate the transcription of a reporter gene construct in cell culture), and the analysis of the regulation of endogenous oestrogen sensitive genes in cell lines. However, in vitro assays do not always reliably predict the outcome in vivo due to differences in metabolic capabilities of the test systems used and the diverse range of mechanisms by which endocrine disrupting chemicals may act. Therefore a complementary battery of short- and long-term in vitro and in vivo assays (that assess both receptor and non-receptor mediated mechanisms of action) seems the most appropriate way at present of assessing the potential endocrine disrupting activities of chemicals. At Unilever we have used a combination of in vitro assays (receptor binding, reporter gene and cell proliferation assays) together with short-term in vivo tests (uterotrophic assay in immature rodents) to examine the oestrogenic potential of a large number of chemicals. An evaluation of the advantages and limitations of these methods is provided. Finally, any potential test system needs to be validated and standardized before the information generated can be for the identification of hazard, and possibly for risk assessment purposes.

Adaptive Focused Acoustics (AFA) Improves the Performance of Microtiter Plate ELISAs.

PubMed

Green, David J; Rudd, Edwin A; Laugharn, James A

2014-08-01

We investigated the use of Adaptive Focused Acoustics (AFA) technology to improve the performance of microtiter plate enzyme-linked immunosorbent assays (ELISAs). Experiments were performed with commercially available AFA instrumentation and off-the-shelf 96-well microtiter plate sandwich ELISAs. AFA was applied over a range of acoustic energies, temperatures, and durations to the antigen/antibody binding step of an ELISA for measuring HIV-1 p24 in tissue culture samples. AFA-mediated antigen/antibody binding was enhanced up to 2-fold over passive binding at comparable temperatures and was superior or comparable at low temperature (8-10 °C) to passive binding at 37 °C. Lower nonspecific binding (NSB), lower inter- and intra-assay coefficients of variation (CVs), higher Z' factors, and lower limits of detection (LODs) were measured in AFA-mediated assays compared with conventional passive binding. In a more limited study, AFA enhancement of antigen/antibody binding and lower NSB was measured in an ELISA for measuring IGFBP-3 in human plasma. We conclude from this study that application of AFA to antigen/antibody binding steps in microtiter plate ELISAs can enhance key assay performance parameters, particularly Z' factors and LODs. These features render AFA-mediated binding assays potentially more useful in applications such as high-throughput screening and in vitro diagnostics than assays processed with conventional passive antigen/antibody binding steps. © 2014 Society for Laboratory Automation and Screening.

Integrated Summary Report: Validation of Two Binding Assays ...

EPA Pesticide Factsheets

This Integrated Summary Report (ISR) summarizes, in a single document, the results from an international multi-laboratory validation study conducted for two in vitro estrogen receptor (ER) binding assays. These assays both use human recombinant estrogen receptor, alpha subtype (hrERα), to identify chemicals that may impact estrogen signaling through binding to the ER. The purpose of the ISR is to support the peer review of the findings obtained during the validation process.The two assays evaluated during this validation process are: The Freyberger-Wilson Assay (FW) using a full length human ER, and The Chemical Evaluation and Research Institute (CERI) Assay using a ligand-binding domain of the human ER.The two assays are mechanistically and functionally similar in that each measures the ability of a test chemical to competitively inhibit binding of [3H]17β-estradiol to the human recombinant ER. The essential elements of the FW and the CERI assays were developed at the laboratories of Bayer Pharma AG, Wuppertal, Germany (Freyberger et al., 2010) and CERI, Tokyo, Japan (Akahori et al., 2008), respectively.The ER competitive binding assay has long been in use, and is a well characterized approach, but historically uses rodent or other animal tissues as a source of the ER. Validation of the FW and CERI assays using human recombinant estrogen receptors ( subtype) will provide an updated alternative for the Agency’s current test guideline (OPPTS 89

SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

PubMed

Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A; Twiss, Jeffery L; Heise, Tilman

2016-01-01

The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5' untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells

PubMed Central

Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A.; Twiss, Jeffery L.

2016-01-01

The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5’ untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5’ UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality. PMID:27224031

Identifying Metabolically Active Chemicals Using a Consensus ...

EPA Pesticide Factsheets

Traditional toxicity testing provides insight into the mechanisms underlying toxicological responses but requires a high investment in a large number of resources. The new paradigm of testing approaches involves rapid screening studies able to evaluate thousands of chemicals across hundreds of biological targets through use of in vitro assays. Endocrine disrupting chemicals (EDCs) are of concern due to their ability to alter neurodevelopment, behavior, and reproductive success of humans and other species. A recent integrated computational model examined results across 18 ER-related assays in the ToxCast in vitro screening program to eliminate chemicals that produce a false signal by possibly interfering with the technological attributes of an individual assay. However, in vitro assays can also lead to false negatives when the complex metabolic processes that render a chemical bioactive in a living system might be unable to be replicated in an in vitro environment. In the current study, the influence of metabolism was examined for over 1,400 chemicals considered inactive using the integrated computational model. Over 2,000 first-generation and over 4,000 second-generation metabolites were generated for the inactive chemicals using in silico techniques. Next, a consensus model comprised of individual structure activity relationship (SAR) models was used to predict ER-binding activity for each of the metabolites. Binding activity was predicted for 8-10% of the meta

Development and Validation of a Computational Model for Androgen Receptor Activity

PubMed Central

2016-01-01

Testing thousands of chemicals to identify potential androgen receptor (AR) agonists or antagonists would cost millions of dollars and take decades to complete using current validated methods. High-throughput in vitro screening (HTS) and computational toxicology approaches can more rapidly and inexpensively identify potential androgen-active chemicals. We integrated 11 HTS ToxCast/Tox21 in vitro assays into a computational network model to distinguish true AR pathway activity from technology-specific assay interference. The in vitro HTS assays probed perturbations of the AR pathway at multiple points (receptor binding, coregulator recruitment, gene transcription, and protein production) and multiple cell types. Confirmatory in vitro antagonist assay data and cytotoxicity information were used as additional flags for potential nonspecific activity. Validating such alternative testing strategies requires high-quality reference data. We compiled 158 putative androgen-active and -inactive chemicals from a combination of international test method validation efforts and semiautomated systematic literature reviews. Detailed in vitro assay information and results were compiled into a single database using a standardized ontology. Reference chemical concentrations that activated or inhibited AR pathway activity were identified to establish a range of potencies with reproducible reference chemical results. Comparison with existing Tier 1 AR binding data from the U.S. EPA Endocrine Disruptor Screening Program revealed that the model identified binders at relevant test concentrations (<100 μM) and was more sensitive to antagonist activity. The AR pathway model based on the ToxCast/Tox21 assays had balanced accuracies of 95.2% for agonist (n = 29) and 97.5% for antagonist (n = 28) reference chemicals. Out of 1855 chemicals screened in the AR pathway model, 220 chemicals demonstrated AR agonist or antagonist activity and an additional 174 chemicals were predicted to have potential weak AR pathway activity. PMID:27933809

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

PubMed

Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

2017-01-01

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Avoiding false positives and optimizing identification of true ...

EPA Pesticide Factsheets

The potential for chemicals to affect endocrine signaling is commonly evaluated via in vitro receptor binding and gene activation, but these assays, especially antagonism assays, have potential artifacts that must be addressed for accurate interpretation. Results are presented from screening 94 chemicals from 54 chemical groups for estrogen receptor (ER) activation in a competitive rainbow trout ER (rtER) binding assay and a trout liver slice vitellogenin mRNA expression assay. Results from true competitive agonists and antagonists, and inactive chemicals with little or no indication of ER binding or gene activation were easily interpreted. However, results for numerous industrial chemicals were more challenging to interpret, including chemicals with: (1) apparent competitive binding curves but no gene activation, (2) apparent binding and gene inhibition with evidence of either cytotoxicity or changes in assay media pH, (3) apparent binding but non-competitive gene inhibition of unknown cause, or (4) no rtER binding and gene inhibition not due to competitive ER interaction but due to toxicity, pH change, or some unknown cause. The use of endpoints such as toxicity, pH, precipitate formation, and determination of inhibitor dissociation constants (Ki) for interpreting the results of antagonism and binding assays for diverse chemicals is presented. Of the 94 chemicals tested for antagonism only two, tamoxifen and ICI-182,780, were found to be true competitive

Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors.

PubMed

Takahashi, Hirotaka; Takahashi, Chikako; Moreland, Nicole J; Chang, Young-Tae; Sawasaki, Tatsuya; Ryo, Akihide; Vasudevan, Subhash G; Suzuki, Youichi; Yamamoto, Naoki

2012-12-01

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

PubMed

Schulz, Sebastian; Doller, Anke; Pendini, Nicole R; Wilce, Jacqueline A; Pfeilschifter, Josef; Eberhardt, Wolfgang

2013-12-01

The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA. © 2013.

In vitro toxicity testing with microplate cell cultures: Impact of cell binding.

PubMed

Gülden, Michael; Schreiner, Jeannine; Seibert, Hasso

2015-06-05

In vitro generated data on toxic potencies are generally based on nominal concentrations. However, cellular and extracellular binding and elimination processes may reduce the available free fraction of a compound. Then, nominal effective concentrations do not represent appropriate measures of toxic exposure in vitro and underestimate toxic potencies. In this study it was investigated whether cell binding can affect the availability of chemicals in microplate based toxicity assays. To this end the cytotoxicity of compounds like mercury chloride, digitonin and alcohol ethoxylates, accumulated by cells via different modes, was investigated in 96-well microplate cultures with varying concentrations of Balb/c 3T3 cells. The median effective nominal concentrations of all but one of the tested compounds depended linearly from the cell concentration. Applying a previously developed equilibrium distribution model cell concentration-independent median effective extracellular concentrations and cell burdens, respectively, could be calculated. The compounds were accumulated by the cells with bioconcentration factors, BCF, between 480 and ≥ 25,000. Cell binding of the alcohol ethoxylates was correlated with their lipophilicity. The results show that significant cell binding can occur even at the small cell volume fractions (∼ 1 × 10(-5) to 3 × 10(-3) L/L) encountered in microplate assays. To what extent cell binding affects the bioavailability depends on the BCF and the cell volume fraction. EC50 measurements in the presence of at least two different cell concentrations allow for excluding or detecting significant cell binding and for determining more appropriate measures of toxic exposure in vitro like median effective extracellular (free) concentrations or cell burdens. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Relative Impact of Incorporating Pharmacokinetics on ...

EPA Pesticide Factsheets

The use of high-throughput in vitro assays has been proposed to play a significant role in the future of toxicity testing. In this study, rat hepatic metabolic clearance and plasma protein binding were measured for 59 ToxCast phase I chemicals. Computational in vitro-to-in vivo extrapolation was used to estimate the daily dose in a rat, called the oral equivalent dose, which would result in steady-state in vivo blood concentrations equivalent to the AC50 or lowest effective concentration (LEC) across more than 600 ToxCast phase I in vitro assays. Statistical classification analysis was performed using either oral equivalent doses or unadjusted AC50/LEC values for the in vitro assays to predict the in vivo effects of the 59 chemicals. Adjusting the in vitro assays for pharmacokinetics did not improve the ability to predict in vivo effects as either a discrete (yes or no) response or a low effect level (LEL) on a continuous dose scale. Interestingly, a comparison of the in vitro assay with the lowest oral equivalent dose with the in vivo endpoint with the lowest LEL suggested that the lowest oral equivalent dose may provide a conservative estimate of the point of departure for a chemical in a dose-response assessment. Furthermore, comparing the oral equivalent doses for the in vitro assays with the in vivo dose range that resulted in adverse effects identified more coincident in vitro assays across chemicals than expected by chance, suggesting that the approach ma

Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies.

PubMed

Wu, Qing-Ping; Zhang, Lei; Shao, Xiao-Xia; Wang, Jia-Hui; Gao, Yu; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

2016-04-01

Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-receptor binding assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1.

Medicinal activities of the leaves of Musa sapientum var. sylvesteris in vitro

PubMed Central

Sahaa, Repon Kumer; Acharyaa, Srijan; Shovon, Syed Sohidul Haque; Royb, Priyanka

2013-01-01

Objective This study is to investigate the medicinal value of methanolic extract of the leaves of Musa sapientum var. sylvesteris in Bangladesh. Methods Several biochemical assays, thin layer chormatogarphy and ultra-violet spectroscopy were used to detect the presence of various types of compounds in this extract. Antioxidant effects were measured by DPPH scavenging assay, total reducing assay and hydrogen peroxide scavenging assay. Receptor binding activities and hydrogen peroxide induced hemolysis assay were performed by hemagglutination assay and hemolysis assay using erythrocytes. Disk diffusion assay was performed to show the antibacterial effect of the extract. Results Methanolic extract of the leaves showed antioxidant and antibacterial activity in vitro. The extract showed hemaglutination inhibition activities and hydrogen peroxide induced hemolysis inhibition activity of human red blood cells. Conclusion Musa sapientum var. sylvesteris can be an useful medicinal plant. PMID:23730561

Integrated Model of Chemical Perturbations of a Biological Pathway Using 18 In Vitro High-Throughput Screening Assays for the Estrogen Receptor

PubMed Central

Judson, Richard S.; Magpantay, Felicia Maria; Chickarmane, Vijay; Haskell, Cymra; Tania, Nessy; Taylor, Jean; Xia, Menghang; Huang, Ruili; Rotroff, Daniel M.; Filer, Dayne L.; Houck, Keith A.; Martin, Matthew T.; Sipes, Nisha; Richard, Ann M.; Mansouri, Kamel; Setzer, R. Woodrow; Knudsen, Thomas B.; Crofton, Kevin M.; Thomas, Russell S.

2015-01-01

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation, and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform (“assay interference”). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 111 (6.1%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in vivo endocrine testing. Finally, this model is generalizable to any molecular pathway for which there are multiple upstream and downstream assays available. PMID:26272952

Nuclear binding of progesterone in hen oviduct. Binding to multiple sites in vitro.

PubMed Central

Pikler, G M; Webster, R A; Spelsberg, T C

1976-01-01

Steroid hormones, including progesterone, are known to bind with high affinity (Kd approximately 1x10(-10)M) to receptor proteins once they enter target cells. This complex (the progesterone-receptor) then undergoes a temperature-and/or salt-dependent activation which allows it to migrate to the cell nucleus and to bind to the deoxyribonucleoproteins. The present studies demonstrate that binding the hormone-receptor complex in vitro to isolated nuclei from the oviducts of laying hens required the same conditions as do other studies of bbinding in vitro reported previously, e.g. the hormone must be complexed to intact and activated receptor. The assay of the nuclear binding by using multiple concentrations of progesterone receptor reveals the presence of more than one class of binding site in the oviduct nuclei. The affinity of each of these classes of binding sites range from Kd approximately 1x10(-9)-1x10(-8)M. Assays using free steroid (not complexed with receptor) show no binding to these sites. The binding to each of the classes of sites, displays a differential stability to increasing ionic concentrations, suggesting primarily an ionic-type interaction for all classes. Only the highest-affinity class of binding site is capable of binding progesterone receptor under physioligical-saline conditions. This class represent 6000-10000 sites per cell nucleus and resembles the sites detected in vivo (Spelsberg, 1976, Biochem. J. 156, 391-398) which cause maximal transcriptional response when saturated with the progesterone receptor. The multiple binding sites for the progesterone receptor either are not present or are found in limited numbers in the nuclei of non-target organs. Differences in extent of binding to the nuclear material between a target tissue (oviduct) and other tissues (spleen or erythrocyte) are markedly dependent on the ionic conditions, and are probably due to binding to different classes of sites in the nuclei. PMID:182147

Replacing antibodies with modified DNA aptamers in vaccine potency assays.

PubMed

Trausch, Jeremiah J; Shank-Retzlaff, Mary; Verch, Thorsten

2017-10-04

Vaccine in vitro potency assays are vital regulatory tests that are used to confirm the presence and concentration of an antigen of interest in a form that directly or indirectly relates to protective activity in patients. Current assays come in many forms, but they almost exclusively use antibody reagents for selective detection of the target antigen. Antibodies provide specific recognition of vaccine antigens but also exhibit drawbacks such as stability limitations, cost, and lot-to-lot variation, which can make it challenging to maintain the reagent throughout the lifetime of the vaccine. We explored replacing antibodies with aptamers. Aptamers are macromolecules, such as nucleic acids, which can bind to their targets with high specificity and affinity, similar to that of antibodies. Some of the advantages of using aptamers over antibodies is that aptamers can be more stable, smaller, less expensive to produce, synthesized in vitro, and logistically easier to supply throughout the multi-decade lifespan of a commercial vaccine. We created modified DNA aptamers against the common vaccine carrier protein, CRM 197 . Several aptamers were discovered and one was chosen for further characterization. The binding kinetics of the aptamer revealed an off-rate 16-fold slower than anti-CRM 197 antibodies used for comparison. The aptamers were more sensitive than available antibodies in some assay formats and comparable in others. The aptamer epitope was mapped to the receptor-binding domain of CRM 197 , a site adjacent to a known antibody binding site. These data address some key aspects for a path forward in replacing antibodies with aptamers for use as critical reagents in vaccine assays. We further highlight the possibility of using nucleic acid reagents to develop next generation potency assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

HPV binding assay to Laminin-332/integrin α6β4 on human keratinocytes.

PubMed

Brendle, Sarah A; Christensen, Neil D

2015-01-01

Human papillomaviruses (HPVs) have been shown to bind to Laminin-332 (Ln-332) on the extracellular matrix (ECM) secreted by human keratinocytes. The assay described here is an important tool to study HPV receptor binding to the ECM. The assay can also be modified to study the receptors required for HPV infection and for binding to tissues. We previously showed that Ln-332 is essential for the binding of HPV11 to human keratinocytes and that infectious entry of HPV11 requires α6β4 integrin for the transfer of HPV11 from ECM to host cells (Culp et al., J Virol 80:8940-8950, 2006). We also demonstrated that several of the high-risk HPV types (16, 18, 31 and 45) bind to Ln-332 and/or other components of the ECM in vitro (Broutian et al., J Gen Virol 91:531-540, 2010). The exact binding and internalization mechanism(s) for HPV are still under investigation. A better understanding of these mechanisms will aid in the design of therapeutics against HPVs and ultimately help prevent many cancers. In this chapter, we describe the HPV binding assay to Ln-332/integrin α6β4 on human keratinocytes (ECM). We also present data and suggestions for modifying the assay for testing the specificity of HPV for receptors (by blocking receptors) and binding to human tissues (basement membrane, BM) in order to study binding mechanisms.

High Throughput Determination of Critical Human Dosing ...

EPA Pesticide Factsheets

High throughput toxicokinetics (HTTK) is a rapid approach that uses in vitro data to estimate TK for hundreds of environmental chemicals. Reverse dosimetry (i.e., reverse toxicokinetics or RTK) based on HTTK data converts high throughput in vitro toxicity screening (HTS) data into predicted human equivalent doses that can be linked with biologically relevant exposure scenarios. Thus, HTTK provides essential data for risk prioritization for thousands of chemicals that lack TK data. One critical HTTK parameter that can be measured in vitro is the unbound fraction of a chemical in plasma (Fub). However, for chemicals that bind strongly to plasma, Fub is below the limits of detection (LOD) for high throughput analytical chemistry, and therefore cannot be quantified. A novel method for quantifying Fub was implemented for 85 strategically selected chemicals: measurement of Fub was attempted at 10%, 30%, and 100% of physiological plasma concentrations using rapid equilibrium dialysis assays. Varying plasma concentrations instead of chemical concentrations makes high throughput analytical methodology more likely to be successful. Assays at 100% plasma concentration were unsuccessful for 34 chemicals. For 12 of these 34 chemicals, Fub could be quantified at 10% and/or 30% plasma concentrations; these results imply that the assay failure at 100% plasma concentration was caused by plasma protein binding for these chemicals. Assay failure for the remaining 22 chemicals may

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

PubMed Central

Murphy, James M.; Zhang, Qingwei; Young, Samuel N.; Reese, Michael L.; Bailey, Fiona P.; Eyers, Patrick A.; Ungureanu, Daniela; Hammaren, Henrik; Silvennoinen, Olli; Varghese, Leila N.; Chen, Kelan; Tripaydonis, Anne; Jura, Natalia; Fukuda, Koichi; Qin, Jun; Nimchuk, Zachary; Mudgett, Mary Beth; Elowe, Sabine; Gee, Christine L.; Liu, Ling; Daly, Roger J.; Manning, Gerard; Babon, Jeffrey J.; Lucet, Isabelle S.

2017-01-01

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains. PMID:24107129

RECOMBINANT ANDROGEN RECEPTOR (AR) BINDING ACROSS VERTEBRATE SPECIES: COMPARISON OF BINDING OF ENVIRONMENTAL COMPOUNDS TO HUMAN, RAINBOW TROUT AND FATHEAD MINNOW AR.

EPA Science Inventory

In vitro screening assays designed to identify androgen mimics or antagonists typically use mammalian (rat, human) androgen receptors (AR). Although the amino acid sequences of receptors from nonmammalian vertebrates are not identical to the mammalian receptors, it is uncertain ...

Muscarinic and alpha 1-adrenergic receptor binding characteristics of saw palmetto extract in rat lower urinary tract.

PubMed

Suzuki, Mayumi; Oki, Tomomi; Sugiyama, Tomomi; Umegaki, Keizo; Uchida, Shinya; Yamada, Shizuo

2007-06-01

To elucidate the in vitro and ex vivo effects of saw palmetto extract (SPE) on autonomic receptors in the rat lower urinary tract. The in vitro binding affinities for alpha 1-adrenergic, muscarinic, and purinergic receptors in the rat prostate and bladder were measured by radioligand binding assays. Rats received vehicle or SPE (0.6 to 60 mg/kg/day) orally for 4 weeks, and alpha 1-adrenergic and muscarinic receptor binding in tissues of these rats were measured. Saw palmetto extract inhibited specific binding of [3H]prazosin and [N-methyl-3H]scopolamine methyl chloride (NMS) but not alpha, beta-methylene adenosine triphosphate [2,8-(3)H]tetrasodium salt in the rat prostate and bladder. The binding activity of SPE for muscarinic receptors was four times greater than that for alpha 1-adrenergic receptors. Scatchard analysis revealed that SPE significantly reduced the maximal number of binding sites (Bmax) for each radioligand in the prostate and bladder under in vitro condition. Repeated oral administration of SPE to rats brought about significant alteration in Bmax for prostatic [3H]prazosin binding and for bladder [3H]NMS binding. Such alteration by SPE was selective to the receptors in the lower urinary tract. Saw palmetto extract exerts significant binding activity on autonomic receptors in the lower urinary tract under in vitro and in vivo conditions.

Comprehensive analysis of T cell epitope discovery strategies using 17DD yellow fever virus structural proteins and BALB/c (H2d) mice model.

PubMed

Maciel, Milton; Kellathur, Srinivasan N; Chikhlikar, Pryia; Dhalia, Rafael; Sidney, John; Sette, Alessandro; August, Thomas J; Marques, Ernesto T A

2008-08-15

Immunomics research uses in silico epitope prediction, as well as in vivo and in vitro approaches. We inoculated BALB/c (H2d) mice with 17DD yellow fever vaccine to investigate the correlations between approaches used for epitope discovery: ELISPOT assays, binding assays, and prediction software. Our results showed a good agreement between ELISPOT and binding assays, which seemed to correlate with the protein immunogenicity. PREDBALB/c prediction software partially agreed with the ELISPOT and binding assay results, but presented low specificity. The use of prediction software to exclude peptides containing no epitopes, followed by high throughput screening of the remaining peptides by ELISPOT, and the use of MHC-biding assays to characterize the MHC restrictions demonstrated to be an efficient strategy. The results allowed the characterization of 2 MHC class I and 17 class II epitopes in the envelope protein of the YF virus in BALB/c (H2d) mice.

Integrated Model of Chemical Perturbations of a Biological Pathway Using 18 In Vitro High-Throughput Screening Assays for the Estrogen Receptor.

PubMed

Judson, Richard S; Magpantay, Felicia Maria; Chickarmane, Vijay; Haskell, Cymra; Tania, Nessy; Taylor, Jean; Xia, Menghang; Huang, Ruili; Rotroff, Daniel M; Filer, Dayne L; Houck, Keith A; Martin, Matthew T; Sipes, Nisha; Richard, Ann M; Mansouri, Kamel; Setzer, R Woodrow; Knudsen, Thomas B; Crofton, Kevin M; Thomas, Russell S

2015-11-01

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation, and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform ("assay interference"). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 111 (6.1%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in vivo endocrine testing. Finally, this model is generalizable to any molecular pathway for which there are multiple upstream and downstream assays available. Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.

Integrated Model of Chemical Perturbations of a Biological ...

EPA Pesticide Factsheets

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform (“”assay interference”). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 52 (2.8%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in v

Aptamers and methods for their in vitro selection and uses thereof

DOEpatents

Doyle, Sharon A [Walnut Creek, CA; Murphy, Michael B [Severna Park, MD

2008-02-12

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

Aptamers and methods for their in vitro selection and uses thereof

DOEpatents

Doyle, Sharon A [Walnut Creek, CA; Murphy, Michael B [Severna Park, MD

2012-01-31

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

Binding of environmental carcinogens to asbestos and mineral fibres.

PubMed Central

Harvey, G; Pagé, M; Dumas, L

1984-01-01

A rapid method has been developed for measuring the binding capacity of asbestos and other mineral fibres for environmental carcinogens. Benzo(alpha)pyrene (B(alpha)P), nitrosonornicotine (NNN), and N-acetyl-2-aminofluorene (NAAF) were assayed in the presence of Canadian grade 4T30 chrysotile, chrysotile A, amosite, crocidolite, glass microfibres, glasswool, attapulgite, and titanium dioxide. Chrysotile binds significantly more carcinogens than the other mineral fibres. This binding assay is reproducible with coefficients of variation of less than 8% and 6% respectively for inter and intra assay. The influence of pH was also studied, and there is good correlation between the carcinogen binding and the charge of the tested mineral fibres. The in vitro cytotoxicity on macrophage like cell line P388D1 and the haemolytic activity of various mineral fibres were also measured; a good correlation was found between the binding capacity and the cytotoxicity of tested mineral fibres on P388D1 cells. These results give some explanations for the reported synergism between exposure to asbestos and the smoking habits of workers. PMID:6331497

THE DEVELOPMENT OF AN IN VITRO ASSAY FOR EVALUATING THE BINDING OF PERFLUOROALKYL ACIDS (PFAAS) TO THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARS)

EPA Science Inventory

The purpose of this work was to evaluate the binding of PFAAs to PPAR receptors and determine the potential for activation or antagonism of the pathway during embryonic development. Activation of mouse and human PPAR isoforms by perfluorooctanoic acid (PFOA) and perfluorooctanes...

In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality

PubMed Central

Yu, Haixiang; Yang, Weijuan; Alkhamis, Obtin; Canoura, Juan; Yang, Kyung-Ae; Xiao, Yi

2018-01-01

Abstract Aptamer-based sensors offer a powerful tool for molecular detection, but the practical implementation of these biosensors is hindered by costly and laborious sequence engineering and chemical modification procedures. We report a simple strategy for directly isolating signal-reporting aptamers in vitro through systematic evolution of ligands by exponential enrichment (SELEX) that transduce binding events into a detectable change of absorbance via target-induced displacement of a small-molecule dye. We first demonstrate that diethylthiatricarbocyanine (Cy7) can stack into DNA three-way junctions (TWJs) in a sequence-independent fashion, greatly altering the dye's absorbance spectrum. We then design a TWJ-containing structured library and isolate an aptamer against 3,4-methylenedioxypyrovalerone (MDPV), a synthetic cathinone that is an emerging drug of abuse. This aptamer intrinsically binds Cy7 within its TWJ domain, but MDPV efficiently displaces the dye, resulting in a change in absorbance within seconds. This assay is label-free, and detects nanomolar concentrations of MDPV. It also recognizes other synthetic cathinones, offering the potential to detect newly-emerging designer drugs, but does not detect structurally-similar non-cathinone compounds or common cutting agents. Moreover, we demonstrate that the Cy7-displacement colorimetric assay is more sensitive than a conventional strand-displacement fluorescence assay. We believe our strategy offers an effective generalized approach for the development of sensitive dye-displacement colorimetric assays for other small-molecule targets. PMID:29361056

The transcription factor CCAAT-binding factor CBF/NF-Y regulates the proximal promoter activity in the human alpha 1(XI) collagen gene (COL11A1).

PubMed

Matsuo, Noritaka; Yu-Hua, Wang; Sumiyoshi, Hideaki; Sakata-Takatani, Keiko; Nagato, Hitoshi; Sakai, Kumiko; Sakurai, Mami; Yoshioka, Hidekatsu

2003-08-29

We have characterized the proximal promoter region of the human COL11A1 gene. Transient transfection assays indicate that the segment from -199 to +1 is necessary for the activation of basal transcription. Electrophoretic mobility shift assays (EMSAs) demonstrated that the ATTGG sequence, within the -147 to -121 fragment, is critical to bind nuclear proteins in the proximal COL11A1 promoter. We demonstrated that the CCAAT binding factor (CBF/NF-Y) bound to this region using an interference assay with consensus oligonucleotides and a supershift assay with specific antibodies in an EMSA. In a chromatin immunoprecipitation assay and EMSA using DNA-affinity-purified proteins, CBF/NF-Y proteins directly bound this region in vitro and in vivo. We also showed that four tandem copies of the CBF/NF-Y-binding fragment produced higher transcriptional activity than one or two copies, whereas the absence of a CBF/NF-Y-binding fragment suppressed the COL11A1 promoter activity. Furthermore, overexpression of a dominant-negative CBF-B/NF-YA subunit significantly inhibited promoter activity in both transient and stable cells. These results indicate that the CBF/NF-Y proteins regulate the transcription of COL11A1 by directly binding to the ATTGG sequence in the proximal promoter region.

Lactobacillus acidophilus binds to MUC3 component of cultured intestinal epithelial cells with highest affinity.

PubMed

Das, Jugal Kishore; Mahapatra, Rajani Kanta; Patro, Shubhransu; Goswami, Chandan; Suar, Mrutyunjay

2016-04-01

Lactobacillus strains have been shown to adhere to the mucosal components of intestinal epithelial cells. However, established in vitro adhesion assays have several drawbacks in assessing the adhesion of new Lactobacillus strains. The present study aimed to compare the adhesion of four different Lactobacillus strains and select the most adherent microbe, based on in silico approach supported by in vitro results. The mucus-binding proteins in Lactobacillus acidophilus, L. plantarum, L. brevis and L. fermentum were identified and their capacities to interact with intestinal mucin were compared by molecular docking analysis. Lactobacillus acidophilus had the maximal affinity of binding to mucin with predicted free energy of -6.066 kcal mol(-1) Further, in vitro experimental assay of adhesion was performed to validate the in silico results. The adhesion of L. acidophilus to mucous secreting colon epithelial HT-29 MTX cells was highest at 12%, and it formed biofilm with maximum depth (Z = 84 μm). Lactobacillus acidophilus was determined to be the most adherent strain in the study. All the Lactobacillus strains tested in this study, displayed maximum affinity of binding to MUC3 component of mucus as compared to other gastrointestinal mucins. These findings may have importance in the design of probiotics and health care management. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

3,3',5-Triiodo-L-thyronine-like activity in effluents from domestic sewage treatment plants detected by in vitro and in vivo bioassays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murata, Tomonori; Yamauchi, Kiyoshi

2008-02-01

Thyroid system-disrupting activity in effluents from municipal domestic sewage treatment plants was detected using three in vitro assays and one in vivo assay. Contaminants in the effluents were extracted by solid-phase extraction (SPE) and eluted stepwise with different organic solvents. The majority of the thyroid system-disrupting activity was detected in the dichloromethane/methanol (1/1) fraction after SPE in all three in vitro assays: competitive assays of 3,3',5-[{sup 125}I]triiodo-L-thyronine ([{sup 125}I]T{sub 3}) binding to the plasma protein transthyretin (TTR assay) and thyroid hormone receptor (TR assay) and T{sub 3}-dependent luciferase assay (Luc assay). Subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) of the dichloromethane/methanolmore » (1/1) fraction separated contaminants potent in the TR and Luc assays from those potent in the TTR assay. The contaminants potent in the TR and Luc assays were also potent in an in vivo short-term gene expression assay in Xenopus laevis (Tadpole assay). The present study demonstrated that the effluents from domestic sewage treatment plants contain contaminants with T{sub 3}-like activity of {approx} 10{sup -10} M T{sub 3}-equivalent concentration (T{sub 3}EQ) and that the TR and Luc assays are powerful in vitro bioassays for detecting thyroid system-disrupting activity in effluents. The availability and applicability of these bioassays for screening contaminants with thyroid system-disrupting activity in the water environment are discussed.« less

Ca(2+)-triggered coelenterazine-binding protein from Renilla as an enzyme-dependent label for binding assay.

PubMed

Krasitskaya, V V; Korneeva, S I; Kudryavtsev, A N; Markova, S V; Stepanyuk, G A; Frank, L A

2011-11-01

The recombinant Ca(2+)-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems.

Vaccination of rhesus macaques with the anthrax vaccine adsorbed vaccine produces a serum antibody response that effectively neutralizes receptor-bound protective antigen in vitro.

PubMed

Clement, Kristin H; Rudge, Thomas L; Mayfield, Heather J; Carlton, Lena A; Hester, Arelis; Niemuth, Nancy A; Sabourin, Carol L; Brys, April M; Quinn, Conrad P

2010-11-01

Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx). They have separate effector proteins (edema factor and lethal factor) but have the same binding protein, protective antigen (PA). PA is the primary immunogen in the current licensed vaccine anthrax vaccine adsorbed (AVA [BioThrax]). AVA confers protective immunity by stimulating production of ATx-neutralizing antibodies, which could block the intoxication process at several steps (binding of PA to the target cell surface, furin cleavage, toxin complex formation, and binding/translocation of ATx into the cell). To evaluate ATx neutralization by anti-AVA antibodies, we developed two low-temperature LTx neutralization activity (TNA) assays that distinguish antibody blocking before and after binding of PA to target cells (noncomplexed [NC] and receptor-bound [RB] TNA assays). These assays were used to investigate anti-PA antibody responses in AVA-vaccinated rhesus macaques (Macaca mulatta) that survived an aerosol challenge with Bacillus anthracis Ames spores. Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation anthrax vaccines.

DNA Length Modulates the Affinity of Fragments of Genomic DNA for the Nuclear Matrix In Vitro.

PubMed

García-Vilchis, David; Aranda-Anzaldo, Armando

2017-12-01

Classical observations have shown that during the interphase the chromosomal DNA of metazoans is organized in supercoiled loops attached to a compartment known as the nuclear matrix (NM). Fragments of chromosomal DNA able to bind the isolated NM in vitro are known as matrix associated/attachment/addressed regions or MARs. No specific consensus sequence or motif has been found that may constitute a universal, defining feature of MARs. On the other hand, high-salt resistant DNA-NM interactions in situ define true DNA loop anchorage regions or LARs, that might correspond to a subset of the potential MARs but are not necessarily identical to MARs characterized in vitro, since there are several examples of MARs able to bind the NM in vitro but which are not actually bound to the NM in situ. In the present work we assayed the capacity of two LARs, as well as of shorter fragments within such LARs, for binding to the NM in vitro. Paradoxically the isolated (≈2 kb) LARs cannot bind to the NM in vitro while their shorter (≈300 pb) sub-fragments and other non-related but equally short DNA fragments, bind to the NM in a high-salt resistant fashion. Our results suggest that the ability of a given DNA fragment for binding to the NM in vitro primarily depends on the length of the fragment, suggesting that binding to the NM is modulated by the local topology of the DNA fragment in suspension that it is known to depend on the DNA length. J. Cell. Biochem. 118: 4487-4497, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

PubMed Central

Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

2013-01-01

WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

Noni as an anxiolytic and sedative: a mechanism involving its gamma-aminobutyric acidergic effects.

PubMed

Deng, S; West, B J; Palu, A K; Zhou, B-N; Jensen, C J

2007-08-01

Noni (Morinda citrifolia) is increasing in worldwide popularity as a food or dietary supplement with versatile health benefits. The aim of this study was to investigate the effects of Noni fruit on anxiety symptoms in vitro. To this end, a competitive GABAa receptor-binding assay was developed. Our preliminary study indicates that the methanol crude extract of Noni fruit showed significant affinity to the gamma-aminobutyric acid A (GABAa) inhibitory neurotransmitter receptors, and displayed 75% binding inhibition of the agonist radioligand [3H] muscimol at a concentration of 100 microg/ml. Further experiments demonstrated that the MeOH extract, and its BuOH and H2O partitions, exhibited IC50 values of 22.8, 27.2, and 17.1 microg/ml, respectively, in the GABAa-binding assay. Experimental results with Noni fruit indicate the presence of competitive ligand(s), which may bind to the GABAa receptor as an agonist, and thus induce its anxiolytic and sedative effects. The study provides an in vitro rationale for one of Noni's versatile and traditional uses. In addition, an HPLC fingerprint profile of the methanolic extract of Noni fruit has been established for quality control purpose.

dsRNA binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35.

PubMed

Zinzula, Luca; Esposito, Francesca; Pala, Daniela; Tramontano, Enzo

2012-03-01

The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)-dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC(50) value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K(d) values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35-dsRNA binding functions. Copyright © 2012 Elsevier B.V. All rights reserved.

The MTA family proteins as novel histone H3 binding proteins.

PubMed

Wu, Meng; Wang, Lina; Li, Qian; Li, Jiwen; Qin, Jun; Wong, Jiemin

2013-01-03

The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined. In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail.

The MTA family proteins as novel histone H3 binding proteins

PubMed Central

2013-01-01

Background The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined. Results In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Conclusions Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail. PMID:23286669

Assessment of phosphate binding by sevelamer carbonate powder for oral suspension mixed in foods.

PubMed

Hanus, Martin; Zhorov, Eugene; Brommage, Deborah; Plone, Melissa; Holmes-Farley, Stephen Randall

2012-01-01

This study investigated mixing sevelamer carbonate powder with foods and beverages other than water. Food samples, including applesauce, oatmeal, chicken, protein powder, scrambled eggs, ginger ale, and diet ginger ale, were subjected to an in vitro assay, and the difference in the amount of phosphate bound between samples pre-exposed to foods and samples where the drug was exposed to foods concurrently was determined Under these assay conditions, pre-exposure to sevelamer carbonate powder had no effect on the ability to bind phosphate. Clinical testing is needed to further evaluate this finding.

Human monoclonal antibody homodimers. Effect of valency on in vitro and in vivo antibacterial activity.

PubMed

Wolff, E A; Esselstyn, J; Maloney, G; Raff, H V

1992-04-15

Human IgG1 mAb dimers specific for either group B streptococci or Escherichia coli K1 bacteria were formed using chemical cross-linkers. The effect of antibody valency on biologic efficacy was investigated by comparing the IgG dimers against the corresponding IgG monomers. Binding activity and relative avidity were assessed using Ag binding and competition ELISA, and functional activity was analyzed using opsonophagocytic assays. These in vitro assays revealed that the dimers were greater than or equal to 50-fold more active than the monomers. A neonatal rat infection model showed the in vivo protective efficacy of the dimers was greater than or equal to 20-fold greater than that of the monomers. Enhancing the activity of mAb by chemical cross-linking may be a useful strategy for salvaging low affinity IgG mAb that possess poor functional properties.

Manuka honey inhibits adhesion and invasion of medically important wound bacteria in vitro.

PubMed

Maddocks, Sarah Elizabeth; Jenkins, Rowena Eleri; Rowlands, Richard Samuel; Purdy, Kevin John; Cooper, Rose Agnes

2013-12-01

To characterize the effect of manuka honey on medically important wound bacteria in vitro, focusing on its antiadhesive properties. Crystal violet biofilm assays, fluorescent microscopy, protein adhesion assay and gentamicin protection assay were used to determine the impact of manuka honey on biofilm formation, human protein binding and adherence to/invasion into human keratinocytes. Manuka honey effectively disrupted and caused extensive cell death in biofilms of Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes. Sublethal doses of manuka honey inhibited bacterial adhesion to the fibronectin, fibrinogen and collagen. Manuka honey impaired adhesion of laboratory and clinical isolates of S. aureus, P. aeruginosa and S. pyogenes to human keratinocytes in vitro, and inhibited invasion by S. pyogenes and homogeneous vancomycin intermediate S. aureus. Manuka honey can directly affect bacterial cells embedded in a biofilm and exhibits antiadhesive properties against three common wound pathogens.

Laboratory Testing for von Willebrand Disease: The Past, Present, and Future State of Play for von Willebrand Factor Assays that Measure Platelet Binding Activity, with or without Ristocetin.

PubMed

Just, Sarah

2017-02-01

von Willebrand disease (VWD) was first described nearly a century ago in 1924 by Erik Adolf von Willebrand. Diagnostic testing at the time was very limited and it was not until the mid to late 1900s that more tests became available to assist with the diagnosis and classification of VWD. Two of these tests are based on ristocetin, one being ristocetin-induced platelet aggregation (RIPA) and the other the von Willebrand factor (VWF) ristocetin cofactor assay (VWF:RCo). The VWF:RCo assay provides functional assessment of in vitro VWF binding to the platelet glycoprotein (Gp) complex, GPIb-IX-V. Despite some advancements and newer technologies utilizing the principles of the original VWF:RCo assay, the original assay is still referred to as the gold standard for measurement of VWF activity. This article will review the history of VWD diagnostic assays, including RIPA and VWF:RCo over the past 40 years, as well as the newer assays that measure platelet binding with or without ristocetin, and which have been developed with the aim to potentially replace platelet-based ristocetin-dependent assays. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Novel Multiplexed Assay for Identifying SH2 Domain Antagonists of STAT Family Proteins

PubMed Central

Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z’ values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors. PMID:23977103

Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

PubMed

Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

Oleamide activates peroxisome proliferator-activated receptor gamma (PPARγ) in vitro.

PubMed

Dionisi, Mauro; Alexander, Stephen P H; Bennett, Andrew J

2012-05-14

Oleamide (ODA) is a fatty acid primary amide first identified in the cerebrospinal fluid of sleep-deprived cats, which exerts effects on vascular and neuronal tissues, with a variety of molecular targets including cannabinoid receptors and gap junctions. It has recently been reported to exert a hypolipidemic effect in hamsters. Here, we have investigated the nuclear receptor family of peroxisome proliferator-activated receptors (PPARs) as potential targets for ODA action. Activation of PPARα, PPARβ and PPARγ was assessed using recombinant expression in Chinese hamster ovary cells with a luciferase reporter gene assay. Direct binding of ODA to the ligand binding domain of each of the three PPARs was monitored in a cell-free fluorescent ligand competition assay. A well-established assay of PPARγ activity, the differentiation of 3T3-L1 murine fibroblasts into adipocytes, was assessed using an Oil Red O uptake-based assay. ODA, at 10 and 50 μM, was able to transactivate PPARα, PPARβ and PPARγ receptors. ODA bound to the ligand binding domain of all three PPARs, although complete displacement of fluorescent ligand was only evident for PPARγ, at which an IC50 value of 38 μM was estimated. In 3T3-L1 cells, ODA, at 10 and 20 μM, induced adipogenesis. We have, therefore, identified a novel site of action of ODA through PPAR nuclear receptors and shown how ODA should be considered as a weak PPARγ ligand in vitro.

Surface Display of the Receptor-Binding Region of the Lactobacillus brevis S-Layer Protein in Lactococcus lactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells

PubMed Central

Åvall-Jääskeläinen, Silja; Lindholm, Agneta; Palva, Airi

2003-01-01

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium. PMID:12676705

Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells.

PubMed

Avall-Jääskeläinen, Silja; Lindholm, Agneta; Palva, Airi

2003-04-01

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.

Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs.

PubMed

Allevato, Michael; Bolotin, Eugene; Grossman, Mark; Mane-Padros, Daniel; Sladek, Frances M; Martinez, Ernest

2017-01-01

The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX) bind Enhancer box (E-box) DNA elements (CANNTG) and have the greatest affinity for the canonical MYC E-box (CME) CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a "non-specific" fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87%) of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

Novel multiple opioid ligands based on 4-aminobenzazepinone (Aba), azepinoindole (Aia) and tetrahydroisoquinoline (Tic) scaffolds

PubMed Central

Ballet, Steven; Marczak, Ewa D.; Feytens, Debby; Salvadori, Severo; Sasaki, Yusuke; Abell, Andrew D.; Lazarus, Lawrence H.; Balboni, Gianfranco; Tourwé, Dirk

2010-01-01

The dimerization and trimerization of the Dmt-Tic, Dmt-Aia and Dmt-Aba pharmacophores provided multiple ligands which were evaluated in vitro for opioid receptor binding and functional activity. Whereas the Tic- and Aba multimers proved to be dual and balanced δ/μ antagonists, as determined by the functional [S35]GTPγS binding assay, the dimerization of potent Aia-based ‘parent’ ligands unexpectedly resulted in substantial less efficient receptor binding and non-active dimeric compounds. PMID:20137938

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taxvig, Camilla, E-mail: camta@food.dtu.dk; Olesen, Pelle Thonning; Nellemann, Christine

2011-02-01

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenousmore » metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.« less

In Silico Prediction and In Vitro Characterization of Multifunctional Human RNase3

PubMed Central

Kuo, Ping-Hsueh; Chen, Chien-Jung; Chang, Hsiu-Hui; Fang, Shun-lung; Wu, Wei-Shuo; Lai, Yiu-Kay; Pai, Tun-Wen; Chang, Margaret Dah-Tsyr

2013-01-01

Human ribonucleases A (hRNaseA) superfamily consists of thirteen members with high-structure similarities but exhibits divergent physiological functions other than RNase activity. Evolution of hRNaseA superfamily has gained novel functions which may be preserved in a unique region or domain to account for additional molecular interactions. hRNase3 has multiple functions including ribonucleolytic, heparan sulfate (HS) binding, cellular binding, endocytic, lipid destabilization, cytotoxic, and antimicrobial activities. In this study, three putative multifunctional regions, 34RWRCK38 (HBR1), 75RSRFR79 (HBR2), and 101RPGRR105 (HBR3), of hRNase3 have been identified employing in silico sequence analysis and validated employing in vitro activity assays. A heparin binding peptide containing HBR1 is characterized to act as a key element associated with HS binding, cellular binding, and lipid binding activities. In this study, we provide novel insights to identify functional regions of hRNase3 that may have implications for all hRNaseA superfamily members. PMID:23484086

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

PubMed Central

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

PubMed

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

Efficient Identification of Novel Hla-A*0201–Presented Cytotoxic T Lymphocyte Epitopes in the Widely Expressed Tumor Antigen Prame by Proteasome-Mediated Digestion Analysis

PubMed Central

Kessler, Jan H.; Beekman, Nico J.; Bres-Vloemans, Sandra A.; Verdijk, Pauline; van Veelen, Peter A.; Kloosterman-Joosten, Antoinette M.; Vissers, Debby C.J.; ten Bosch, George J.A.; Kester, Michel G.D.; Sijts, Alice; Drijfhout, Jan Wouter; Ossendorp, Ferry; Offringa, Rienk; Melief, Cornelis J.M.

2001-01-01

We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A*0201–presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved “reverse immunology” strategy. Next to motif-based HLA-A*0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A*0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA100–108; SLYSFPEPEA, PRA142–151; ALYVDSLFFL, PRA300–309; and SLLQHLIGL, PRA425–433) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A*0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer. PMID:11136822

Impact of cadmium, cobalt and nickel on sequence-specific DNA binding of p63 and p73 in vitro and in cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adámik, Matej; Bažantová, Pavla; Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 701 03 Ostrava

Highlights: • DNA binding of p53 family core domains is inhibited by cadmium, cobalt and nickel. • Binding to DNA protects p53 family core domains from metal induced inhibition. • Cadmium, cobalt and nickel induced inhibition was reverted by EDTA in vitro. - Abstract: Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt,more » which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50 μM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA–protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA–p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed.« less

Brittle Culm1, a COBRA-Like Protein, Functions in Cellulose Assembly through Binding Cellulose Microfibrils

PubMed Central

Zhang, Baocai; Liu, Xiangling; Yan, Meixian; Zhang, Lanjun; Shi, Yanyun; Zhang, Mu; Qian, Qian; Li, Jiayang; Zhou, Yihua

2013-01-01

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity. PMID:23990797

Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils.

PubMed

Liu, Lifeng; Shang-Guan, Keke; Zhang, Baocai; Liu, Xiangling; Yan, Meixian; Zhang, Lanjun; Shi, Yanyun; Zhang, Mu; Qian, Qian; Li, Jiayang; Zhou, Yihua

2013-01-01

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.

SH2 Binding Site Protection Assay: A Method for Identification of SH2 Domain Interaction Partners by Exploiting SH2 Mediated Phosphosite Protection.

PubMed

Jadwin, Joshua A

2017-01-01

Over the last two decades there has been a significant effort in the field to characterize the phosphosite binding specificities of SH2 domains with the goal of deciphering the pY signaling code. Although high throughput studies in various formats using most SH2 domains have collectively provided a rich resource of in vitro SH2-pTyr site specificity maps, this data can only be used approximate what is happening in the cell where protein concentrations and localization are not homogenous, as they are for in vitro experiments. Here we describe an in vivo approach, SH2 site protection assay, which can capture the pTyr binding specificity of SH2 domains in the cell. The basis of this approach is SH2-pY site protection, the ability of SH2 domains to prevent the PTP-dependent dephosphorylation of their pY site binding partners. We overexpress a tracer SH2 domain in cells and quantify the change in abundance of tyrosine phosphorylated sites using MS. Since the method is performed in vivo, it has the advantage of identifying SH2-pY interactions as they occur within in the cell.

Bisphenol A alternatives bisphenol S and bisphenol F interfere with thyroid hormone signaling pathway in vitro and in vivo.

PubMed

Zhang, Yin-Feng; Ren, Xiao-Min; Li, Yuan-Yuan; Yao, Xiao-Fang; Li, Chuan-Hai; Qin, Zhan-Fen; Guo, Liang-Hong

2018-06-01

The wide use of the alternatives to bisphenol A (BPA) has raised concerns about their potential toxicities. Considering the disrupting activity of BPA on thyroid hormone (TH) signaling, we investigated whether bisphenol S (BPS) and bisphenol F (BPF), two leading alternatives, could interfere with TH signaling pathway using a series of assays in vitro and in vivo. In the fluorescence competitive binding assay, we found BPS and BPF, like BPA, bound to TH receptors (TRα and TRβ), with the binding potencies an order of magnitude lower than BPA (BPA > BPF > BPS). Molecular docking data also show their binding potencies to TRs. In the coactivator recruitment assay, BPS and BPF recruited coactivator to TRβ but not TRα, with weaker potencies than BPA. Correspondingly, agonistic actions of the three bisphenols in the absence or presence of T3 were observed in the TR-mediated reporter gene transcription assay. Also, all the three bisphenols induced TH-dependent GH3 cell proliferation, whereas BPA and BPF inhibited T3 induction in the presence of T3. As for in vivo assay, the three bisphenols like T3 induced TH-response gene transcription in Pelophylax nigromaculatus tadpoles, but in the presence of T3 altered T3-induced gene transcription in a biphasic concentration-response manner. These results for the first time demonstrate that BPS and BPF, like BPA, have potential to interfere with TH signaling pathway, i.e., they generally activate TH signaling in the absence of T3, but in the presence of TH, display agonistic or/and antagonistic actions under certain condition. Our study highlights the potential risks of BPS and BPF as BPA alternatives. Copyright © 2017 Elsevier Ltd. All rights reserved.

An assay to image neuronal microtubule dynamics in mice.

PubMed

Kleele, Tatjana; Marinković, Petar; Williams, Philip R; Stern, Sina; Weigand, Emily E; Engerer, Peter; Naumann, Ronald; Hartmann, Jana; Karl, Rosa M; Bradke, Frank; Bishop, Derron; Herms, Jochen; Konnerth, Arthur; Kerschensteiner, Martin; Godinho, Leanne; Misgeld, Thomas

2014-09-12

Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.

Streptomyces coelicolor SCO4226 Is a Nickel Binding Protein

PubMed Central

Jin, Hua; Zhang, Rong-Guang; Virolle, Marie-Joelle; Chen, Yuxing; Zhou, Cong-Zhao

2014-01-01

The open reading frame SCO4226 of Streptomyces coelicolor A3(2) encodes an 82-residue hypothetical protein. Biochemical assays revealed that each SCO4226 dimer binds four nickel ions. To decipher the molecular function, we solved the crystal structures of SCO4226 in both apo- and nickel-bound (Ni-SCO4226) forms at 1.30 and 2.04 Å resolution, respectively. Each subunit of SCO4226 dimer adopts a canonical ferredoxin-like fold with five β-strands flanked by two α-helices. In the structure of Ni-SCO4226, four nickel ions are coordinated at the surface of the dimer. Further biochemical assays suggested that the binding of Ni2+ triggers the self-aggregation of SCO4226 in vitro. In addition, RT-qPCR assays demonstrated that the expression of SCO4226 gene in S. coelicolor is specifically up-regulated by the addition of Ni2+, but not other divalent ions such as Cu2+, Mn2+ or Co2+. All these results suggested that SCO4226 acts as a nickel binding protein, probably required for nickel sequestration and/or detoxification. PMID:25285530

Deformability in the cleavage site of primary microRNA is not sensed by the double-stranded RNA binding domains in the microprocessor component DGCR8.

PubMed

Quarles, Kaycee A; Chadalavada, Durga; Showalter, Scott A

2015-06-01

The prevalence of double-stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA-binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD-containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8-Core, consists of its two dsRBDs and a C-terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N-terminal dsRBD of DGCR8 in isolation nor the DGCR8-Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single-stranded segments flanking the stem. Extending DGCR8-Core to include an N-terminal heme-binding region does not change our conclusions. Thus, our data suggest that although the DGCR8-Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency. © 2015 Wiley Periodicals, Inc.

The minor house dust mite allergen Der p 13 is a fatty acid-binding protein and an activator of a TLR2-mediated innate immune response.

PubMed

Satitsuksanoa, P; Kennedy, M; Gilis, D; Le Mignon, M; Suratannon, N; Soh, W T; Wongpiyabovorn, J; Chatchatee, P; Vangveravong, M; Rerkpattanapipat, T; Sangasapaviliya, A; Piboonpocanun, S; Nony, E; Ruxrungtham, K; Jacquet, A

2016-10-01

The house dust mite (HDM) allergen Der p 13 could be a lipid-binding protein able to activate key innate signaling pathways in the initiation of the allergic response. We investigated the IgE reactivity of recombinant Der p 13 (rDer p 13), its lipid-binding activities, and its capacity to stimulate airway epithelium cells. Purified rDer p 13 was characterized by mass spectrometry, circular dichroism, fluorescence-based lipid-binding assays, and in silico structural prediction. IgE-binding activity and allergenic potential of Der p 13 were examined by ELISA, basophil degranulation assays, and in vitro airway epithelial cell activation assays. Protein modeling and biophysical analysis indicated that Der p 13 adopts a β-barrel structure with a predominately apolar pocket representing a potential binding site for hydrophobic ligands. Fluorescent lipid-binding assays confirmed that the protein is highly selective for ligands and that it binds a fatty acid with a dissociation constant typical of lipid transporter proteins. The low IgE-binding frequency (7%, n = 224) in Thai HDM-allergic patients as well as the limited propensity to activate basophil degranulation classifies Der p 13 as a minor HDM allergen. Nevertheless, the protein with its presumptively associated lipid(s) triggered the production of IL-8 and GM-CSF in respiratory epithelial cells through a TLR2-, MyD88-, NF-kB-, and MAPK-dependent signaling pathway. Although a minor allergen, Der p 13 may, through its lipid-binding capacity, play a role in the initiation of the HDM-allergic response through TLR2 activation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein

PubMed Central

Yamada, Kazunori; Kondoh, Yasumitsu; Hikono, Hirokazu; Osada, Hiroyuki; Tomii, Kentaro; Saito, Takehiko; Aida, Yoko

2015-01-01

Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP. PMID:26222066

EMSA Analysis of DNA Binding By Rgg Proteins

PubMed Central

LaSarre, Breah; Federle, Michael J.

2016-01-01

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function (e.g. interruption of DNA-binding in some cases). PMID:27430004

EMSA Analysis of DNA Binding By Rgg Proteins.

PubMed

LaSarre, Breah; Federle, Michael J

2013-08-20

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function ( e.g. interruption of DNA-binding in some cases).

A Small-Molecule Inhibitor of BCL6 Kills DLBCL Cells In Vitro and In Vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cerchietti, L.C.; Ghetu, A.F.; Zhu, X.

2010-09-22

The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low-molecular-weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was nontoxic and potently suppressed DLBCL tumors in vivo. The compoundmore » also killed primary DLBCLs from human patients.« less

Two antibacterial C-type lectins from crustacean, Eriocheir sinensis, stimulated cellular encapsulation in vitro.

PubMed

Jin, Xing-Kun; Li, Shuang; Guo, Xiao-Nv; Cheng, Lin; Wu, Min-Hao; Tan, Shang-Jian; Zhu, You-Ting; Yu, Ai-Qing; Li, Wei-Wei; Wang, Qun

2013-12-01

The first step of host fighting against pathogens is that pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. In the present study, we investigated how invertebrate pattern recognition receptor (PRR) C-type lectins might be involved in the antimicrobial response in crustacean. Based on our previously obtained completed coding regions of EsLecA and EsLecG in Eriocheir sinensis, the recombinant EsLectin proteins were produced via prokaryotic expression system and affinity chromatography. Subsequently, both rEsLecA and rEsLecG were discovered to have wide spectrum binding activities towards microorganisms, and their microbial-binding was calcium-independent. Moreover, the binding activities of both rEsLecA and rEsLecG induced the aggregation against microbial pathogens. Both microorganism growth inhibitory activities assays and antibacterial activities assays revealed their capabilities of suppressing microorganisms growth and directly killing microorganisms respectively. Furthermore, the encapsulation assays signified that both rEsLecA and rEsLecG could stimulate the cellular encapsulation in vitro. Collectively, data presented here demonstrated the successful expression and purification of two C-type lectins proteins in the Chinese mitten crab, and their critical role in the innate immune system of an invertebrate. Copyright © 2013 Elsevier Ltd. All rights reserved.

Studying the Salt Dependence of the Binding of σ70 and σ32 to Core RNA Polymerase Using Luminescence Resonance Energy Transfer

PubMed Central

Glaser, Bryan T.; Bergendahl, Veit; Anthony, Larry C.; Olson, Brian; Burgess, Richard R.

2009-01-01

The study of protein-protein interactions is becoming increasingly important for understanding the regulation of many cellular processes. The ability to quantify the strength with which two binding partners interact is desirable but the accurate determination of equilibrium binding constants is a difficult process. The use of Luminescence Resonance Energy Transfer (LRET) provides a homogeneous binding assay that can be used for the detection of protein-protein interactions. Previously, we developed an LRET assay to screen for small molecule inhibitors of the interaction of σ70 with theβ' coiled-coil fragment (amino acids 100–309). Here we describe an LRET binding assay used to monitor the interaction of E. coli σ70 and σ32 with core RNA polymerase along with the controls to verify the system. This approach generates fluorescently labeled proteins through the random labeling of lysine residues which enables the use of the LRET assay for proteins for which the creation of single cysteine mutants is not feasible. With the LRET binding assay, we are able to show that the interaction of σ70 with core RNAP is much more sensitive to NaCl than to potassium glutamate (KGlu), whereas the σ32 interaction with core RNAP is insensitive to both salts even at concentrations >500 mM. We also find that the interaction of σ32 with core RNAP is stronger than σ70 with core RNAP, under all conditions tested. This work establishes a consistent set of conditions for the comparison of the binding affinities of the E.coli sigma factors with core RNA polymerase. The examination of the importance of salt conditions in the binding of these proteins could have implications in both in vitro assay conditions and in vivo function. PMID:19649256

Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization

PubMed Central

Johansen, Jesper; Alfaro, Gabriel; Beh, Christopher T.

2016-01-01

Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott—Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization. PMID:27526190

[Methods of testing inactivated antirabies vaccines].

PubMed

Nedosekov, V V; Vishniakov, I F; Gruzdev, K N

2001-01-01

Methods for evaluating the potency of inactivated rabies vaccines are reviewed. Shortcomings of the traditional NIH method and advantages of modern rapid immunological in vitro methods (antibody binding test, radial immunodiffusion test, enzyme linked immunoadsorbent assay) for estimation of antigenic activity of vaccines are discussed.

COMPARISON OF FATHEAD MINNOW AND HUMAN ESTROGEN RECEPTOR BINDING TO ENDOCRINE DISRUPTING COMPOUNDS

EPA Science Inventory

Environmental estrogens have the potential to disrupt endocrine function in a myriad of species. However, in vitro assays designed to detect and characterize endocrine disrupting chemicals (EDCs) typically utilize mammalian estrogen receptors. Our overall objective is to charac...

High throughput screening technologies for ion channels

PubMed Central

Yu, Hai-bo; Li, Min; Wang, Wei-ping; Wang, Xiao-liang

2016-01-01

Ion channels are involved in a variety of fundamental physiological processes, and their malfunction causes numerous human diseases. Therefore, ion channels represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. Chronologically, HTS methods for ion channels include the ligand binding assay, flux-based assay, fluorescence-based assay, and automated electrophysiological assay. In this review we summarize the current HTS technologies for different ion channel classes and their applications. PMID:26657056

Discovery of 5-substituted tetrahydronaphthalen-2yl-methyl with N-phenyl-N-(piperidin-4-yl)propionamide derivatives as potent opioid receptor ligands.

PubMed

Deekonda, Srinivas; Wugalter, Lauren; Kulkarni, Vinod; Rankin, David; Largent-Milnes, Tally M; Davis, Peg; Bassirirad, Neemah M; Lai, Josephine; Vanderah, Todd W; Porreca, Frank; Hruby, Victor J

2015-09-15

A new series of novel opioid ligands have been designed and synthesized based on the 4-anilidopiperidine scaffold containing a 5-substituted tetrahydronaphthalen-2yl)methyl group with different N-phenyl-N-(piperidin-4-yl)propionamide derivatives to study the biological effects of these substituents on μ and δ opioid receptor interactions. Recently our group reported novel 4-anilidopiperidine analogues, in which several aromatic ring-contained amino acids were conjugated with N-phenyl-N-(piperidin-4-yl)propionamide and examined their biological activities at the μ and δ opioid receptors. In continuation of our efforts in these novel 4-anilidopiperidine analogues, we took a peptidomimetic approach in the present design, in which we substituted aromatic amino acids with tetrahydronaphthalen-2yl methyl moiety with amino, amide and hydroxyl substitutions at the 5th position. In in vitro assays these ligands, showed very good binding affinity and highly selective toward the μ opioid receptor. Among these, the lead ligand 20 showed excellent binding affinity (2 nM) and 5000 fold selectivity toward the μ opioid receptor, as well as functional selectivity in GPI assays (55.20 ± 4.30 nM) and weak or no agonist activities in MVD assays. Based on the in vitro bioassay results the lead compound 20 was chosen for in vivo assessment for efficacy in naïve rats after intrathecal administration. Compound 20 was not significantly effective in alleviating acute pain. This discrepancy between high in vitro binding affinity, moderate in vitro activity, and low in vivo activity may reflect differences in pharmacodynamics (i.e., engaging signaling pathways) or pharmacokinetics (i.e., metabolic stability). In sum, our data suggest that further optimization of this compound 20 is required to enhance in vivo activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

An In Vitro TORC1 Kinase Assay That Recapitulates the Gtr-Independent Glutamine-Responsive TORC1 Activation Mechanism on Yeast Vacuoles

PubMed Central

Tanigawa, Mirai

2017-01-01

ABSTRACT Evolutionarily conserved target of rapamycin (TOR) complex 1 (TORC1) responds to nutrients, especially amino acids, to promote cell growth. In the yeast Saccharomyces cerevisiae, various nitrogen sources activate TORC1 with different efficiencies, although the mechanism remains elusive. Leucine, and perhaps other amino acids, was reported to activate TORC1 via the heterodimeric small GTPases Gtr1-Gtr2, the orthologues of the mammalian Rag GTPases. More recently, an alternative Gtr-independent TORC1 activation mechanism that may respond to glutamine was reported, although its molecular mechanism is not clear. In studying the nutrient-responsive TORC1 activation mechanism, the lack of an in vitro assay hinders associating particular nutrient compounds with the TORC1 activation status, whereas no in vitro assay that shows nutrient responsiveness has been reported. In this study, we have developed a new in vitro TORC1 kinase assay that reproduces, for the first time, the nutrient-responsive TORC1 activation. This in vitro TORC1 assay recapitulates the previously predicted Gtr-independent glutamine-responsive TORC1 activation mechanism. Using this system, we found that this mechanism specifically responds to l-glutamine, resides on the vacuolar membranes, and involves a previously uncharacterized Vps34-Vps15 phosphatidylinositol (PI) 3-kinase complex and the PI-3-phosphate [PI(3)P]-binding FYVE domain-containing vacuolar protein Pib2. Thus, this system was proved to be useful for dissecting the glutamine-responsive TORC1 activation mechanism. PMID:28483912

Engineered Single-Chain, Antiparallel, Coiled Coil Mimics the MerR Metal Binding Site

PubMed Central

Song, Lingyun; Caguiat, Jonathan; Li, Zhongrui; Shokes, Jacob; Scott, Robert A.; Olliff, Lynda; Summers, Anne O.

2004-01-01

The repressor-activator MerR that controls transcription of the mercury resistance (mer) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays. The metal-recognition domain of MerR resides at the homodimer interface in a novel antiparallel arrangement of α-helix 5 that forms a coiled-coil motif. To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue α-helix 5 of MerR. The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine structure data showed that MBD has essentially the same Hg(II) coordination environment as MerR. In vivo, cells overexpressing MBD accumulated 70 to 100% more 203Hg(II) than cells bearing the vector alone, without deleterious effects on cell growth. Both MerR and MBD variously bound other thiophilic metal ions, including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We conclude that (i) it is possible to simulate in a single polypeptide chain the in vitro and in vivo metal-binding ability of dimeric, full-length MerR and (ii) MerR's specificity in transcriptional activation does not reside solely in the metal-binding step. PMID:14996817

Galline Ex-FABP is an Antibacterial Siderocalin and a Lysophosphatidic Acid Sensor Functioning through Dual Ligand Specificities

PubMed Central

Correnti, Colin; Clifton, Matthew C.; Abergel, Rebecca J.; Allred, Ben; Hoette, Trisha M.; Ruiz, Mario; Cancedda, Ranieri; Raymond, Kenneth N.; Descalzi, Fiorella; Strong, Roland K.

2011-01-01

SUMMARY Galline Ex-FABP was identified as another candidate antibacterial, catecholate siderophore binding lipocalin (siderocalin) based on structural parallels with the family archetype, mammalian Siderocalin. Binding assays show that Ex-FABP retains iron in a siderophore-dependent manner in both hypertrophic and dedifferentiated chondrocytes, where Ex-FABP expression is induced after treatment with proinflammatory agents, and specifically binds ferric complexes of enterobactin, parabactin, bacillibactin and, unexpectedly, monoglucosylated enterobactin, which does not bind to Siderocalin. Growth arrest assays functionally confirm the bacteriostatic effect of Ex-FABP in vitro under iron-limiting conditions. The 1.8Å crystal structure of Ex-FABP explains the expanded specificity, but also surprisingly reveals an extended, multi-chambered cavity extending through the protein and encompassing two separate ligand specificities, one for bacterial siderophores (as in Siderocalin) at one end and one specifically binding co-purified lysophosphatidic acid, a potent cell signaling molecule, at the other end, suggesting Ex-FABP employs dual functionalities to explain its diverse endogenous activities. PMID:22153502

Purification of ribonucleoproteins by a novel approach: isolation of the SSB1 ribonucleoprotein from yeast and demonstration that it has no role in mRNA splicing.

PubMed

Cusick, M E

1992-12-29

A novel approach is described to purify potential ribonucleoproteins (RNP) of yeast. The method assays a yeast RNP complex, assembled in vitro on actin pre-mRNA, by low-ionic strength acrylamide gel electrophoresis. The minimal protein components of this RNP complex were three proteins, one of 30 kDa and two at 42-44 kDa, defined by formation of the complex on biotinylated-RNA, binding of this complex to avidin-agarose, and salt elution of the protein in the biotinylated-RNP complex. Using the assay for RNP complex formation, an RNP protein was purified to homogeneity on the basis of its affinity towards single-stranded DNA and RNA. This RNP protein turned out to be identical to a known RNP protein, the single-stranded binding protein 1 (ssb1) of yeast, on the basis of identical gel electrophoretic migration, antibody cross-reactivity, and identical properties on the gel complex formation assay. In vitro mRNA splicing was normal in extracts made from a yeast strain missing ssb1 (ssb1- strain). Addition of anti-ssb1 antibody to splicing extracts made from a wild type strain did not inhibit or diminish splicing. Instead, mRNA splicing was reproducibly stimulated several fold, indicating competition between ssb1 and splicing factors for binding to single-stranded RNA in the extracts. RNP complexes still formed in the ssb1- strain, demonstrating that it would be possible to purify other RNP proteins from this strain using the gel complex formation assay.

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay.

PubMed

Taxvig, Camilla; Olesen, Pelle Thonning; Nellemann, Christine

2011-02-01

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects. Copyright © 2010 Elsevier Inc. All rights reserved.

Identification of Escherichia coli enterotoxin inhibitors from traditional medicinal herbs by in silico, in vitro, and in vivo analyses.

PubMed

Chen, Jaw-Chyun; Ho, Tin-Yun; Chang, Yuan-Shiun; Wu, Shih-Lu; Li, Chia-Cheng; Hsiang, Chien-Yun

2009-01-30

Glycyrrhiza uralensis has been used for the treatment of gastrointestinal disorders, such as diarrhea, in several ancient cultures. Glycyrrhizin is the principal component of liquorice and lots of pharmacological effects have been demonstrated. Heat-labile enterotoxin (LT), the virulence factor of enterotoxigenic Escherichia coli, induces diarrhea by initially binding to the GM1 on the surfaces of intestinal epithelial cells and consequently leading to the massive loss of fluid and ions from cells. Therefore, we evaluated the inhibitory effects of traditional medicinal herbs (TMH) on the B subunit of LT (LTB) and GM1 interaction. The inhibitory effects of TMH on LTB-GM1 interaction were evaluated by GM1-enzyme-linked immunosorbent assay (ELISA). The likely active phytochemicals of these TMH were then predicted by in silico model (docking) and analyzed by in vitro (GM1-ELISA) and in vivo (patent mouse gut assay) models. We found that various TMH, which have been ethnomedically used for the treatment of diarrhea, inhibited the LTB-GM1 interaction. Docking data showed that triterpenoids were the most active phytochemicals and the oleanane-type triterpenoids presented better LTB-binding abilities than other types of triterpenoids. Moreover, by in vitro and in vivo models, we demonstrated that glycyrrhizin was the most effective oleanane-type triterpenoid that significantly suppressed both the LTB-binding ability (IC50=3.26+/-0.17 mM) and the LT-induced fluid accumulation in mice. We found an LT inhibitor, glycyrrhizin, from TMH by in silico, in vitro, and in vivo analyses.

Synthesis and in vitro characterization of a P2X7 radioligand [123I]TZ6019 and its response to neuroinflammation in a mouse model of Alzheimer disease.

PubMed

Jin, Hongjun; Han, Junbin; Resing, Derek; Liu, Hui; Yue, Xuyi; Miller, Rebecca L; Schoch, Kathleen M; Miller, Timothy M; Perlmutter, Joel S; Egan, Terrance M; Tu, Zhude

2018-02-05

The purinergic receptor P2X ligand-gated ion channel 7 (P2X7 receptor) is a promising imaging target to detect neuroinflammation. Herein, we report development of a potent iodinated radiotracer for P2X7 receptor, [ 123 I]TZ6019. The radiosynthesis of [ 123 I]TZ6019 was accomplished by allylic-tin precursor iodination using [ 123 I]NaI with good radiochemical yield of 85% and high radiochemical purity of > 99%. Human embryonic kidney 293 (HEK-293) cell line stably transfected with the human P2X7 receptor was used to characterize the binding affinity of TZ6019 by fluorescence, radioactive competitive, and saturation binding assays. A radioligand competitive binding assay with [ 123 I]TZ6019 demonstrated that the nonradioactive compound TZ6019 has an IC 50 value of 9.49 ± 1.4nM, and the known P2X7 receptor compound GSK1482160 has an IC 50 value of 4.30 ± 0.86nM, consistent with previous reports. The radioligand saturation binding assay and competitive assay revealed that [ 123 I]TZ6019 specifically bound to the human P2X7 receptor with high affinity (K i = 6.3 ± 0.9nM). In vitro autoradiography quantification with brain slices collected from 9-month old P301S tau transgenic mice along with wild type controls, revealed higher binding of [ 123 I]TZ6019 (35% increase) in the brain of P301S transgenic mice (n = 3, p = 0.04) compared to wild type controls. The immunofluorescence microscopy confirmed that expression of P2X7 receptor was colocalized with astrocytes in the tauopathy P301S transgenic mice. [ 123 I]TZ6019 has specific binding for P2X7 receptor and has great potential to be a radiotracer for screening new compounds and quantifying expression of P2X7 receptor in neuroinflammation related diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Transcriptional regulation of human MUC4 gene: identification of a novel inhibitory element and its nuclear binding protein.

PubMed

Zhang, Jing-Jing; Zhu, Yi; Zhang, Xiong-Fei; Liang, Wen-Biao; Xie, Kun-Ling; Tao, Jin-Qiu; Peng, Yun-Peng; Xu, Ze-Kuan; Miao, Yi

2013-08-01

The human mucin 4 (MUC4) is aberrantly expressed in pancreatic adenocarcinoma and tumor cell lines, while remaining undetectable in normal pancreas, indicating its important role in pancreatic cancer development. Although its transcriptional regulation has been investigated in considerable detail, some important elements remain unknown. The aim of the present study was to demonstrate the existence of a novel inhibitory element in the MUC4 promoter and characterize some of its binding proteins. By luciferase reporter assay, we located the inhibitory element between nucleotides -2530 and -2521 in the MUC4 promoter using a series of deletion and mutant reporter constructs. Electrophoretic mobility shift assay (EMSA) with Bxpc-3 cell nuclear extracts revealed that one protein or protein complex bind to this element. The proteins binding to this element were purified and identified as Yin Yang 1 (YY1) by mass spectrometry. Supershift assay and chromatin immunoprecipitation (ChIP) assay confirmed that YY1 binds to this element in vitro and in vivo. Moreover, transient YY1 overexpression significantly inhibited MUC4 promoter activity and endogenous MUC4 protein expression. In conclusion, we reported here a novel inhibitory element in the human MUC4 promoter. This provides additional data on MUC4 gene regulation and indicates that YY1 may be a potential target for abnormal MUC4 expression.

Method for estimating protein binding capacity of polymeric systems.

PubMed

Sharma, Vaibhav; Blackwood, Keith A; Haddow, David; Hook, Lilian; Mason, Chris; Dye, Julian F; García-Gareta, Elena

2015-01-01

Composite biomaterials made from synthetic and protein-based polymers are extensively researched in tissue engineering. To successfully fabricate a protein-polymer composite, it is critical to understand how strongly the protein binds to the synthetic polymer, which occurs through protein adsorption. Currently, there is no cost-effective and simple method for characterizing this interfacial binding. To characterize this interfacial binding, we introduce a simple three-step method that involves: 1) synthetic polymer surface characterisation, 2) a quick, inexpensive and robust novel immuno-based assay that uses protein extraction compounds to characterize protein binding strength followed by 3) an in vitro 2D model of cell culture to confirm the results of the immuno-based assay. Fibrinogen, precursor of fibrin, was adsorbed (test protein) on three different polymeric surfaces: silicone, poly(acrylic acid)-coated silicone and poly(allylamine)-coated silicone. Polystyrene surface was used as a reference. Characterisation of the different surfaces revealed different chemistry and roughness. The novel immuno-based assay showed significantly stronger binding of fibrinogen to both poly(acrylic acid) and poly(allylamine) coated silicone. Finally, cell studies showed that the strength of the interaction between the protein and the polymer had an effect on cell growth. This novel immuno-based assay is a valuable tool in developing composite biomaterials of synthetic and protein-based polymers with the potential to be applied in other fields of research where protein adsorption onto surfaces plays an important role.

Kinetic assay for high-throughput screening of in vitro transthyretin amyloid fibrillogenesis inhibitors.

PubMed

Dolado, Ignacio; Nieto, Joan; Saraiva, Maria João M; Arsequell, Gemma; Valencia, Gregori; Planas, Antoni

2005-01-01

Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies, such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay is developed for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose-response curve from which inhibition parameters are calculated. For the assay development, thyroid hormones and nonsteroidal antiinflamatory drugs were chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors.

Light-controlled cellular internalization and cytotoxicity of nucleic acid-binding agents. Studies in vitro and in zebrafish embryos

PubMed Central

Penas, Cristina; Sánchez, Mateo I.; Guerra-Varela, Jorge; Sanchez-Piñón, Laura; Vázquez, M. Eugenio; Mascareñas, José L.

2016-01-01

We have synthesized oligoarginine conjugates of selected DNA-binding agents (a bisbenzamidine, acridine and thiazole orange) and demonstrated that the DNA binding and cell internalization properties of such conjugates can be inhibited by appending a negatively charged oligoglutamic tail through a photolabile linker. Irradiation with UV light releases the parent octaarginine conjugates, thus restoring their cell internalization and biological activity. Preliminary assays using zebrafish embryos demonstrates the potential of this prodrug strategy for controlling in vivo cytotoxicity. PMID:26534774

Comparison of 99mTc-UBI 29-41, 99mTc-Ciprofloxacin, 99mTc-Ciprofloxacin dithiocarbamate and 111In-biotin for targeting experimental Staphylococcus aureus and Escherichia coli foreign-body infections: an ex-vivo study.

PubMed

Auletta, Sveva; Baldoni, Daniela; Varani, Michela; Galli, Filippo; Hajar, Iman A; Duatti, Adriano; Ferro-Flores, Guillermina; Trampuz, Andrej; Signore, Alberto

2017-08-28

Diagnosis of implant-associated infection is challenging. Several radiopharmaceuticals have been described but direct comparisons are limited. Here we compared in vitro and in an animal model 99mTc-UBI, 99mTc-Ciprofloxacin, 99mTcN-CiproCS2 and 111In-DTPA-biotin for targeting E. coli (ATCC 25922) and S. aureus (ATCC 43335). Stability controls were performed with the labelled radiopharmaceuticals during 6 h in saline and serum. The in vitro binding to viable or killed bacteria was evaluated at 37 °C and 4 °C. For in vivo studies, Teflon cages were subcutaneously implanted in mice, followed by percutaneous infection. Biodistribution of i.v. injected radiolabelled radiopharmaceuticals were evaluated during 24 h in cages and dissected tissues. Labelling efficiency of all radiopharmaceuticals ranged between 94% and 98%, with high stability both in saline and in human serum. In vitro binding assays displayed a rapid but poor bacterial binding for all tested agents. Similar binding kinetic occurred also with heat-killed and ethanol-killed bacteria. In the tissue cage model, infection was detected at different time points: 99mTc-UBI and 99mTcN-CiproCS2 showed higher infected cage/sterile cage ratio at 24 h for both E. coli and S. aureus; 99mTc-Ciprofloxacin at 24 h for both E. coli and at 4 h for S. aureus; 111In-DTPA-biotin accumulates faster in both E. coli and S. aureus infected cages. 99mTc-UBI, 99mTcN-CiproCS2 showed poor in vitro binding but good in vivo binding to E. coli only. 111In-DTPA-biotin showed poor in vitro binding but good in vivo binding to S. aureus and poor to E. coli. 99mTc-Ciprofloxacin showed poor in vitro binding but good in vivo binding to all tested bacteria. The mechanism of accumulation in infected sites remains to be elucidated.

Two novel assays for the detection of haemin-binding properties of antimalarials evaluated with compounds isolated from medicinal plants.

PubMed

Steele, J C P; Phelps, R J; Simmonds, M S J; Warhurst, D C; Meyer, D J

2002-07-01

Forty-two compounds isolated from nine plants used within South America for the treatment of malaria were tested for haemin binding using two novel, rapid screening methods. The data obtained were analysed with respect to IC(50) values for in vitro toxicity to Plasmodium falciparum trophozoites. One method, a multiwell assay based on the inhibition of the interaction of haemin with glutathione (GSH), is sensitive in the 10 microM range, takes c. 1 h and is suitable for either a high throughput screen or rapid assay during natural product isolation. Of 19 compounds showing antiplasmodial activity (IC(50) < 40 microM), 16 (84%) showed >40% inhibition of GSH-haemin reaction. The sensitivity and specificity of the assay were 0.85 and 0.82, respectively. The positive predictive value was 0.81 and the negative predictive value 0.86. A more sensitive assay (0.1 microM range) is based on the reversal by haemin-binding compounds of the haemin inhibition of the L-dopachrome-methyl ester tautomerase activity of human macrophage migration inhibitory factor. This assay gives a better idea of the affinity of interaction and uses very small amounts of test compound. The log[RI(50)] of eight of the compounds that tested positive in the above assays together with those of quinine and chloroquine showed a positive correlation with log[antiplasmodial IC(50)] for strain T9-96 (r = 0.824) and strain K1 (r = 0.904). Several of the antimalarial compounds that bind haemin are isoquinolines, a class not shown previously to interact with haemin.

Two complementary fluorimetric assays for the determination of aminoquinoline binding and uptake by human erythrocytes in vitro.

PubMed

Basilico, Nicoletta; Cortelezzi, Lucia; Serpellini, Chiara; Taramelli, Donatella; Omodeo-Salè, Fausta; Salè, Fausta

2009-02-15

We provide two simple low-cost and low-tech procedures to measure with good precision and accuracy the binding and internalization into human erythrocytes of chloroquine and other aminoquinolines. The methods are based on the high fluorescence of the quinoline ring and are complementary. Method A evaluates residual drugs in the supernatants of treated erythrocytes, whereas method B quantifies the total uptake by whole cells and the fraction bound to the membranes. Drug uptake is dose dependent and related to the number of erythrocytes. These assays could be useful when studying the cell interaction of quinoline-type compounds not available in the radioactive form.

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

PubMed Central

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system. PMID:26222440

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

PubMed

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.

Binding interactions of halogenated bisphenol A with mouse PPARα: In vitro investigation and molecular dynamics simulation.

PubMed

Zhang, Jie; Li, Tiezhu; Wang, Tuoyi; Guan, Tianzhu; Yu, Hansong; Li, Zhuolin; Wang, Yongzhi; Wang, Yongjun; Zhang, Tiehua

2018-02-01

The binding of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs) to mouse peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD) was examined by a combination of in vitro investigation and in silico simulation. Fluorescence polarization (FP) assay showed that halogenated BPAs could bind to mPPARα-LBD* as the affinity ligands. The calculated electrostatic potential (ESP) illustrated the different charge distributions of halogenated BPAs with altered halogenation patterns. As electron-attracting substituents, halogens decrease the positive electrostatic potential and thereby have a significant influence on the electrostatic interactions of halogenated BPAs with mPPARα-LBD*. The docking results elucidated that hydrophobic and hydrogen-bonding interactions may also contribute to stabilize the binding of the halogenated BPAs to their receptor molecule. Comparison of the calculated binding energies with the experimentally determined affinities yielded a good correlation (R 2 =0.6659) that could provide a rational basis for designing environmentally benign chemicals with reduced toxicities. This work can potentially be used for preliminary screening of halogenated BPAs. Copyright © 2017 Elsevier B.V. All rights reserved.

INCREASED ENDOCRINE ACTIVITY OF XENOBIOTIC CHEMICALS AS MEDIATED BY METABOLIC ACTIVATION

EPA Science Inventory

This research is part of an effort to develop in vitro assays and QSARs applicable to untested chemicals on EPA inventories through study of estrogen receptor (ER) binding and estrogen mediated gene expression in fish. The current effort investigates metabolic activation of chemi...

RNA binding and replication by the poliovirus RNA polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oberste, M.S.

1988-01-01

RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to {sup 32}P-labeled ribohomopolymeric RNAs wasmore » examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K{sub a} for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 {times} 10{sup 9} M{sup {minus}1}. The polymerase binds to a subgenomic RNAs which contain the 3{prime} end of the genome with a K{sub a} similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3{prime} noncoding region.« less

SKLB060 Reversibly Binds to Colchicine Site of Tubulin and Possesses Efficacy in Multidrug-Resistant Cell Lines.

PubMed

Yan, Wei; Yang, Tao; Yang, Jianhong; Wang, Taijin; Yu, Yamei; Wang, Yuxi; Chen, Qiang; Bai, Peng; Li, Dan; Ye, Haoyu; Qiu, Qiang; Zhou, Yongzhao; Hu, Yiguo; Yang, Shengyong; Wei, Yuquan; Li, Weimin; Chen, Lijuan

2018-05-22

Many tubulin inhibitors are in clinical use as anti-cancer drugs. In our previous study, a novel series of 4-substituted coumarins derivatives were identified as novel tubulin inhibitors. Here, we report the anti-cancer activity and underlying mechanism of one of the 4-substituted coumarins derivatives (SKLB060). The anti-cancer activity of SKLB060 was tested on 13 different cancer cell lines and four xenograft cancer models. Immunofluorescence staining, cell cycle analysis, and tubulin polymerization assay were employed to study the inhibition of tubulin. N, N '-Ethylenebis(iodoacetamide) assay was used to measure binding to the colchicine site. Wound-healing migration and tube formation assays were performed on human umbilical vascular endothelial cells to study anti-vascular activity (the ability to inhibit blood vessel growth). Mitotic block reversibility and structural biology assays were used to investigate the SKLB060-tubulin bound model. SKLB060 inhibited tubulin polymerization and subsequently induced G2/M cell cycle arrest and apoptosis in cancer cells. SKLB060 bound to the colchicine site of β-tubulin and showed antivascular activity in vitro. Moreover, SKLB060 induced reversible cell cycle arrest and reversible inhibition of tubulin polymerization. A mitotic block reversibility assay showed that the effects of SKLB060 have greater reversibility than those of colcemid (a reversible tubulin inhibitor), indicating that SKLB060 binds to tubulin in a totally reversible manner. The crystal structures of SKLB060-tubulin complexes confirmed that SKLB060 binds to the colchicine site, and the natural coumarin ring in SKLB060 enables reversible binding. These results reveal that SKLB060 is a powerful and reversible microtubule inhibitor that binds to the colchicine site and is effective in multidrug-resistant cell lines. © 2018 The Author(s). Published by S. Karger AG, Basel.

Development of a homogeneous, fluorescence resonance energy transfer-based in vitro recruitment assay for peroxisome proliferator-activated receptor delta via selection of active LXXLL coactivator peptides.

PubMed

Drake, Katherine A; Zhang, Ji-Hu; Harrison, Richard K; McGeehan, Gerald M

2002-05-01

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors activated by fatty acids and their metabolites. The PPARdelta subtype is believed to be involved in lipoprotein regulation and may have a role in reverse cholesterol transport. While the range of biological roles of PPARdelta still remains unclear, it is of therapeutic interest in cardiovascular diseases. Here we report a homogeneous in vitro assay for studying ligand activation of PPARdelta. We surveyed a panel of peptides containing the LXXLL motifs derived from coactivator protein sequences. Peptides with the best response were used to develop a sensitive and homogeneous recruitment assay for PPARdelta. The optimized assay has a signal-to-background ratio of about 8:1 and an assay quality parameter Z'-factor value of 0.8. The assay signal generated is stable for hours to even overnight. This simple recruitment assay can provide agonist and/or antagonist information that cannot be assessed by receptor-binding assay, and can be used for characterization and screening of ligands that modulate the activation of PPARdelta. (c)2002 Elsevier Science (USA).

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) γ Activators and Pan-PPAR Partial Agonists

PubMed Central

Ayers, Steven D.; Lin, Jean Z.; Cvoro, Aleksandra; Silveira, Rodrigo L.; Martínez, Leandro; Souza, Paulo C. T.; Saidemberg, Daniel; Deng, Tuo; Amato, Angela Angelica; Togashi, Marie; Hsueh, Willa A.; Phillips, Kevin; Palma, Mário Sérgio; Neves, Francisco A. R.; Skaf, Munir S.; Webb, Paul; Polikarpov, Igor

2012-01-01

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8–C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products. PMID:22649490

Medium chain fatty acids are selective peroxisome proliferator activated receptor (PPAR) γ activators and pan-PPAR partial agonists.

PubMed

Liberato, Marcelo Vizoná; Nascimento, Alessandro S; Ayers, Steven D; Lin, Jean Z; Cvoro, Aleksandra; Silveira, Rodrigo L; Martínez, Leandro; Souza, Paulo C T; Saidemberg, Daniel; Deng, Tuo; Amato, Angela Angelica; Togashi, Marie; Hsueh, Willa A; Phillips, Kevin; Palma, Mário Sérgio; Neves, Francisco A R; Skaf, Munir S; Webb, Paul; Polikarpov, Igor

2012-01-01

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8-C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products.

In vitro expressed GPCR inserted in polymersome membranes for ligand-binding studies.

PubMed

May, Sylvia; Andreasson-Ochsner, Mirjam; Fu, Zhikang; Low, Ying Xiu; Tan, Darren; de Hoog, Hans-Peter M; Ritz, Sandra; Nallani, Madhavan; Sinner, Eva-Kathrin

2013-01-07

The dopamine receptor D2 (DRD2), a G-protein coupled receptor is expressed into PBd(22)-PEO(13) and PMOXA(20)-PDMS(54)-PMOXA(20) block copolymer vesicles. The conformational integrity of the receptor is confirmed by antibody- and ligand-binding assays. Replacement of bound dopamine is demonstrated on surface-immobilized polymersomes, thus making this a promising platform for drug screening. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The calcium binding properties and structure prediction of the Hax-1 protein.

PubMed

Balcerak, Anna; Rowinski, Sebastian; Szafron, Lukasz M; Grzybowska, Ewa A

2017-01-01

Hax-1 is a protein involved in regulation of different cellular processes, but its properties and exact mechanisms of action remain unknown. In this work, using purified, recombinant Hax-1 and by applying an in vitro autoradiography assay we have shown that this protein binds Ca 2+ . Additionally, we performed structure prediction analysis which shows that Hax-1 displays definitive structural features, such as two α-helices, short β-strands and four disordered segments.

Blocking CLEC14A-MMRN2 binding inhibits sprouting angiogenesis and tumour growth

PubMed Central

PJ, Noy; P, Lodhia; K, Khan; X, Zhuang; DG, Ward; AR, Verissimo; A, Bacon; R, Bicknell

2015-01-01

We previously identified CLEC14A as a tumour endothelial marker. Here we show CLEC14A is a regulator of sprouting angiogenesis in vitro and in vivo. Using a HUVEC spheroid sprouting assay we found CLEC14A to be a regulator of sprout initiation. Analysis of endothelial sprouting in aortic ring and in vivo subcutaneous sponge assays from clec14a+/+ and clec14a−/− mice revealed defects in sprouting angiogenesis in CLEC14A deficient animals. Tumour growth was retarded and vascularity reduced in clec14a−/− mice. Pulldown and co-immunoprecipitation experiments confirmed MMRN2 binds to the extracellular region of CLEC14A. The CLEC14A-MMRN2 interaction was interrogated using mouse monoclonal antibodies. Monoclonal antibodies were screened for their ability to block this interaction. Clone C4 but not C2 blocked CLEC14A-MMRN2 binding. C4 antibody perturbed tube formation and endothelial sprouting in vitro and in vivo, with a similar phenotype to loss of CLEC14A. Significantly, tumour growth was impaired in C4 treated animals and vascular density was also reduced in the C4 treated group. We conclude that CLEC14A-MMRN2 binding has a role in inducing sprouting angiogenesis during tumour growth, that has the potential to be manipulated in future anti-angiogenic therapy design. PMID:25745997

POZ domain transcription factor, FBI-1, represses transcription of ADH5/FDH by interacting with the zinc finger and interfering with DNA binding activity of Sp1.

PubMed

Lee, Dong-Kee; Suh, Dongchul; Edenberg, Howard J; Hur, Man-Wook

2002-07-26

The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp -38 to -22) located just upstream of the core Sp1 binding sites (bp -22 to +22) of the ADH5/FDH minimal promoter (bp -38 to +61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.

Identification of berberine as a direct thrombin inhibitor from traditional Chinese medicine through structural, functional and binding studies

NASA Astrophysics Data System (ADS)

Wang, Xing; Zhang, Yuxin; Yang, Ying; Wu, Xia; Fan, Hantian; Qiao, Yanjiang

2017-03-01

Thrombin acts as a key enzyme in the blood coagulation cascade and represents a potential drug target for the treatment of several cardiovascular diseases. The aim of this study was to identify small-molecule direct thrombin inhibitors from herbs used in traditional Chinese medicine (TCM). A pharmacophore model and molecular docking were utilized to virtually screen a library of chemicals contained in compositions of traditional Chinese herbs, and these analyses were followed by in vitro bioassay validation and binding studies. Berberine (BBR) was first confirmed as a thrombin inhibitor using an enzymatic assay. The BBR IC50 value for thrombin inhibition was 2.92 μM. Direct binding studies using surface plasmon resonance demonstrated that BBR directly interacted with thrombin with a KD value of 16.39 μM. Competitive binding assay indicated that BBR could bind to the same argartroban/thrombin interaction site. A platelet aggregation assay demonstrated that BBR had the ability to inhibit thrombin-induced platelet aggregation in washed platelets samples. This study proved that BBR is a direct thrombin inhibitor that has activity in inhibiting thrombin-induced platelet aggregation. BBR may be a potential candidate for the development of safe and effective thrombin-inhibiting drugs.

Assessment and characterisation of yeast-based products intended to mitigate ochratoxin exposure using in vitro and in vivo models.

PubMed

Pfohl-Leszkowicz, A; Hadjeba-Medjdoub, K; Ballet, N; Schrickx, J; Fink-Gremmels, J

2015-01-01

The aim of this paper was to evaluate the capacity of several yeast-based products, derived from baker's and brewer's yeasts, to sequester the mycotoxin ochratoxin A (OTA) and to decrease its rate of absorption and DNA adduct formation in vivo. The experimental protocol included in vitro binding studies using isotherm models, in vivo chicken experiments, in which the serum and tissue concentrations of OTA were analysed in the absence and presence of the test compounds, and the profile of OTA-derived metabolites and their associated DNA adducts were determined. Additionally in vitro cell culture studies (HK2 cells) were applied to assess further the effects for yeast cell product enriched with glutathione (GSH) or selenium. Results of the in vitro binding assay in a buffer system indicated the ability of the yeast-based products, as sequester of OTA, albeit at a different level. In the in vitro experiments in chickens, decreased serum and tissue concentrations of treated animals confirmed that yeast-based products are able to prevent the absorption of OTA. A comparison of the binding affinity in a standard in vitro binding assay with the results obtained in an in vivo chicken experiment, however, showed a poor correlation and resulted in a different ranking of the products. More importantly, we could show that yeast-based products actively modulate the biotransformation of OTA in vivo as well as in vitro in a cell culture model. This effect seems to be attributable to residual enzymatic activities in the yeast-based products. An enrichment of yeast cell wall products with GSH or selenium further modulated the profile of the generated OTA metabolites and the associated pattern of OTA-induced DNA adducts by increasing the conversion of OTA into less toxic metabolites such as OTA, OTB and 4-OH-OTA. A reduced absorption and DNA adduct formation was particularly observed with GSH-enriched yeast, whereas selenium-enriched yeasts could counteract the OTA-induced decrease in cell viability, but at the same time increased the OTA-DNA adducts formation. These findings indicate the need for an in-depth characterisation of yeast-based products used as mycotoxin-mitigating feed additives, in in vivo models with target animal species taking into account not only their ability to sequester toxins in the gastrointestinal tract but also their potential effects on the biotransformation of mycotoxins.

Analysis of the actions of the novel dopamine receptor-directed compounds (S)-OSU6162 and ACR16 at the D2 dopamine receptor

PubMed Central

Kara, Elodie; Lin, Hong; Svensson, Kjell; Johansson, Anette M; Strange, Philip G

2010-01-01

BACKGROUND AND PURPOSE The two phenylpiperidines, OSU6162 and ACR16, have been proposed as novel drugs for the treatment of brain disorders, including schizophrenia and Huntington's disease, because of their putative dopamine stabilizing effects. Here we evaluated the activities of these compounds in a range of assays for the D2 dopamine receptor in vitro. EXPERIMENTAL APPROACH The affinities of these compounds for the D2 dopamine receptor were evaluated in competition with [3H]spiperone and [3H]NPA. Agonist activity of these compounds was evaluated in terms of their ability to stimulate [35S]GTPγS binding. KEY RESULTS Both compounds had low affinities for inhibition of [3H]spiperone binding (pKi vs. [3H]spiperone, ACR16: <5, OSU6162: 5.36). Neither compound was able to stimulate [35S]GTPγS binding when assayed in the presence of Na+ ions, but if the Na+ ions were removed, both compounds were low-affinity, partial agonists (Emax relative to dopamine: ACR16: 10.2%, OSU6162:54.3%). Schild analysis of the effects of OSU6162 to inhibit dopamine-stimulated [35S]GTPγS binding indicated Schild slopes of ∼0.9, suggesting little deviation from competitive inhibition. OSU6162 was, however, able to accelerate [3H]NPA dissociation from D2 dopamine receptors, indicating some allosteric effects of this compound. CONCLUSIONS AND IMPLICATIONS The two phenylpiperidines were low-affinity, low-efficacy partial agonists at the D2 dopamine receptor in vitro, possibly exhibiting some allosteric effects. Comparing their in vitro and in vivo effects, the in vitro affinities were a reasonable guide to potencies in vivo. However, the lack of in vitro–in vivo correlation for agonist efficacy needs to be further addressed. PMID:20804495

Can penicillins and other beta-lactam antibiotics be used to treat tuberculosis?

PubMed Central

Chambers, H F; Moreau, D; Yajko, D; Miick, C; Wagner, C; Hackbarth, C; Kocagöz, S; Rosenberg, E; Hadley, W K; Nikaido, H

1995-01-01

An increase in the number of tuberculosis cases caused by multiple-drug-resistant strains of Mycobacterium tuberculosis has stimulated search for new antituberculous agents. Beta-lactam antibiotics, traditionally regarded as ineffective against tuberculosis, merit consideration. Four major penicillin-binding proteins (PBPs) with approximate molecular sizes of 94, 82, 52, and 37 kDa were detected by fluorography of [3H]penicillin-radiolabeled membrane proteins prepared from M. tuberculosis H37Ra. The presence of membrane-associated beta-lactamase precluded the use of membranes for assaying the binding affinities of beta-lactam antibiotics. Therefore, ampicillin affinity chromatography was used to purify these four PBPs from crude membranes in order to assay the binding affinities of beta-lactam antibiotics. Ampicillin, amoxicillin, and imipenem, beta-lactam antibiotics previously reported to be active in vitro against M. tuberculosis, bound to M. tuberculosis PBPs at therapeutically achievable concentrations. Binding of the 94-, 82-, and 52-kDa PBPs, but not the 37-kDa PBP, was associated with antibacterial activity, suggesting that these PBPs are the critical targets. Studies of mycobacterial cell wall permeability, which was assayed with a panel of reference cephalosporins and penicillins with different charge positivities, indicated that the rate of penetration of beta-lactam antibiotics to the target PBPs could not account for resistance. Resistance could be reversed with the beta-lactamase inhibitors clavulanate or sulbactam or could be circumvented by the use of a beta-lactamase-stable drug, imipenem, indicating that mycobacterial beta-lactamase, probably in conjunction with slow penetration, is a major determinant of M. tuberculosis resistance to beta-lactam antibiotics. These findings confirm in vitro data that M. tuberculosis is susceptible to some beta-lactam antibiotics. Further evaluation of these drugs for the treatment of tuberculosis in animal models and in clinical trials is warranted. PMID:8592990

Spectroscopic and molecular modeling approaches to investigate the interaction of bisphenol A, bisphenol F and their diglycidyl ethers with PPARα.

PubMed

Zhang, Jie; Zhang, Tiehua; Guan, Tianzhu; Ruan, Ping; Ren, Dayong; Dai, Weichang; Yu, Hansong; Li, Tiezhu

2017-08-01

A fluorescence polarization (FP) assay for the simultaneous determination of bisphenol A (BPA), bisphenol F (BPF), bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) was developed. The method was based on the competition between bisphenols (BPs) and fluorescein-labeled dexamethasone derivative (Dex-fl) for mouse peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD). A recombinant soluble protein derivative mPPARα-LBD* was prepared, then in vitro binding of 4 BPs to mPPARα-LBD* was investigated. Fluorescence polarization assay showed that these compounds exhibited different binding potencies with mPPARα-LBD*. Additionally, molecular dynamics simulations were performed to further understand the mechanism of BPs binding affinity for mPPARα-LBD*. Docking results elucidated that the driving forces for the binding of BPs to mPPARα-LBD* were predominantly dependent on hydrophobic and hydrogen-bonding interactions. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation (R 2  = 0.7258). The proposed method has potential for multi-residue detection of BPA, BPF, BADGE, and BFDGE. Copyright © 2017 Elsevier Ltd. All rights reserved.

Alkylating derivative of oxotremorine interacts irreversibly with the muscarinic receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ehlert, F.J.; Jenden, D.J.; Ringdahl, B.

A 2-chloroethylamine derivative of oxotremorine was studied in pharmacological experiments and muscarinic receptor binding assays. The compound, N-(4-(2-chloroethylmethylamino)-2-butynyl)-2-pyrrolidone (BM 123), forms an aziridinium ion in aqueous solution at neutral pH that stimulates contractions of guinea pig ileum with a potency similar to that of oxotremorine. Following the initial stimulation, there is a long lasting period of lack of sensitivity of the guinea pig ileum to muscarinic agonists. BM 123 also produces muscarinic effects in vivo. When homogenates of the rat cerebral cortex were incubated with BM 123 and assayed subsequently in muscarinic receptor binding assays, a loss of binding capacitymore » for the muscarinic antagonist, (/sup 3/H)N-methylscopolamine ((/sup 3/H)NMS), was noted without a change in affinity. Similar observations were made in (/sup 3/H)1-3-quinuclidinyl benzilate ((/sup 3/H)-QNB) binding assays on the forebrains of mice that had been injected with BM 123 24 hr earlier. The loss in receptor capacity for both (/sup 3/H)NMS and (/sup 3/H)-QNB was prevented by atropine treatment. Kinetic studies of the interaction of BM 123 with homogenates of the rat cerebral cortex in vitro showed that the half-time for the loss of (/sup 3/H)-QNB binding sites increased from 10 to 45 min as the concentration of BM 123 decreased from 10 to 1 ..mu..M. In contrast to the aziridinium ion, the parent 2-chloroethylamine compound and the alcoholic hydrolysis product were largely devoid of pharmacological and binding activity.« less

Pull-down Assay to Characterize Ca2+/Calmodulin Binding to Plant Receptor Kinases.

PubMed

Kaufmann, Christine; Sauter, Margret

2017-01-01

Plant receptor-like kinases (RLKs) are regulated by posttranscriptional modification and by interaction with regulatory proteins. A common modification of RLKs is (auto)phosphorylation, and a common regulatory protein is the calcium sensor calmodulin (CaM). We have developed protocols to detect the interaction of an RLK with CaM. The interaction with CaM was shown by bimolecular fluorescence complementation (BiFC) (see Chapter 14) and pull-down assay (this chapter). Both methods offer unique advantages. BiFC is useful in showing interaction of soluble as well as of membrane-bound proteins in planta. Pull-down assays are restricted to soluble proteins and provide in vitro data. The pull-down assay provides the advantage that proteins can be modified prior to binding and that experimental conditions such as the concentration of Ca 2+ or other divalent cations can be controlled. This chapter provides a pull-down protocol to study RLK-CaM interaction with optional steps to investigate the impact of RLK phosphorylation or of Ca 2+ .

Engineering Tocopherol Selectivity in α-TTP: A Combined In Vitro/In Silico Study

PubMed Central

Helbling, Rachel E.; Aeschimann, Walter; Simona, Fabio; Stocker, Achim; Cascella, Michele

2012-01-01

We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of -tocopherol transfer protein (-TTP) towards -tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for -tocopherol to -TTP 8.262.13 kcal mol lower than that of -tocopherol. Our calculations show that -tocopherol binds to -TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of -TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to -tocopherol, with striking structural similarities to the wild-type--tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of -TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover, the identification of a -tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for -tocopherol selection in omnivorous animals. PMID:23152872

Characterization of Lactic Acid Bacteria as Poultry Probiotic Candidates with Aflatoxin B1 Binding Activities

NASA Astrophysics Data System (ADS)

Damayanti, E.; Istiqomah, L.; Saragih, J. E.; Purwoko, T.; Sardjono

2017-12-01

Our previous studies have selected lactic acid bacteria (LAB) with antifungal activities from traditional fermented foods made from cassava (G7) and silage feed palm leaf (PDS5 and PDS3). In this study we evaluated their ability to bind aflatoxin B1 (AFB1) and probiotic characteristic. The probiotic characteristic assays of LAB consisted of resistance to acidic conditions (pH 3), gastric juice and bile salts 0.3%. We also carried out an in vitro evaluation of LAB aflatoxin binding ability in viable and non-viable cell for 24 and 48 hours of incubation. The measurement of aflatoxin content was performed by ELISA method using AgraQuant Total Aflatoxin Assay kit. The results showed that all isolates were potential as probiotics and the G7 isolate had the highest viability among other isolates in pH 3 (92.61 %) and the bile salts assay (97.71 %). The percentage of aflatoxin reduction between viable and non-viable cell from each LAB isolate were different. The highest aflatoxin reduction in viable cell assay was performed by G7 isolate (69.11 %) whereas in non-viable cell assay was performed by PDS3 isolate (73.75 %) during incubation time 48 hours. In this study, G7 isolate performed the best probiotic characteristics with the highest viability in acid pH assay, bile salt 0.3% assay and percentage of aflatoxin B1 reduction in viable cell condition. Molecular identification using 16S rRNA sequence analysis showed that G7 isolate had homology with Lactobacillus plantarum (99.9%). It was concluded that Lactobacillus plantarum G7 was potential as probiotic with aflatoxin binding activities.

Characterization and antifungal properties of wheat nonspecific lipid transfer proteins.

PubMed

Sun, Jin-Yue; Gaudet, Denis A; Lu, Zhen-Xiang; Frick, Michele; Puchalski, Byron; Laroche, André

2008-03-01

This study simultaneously considered the phylogeny, fatty acid binding ability, and fungal toxicity of a large number of monocot nonspecific lipid transfer proteins (ns-LTP). Nine novel full-length wheat ns-LTP1 clones, all possessing coding sequences of 348 bp, isolated from abiotic- and biotic-stressed cDNA libraries from aerial tissues, exhibited highly conserved coding regions with 78 to 99 and 71 to 100% identity at the nucleotide and amino acid levels, respectively. Phylogenetic analyses revealed two major ns-LTP families in wheat. Eight wheat ns-LTP genes from different clades were cloned into the expression vector pPICZalpha and transformed into Pichia pastoris. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and in vitro lipid binding activity assay confirmed that the eight ns-LTP were all successfully expressed and capable of in vitro binding fatty acid molecules. A comparative in vitro study on the toxicity of eight wheat ns-LTP to mycelium growth or spore germination of eight wheat pathogens and three nonwheat pathogens revealed differential toxicities among different ns-LTP. Values indicating 50% inhibition of fungal growth or spore germination of three selected ns-LTP against six fungi ranged from 1 to 7 microM. In vitro lipid-binding activity of ns-LTP was not correlated with their antifungal activity. Using the fluorescent probe SYTOX Green as an indicator of fungal membrane integrity, the in vitro toxicity of wheat ns-LTP was associated with alteration in permeability of fungal membranes.

The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

NASA Astrophysics Data System (ADS)

Zuo, Ping; Manley, James L.

1994-04-01

ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

Live Cell Imaging Confocal Microscopy Analysis of HBV Myr-PreS1 Peptide Binding and Uptake in NTCP-GFP Expressing HepG2 Cells.

PubMed

König, Alexander; Glebe, Dieter

2017-01-01

To obtain basic knowledge about specific molecular mechanisms involved in the entry of pathogens into cells is the basis for establishing pharmacologic substances blocking initial viral binding, infection, and subsequent viral spread. Lack of information about key cellular factors involved in the initial steps of HBV infection has hampered the characterization of HBV binding and entry for decades. However, recently, the liver-specific sodium-dependent taurocholate cotransporting polypeptide (NTCP) has been discovered as a functional receptor for HBV and HDV, thus opening the field for new concepts of basic binding and entry of HBV and HDV. Here, we describe practical issues of a basic in vitro assay system to examine kinetics and mechanisms of receptor-dependent HBV binding, uptake, and intracellular trafficking by live-cell imaging confocal microscopy. The assay system is comprised of HepG2 cells expressing a NTCP-GFP fusion-protein and chemically synthesized, fluorophore-labeled part of HBV surface protein, spanning the first N-terminal 48 amino acids of preS1 of the large hepatitis B virus surface protein.

In vitro assessment of the effects of vedolizumab binding on peripheral blood lymphocytes

PubMed Central

Wyant, Tim; Yang, Lili; Fedyk, Eric

2013-01-01

Vedolizumab (VDZ) is a humanized monoclonal antibody in development for the treatment of inflammatory bowel disease. VDZ binds to the α4β7 integrin complex and inhibits its binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), thus preventing lymphocyte extravasation to gut mucosal tissues. To understand whether VDZ has additional effects that may affect its overall safety as a therapeutic molecule, we examined other potential actions of VDZ. In vitro assays with human peripheral blood lymphocytes demonstrated that VDZ fails to elicit cytotoxicity, lymphocyte activation, and cytokine production from memory T lymphocytes and does not interfere with the suppressive ability of regulatory T cells. Furthermore, we demonstrated that VDZ induces internalization of α4β7 and that the integrin is rapidly re-expressed and fully functional after VDZ withdrawal. These studies provide insight into the mechanisms underlying the observed safety profile of VDZ in clinical trials. PMID:24492340

Enhanced Peptide Radiotherapy of Prostate Cancer Using Targeted Adenoviral Vectors

DTIC Science & Technology

2004-06-01

regard to binding of 64Cu - octreotide. In vitro experiments were performed with DU-145 and PC-3 human prostate cancer cells. Expression levels of SSTR2...were determined using a 64Cu -octreotide saturation binding assay on cell membrane preparations. In vivo experiments were conducted in scid mice bearing...subcutaneous DU-l45 or PC-3 cells. AdSSTR2 was injected intratumorally followed 48 h later by an i.v. injection of 64Cu -octreotide. The mice were

Fluorescent rhodanine-3-acetic acids visualize neurofibrillary tangles in Alzheimer's disease brains.

PubMed

Anumala, Upendra Rao; Gu, Jiamin; Lo Monte, Fabio; Kramer, Thomas; Heyny-von Haußen, Roland; Hölzer, Jana; Goetschy-Meyer, Valerie; Schön, Christian; Mall, Gerhard; Hilger, Ingrid; Czech, Christian; Herms, Jochen; Schmidt, Boris

2013-09-01

There is a high demand for the development of an imaging agent for neurofibrillary tangles (NFTs) detection in Alzheimer's diagnosis. In the present study, a series of rhodanine-3-acetic acids was synthesized and evaluated for fluorescence imaging of NFTs in brain tissues of AD patients. Five out of seven probes have shown excellent binding affinity to NFTs over amyloid plaques in the Thiazine red R displacement assay. However, the selectivity in this in vitro assay is not confirmed by the histopathological evaluation, which indicates significant differences in the binding sites in the assays. Probe 6 showed binding affinity (IC50=19nM) to tau aggregates which is the highest among this series. Probes 2, 3, 4 and 5 display IC50 values of lower than 100nM to tau aggregates to displace Thiazine red R. Evaluation of the cytotoxicity of these five probes with human liver carcinoma cells revealed that these compounds excert negligible cytotoxicity. The in vivo studies with zebrafish embryos confirmed negligible cytotoxicity at 24 and 72h post fertilization. Copyright © 2013 Elsevier Ltd. All rights reserved.

c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro.

PubMed

Lively, T N; Ferguson, H A; Galasinski, S K; Seto, A G; Goodrich, J A

2001-07-06

c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

Synthesis and biological evaluation of Santacruzamate-A based analogues.

PubMed

Randino, Rosario; Gazzerro, Patrizia; Mazitschek, Ralph; Rodriquez, Manuela

2017-12-15

Several derivatives of Santacruzamate-A, a natural product that is structurally related to SAHA, were synthesized to explore the potential of carbamates and oxalylamides as novel biasing element for targeting the catalytic site of zinc-dependent histone deacetylases (HDACs). An additional class of Santacruzamate-A derivatives was synthesized to investigate the influence of the cap group and the linker element on HDAC inhibitory activity. All compounds were evaluated in dose response for their in vitro cytotoxic activity in MTT assay in HCT116 cells. HDAC inhibitory activity was evaluated in vitro by western blot analysis for histone hyperacetylation assay and biochemically for representative human HDACs isoforms. Two novel compounds were identified to exhibit potent time dependent anti proliferative activity. However, unlike hydroxamic acid analogues, the tested Santacruzamate-A derivatives showed no noticeable HDAC inhibitory activity. The ethylcarbamate moiety as unusual zinc-binding group displayed no ability to coordinate the zinc ion and thus, presumably, was not able to reproduce known inhibitor-substrate zinc-binding group interactions with the HDAC catalytic site. This study confirmed that the accommodation of the zinc-binding group is deeply critical of the positioning of the linker and the projection of the cap group toward the different surface pockets of the enzyme. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multiple protein–protein interactions converging on the Prp38 protein during activation of the human spliceosome

PubMed Central

Schütze, Tonio; Ulrich, Alexander K.C.; Apelt, Luise; Will, Cindy L.; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C.

2016-01-01

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein–protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein–protein interaction platform that might organize the relative positioning of other proteins during splicing. PMID:26673105

Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform

PubMed Central

Wagner, Tobias; Greschik, Holger; Burgahn, Teresa; Schmidtkunz, Karin; Schott, Anne-Kathrin; McMillan, Joel; Baranauskienė, Lina; Xiong, Yan; Fedorov, Oleg; Jin, Jian; Oppermann, Udo; Matulis, Daumantas; Schüle, Roland; Jung, Manfred

2016-01-01

Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein–protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader. PMID:26893353

Carotenoid biosynthesis in bacteria: In vitro studies of a crt/bch transcription factor from Rhodobacter capsulatus and carotenoid enzymes from Erwinia herbicola

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Brien, D.A.

1992-11-01

A putative transcription factor in Rhodobactor capsulatus which binds upstream of the crt and bch pigment biosynthesis operons and appears to play a role in the adaptation of the organism from the aerobic to the anaerobic-photosynthetic growth mode was characterized. Chapter 2 describes the identification of this factor through an in vitro mobility shift assay, as well as the determination of its binding properties and sequence specificity. Chapter 3 focuses on the isolation of this factor. Biochemistry of later carotenoid biosynthesis enzymes derived from the non-photosynthetic bacterium, Erwinia herbicola. Chapter 4 describes the separate overexpression and in vitro analysis ofmore » two enzymes involved in the main sequence of the carotenoid biosynthesis pathway, lycopene cyclase and 5-carotene hydroxylase. Chapter 5 examines the overexpression and enzymology of functionally active zeaxanthin glucosyltransferase, an enzyme which carries out a more unusual transformation, converting a carotenoid into its more hydrophilic mono- and diglucoside derivatives. In addition, amino acid homology with other glucosyltransferases suggests a putative binding site for the UDP-activated glucose substrate.« less

Deriving an explicit hepatic clearance equation accounting for plasma protein binding and hepatocellular uptake.

PubMed

Yoon, Miyoung; Clewell, Harvey J; Andersen, Melvin E

2013-02-01

High throughput in vitro biochemical and cell-based assays have the promise to provide more mechanism-based assessments of the adverse effects of large numbers of chemicals. One of the most challenging hurdles for interpreting in vitro toxicity findings is the need for reverse dosimetry tools that estimate the exposures that will give concentrations in vivo similar to the active concentrations in vitro. Recent experience using IVIVE approaches to estimate in vivo pharmacokinetics (Wetmore et al., 2012) identified the need to develop a hepatic clearance equation that explicitly accounted for a broader set of protein binding and membrane transport processes and did not depend on a well-mixed description of the liver compartment. Here we derive an explicit steady-state hepatic clearance equation that includes these factors. In addition to the derivation, we provide simple computer code to calculate steady-state extraction for any combination of blood flow, membrane transport processes and plasma protein-chemical binding rates. This expanded equation provides a tool to estimate hepatic clearance for a more diverse array of compounds. Copyright © 2012 Elsevier Ltd. All rights reserved.

Evolution of a Histone H4-K16 Acetyl-Specific DNA Aptamer

PubMed Central

Williams, Berea A. R.; Lin, Liyun; Lindsay, Stuart M.; Chaput, John C.

2009-01-01

We report the in vitro selection of DNA aptamers that bind to histone H4 proteins acetylated at lysine 16. The best aptamer identified in this selection binds to the target protein with a Kd of 21 nM, and discriminates against both the non-acetylated protein and histone H4 proteins acetylated at lysine 8. Comparative binding assays performed with a chip-quality antibody reveal that this aptamer binds to the acetylated histone target with similar affinity to a commercial antibody, but shows significantly greater specificity (15-fold versus 2,400-fold) for the target molecule. This result demonstrates that aptamers that are both modification and location specific can be generated to bind specific protein post-translational modifications. PMID:19385619

Structure dependent selective efficacy of pyridine and pyrrole based Cu(II) Schiff base complexes towards in vitro cytotoxicity, apoptosis and DNA-bases binding in ground and excited state.

PubMed

Koley Seth, Banabithi; Saha, Arpita; Haldar, Srijan; Chakraborty, Partha Pratim; Saha, Partha; Basu, Samita

2016-09-01

This work highlights a systematic and comparative study of the structure-dependent influence of a series of biologically active Cu(II) Schiff base complexes (CSCs) on their in vitro cytotoxicity, apoptosis and binding with polymeric DNA-bases in ground and photo-excited states. The structure-activity relationship of the closely resembled CSCs towards in vitro cytotoxicity and apoptosis against cervical cancerous HeLa and normal human diploid WI-38 cell lines has been investigated by MTT assay and FACS techniques respectively. The steady-state and time-resolved spectroscopic studies have also been carried out to explore the selective binding affinities of the potential complexes towards different polymeric nucleic acid bases (poly d(A), poly d(T), poly d(G), poly d(C), Poly d(G)-Poly d(C)), which enlighten the knowledge regarding their ability in controlling the structure and medium dependent interactions in 'ground' and 'excited' states. The pyridine containing water soluble complexes (CuL(1) and CuL(3)) are much more cytotoxic than the corresponding pyrrole counterparts (CuL(2) and CuL(4)). Moreover the acidic hydrogens in CuL(1) increase its cytotoxicity much more than methyl substitution as in CuL(3). The results of MTT assay and double staining FACS experiments indicate selective inhibition of cell growth (cell viability 39% (HeLa) versus 85% (WI-38)) and occurrence of apoptosis rather than necrosis. The ground state binding of CuL(1) with polymeric DNA bases, especially with guanine rich DNA (Kb=6.41±0.122×10(5)), that enhances its cytotoxic activity, is further confirmed from its binding isotherms. On the other hand the pyrrole substituted CuL(4) complex exhibits the structure and medium dependent selective electron-transfer in triplet state as observed in laser flash photolysis studies followed by magnetic field (MF) effect. Copyright © 2016 Elsevier B.V. All rights reserved.

Filling the concept with data: integrating data from different in vitro and in silico assays on skin sensitizers to explore the battery approach for animal-free skin sensitization testing.

PubMed

Natsch, Andreas; Emter, Roger; Ellis, Graham

2009-01-01

Tests for skin sensitization are required prior to the market launch of new cosmetic ingredients. Significant efforts are made to replace the current animal tests. It is widely recognized that this cannot be accomplished with a single in vitro test, but that rather the integration of results from different in vitro and in silico assays will be needed for the prediction of the skin sensitization potential of chemicals. This has been proposed as a theoretical scheme so far, but no attempts have been made to use experimental data to prove the validity of this concept. Here we thus try for the first time to fill this widely cited concept with data. To this aim, we integrate and report both novel and literature data on 116 chemicals of known skin sensitization potential on the following parameters: (1) peptide reactivity as a surrogate for protein binding, (2) induction of antioxidant/electrophile responsive element dependent luciferase activity as a cell-based assay; (3) Tissue Metabolism Simulator skin sensitization model in silico prediction; and (4) calculated octanol-water partition coefficient. The results of the in vitro assays were scaled into five classes from 0 to 4 to give an in vitro score and compared to the local lymph node assay (LLNA) data, which were also scaled from 0 to 4 (nonsensitizer/weak/moderate/strong/extreme). Different ways of evaluating these data have been assessed to rate the hazard of chemicals (Cooper statistics) and to also scale their potency. With the optimized model an overall accuracy for predicting sensitizers of 87.9% was obtained. There is a linear correlation between the LLNA score and the in vitro score. However, the correlation needs further improvement as there is still a relatively high variation in the in vitro score between chemicals belonging to the same sensitization potency class.

SPECIES DIFFERENCES IN ANDROGEN AND ESTROGEN RECEPTOR STRUCTURE AND FUNCTION AMONG VERTEBRATES AND INVERTEBRATES FOR INTERSPECIES EXTRAPOLATION OF ENDOCRINE DISRUPTING CHEMICALS

EPA Science Inventory

In vitro screening assays designed to identify hormone minics or antagonists, including the EDSTAC Tier 1 Screening (TIS) Battery, typically use only mammalian estrogen (ER) and androgen receptors (AR). However, there is uncertainty concerning species differences in binding affin...

Toxicity challenges in environmental chemicals: Prediction of human plasma protein binding through quantitative structure-activity relationship (QSAR) models (2016 IVIVE Workshop Proceedings)

EPA Science Inventory

Physiologically based pharmacokinetic (PBPK) models bridge the gap between in vitro assays and in vivo effects by accounting for the adsorption, distribution, metabolism, and excretion of xenobiotics, which is especially useful in the assessment of human toxicity. Quantitative st...

Identification of cis-elements for ethylene and circadian regulation of the Solanum melongena gene encoding cysteine proteinase.

PubMed

Rawat, Reetika; Xu, Zeng-Fu; Yao, Kwok-Ming; Chye, Mee-Len

2005-03-01

We have previously shown that the expression of SmCP which encodes Solanum melongena cysteine proteinase is ethylene-inducible and is under circadian control. To understand the regulation of SmCP, a 1.34-kb SmCP 5'-flanking region and its deletion derivatives were analyzed for cis-elements using GUS and luc fusions and by in vitro binding assays. Analysis of transgenic tobacco transformed with SmCP promoter-GUS constructs confirmed that the promoter region -415/+54 containing Ethylene Responsive Element ERE(-355/-348) conferred threefold ethylene-induction of GUS expression, while -827/+54 which also contains ERE(-683/-676), produced fivefold induction. Using gel mobility shift assays, we demonstrated that each ERE binds nuclear proteins from both ethephon-treated and untreated 5-week-old seedlings, suggesting that different transcriptions factors bind each ERE under varying physiological conditions. Binding was also observed in extracts from senescent, but not young, fruits. The variation in binding at the EREs in fruits and seedlings imply that organ-specific factors may participate in binding. Analysis of transgenic tobacco expressing various SmCP promoter-luc constructs containing wild-type or mutant Evening Elements (EEs) confirmed that both conserved EEs at -795/-787 and -785/-777 are important in circadian control. We confirmed the binding of total nuclear proteins to EEs in gel mobility shift assays and in DNase I footprinting. Our results suggest that multiple proteins bind the EEs which are conserved in plants other than Arabidopsis and that functional EEs and EREs are present in the 5'-flanking region of a gene encoding cysteine proteinase.

Elucidating pathways of Toxoplasma gondii invasion in the gastrointestinal tract: involvement of the tight junction protein occludin.

PubMed

Weight, Caroline M; Jones, Emily J; Horn, Nikki; Wellner, Nikolaus; Carding, Simon R

2015-10-01

Toxoplasma gondii is an obligate intracellular parasite infecting one third of the world's population. The small intestine is the parasite's primary route of infection, although the pathway of epithelium transmigration remains unclear. Using an in vitro invasion assay and live imaging we showed that T. gondii (RH) tachyzoites infect and transmigrate between adjacent intestinal epithelial cells in polarized monolayers without altering barrier integrity, despite eliciting the production of specific inflammatory mediators and chemokines. During invasion, T. gondii co-localized with occludin. Reducing the levels of endogenous cellular occludin with specific small interfering RNAs significantly reduced the ability of T. gondii to penetrate between and infect epithelial cells. Furthermore, an in vitro invasion and binding assays using recombinant occludin fragments established the capacity of the parasite to bind occludin and in particular to the extracellular loops of the protein. These findings provide evidence for occludin playing a role in the invasion of T. gondii in small intestinal epithelial cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Profiling Heparin-Chemokine Interactions Using Synthetic Tools

PubMed Central

de Paz, Jose L.; Moseman, E. Ashley; Noti, Christian; Polito, Laura; von Andrian, Ulrich H.; Seeberger, Peter H.

2009-01-01

Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity. PMID:18030990

Comparative effectiveness of Calabadion and sugammadex to reverse non-depolarizing neuromuscular blocking agents

PubMed Central

Haerter, Friederike; Simons, Jeroen Cedric Peter; Foerster, Urs; Duarte, Ingrid Moreno; Diaz-Gil, Daniel; Ganapati, Shweta; Eikermann-Haerter, Katharina; Ayata, Cenk; Zhang, Ben; Blobner, Manfred; Isaacs, Lyle; Eikermann, Matthias

2015-01-01

Background We evaluated the comparative effectiveness of calabadion 2 to reverse non-depolarizing neuromuscular blocking agents (NMBAs) by binding and inactivation. Methods The dose-response relationship of drugs to reverse vecuronium, rocuronium, and cisatracurium-induced neuromuscular block (NMB) was evaluated in vitro (competition binding assays and urine analysis), ex vivo (n=34; phrenic nerve hemidiaphragm preparation) and in vivo (n=108; quadriceps femoris muscle of the rat). Cumulative dose-response curves of calabadions, neostigmine, or sugammadex were created ex vivo at steady-state deep NMB. In living rats, we studied the dose-response relationship of the test drugs to reverse deep block under physiological conditions and we measured the amount of calabadion 2 excreted in the urine. Results In vitro experiments showed that calabadion 2 binds rocuronium with 89 times the affinity of sugammadex (Ka = 3.4 × 109 M−1 and Ka = 3.8 × 107 M−1). Urine analysis (proton nuclear magnetic resonance), competition binding assays and ex vivo study results obtained in the absence of metabolic deactivation are in accordance with an 1:1 binding ratio of sugammadex and calabadion 2 toward rocuronium. In living rats, calabadion 2 dose-dependently and rapidly reversed all NMBAs tested. The molar potency of calabadion 2 to reverse vecuronium and rocuronium was higher compared to sugammadex. Calabadion 2 was eliminated renally, and did not affect blood pressure or heart rate. Conclusion Calabadion 2 reverses NMB-induced by benzylisoquinolines and steroidal NMBAs in rats more effectively, i.e. faster, than sugammadex. Calabadion 2 is eliminated in the urine and well tolerated in rats. PMID:26418697

Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9

PubMed Central

Capitanio, Juliana S; Montpetit, Ben; Wozniak, Richard W

2017-01-01

Beyond their role at nuclear pore complexes, some nucleoporins function in the nucleoplasm. One such nucleoporin, Nup98, binds chromatin and regulates gene expression. To gain insight into how Nup98 contributes to this process, we focused on identifying novel binding partners and understanding the significance of these interactions. Here we report on the identification of the DExH/D-box helicase DHX9 as an intranuclear Nup98 binding partner. Various results, including in vitro assays, show that the FG/GLFG region of Nup98 binds to N- and C-terminal regions of DHX9 in an RNA facilitated manner. Importantly, binding of Nup98 stimulates the ATPase activity of DHX9, and a transcriptional reporter assay suggests Nup98 supports DHX9-stimulated transcription. Consistent with these observations, our analysis revealed that Nup98 and DHX9 bind interdependently to similar gene loci and their transcripts. Based on our results, we propose that Nup98 functions as a co-factor that regulates DHX9 and, potentially, other RNA helicases. DOI: http://dx.doi.org/10.7554/eLife.18825.001 PMID:28221134

The LacI family protein GlyR3 co-regulates the celC operon and manB in Clostridium thermocellum

DOE PAGES

Choi, Jinlyung; Klingeman, Dawn M.; Brown, Steven D.; ...

2017-06-24

In this paper, we demonstrate that the GlyR3 protein mediates the regulation of manB. We first identify putative GlyR3 binding sites within or just upstream of the coding regions of manB and celT. Using an electrophoretic mobility shift assay (EMSA), we determined that a higher concentration of GlyR3 is required to effectively bind to the putative manB site in comparison to the celC site. Neither the putative celT site nor random DNA significantly binds GlyR3. While laminaribiose interfered with GlyR3 binding to the celC binding site, binding to the manB site was unaffected. In the presence of laminaribiose, in vivomore » transcription of the celC–glyR3–licA gene cluster increases, while manB expression is repressed, compared to in the absence of laminaribiose, consistent with the results from the EMSA. An in vitro transcription assay demonstrated that GlyR3 and laminaribiose interactions were responsible for the observed patters of in vivo transcription.« less

Sa-Lrp from Sulfolobus acidocaldarius is a versatile, glutamine-responsive, and architectural transcriptional regulator

PubMed Central

Vassart, Amelia; Wolferen, Marleen; Orell, Alvaro; Hong, Ye; Peeters, Eveline; Albers, Sonja-Verena; Charlier, Daniel

2013-01-01

Sa-Lrp is a member of the leucine-responsive regulatory protein (Lrp)-like family of transcriptional regulators in Sulfolobus acidocaldarius. Previously, we demonstrated the binding of Sa-Lrp to the control region of its own gene in vitro. However, the function and cofactor of Sa-Lrp remained an enigma. In this work, we demonstrate that glutamine is the cofactor of Sa-Lrp by inducing the formation of octamers and increasing the DNA-binding affinity and sequence specificity. In vitro protein-DNA interaction assays indicate that Sa-Lrp binds to promoter regions of genes with a variety of functions including ammonia assimilation, transcriptional control, and UV-induced pili synthesis. DNA binding occurs with a specific affinity for AT-rich binding sites, and the protein induces DNA bending and wrapping upon binding, indicating an architectural role of the regulator. Furthermore, by analyzing an Sa-lrp deletion mutant, we demonstrate that the protein affects transcription of some of the genes of which the promoter region is targeted and that it is an important determinant of the cellular aggregation phenotype. Taking all these results into account, we conclude that Sa-Lrp is a glutamine-responsive global transcriptional regulator with an additional architectural role. PMID:23255531

A Receptor-targeted Fluorescent Radiopharmaceutical for Multireporter Sentinel Lymph Node Imaging

PubMed Central

Emerson, Derek K.; Limmer, Karl K.; Hall, David J.; Han, Sung-Ho; Eckelman, William C.; Kane, Christopher J.; Wallace, Anne M.

2012-01-01

Purpose: To determine the imaging and receptor-binding properties of a multireporter probe designed for sentinel lymph node (SLN) mapping via nuclear and fluorescence detection. Materials and Methods: The animal experiments were approved by the institutional animal care and use committee. A multireporter probe was synthesized by covalently attaching cyanine 7 (Cy7), a near-infrared cyanine dye, to tilmanocept, a radiopharmaceutical that binds to a receptor specific to recticuloendothelial cells. In vitro binding assays of technetium 99m (99mTc) -labeled Cy7 tilmanocept were conducted at 4°C by using receptor-bearing macrophages. Optical SLN imaging after foot pad administration was performed by using two molar doses of Cy7 tilmanocept. Six mice were injected with 0.11 nmol of 99mTc-labeled Cy7 tilmanocept (low-dose group); an additional six mice were injected with 31 nmol of 99mTc-labeled Cy7 tilmanocept (high-dose group) to saturate the receptor sites within the SLN. After 2.5 hours of imaging, the mice were euthanized, and the sentinel and distal lymph nodes were excised and assayed for radioactivity for calculation of SLN percentage of injected dose and extraction. Four mice were used as controls for autofluorescence. Standard optical imaging software was used to plot integrated fluorescence intensity against time for calculation of the SLN uptake rate constant and scaled peak intensity. Significance was calculated by using the Student t test. Results: In vitro binding assays showed subnanomolar affinity (mean dissociation constant, 0.25 nmol/L ± 0.10 [standard deviation]). Fluorescence imaging showed a detection sensitivity of 1.6 × 103 counts · sec−1 · μW−1 per picomole of Cy7. All four imaging metrics (percentage of injected dose, SLN extraction, SLN uptake rate constant, and expected peak fluorescence intensity) exhibited higher values (P = .005 to P = .042) in the low-dose group than in the high-dose group; this finding was consistent with receptor-mediated image formation. Conclusion: The multireporter probe 99mTc-labeled Cy7 tilmanocept exhibits in vitro and in vivo receptor-binding properties for successful receptor-targeted SLN mapping with nuclear and optical imaging. © RSNA, 2012 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12120638/-/DC1 PMID:22753678

Discovery of a Series of Acridinones as Mechanism-Based Tubulin Assembly Inhibitors with Anticancer Activity

PubMed Central

Magalhaes, Luma G.; Marques, Fernando B.; da Fonseca, Marina B.; Rogério, Kamilla R.; Graebin, Cedric S.; Andricopulo, Adriano D.

2016-01-01

Microtubules play critical roles in vital cell processes, including cell growth, division, and migration. Microtubule-targeting small molecules are chemotherapeutic agents that are widely used in the treatment of cancer. Many of these compounds are structurally complex natural products (e.g., paclitaxel, vinblastine, and vincristine) with multiple stereogenic centers. Because of the scarcity of their natural sources and the difficulty of their partial or total synthesis, as well as problems related to their bioavailability, toxicity, and resistance, there is an urgent need for novel microtubule binding agents that are effective for treating cancer but do not have these disadvantages. In the present work, our lead discovery effort toward less structurally complex synthetic compounds led to the discovery of a series of acridinones inspired by the structure of podophyllotoxin, a natural product with important microtubule assembly inhibitory activity, as novel mechanism-based tubulin assembly inhibitors with potent anticancer properties and low toxicity. The compounds were evaluated in vitro by wound healing assays employing the metastatic and triple negative breast cancer cell line MDA-MB-231. Four compounds with IC50 values between 0.294 and 1.7 μM were identified. These compounds showed selective cytotoxicity against MDA-MB-231 and DU-145 cancer cell lines and promoted cell cycle arrest in G2/M phase and apoptosis. Consistent with molecular modeling results, the acridinones inhibited tubulin assembly in in vitro polymerization assays with IC50 values between 0.9 and 13 μM. Their binding to the colchicine-binding site of tubulin was confirmed through competitive assays. PMID:27508497

Evidence of the neuron-restrictive silencer factor (NRSF) interaction with Sp3 and its synergic repression to the mu opioid receptor (MOR) gene

PubMed Central

Kim, Chun Sung; Choi, Hack Sun; Hwang, Cheol Kyu; Song, Kyu Young; Lee, Byung-Kwon; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

2006-01-01

Previously, we reported that the neuron-restrictive silencer element (NRSE) of mu opioid receptor (MOR) functions as a critical regulator to repress the MOR transcription in specific neuronal cells, depending on neuron-restriction silence factor (NRSF) expression levels [C.S.Kim, C.K.Hwang, H.S.Choi, K.Y.Song, P.Y.Law, L.N.Wei and H.H.Loh (2004) J. Biol. Chem., 279, 46464–46473]. Herein, we identify a conserved GC sequence next to NRSE region in the mouse MOR gene. The inhibition of Sp family factors binding to this GC box by mithramycin A led to a significant increase in the endogenous MOR transcription. In the co-immunoprecipitation experiment, NRSF interacted with the full-length Sp3 factor, but not with Sp1 or two short Sp3 isoforms. The sequence specific and functional binding by Sp3 at this GC box was confirmed by in vitro gel-shift assays using either in vitro translated proteins or nuclear extract, and by in vivo chromatin immunoprecipitation assays. Transient transfection assays showed that Sp3-binding site of the MOR gene is a functionally synergic repressor element with NRSE in NS20Y cells, but not in the NRSF negative PC12 cells. The results suggest that the synergic interaction between NRSF and Sp3 is required to negatively regulate MOR gene transcription and that transcription of MOR gene would be governed by the context of available transcription factors rather than by a master regulator. PMID:17130167

A 3'-untranslated region (3'UTR) induces organ adhesion by regulating miR-199a* functions.

PubMed

Lee, Daniel Y; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W; Deng, Zhaoqun; Yang, Burton B

2009-01-01

Mature microRNAs (miRNAs) are single-stranded RNAs of 18-24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3'UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3'UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3'UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3'UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3'UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3'UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3'UTR may be an approach in the development of gene therapy.

Role of the Adenovirus DNA-Binding Protein in In Vitro Adeno-Associated Virus DNA Replication

PubMed Central

Ward, Peter; Dean, Frank B.; O’Donnell, Michael E.; Berns, Kenneth I.

1998-01-01

A basic question in adeno-associated virus (AAV) biology has been whether adenovirus (Ad) infection provided any function which directly promoted replication of AAV DNA. Previously in vitro assays for AAV DNA replication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demonstrated that Ad infection provides a direct helper effect for AAV DNA replication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of direct participation of Ad proteins in AAV DNA replication. In this report, we show that in the in vitro assay, enhancement of processivity occurs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [RPA]). Clearly Ad-DBP is present after Ad infection but not before, whereas the cellular level of RPA is not apparently affected by Ad infection. However, we have not measured possible modifications of RPA which might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmid vector, RPA was not an effective substitute for Ad-DBP. Extracts supplemented with Ad-DBP preferentially replicated AAV sequences rather than adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences. PMID:9420241

High-Throughput Screens To Identify Autophagy Inducers That Function by Disrupting Beclin 1/Bcl-2 Binding.

PubMed

Chiang, Wei-Chung; Wei, Yongjie; Kuo, Yi-Chun; Wei, Shuguang; Zhou, Anwu; Zou, Zhongju; Yehl, Jenna; Ranaghan, Matthew J; Skepner, Adam; Bittker, Joshua A; Perez, Jose R; Posner, Bruce A; Levine, Beth

2018-06-21

Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. We sought to establish a method for developing new chemical compounds that specifically induce autophagy. To do this, we developed two assays to identify compounds that target a key regulatory node of autophagy induction-specifically, the binding of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation). These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2 binding in vitro. We screened two different chemical compound libraries, comprising ∼300 K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were identified that directly inhibit Beclin 1/Bcl-2 interaction with an IC 50 in the micromolar range and increase autophagic flux. These compounds do not demonstrate significant cytotoxicity, and they exert selectivity for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared with the BH3 domain of the pro-apoptotic Bcl-2 family members, Bax and Bim. Thus, we have identified candidate molecules that serve as lead templates for developing potent and selective Beclin 1/Bcl-2 inhibitors that may be clinically useful as autophagy-inducing agents.

A mutagenic analysis of the RNase mechanism of the bacterial Kid toxin by mass spectrometry.

PubMed

Diago-Navarro, Elizabeth; Kamphuis, Monique B; Boelens, Rolf; Barendregt, Arjan; Heck, Albert J; van den Heuvel, Robert H; Díaz-Orejas, Ramón

2009-09-01

Kid, the toxin of the parD (kis, kid) maintenance system of plasmid R1, is an endoribonuclease that preferentially cleaves RNA at the 5' of A in the core sequence 5'-UA(A/C)-3'. A model of the Kid toxin interacting with the uncleavable mimetic 5'-AdUACA-3' is available. To evaluate this model, a significant collection of mutants in some of the key residues proposed to be involved in RNA binding (T46, A55, T69 and R85) or RNA cleavage (R73, D75 and H17) were analysed by mass spectrometry in RNA binding and cleavage assays. A pair of substrates, 5'-AUACA-3', and its uncleavable mimetic 5'-AdUACA-3', used to establish the model and structure of the Kid-RNA complex, were used in both the RNA cleavage and binding assays. A second RNA substrate, 5'-UUACU-3' efficiently cleaved by Kid both in vivo and in vitro, was also used in the cleavage assays. Compared with the wild-type protein, mutations in the residues of the catalytic site abolished RNA cleavage without substantially altering RNA binding. Mutations in residues proposed to be involved in RNA binding show reduced binding efficiency and a corresponding decrease in RNA cleavage efficiency. The cleavage profiles of the different mutants were similar with the two substrates used, but RNA cleavage required much lower protein concentrations when the 5'-UUACU-3' substrate was used. Protein synthesis and growth assays are consistent with there being a correlation between the RNase activity of Kid and its inhibitory potential. These results give important support to the available models of Kid RNase and the Kid-RNA complex.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

PubMed

Nuñez, S B; Medin, J A; Braissant, O; Kemp, L; Wahli, W; Ozato, K; Segars, J H

1997-03-14

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

The Activation of the Rat Insulin Gene II by BETA2 and PDX-1 in Rat Insulinoma Cells Is Repressed by Pax6

PubMed Central

Wolf, Gabriele; Hessabi, Behnam; Karkour, Anke; Henrion, Ulrike; Dahlhaus, Meike; Ostmann, Annett; Giese, Bernd; Fraunholz, Martin; Grabarczyk, Piotr; Jack, Robert; Walther, Reinhard

2010-01-01

The transcriptional transactivator Pax6 binds the pancreatic islet cell-specific enhancer sequence (PISCES) of the rat insulin I gene. However the human, mouse, and rat insulin gene II promoters do not contain a PISCES element. To analyze the role of Pax6 in those PISCES-less promoters, we investigated its influence on rat insulin gene II expression and included in our studies the main activators: pancreatic and duodenal homeobox protein-1 (PDX-1) and BETA2/E47. Luciferase assays, Northern blots, and RIA were used to study effects of Pax6 overexpression, gel shift and chromatin precipitation assays to study its binding to the DNA, and yeast two-hybrid assays and glutathione S transferase capture assays to investigate its interactions with PDX-1 and BETA2. Finally, glucose-dependent intracellular transport of Pax6 was demonstrated by fluorescence microscopy. Overexpression of Pax6 prevents activation of the rat insulin II gene by BETA2 and PDX-1 and hence suppresses insulin synthesis and secretion. In vitro, Pax6 binds to the A-boxes, thereby blocking binding of PDX-1, and at the same time, its paired domain interacts with BETA2. Fluorescence microscopy demonstrated that the nuclear-cytoplasmic localization of Pax6 and PDX-1 are oppositely regulated by glucose. From the results, it is suggested that at low concentrations of glucose, Pax6 is localized in the nucleus and prevents the activation of the insulin gene by occupying the PDX-1 binding site and by interacting with BETA2. PMID:20943817

Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

PubMed Central

Hansen, Randi Westh; Wang, Xiaole; Golab, Agnieszka; Bornert, Olivier; Oswald, Christine; Wagner, Renaud; Martinez, Karen Laurence

2016-01-01

Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors—a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption. PMID:27035823

Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization.

PubMed

Okamoto-Uchida, Yoshimi; Nakamura, Ryosuke; Matsuzawa, Yumiko; Soma, Megumi; Kawakami, Hiroshi; Ishii-Watabe, Akiko; Nishimaki-Mogami, Tomoko; Teshima, Reiko; Saito, Yoshiro

2016-01-01

The physicochemical nature of allergen molecules differ from the liquid phase to the solid phase. However, conventional allergy tests are based on the detection of immunoglobulin (Ig)E binding to immobilized allergens. We recently developed an in vitro allergy testing method using a luciferase-reporting humanized rat mast cell line to detect IgE crosslinking-induced luciferase expression (EXiLE test). The aim of the present study was to evaluate the effects of antigen immobilization on the results of different in vitro allergy tests using two anti-ovalbumin (OVA) antibodies (Abs), E-C1 and E-G5, with different properties in the OVA-induced allergic reaction. Both Abs showed clear binding to OVA with an enzyme-linked immunosorbent assay and by BIAcore analysis. However, only E-C1 potentiated EXiLE response for the liquid-phase OVA. On the other hand, OVA immobilized on solid-phase induced EXiLE responses in both E-C1 Ab- and E-G5 Ab-sensitized mast cells. Western blotting of OVA indicated that E-C1 Ab binds both to OVA monomers and dimers, unlike E-G5 Ab, which probably binds only to the OVA dimer. These results suggest that antigen immobilization enhanced IgE crosslinking ability through multimerization of allergen molecules in the solid phase, resulting in an increase in false positives in IgE binding-based conventional in vitro allergy tests. These findings shed light on the physicochemical nature of antigens as an important factor for the development and evaluation of in vitro allergy tests and suggest that mast cell activation-based allergy testing with liquid-phase allergens is a promising strategy to evaluate the physiological interactions of IgE and allergens.

In vitro selection using a dual RNA library that allows primerless selection

PubMed Central

Jarosch, Florian; Buchner, Klaus; Klussmann, Sven

2006-01-01

High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC50 of 4.5 nM at 37°C. PMID:16855281

Protein and lipid binding parameters in rainbow trout (Oncorhynchus mykiss) blood and liver fractions to extrapolate from an in vitro metabolic degradation assay to in vivo bioaccumulation potential of hydrophobic organic chemicals.

PubMed

Escher, Beate I; Cowan-Ellsberry, Christina E; Dyer, Scott; Embry, Michelle R; Erhardt, Susan; Halder, Marlies; Kwon, Jung-Hwan; Johanning, Karla; Oosterwijk, Mattheus T T; Rutishauser, Sibylle; Segner, Helmut; Nichols, John

2011-07-18

Binding of hydrophobic chemicals to colloids such as proteins or lipids is difficult to measure using classical microdialysis methods due to low aqueous concentrations, adsorption to dialysis membranes and test vessels, and slow kinetics of equilibration. Here, we employed a three-phase partitioning system where silicone (polydimethylsiloxane, PDMS) serves as a third phase to determine partitioning between water and colloids and acts at the same time as a dosing device for hydrophobic chemicals. The applicability of this method was demonstrated with bovine serum albumin (BSA). Measured binding constants (K(BSAw)) for chlorpyrifos, methoxychlor, nonylphenol, and pyrene were in good agreement with an established quantitative structure-activity relationship (QSAR). A fifth compound, fluoxypyr-methyl-heptyl ester, was excluded from the analysis because of apparent abiotic degradation. The PDMS depletion method was then used to determine partition coefficients for test chemicals in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (K(S9w)) and blood plasma (K(bloodw)). Measured K(S9w) and K(bloodw) values were consistent with predictions obtained using a mass-balance model that employs the octanol-water partition coefficient (K(ow)) as a surrogate for lipid partitioning and K(BSAw) to represent protein binding. For each compound, K(bloodw) was substantially greater than K(S9w), primarily because blood contains more lipid than liver S9 fractions (1.84% of wet weight vs 0.051%). Measured liver S9 and blood plasma binding parameters were subsequently implemented in an in vitro to in vivo extrapolation model to link the in vitro liver S9 metabolic degradation assay to in vivo metabolism in fish. Apparent volumes of distribution (V(d)) calculated from the experimental data were similar to literature estimates. However, the calculated binding ratios (f(u)) used to relate in vitro metabolic clearance to clearance by the intact liver were 10 to 100 times lower than values used in previous modeling efforts. Bioconcentration factors (BCF) predicted using the experimental binding data were substantially higher than the predicted values obtained in earlier studies and correlated poorly with measured BCF values in fish. One possible explanation for this finding is that chemicals bound to proteins can desorb rapidly and thus contribute to metabolic turnover of the chemicals. This hypothesis remains to be investigated in future studies, ideally with chemicals of higher hydrophobicity. © 2011 American Chemical Society

Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets.

PubMed

Nelson, Christopher S; Fuller, Chris K; Fordyce, Polly M; Greninger, Alexander L; Li, Hao; DeRisi, Joseph L

2013-07-01

The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein's DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2's-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved.

Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets

PubMed Central

Nelson, Christopher S.; Fuller, Chris K.; Fordyce, Polly M.; Greninger, Alexander L.; Li, Hao; DeRisi, Joseph L.

2013-01-01

The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein’s DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2’s-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved. PMID:23625967

The gammaPE complex contains both SATB1 and HOXB2 and has positive and negative roles in human gamma-globin gene regulation.

PubMed

Case, S S; Huber, P; Lloyd, J A

1999-11-01

A large nuclear protein complex, termed gammaPE (for gamma-globin promoter and enhancer binding factor), binds to five sites located 5' and 3' of the human y-globin gene. Two proteins, SATB1 (special A-T-rich binding protein 1) and HOXB2, can bind to yPE binding sites. SATB1 binds to nuclear matrix-attachment sites, and HOXB2 is a homeodomain protein important in neural development that is also expressed during erythropoiesis. The present work showed that antisera directed against either SATB1 or HOXB2 reacted specifically with the entire gammaPE complex in electrophoretic mobility shift assays (EMSAs), suggesting that the two proteins can bind to the gammaPE binding site simultaneously. When SATB1 or HOXB2 was expressed in vitro, they could bind independently to gammaPE binding sites in EMSA. Interestingly, the proteins expressed in vitro competed effectively with each other for the gammaPE binding site, suggesting that this may occur under certain conditions in vivo. Transient cotransfections of a HOXB2 cDNA and a y-globin-luciferase reporter gene construct into cells expressing SATB1 suggested that SATB1 has a positive and HOXB2 a negative regulatory effect on transcription. Taking into account their potentially opposing effects and binding activities, SATB1 and HOXB2 may modulate the amount of gamma-globin mRNA expressed during development and differentiation.

Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

PubMed Central

Brázdová, Marie; Navrátilová, Lucie; Tichý, Vlastimil; Němcová, Kateřina; Lexa, Matej; Hrstka, Roman; Pečinka, Petr; Adámik, Matej; Vojtesek, Borivoj; Paleček, Emil; Deppert, Wolfgang; Fojta, Miroslav

2013-01-01

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed. PMID:23555710

Progranulin Directly Binds to the CRD 2 and CRD3 of TNFR Extracellular Domains

PubMed Central

Jian, Jinlong; Zhao, Shuai; Tian, Qingyun; Gonzalez-Gugel, Elena; Mundra, Jyoti Joshi; Uddin, Sardar MZ; Liu, Ben; Richbourgh, Brendon; Brunetti, Ryan; Liu, Chuan-ju

2013-01-01

We previously reported that PGRN directly bound to TNF receptors (TNFR) in vitro and in chondrocytes (Tang, et al, Science, 2011). Here we report that PGRN also associated with TNFR in splenocytes, and inhibited the binding of TNFα to immune cells. Proper folding of PGRN is essential for its binding to TNFR, as DTT treatment abolished its binding to TNFR. In contrast, the binding of PGRN to Sortilin was enhanced by DTT. Protein interaction assays with mutants of the TNFR extracellular domain demonstrated that CRD2 and CRD3 of TNFR are important for the interaction with PGRN, similar to the binding to TNFα. Taken together, these findings provide the molecular basis underlying PGRN/TNFR interaction and PGRN-mediated anti-inflammatory activity in various autoimmune diseases and conditions. PMID:24070898

In vitro CPC retention and VSC adsorption by IPM oil droplets: possible mechanisms of action of a two phase mouthwash.

PubMed

Sterer, N; Slutzky, H; Kohavi, D; Matalon, S

2013-09-01

Two phase oil-water mouthwash has been previously shown to efficiently bind oral microorganisms, relying on their cell surface hydrophobicity. The aim of the present in vitro study was to test the cetylpyridinium chloride (CPC) retention and volatile sulfide compounds (VSCs) adsorption abilities of the oil droplets created by mixing of a two phase oil-water solution. VSC adsorption was assayed using a salivary incubation assay and garlic powder solutions, and demonstrated using microscopic sulfide assay. CPC retention was assayed by kinetic and endpoint measurement of Streptococcus salivarius outgrowth using microplate (ELISA) reader. Results showed that the isopropyl myristate (IPM) oil droplets in the two phase solutions were able to adsorb 68-80% of VSCs. CPC at a concentration of 0.05% was most affectively retained by the oil droplets showing a significantly increase in residual antibacterial activity against Streptococcus salivarius. These results taken together, suggests that VSC adsorption and CPC retention by IPM oil droplets may be two additional mechanisms in the activity of the two phase mouthwash formulation.

In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors.

PubMed

García-Garayoa, E; Allemann-Tannahill, L; Bläuenstein, P; Willmann, M; Carrel-Rémy, N; Tourwé, D; Iterbeke, K; Conrath, P; Schubiger, P A

2001-01-01

The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [99mTc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein.

PubMed

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S; Prasad, Manoj

2011-10-01

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein

PubMed Central

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S

2011-01-01

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncation of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T] [T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein. PMID:21918373

Radioreceptor assay for analysis of fentanyl and its analogs in biological samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alburges, M.E.

The assay is based on the competition of these drugs with ({sup 3}H) fentanyl for opioid receptors in membrane preparations of rat forebrain in vitro. The binding in stereospecific, reversible and saturable. Scatchard plots of saturation suggest the presence of high and low affinity binding sites. Morphine and hydromorphone complete with ({sup 3}H)fentanyl for the opioid receptor, but other morphine-like compounds were relatively weak displacers of ({sup 3}H)fentanyl. Many other commonly abused drugs do not compete with ({sup 3}H)fentanyl for the opioid receptors. Urine samples from animals injected with fentanyl, ({plus minus})-cis-3-methylfentanyl, alpha-methylfentanyl, butyrylfentanyl and benzylfentanyl were analyzed by radioreceptormore » assay, radioimmunoassay, and gas chromatography/mass spectrometry. Urinary analysis of fentanyl showed a good correlation with these three methods; however, discrepancies were observed in the analysis of fentanyl analogs. This radioreceptor assay is well-suited as an initial assay for the detection of active analogs of fentanyl in urine with good correlation with other techniques in the analysis of fentanyl; however, there is substantial disagreement between techniques in the quantitation of fentanyl analogs. The implications of these discrepancies are discussed.« less

A Second Las17 Monomeric Actin-Binding Motif Functions in Arp2/3-Dependent Actin Polymerization During Endocytosis

PubMed Central

Feliciano, Daniel; Tolsma, Thomas O.; Farrell, Kristen B.; Aradi, Al; Di Pietro, Santiago M.

2018-01-01

During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME. PMID:25615019

Crohn's Disease Variants of Nod2 Are Stabilized by the Critical Contact Region of Hsp70.

PubMed

Schaefer, Amy K; Wastyk, Hannah C; Mohanan, Vishnu; Hou, Ching-Wen; Lauro, Mackenzie L; Melnyk, James E; Burch, Jason M; Grimes, Catherine L

2017-08-29

Nod2 is a cytosolic, innate immune receptor responsible for binding to bacterial cell wall fragments such as muramyl dipeptide (MDP). Upon binding, subsequent downstream activation of the NF-κB pathway leads to an immune response. Nod2 mutations are correlated with an increased susceptibility to Crohn's disease (CD) and ultimately result in a misregulated immune response. Previous work had demonstrated that Nod2 interacts with and is stabilized by the molecular chaperone Hsp70. In this work, it is shown using purified protein and in vitro biochemical assays that the critical Nod2 CD mutations (G908R, R702W, and 1007fs) preserve the ability to bind bacterial ligands. A limited proteolysis assay and luciferase reporter assay reveal regions of Hsp70 that are capable of stabilizing Nod2 and rescuing CD mutant activity. A minimal 71-amino acid subset of Hsp70 that stabilizes the CD-associated variants of Nod2 and restores a proper immune response upon activation with MDP was identified. This work suggests that CD-associated Nod2 variants could be stabilized in vivo with a molecular chaperone.

Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

PubMed

Schütze, Tonio; Ulrich, Alexander K C; Apelt, Luise; Will, Cindy L; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C

2016-02-01

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing. © 2016 Schütze et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG).

PubMed

Chen, Hanyong; Yao, Ke; Chang, Xiaoyu; Shim, Jung-Hyun; Kim, Hong-Gyum; Malakhova, Margarita; Kim, Dong-Joon; Bode, Ann M; Dong, Zigang

2015-01-01

The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.

Integrated In Silico-In Vitro Identification and Characterization of the SH3-Mediated Interaction between Human PTTG and its Cognate Partners in Medulloblastoma.

PubMed

Liu, Jiangang; Wang, Dapeng; Li, Yanyan; Yao, Hui; Zhang, Nan; Zhang, Xuewen; Zhong, Fangping; Huang, Yulun

2018-06-01

The human pituitary tumor-transforming gene is an oncogenic protein which serves as a central hub in the cellular signaling network of medulloblastoma. The protein contains two vicinal PxxP motifs at its C terminus that are potential binding sites of peptide-recognition SH3 domains. Here, a synthetic protocol that integrated in silico analysis and in vitro assay was described to identify the SH3-binding partners of pituitary tumor-transforming gene in the gene expression profile of medulloblastoma. In the procedure, a variety of structurally diverse, non-redundant SH3 domains with high gene expression in medulloblastoma were compiled, and their three-dimensional structures were either manually retrieved from the protein data bank database or computationally modeled through bioinformatics technique. The binding capability of these domains towards the two PxxP-containing peptides m1p: 161 LGPPSPVK 168 and m2p: 168 KMPSPPWE 175 of pituitary tumor-transforming gene were ranked by structure-based scoring and fluorescence-based assay. Consequently, a number of SH3 domains, including MAP3K and PI3K, were found to have moderate or high affinity for m1p and/or m2p. Interestingly, the two overlapping peptides exhibits a distinct binding profile to these identified domain partners, suggesting that the binding selectivity of m1p and m2p is optimized across the medulloblastoma expression spectrum by competing for domain candidates. In addition, two redesigned versions of m1p peptide ware obtained via a structure-based rational mutation approach, which exhibited an increased affinity for the domain as compared to native peptide.

Brevenal is a natural inhibitor of brevetoxin action in sodium channel receptor binding assays.

PubMed

Bourdelais, Andrea J; Campbell, Susan; Jacocks, Henry; Naar, Jerome; Wright, Jeffery L C; Carsi, Jigani; Baden, Daniel G

2004-08-01

1. Florida red tides produce profound neurotoxicity that is evidenced by massive fish kills, neurotoxic shellfish poisoning, and respiratory distress. Red tides vary in potency, potency that is not totally governed by toxin concentration. The purpose of the study was to understand the variable potency of red tides by evaluating the potential for other natural pharmacological agents which could modulate or otherwise reduce the potency of these lethal environmental events. 2. A synaptosome binding preparation with 3-fold higher specific brevetoxin binding was developed to detect small changes in toxin binding in the presence of potential antagonists. Rodent brain labeled in vitro with tritiated brevetoxin shows high specific binding in the cerebellum as evidenced by autoradiography. Synaptosome binding assays employing cerebellum-derived synaptosomes illustrate 3-fold increased specific binding. 3. A new polyether natural product from Florida's red tide dinoflagellate Karenia brevis, has been isolated and characterized. Brevenal, as the nontoxic natural product is known, competes with tritiated brevetoxin for site 5 associated with the voltage-sensitive sodium channel (VSSC). Brevenal displacement of specific brevetoxin binding is purely competitive in nature. 4. Brevenal, obtained from either laboratory cultures or field collections during a red tide, protects fish from the neurotoxic effects of brevetoxin exposure. 5. Brevenal may serve as a model compound for the development of therapeutics to prevent or reverse intoxication in red tide exposures.

Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor

PubMed Central

Assadi-Porter, Fariba M.; Maillet, Emeline L.; Radek, James T.; Quijada, Jeniffer; Markley, John L.; Max, Marianna

2010-01-01

The sweet protein brazzein activates the human sweet receptor, a heterodimeric G-protein coupled receptor (GPCR) composed of subunits T1R2 and T1R3. In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by the in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: Site 1 (Loop43), Site 2 (N- and C-terminus and adjacent Glu36, Loop33), and Site 3 (Loop9–19). Basic residues in Site 1 and acidic residues in Site 2 were essential for positive responses from each assay. Mutation of Y39A (Site 1) greatly reduced positive responses. A bulky side chain at position 54 (Site 2), rather than a side chain with hydrogen bonding potential, was required for positive responses as was the presence of the native disulfide bond in Loop 9–19 (Site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus fly trap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in the brazzein response. The exception, hT1R2:R217A-hT1R3, which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site in involved in subunit-subunit interaction rather than direct brazzein binding. Results from this study support a multipoint interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models. PMID:20302879

Use of a benzimidazole derivative BF-188 in fluorescence multispectral imaging for selective visualization of tau protein fibrils in the Alzheimer's disease brain.

PubMed

Harada, Ryuichi; Okamura, Nobuyuki; Furumoto, Shozo; Yoshikawa, Takeo; Arai, Hiroyuki; Yanai, Kazuhiko; Kudo, Yukitsuka

2014-02-01

Selective visualization of amyloid-β and tau protein deposits will help to understand the pathophysiology of Alzheimer's disease (AD). Here, we introduce a novel fluorescent probe that can distinguish between these two deposits by multispectral fluorescence imaging technique. Fluorescence spectral analysis was performed using AD brain sections stained with novel fluorescence compounds. Competitive binding assay using [(3)H]-PiB was performed to evaluate the binding affinity of BF-188 for synthetic amyloid-β (Aβ) and tau fibrils. In AD brain sections, BF-188 clearly stained Aβ and tau protein deposits with different fluorescence spectra. In vitro binding assays indicated that BF-188 bound to both amyloid-β and tau fibrils with high affinity (K i  < 10 nM). In addition, BF-188 showed an excellent blood-brain barrier permeability in mice. Multispectral imaging with BF-188 could potentially be used for selective in vivo imaging of tau deposits as well as amyloid-β in the brain.

GATA-1 directly regulates Nanog in mouse embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Wen-Zhong; Ai, Zhi-Ying; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling 712100

2015-09-25

Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation.more » Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression. - Highlights: • The Nanog proximal promoter conceives functional element for GATA-1. • GATA-1 occupies the Nanog proximal promoter in vitro and in vivo. • GATA-1 transcriptionally suppresses Nanog.« less

Sequence, Structure, and Context Preferences of Human RNA Binding Proteins.

PubMed

Dominguez, Daniel; Freese, Peter; Alexis, Maria S; Su, Amanda; Hochman, Myles; Palden, Tsultrim; Bazile, Cassandra; Lambert, Nicole J; Van Nostrand, Eric L; Pratt, Gabriel A; Yeo, Gene W; Graveley, Brenton R; Burge, Christopher B

2018-06-07

RNA binding proteins (RBPs) orchestrate the production, processing, and function of mRNAs. Here, we present the affinity landscapes of 78 human RBPs using an unbiased assay that determines the sequence, structure, and context preferences of these proteins in vitro by deep sequencing of bound RNAs. These data enable construction of "RNA maps" of RBP activity without requiring crosslinking-based assays. We found an unexpectedly low diversity of RNA motifs, implying frequent convergence of binding specificity toward a relatively small set of RNA motifs, many with low compositional complexity. Offsetting this trend, however, we observed extensive preferences for contextual features distinct from short linear RNA motifs, including spaced "bipartite" motifs, biased flanking nucleotide composition, and bias away from or toward RNA structure. Our results emphasize the importance of contextual features in RNA recognition, which likely enable targeting of distinct subsets of transcripts by different RBPs that recognize the same linear motif. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Discovery of DNA dyes Hoechst 34580 and 33342 as good candidates for inhibiting amyloid beta formation: in silico and in vitro study

NASA Astrophysics Data System (ADS)

Thai, Nguyen Quoc; Tseng, Ning-Hsuan; Vu, Mui Thi; Nguyen, Tin Trung; Linh, Huynh Quang; Hu, Chin-Kun; Chen, Yun-Ru; Li, Mai Suan

2016-08-01

Combining Lipinski's rule with the docking and steered molecular dynamics simulations and using the PubChem data base of about 1.4 million compounds, we have obtained DNA dyes Hoechst 34580 and Hoechst 33342 as top-leads for the Alzheimer's disease. The binding properties of these ligands to amyloid beta (Aβ) fibril were thoroughly studied by in silico and in vitro experiments. Hoechst 34580 and Hoechst 33342 prefer to locate near hydrophobic regions with binding affinity mainly governed by the van der Waals interaction. By the Thioflavin T assay, it was found that the inhibition constant IC50 ≈ 0.86 and 0.68 μM for Hoechst 34580 and Hoechst 33342, respectively. This result qualitatively agrees with the binding free energy estimated using the molecular mechanic-Poisson Boltzmann surface area method and all-atom simulations with the AMBER-f99SB-ILDN force field and water model TIP3P. In addition, DNA dyes have the high capability to cross the blood brain barrier. Thus, both in silico and in vitro experiments have shown that Hoechst 34580 and 33342 are good candidates for treating the Alzheimer's disease by inhibiting Aβ formation.

Identification of a 27.8 kDa protein from flounder gill cells involved in lymphocystis disease virus binding and infection.

PubMed

Wang, Mu; Sheng, Xiu-Zhen; Xing, Jing; Tang, Xiao-Qian; Zhan, Wen-Bin

2011-03-16

In vitro, lymphocystis disease virus (LCDV) infection of flounder gill (FG) cell cultures causes obvious cytopathic effect (CPE). We describe attempts to isolate and characterize the LCDV-binding molecule(s) on the plasma membrane of FG cells that were responsible for virus entry. The results showed that the co-immunoprecipitation assay detected a 27.8 kDa molecule from FG cells that bound to LCDV. In a blocking ELISA, pre-incubation of FG cell membrane proteins with the specific antiserum developed against the 27.8 kDa protein could block LCDV binding. Similarly, antiserum against 27.8 kDa protein could also inhibit LCDV infection of FG cells in vitro. Mass spectrometric analysis established that the 27.8 kDa protein and beta-actin had a strong association. These results strongly supported the possibility that the 27.8 kDa protein was the putative receptor specific for LCDV infection of FG cells.

Pulse seed germination improves antioxidative activity of phenolic compounds in stripped soybean oil-in-water emulsions.

PubMed

Xu, Minwei; Jin, Zhao; Peckrul, Allen; Chen, Bingcan

2018-06-01

The purpose of this study was to investigate antioxidative activity of phenolic compounds extracted from germinated pulse seed including chickpeas, lentils and yellow peas. Phenolic compounds were extracted at different germination time and total phenolic content was examined by Folin Ciocalteu's reaction. Antioxidative activity of extracts was characterized by in vitro assay including 2, 2-diphenyl-1-picrylhydrazyl radical scavenging capacity (DPPH), oxygen radical absorbance capacity (ORAC), iron-binding assay, and in stripped soybean oil-in-water emulsions. The results suggested that germination time is critical for phenolic compounds production. The form variation of phenolic compounds influenced the antioxidative activity of phenolic compounds both in vitro assay and in emulsion systems. Soluble bound phenolic compounds showed higher antioxidative ability in emulsion system with the order of chickpea > yellow pea > lentil. On the basis of these results, soluble bound phenolic compounds may be considered as a promising natural antioxidant to prevent lipid oxidation in foods. Copyright © 2018 Elsevier Ltd. All rights reserved.

Tn552 transposase purification and in vitro activities.

PubMed Central

Rowland, S J; Sherratt, D J; Stark, W M; Boocock, M R

1995-01-01

The Staphylococcus aureus transposon Tn552 encodes a protein (p480) containing the 'D,D(35)E' motif common to retroviral integrases and the transposases of a number of bacterial elements, including phage Mu, the integron-containing element Tn5090, Tn7 and IS3. p480 and a histidine-tagged derivative were overexpressed in Escherichia coli and purified by methods involving denaturation and renaturation. DNase I footprinting and gel binding assays demonstrated that p480 binds to two adjacent, directly repeated 23 bp motifs at each end of Tn552. Although donor strand cleavage by p480 was not detected, in vitro conditions were defined for strand transfer activity with transposon end fragments having pre-cleaved 3' termini. Strand transfer was Mn(2+)-dependent and appeared to join a single left or right end fragment to target DNA. The importance of the terminal dinucleotide CA-3' was demonstrated by mutation. The in vitro activities of p480 are consistent with its proposed function as the Tn552 transposase. Images PMID:7828593

The role of dimension in multivalent binding events: structure-activity relationship of dendritic polyglycerol sulfate binding to L-selectin in correlation with size and surface charge density.

PubMed

Weinhart, Marie; Gröger, Dominic; Enders, Sven; Riese, Sebastian B; Dernedde, Jens; Kainthan, Rajesh K; Brooks, Donald E; Haag, Rainer

2011-08-11

L-, P-, and E-Selectin are cell adhesion molecules that play a crucial role in leukocyte recruitment from the blood stream to the afflicted tissue in an acute and chronic inflammatory setting. Since selectins mediate the initial contact of leukocytes to the vascular endothelium, they have evolved as a valuable therapeutic target in diseases related to inflammation by inhibition of the physiological selectin-ligand interactions. In a previous study, it was demonstrated that dPGS, a fully synthetic heparin analogue, works as an efficient inhibitor towards L- and P-selectin in vitro as well as in vivo. Herein, the focus is directed towards the effect of size and charge density of the polyanion. The efficiency of L-selectin inhibition via an SPR-based in vitro assay and a cell-based flow chamber assay is investigated with dPGS ranging from approximately 4 to 2000 kDa. SPR measurements show that the inhibitory potential of highly sulfated dPGS increases with size and charge density. Thereby, IC(50) values from the micromolar to the low picomolar range are determined. The same tendency could be observed in a cell-based flow chamber assay with three representative dPGS samples. This structure-affinity relationship of dPGS suggests that the strong inhibitory potential of dPGS is not only based on the strong electrostatic interaction with areas of cationic surface potential on L-selectin but is also due to a steric shielding of the carbohydrate binding site by large, flexible dPGS particles. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay

PubMed Central

Sharma, Vasundhara; Jordan, Jennifer J.; Ciribilli, Yari; Resnick, Michael A.; Bisio, Alessandra; Inga, Alberto

2015-01-01

The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD), a chimeric construct containing the TAD derived from p65 was also generated (p50TAD) to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs) where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators. PMID:26147604

A 3′-Untranslated Region (3′UTR) Induces Organ Adhesion by Regulating miR-199a* Functions

PubMed Central

Lee, Daniel Y.; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W.; Deng, Zhaoqun; Yang, Burton B.

2009-01-01

Mature microRNAs (miRNAs) are single-stranded RNAs of 18–24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3′UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3′UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3′UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3′UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3′UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3′UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3′UTR may be an approach in the development of gene therapy. PMID:19223980

New PAH gene promoter KLF1 and 3'-region C/EBPalpha motifs influence transcription in vitro.

PubMed

Klaassen, Kristel; Stankovic, Biljana; Kotur, Nikola; Djordjevic, Maja; Zukic, Branka; Nikcevic, Gordana; Ugrin, Milena; Spasovski, Vesna; Srzentic, Sanja; Pavlovic, Sonja; Stojiljkovic, Maja

2017-02-01

Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50 % in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60 % of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

In vitro and in vivo potency of insulin analogues designed for clinical use.

PubMed

Vølund, A; Brange, J; Drejer, K; Jensen, I; Markussen, J; Ribel, U; Sørensen, A R; Schlichtkrull, J

1991-11-01

Analogues of human insulin designed to have improved absorption properties after subcutaneous injection have been prepared by recombinant DNA technology. Five rapidly absorbed analogues, being predominantly in mono- or di-meric states in the pharmaceutical preparation, and a hexameric analogue with very low solubility at neutral pH and slow absorption, were studied. Receptor binding assays with HEP-G2 cells showed overall agreement with mouse free adipocyte assays. Two analogues, B28Asp and A21Gly + B27Arg + B30Thr-NH2, had nearly the same molar in vitro potency as human insulin. Another two showed increased adipocyte potency and receptor binding, B10Asp 194% and 333% and A8His + B4His + B10Glu + B27His 575% and 511%, while B9Asp + B27Glu showed 29% and 18% and the B25Asp analogue only 0.12% and 0.05% potency. Bioassays in mice or rabbits of the analogues except B25Asp showed that they had the same in vivo potency as human insulin 1.00 IU = 6.00 nmol. Thus the variation had the same in vivo potency as human insulin 1.00 IU = 6.00 nmol. Thus the variation in in vivo potency reflects the differences in receptor binding affinity. Relative to human insulin a low concentration is sufficient for a high affinity analogue to produce a given receptor complex formation and metabolic response. In conclusion, human insulin and analogues with markedly different in vitro potencies were equipotent in terms of hypoglycaemic effect. This is in agreement with the concept that elimination of insulin from blood and its subsequent degradation is mediated by insulin receptors.

Is Congo red an amyloid-specific dye?

PubMed

Khurana, R; Uversky, V N; Nielsen, L; Fink, A L

2001-06-22

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.

Structure-5-HT/D2 Receptor Affinity Relationship in a New Group of 1-Arylpiperazynylalkyl Derivatives of 8-Dialkylamino-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione.

PubMed

Żmudzki, Paweł; Satała, Grzegorz; Chłoń-Rzepa, Grażyna; Bojarski, Andrzej J; Kazek, Grzegorz; Siwek, Agata; Gryboś, Anna; Głuch-Lutwin, Monika; Wesołowska, Anna; Pawłowski, Maciej

2016-10-01

In our previous papers, we have reported that some 8-amino-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione derivatives possessed high affinity and displayed agonistic, partial agonistic, or antagonistic activity for serotonin 5-HT 1A and dopamine D 2 receptors. In order to examine further the influence of the substituent in the position 8 of the purine moiety and the influence of the xanthine core on the affinity for serotonin 5-HT 1A , 5-HT 2A , 5-HT 6 , 5-HT 7 , and dopamine D 2 receptors, two series of 1-arylpiperazynylalkyl derivatives of 8-amino-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione were synthesized. All the final compounds were investigated in in vitro competition binding experiments for the serotonin 5-HT 1A , 5-HT 2A , 5-HT 6 , 5-HT 7 , and dopamine D 2 receptors. The structure-affinity relationships for this group of compounds were discussed. For selected compounds, the functional assays for the 5-HT 1A and D 2 receptors were carried out. The results of the assays indicated that these groups of derivatives possessed antagonistic activity for 5-HT 1A receptors and agonistic, partial agonistic, or antagonistic activity for D 2 receptors. In total, 26 new compounds were synthesized, 20 of which were tested in in vitro binding experiments and 5 were tested in in vitro functional assays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein and Lipid Binding Parameters in Rainbow Trout (Oncorhynchus mykiss) Blood and Liver Fractions to Extrapolate from an in Vitro metabolic Degradation Assay to in Vivo Bioaccumulation Potential of Hydrophobic Organic Chemicals

EPA Science Inventory

Biotransformation reduces the extent to which environmental contaminants accumulate in fish and other aquatic biota. Unfortunately, the tendency for compounds to be metabolized is not easily predicted from physico-chemical properties (e.g., octanol:water partitioning) or an exam...

Novel dengue virus NS2B/NS3 protease inhibitors.

PubMed

Wu, Hongmei; Bock, Stefanie; Snitko, Mariya; Berger, Thilo; Weidner, Thomas; Holloway, Steven; Kanitz, Manuel; Diederich, Wibke E; Steuber, Holger; Walter, Christof; Hofmann, Daniela; Weißbrich, Benedikt; Spannaus, Ralf; Acosta, Eliana G; Bartenschlager, Ralf; Engels, Bernd; Schirmeister, Tanja; Bodem, Jochen

2015-02-01

Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication

PubMed Central

Choong, Oi Kuan; Tejo, Bimo Ario; Omar, Abdul Rahman

2014-01-01

Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 1014 in the virus-inoculated cells to 109 in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. PMID:24707494

Nuclear blebbing of biologically active organoselenium compound towards human cervical cancer cell (HeLa): in vitro DNA/HSA binding, cleavage and cell imaging studies.

PubMed

Rizvi, Masood Ahmad; Zaki, Mehvash; Afzal, Mohd; Mane, Manoj; Kumar, Manjeet; Shah, Bhahwal Ali; Srivastav, Saurabh; Srikrishna, Saripella; Peerzada, Ghulam Mustafa; Tabassum, Sartaj

2015-01-27

New pharmacophore organoselenium compound (1) was designed, synthesized and characterized by various spectroscopic methods (IR, ESI-MS, (1)H, (13)C and (77)Se NMR) and further confirmed by X-ray crystallography. Compound 1 consists of two 3,5-bis(trifluoromethyl)phenyl units which are connected to the selenium atom via the organometallic C-Se bond. In vitro DNA binding studies of 1 was investigated by absorption and emission titration methods which revealed that 1 recognizes the minor groove of DNA in accordance with molecular docking studies with the DNA duplex. Gel electrophoretic assay demonstrates the ability of 1 to cleave pBR322 DNA through hydrolytic process which was further validated by T4 religation assay. To understand the drug-protein interaction of which ultimate molecular target was DNA, the affinity of 1 towards HSA was also investigated by the spectroscopic and molecular modeling techniques which showed hydrophobic interaction in the subdomain IIA of HSA. Furthermore, the intracellular localization of 1 was evidenced by cell imaging studies using HeLa cells. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Solubilization of a membrane protein by combinatorial supercharging.

PubMed

Hajduczki, Agnes; Majumdar, Sudipta; Fricke, Marie; Brown, Isola A M; Weiss, Gregory A

2011-04-15

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

IgMk paraprotein from gammopathy patient can bind to cardiolipin and interfere with coagulation assay: a case report.

PubMed

Wu, Xin-Yao; Yin, Yu-Feng; Teng, Jia-Lin; Zhang, Li-Wei; Yang, Cheng-de

2017-06-23

The monoclonal gammopathies are a group of plasma-cell proliferative disorders characterized by the secretion of monoclonal immunoglobulin (M protein or paraprotein). Some rare cases have revealed the specific affinity of paraprotein as autoantibody. Here we report a patient with monoclonal gammopathy of undetermined significance (MGUS) accompanied by a remarkable increase of anticardiolipin antibody (aCL) and an extensively decreased coagulation factor activity, however, without any clinical signs of antiphospholipid syndrome (APS) and bleeding. Our further investigation indicated that IgMκ paraprotein of this patient possessed an antibody activity against phospholipids so as to bind to cardiolipin and interfere with coagulation assay in vitro. This case might be indicative that an abnormality of coagulation tests, disturbed by IgMκ paraprotein, does not predict a risk of bleeding in this patient.

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis.

PubMed

Wilson, Kris; Webster, Scott P; Iredale, John P; Zheng, Xiaozhong; Homer, Natalie Z; Pham, Nhan T; Auer, Manfred; Mole, Damian J

2017-12-15

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis

NASA Astrophysics Data System (ADS)

Wilson, Kris; Webster, Scott P.; Iredale, John P.; Zheng, Xiaozhong; Homer, Natalie Z.; Pham, Nhan T.; Auer, Manfred; Mole, Damian J.

2018-01-01

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

Automated On-tip Affinity Capture Coupled with Mass Spectrometry to Characterize Intact Antibody-Drug Conjugates from Blood

NASA Astrophysics Data System (ADS)

Li, Ke Sherry; Chu, Phillip Y.; Fourie-O'Donohue, Aimee; Srikumar, Neha; Kozak, Katherine R.; Liu, Yichin; Tran, John C.

2018-05-01

Antibody-drug conjugates (ADCs) present unique challenges for ligand-binding assays primarily due to the dynamic changes of the drug-to-antibody ratio (DAR) distribution in vivo and in vitro. Here, an automated on-tip affinity capture platform with subsequent mass spectrometry analysis was developed to accurately characterize the DAR distribution of ADCs from biological matrices. A variety of elution buffers were tested to offer optimal recovery, with trastuzumab serving as a surrogate to the ADCs. High assay repeatability (CV 3%) was achieved for trastuzumab antibody when captured below the maximal binding capacity of 7.5 μg. Efficient on-tip deglycosylation was also demonstrated in 1 h followed by affinity capture. Moreover, this tip-based platform affords higher throughput for DAR characterization when compared with a well-characterized bead-based method.

Rational design of class I MHC ligands

NASA Astrophysics Data System (ADS)

Rognan, D.; Scapozza, L.; Folkers, G.; Daser, Angelika

1995-04-01

From the knowledge of the three-dimensional structure of a class I MHC protein, several non natural peptides were designed in order to either optimize the interactions of one secondary anchor amino acid with its HLA binding pocket or to substitute the non interacting part with spacer residues. All peptides were synthesized and tested for binding to the class I MHC protein in an in vitro reconstitution assay. As predicted, the non natural peptides present an enhanced binding to the HLA-B27 molecule with respect to their natural parent peptides. This study constitutes the first step towards the rational design of non peptidic MHC ligands that should be very promising tools for the selective immunotherapy of autoimmune diseases.

Docking simulations and in vitro assay unveil potent inhibitory action of papaverine against protein tyrosine phosphatase 1B.

PubMed

Bustanji, Yasser; Taha, Mutasem Omar; Al-Masri, Ihab Mustafa; Mohammad, Mohammad Khalil

2009-04-01

The structural similarity between papaverine and berberine, a known inhibitor of human protein tyrosine phosphatase 1B (h-PTP 1B), prompted us to investigate the potential of papaverine as h-PTP 1B inhibitor. The investigation included simulated docking experiments to fit papaverine into the binding pocket of h-PTP 1B. Papaverine was found to readily dock within the binding pocket of h-PTP 1B in a low energy orientation via an optimal set of attractive interactions. Experimentally, papaverine illustrated potent in vitro inhibitory effect against recombinant h-PTP 1B (IC(50)=1.20 microM). In vivo, papaverine significantly decreased fasting blood glucose level of Balb/c mice. Our findings should encourage screening of other natural alkaloids for possible anti-h-PTP 1B activities.

Evaluation of Phosphatidylserine-Binding Peptides Radiolabeled with Fluorine 18 for in vivo Imaging of Apoptosis

NASA Astrophysics Data System (ADS)

Kapty, Janice Sarah

We currently do not have a clinical method to directly assess apoptosis induced by cancer therapies. Phosphatidylserine (PS) is an attractive target for imaging apoptosis since it is on the exterior of the apoptotic cells and PS externalization is an early marker of apoptosis. PS-binding peptides are an attractive option for developing an imaging probe to detect apoptosis using positron emission tomography. In this study we evaluated binding characteristics of PS-binding peptides for ability to bind to PS, radiolabeled PS-binding peptides with fluorine-18, and performed in vitro and in vivo analysis of 18F radiolabeled PS-binding peptides including biodistribution analysis and dynamic PET imaging in a murine tumor model of apoptosis. Four peptides were evaluated for PS binding characteristics using a plate based assay system, a liposome mimic of cell membrane PS presentation, and a cell assay of apoptosis. The results indicate that all four peptides bind to PS and are specific to apoptotic cells. The widely used 18 F prosthetic group N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) and the recently developed N-[6-(4-[ 18F]fluorobenzylidene) aminooxyhexyl]maleimide ([18F]FBAM) were investigated for radiolabeling of two representative phosphatidylserine-binding peptides. The prosthetic groups were compared with respect to required reaction conditions for optimum labeling, radiolabeling yield and chemoselectivity. The N-terminus labeled product produced by reaction of [18F]SFB with binding peptide LIKKPF was produced in 18% radiochemical yield while no N-terminus labeled product could be isolated following [18F]SFB reaction with PDGLSR. When the peptides were modified by addition of a cysteine residue at the N-terminus they provided almost quantitative radiochemical yields with [18F]FBAM. Results indicate that for the peptides in this study, [18F]FBAM is a more useful prosthetic group compared to [18F]SFB due to its excellent chemo-selectivity and high radiochemical yield. We report the first experiments where PS-binding peptides were radiolabeled with 18F and evaluated as possible radiotracers for imaging apoptosis. We investigated two radio-peptides ([ 18F]FBAM-CLIKKPF and [18F]FBAM-CPGDLSR) in vitro and in vivo as possible radiotracers able to bind to apoptotic cells and to image chemotherapy induced apoptosis.

Fate of wastewater effluent hER-agonists and hER-antagonists during soil aquifer treatment.

PubMed

Otakuye, Conroy; Quanrud, David M; Ela, Wendell P; Wicke, Daniel; Lansey, Kevin E; Arnold, Robert G

2005-04-01

Estrogen activity was measured in wastewater effluent before and after polishing via soil-aquifer treatment (SAT) using both a (hER-beta) competitive binding assay and a transcriptional activation (yeast estrogen screen, YES) assay. From the competitive binding assay, the equivalent 17alpha-ethinylestradiol (EE2) concentration in secondary effluent was 4.7 nM but decreased to 0.22 nM following SAT. The YES assay indicated that the equivalent EE2 concentration in the same effluent sample was below the method-detection limit (<2.5 x 10(-3) nM) but increased to 0.68 nM in effluent polished via SAT processes. It was hypothesized thattest-dependent differences arose because the competitive binding assay responds positively to both estrogen mimics and anti-estrogens; the YES assay responds to estrogen mimics, but test response is inhibited by anti-estrogens. The hypothesis was supported when organics extracted from wastewater effluent inhibited the YES test response to EE2 (anti-estrogenic effect). A similar extract prepared from SAT-polished effluent augmented the EE2 curve (agonist response). When hydrophobic organics in secondary effluent were fractionated, assay results indicated that several physically distinct anti-estrogens were present in the sample. From this work, it is evident that transcription-activation bioassays alone should not be relied upon to measure estrogenic activity in complex environmental samples because the simultaneous presence of both agonists and antagonist compounds can yield false negatives. Multiple in vitro bioassays, sample fractionation or tests designed to measure anti-estrogenic activity can be used to overcome this problem. It is also clear that there are circumstances under which SAT does not completely remove estrogenic activity during municipal wastewater effluent polishing.

Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor.

PubMed

Yin, Donghua; He, Yali; Perera, Minoli A; Hong, Seoung Soo; Marhefka, Craig; Stourman, Nina; Kirkovsky, Leonid; Miller, Duane D; Dalton, James T

2003-01-01

The purposes of the present studies were to examine the androgen receptor (AR) binding ability and in vitro functional activity of multiple series of nonsteroidal compounds derived from known antiandrogen pharmacophores and to investigate the structure-activity relationships (SARs) of these nonsteroidal compounds. The AR binding properties of sixty-five nonsteroidal compounds were assessed by a radioligand competitive binding assay with the use of cytosolic AR prepared from rat prostates. The AR agonist and antagonist activities of high-affinity ligands were determined by the ability of the ligand to regulate AR-mediated transcriptional activation in cultured CV-1 cells, using a cotransfection assay. Nonsteroidal compounds with diverse structural features demonstrated a wide range of binding affinity for the AR. Ten compounds, mainly from the bicalutamide-related series, showed a binding affinity superior to the structural pharmacophore from which they were derived. Several SARs regarding nonsteroidal AR binding were revealed from the binding data, including stereoisomeric conformation, steric effect, and electronic effect. The functional activity of high-affinity ligands ranged from antagonist to full agonist for the AR. Several structural features were found to be determinative of agonist and antagonist activities. The nonsteroidal AR agonists identified from the present studies provided a pool of candidates for further development of selective androgen receptor modulators (SARMs) for androgen therapy. Also, these studies uncovered or confirmed numerous important SARs governing AR binding and functional properties by nonsteroidal molecules, which would be valuable in the future structural optimization of SARMs.

Cytokinin activity of N6-benzyladenine derivatives assayed by interaction with the receptors in planta, in vitro, and in silico.

PubMed

Savelieva, Ekaterina M; Oslovsky, Vladimir E; Karlov, Dmitry S; Kurochkin, Nikolay N; Getman, Irina A; Lomin, Sergey N; Sidorov, Georgy V; Mikhailov, Sergey N; Osolodkin, Dmitry I; Romanov, Georgy A

2018-05-01

Biological effects of hormones in both plants and animals are based on high-affinity interaction with cognate receptors resulting in their activation. The signal of cytokinins, classical plant hormones, is perceived in Arabidopsis by three homologous membrane receptors: AHK2, AHK3, and CRE1/AHK4. To study the cytokinin-receptor interaction, we used 25 derivatives of potent cytokinin N 6 -benzyladenine (BA) with substituents in the purine heterocycle and/or in the side chain. The study was focused primarily on individual cytokinin receptors from Arabidopsis. The main in planta assay system was based on Arabidopsis double mutants retaining only one isoform of cytokinin receptors and harboring cytokinin-sensitive reporter gene. Classical cytokinin biotest with Amaranthus seedlings was used as an additional biotest. In parallel, the binding of ligands to individual cytokinin receptors was assessed in the in vitro test system. Quantitative comparison of results of different assays confirmed the partial similarity of ligand-binding properties of receptor isoforms. Substituents at positions 8 and 9 of adenine moiety, elongated linker up to 4 methylene units, and replacement of N 6 by sulfur or oxygen have resulted in the suppression of cytokinin activity of the derivative toward all receptors. Introduction of a halogen into position 2 of adenine moiety, on the contrary, often increased the ligand activity, especially toward AHK3. Features both common and distinctive of cytokinin receptors in Arabidopsis and Amaranthus were revealed, highlighting species specificity of the cytokinin perception apparatus. Correlations between the extent to which a compound binds to a receptor in vitro and its ability to activate the same receptor in planta were evaluated for each AHK protein. Interaction patterns between individual receptors and ligands were rationalized by structure analysis and molecular docking in sensory modules of AHK receptors. The best correlation between docking scores and specific binding was observed for AHK3. In addition, receptor-specific ligands have been discovered with unique properties to predominantly activate or block distinct cytokinin receptors. These ligands are promising for practical application and as molecular tools in the study of the cytokinin perception by plant cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

PubMed Central

de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; Gomes da Silva, Lúcia Patrícia Bezerra; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Gois Ruiz, Ana Lucia Tasca; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

2015-01-01

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

Participation of Water in the Binding of Estrogen Receptor with Estrogen Responsive Element in vitro.

PubMed

Zhu, Guo-Zhang; Tang, Guo-Qing; Ruan, Kang-Cheng; Gong, Yue-Ting; Zhang, Yong-Lian

1998-01-01

Many reports have showed that bound water was involved in the interaction between/among the macromolecules. However, it has not been reported whether bound water is also involved in the binding of trans-factors and cis-elements in the regulation of the eukaryotic gene trans-cription or not. Preliminary studies have been made on the effect of bound water on the binding of estrogen receptor with estrogen responsive element in vitro. In the gel retardation assay using the cytosol extract of rat uterus as the supplier of estrogen receptor and 32 bp oligonucleotide containing a concensus vitellogenin A(2) ERE as the probe, various cosolvents, such as glycerol, sucrose, N-dimethylformamide and dimethylsulfoxide, were added respectively to the reaction mixture in varying concentrations to regulate the osmotic pressure. The results indicated that the binding of ER-ERE was enhanced with the increase in the final concentration of these individual cosolvents. On the other hand, when the reaction was carried out under an increasing hydrostatic pressure, the ER-ERE binding was decreased sharply. After decompression the binding of ER-ERE was gradually restored to the normal level with the lapse of time. These results suggested that bound water was directly involved in the binding of ER-ERE and may play an important role in the regulation of the eukaryotic gene transcription.

Stability and in vitro toxicity of an infliximab eye drop formulation.

PubMed

Robert, Marie-Claude; Spurr-Michaud, Sandra; Frenette, Mathieu; Young, David; Gipson, Ilene K; Dohlman, Claes H

2014-01-01

The purpose of this study was to develop a novel 10-mg/mL infliximab eye drop, to characterize its physical and biological stability under recommended storage conditions, and to assess the formulation's toxicity to ocular surface epithelium in vitro. Infliximab (10 mg/mL) was reconstituted using equal volumes of sterile water and 1% carboxymethylcellulose artificial tears. Aliquots were stored in either a 4 degrees C refrigerator or -20 degrees C freezer for up to 45 days. Physical stability was assessed through monitoring the solution's appearance, pH, ultraviolet-visible-near infrared absorbance and scattering, as well as protein gel electrophoresis. Biological stability was assayed through binding to tumor necrosis factor-alpha using an enzyme-linked immunosorbent assay. In vitro cytotoxicity to human corneal-limbal epithelial cells was examined following a 4-hour exposure to the study drug. Refrigerated and frozen infliximab eye drops remained clear and colorless for the duration of study. The formulation's pH (7.0) was comparable to that of the artificial tear vehicle alone. Low levels of ultraviolet-visible-near infrared light absorbance and scattering established the lack of protein precipitate after refrigeration or freezing. Protein gel electrophoresis performed under reducing conditions revealed the presence of two main protein bands of approximately 50 kDa and 25 kDa, representing immunoglobulin G heavy and light chains. The migration pattern of the proteins did not change under the different storage conditions and between day 10 and 45 after formulation. Infliximab binding to tumor necrosis factor-alpha remained stable for up to 45 days, with conservation of 101% and 102% of its initial binding activity when refrigerated or frozen, respectively. In vitro human corneal-limbal epithelial cultures showed no increase in cytotoxicity with infliximab treatment when compared to vehicle and culture media controls (P > 0.05). Infliximab can be formulated as an eye drop and remains stable when stored in accordance with current regulations regarding compounded eye drops. The demonstrated physical and biological stability as well as in vitro innocuity of this infliximab eye drop formulation may facilitate future clinical investigation targeting tumor necrosis factor-alpha as a modulator of various ocular surface diseases.

CroFabtrade mark total anti-venom activity measured by SE-HPLC, and anti-PLA(2) activity assayed in vitro at physiological pH.

PubMed

Price, Joseph A; Sanny, Charles G

2007-05-01

One problem in the development and refinement of anti-venoms is ascertaining both overall anti-venom reactivity and which key toxins are neutralized. Here we show by SE-HPLC that the in vitro reaction of CroFab anti-venin with Crotalus atrox venom asymptotically nears completion (>95%) by 11 min at 4 degrees C by following the change in area under chromatographic peaks. The peaks for reactants decrease and the formation of high molecular weight complexes increases with time. To assay the large number of samples a new microplate format phospholipase A(2) (PLA(2)) assay at an initial pH of 7.5 was developed using phosphotidyl choline as the substrate. The change in absorbance is due to the pH change caused by release of fatty acids, and is linear with dilution of enzyme. This choice of substrate limits detection to PLA(2) and nonspecific esterase (if any) activities. The neutralization mixtures show a dose dependent (CroFab anti-venin) inactivation of C. atrox PLA(2) activity approaching a maximum of 85% neutralization. This approach of revealing antibody binding to venom components coupled with enzyme activity measurements is effective and may lead to greater in vitro assessment of antivenin activity in product development, and less routine use of mouse lethality assays.

An exposure-response analysis based on rifampin suggests CYP3A4 induction is driven by AUC: an in vitro investigation.

PubMed

Chang, Cheng; Yang, Xin; Fahmi, Odette A; Riccardi, Keith A; Di, Li; Obach, R Scott

2017-08-01

1. Induction is an important mechanism contributing to drug-drug interactions. It is most commonly evaluated in the human hepatocyte assay over 48-h or 72-h incubation period. However, whether the overall exposure (i.e. Area Under the Curve (AUC) or C ave ) or maximum exposure (i.e. C max ) of the inducer is responsible for the magnitude of subsequent induction has not been thoroughly investigated. Additionally, in vitro induction assays are typically treated as static systems, which could lead to inaccurate induction potency estimation. Hence, European Medicines Agency (EMA) guidance now specifies quantitation of drug levels in the incubation. 2. This work treated the typical in vitro evaluation of rifampin induction as an in vivo system by generating various target engagement profiles, measuring free rifampin concentration over 3 d of incubation and evaluating the impact of these factors on final induction response. 3. This rifampin-based analysis demonstrates that the induction process is driven by time-averaged target engagement (i.e. AUC-driven). Additionally, depletion of rifampin in the incubation medium over 3 d as well as non-specific/specific binding were observed. 4. These findings should help aid the discovery of clinical candidates with minimal induction liability and further expand our knowledge in the quantitative translatability of in vitro induction assays.

Molecular characterization and functional analysis of pheromone binding protein 1 from Cydia pomonella (L.).

PubMed

Tian, Z; Zhang, Y

2016-12-01

A full-length cDNA encoding Cydia pomonella pheromone binding protein 1 (CpomPBP1) was cloned and characterized. CpomPBP1, possessing the typical characteristics of lepidopteran odorant binding proteins, was detected to be specifically expressed in the antennae of male and female moths at the mRNA and protein level. Soluble recombinant CpomPBP1 was subjected to in vitro binding to analyse its binding properties and to search for potentially active semiochemicals. A competitive binding assay showed that three 12-carbon ligands, codlemone, 1-dodecanol and E,E-2,4-dodecadienal, were able to bind to CpomPBP1 in decreasing order of affinity. Moreover, unlike the wild-type CpomPBP1, the C-terminus truncated CpomPBP1 exhibited high affinity to ligands even in an acidic environment, suggesting that the C-terminus plays a role in preventing ligands from binding to CpomPBP1 in a lower pH environment. © 2016 The Royal Entomological Society.

Special AT-rich sequence binding protein 1 promotes tumor growth and metastasis of esophageal squamous cell carcinoma.

PubMed

Ma, Jun; Wu, Kaiming; Zhao, Zhenxian; Miao, Rong; Xu, Zhe

2017-03-01

Esophageal squamous cell carcinoma is one of the most aggressive malignancies worldwide. Special AT-rich sequence binding protein 1 is a nuclear matrix attachment region binding protein which participates in higher order chromatin organization and tissue-specific gene expression. However, the role of special AT-rich sequence binding protein 1 in esophageal squamous cell carcinoma remains unknown. In this study, western blot and quantitative real-time polymerase chain reaction analysis were performed to identify differentially expressed special AT-rich sequence binding protein 1 in a series of esophageal squamous cell carcinoma tissue samples. The effects of special AT-rich sequence binding protein 1 silencing by two short-hairpin RNAs on cell proliferation, migration, and invasion were assessed by the CCK-8 assay and transwell assays in esophageal squamous cell carcinoma in vitro. Special AT-rich sequence binding protein 1 was significantly upregulated in esophageal squamous cell carcinoma tissue samples and cell lines. Silencing of special AT-rich sequence binding protein 1 inhibited the proliferation of KYSE450 and EC9706 cells which have a relatively high level of special AT-rich sequence binding protein 1, and the ability of migration and invasion of KYSE450 and EC9706 cells was distinctly suppressed. Special AT-rich sequence binding protein 1 could be a potential target for the treatment of esophageal squamous cell carcinoma and inhibition of special AT-rich sequence binding protein 1 may provide a new strategy for the prevention of esophageal squamous cell carcinoma invasion and metastasis.

Synthesis and pharmacological evaluation of indole-based sigma receptor ligands

PubMed Central

Mésangeau, Christophe; Amata, Emanuele; Alsharif, Walid; Seminerio, Michael J.; Robson, Matthew J.; Matsumoto, Rae R.; Poupaert, Jacques H.; McCurdy, Christopher R.

2011-01-01

A series of novel indole-based analogues were prepared and their affinities for sigma receptors were determined using in vitro radioligand binding assays. The results of this study identified several compounds with nanomolar sigma-2 affinity and significant selectivity over sigma-1 receptors. In particular, 2-(4-(3-(4-fluorophenyl)indol-1-yl)butyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (9f) was found to display high affinity at sigma-2 receptors with good selectivity (σ-1/σ-2 = 395). The pharmacological binding profile for this compound was established with other relevant nonsigma sites. PMID:21899931

Interaction between the cellular protein eEF1A and the 3'-terminal stem-loop of West Nile virus genomic RNA facilitates viral minus-strand RNA synthesis.

PubMed

Davis, William G; Blackwell, Jerry L; Shi, Pei-Yong; Brinton, Margo A

2007-09-01

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles

PubMed Central

Labonté, Eric D.; Howles, Philip N.; Granholm, Norman A.; Rojas, Juan C.; Davies, Joanna P.; Ioannou, Yiannis A.; Hui, David Y.

2007-01-01

Recent studies have documented the importance of Niemann Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1−/− mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1−/− mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1−/− mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1. PMID:17442616

Isolation of serotype-specific antibodies against dengue virus non-structural protein 1 using phage display and application in a multiplexed serotyping assay.

PubMed

Lebani, Kebaneilwe; Jones, Martina L; Watterson, Daniel; Ranzoni, Andrea; Traves, Renee J; Young, Paul R; Mahler, Stephen M

2017-01-01

The multidimensional nature of dengue virus (DENV) infections, which can be caused by four distinct serotypes of the virus, complicates the sensitivity of assays designed for the diagnosis of infection. Different viral markers can be optimally detected at different stages of infection. Of particular clinical importance is the early identification of infection, which is pivotal for disease management and the development of blood screening assays. Non-structural protein 1 (NS1) is an early surrogate marker of infection and its detection in serum coincides with detectable viraemia. The aim of this work was to isolate and characterise serotype-specific monoclonal antibodies that bind to NS1 for each of the four DENV serotypes. This was achieved using phage display and a subtractive biopanning strategy to direct the antibody selection towards serotype-specific epitopes. This antibody isolation strategy has advantages over immunisation techniques where it is difficult to avoid antibody responses to cross-reactive, immunodominant epitopes. Serotype specificity to recombinant antigen for each of the antibodies was confirmed by Enzyme Linked Immunosorbent Assay (ELISA) and Surface Plasmon Resonance. Confirmation of binding to native DENV NS1 was achieved using ELISA and immunofluorescence assay on DENV infected Vero cells. No cross-reactivity with Zika or Kunjin viruses was observed. A previously isolated pan-reactive antibody that binds to an immunodominant epitope was able to pair with each of the serotype-specific antibodies in a sandwich ELISA, indicating that the serotype specific antibodies bind to epitopes which are all spatially distinct from the immunodominant epitope. These antibodies were suitable for use in a multiplexed assay for simultaneous detection and serotyping of DENV NS1 in human serum. This work demonstrates that phage display coupled with novel biopanning strategies is a valuable in vitro methodology for isolation of binders that can discern amongst antigens with high homology for diagnostic applicability.

An easily accessible sulfated saccharide mimetic inhibits in vitro human tumor cell adhesion and angiogenesis of vascular endothelial cells

PubMed Central

Marano, Grazia; Gronewold, Claas; Frank, Martin; Merling, Anette; Kliem, Christian; Sauer, Sandra; Wiessler, Manfred; Frei, Eva

2012-01-01

Summary Oligosaccharides aberrantly expressed on tumor cells influence processes such as cell adhesion and modulation of the cell’s microenvironment resulting in an increased malignancy. Schmidt’s imidate strategy offers an effective method to synthesize libraries of various oligosaccharide mimetics. With the aim to perturb interactions of tumor cells with extracellular matrix proteins and host cells, molecules with 3,4-bis(hydroxymethyl)furan as core structure were synthesized and screened in biological assays for their abilities to interfere in cell adhesion and other steps of the metastatic cascade, such as tumor-induced angiogenesis. The most active compound, (4-{[(β-D-galactopyranosyl)oxy]methyl}furan-3-yl)methyl hydrogen sulfate (GSF), inhibited the activation of matrix-metalloproteinase-2 (MMP-2) as well as migration of the human melanoma cells of the lines WM-115 and WM-266-4 in a two-dimensional migration assay. GSF inhibited completely the adhesion of WM-115 cells to the extracellular matrix (ECM) proteins, fibrinogen and fibronectin. In an in vitro angiogenesis assay with human endothelial cells, GSF very effectively inhibited endothelial tubule formation and sprouting of blood vessels, as well as the adhesion of endothelial cells to ECM proteins. GSF was not cytotoxic at biologically active concentrations; neither were 3,4-bis{[(β-D-galactopyranosyl)oxy]methyl}furan (BGF) nor methyl β-D-galactopyranoside nor 3,4-bis(hydroxymethyl)furan, which were used as controls, eliciting comparable biological activity. In silico modeling experiments, in which binding of GSF to the extracellular domain of the integrin αvβ3 was determined, revealed specific docking of GSF to the same binding site as the natural peptidic ligands of this integrin. The sulfate in the molecule coordinated with one manganese ion in the binding site. These studies show that this chemically easily accessible molecule GSF, synthesized in three steps from 3,4-bis(hydroxymethyl)furan and benzoylated galactose imidate, is nontoxic and antagonizes cell physiological processes in vitro that are important for the dissemination and growth of tumor cells in vivo. PMID:23015827

Efavirenz directly modulates the oestrogen receptor and induces breast cancer cell growth.

PubMed

Sikora, M J; Rae, J M; Johnson, M D; Desta, Z

2010-10-01

Efavirenz-based HIV therapy is associated with breast hypertrophy and gynaecomastia. Here, we tested the hypothesis that efavirenz induces gynaecomastia through direct binding and modulation of the oestrogen receptor (ER). To determine the effect of efavirenz on growth, the oestrogen-dependent, ER-positive breast cancer cell lines MCF-7, T47D and ZR-75-1 were treated with efavirenz under oestrogen-free conditions in the presence or absence of the anti-oestrogen ICI 182,780. Cells treated with 17β-oestradiol in the absence or presence of ICI 182,780 served as positive and negative controls, respectively. Cellular growth was assayed using the crystal violet staining method and an in vitro receptor binding assay was used to measure the ER binding affinity of efavirenz. Efavirenz induced growth in MCF-7 cells with an estimated effective concentration for half-maximal growth (EC(50)) of 15.7 μM. This growth was reversed by ICI 182,780. Further, efavirenz binds directly to the ER [inhibitory concentration for half maximal binding (IC(50)) of ∼52 μM] at a roughly 1000-fold higher concentration than observed with 17β-oestradiol. Our data suggest that efavirenz-induced gynaecomastia may be caused, at least in part, by drug-induced ER activation in breast tissues.

Efavirenz directly modulates estrogen receptor and induces breast cancer cell growth

PubMed Central

Sikora, Matthew J.; Rae, James M.; Johnson, Michael D.; Desta, Zeruesenay

2010-01-01

Objectives Efavirenz-based HIV therapy is associated with breast hypertrophy and gynecomastia. Here, we tested the hypothesis that efavirenz induces gynecomastia through direct binding and modulation of estrogen receptor (ER). Methods To determine the effect of efavirenz on growth, the estrogen-dependent, ER-positive breast cancer cell lines MCF-7, T47D and ZR-75-1 were treated with efavirenz under estrogen-free conditions in the presence or absence of the anti-estrogen ICI 182,780. Cells treated with 17β-estradiol in the absence or presence of ICI 182,780 served as positive and negative controls, respectively. Cellular growth was assayed using the crystal violet staining method and an in vitro receptor binding assay was used to measure efavirenz’s ER binding affinity. Results Efavirenz induced growth in MCF-7 cells with an estimated EC50 of 15.7µM. This growth was reversed by ICI 182,780. Further, efavirenz binds directly to ER (IC50 of ~52µM) at roughly 1000-fold higher concentration than observed with E2. Conclusions Our data suggest that efavirenz-induced gynecomastia may be due, at least in part, to drug-induced ER activation in breast tissues. PMID:20408889

From receptor binding kinetics to signal transduction; a missing link in predicting in vivo drug-action.

PubMed

Nederpelt, Indira; Kuzikov, Maria; de Witte, Wilbert E A; Schnider, Patrick; Tuijt, Bruno; Gul, Sheraz; IJzerman, Adriaan P; de Lange, Elizabeth C M; Heitman, Laura H

2017-10-26

An important question in drug discovery is how to overcome the significant challenge of high drug attrition rates due to lack of efficacy and safety. A missing link in the understanding of determinants for drug efficacy is the relation between drug-target binding kinetics and signal transduction, particularly in the physiological context of (multiple) endogenous ligands. We hypothesized that the kinetic binding parameters of both drug and endogenous ligand play a crucial role in determining cellular responses, using the NK1 receptor as a model system. We demonstrated that the binding kinetics of both antagonists (DFA and aprepitant) and endogenous agonists (NKA and SP) have significantly different effects on signal transduction profiles, i.e. potency values, in vitro efficacy values and onset rate of signal transduction. The antagonistic effects were most efficacious with slowly dissociating aprepitant and slowly associating NKA while the combination of rapidly dissociating DFA and rapidly associating SP had less significant effects on the signal transduction profiles. These results were consistent throughout different kinetic assays and cellular backgrounds. We conclude that knowledge of the relationship between in vitro drug-target binding kinetics and cellular responses is important to ultimately improve the understanding of drug efficacy in vivo.

Characterization of monomeric DNA-binding protein Histone H1 in Leishmania braziliensis.

PubMed

Carmelo, Emma; González, Gloria; Cruz, Teresa; Osuna, Antonio; Hernández, Mariano; Valladares, Basilio

2011-08-01

Histone H1 in Leishmania presents relevant differences compared to higher eukaryote counterparts, such as the lack of a DNA-binding central globular domain. Despite that, it is apparently fully functional since its differential expression levels have been related to changes in chromatin condensation and infectivity, among other features. The localization and the aggregation state of L. braziliensis H1 has been determined by immunolocalization, mass spectrometry, cross-linking and electrophoretic mobility shift assays. Analysis of H1 sequences from the Leishmania Genome Database revealed that our protein is included in a very divergent group of histones H1 that is present only in L. braziliensis. An antibody raised against recombinant L. braziliensis H1 recognized specifically that protein by immunoblot in L. braziliensis extracts, but not in other Leishmania species, a consequence of the sequence divergences observed among Leishmania species. Mass spectrometry analysis and in vitro DNA-binding experiments have also proven that L. braziliensis H1 is monomeric in solution, but oligomerizes upon binding to DNA. Finally, despite the lack of a globular domain, L. braziliensis H1 is able to form complexes with DNA in vitro, with higher affinity for supercoiled compared to linear DNA.

Nerve cell-mimicking liposomes as biosensor for botulinum neurotoxin complete physiological activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weingart, Oliver G., E-mail: Oliver.Weingart@hest.

Botulinum neurotoxins (BoNT) are the most toxic substances known, and their neurotoxic properties and paralysing effects are exploited for medical treatment of a wide spectrum of disorders. To accurately quantify the potency of a pharmaceutical BoNT preparation, its physiological key activities (binding to membrane receptor, translocation, and proteolytic degradation of SNARE proteins) need to be determined. To date, this was only possible using animal models, or, to a limited extent, cell-based assays. We here report a novel in vitro system for BoNT/B analysis, based on nerve-cell mimicking liposomes presenting motoneuronal membrane receptors required for BoNT binding. Following triggered membrane translocationmore » of the toxin's Light Chain, the endopeptidase activity can be quantitatively monitored employing a FRET-based reporter assay within the functionalized liposomes. We were able to detect BoNT/B physiological activity at picomolar concentrations in short time, opening the possibility for future replacement of animal experimentation in pharmaceutical BoNT testing. - Highlights: • A cell-free in vitro system was used to measure BoNT/B physiological function. • The system relies on nerve-cell mimicking liposomes as a novel detection system. • A FRET-based reporter assay is used as final readout of the test system. • BoNT/B physiological activity was detected at picogram quantities in short time. • The method opens the possibility to replace animal experimentation in BoNT testing.« less

The 5-HT1A Receptor PET Radioligand 11C-CUMI-101 Has Significant Binding to α1-Adrenoceptors in Human Cerebellum, Limiting Its Use as a Reference Region.

PubMed

Shrestha, Stal S; Liow, Jeih-San; Jenko, Kimberly; Ikawa, Masamichi; Zoghbi, Sami S; Innis, Robert B

2016-12-01

Prazosin, a potent and selective α 1 -adrenoceptor antagonist, displaces 25% of 11 C-CUMI-101 ([O-methyl- 11 C]2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione) binding in monkey cerebellum. We sought to estimate the percentage contamination of 11 C-CUMI-101 binding to α 1 -adrenoceptors in human cerebellum under in vivo conditions. In vitro receptor-binding techniques were used to measure α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors in human, monkey, and rat cerebellum. Binding potential (maximum number of binding sites × affinity [(1/dissociation constant]) was determined using in vitro homogenate binding assays in human, monkey, and rat cerebellum. 3 H-prazosin was used to determine the maximum number of binding sites, as well as the dissociation constant of 3 H-prazosin and the inhibition constant of CUMI-101. α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors were similar across species. Cerebellar binding potentials were 3.7 for humans, 2.3 for monkeys, and 3.4 for rats. Reasoning by analogy, 25% of 11 C-CUMI-101 uptake in human cerebellum reflects binding to α 1 -adrenoceptors, suggesting that the cerebellum is of limited usefulness as a reference tissue for quantification in human studies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

[The effect of hypoxia preconditioning no binding activity of HIF-1 on the HRE with EPO in the hippocampus of mice].

PubMed

Shao, Guo; Zhou, Wei-Hua; Gao, Cui-Ying; Zhang, Ran; Lu, Guo-Wei

2007-02-01

To observe change of binding activity of HIF-1 with erythropoietin (EPO) hypoxia response element (HRE) in the hippocampus of mice preconditioned to hypoxia and explore relationship between the changes and the preconditioning. The hippocampus was removed from mice exposed to hypoxia for 0 run (control group), 1 run (H1 group) and 4 runs(H4 group). Electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP)and real time PCR were used to detect the change of activity of HIF-1 on HRE of EPO. Both in vitro and in vivo binding tests showed that the HIF-1 DNA-binding activities were increased in group H1 and markedly increased in group H4. The increase of HIF-1 and HRE of EPO binding activities is thought be involved in hypoxic preconditioning.

Beauvericin synthetase contains a calmodulin binding motif in the entomopathogenic fungus Beauveria bassiana.

PubMed

Kim, Jiyoung; Sung, Gi-Ho

2018-03-19

Beauvericin is a mycotoxin which has insecticidal, anti-microbial, anti-viral and anti-cancer activities. Beauvericin biosynthesis is rapidly catalyzed by the beauvericin synthetase (BEAS) in Beauveria bassiana. Ca 2+ plays crucial roles in multiple signaling pathways in eukaryotic cells. These Ca 2+ signals are partially decoded by Ca 2+ sensor calmodulin (CaM). In this report, we describe that B. bassiana BEAS (BbBEAS) can interact with CaM in a Ca 2+ -dependent manner. A synthetic BbBEAS peptide, corresponding to the putative CaM-binding motif, formed a stable complex with CaM in the presence of Ca 2+ . In addition, in vitro CaM-binding assay revealed that the His-tagged BbBEAS (amino acids 2421-2538) binds to CaM in a Ca 2+ -dependent manner. Therefore, this work suggests that BbBEAS is a novel CaM-binding protein in B. bassiana.

The effect of feeding high corn oil on fatty-acid-binding-protein isolated from rat liver.

PubMed

Catalá, A

1987-12-01

Fatty-acid-binding-protein isolated from liver of rats receiving normal or high fat diet was studied by three different methods. The effect of high fat diet on the thermal stability of the protein was determined employing differential scanning calorimetry. Fatty acids have a stabilizing effect on the thermal stability of the protein. In order to determine the relative binding affinity of native and delipidated protein a Sephadex G-50 assay was employed using [1-14C] oleate as ligand. The delipidated protein exhibited greater binding of oleate than did the native material. Increases in the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes in vitro were also observed when protein obtained from both sources were delipidated. The results suggest that high corn oil diet would modify the properties of fatty-acid-binding-protein in the uptake and cytosolic transport of long-chain fatty acids.

In Vitro Binding of [³H]PSB-0413 to P2Y₁₂ Receptors.

PubMed

Dupuis, Arnaud; Heim, Véronique; Ohlmann, Philippe; Gachet, Christian

2015-12-08

The P2Y₁₂/ADP receptor plays a central role in platelet activation. Characterization of this receptor is mandatory for studying disorders associated with a P2Y₁₂ receptor defect and for evaluating P2Y₁₂ receptor agonists and antagonists. In the absence of suitable anti-P2Y₁₂ antibodies, radioligand binding assays are the only way to conduct such studies. While various radioligands were employed in the past for this purpose, none were found to be suitable for routine use. Described in this unit are protocols for quantitatively and qualitatively assessing P2Y₁₂ receptors with [³H]PSB-0413, a selective antagonist for this site. The saturation and competition assays described herein make possible the determination of P2Y₁₂ receptor density on cells, as well as the potencies and affinities of test agents at this site. Copyright © 2015 John Wiley & Sons, Inc.

The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Varnum, Susan M.

2012-03-01

Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was foundmore » to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.« less

In vitro studies on the relationship between the antioxidant activities of some berry extracts and their binding properties to serum albumin.

PubMed

Namiesnik, Jacek; Vearasilp, Kann; Nemirovski, Alina; Leontowicz, Hanna; Leontowicz, Maria; Pasko, Pawel; Martinez-Ayala, Alma Leticia; González-Aguilar, Gustavo A; Suhaj, Milan; Gorinstein, Shela

2014-03-01

The aim of this study was to investigate the possibility to use the bioactive components from cape gooseberry (Physalis peruviana), blueberry (Vaccinium corymbosum), and cranberry (Vaccinium macrocarpon) extracts as a novel source against oxidation in food supplementation. The quantitative analysis of bioactive compounds (polyphenols, flavonoids, flavanols, carotenoids, and chlorophyll) was based on radical scavenging spectrophometric assays and mass spectrometry. The total phenolic content was the highest (P < 0.05) in water extract of blueberries (46.6 ± 4.2 mg GAE/g DW). The highest antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay and Cupric reducing antioxidant capacity were in water extracts of blueberries, showing 108.1 ± 7.2 and 131.1 ± 9.6 μMTE/g DW with correlation coefficients of 0.9918 and 0.9925, and by β-carotene linoleate assay at 80.1 ± 6.6 % with correlation coefficient of 0.9909, respectively. The water extracts of berries exhibited high binding properties with human serum albumin in comparison with quercetin. In conclusion, the bioactive compounds from a relatively new source of gooseberries in comparison with blueberries and cranberries have the potential as food supplementation for human health. The antioxidant and binding activities of berries depend on their bioactive compounds.

Mechanistic insights into the link between visfatin gene C-1535T polymorphism and coronary artery disease: an in vitro study.

PubMed

Wang, Yong-Sheng; Gao, Wei; Li, Hong-Fen; Wang, Ze-Mu; Zhu, Jun; Zhao, Huan; Yan, Jian-Jun; Jia, En-Zhi; Yang, Zhi-Jian; Wang, Lian-Sheng

2012-04-01

Visfatin, a pro-inflammatory cytokine predominantly released from leucocytes, is correlated with coronary artery disease (CAD). We have previously reported that the -1535C>T polymorphism (rs1330082), which located on the promoter region of visfatin, was associated with decreased risk of CAD. Here, we investigated the underlying mechanism by which this polymorphism affects the genetic susceptibility to CAD. The difference of the promoter activities between -1535T variant and -1535C allele was tested by luciferase reporter gene assay. The difference of transcription factor binding activities between T and C allele was evaluated by electrophoretic mobility shift assay. In reporter gene assay, we showed that the T variant had a significantly reduced transcriptional activity compared with the C allele. The T-variant significantly attenuated the promoter binding affinity to nuclear transcription factors and this effect became much obvious after treatment with TNF-α. Moreover, competition experiment revealed that the retarded complex formed by T-1535- or C-1535-probe binding to nuclear extracts was nearly completely inhibited by unlabeled activator protein-1 (AP-1) specific probe, indicating that AP-1 might be the target nuclear effector. Taken together, our data provided potential mechanistic link between the visfatin -1535C>T polymorphism and reduced CAD risk.

A Chrysin Derivative Suppresses Skin Cancer Growth by Inhibiting Cyclin-dependent Kinases*

PubMed Central

Liu, Haidan; Liu, Kangdong; Huang, Zunnan; Park, Chan-Mi; Thimmegowda, N. R.; Jang, Jae-Hyuk; Ryoo, In-Ja; He, Long; Kim, Sun-Ok; Oi, Naomi; Lee, Ki Won; Soung, Nak-Kyun; Bode, Ann M.; Yang, Yifeng; Zhou, Xinmin; Erikson, Raymond L.; Ahn, Jong-Seog; Hwang, Joonsung; Kim, Kyoon Eon; Dong, Zigang; Kim, Bo-Yeon

2013-01-01

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P+ cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P+ cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency. PMID:23888052

Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

PubMed Central

Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G.; Orlova, Albina; Egelman, Edward H.; Beck, Moriah R.

2016-01-01

The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. Here we show that the actin binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro crosslinking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of G-actin, akin to metal ions, either through charge neutralization or conformational changes. PMID:26607837

In vitro and in silico assessment of the structure-dependent binding of bisphenol analogues to glucocorticoid receptor.

PubMed

Zhang, Jie; Zhang, Tiehua; Guan, Tianzhu; Yu, Hansong; Li, Tiezhu

2017-03-01

Widespread use of bisphenol A (BPA) and other bisphenol analogues has attracted increasing attention for their potential adverse effects. As environmental endocrine-disrupting compounds (EDCs), bisphenols (BPs) may activate a variety of nuclear receptors, including glucocorticoid receptor (GR). In this work, the binding of 11 BPs to GR was investigated by fluorescence polarization (FP) assay in combination with molecular dynamics simulations. The human glucocorticoid receptor was prepared as a soluble recombinant protein. A fluorescein-labeled dexamethasone derivative (Dex-fl) was employed as tracer. Competitive displacement of Dex-fl from GR by BPs showed that the binding affinities of bisphenol analogues were largely dependent on their characteristic functional groups. In order to further understand the relationship between BPs structures and their GR-mediated activities, molecular docking was utilized to explore the binding modes at the atomic level. The results confirmed that structural variations of bisphenol analogues contributed to different interactions of BPs with GR, potentially causing distinct toxic effects. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation (R 2  = 0.8266), which might be helpful for the design of environmentally benign materials with reduced toxicities. In addition, the established FP assay based on GR exhibited the potential to offer an alternative to traditional methods for the detection of bisphenols.

Binding of immunoglobulins and immune complexes to cartilage derived extracts.

PubMed Central

Alomari, W R; Archer, J R; Brocklehurst, R; Currey, H L

1983-01-01

Cartilage extracts with affinity for heat aggregated immunoglobulins were prepared from human articular and bovine nasal cartilage. These extracts, containing predominantly collagen, also bound both to immune complexes (IC) prepared in vitro and to immunoglobulins from sera of many patients with rheumatoid arthritis (RA). Cryoprecipitation of rheumatoid sera removed material reacting with the extract and density gradient fractionation of a positive serum showed correlation between binding to the extract and to C1q. These results indicate that the binding materials in rheumatoid sera were likely to be IC. We suggest that some assays which apparently demonstrate anti-collagen autoantibodies in fact measure IC. These findings also have implications for models of the pathogenesis of RA. PMID:6606513

Synthesis and biological evaluation of cyclopropyl analogues of 2-amino-5-phosphonopentanoic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dappen, M.S.; Pellicciari, R.; Natalini, B.

1991-01-01

A series of cyclopropyl analogues related to 2-amino-5-phosphonopentanoic acid (AP5) were synthesized and their biological activity was assessed as competitive antagonists for the N-methyl-D-aspartate (NMDA) receptor. In vitro receptor binding using (3H)-L-glutamate as the radioligand provided affinity data, while modulation of (3H)MK-801 binding was used as a functional assay. The analogues were also evaluated in (3H)kainate binding to assess selectivity over non-NMDA glutamate receptors. Of the compounds tested, 4,5-methano-AP5 analogue 26 was the most potent selective NMDA antagonist; however, potency was lower than that for (((+/-)-2-carboxypiperidin-4-yl)methyl)phosphonic acid (CGS 19755, 5).

A phorbol ester-binding protein is required downstream of Rab5 in endosome fusion.

PubMed

Aballay, A; Barbieri, M A; Colombo, M I; Arenas, G N; Stahl, P D; Mayorga, L S

1998-12-28

Previous observations indicate that a zinc and phorbol ester binding factor is necessary for endosome fusion. To further characterize the role of this factor in the process, we used an in vitro endosome fusion assay supplemented with recombinant Rab5 proteins. Both zinc depletion and addition of calphostin C, an inhibitor of protein kinase C, inhibited endosome fusion in the presence of active Rab5. Addition of the phorbol ester PMA (phorbol 12-myristate 13-acetate) reversed the inhibition of endosome fusion caused by a Rab5 negative mutant. Moreover, PMA stimulated fusion in the presence of Rab5 immunodepleted cytosol. These results suggest that the phorbol ester binding protein is acting downstream of Rab5 in endosome fusion.

Assessment of androgen receptor agonistic/antagonistic effects on 25 chemicals in household applicants by OECD in vitro stably transfected transcriptional activation assays.

PubMed

Lee, Hee-Seok; Park, Eun-Jung; Han, Songyi; Oh, Gyeong-Yong; Kang, Hui-Seung; Suh, Jin-Hyang; Shin, Min-Ki; Oh, Hyun-Suk; Hwang, Myung-Sil; Moon, Guiim; Koh, Young-Ho; Park, Yooheon; Hong, Jin-Hwan; Koo, Yong Eui

2018-01-01

The aim of this study is to assess the androgen receptor (AR) agonistic/antagonistic effects on various chemicals, which are used in household products including cleaning agents and wetted tissues by in vitro OECD test guideline No. 458 (using AR-EcoScreen™ cell line) and the me-too test method (using 22Rv1cell line), which was adopted as OECD project No. 4.99. All chemicals were not determined as AR agonists. However α-dodecyl-ω-hydroxypoly (oxyethylene) and 3-iodo-2-propynyl butylcarbamate have shown a weak AR antagonistic effects with IC 50 values of 2.18 ± 0.12 and 4.26 ± 0.17 μg/ml via binding affinity to AR in only 22Rv1/mouse mammary tumor virus using AR transcriptional activation assay, because of their different cytotoxicity on each applied cell line. This report firstly provides information about agonistic/antagonistic effects against human AR of various chemicals including surfactants and biocides by OECD in vitro stably transfected transcriptional activation assays. However, further in vivo and human model studies are needed to confirm their adverse effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Abrin Toxicity and Bioavailability after Temperature and pH Treatment.

PubMed

Tam, Christina C; Henderson, Thomas D; Stanker, Larry H; He, Xiaohua; Cheng, Luisa W

2017-10-13

Abrin, one of most potent toxins known to man, is derived from the rosary pea (jequirity pea), Abrus precatorius and is a potential bioterror weapon. The temperature and pH stability of abrin was evaluated with an in vitro cell free translation (CFT) assay, a Vero cell culture cytotoxicity assay, and an in vivo mouse bioassay. pH treatment of abrin had no detrimental effect on its stability and toxicity as seen either in vitro or in vivo. Abrin exposure to increasing temperatures did not completely abrogate protein translation. In both the cell culture cytotoxicity model and the mouse bioassay, abrin's toxic effects were completely abrogated if the toxin was exposed to temperatures of 74 °C or higher. In the cell culture model, 63 °C-treated abrin had a 30% reduction in cytotoxicity which was validated in the in vivo mouse bioassay with all mice dying but with a slight time-to-death delay as compared to the non-treated abrin control. Since temperature inactivation did not affect abrin's ability to inhibit protein synthesis (A-chain), we hypothesize that high temperature treatment affected abrin's ability to bind to cellular receptors (affecting B-chain). Our results confirm the absolute need to validate in vitro cytotoxicity assays with in vivo mouse bioassays.

Abrin Toxicity and Bioavailability after Temperature and pH Treatment

PubMed Central

Tam, Christina C.; Henderson, Thomas D.; Stanker, Larry H.; He, Xiaohua; Cheng, Luisa W.

2017-01-01

Abrin, one of most potent toxins known to man, is derived from the rosary pea (jequirity pea), Abrus precatorius and is a potential bioterror weapon. The temperature and pH stability of abrin was evaluated with an in vitro cell free translation (CFT) assay, a Vero cell culture cytotoxicity assay, and an in vivo mouse bioassay. pH treatment of abrin had no detrimental effect on its stability and toxicity as seen either in vitro or in vivo. Abrin exposure to increasing temperatures did not completely abrogate protein translation. In both the cell culture cytotoxicity model and the mouse bioassay, abrin’s toxic effects were completely abrogated if the toxin was exposed to temperatures of 74 °C or higher. In the cell culture model, 63 °C-treated abrin had a 30% reduction in cytotoxicity which was validated in the in vivo mouse bioassay with all mice dying but with a slight time-to-death delay as compared to the non-treated abrin control. Since temperature inactivation did not affect abrin’s ability to inhibit protein synthesis (A-chain), we hypothesize that high temperature treatment affected abrin’s ability to bind to cellular receptors (affecting B-chain). Our results confirm the absolute need to validate in vitro cytotoxicity assays with in vivo mouse bioassays. PMID:29027937

Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics

PubMed Central

King, Amy C.; Kavosi, Mania; Wang, Mengmeng; O'Hara, Denise M.; Tchistiakova, Lioudmila; Katragadda, Madan

2018-01-01

ABSTRACT A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1–5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1–3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics. PMID:28991504

Apple S-RNase interacts with an actin-binding protein, MdMVG, to reduce pollen tube growth by inhibiting its actin-severing activity at the early stage of self-pollination induction.

PubMed

Yang, Qing; Meng, Dong; Gu, Zhaoyu; Li, Wei; Chen, Qiuju; Li, Yang; Yuan, Hui; Yu, Jie; Liu, Chunsheng; Li, Tianzhong

2018-04-18

In S-RNase-mediated self-incompatibility, S-RNase secreted from the style destroys the actin cytoskeleton of the self-pollen tubes, eventually halting their growth, but the mechanism of this process remains unclear. In vitro biochemical assays revealed that S-RNase does not bind or sever filamentous actin (F-actin). In apple (Malus domestica), we identified an actin-binding protein containing myosin, villin and GRAM (MdMVG), that physically interacts with S-RNase and directly binds and severs F-actin. Immunofluorescence assays and total internal reflection fluorescence microscopy indicated that S-RNase inhibits the F-actin-severing activity of MdMVG in vitro. In vivo, the addition of S-RNase to self-pollen tubes increased the fluorescence intensity of actin microfilaments and reduced the severing frequency of microfilaments and the rate of pollen tube growth in self-pollination induction in the presence of MdMVG overexpression. By generating 25 single-, double- and triple-point mutations in the amino acid motif E-E-K-E-K of MdMVG via mutagenesis and testing the resulting mutants with immunofluorescence, we identified a triple-point mutant, MdMVG (E167A/E171A/K185A) , that no longer has F-actin-severing activity or interacts with any of the four S-haplotype S-RNases, indicating that all three amino acids (E167, E171 and K185) are essential for the severing activity of MdMVG and its interaction with S-RNases. We conclude that apple S-RNase interacts with MdMVG to reduce self-pollen tube growth by inhibiting its F-actin-severing activity. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

A sensory complex consisting of an ATP-binding cassette transporter and a two-component regulatory system controls bacitracin resistance in Bacillus subtilis.

PubMed

Dintner, Sebastian; Heermann, Ralf; Fang, Chong; Jung, Kirsten; Gebhard, Susanne

2014-10-03

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis*

PubMed Central

Dintner, Sebastian; Heermann, Ralf; Fang, Chong; Jung, Kirsten; Gebhard, Susanne

2014-01-01

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems. PMID:25118291

Minoxidil Induction of VEGF Is Mediated by Inhibition of HIF-Prolyl Hydroxylase

PubMed Central

Yum, Soohwan; Jeong, Seongkeun; Kim, Dohoon; Lee, Sunyoung; Kim, Wooseong; Yoo, Jin-Wook; Kwon, Oh Sang; Kim, Dae-Duk; Min, Do Sik; Jung, Yunjin

2017-01-01

The topical application of minoxidil may achieve millimolar concentrations in the skin. We investigated whether millimolar minoxidil could induce vascular endothelial growth factor (VEGF), a possible effector for minoxidil-mediated hair growth, and how it occurred at the molecular level. Cell-based experiments were performed to investigate a molecular mechanism underlying the millimolar minoxidil induction of VEGF. The inhibitory effect of minoxidil on hypoxia-inducible factor (HIF) prolyl hydroxylase-2 (PHD-2) was tested by an in vitro von Hippel–Lindau protein (VHL) binding assay. To examine the angiogenic potential of millimolar minoxidil, a chorioallantoic membrane (CAM) assay was used. In human keratinocytes and dermal papilla cells, millimolar minoxidil increased the secretion of VEGF, which was not attenuated by a specific adenosine receptor antagonist that inhibits the micromolar minoxidil induction of VEGF. Millimolar minoxidil induced hypoxia-inducible factor-1α (HIF-1α), and the induction of VEGF was dependent on HIF-1. Moreover, minoxidil applied to the dorsal area of mice increased HIF-1α and VEGF in the skin. In an in vitro VHL binding assay, minoxidil directly inhibited PHD-2, thus preventing the hydroxylation of cellular HIF-1α and VHL-dependent proteasome degradation and resulting in the stabilization of HIF-1α protein. Minoxidil inhibition of PHD-2 was reversed by ascorbate, a cofactor of PHD-2, and the minoxidil induction of cellular HIF-1α was abrogated by the cofactor. Millimolar minoxidil promoted angiogenesis in the CAM assay, an in vivo angiogenic test, and this was nullified by the specific inhibition of VEGF. Our data demonstrate that PHD may be the molecular target for millimolar minoxidil-mediated VEGF induction via HIF-1. PMID:29295567

Development of magnetic resonance imaging based detection methods for beta amyloids via sialic acid-functionalized magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Kouyoumdjian, Hovig

The development of a non-invasive method for the detection of Alzheimer's disease is of high current interest, which can be critical in early diagnosis and in guiding preventive treatment of the disease. The aggregates of beta amyloids are a pathological hallmark of Alzheimer's disease. Carbohydrates such as sialic acid terminated gangliosides have been shown to play significant roles in initiation of amyloid aggregation. Herein, we report a biomimetic approach using sialic acid coated iron oxide superparamagnetic nanoparticles for in vitro detection in addition to the assessment of the in vivo mouse-BBB (Blood brain barrier) crossing of the BSA (bovine serum albumin)-modified ones. The sialic acid functionalized dextran nanoparticles were shown to bind with beta amyloids through several techniques including ELISA (enzyme linked immunosorbent assay), MRI (magnetic resonance imaging), TEM (transmission electron microscopy), gel electrophoresis and tyrosine fluorescence assay. The superparamagnetic nature of the nanoparticles allowed easy detection of the beta amyloids in mouse brains in both in vitro and ex vivo model by magnetic resonance imaging. Furthermore, the sialic acid nanoparticles greatly reduced beta amyloid induced cytotoxicity to SH-SY5Y neuroblastoma cells, highlighting the potential of the glyconanoparticles for detection and imaging of beta amyloids. Sialic acid functionalized BSA (bovine serum albumin) nanoparticles also showed significant binding to beta amyloids, through ELISA and ex vivo mouse brain MRI experiments. Alternatively, the BBB crossing was demonstrated by several techniques such as confocal microscopy, endocytosis, exocytosis assays and were affirmed by nanoparticles transcytosis assays through bEnd.3 endothelial cells. Finally, the BBB crossing was confirmed by analyzing the MRI signal of nanoparticle-injected CD-1 mice.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

PubMed

Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

2018-05-03

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Recombinant human antibody fragment against tetanus toxoid produced by phage display.

PubMed

Neelakantam, B; Sridevi, N V; Shukra, A M; Sugumar, P; Samuel, S; Rajendra, L

2014-03-01

Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen.

Ricin - inhibitor design. Annual report, 15 April 1994-14 April 1995

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schramm, V.L.

1995-05-14

Substrates for ricin A-chain include short RNA stem-loop structures which have been synthesized with radioactive labels for ease of catalytic assay and for kinetic isotope effects. Ricin A-chain from several sources is incapable of completing multiple catalytic cycles using these substrates. A family of ricin substrate analogue molecules have been synthesized and tested which are specific for transition states with oxycarbonium character or for enzymatic mechanisms involving protonation of the adenine leaving group. Formycin analogues were incorporated into RNA oligomeric structures and tested for binding to ricin A-chain or as inhibitors of the ricin-inactivation of in vitro translation using rabbitmore » reticulocyte lysates. Ribo-oxycarbonium ion analogues containing iminoribitol analogues of ribose were synthetically incorporated into RNA oligomeric structures. Neither formycin nor ribo-oxycarbonium analogues, either singly or in RNA oligomers caused significant inhibition of ricin A-chain when assayed in reticulocyte lysate translation assays. The results indicate a novel transition state mechanism for ricin A-chain, or a requirement for additional features of 28s rRNA to bind transition state analogues.« less

Adhesion of MC3T3-E1 cells to bone sialoprotein and bone osteopontin specifically bound to collagen I.

PubMed

Bernards, Matthew T; Qin, Chunlin; Ratner, Buddy D; Jiang, Shaoyi

2008-09-01

Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins commonly found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen. In this work, the orientation of BSP under similar circumstances is examined and compared with OPN. Radiolabeled adsorption isotherms were obtained for BSP bound to both tissue culture polystyrene (TCPS) and collagen-coated TCPS. The results show that collagen has the capacity to bind almost twice as much OPN under identical conditions. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of BSP on either TCPS or collagen-coated TCPS with identical amounts of adsorbed protein. It was found that there is no significant difference in the cell binding ability of BSP on either of the substrates. For cell binding studies on collagen-coated TCPS, it was shown that there are a greater number of cells bound to substrates with adsorbed OPN as compared with BSP. The preferable orientation of OPN for cell binding coupled with the higher binding capability of collagen for OPN indicates that OPN is more important than BSP for osteoblast adhesion to the collagen matrix. In addition, a cell inhibition assay was performed to show that all of the cell binding that occurred throughout these studies was dependent upon integrin interactions with the RGD cell binding moiety.

Adhesion of MC3T3-E1 cells to bone sialoprotein and bone osteopontin specifically bound to collagen I

PubMed Central

Bernards, Matthew T.; Qin, Chunlin; Ratner, Buddy D.; Jiang, Shaoyi

2009-01-01

Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins commonly found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen. In this work, the orientation of BSP under similar circumstances is examined and compared with OPN. Radiolabeled adsorption isotherms were obtained for BSP bound to both tissue culture polystyrene (TCPS) and collagen-coated TCPS. The results show that collagen has the capacity to bind almost twice as much OPN under identical conditions. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of BSP on either TCPS or collagen-coated TCPS with identical amounts of adsorbed protein. It was found that there is no significant difference in the cell binding ability of BSP on either of the substrates. For cell binding studies on collagen-coated TCPS, it was shown that there are a greater number of cells bound to substrates with adsorbed OPN as compared with BSP. The preferable orientation of OPN for cell binding coupled with the higher binding capability of collagen for OPN indicates that OPN is more important than BSP for osteoblast adhesion to the collagen matrix. In addition, a cell inhibition assay was performed to show that all of the cell binding that occurred throughout these studies was dependent upon integrin interactions with the RGD cell binding moiety. PMID:18041732

Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

PubMed Central

Galka, Marek M.; Rajagopalan, Nandhakishore; Buhrow, Leann M.; Nelson, Ken M.; Switala, Jacek; Cutler, Adrian J.; Palmer, David R. J.; Loewen, Peter C.; Abrams, Suzanne R.; Loewen, Michele C.

2015-01-01

Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation. PMID:26197050

Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

PubMed

Galka, Marek M; Rajagopalan, Nandhakishore; Buhrow, Leann M; Nelson, Ken M; Switala, Jacek; Cutler, Adrian J; Palmer, David R J; Loewen, Peter C; Abrams, Suzanne R; Loewen, Michele C

2015-01-01

Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

New modulated design, docking and synthesis of carbohydrate-conjugate heterobimetallic CuII-SnIV complex as potential topoisomerase II inhibitor: in vitro DNA binding, cleavage and cytotoxicity against human cancer cell lines.

PubMed

Tabassum, Sartaj; Afzal, Mohd; Arjmand, Farukh

2014-03-03

New carbohydrate-conjugate heterobimetallic complexes [C₂₂H₅₀N₆O₁₃CuSnCl₂] (3) and [C₂₂H₅₈N₆O₁₇NiSnCl₂] (4) were synthesized from their monometallic analogs [C₂₂H₅₂N₆O₁₃Cu] (1) and [C₂₂H₆₀N₆O₁₇Ni] (2) containing N-glycoside ligand (L). In vitro DNA binding studies of L and complexes (1-4) with CT DNA were carried out by employing various biophysical and molecular docking techniques which revealed that heterobimetallic complex 3 strongly binds to DNA in comparison to 4, monometallic complexes (1 and 2) and the free ligand. Complex 3 cleaves pBR322 DNA via hydrolytic pathway (confirmed by T4 DNA ligase assay) and inhibited Topo-II activity in a dose-dependent manner. Furthermore, complex 3 was docked into the ATPase domain of human-Topo-II in order to probe the possible mechanism of inhibition. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

PubMed

Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

2014-12-10

A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. Copyright © 2014 Elsevier B.V. All rights reserved.

Triazolopyridinyl-acrylonitrile derivatives as antimicrotubule agents: Synthesis, in vitro and in silico characterization of antiproliferative activity, inhibition of tubulin polymerization and binding thermodynamics.

PubMed

Briguglio, Irene; Laurini, Erik; Pirisi, Maria Antonietta; Piras, Sandra; Corona, Paola; Fermeglia, Maurizio; Pricl, Sabrina; Carta, Antonio

2017-12-01

In this paper we report the synthesis, in vitro anticancer activity, and the experimental/computational characterization of mechanism of action of a new series of E isomers of triazolo[4,5-b/c]pyridin-acrylonitrile derivatives (6c-g, 7d-e, 8d-e, 9c-f, 10d-e, 11d-e). All new compounds are endowed with moderate to interesting antiproliferative activity against 9 different cancer cell lines derived from solid and hematological human tumors. Fluorescence-based assays prove that these molecules interfere with tubulin polymerization. Furthermore, isothermal titration calorimetry (ITC) provides full tubulin/compound binding thermodynamics, thereby ultimately qualifying and quantifying the interactions of these molecular series with the target protein. Lastly, the analysis based on the tight coupling of in vitro and in silico modeling of the interactions between tubulin and the title compounds allows to propose a molecular rationale for their biological activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Expanding RNA binding specificity and affinity of engineered PUF domains.

PubMed

Zhao, Yang-Yang; Mao, Miao-Wei; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-05-18

Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way.

Expanding RNA binding specificity and affinity of engineered PUF domains

PubMed Central

Zhao, Yang-Yang; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-01-01

Abstract Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way. PMID:29490074

Odorant-binding protein-based identification of natural spatial repellents for the African malaria mosquito Anopheles gambiae.

PubMed

Kröber, Thomas; Koussis, Konstantinos; Bourquin, Martine; Tsitoura, Panagiota; Konstantopoulou, Maria; Awolola, Taiwo Sam; Dani, Francesca R; Qiao, Huili; Pelosi, Paolo; Iatrou, Kostas; Guerin, Patrick M

2018-05-01

There is increasing interest in the development of effective mosquito repellents of natural origin to reduce transmission of diseases such as malaria and yellow fever. To achieve this we have employed an in vitro competition assay involving odorant-binding proteins (OBPs) of the malaria mosquito, Anopheles gambiae, with a predominantly female expression bias to identify plant essential oils (EOs) containing bioactive compounds that target mosquito olfactory function. EOs and their fractions capable of binding to such OBPs displayed repellence against female mosquitoes in a laboratory repellent assay. Repellent EOs were subjected to gas chromatographic analysis linked to antennogram (EAG) recordings from female A. gambiae to identify the biologically active constituents. Among these compounds cumin alcohol, carvacrol, ethyl cinnamate and butyl cinnamate proved as effective as DEET at an equivalent dose in the repellent assay, and combinations of carvacrol with either butyl cinnamate or cumin alcohol proved to be significantly more effective than DEET in the assay. When tested as spatial repellents in experimental shelters housing sleeping humans in northern Nigeria a binary mixture of carvacrol plus cumin alcohol caused mosquitoes to leave shelters in significantly higher numbers to those induced by DEET in female Anopheles spp. and in numbers equivalent to that of DEET in Culex spp. mosquitoes. These findings indicate an approach for the identification of biologically active molecules of natural origin serving as repellents for mosquitoes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Characterization of binding preference of polyhydroxyalkanoate biosynthesis-related multifunctional protein PhaM from Ralstonia eutropha.

PubMed

Ushimaru, Kazunori; Tsuge, Takeharu

2016-05-01

The binding preference of a polyhydroxyalkanoate (PHA) biosynthesis-related multifunctional protein from Ralstonia eutropha (PhaMRe) was characterized. In vitro activity assay showed that PHA synthase from R. eutropha (PhaCRe) was activated by the presence of PhaMRe but PHA synthase from Aeromonas caviae (PhaCAc) was not. Additionally, in vitro assays of protein-protein interactions demonstrated that PhaMRe interacted with PhaCRe directly, but did not interact with PhaCAc. These results suggest that the protein-protein interaction is important for the activation of PhaC by PhaMRe. Further analyses indicated that PhaMRe has little or no direct interaction with the PHA polymer chain. Subsequently, PHA biosynthesis genes (phaA Re, phaB Re, and phaC Re/phaC Ac) and the phaM Re gene were introduced into recombinant Escherichia coli and cultivated for PHA accumulation. Contrary to our expectations, the expression of PhaMRe decreased PHA accumulation and changed the morphology of PHA granules to be microscopically obscure shape in PhaCRe-expressing E. coli. No change in the amount of P(3HB) or the morphology of granules by PhaMRe expression was observed in PhaCAc-expressing E. coli. These observations suggest that PhaMRe affects cellular physiology through the PhaM-PhaC interaction.

Development and evaluation of a new epitope-blocking ELISA for universal detection of antibodies to West Nile virus.

PubMed

Sotelo, Elena; Llorente, Francisco; Rebollo, Belen; Camuñas, Ana; Venteo, Angel; Gallardo, Carmina; Lubisi, Alison; Rodríguez, María José; Sanz, Antonio J; Figuerola, Jordi; Jiménez-Clavero, Miguel Ángel

2011-06-01

West Nile virus (WNV) is an emerging zoonotic pathogen with a wide range of hosts, including birds, horses and humans. The development and evaluation of the performance of a new enzyme-linked immunosorbent assay (ELISA) are described for rapid detection of WNV-specific antibodies in samples originating from an extensive range of vertebrates susceptible to WNV infection. The assay uses a monoclonal antibody (MAb) which binds whole virus particles and neutralizes infection in vitro by recognizing a neutralizing epitope within the envelope (E) glycoprotein of the virus. This MAb, labelled with horseradish peroxidase, was used to compete with WNV-specific serum antibodies for virus-binding in vitro. The epitope-blocking ELISA was optimized in a manner that enabled its validation with a number of experimental and field sera, from a wide range of wild bird species, and susceptible mammals. The new ELISA exhibited high specificity (79.5-96.5%) and sensitivity (100%), using the virus-neutralization test as reference standard. It also required a much lower volume of sample (10 μl per analysis) compared to other ELISAs available commercially. This new method may be helpful for diagnosis and disease surveillance, particularly when testing samples from small birds, which are available in limited amounts. Copyright © 2011 Elsevier B.V. All rights reserved.

In vitro evidence for the participation of Drosophila melanogaster sperm β-N-acetylglucosaminidases in the interactions with glycans carrying terminal N-acetylglucosamine residues on the egg's envelopes.

PubMed

Intra, Jari; Veltri, Concetta; De Caro, Daniela; Perotti, Maria Elisa; Pasini, Maria Enrica

2017-09-01

Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm-associated β-N-acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid-phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well-defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β-N-acetylglucosamine, but not core β-N-acetylglucosamine residues. The capacity of sperm β-N-acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β-N-acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β-N-acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm-egg interactions in Drosophila. © 2017 Wiley Periodicals, Inc.

Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

PubMed Central

Sterk, Maaike; Romilly, Cédric; Wagner, E Gerhart H

2018-01-01

Abstract Initiation is the rate-limiting step in translation. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. The ribosome standby model of de Smit and van Duin, based on studies of the MS2 phage coat cistron, proposed how high translation rates can be reconciled with stable, inhibitory structures at an RBS. Here, we revisited the coat protein system and assessed the translation efficiency from its sequestered RBS by introducing standby mutations. Further experiments with gfp reporter constructs assessed the effects of 5′-tails—as standby sites—with respect to length and sequence contributions. In particular, combining in vivo and in vitro assays, we can show that tails of CA-dinucleotide repeats—and to a lesser extent, AU-repeats—dramatically increase translation rates. Tails of increasing length reach maximal rate-enhancing effects at 16–18 nucleotides. These standby tails are single-stranded and do not exert their effect by structure changes in the neighboring RBS stem–loop. In vitro translation and toeprinting assays furthermore demonstrate that standby effects are exerted at the level of translation initiation. Finally, as expected, destabilizing mutations within the coat RBS indicate an interplay with the effects of standby tails. PMID:29420821

A novel chalcone derivative which acts as a microtubule depolymerising agent and an inhibitor of P-gp and BCRP in in-vitro and in-vivo glioblastoma models

PubMed Central

2009-01-01

Background Over the past decades, in spite of intensive search, no significant increase in the survival of patients with glioblastoma has been obtained. The role of the blood-brain barrier (BBB) and especially the activity of efflux pumps belonging to the ATP Binding Cassette (ABC) family may, in part, explain this defect. Methods The in-vitro activities of JAI-51 on cell proliferation were assessed by various experimental approaches in four human and a murine glioblastoma cell lines. Using drug exclusion assays and flow-cytometry, potential inhibitory effects of JAI-51 on P-gp and BCRP were evaluated in sensitive or resistant cell lines. JAI-51 activity on in-vitro microtubule polymerization was assessed by tubulin polymerization assay and direct binding measurements by analytical ultracentrifugation. Finally, a model of C57BL/6 mice bearing subcutaneous GL26 glioblastoma xenografts was used to assess the activity of the title compound in vivo. An HPLC method was designed to detect JAI-51 in the brain and other target organs of the treated animals, as well as in the tumours. Results In the four human and the murine glioblastoma cell lines tested, 10 μM JAI-51 inhibited proliferation and blocked cells in the M phase of the cell cycle, via its activity as a microtubule depolymerising agent. This ligand binds to tubulin with an association constant of 2 × 105 M-1, overlapping the colchicine binding site. JAI-51 also inhibited the activity of P-gp and BCRP, without being a substrate of these efflux pumps. These in vitro studies were reinforced by our in vivo investigations of C57BL/6 mice bearing GL26 glioblastoma xenografts, in which JAI-51 induced a delay in tumour onset and a tumour growth inhibition, following intraperitoneal administration of 96 mg/kg once a week. In accordance with these results, JAI-51 was detected by HPLC in the tumours of the treated animals. Moreover, JAI-51 was detected in the brain, showing that the molecule is also able to cross the BBB. Conclusion These in vitro and in vivo data suggest that JAI-51 could be a good candidate for a new treatment of tumours of the CNS. Further investigations are in progress to associate the title compound chemotherapy to radiotherapy in a rat model. PMID:19619277

Saw palmetto extracts potently and noncompetitively inhibit human alpha1-adrenoceptors in vitro.

PubMed

Goepel, M; Hecker, U; Krege, S; Rübben, H; Michel, M C

1999-02-15

We wanted to test whether phytotherapeutic agents used in the treatment of lower urinary tract symptoms have alpha1-adrenoceptor antagonistic properties in vitro. Preparations of beta-sitosterol and extracts of stinging nettle, medicinal pumpkin, and saw palmetto were obtained from several pharmaceutical companies. They were tested for their ability to inhibit [3H]tamsulosin binding to human prostatic alpha1-adrenoceptors and [3H]prazosin binding to cloned human alpha1A- and alpha1B-adrenoceptors. Inhibition of phenylephrine-stimulated [3H]inositol phosphate formation by cloned receptors was also investigated. Up to the highest concentration which could be tested, preparations of beta-sitosterol, stinging nettle, and medicinal pumpkin were without consistent inhibitory effect in all assays. In contrast, all tested saw palmetto extracts inhibited radioligand binding to human alpha1-adrenoceptors and agonist-induced [3H]inositol phosphate formation. Saturation binding experiments in the presence of a single saw palmetto extract concentration indicated a noncompetitive antagonism. The relationship between active concentrations in vitro and recommended therapeutic doses for the saw palmetto extracts was slightly lower than that for several chemically defined alpha1-adrenoceptor antagonists. Saw palmetto extracts have alpha1-adrenoceptor-inhibitory properties. If bioavailability and other pharmacokinetic properties of these ingredients are similar to those of the chemically defined alpha1-adrenoceptor antagonists, alpha1-adrenoceptor antagonism might be involved in the therapeutic effects of these extracts in patients with lower urinary tract symptoms suggestive of benign prostatic obstruction.

Protective effects and mechanisms of curcumin on podophyllotoxin toxicity in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Juan; Dai, Cai-Xia; Sun, Hua

2012-12-01

Podophyllotoxin (POD) is a naturally occurring lignan with pronounced antineoplastic and antiviral properties. POD binds to tubulin and prevents the formation of mitotic spindle. Although cases of overdose or accidental ingestion are quite often, no specific therapy is currently available to treat the POD intoxication. In the current investigation, the protective effects and mechanisms of curcumin (CUR) on podophyllotoxin toxicity were evaluated in vitro and in vivo. The results showed that CUR could protect POD-induced cytotoxicity by recovering the G2/M arrest and decrease the changes of membrane potential and microtubule structure in Vero cells. A significant decrease of mortality ratesmore » was observed in Swiss mice treated by intragastrical administration of POD + CUR as compared with POD alone. The POD + CUR group also exhibited decreases in plasma transaminases, alkaline phosphatase, lactate dehydrogenase, plasma urea, creatinine and malondialdehyde level but elevated superoxide dismutase and glutathione levels as compared to the POD group. Histological examination of the liver and kidney demonstrated less morphological changes in the treatment of POD + CUR as compared with POD alone. The mechanism of the protective effects might be due to the competitive binding of CUR with POD in the same colchicines binding site as revealed by the tubulin polymerization assay and the molecular docking analysis, and the antioxidant activity against the oxidative stress induced by POD. In summary, both in vitro and in vivo data indicated the promising role of CUR as a protective agent against the POD poisoning. Highlights: ► A potential antidote to treat the podophyllotoxin (POD) intoxication is found. ► Curcumin showed promising effects against POD poisoning in vitro and in vivo. ► The mechanisms lie in the antioxidant activity and competitive binding with tubulin.« less

The juxtamembrane domain of the E-cadherin cytoplasmic tail contributes to its interaction with Myosin VI

PubMed Central

Mangold, Sabine; Norwood, Suzanne J.; Yap, Alpha S.; Collins, Brett M.

2012-01-01

We recently identified the atypical myosin, Myosin VI, as a component of epithelial cell-cell junctions that interacts with E-cadherin. Recombinant proteins bearing the cargo-binding domain of Myosin VI (Myo VI-CBD) or the cytoplasmic tail of E-cadherin can interact directly with one another. In this report we further investigate the molecular requirements of the interaction between Myo VI-CBD and E-cadherin combining truncation mutation analysis with in vitro binding assays. We report that a short (28 amino acid) juxtamembrane region of the cadherin cytoplasmic tail is sufficient to bind Myo VI-CBD. However, central regions of the cadherin tail adjacent to the juxtamembrane sequence also display binding activity for Myo VI-CBD. It is therefore possible that the cadherin tail bears two binding sites for Myosin VI, or an extended binding site that includes the juxtamembrane region. Nevertheless, our biochemical data highlight the capacity for the juxtamembrane region to interact with functionally-significant cytoplasmic proteins. PMID:23007415

Alanine analogues of [D-Trp]CJ-15,208: novel opioid activity profiles and prevention of drug- and stress-induced reinstatement of cocaine-seeking behaviour.

PubMed

Aldrich, J V; Senadheera, S N; Ross, N C; Reilley, K A; Ganno, M L; Eans, S E; Murray, T F; McLaughlin, J P

2014-07-01

The novel macrocyclic peptide cyclo[Phe-D-Pro-Phe-D-Trp] ([D-Trp]CJ-15,208) exhibits κ opioid (KOP) receptor antagonist activity in both in vitro and in vivo assays. The four alanine analogues of this peptide were synthesized and characterized both in vitro and in vivo to assess the contribution of different amino acid residues to the activity of [D-Trp]CJ-15,208. The peptides were synthesized by a combination of solid phase peptide synthesis and cyclization in solution. The analogues were evaluated in vitro in receptor binding and functional assays, and in vivo with mice using a tail-withdrawal assay for antinociceptive and opioid antagonist activity. Mice demonstrating extinction of cocaine conditioned-place preference (CPP) were pretreated with selected analogues to evaluate prevention of stress or cocaine-induced reinstatement of CPP. The alanine analogues displayed pharmacological profiles in vivo distinctly different from [D-Trp]CJ-15,208. While the analogues exhibited varying opioid receptor affinities and κ and μ opioid receptor antagonist activity in vitro, they produced potent opioid receptor-mediated antinociception (ED50 = 0.28-4.19 nmol, i.c.v.) in vivo. Three of the analogues also displayed KOP receptor antagonist activity in vivo. Pretreatment with an analogue exhibiting both KOP receptor agonist and antagonist activity in vivo prevented both cocaine- and stress-induced reinstatement of cocaine-seeking behaviour in the CPP assay in a time-dependent manner. These unusual macrocyclic peptides exhibit in vivo opioid activity profiles different from the parent compound and represent novel compounds for potential development as therapeutics for drug abuse and possibly as analgesics. © 2014 The British Pharmacological Society.

Alanine analogues of [D-Trp]CJ-15,208: novel opioid activity profiles and prevention of drug- and stress-induced reinstatement of cocaine-seeking behaviour

PubMed Central

Aldrich, J V; Senadheera, S N; Ross, N C; Reilley, K A; Ganno, M L; Eans, S E; Murray, T F; McLaughlin, J P

2014-01-01

BACKGROUND AND PURPOSE The novel macrocyclic peptide cyclo[Phe-D-Pro-Phe-D-Trp] ([D-Trp]CJ-15,208) exhibits κ opioid (KOP) receptor antagonist activity in both in vitro and in vivo assays. The four alanine analogues of this peptide were synthesized and characterized both in vitro and in vivo to assess the contribution of different amino acid residues to the activity of [D-Trp]CJ-15,208. EXPERIMENTAL APPROACH The peptides were synthesized by a combination of solid phase peptide synthesis and cyclization in solution. The analogues were evaluated in vitro in receptor binding and functional assays, and in vivo with mice using a tail-withdrawal assay for antinociceptive and opioid antagonist activity. Mice demonstrating extinction of cocaine conditioned-place preference (CPP) were pretreated with selected analogues to evaluate prevention of stress or cocaine-induced reinstatement of CPP. KEY RESULTS The alanine analogues displayed pharmacological profiles in vivo distinctly different from [D-Trp]CJ-15,208. While the analogues exhibited varying opioid receptor affinities and κ and μ opioid receptor antagonist activity in vitro, they produced potent opioid receptor-mediated antinociception (ED50 = 0.28–4.19 nmol, i.c.v.) in vivo. Three of the analogues also displayed KOP receptor antagonist activity in vivo. Pretreatment with an analogue exhibiting both KOP receptor agonist and antagonist activity in vivo prevented both cocaine- and stress-induced reinstatement of cocaine-seeking behaviour in the CPP assay in a time-dependent manner. CONCLUSIONS AND IMPLICATIONS These unusual macrocyclic peptides exhibit in vivo opioid activity profiles different from the parent compound and represent novel compounds for potential development as therapeutics for drug abuse and possibly as analgesics. PMID:24588614

The novel BH3 α-helix mimetic JY-1-106 induces apoptosis in a subset of cancer cells (lung cancer, colon cancer and mesothelioma) by disrupting Bcl-xL and Mcl-1 protein–protein interactions with Bak

PubMed Central

2013-01-01

Background It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-2/Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. We designed the BH3 α-helix mimetic JY-1-106 to engage the hydrophobic BH3-binding grooves on the surfaces of both Bcl-xL and Mcl-1. Methods JY-1-106–protein complexes were studied using molecular dynamics (MD) simulations and the SILCS methodology. We have evaluated the in vitro effects of JY-1-106 by using a fluorescence polarization (FP) assay, an XTT assay, apoptosis assays, and immunoprecipitation and western-blot assays. A preclinical human cancer xenograft model was used to test the efficacy of JY-1-106 in vivo. Results MD and SILCS simulations of the JY-1-106–protein complexes indicated the importance of the aliphatic side chains of JY-1-106 to binding and successfully predicted the improved affinity of the ligand for Bcl-xL over Mcl-1. Ligand binding affinities were measured via an FP assay using a fluorescently labeled Bak-BH3 peptide in vitro. Apoptosis induction via JY-1-106 was evidenced by TUNEL assay and PARP cleavage as well as by Bax–Bax dimerization. Release of multi-domain Bak from its inhibitory binding to Bcl-2/Bcl-xL and Mcl-1 using JY-1-106 was detected via immunoprecipitation (IP) western blotting. At the cellular level, we compared the growth proliferation IC50s of JY-1-106 and ABT-737 in multiple cancer cell lines with various Bcl-xL and Mcl-1 expression levels. JY-1-106 effectively induced cell death regardless of the Mcl-1 expression level in ABT-737 resistant solid tumor cells, whilst toxicity toward normal human endothelial cells was limited. Furthermore, synergistic effects were observed in A549 cells using a combination of JY-1-106 and multiple chemotherapeutic agents. We also observed that JY-1-106 was a very effective agent in inducing apoptosis in metabolically stressed tumors. Finally, JY-1-106 was evaluated in a tumor-bearing nude mouse model, and was found to effectively repress tumor growth. Strong TUNEL signals in the tumor cells demonstrated the effectiveness of JY-1-106 in this animal model. No significant side effects were observed in mouse organs after multiple injections. Conclusions Taken together, these observations demonstrate that JY-1-106 is an effective pan-Bcl-2 inhibitor with very promising clinical potential. PMID:23680104

Evidence for zinc binding by two structural proteins of Plodia interpunctella granulosis virus

NASA Technical Reports Server (NTRS)

Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

Workers in our laboratory previously reported the possibility of cation involvement in the in vitro dissociation of the Plodia interpunctella granulosis virus nucleocapsids (K. A. Tweeten, L. A. Bulla, Jr., and R. A. Consigli, J. Virol. 33:866-876, 1980; M. E. Wilson and R. A. Consigli, Virology 143:516-525, 1985). The current study found zinc associated with both granulosis virus nucleocapsids and granulin by atomic absorption analysis. A blotting assay with 65Zn2+ specifically identified the radioactive cation as binding to two viral structural proteins, granulin and VP12. These findings indicate that zinc may have a critical role in maintaining virus stability.

Synaptotagmin C2B Domain Regulates Ca2+-triggered Fusion in Vitro

PubMed Central

Gaffaney, Jon D.; Dunning, F. Mark; Wang, Zhao; Hui, Enfu; Chapman, Edwin R.

2008-01-01

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC for Ca2+ 50-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B. PMID:18784080

Identification of a divalent metal cation binding site in herpes simplex virus 1 (HSV-1) ICP8 required for HSV replication.

PubMed

Bryant, Kevin F; Yan, Zhipeng; Dreyfus, David H; Knipe, David M

2012-06-01

Herpes simplex virus 1 (HSV-1) ICP8 is a single-stranded DNA-binding protein that is necessary for viral DNA replication and exhibits recombinase activity in vitro. Alignment of the HSV-1 ICP8 amino acid sequence with ICP8 homologs from other herpesviruses revealed conserved aspartic acid (D) and glutamic acid (E) residues. Amino acid residue D1087 was conserved in every ICP8 homolog analyzed, indicating that it is likely critical for ICP8 function. We took a genetic approach to investigate the functions of the conserved ICP8 D and E residues in HSV-1 replication. The E1086A D1087A mutant form of ICP8 failed to support the replication of an ICP8 mutant virus in a complementation assay. E1086A D1087A mutant ICP8 bound DNA, albeit with reduced affinity, demonstrating that the protein is not globally misfolded. This mutant form of ICP8 was also recognized by a conformation-specific antibody, further indicating that its overall structure was intact. A recombinant virus expressing E1086A D1087A mutant ICP8 was defective in viral replication, viral DNA synthesis, and late gene expression in Vero cells. A class of enzymes called DDE recombinases utilize conserved D and E residues to coordinate divalent metal cations in their active sites. We investigated whether the conserved D and E residues in ICP8 were also required for binding metal cations and found that the E1086A D1087A mutant form of ICP8 exhibited altered divalent metal binding in an in vitro iron-induced cleavage assay. These results identify a novel divalent metal cation-binding site in ICP8 that is required for ICP8 functions during viral replication.

Krit 1 interactions with microtubules and membranes are regulated by Rap1 and integrin cytoplasmic domain associated protein-1

PubMed Central

Béraud-Dufour, Sophie; Gautier, Romain; Albiges-Rizo, Corinne; Chardin, Pierre; Faurobert, Eva

2007-01-01

The small G protein Rap1 regulates diverse cellular processes such as integrin activation, cell adhesion, cell-cell junction formation and cell polarity. It is crucial to identify Rap1 effectors to better understand signalling pathways controlling these processes. Krit1, a FERM protein, was identified as a Rap1 partner in a yeast two-hydrid screen, but this interaction was not confirmed in subsequent studies. As evidence suggests a role for Krit1 in Rap1-dependent pathways, we readdressed this question. Here, we demonstrate by biochemical assays that Krit1 is a specific Rap1 effector. We show that, like other FERM proteins, Krit1 adopts two conformations: a closed conformation in which its N-terminal NPAY motif interacts with its C-terminus and an opened conformation bound to ICAP-1, a negative regulator of focal adhesion assembly. We show that a ternary complex can form in vitro between Krit1, Rap1 and ICAP-1 and that Rap1 binds Krit1 FERM domain in both closed and opened conformations. Unlike ICAP-1, Rap1 does not open Krit1. Using sedimentation assays, we show that Krit1 binds in vitro to microtubules through its N and C-termini and that Rap1 and ICAP-1 inhibit Krit1 binding to microtubules. Consistently, YFP-Krit1 localizes on CFP-labelled microtubules in BHK cells and is delocalized from microtubules upon co-expression with activated Rap1V12. Finally, we show that Krit1 binds to PIP2 containing liposomes and that Rap1 enhances this binding. Based on these results, we propose a model in which Krit1 would be delivered by microtubules to the plasma membrane where it would be captured by Rap1 and ICAP-1. PMID:17916086

Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

PubMed

Lee, Gregory; Ge, Bixia

2010-07-01

As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd < or = 1 x 10(-8) M) and specificity to GnRH receptor as judged by the whole cell binding immunoassay and Western blot assay. Both anti-GnRH receptor monoclonal antibodies and GnRH were shown to compete for the same binding site of GnRH receptor on the surface of cultured cancer cells. Growth inhibitions of cancer cells cultured in vitro were demonstrated by cellular apoptosis experiments (TUNEL and MTT assays) under different conditions of treatment with GHR-106 monoclonal antibody or GnRH analogs. It was generally observed that both GnRH I and GHR-106 effectively induce the apoptosis of cultured cancer cells as determined by TUNEL and MTT assays. Consistently, suppressions of gene expressions at mRNA levels were demonstrated with several ribosomal proteins (P0, P1, P2 and L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

Arginine methylation of translocated in liposarcoma (TLS) inhibits its binding to long noncoding RNA, abrogating TLS-mediated repression of CBP/p300 activity.

PubMed

Cui, Wei; Yoneda, Ryoma; Ueda, Naomi; Kurokawa, Riki

2018-05-21

Translocated in liposarcoma (TLS) is an RNA-binding protein and a transcription-regulatory sensor of DNA damage. TLS binds promoter-associated noncoding RNA (pncRNA) and inhibits histone acetyltransferase (HAT) activity of CREB-binding protein (CBP)/E1A-binding protein P300 (p300) on the cyclin D1 (CCND1) gene. Although post-translational modifications of TLS, such as arginine methylation, are known to regulate TLS's nucleocytoplasmic shuttling and assembly in stress granules, its interactions with RNAs remain poorly characterized. Herein, using various biochemical assays, we confirmed the earlier observations that TLS is methylated by protein arginine methyltransferase 1 (PRMT1) in vitro. The arginine methylation of TLS disrupted binding to pncRNA and also prevented binding of TLS to and inhibition of CBP/p300. This result indicated that arginine methylation of TLS abrogates both binding to pncRNA and TLS-mediated inhibition of CBP/p300 HAT activities. We also report that an arginine residue within the Arg-Gly-Gly domain of TLS, Arg-476, serves as the major determinant for binding to pncRNA. Either methylation or mutation of Arg-476 of TLS significantly decreased pncRNA binding and thereby prevented a pncRNA-induced allosteric alteration in TLS that is required for its interaction with CBP/p300. Moreover, unlike wildtype TLS, an R476A TLS mutant did not inhibit CCND1 promoter activity in luciferase reporter assays. Taken together, we propose the hypothesis that arginine methylation of TLS regulates both TLS-nucleic acid and TLS-protein interactions and thereby participates in transcriptional regulation. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Myopodin is an F-actin bundling protein with multiple independent actin-binding regions.

PubMed

Linnemann, Anja; Vakeel, Padmanabhan; Bezerra, Eduardo; Orfanos, Zacharias; Djinović-Carugo, Kristina; van der Ven, Peter F M; Kirfel, Gregor; Fürst, Dieter O

2013-02-01

The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric α-actinin, as well as its binding to actin, α-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of α-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin's ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.

Bmh1p (14-3-3) mediates pathways associated with virulence in Candida albicans

PubMed Central

Kelly, Michelle N.; Johnston, Douglas A.; Peel, Bethany A.; Morgan, Timothy W.; Palmer, Glen E.; Sturtevant, Joy E.

2009-01-01

The ability of the pathogenic fungus Candida albicans to cause disease requires rapid adaptation to changes in the host environment and to an evolving host immune response. The identification of ‘virulence factors’ using in vitro characterization of mutant strains has traditionally relied on a common set of phenotypic and biochemical assays (most often performed at 30 °C) and the subsequent correlation with their corresponding virulence in mouse models of disease. Utilizing a panel of isogenic mutants for the multifunctional signal-modulating 14-3-3 protein (Bmh1p), we have found that specific mutations affect a variety of different pathways currently associated with virulence, including those involved with the formation of filaments, as well as interaction with host immune cells. Surprisingly, our studies revealed that deficiencies in many of these pathways do not always correlate with virulence in a mouse model of disseminated infection. Mutations within the binding pocket of Bmh1p that affect the ability of the protein to efficiently bind ligand had varying effects on the results of a number of in vitro and in vivo assays. The capability, in vitro, to filament in embedment conditions, and to filament and form chlamydospores under microaerophilic conditions on cornmeal agar, does not correlate with virulence. It is likely that only a subset of hyphal signalling pathways is actually required for the establishment of infection in the disseminated mouse model. Most importantly, our results suggest that the delayed onset of lag-phase growth in vitro at 37 °C, and not at 30 °C, results in an inability of these mutants to rapidly adjust to environmental changes in vivo and may be responsible for their increased clearance and reduced virulence. It is critical, therefore, that future in vitro studies of putative virulence factors in C. albicans include careful characterization at physiological temperatures. PMID:19372164

Bmh1p (14-3-3) mediates pathways associated with virulence in Candida albicans.

PubMed

Kelly, Michelle N; Johnston, Douglas A; Peel, Bethany A; Morgan, Timothy W; Palmer, Glen E; Sturtevant, Joy E

2009-05-01

The ability of the pathogenic fungus Candida albicans to cause disease requires rapid adaptation to changes in the host environment and to an evolving host immune response. The identification of 'virulence factors' using in vitro characterization of mutant strains has traditionally relied on a common set of phenotypic and biochemical assays (most often performed at 30 degrees C) and the subsequent correlation with their corresponding virulence in mouse models of disease. Utilizing a panel of isogenic mutants for the multifunctional signal-modulating 14-3-3 protein (Bmh1p), we have found that specific mutations affect a variety of different pathways currently associated with virulence, including those involved with the formation of filaments, as well as interaction with host immune cells. Surprisingly, our studies revealed that deficiencies in many of these pathways do not always correlate with virulence in a mouse model of disseminated infection. Mutations within the binding pocket of Bmh1p that affect the ability of the protein to efficiently bind ligand had varying effects on the results of a number of in vitro and in vivo assays. The capability, in vitro, to filament in embedment conditions, and to filament and form chlamydospores under microaerophilic conditions on cornmeal agar, does not correlate with virulence. It is likely that only a subset of hyphal signalling pathways is actually required for the establishment of infection in the disseminated mouse model. Most importantly, our results suggest that the delayed onset of log-phase [corrected] growth in vitro at 37 degrees C, and not at 30 degrees C, results in an inability of these mutants to rapidly adjust to environmental changes in vivo and may be responsible for their increased clearance and reduced virulence. It is critical, therefore, that future in vitro studies of putative virulence factors in C. albicans include careful characterization at physiological temperatures.

New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics.

PubMed

Zeilinger, Markus; Pichler, Florian; Nics, Lukas; Wadsak, Wolfgang; Spreitzer, Helmut; Hacker, Marcus; Mitterhauser, Markus

2017-12-01

Resolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A 3 R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the K i of eight different A 3 R antagonists, using CHO-K1 cells stably expressing the hA 3 R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants k on and k off , as well as the dedicated K d of the A 3 R agonist [ 125 I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds. Two novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A 3 R antagonists, no influences on the experimental performance and the resulting affinity were investigated. Both kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

Insight into the recognition, binding, and reactivity of catalytic metallodrugs targeting stem loop IIb of hepatitis C IRES RNA.

PubMed

Bradford, Seth S; Ross, Martin James; Fidai, Insiya; Cowan, James A

2014-06-01

The complex Cu-GGHYrFK-amide (1-Cu) was previously reported as a novel metallotherapeutic that catalytically inactivates stem loop IIb (SLIIb) of the hepatitis C virus (HCV) internal ribosomal entry site (IRES) RNA and demonstrates significant antiviral activity in a cellular HCV replicon assay. Herein we describe additional studies focused on understanding the cleavage mechanism as well as the relationship of catalyst configuration to structural recognition and site-selective cleavage of the structured RNA motif. These are advanced by use of a combination of MALDI-TOF mass spectrometry, melting temperature determinations, and computational analysis to develop a structural model for binding and reactivity toward SLIIb of the IRES RNA. In addition, the binding, reactivity, and structural chemistry of the all-D-amino acid form of this metallopeptide, complex 2-Cu, are reported and compared with those of complex 1-Cu. In vitro RNA binding and cleavage assays for complex 2-Cu show a KD value of 76 ± 3 nM, and Michaelis-Menten parameters of kcat =0.14 ± 0.01 min(-1) and KM =7.9 ± 1.2 μM, with a turnover number exceeding 40. In a luciferase-based cellular replicon assay Cu-GGhyrfk-amide shows activity similar to that of the 1-Cu parent peptide, with an IC50 value of 1.9 ± 0.4 μM and cytotoxicity exceeding 100 μM. RT-PCR experiments confirm a significant decrease in HCV RNA levels in replicon assays for up to nine days when treated with complex 1-Cu in three-day dosing increments. This study shows the influence that the α-carbon stereocenter has for this new class of compounds, while detailed mass spectrometry and computational analyses provide new insight into the mechanisms of recognition, binding, and reactivity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In vitro screening of major neurotransmitter systems possibly involved in the mechanism of action of antibodies to S100 protein in released-active form

PubMed Central

Gorbunov, Evgeniy A; Ertuzun, Irina A; Kachaeva, Evgeniya V; Tarasov, Sergey A; Epstein, Oleg I

2015-01-01

Experimentally and clinically, it was shown that released-active form of antibodies to S100 protein (RAF of Abs to S100) exerts a wide range of pharmacological activities: anxiolytic, antiasthenic, antiaggressive, stress-protective, antihypoxic, antiischemic, neuroprotective, and nootropic. The purpose of this study was to determine the influence of RAF of Abs to S100 on major neurotransmitter systems (serotoninergic, GABAergic, dopaminergic, and on sigma receptors as well) which are possibly involved in its mechanism of pharmacological activity. Radioligand binding assays were used for assessment of the drug influence on ligand–receptor interaction. [35S]GTPγS binding assay, cyclic adenosine monophosphate HTRF™, cellular dielectric spectroscopy assays, and assays based on measurement of intracellular concentration of Ca2+ ions were used for assessment of agonist or antagonist properties of the drug toward receptors. RAF of Abs to S100 increased radioligand binding to 5-HT1F, 5-HT2B, 5-HT2Cedited, 5-HT3, and to D3 receptors by 142.0%, 131.9%, 149.3%, 120.7%, and 126.3%, respectively. Also, the drug significantly inhibited specific binding of radioligands to GABAB1A/B2 receptors by 25.8%, and to both native and recombinant human sigma1 receptors by 75.3% and 40.32%, respectively. In the functional assays, it was shown that the drug exerted antagonism at 5-HT1B, D3, and GABAB1A/B2 receptors inhibiting agonist-induced responses by 23.24%, 32.76%, and 30.2%, respectively. On the contrary, the drug exerted an agonist effect at 5-HT1A receptors enhancing receptor functional activity by 28.0%. The pharmacological profiling of RAF of Abs to S100 among 27 receptor provides evidence for drug-related modification of major neurotransmitter systems. PMID:26604768

In vitro and in vivo comparison of binding of 99m-Tc-labeled anti-CEA MAb F33-104 with 99m-Tc-labeled anti-CEA MAb BW431/26.

PubMed

Watanabe, N; Oriuchi, N; Sugiyama, S; Kuroki, M; Matsuoka, Y; Tanada, S; Murata, H; Inoue, T; Sasaki, Y

1999-01-01

The purpose of this study was to assess the potential for radio-immunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tc-labeled anti-CEA MAb BW431/261 (31.4 +/- 0.95% vs. 11.9 +/- 0.55% at 100 ng/mL of soluble CEA, 83.5 +/- 2.84% vs. 54.0 +/- 2.54% at 10(7) of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 +/- 3.50% ID/g vs. 14.4 +/- 3.30% ID/g). 99m-Tc-activity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 +/- 2.10% ID/g vs. 8.01 +/- 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 +/- 1.70% ID/g vs. 8.10 +/- 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

Cyquant cell proliferation assay as a fluorescence-based method for in vitro screening of antimalarial activity.

PubMed

Sriwilaijaroen, Nongluk; Kelly, Jane Xu; Riscoe, Michael; Wilairat, Prapon

2004-12-01

The appearance of drug resistant parasites and the absence of an effective vaccine have resulted in the need for new effective antimalarial drugs. Consequently, a convenient method for in vitro screening of large numbers of antimalarial drug candidates has become apparent. The CyQUANT cell proliferation assay is a highly sensitive fluorescence-based method for quantitation of cell number by measuring the strong fluorescence produced when green GR dye binds to nucleic acids. We have applied the CyQUANT assay method to evaluate the growth of Plasmodium falciparum D6 strain in culture. The GR-nucleic acid fluorescence linearly correlated with percent parasitemia at both 0.75 or 1 percent hematocrit with the same correlation coefficient of r2 = 0.99. The sensitivity of P. falciparum D6 strain to chloroquine and to 3,6-bis-omega-diethylaminoamyloxyxanthone, a novel antimalarial, determined by the CyQUANT assay were comparable to those obtained by the traditional [3H]-ethanolamine assay: IC50 value of chloroquine was 54 nM and 51 nM by the CyQUANT and [3H]-ethanolamine assay, respectively; IC50 value for 3,6-bis-omega-diethylaminoamyloxyxanthone was 254 nM and 223 nM by the CyQUANT and [3H]-ethanolamine assay, respectively. This procedure requires no radioisotope, uses simple equipment, and is an easy and convenient procedure, with no washing and harvesting steps. Moreover, all procedures can be set up continuously and thus, the CyQUANT assay is suitable in automatic high through-put drug screening of antimalarial drugs.

Production and characterization of monoclonal antibodies to the protective antigen component of Bacillus anthracis toxin.

PubMed Central

Little, S F; Leppla, S H; Cora, E

1988-01-01

Thirty-six monoclonal antibodies to the protective antigen protein of Bacillus anthracis exotoxin have been characterized for affinity, antibody subtype, competitive binding to antigenic regions, and ability to neutralize lethal and edema toxin activities. At least 23 antigenic regions were detected on protective antigen by a blocking, enzyme-linked immunosorbent assay. Two clones, 3B6 and 14B7, competed for a single antigenic region and neutralized the activity of both the lethal toxin in vivo (Fisher 344 rat) and the edema toxin in vitro (CHO cells). These two antibodies blocked the binding of 125I-labeled protective antigen to FRL-103 cells. Our results support the proposal that binding of protective antigen to cell receptors is required for expression of toxicity. Images PMID:3384478

A substitutional mutation in the DNA binding domain of the androgen receptor causes complete androgen insensitivity syndrome.

PubMed

Komori, S; Sakata, K; Kasumi, H; Tsuji, Y; Hamada, K; Koyama, K

1999-10-01

DNA analysis of the androgen receptor gene in a patient with complete androgen insensitivity syndrome identified a substitutional mutation (tyrosine converted to cysteine at position 571) in the DNA binding domain. In vitro transfection experiments with the patients' androgen receptor gene, indicated normal expression of the androgen receptor in transfected COS-7 cells compared to the wild type gene. There was also no evidence of impaired thermal stability of the 5 alpha-dihydrotestosterone-androgen receptor complex. However, the capacity of the androgen receptor to activate target gene transcription was found to be completely disrupted in a luciferase assay. These results confirmed that only one substitutional mutation in the DNA binding domain was related to the pathogenesis of the complete androgen insensitivity syndrome.

Structural Characterization of an Alternative Mode of Tigecycline Binding to the Bacterial Ribosome

PubMed Central

Schedlbauer, Andreas; Kaminishi, Tatsuya; Ochoa-Lizarralde, Borja; Dhimole, Neha; Zhou, Shu; López-Alonso, Jorge P.

2015-01-01

Although both tetracycline and tigecycline inhibit protein synthesis by sterically hindering the binding of tRNA to the ribosomal A site, tigecycline shows increased efficacy in both in vitro and in vivo activity assays and escapes the most common resistance mechanisms associated with the tetracycline class of antibiotics. These differences in activities are attributed to the tert-butyl-glycylamido side chain found in tigecycline. Our structural analysis by X-ray crystallography shows that tigecycline binds the bacterial 30S ribosomal subunit with its tail in an extended conformation and makes extensive interactions with the 16S rRNA nucleotide C1054. These interactions restrict the mobility of C1054 and contribute to the antimicrobial activity of tigecycline, including its resistance to the ribosomal protection proteins. PMID:25753625

Abalone Hemocyanin Blocks the Entry of Herpes Simplex Virus 1 into Cells: a Potential New Antiviral Strategy

PubMed Central

Talaei Zanjani, Negar; Miranda-Saksena, Monica; Valtchev, Peter; Hueston, Linda; Diefenbach, Eve; Sairi, Fareed; Gomes, Vincent G.

2015-01-01

A marine-derived compound, abalone hemocyanin, from Haliotis rubra was shown to have a unique mechanism of antiviral activity against herpes simplex virus 1 (HSV-1) infections. In vitro assays demonstrated the dose-dependent and inhibitory effect of purified hemocyanin against HSV-1 infection in Vero cells with a 50% effective dose (ED50) of 40 to 50 nM and no significant toxicity. In addition, hemocyanin specifically inhibited viral attachment and entry by binding selectively to the viral surface glycoproteins gD, gB, and gC, probably by mimicking their receptors. However, hemocyanin had no effect on postentry events and did not block infection by binding to cellular receptors for HSV. By the use of different mutants of gD and gB and a competitive heparin binding assay, both protein charge and conformation were shown to be the driving forces of the interaction between hemocyanin and viral glycoproteins. These findings also suggested that hemocyanin may have different motifs for binding to each of the viral glycoproteins B and D. The dimer subunit of hemocyanin with a 10-fold-smaller molecular mass exhibited similar binding to viral surface glycoproteins, showing that the observed inhibition did not require the entire multimer. Therefore, a small hemocyanin analogue could serve as a new antiviral candidate for HSV infections. PMID:26643336

DNA hypomethylation of a transcription factor binding site within the promoter of a gout risk gene NRBP1 upregulates its expression by inhibition of TFAP2A binding.

PubMed

Zhu, Zaihua; Meng, Weida; Liu, Peiru; Zhu, Xiaoxia; Liu, Yun; Zou, Hejian

2017-01-01

Genome-wide association studies (GWASs) have identified dozens of loci associated with gout, but for most cases, the risk genes and the underlying molecular mechanisms contributing to these associations are unknown. This study sought to understand the molecular mechanism of a common genetic variant, rs780093, in the development of gout, both in vitro and in vivo. Nuclear receptor binding protein 1 ( NRBP1 ), as a gout risk gene, and its regulatory region, 72 bp upstream of the transcription start site, designated as B1, were identified through integrative analyses of genome-wide genotype and DNA methylation data. We observed elevated NRBP1 expression in human peripheral blood mononuclear cells (PBMCs) from gout patients. In vitro luciferase reporter and protein pulldown assay results showed that DNA methylation could increase the binding of the transcription factor TFAP2A to B1, leading to suppressed gene expression. There results were further confirmed by in vivo bisulfite pyrosequencing showing that hypomethylation on B1 is associated with increased NRBP1 expression in gout patients. Hypomethylation at the promoter region of NRBP1 reduces the binding of TFAP2A and thus leads to elevated NRBP1 expression, which might contribute to the development of gout.

Structure-Function Relationships in Human Testis-determining Factor SRY

PubMed Central

Racca, Joseph D.; Chen, Yen-Shan; Maloy, James D.; Wickramasinghe, Nalinda; Phillips, Nelson B.; Weiss, Michael A.

2014-01-01

Human testis determination is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in SRY cause 46 XY gonadal dysgenesis with female somatic phenotype (Swyer syndrome) and confer a high risk of malignancy (gonadoblastoma). Such mutations cluster in the SRY high mobility group (HMG) box, a conserved motif of specific DNA binding and bending. To explore structure-function relationships, we constructed all possible substitutions at a site of clinical mutation (W70L). Our studies thus focused on a core aromatic residue (position 15 of the consensus HMG box) that is invariant among SRY-related HMG box transcription factors (the SOX family) and conserved as aromatic (Phe or Tyr) among other sequence-specific boxes. In a yeast one-hybrid system sensitive to specific SRY-DNA binding, the variant domains exhibited reduced (Phe and Tyr) or absent activity (the remaining 17 substitutions). Representative nonpolar variants with partial or absent activity (Tyr, Phe, Leu, and Ala in order of decreasing side-chain volume) were chosen for study in vitro and in mammalian cell culture. The clinical mutation (Leu) was found to markedly impair multiple biochemical and cellular activities as respectively probed through the following: (i) in vitro assays of specific DNA binding and protein stability, and (ii) cell culture-based assays of proteosomal degradation, nuclear import, enhancer DNA occupancy, and SRY-dependent transcriptional activation. Surprisingly, however, DNA bending is robust to this or the related Ala substitution that profoundly impairs box stability. Together, our findings demonstrate that the folding, trafficking, and gene-regulatory function of SRY requires an invariant aromatic “buttress” beneath its specific DNA-bending surface. PMID:25258310

Naphthol AS-E Phosphate Inhibits the Activity of the Transcription Factor Myb by Blocking the Interaction with the KIX Domain of the Coactivator p300.

PubMed

Uttarkar, Sagar; Dukare, Sandeep; Bopp, Bertan; Goblirsch, Michael; Jose, Joachim; Klempnauer, Karl-Heinz

2015-06-01

The transcription factor c-Myb is highly expressed in hematopoietic progenitor cells and controls the transcription of genes important for lineage determination, cell proliferation, and differentiation. Deregulation of c-Myb has been implicated in the development of leukemia and certain other types of human cancer. c-Myb activity is highly dependent on the interaction of the c-Myb with the KIX domain of the coactivator p300, making the disruption of this interaction a reasonable strategy for the development of Myb inhibitors. Here, we have used bacterial Autodisplay to develop an in vitro binding assay that mimics the interaction of Myb and the KIX domain of p300. We have used this binding assay to investigate the potential of Naphthol AS-E phosphate, a compound known to bind to the KIX domain, to disrupt the interaction between Myb and p300. Our data show that Naphthol AS-E phosphate interferes with the Myb-KIX interaction in vitro and inhibits Myb activity in vivo. By using several human leukemia cell lines, we demonstrate that Naphthol AS-E phosphate suppresses the expression of Myb target genes and induces myeloid differentiation and apoptosis. Our work identifies Naphthol AS-E phosphate as the first low molecular weight compound that inhibits Myb activity by disrupting its interaction with p300, and suggests that inhibition of the Myb-KIX interaction might be a useful strategy for the treatment of leukemia and other tumors caused by deregulated c-Myb. ©2015 American Association for Cancer Research.

Modifications of nano-titania surface for in vitro evaluations of hemolysis, cytotoxicity, and nonspecific protein binding

NASA Astrophysics Data System (ADS)

Datta, Aparna; Dasgupta, Sayantan; Mukherjee, Siddhartha

2017-04-01

In the past decade, a variety of drug carriers based on mesoporous silica nanoparticles has been extensively reported. However, their biocompatibility still remains debatable, which motivated us to explore the porous nanostructures of other metal oxides, for example titanium dioxide (TiO2), as potential drug delivery vehicles. Herein, we report the in vitro hemolysis, cytotoxicity, and protein binding of TiO2 nanoparticles, synthesized by a sol-gel method. The surface of the TiO2 nanoparticles was modified with hydroxyl, amine, or thiol containing moieties to examine the influence of surface functional groups on the toxicity and protein binding aspects of the nanoparticles. Our study revealed the superior hemocompatibility of pristine, as well as functionalized TiO2 nanoparticles, compared to that of mesoporous silica, the present gold standard. Among the functional groups studied, aminosilane moieties on the TiO2 surface substantially reduced the degree of hemolysis (down to 5%). Further, cytotoxicity studies by MTT assay suggested that surface functional moieties play a crucial role in determining the biocompatibility of the nanoparticles. The presence of NH2- functional groups on the TiO2 nanoparticle surface enhanced the cell viability by almost 28% as compared to its native counterpart (at 100 μg/ml), which was in agreement with the hemolysis assay. Finally, nonspecific protein adsorption on functionalized TiO2 surfaces was examined using human serum albumin and it was found that negatively charged surface moieties, like -OH and -SH, could mitigate protein adsorption to a significant extent.

Bacteroides gingivalis-Actinomyces viscosus cohesive interactions as measured by a quantitative binding assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwarz, S.; Ellen, R.P.; Grove, D.A.

1987-10-01

There is limited evidence, mostly indirect, to suggest that the adherence of Bacteroides gingivalis to teeth may be enhanced by the presence of gram-positive dental plaque bacteria like Actinomyces viscosus. The purpose of this study was to carry out direct quantitative assessments of the cohesion of B gingivalis and A. viscosus by using an in vitro assay modeled on the natural sequence in which these two species colonize the teeth. The assay allowed comparisons to be made of the adherence of /sup 3/H-labeled B. gingivalis 2561 and 381 to saliva-coated hydroxyapatite beads (S-HA) and A. viscosus WVU627- or T14V-coated S-HAmore » (actinobeads) in equilibrium and kinetics binding studies. A series of preliminary binding studies with 3H-labeled A. viscosus and parallel studies by scanning electron microscopy with unlabeled A. viscosus were conducted to establish a protocol by which actinobeads suitable for subsequent Bacteroides adherence experiments could be prepared. By scanning electron microscopy, the actinobeads had only small gaps of exposed S-HA between essentially irreversibly bound A. viscosus cells. Furthermore, B. gingivalis cells appeared to bind preferentially to the Actinomyces cells instead of the exposed S-HA. B. gingivalis binding to both S-HA and actinobeads was saturable with at least 2 X 10(9) to 3 X 10(9) cells per ml, and equilibrium with saturating concentrations was reached within 10 to 20 min. B. gingivalis always bound in greater numbers to the actinobeads than to S-HA. These findings provide direct measurements supporting the concept that cohesion with dental plaque bacteria like A. viscosus may foster the establishment of B. gingivalis on teeth by enhancing its adherence.« less

Specific repression of β-globin promoter activity by nuclear ferritin

PubMed Central

Broyles, Robert H.; Belegu, Visar; DeWitt, Christina R.; Shah, Sandeep N.; Stewart, Charles A.; Pye, Quentin N.; Floyd, Robert A.

2001-01-01

Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult β-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the β-globin promoter at −153 to −148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress β-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the β-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant β-globin promoter for the −150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the −150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human β-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases. PMID:11481480

Specific detection of tetanus toxoid using an aptamer-based matrix.

PubMed

Modh, Harshvardhan B; Bhadra, Ankan K; Patel, Kinjal A; Chaudhary, Rajeev K; Jain, Nishant K; Roy, Ipsita

2016-11-20

Batch-to-batch variation of therapeutic proteins produced by biological means requires rigorous monitoring at all stages of the production process. A large number of animals are employed for risk assessment of biologicals, which has low ethical and economic acceptability. Research is now focussed on the validation of in vitro and ex vivo tests to replace live challenges. Among in vitro methods, enzyme-linked immunosorbent assay (ELISA) is considered to be the gold standard for estimation of integrity of tetanus toxoid. ELISA utilizes antibodies for detection, which, because of their biological origin and limited modifiability, may have low stability and result in irreproducibility. We have developed a method using highly specific and selective RNA aptamers for detection of tetanus toxoid. Using displacement assay, we first identified aptamers which bind to different aptatopes on the surface of the toxoid. Pairs of these aptamers were employed as capture-detection ligands in a sandwich-ALISA (aptamer-linked immobilized sorbent assay) format. The binding efficiency was confirmed by the fluorescence intensity in each microtire plate well. Using aptamers alone, detection of tetanus toxoid was possible with the same level of sensitivity as antibody. Aptamers were also used in the capture ALISA format. Adjuvanted tetanus toxoid was subjected to accelerated stress testing, including thermal, mechanical and freeze-thawing stress conditions. The loss in antigenicity of the preparation determined by ALISA in each case was found to be similar to that determined by conventional ELISA. Thus, it is possible to replace antibodies with aptamers to develop a more robust detection tool for tetanus toxoid. Copyright © 2016 Elsevier B.V. All rights reserved.

Application of an in vitro DNA protection assay to visualize stress mediation properties of the Dps protein.

PubMed

Karas, Vlad O; Westerlaken, Ilja; Meyer, Anne S

2013-05-31

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage. The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

A cytoplasmic protein, bystin, interacts with trophinin, tastin, and cytokeratin and may be involved in trophinin-mediated cell adhesion between trophoblast and endometrial epithelial cells

PubMed Central

Suzuki, Nao; Zara, Jane; Sato, Takaaki; Ong, Edgar; Bakhiet, Nouna; Oshima, Robert G.; Watson, Kellie L.; Fukuda, Michiko N.

1998-01-01

Trophinin and tastin form a cell adhesion molecule complex that potentially mediates an initial attachment of the blastocyst to uterine epithelial cells at the time of implantation. Trophinin and tastin, however, do not directly bind to each other, suggesting the presence of an intermediary protein. The present study identifies a cytoplasmic protein, named bystin, that directly binds trophinin and tastin. Bystin consists of 306 amino acid residues and is predicted to contain tyrosine, serine, and threonine residues in contexts conforming to motifs for phosphorylation by protein kinases. Database searches revealed a 53% identity of the predicted peptide sequence with the Drosophila bys (mrr) gene. Direct protein–protein interactions of trophinin, tastin, and bystin analyzed by yeast two-hybrid assays and by in vitro protein binding assays indicated that binding between bystin and trophinin and between bystin and tastin is enhanced when cytokeratin 8 and 18 are present as the third molecule. Immunocytochemistry of bystin showed that bystin colocalizes with trophinin, tastin, and cytokeratins in a human trophoblastic teratocarcinoma cell, HT-H. It is therefore possible that these molecules form a complex and thus are involved in the process of embryo implantation. PMID:9560222

DNA Replication Arrest and DNA Damage Responses Induced by Alkylating Minor Groove Binders

DTIC Science & Technology

2001-05-01

We are interested in the molecular mechanisms involved in DNA replication arrest by the S phase DNA damage checkpoints. Using in vitro simian virus...40 DNA replication assays, we have found three factors that directly contribute to DNA damage-induced DNA replication arrest: Replication Protein A...trans-acting inhibitors. RPA is the major eukaryotic single-stranded DNA binding protein required for DNA replication , repair and recombination. Upon DNA

p53 Specifically Binds Triplex DNA In Vitro and in Cells

PubMed Central

Brázdová, Marie; Tichý, Vlastimil; Helma, Robert; Bažantová, Pavla; Polášková, Alena; Krejčí, Aneta; Petr, Marek; Navrátilová, Lucie; Tichá, Olga; Nejedlý, Karel; Bennink, Martin L.; Subramaniam, Vinod; Bábková, Zuzana; Martínek, Tomáš; Lexa, Matej; Adámik, Matej

2016-01-01

Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed. PMID:27907175

Real-Time Protein and Cell Binding Measurements on Hydroxyapatite Coatings

PubMed Central

Vilardell, A. M.; Cinca, N.; Jokinen, A.; Garcia-Giralt, N.; Dosta, S.; Cano, I. G.; Guilemany, J. M.

2016-01-01

Although a lot of in vitro and in vivo assays have been performed during the last few decades years for hydroxyapatite bioactive coatings, there is a lack of exploitation of real-time in vitro interaction measurements. In the present work, real-time interactions for a plasma sprayed hydroxyapatite coating were measured by a Multi-Parametric Surface Plasmon Resonance (MP-SPR), and the results were compared with standard traditional cell viability in vitro assays. MP-SPR is proven to be suitable not only for measurement of molecule–molecule interactions but also molecule–material interaction measurements and cell interaction. Although SPR is extensively utilized in interaction studies, recent research of protein or cell adsorption on hydroxyapatite coatings for prostheses applications was not found. The as-sprayed hydroxyapatite coating resulted in 62.4% of crystalline phase and an average thickness of 24 ± 6 μm. The MP-SPR was used to measure lysozyme protein and human mesenchymal stem cells interaction to the hydroxyapatite coating. A comparison between the standard gold sensor and Hydroxyapatite (HA)-plasma coated sensor denoted a clearly favourable cell attachment on HA coated sensor as a significantly higher signal of cell binding was detected. Moreover, traditional cell viability and proliferation tests showed increased activity with culture time indicating that cells were proliferating on HA coating. Cells show homogeneous distribution and proliferation along the HA surface between one and seven days with no significant mortality. Cells were flattened and spread on rough surfaces from the first day, with increasing cytoplasmatic extensions during the culture time. PMID:27618911

In vitro bioactivity of 17alpha-estradiol.

PubMed

Sievernich, André; Wildt, Ludwig; Lichtenberg-Fraté, Hella

2004-12-01

A miniaturised short-term in vitro assay based on the activation of the human estrogen receptor alpha and genetically modified yeast (Saccharomyces cerevisiae) cells was performed to explore the capacity of this system to monitor the bioactivity of estrogenic compounds, particularly 17alpha- and 17beta-estradiol. Together with the human estrogen receptor (hER)-alpha plasmid, the reporter plasmid containing a yeast-optimised version of the green fluorescent protein (yEGFP) linked to three repeats of the cis-acting estrogen hormone-responsive element (ERE) were expressed in a strain being deleted in the pleiotropic drug resistance transporters Pdr5, Snq2 and Yor1, known to facilitate efflux of organic compounds including steroids and chemotherapeutics. Agonists that bind to hER in vitro trigger estrogen receptor-mediated transcriptional activation of the GFP reporter gene monitored by fluorescence emission at 535 nm. The sensitivity of the assay was tested with various 17alpha- and 17beta-estradiol concentrations, yielding a detection limit of 5 pg/ml (0.018 nM) for the agonist 17beta-E2 in solvent and in human charcoal-stripped serum using a S. cerevisiae pdr5, snq2 and yor1 mutant strain. For 17alpha-estradiol only, at approximately 1500 pg/ml a similar fluorescence response compared to 100 pg/ml 17beta-E2 was observed implicating a much weaker potency of this stereoisomer. The specificity of the system was tested by expression of a truncated hER lacking the ligand-binding domain E and by administration of the androgen, 4-androsten 3,17 dione. Both controls did not yield an increase in fluorescence emission. This fluorescence emission assay enables detection of estrogenic biological activity induced by direct agonists, such as 17beta-E2 at concentrations similar to those found in human sera or by estrogen-like chemicals.

Green tea polyphenol epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-alpha and matrix metalloproteinase-13 in human chondrocytes.

PubMed

Rasheed, Zafar; Anbazhagan, Arivarasu N; Akhtar, Nahid; Ramamurthy, Sangeetha; Voss, Frank R; Haqqi, Tariq M

2009-01-01

The major risk factor for osteoarthritis (OA) is aging, but the mechanisms underlying this risk are only partly understood. Age-related accumulation of advanced glycation end products (AGEs) can activate chondrocytes and induce the production of proinflammatory cytokines and matrix metalloproteinases (MMPs). In the present study, we examined the effect of epigallocatechin-3-gallate (EGCG) on AGE-modified-BSA (AGE-BSA)-induced activation and production of TNFalpha and MMP-13 in human OA chondrocytes. Human chondrocytes were derived from OA cartilage by enzymatic digestion and stimulated with in vitro-generated AGE-BSA. Gene expression of TNFalpha and MMP-13 was measured by quantitative RT-PCR. TNFalpha protein in culture medium was determined using cytokine-specific ELISA. Western immunoblotting was used to analyze the MMP-13 production in the culture medium, phosphorylation of mitogen-activated protein kinases (MAPKs), and the activation of NF-kappaB. DNA binding activity of NF-kappaB p65 was determined using a highly sensitive and specific ELISA. IkappaB kinase (IKK) activity was determined using an in vitro kinase activity assay. MMP-13 activity in the culture medium was assayed by gelatin zymography. EGCG significantly decreased AGE-stimulated gene expression and production of TNFalpha and MMP-13 in human chondrocytes. The inhibitory effect of EGCG on the AGE-BSA-induced expression of TNFalpha and MMP-13 was mediated at least in part via suppression of p38-MAPK and JNK activation. In addition, EGCG inhibited the phosphorylating activity of IKKbeta kinase in an in vitro activity assay and EGCG inhibited the AGE-mediated activation and DNA binding activity of NF-kappaB by suppressing the degradation of its inhibitory protein IkappaBalpha in the cytoplasm. These novel pharmacological actions of EGCG on AGE-BSA-stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG-derived compounds may inhibit cartilage degradation by suppressing AGE-mediated activation and the catabolic response in human chondrocytes.

In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2

PubMed Central

Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R

2018-01-01

Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V1) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys8]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg8]-vasopressin (AVP) at V1 and vasopressin-2 (V2) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V1 and V2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [3H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V1) and cyclic adenosine monophosphate (V2). Binding potency at V1 and V2 was AVP>LVP>>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V1 than for V2. Cellular activity potency was also AVP>LVP>>terlipressin. Terlipressin was a partial agonist at V1 and a full agonist at V2; LVP was a full agonist at both V1 and V2. The in vivo response to terlipressin is likely due to the partial V1 agonist activity of terlipressin and full V1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors. PMID:29302194

In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2.

PubMed

Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R

2018-01-01

Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V 1 ) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys 8 ]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg 8 ]-vasopressin (AVP) at V 1 and vasopressin-2 (V 2 ) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V 1 and V 2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [ 3 H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V 1 ) and cyclic adenosine monophosphate (V 2 ). Binding potency at V 1 and V 2 was AVP>LVP>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V 1 than for V 2 . Cellular activity potency was also AVP>LVP>terlipressin. Terlipressin was a partial agonist at V 1 and a full agonist at V 2 ; LVP was a full agonist at both V 1 and V 2 . The in vivo response to terlipressin is likely due to the partial V 1 agonist activity of terlipressin and full V 1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors.

Regulation of Son of sevenless by the membrane-actin linker protein ezrin

PubMed Central

Geißler, Katja J.; Jung, M. Juliane; Riecken, Lars Björn; Sperka, Tobias; Cui, Yan; Schacke, Stephan; Merkel, Ulrike; Markwart, Robby; Rubio, Ignacio; Than, Manuel E.; Breithaupt, Constanze; Peuker, Sebastian; Seifert, Reinhard; Kaupp, Ulrich Benjamin; Herrlich, Peter; Morrison, Helen

2013-01-01

Receptor tyrosine kinases participate in several signaling pathways through small G proteins such as Ras (rat sarcoma). An important component in the activation of these G proteins is Son of sevenless (SOS), which catalyzes the nucleotide exchange on Ras. For optimal activity, a second Ras molecule acts as an allosteric activator by binding to a second Ras-binding site within SOS. This allosteric Ras-binding site is blocked by autoinhibitory domains of SOS. We have reported recently that Ras activation also requires the actin-binding proteins ezrin, radixin, and moesin. Here we report the mechanism by which ezrin modulates SOS activity and thereby Ras activation. Active ezrin enhances Ras/MAPK signaling and interacts with both SOS and Ras in vivo and in vitro. Moreover, in vitro kinetic assays with recombinant proteins show that ezrin also is important for the activity of SOS itself. Ezrin interacts with GDP-Ras and with the Dbl homology (DH)/pleckstrin homology (PH) domains of SOS, bringing GDP-Ras to the proximity of the allosteric site of SOS. These actions of ezrin are antagonized by the neurofibromatosis type 2 tumor-suppressor protein merlin. We propose an additional essential step in SOS/Ras control that is relevant for human cancer as well as all physiological processes involving Ras. PMID:24297905

Structural insights into the recognition of the internal A-rich linker from OxyS sRNA by Escherichia coli Hfq

PubMed Central

Wang, Lijun; Wang, Weiwei; Li, Fudong; Zhang, Jiahai; Wu, Jihui; Gong, Qingguo; Shi, Yunyu

2015-01-01

Small RNA OxyS is induced during oxidative stress in Escherichia coli and it is an Hfq-dependent negative regulator of mRNA translation. OxyS represses the translation of fhlA and rpoS mRNA, which encode the transcriptional activator and σs subunit of RNA polymerase, respectively. However, little is known regarding how Hfq, an RNA chaperone, interacts with OxyS at the atomic level. Here, using fluorescence polarization and tryptophan fluorescence quenching assays, we verified that the A-rich linker region of OxyS sRNA binds Hfq at its distal side. We also report two crystal structures of Hfq in complex with A-rich RNA fragments from this linker region. Both of these RNA fragments bind to the distal side of Hfq and adopt a different conformation compared with those previously reported for the (A-R-N)n tripartite recognition motif. Furthermore, using fluorescence polarization, electrophoresis mobility shift assays and in vivo translation assays, we found that an Hfq mutant, N48A, increases the binding affinity of OxyS for Hfq in vitro but is defective in the negative regulation of fhlA translation in vivo, suggesting that the normal function of OxyS depends on the details of the interaction with Hfq that may be related to the rapid recycling of Hfq in the cell. PMID:25670676

Screening for lead compounds and herbal extracts with potential anti-influenza viral activity.

PubMed

Klaywong, Konrapob; Khutrakul, Gachagorn; Choowongkomon, Kiattawee; Lekcharoensuk, Chalermpol; Petcharat, Nantawan; Leckcharoensuk, Porntippa; Ramasoota, Pongrama

2014-01-01

Nonstructural protein 1 (NS1) of the highly pathogenic avian influenza virus (H5N1) contains a conserved RNA binding domain (RBD) that inhibits antiviral functions of host-innate immune response. Dimerization of NS1 forms a central groove and binds to double stranded (ds) RNA. This region might serve as a potential drug target. In this study, three dimensional structure model of NS1 RBD protein was constructed and virtual screening was performed to identify lead compounds that bound within and around the central groove. The virtual screening showed that 5 compounds bound within the central groove with binding energy ranging between -16.05 and -17.36 Kcal/mol. Two commercially available compounds, estradiol and veratridine, were selected for using in an in vitro screening assay. The results showed that neither of the compounds could inhibit the association between dsRNA and NS1 RBD protein. In addition, 34 herbal extracts were examined for their inhibitory effects. Five of them were able to inhibit association between NS1 RBD and dsRNA in electrophoresis mobility shift assay. Four herbs, Terminalia belirica, Salacia chinensis, Zingiber montanum and Peltophorum pterocarpum, could reduce > 50% of infectivity of H5N1 in a cell-based assay, and it is worth further studying their potential use as source of antiviral drugs.

In Vitro Assays for the Discovery of PCSK9 Autoprocessing Inhibitors.

PubMed

Salowe, Scott P; Zhang, Lei; Zokian, Hratch J; Gesell, Jennifer J; Zink, Deborah L; Wiltsie, Judyann; Ai, Xi; Kavana, Michael; Pinto, Shirly

2016-12-01

PCSK9 plays a significant role in regulating low-density lipoprotein (LDL) cholesterol levels and has become an important drug target for treating hypercholesterolemia. Although a member of the serine protease family, PCSK9 only catalyzes a single reaction, the autocleavage of its prodomain. The maturation of the proprotein is an essential prerequisite for the secretion of PCSK9 to the extracellular space where it binds the LDL receptor and targets it for degradation. We have found that a construct of proPCSK9 where the C-terminal domain has been truncated has sufficient stability to be expressed and purified from Escherichia coli for the in vitro study of autoprocessing. Using automated Western analysis, we demonstrate that autoprocessing exhibits the anticipated first-order kinetics. A high-throughput time-resolved fluorescence resonance energy transfer assay for autocleavage has been developed using a PCSK9 monoclonal antibody that is sensitive to the conformational changes that occur upon maturation of the proprotein. Kinetic theory has been developed that describes the behavior of both reversible and irreversible inhibitors of autocleavage. The analysis of an irreversible lactone inhibitor validates the expected relationship between potency and the reaction end point. An orthogonal liquid chromatography-mass spectrometry assay has also been implemented for the confirmation of hits from the antibody-based assays.

Food proteins and maturation of small intestinal microvillus membranes (MVM). II. Binding of gliadin hydrolysate fractions and of the gliadin peptide B3142.

PubMed

Stern, M; Gellermann, B; Belitz, H D; Wieser, H

1988-01-01

To investigate in vitro interactions between gliadin peptide fractions that have been shown to be toxic to celiac small intestinal mucosa in humans and small intestinal microvillus membranes (MVM) from rats during postnatal maturation, MVM were prepared from newborn, 18-day-old preweanling, and adult rats. Partially hydrolyzed gliadin peptide fractions B1-B4, and the pure gliadin peptide B3142 were radioiodinated and used for binding assays. Miniature ultracentrifugation was used for separation of unbound material. Binding of gliadin fractions to MVM was weak and nonspecific in terms of lacking saturation and inhibition. There was no inhibition of binding by mannan. Enzyme pretreatment of MVM (trypsin, neuraminidase, phospholipase C) did not result in any significant change of binding. Compared with peptides prepared from bovine serum albumin as a control, there was no significant difference in binding of gliadin peptide fractions to MVM. Thus, a lectin-like effect of gliadin peptides toward MVM, or the existence of a specific intestinal surface receptor for gliadin peptides appeared improbable. There were, however, consistent maturational changes in MVM binding in that newborn MVM bound more B1-B4 and B3142 compared with adult controls (p less than 0.001). Nonspecific binding of gliadin fractions to MVM might be related to the initiation of nonspecific in vitro effects of gliadin, particularly toward the immature small intestine. The MVM binding model in the rat clearly does not provide a system for studying celiac disease pathogenesis, but it might help clarify basic processes in the interaction between food-derived substances and elements of the gastrointestinal mucosal barrier.

Functionality of the STNV translational enhancer domain correlates with affinity for two wheat germ factors

PubMed Central

Lipzig, Rosalinde van; Montagu, Marc Van; Cornelissen, Marc; Meulewaeter, Frank

2001-01-01

The satellite tobacco necrosis virus RNA is uncapped and requires a 3′ translational enhancer domain (TED) for translation. Both in the wheat germ extract and in tobacco, TED stimulates in cis translation of heterologous, uncapped RNAs. In this study we investigated to what extent translation stimulation by TED depends on binding to wheat germ factors. We show that in vitro TED binds at least seven wheat germ proteins. Translation and crosslinking assays, to which TED or TED derivatives with reduced functionality were included as competitor, showed that TED function correlates with binding to a 28 kDa protein (p28). One particular condition of competition revealed that p28 binding is not obligatory for TED function. Under this condition, a 30 kDa protein (p30) binds to TED. Importantly, affinity of p30 correlates with functionality of TED. These results strongly suggest that TED has the capacity to stimulate translation by recruiting the translational machinery either via binding to p28 or via binding to p30. PMID:11222757

Prediction of phospholipidosis-inducing potential of drugs by in vitro biochemical and physicochemical assays followed by multivariate analysis.

PubMed

Kuroda, Yukihiro; Saito, Madoka

2010-03-01

An in vitro method to predict phospholipidosis-inducing potential of cationic amphiphilic drugs (CADs) was developed using biochemical and physicochemical assays. The following parameters were applied to principal component analysis, as well as physicochemical parameters: pK(a) and clogP; dissociation constant of CADs from phospholipid, inhibition of enzymatic phospholipid degradation, and metabolic stability of CADs. In the score plot, phospholipidosis-inducing drugs (amiodarone, propranolol, imipramine, chloroquine) were plotted locally forming the subspace for positive CADs; while non-inducing drugs (chlorpromazine, chloramphenicol, disopyramide, lidocaine) were placed scattering out of the subspace, allowing a clear discrimination between both classes of CADs. CADs that often produce false results by conventional physicochemical or cell-based assay methods were accurately determined by our method. Basic and lipophilic disopyramide could be accurately predicted as a nonphospholipidogenic drug. Moreover, chlorpromazine, which is often falsely predicted as a phospholipidosis-inducing drug by in vitro methods, could be accurately determined. Because this method uses the pharmacokinetic parameters pK(a), clogP, and metabolic stability, which are usually obtained in the early stages of drug development, the method newly requires only the two parameters, binding to phospholipid, and inhibition of lipid degradation enzyme. Therefore, this method provides a cost-effective approach to predict phospholipidosis-inducing potential of a drug. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Anticoagulant Effect of Sugammadex: Just an In Vitro Artifact.

PubMed

Dirkmann, Daniel; Britten, Martin W; Pauling, Henning; Weidle, Juliane; Volbracht, Lothar; Görlinger, Klaus; Peters, Jürgen

2016-06-01

Sugammadex prolongs activated partial thromboplastin time (aPTT) and prothrombin time (PT) suggestive of anticoagulant effects. To pinpoint its presumed anticoagulant site of action, the authors assessed Sugammadex's impact on a panel of coagulation assays. Sugammadex, Rocuronium, Sugammadex and Rocuronium combined, or saline were added to blood samples from healthy volunteers and analyzed using plasmatic (i.e., aPTT, thrombin time, and fibrinogen concentration) (n = 8 each), PT (quick), activities of plasmatic coagulation factors, and whole blood (extrinsically and intrinsically activated thromboelastometry) assays (n = 18 each). Furthermore, dose-dependent effects of Sugammadex were also assessed (n = 18 each) in diluted Russel viper venom time (DRVVT) assays with low (DRVVT1) and high (DRVVT2) phospholipid concentrations and in a highly phospholipid-sensitive aPTT assay. Sugammadex increased PT (+9.1%; P < 0.0001), aPTT (+13.1%; P = 0.0002), and clotting time in extrinsically (+33.1%; P = 0.0021) and intrinsically (+22.4%; P < 0.0001) activated thromboelastometric assays. Furthermore, activities of factors VIII, IX, XI, and XII decreased (-7%, P = 0.009; -7.8%, P < 0.0001; -6.9%, P < 0.0001; and -4.3%, P = 0.011, respectively). Sugammadex dose-dependently prolonged both DRVVT1 and the highly phospholipid-sensitive aPTT assays, but additional phospholipids in the DRVVT2 assay almost abolished these prolongations. Thrombin time, a thromboelastometric thrombin generation assay, clot firmness, clot lysis, fibrinogen concentration, and activities of other coagulation factors were unaltered. Rocuronium, Sugammadex and Rocuronium combined, and saline exerted no effects. Sugammadex significantly affects various coagulation assays, but this is explainable by an apparent phospholipid-binding effect, suggesting that Sugammadex`s anticoagulant effects are likely an in vitro artifact.

Acteoside and Acyl-Migrated Acteoside, Compounds in Chinese Kudingcha Tea, Inhibit α-Amylase In Vitro.

PubMed

Lu, Yuqin; Zhou, Wenyu; Feng, Yue; Li, Yao; Liu, Ke; Liu, Lizhong; Lin, Dongxu; He, Zhendan; Wu, Xuli

2017-06-01

Acteoside, the predominant polyphenol of small-leaved kudingcha, the Chinese tea, has various biological activities. In this study, we examined the acyl migration of acteoside to isoacteoside with high-temperature treatment of acteoside. The inhibitory effects of acyl-migrated acteoside and acteoside on α-amylase were investigated, as were their binding interaction with α-amylase. The binding of acteoside and isoacteoside to α-amylase was investigated by using the fluorescence spectra assay, circular dichroism, and protein-ligand docking studies. Acteoside was more effective than preheated acteoside and isoacteoside in inhibiting α-amylase activity. Acteoside and isoacteoside binding to α-amylase may induce conformational changes to α-amylase, and the binding site of acteoside and isoacteoside being near the active site pocket of α-amylase may explain the decreased activity of α-amylase. The different affinities and binding sites of acteoside and isoacteoside for α-amylase resulted in different inhibition rates, which may be due to structural differences between acteoside and isoacteoside.

An RNA-Binding Multimer Specifies Nematode Sperm Fate.

PubMed

Aoki, Scott T; Porter, Douglas F; Prasad, Aman; Wickens, Marvin; Bingman, Craig A; Kimble, Judith

2018-06-26

FOG-3 is a master regulator of sperm fate in Caenorhabditis elegans and homologous to Tob/BTG proteins, which in mammals are monomeric adaptors that recruit enzymes to RNA binding proteins. Here, we determine the FOG-3 crystal structure and in vitro demonstrate that FOG-3 forms dimers that can multimerize. The FOG-3 multimeric structure has a basic surface potential, suggestive of binding nucleic acid. Consistent with that prediction, FOG-3 binds directly to nearly 1,000 RNAs in nematode spermatogenic germ cells. Most binding is to the 3' UTR, and most targets (94%) are oogenic mRNAs, even though assayed in spermatogenic cells. When tethered to a reporter mRNA, FOG-3 represses its expression. Together these findings elucidate the molecular mechanism of sperm fate specification and reveal the evolution of a protein from monomeric to multimeric form with acquisition of a distinct mode of mRNA repression. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

CLASPs are required for proper microtubule localization of End-binding proteins

PubMed Central

Grimaldi, Ashley D.; Maki, Takahisa; Fitton, Benjamin P.; Roth, Daniel; Yampolsky, Dmitry; Davidson, Michael W.; Svitkina, Tatyana; Straube, Anne; Hayashi, Ikuko; Kaverina, Irina

2014-01-01

Summary Microtubule (MT) plus-end tracking proteins (+TIPs) preferentially localize to MT plus-ends. End-binding proteins (EBs) are master regulators of the +TIP complex; however, it is unknown whether EBs are regulated by other +TIPs. Here, we show that Cytoplasmic linker associated proteins (CLASPs) modulate EB localization at MTs. In CLASP-depleted cells, EBs localized along the MT lattice in addition to plus-ends. The MT-binding region of CLASP was sufficient for restoring normal EB localization, while neither EB-CLASP interactions nor EB tail-binding proteins are involved. In vitro assays revealed that CLASP directly functions to remove EB from MTs. Importantly, this effect occurs specifically during MT polymerization, but not at pre-formed MTs. Increased GTP-tubulin content within MTs in CLASP-depleted cells suggests that CLASPs facilitate GTP-hydrolysis to reduce EB lattice binding. Together, these findings suggest that CLASPs influence the MT lattice itself to regulate EB and determine exclusive plus-end localization of EBs in cells. PMID:25117684

In vitro and in vivo binding of (E)- and (Z)-N-(iodoallyl)spiperone to dopamine D sub 2 and serotonin 5-HT sub 2 neuroreceptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lever, J.R.; Scheffel, U.A.; Stathis, M.

1990-01-01

Apparent affinities (K{sub i}) of (E)- and (Z)-N-(iodoallyl)spiperone ((E)- and (Z)- NIASP) for dopamine D{sub 2} and serotonin 5-HT{sub 2} receptors were determined in competition binding assays. (Z)-NIASP (K{sub i} 0.35 nM, D{sub 2}; K{sub i} 1.75 nM, 5-HT{sub 2}) proved slightly more potent and selective for D{sub 2} sites in vitro than (E)-NIASP (K{sub i} 0.72 nM, D{sub 2}; K{sub i} 1.14 nM, 5-HT{sub 2}). In vivo, radioiodinated (E)- and (Z)-({sup 125}I)-NIASP showed regional distributions in mouse brain which are consonant with prolonged binding to dopamine D{sub 2} receptors accompanied by a minor serotonergic component of shorter duration. Stereoselective,more » dose-dependent blockade of (E)-({sup 125}I)-NIASP uptake was found for drugs binding to dopamine D{sub 2} sites, while drugs selective for serotonin 5-HT{sub 2}, {alpha}{sub 1}-adrenergic and dopamine D{sub 1} receptors did not inhibit radioligand binding 2 hr postinjection. Specific binding in striatal tissue was essentially irreversible over the time course of the study, and (E)-({sup 125}I)-NIASP gave a striatal to cerebellar tissue radioactivity concentration of 16.9 to 1 at 6 hr postinjection. Thus, (E)-({sup 125}I)-NIASP binds with high selectivity and specificity to dopamine D{sub 2} sites in vivo.« less

Discovery and Optimization of Novel 5-Indolyl-7-arylimidazo[1,2-a]pyridine-8-carbonitrile Derivatives as Potent Antitubulin Agents Targeting Colchicine-binding Site

NASA Astrophysics Data System (ADS)

Zhai, Xin; Wang, Xiaoqiang; Wang, Jiao; Liu, Jin; Zuo, Daiying; Jiang, Nan; Zeng, Tianfang; Yang, Xiuxiu; Jing, Tongfei; Gong, Ping

2017-02-01

Aiming at development of potent antitubulin agents targeting colchicine-binding site, a series of novel 5-indolyl-7-arylimidazo[1,2-a]pyridine-8-carbonitrilederivatives (5a-5v and 7a-7h) were designed based on bioisosterism and hybridization strategies. All these compounds were concisely synthesized via a three-step process and examined against five human cancer cell lines (HT-29, A549, MKN-45, MDA-MB-231 and SMMC-7721) along with a normal human cell (L02) in vitro. A structure-activity relationships (SARs) study was carried out and optimization towards this series of compounds in cellular assay resulted in the discovery of 5k, which displayed similar or better antitumor potency against the tested cancer cells with IC50 value ranging from 0.02 to 1.22 μM superior to CA-4 and Crolibulin. Significantly, a cell cycle study disclosed the ability of 5k to arrest cell cycle at the G2/M phase, and immunofluorescence assay as well as a colchicine competition assay revealed that tubulin polymerization was disturbed by 5k by binding to the colchicine site. Moreover, the molecular modeling mode showed the posture of 5k and Crolibulin was similar in the colchcine-binding pocket of tubulin as identified with the SARs and pharmacological results. Together, all these results rationalized 5k might serve as a promising lead for a novel class of antitubulin agents for cancer treatments.

Mouse pharmacokinetics and metabolism of the curcumin analog, 4-piperidinone,3,5-bis[(2-fluorophenyl)methylene]-acetate(3E,5E) (EF-24; NSC 716993).

PubMed

Reid, Joel M; Buhrow, Sarah A; Gilbert, Judith A; Jia, Lee; Shoji, Mamoru; Snyder, James P; Ames, Matthew M

2014-06-01

Curcumin, a keto-enol constituent of turmeric, has in vitro and in vivo antitumor activity. However, in vivo potency is low due to poor oral absorption. The mono-carbonyl analog, 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone acetate (EF-24, NSC 716993), exhibited broad-spectrum activity in the NCI anticancer cell line screen and potent antiangiogenesis activity in a HUVEC cell migration assay. The purpose of this study was to characterize the preclinical pharmacology of EF-24 in mice. EF-24 plasma stability, protein binding, pharmacokinetics, and metabolism were characterized utilizing an LC/MS/MS assay. An LC/MS/MS assay incorporated protein precipitation with methanol, reverse-phase HPLC separation under gradient elution using an aqueous methanol mobile phase containing 0.1 % formic acid, and positive electrospray ionization detection of the m/z 312 > 149 transition for EF-24. The assay was linear over the range 7.8-1,000 nM. Plasma protein binding was >98 % with preferential binding to albumin. EF-24 plasma disposition in mice after i.v. administration of a 10 mg/kg dose was best fit to a 3-compartment open model. The terminal elimination half-life and plasma clearance values were 73.6 min and 0.482 L/min/kg, respectively. EF-24 bioavailability was 60 and 35 % after oral and i.p. administration, respectively. NADPH-dependent metabolism of EF-24 loss in liver microsomal preparations yielded several metabolites consistent with EF-24 hydroxylation and reduction.

Integration of cell-free protein coexpression with an enzyme-linked immunosorbent assay enables rapid analysis of protein–protein interactions directly from DNA

PubMed Central

Layton, Curtis J; Hellinga, Homme W

2011-01-01

Assays that integrate detection of binding with cell-free protein expression directly from DNA can dramatically increase the pace at which protein–protein interactions (PPIs) can be analyzed by mutagenesis. In this study, we present a method that combines in vitro protein production with an enzyme-linked immunosorbent assay (ELISA) to measure PPIs. This method uses readily available commodity instrumentation and generic antibody–affinity tag interactions. It is straightforward and rapid to execute, enabling many interactions to be assessed in parallel. In traditional ELISAs, reporter complexes are assembled stepwise with one layer at a time. In the method presented here, all the members of the reporter complex are present and assembled together. The signal strength is dependent on all the intercomponent interaction affinities and concentrations. Although this assay is straightforward to execute, establishing proper conditions and analysis of the results require a thorough understanding of the processes that determine the signal strength. The formation of the fully assembled reporter sandwich can be modeled as a competition between Langmuir adsorption isotherms for the immobilized components and binding equilibria of the solution components. We have shown that modeling this process provides semiquantitative understanding of the effects of affinity and concentration and can guide strategies for the development of experimental protocols. We tested the method experimentally using the interaction between a synthetic ankyrin repeat protein (Off7) and maltose-binding protein. Measurements obtained for a collection of alanine mutations in the interface between these two proteins demonstrate that a range of affinities can be analyzed. PMID:21674663

Mouse pharmacokinetics and metabolism of the curcumin analog, 4-Piperidione,3,5-bis[(2-fluorophenyl)methylene]-acetate(3E,5E) (EF-24; NSC 716993)

PubMed Central

Reid, Joel M.; Buhrow, Sarah A.; Gilbert, Judith A.; Jia, Lee; Shoji, Mamoru; Snyder, James P.; Ames, Matthew M.

2015-01-01

Purpose Curcumin, a keto-enol constituent of turmeric, has in vitro and in vivo antitumor activity. However, in vivo potency is low due to poor oral absorption. The mono-carbonyl analog, 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone acetate (EF-24, NSC 716993), exhibited broad spectrum activity in the NCI anti-cancer cell line screen and potent anti-angiogenesis activity in a HUVEC cell migration assay. The purpose of this study was to characterize the preclinical pharmacology of EF-24 in mice. Methods EF-24 plasma stability, protein binding, pharmacokinetics and metabolism were characterized utilizing an LC/MS/MS assay. Results An LC/MS/MS assay that incorporated protein precipitation with methanol, reverse-phase HPLC separation under gradient elution using an aqueous methanol mobile phase containing 0.1% formic acid and positive electrospray ionization detection of the m/z 312 > 149 transition for EF-24. The assay was linear over the range 7.8-1000 nM. Plasma protein binding was > 98% with preferential binding to albumin. EF-24 plasma disposition in mice after i.v. administration of a 10 mg/kg dose was best fit to a 3-compartment open model. The terminal elimination half-life and plasma clearance values were 73.6 min and 0.482 L/min/kg, respectively. EF-24 bioavailability was 60% and 35% after oral and i.p. administration, respectively. NADPH-dependent metabolism of EF-24 loss in liver microsomal preparations yielded several metabolites consistent with EF-24 hydroxylation and reduction. PMID:24760417

In Silico and In Vitro Analysis of Bacoside A Aglycones and Its Derivatives as the Constituents Responsible for the Cognitive Effects of Bacopa monnieri

PubMed Central

Ramasamy, Seetha; Chin, Sek Peng; Sukumaran, Sri Devi; Buckle, Michael James Christopher; Kiew, Lik Voon; Chung, Lip Yong

2015-01-01

Bacopa monnieri has been used in Ayurvedic medicine to improve memory and cognition. The active constituent responsible for its pharmacological effects is bacoside A, a mixture of dammarane-type triterpenoid saponins containing sugar chains linked to a steroid aglycone skeleton. Triterpenoid saponins have been reported to be transformed in vivo to metabolites that give better biological activity and pharmacokinetic characteristics. Thus, the activities of the parent compounds (bacosides), aglycones (jujubogenin and pseudojujubogenin) and their derivatives (ebelin lactone and bacogenin A1) were compared using a combination of in silico and in vitro screening methods. The compounds were docked into 5-HT1A, 5-HT2A, D1, D2, M1 receptors and acetylcholinesterase (AChE) using AutoDock and their central nervous system (CNS) drug-like properties were determined using Discovery Studio molecular properties and ADMET descriptors. The compounds were screened in vitro using radioligand receptor binding and AChE inhibition assays. In silico studies showed that the parent bacosides were not able to dock into the chosen CNS targets and had poor molecular properties as a CNS drug. In contrast, the aglycones and their derivatives showed better binding affinity and good CNS drug-like properties, were well absorbed through the intestines and had good blood brain barrier (BBB) penetration. Among the compounds tested in vitro, ebelin lactone showed binding affinity towards M1 (Ki = 0.45 μM) and 5-HT2A (4.21 μM) receptors. Bacoside A and bacopaside X (9.06 μM) showed binding affinity towards the D1 receptor. None of the compounds showed any inhibitory activity against AChE. Since the stimulation of M1 and 5-HT2A receptors has been implicated in memory and cognition and ebelin lactone was shown to have the strongest binding energy, highest BBB penetration and binding affinity towards M1 and 5-HT2A receptors, we suggest that B. monnieri constituents may be transformed in vivo to the active form before exerting their pharmacological activity. PMID:25965066

In Silico and In Vitro Analysis of Bacoside A Aglycones and Its Derivatives as the Constituents Responsible for the Cognitive Effects of Bacopa monnieri.

PubMed

Ramasamy, Seetha; Chin, Sek Peng; Sukumaran, Sri Devi; Buckle, Michael James Christopher; Kiew, Lik Voon; Chung, Lip Yong

2015-01-01

Bacopa monnieri has been used in Ayurvedic medicine to improve memory and cognition. The active constituent responsible for its pharmacological effects is bacoside A, a mixture of dammarane-type triterpenoid saponins containing sugar chains linked to a steroid aglycone skeleton. Triterpenoid saponins have been reported to be transformed in vivo to metabolites that give better biological activity and pharmacokinetic characteristics. Thus, the activities of the parent compounds (bacosides), aglycones (jujubogenin and pseudojujubogenin) and their derivatives (ebelin lactone and bacogenin A1) were compared using a combination of in silico and in vitro screening methods. The compounds were docked into 5-HT1A, 5-HT2A, D1, D2, M1 receptors and acetylcholinesterase (AChE) using AutoDock and their central nervous system (CNS) drug-like properties were determined using Discovery Studio molecular properties and ADMET descriptors. The compounds were screened in vitro using radioligand receptor binding and AChE inhibition assays. In silico studies showed that the parent bacosides were not able to dock into the chosen CNS targets and had poor molecular properties as a CNS drug. In contrast, the aglycones and their derivatives showed better binding affinity and good CNS drug-like properties, were well absorbed through the intestines and had good blood brain barrier (BBB) penetration. Among the compounds tested in vitro, ebelin lactone showed binding affinity towards M1 (Ki = 0.45 μM) and 5-HT2A (4.21 μM) receptors. Bacoside A and bacopaside X (9.06 μM) showed binding affinity towards the D1 receptor. None of the compounds showed any inhibitory activity against AChE. Since the stimulation of M1 and 5-HT2A receptors has been implicated in memory and cognition and ebelin lactone was shown to have the strongest binding energy, highest BBB penetration and binding affinity towards M1 and 5-HT2A receptors, we suggest that B. monnieri constituents may be transformed in vivo to the active form before exerting their pharmacological activity.

Application of the SPI (Saliva Precipitation Index) to the evaluation of red wine astringency.

PubMed

Rinaldi, Alessandra; Gambuti, Angelita; Moio, Luigi

2012-12-15

The aim of this work was to evaluate the astringency of red wines by means of a SDS-PAGE based-method. The optimization of the in vitro assay, named SPI (Saliva Precipitation Index) that measured the reactivity of salivary proteins towards wine polyphenols, has been performed. Improvements included the choice of saliva:wine ratio, saliva typology (resting or stimulated saliva), and temperature of binding. The LOD (0.05 g/L of condensed tannin) and LOQ (0.1g/L of condensed tannin) for the binding reaction between salivary proteins and tannins added in white wine were also determined. Fifty-seven red wines were analysed by the optimised SPI, the Folin-Ciocalteu Index, the gelatine index, the content of total tannins and the sensory quantitative evaluation of astringency. A significant correlation between the SPI and the astringency of red wines was found (R(2)=0.969), thus indicating that this assay may be useful as estimator of astringency. Copyright © 2012 Elsevier Ltd. All rights reserved.

Ligand and membrane-binding behavior of the phosphatidylinositol transfer proteins PITPα and PITPβ.

PubMed

Baptist, Matilda; Panagabko, Candace; Cockcroft, Shamshad; Atkinson, Jeffrey

2016-12-01

Phosphatidylinositol transfer proteins (PITPs) are believed to be lipid transfer proteins because of their ability to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments, in vitro. However, the detailed mechanism of this transfer process is not fully established. To further understand the transfer mechanism of PITPs we examined the interaction of PITPs with membranes using dual polarization interferometry (DPI), which measures protein binding affinity on a flat immobilized lipid surface. In addition, a fluorescence resonance energy transfer (FRET)-based assay was also employed to monitor how quickly PITPs transfer their ligands to lipid vesicles. DPI analysis revealed that PITPβ had a higher affinity to membranes compared with PITPα. Furthermore, the FRET-based transfer assay revealed that PITPβ has a higher ligand transfer rate compared with PITPα. However, both PITPα and PITPβ demonstrated a preference for highly curved membrane surfaces during ligand transfer. In other words, ligand transfer rate was higher when the accepting vesicles were highly curved.

Alkyne-substituted diminazene as G-quadruplex binders with anticancer activities.

PubMed

Wang, Changhao; Carter-Cooper, Brandon; Du, Yixuan; Zhou, Jie; Saeed, Musabbir A; Liu, Jinbing; Guo, Min; Roembke, Benjamin; Mikek, Clinton; Lewis, Edwin A; Lapidus, Rena G; Sintim, Herman O

2016-08-08

G-quadruplex ligands have been touted as potential anticancer agents, however, none of the reported G-quadruplex-interactive small molecules have gone past phase II clinical trials. Recently it was revealed that diminazene (berenil, DMZ) actually binds to G-quadruplexes 1000 times better than DNA duplexes, with dissociation constants approaching 1 nM. DMZ however does not have strong anticancer activities. In this paper, using a panel of biophysical tools, including NMR, FRET melting assay and FRET competition assay, we discovered that monoamidine analogues of DMZ bearing alkyne substitutes selectively bind to G-quadruplexes. The lead DMZ analogues were shown to be able to target c-MYC G-quadruplex both in vitro and in vivo. Alkyne DMZ analogues display respectable anticancer activities (single digit micromolar GI50) against ovarian (OVCAR-3), prostate (PC-3) and triple negative breast (MDA-MB-231) cancer cell lines and represent interesting new leads to develop anticancer agents. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Crosslinked self-assemblies of lipoid acid-substituted low molecular weight (1800 Da) polyethylenimine as reductive-sensitive non-viral gene vectors

NASA Astrophysics Data System (ADS)

Chen, Xiaojiao; Yuan, Zhefan; Yi, Xiaoqing; Zhuo, Renxi; Li, Feng

2012-10-01

In this study, amphiphilic polyethylenimine-graft-thioctic acid (PEI-TA) and polyethylenimine-graft-lauric acid (PEI-LA) were synthesized. Both PEI-TA and PEI-LA could self-assemble into micelles. Due to the existence of disulfide-linked rings at the end of hydrophobic moieties, PEI-TA could form stable micelles with disulfide crosslinked cores (PEI-TA-SS). In comparison with the PEI-LA micelle, PEI-TA-SS possessed higher DNA binding ability according to the gel retardation assay and heparin replacement assay. In vitro transfection experiments indicated that PEI-TA-SS showed comparably high transfection efficiency as compared to 25 kDa PEI. More interestingly, the luciferase expression of PEI-TA-SS was superior to that of PEI-LA at low N/P ratio, which might be ascribed to the stronger binding capacity of PEI-TA-SS facilitating the entering of PEI-TA-SS/pDNA complexes into cells.

Recombinant human antibody fragment against tetanus toxoid produced by phage display

PubMed Central

Neelakantam, B.; Sridevi, N. V.; Shukra, A. M.; Sugumar, P.; Samuel, S.

2014-01-01

Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen. PMID:24678405

Conserved and Variant Epitopes of Plasmodium vivax Duffy Binding Protein as Targets of Inhibitory Monoclonal Antibodies

PubMed Central

Ntumngia, Francis B.; Schloegel, Jesse; Barnes, Samantha J.; McHenry, Amy M.; Singh, Sanjay; King, Christopher L.

2012-01-01

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains. PMID:22215740

Conserved and variant epitopes of Plasmodium vivax Duffy binding protein as targets of inhibitory monoclonal antibodies.

PubMed

Ntumngia, Francis B; Schloegel, Jesse; Barnes, Samantha J; McHenry, Amy M; Singh, Sanjay; King, Christopher L; Adams, John H

2012-03-01

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains.

Pirfenidone induces intercellular adhesion molecule-1 (ICAM-1) down-regulation on cultured human synovial fibroblasts

PubMed Central

Kaneko, M; Inoue, H; Nakazawa, R; Azuma, N; Suzuki, M; Yamauchi, S; Margolin, S B; Tsubota, K; Saito, I

1998-01-01

Pirfenidone has been shown to modify some cytokine regulatory actions and inhibit fibroblast biochemical reactions resulting in inhibition of proliferation and collagen matrix synthesis by fibroblast. We have investigated the effect of pirfenidone on the expression of cell adhesion molecules. The synovial fibroblasts were treated with IL-1α in the presence or absence of pirfenidone (range 0–1000 μm), and assayed for the expression of adhesion molecules such as ICAM-1 and endothelial-leucocyte adhesion molecule-1 (E-selectin) by cell ELISA. Pirfenidone significantly down-regulated the expression of ICAM-1 on cultured synovial fibroblasts in a dose-dependent manner. In contrast, expression of E-selectin was not affected. Furthermore, we examined whether pirfenidone affects the cellular binding between cultured lymphocytes and IL-1α-stimulated synovial fibroblasts by in vitro binding assay and found their mutual binding was significantly suppressed in a dose-dependent manner by pirfenidone. It is speculated that down-regulation of ICAM-1 might be one of the novel mechanisms of action of pirfenidone. These data indicate a novel mechanism of action for pirfenidone to reduce the activation of synovial fibroblasts. PMID:9697986

Selection of homeotic proteins for binding to a human DNA replication origin.

PubMed

de Stanchina, E; Gabellini, D; Norio, P; Giacca, M; Peverali, F A; Riva, S; Falaschi, A; Biamonti, G

2000-06-09

We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation. Copyright 2000 Academic Press.

Targeting of Arabidopsis KNL2 to Centromeres Depends on the Conserved CENPC-k Motif in Its C Terminus.

PubMed

Sandmann, Michael; Talbert, Paul; Demidov, Dmitri; Kuhlmann, Markus; Rutten, Twan; Conrad, Udo; Lermontova, Inna

2017-01-01

KINETOCHORE NULL2 (KNL2) is involved in recognition of centromeres and in centromeric localization of the centromere-specific histone cenH3. Our study revealed a cenH3 nucleosome binding CENPC-k motif at the C terminus of Arabidopsis thaliana KNL2, which is conserved among a wide spectrum of eukaryotes. Centromeric localization of KNL2 is abolished by deletion of the CENPC-k motif and by mutating single conserved amino acids, but can be restored by insertion of the corresponding motif of Arabidopsis CENP-C. We showed by electrophoretic mobility shift assay that the C terminus of KNL2 binds DNA sequence-independently and interacts with the centromeric transcripts in vitro. Chromatin immunoprecipitation with anti-KNL2 antibodies indicated that in vivo KNL2 is preferentially associated with the centromeric repeat pAL1 Complete deletion of the CENPC-k motif did not influence its ability to interact with DNA in vitro. Therefore, we suggest that KNL2 recognizes centromeric nucleosomes, similar to CENP-C, via the CENPC-k motif and binds adjoining DNA. © 2017 American Society of Plant Biologists. All rights reserved.

Synthesis of aryl pyrazole via Suzuki coupling reaction, in vitro mushroom tyrosinase enzyme inhibition assay and in silico comparative molecular docking analysis with Kojic acid.

PubMed

Channar, Pervaiz Ali; Saeed, Aamer; Larik, Fayaz Ali; Batool, Bakhtawar; Kalsoom, Saima; Hasan, M M; Erben, Mauricio F; El-Seedi, Hesham R; Ali, Musrat; Ashraf, Zaman

2018-04-30

Aryl pyrazoles are well recognized class of heterocyclic compounds found in several commercially available drugs. Owing to their significance in medicinal chemistry, in this current account we have synthesized a series of suitably substituted aryl pyrazole by employing Suzuki cross-coupling reaction. All compounds were evaluated for inhibition of mushroom tyrosinase enzyme both in vitro and in silico. Compound 3f (IC 50  = 1.568 ± 0.01 µM) showed relatively better potential compared to reference kojic acid (IC 50  = 16.051 ± 1.27 µM). A comparative docking studies showed that compound 3f have maximum binding affinity against mushroom tyrosinase (PDBID: 2Y9X) with binding energy value (-6.90 kcal/mol) as compared to Kojic acid. The 4-methoxy group in compound 3f shows 100% interaction with Cu. Compound 3f displayed hydrogen binding interaction with His61 and His94 at distance of 1.71 and 1.74 Å which might be responsible for higher activity compared to Kojic acid. Copyright © 2018 Elsevier Inc. All rights reserved.

DNA Replication Arrest and DNA Damage Responses Induced by Alkylating Minor Groove Binders

DTIC Science & Technology

2003-05-01

Alkylating minor groove DNA binder adozelesin is capable of inhibiting DNA replication in treated cells through a trans-acting mechanism. The trans... replication in vitro. Using purified proteins in DNA replication initiation assays, we found that RPA purified from cells treated with adozelesin in not...adozelesin has the same single-stranded DNA binding activity and support nucleotide excision repair as normal RPA, but is not able to support SV40 DNA

Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

PubMed

Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

2017-04-15

RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

Epigallocatechin gallate (EGCG) inhibits adhesion and migration of neural progenitor cells in vitro.

PubMed

Barenys, Marta; Gassmann, Kathrin; Baksmeier, Christine; Heinz, Sabrina; Reverte, Ingrid; Schmuck, Martin; Temme, Thomas; Bendt, Farina; Zschauer, Tim-Christian; Rockel, Thomas Dino; Unfried, Klaus; Wätjen, Wim; Sundaram, Sivaraj Mohana; Heuer, Heike; Colomina, Maria Teresa; Fritsche, Ellen

2017-02-01

Food supplements based on herbal products are widely used during pregnancy as part of a self-care approach. The idea that such supplements are safe and healthy is deeply seated in the general population, although they do not underlie the same strict safety regulations than medical drugs. We aimed to characterize the neurodevelopmental effects of the green tea catechin epigallocatechin gallate (EGCG), which is now commercialized as high-dose food supplement. We used the "Neurosphere Assay" to study the effects and unravel underlying molecular mechanisms of EGCG treatment on human and rat neural progenitor cells (NPCs) development in vitro. EGCG alters human and rat NPC development in vitro. It disturbs migration distance, migration pattern, and nuclear density of NPCs growing as neurospheres. These functional impairments are initiated by EGCG binding to the extracellular matrix glycoprotein laminin, preventing its binding to β1-integrin subunits, thereby prohibiting cell adhesion and resulting in altered glia alignment and decreased number of migrating young neurons. Our data raise a concern on the intake of high-dose EGCG food supplements during pregnancy and highlight the need of an in vivo characterization of the effects of high-dose EGCG exposure during neurodevelopment.

Anti-angiogenic activity and antitumor efficacy of amphiphilic twin drug from ursolic acid and low molecular weight heparin

NASA Astrophysics Data System (ADS)

Cheng, Wenming; Zohra Dahmani, Fatima; Zhang, Juan; Xiong, Hui; Wu, Yuanyuan; Yin, Lifang; Zhou, Jianping; Yao, Jing

2017-02-01

Heparin, a potential blood anti-coagulant, is also known for its binding ability to several angiogenic factors through electrostatic interactions due to its polyanionic character. However, the clinical application of heparin for cancer treatment is limited by several drawbacks, such as unsatisfactory therapeutic effects and severe anticoagulant activity that could induce hemorrhaging. Herein, low molecular weight heparin (LMWH) was conjugated to ursolic acid (UA), which is also an angiogenesis inhibitor, by binding the amine group of aminoethyl-UA (UA-NH2) with the carboxylic groups of LMWH. The resulting LMWH-UA conjugate as an amphiphilic twin drug showed reduced anticoagulant activity and could also self-assemble into nanomicelles with a mean particle size ranging from 200-250 nm. An in vitro endothelial tubular formation assay and an in vivo Matrigel plug assay were performed to verify the anti-angiogenic potential of LMWH-UA. Meanwhile, the in vivo antitumor effect of LMWH-UA was also evaluated using a B16F10 mouse melanoma model. LMWH-UA nanomicelles were shown to inhibit angiogenesis both in vitro and in vivo. In addition, the i.v. administration of LMWH-UA to the B16F10 tumor-bearing mice resulted in a significant inhibition of tumor growth as compared to the free drug solutions. These findings demonstrate the therapeutic potential of LMWH-UA as a new therapeutic remedy for cancer therapy.

Bacterial adhesion to conventional hydrogel and new silicone-hydrogel contact lens materials.

PubMed

Kodjikian, Laurent; Casoli-Bergeron, Emmanuelle; Malet, Florence; Janin-Manificat, Hélène; Freney, Jean; Burillon, Carole; Colin, Joseph; Steghens, Jean-Paul

2008-02-01

As bacterial adhesion to contact lenses may contribute to the pathogenesis of keratitis, the aim of our study was to investigate in vitro adhesion of clinically relevant bacteria to conventional hydrogel (standard HEMA) and silicone-hydrogel contact lenses using a bioluminescent ATP assay. Four types of unworn contact lenses (Etafilcon A, Galyfilcon A, Balafilcon A, Lotrafilcon B) were incubated with Staphylococcus epidermidis (two different strains) and Pseudomonas aeruginosa suspended in phosphate buffered saline (PBS). Lenses were placed with the posterior surface facing up and were incubated in the bacterial suspension for 4 hours at 37 degrees C. Bacterial binding was then measured and studied by bioluminescent ATP assay. Six replicate experiments were performed for each lens and strain. Adhesion of all species of bacteria to standard HEMA contact lenses (Etafilcon A) was found to be significantly lower than that of three types of silicone-hydrogel contact lenses, whereas Lotrafilcon B material showed the highest level of bacterial binding. Differences between species in the overall level of adhesion to the different types of contact lenses were observed. Adhesion of P. aeruginosa was typically at least 20 times greater than that observed with both S. epidermidis strains. Conventional hydrogel contact lenses exhibit significantly lower bacterial adhesion in vitro than silicone-hydrogel ones. This could be due to the greater hydrophobicity but also to the higher oxygen transmissibility of silicone-hydrogel lenses.

A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

PubMed Central

Hoggard, Timothy; Shor, Erika; Müller, Carolin A.; Nieduszynski, Conrad A.; Fox, Catherine A.

2013-01-01

Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time. PMID:24068963

Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging.

PubMed

Sun, Meng; Grigsby, Iwen F; Gorelick, Robert J; Mansky, Louis M; Musier-Forsyth, Karin

2014-01-01

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

The Sequence-specific Peptide-binding Activity of the Protein Sulfide Isomerase AGR2 Directs Its Stable Binding to the Oncogenic Receptor EpCAM.

PubMed

Mohtar, M Aiman; Hernychova, Lenka; O'Neill, J Robert; Lawrence, Melanie L; Murray, Euan; Vojtesek, Borek; Hupp, Ted R

2018-04-01

AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

PubMed

Verrier, C S; Roodi, N; Yee, C J; Bailey, L R; Jensen, R A; Bustin, M; Parl, F F

1997-07-01

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

Mass spectrometry for identification of proteins that specifically bind to a distal enhancer of the Oct4 gene

NASA Astrophysics Data System (ADS)

Bakhmet, E. I.; Nazarov, I. B.; Artamonova, T. O.; Khodorkovsky, M. A.; Tomilin, A. N.

2017-11-01

Transcription factor Oct4 is a marker of pluripotent stem cells and has a significant role in their self-renewal. Oct4 gene is controlled by three cis-regulatory elements - proximal promoter, proximal enhancer and distal enhancer. All of these elements are targets for binding of regulatory proteins. Distal enhancer is in our research focus because of its activity in early stages of embryonic development. There are two main sequences called site 2A and site 2B that are presented in distal enhancer. For this moment proteins which bind to a site 2A (CCCCTCCCCCC) remain unknown. Using combination of in vitro method electrophoretic mobility shift assay (EMSA) and mass spectromery we identified several candidates that can regulate Oct4 gene expression through site 2A.

Targeting the Allosteric Site of Oncoprotein BCR-ABL as an Alternative Strategy for Effective Target Protein Degradation.

PubMed

Shimokawa, Kenichiro; Shibata, Norihito; Sameshima, Tomoya; Miyamoto, Naoki; Ujikawa, Osamu; Nara, Hiroshi; Ohoka, Nobumichi; Hattori, Takayuki; Cho, Nobuo; Naito, Mikihiko

2017-10-12

Protein degradation technology based on hybrid small molecules is an emerging drug modality that has significant potential in drug discovery and as a unique method of post-translational protein knockdown in the field of chemical biology. Here, we report the first example of a novel and potent protein degradation inducer that binds to an allosteric site of the oncogenic BCR-ABL protein. BCR-ABL allosteric ligands were incorporated into the SNIPER (Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) platform, and a series of in vitro biological assays of binding affinity, target protein modulation, signal transduction, and growth inhibition were carried out. One of the designed compounds, 6 (SNIPER(ABL)-062), showed desirable binding affinities against ABL1, cIAP1/2, and XIAP and consequently caused potent BCR-ABL degradation.

Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis

PubMed Central

Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

2016-01-01

Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high-affinity conformation with an ‘open' headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation. PMID:27578049

In Vitro and In Vivo Single Myosin Step-Sizes in Striated Muscle a

PubMed Central

Burghardt, Thomas P.; Sun, Xiaojing; Wang, Yihua; Ajtai, Katalin

2016-01-01

Myosin in muscle transduces ATP free energy into the mechanical work of moving actin. It has a motor domain transducer containing ATP and actin binding sites, and, mechanical elements coupling motor impulse to the myosin filament backbone providing transduction/mechanical-coupling. The mechanical coupler is a lever-arm stabilized by bound essential and regulatory light chains. The lever-arm rotates cyclically to impel bound filamentous actin. Linear actin displacement due to lever-arm rotation is the myosin step-size. A high-throughput quantum dot labeled actin in vitro motility assay (Qdot assay) measures motor step-size in the context of an ensemble of actomyosin interactions. The ensemble context imposes a constant velocity constraint for myosins interacting with one actin filament. In a cardiac myosin producing multiple step-sizes, a “second characterization” is step-frequency that adjusts longer step-size to lower frequency maintaining a linear actin velocity identical to that from a shorter step-size and higher frequency actomyosin cycle. The step-frequency characteristic involves and integrates myosin enzyme kinetics, mechanical strain, and other ensemble affected characteristics. The high-throughput Qdot assay suits a new paradigm calling for wide surveillance of the vast number of disease or aging relevant myosin isoforms that contrasts with the alternative model calling for exhaustive research on a tiny subset myosin forms. The zebrafish embryo assay (Z assay) performs single myosin step-size and step-frequency assaying in vivo combining single myosin mechanical and whole muscle physiological characterizations in one model organism. The Qdot and Z assays cover “bottom-up” and “top-down” assaying of myosin characteristics. PMID:26728749

STANDARDIZATION OF A FLUORESCENT-BASED QUANTITATIVE ADHESION ASSAY TO STUDY ATTACHMENT OF Taenia solium ONCOSPHERE TO EPITHELIAL CELLS In Vitro

PubMed Central

Chile, Nancy; Evangelista, Julio; Gilman, Robert H.; Arana, Yanina; Palma, Sandra; Sterling, Charles R; Garcia, Hector H.; Gonzalez, Armando; Verastegui, Manuela

2012-01-01

To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment. PMID:22178422

[The effects of epinephrine and adrenergic antagonists on adenosine 3', 5'-monophosphate level of bovine trabecular cells in vitro].

PubMed

Lu, Y; Li, M; Shen, Y

1998-03-01

To determine the effects of epinephrine (EPI) and adrenergic antagonists on adenosine 3', 5'-monophosphate (cAMP) level of bovine trabecular cells (BTC) in vitro. (3)H-cAMP was used in protein binding assay for measuring the intracellular level of cAMP. (1) 10(-5) mol/L EPI induced a fold increase of cAMP in cultured BTC in vitro; (2) Timilol and ICI 118, 551 blocked efficiently the effect of EPI at a lower concentration (10(-6) mol/L). (3) Bisoprolol did not efficiently block the effect of EPI unless at high concentrations (>or= 10(-5) mol/L). The effects of EPI increasing outflow facility may be associated with its increase of cAMP in trabecular cells; BTC contains beta-adrenergic receptors, and beta(2)-adrenergic receptors are dominant.

Critical ligand binding reagent preparation/selection: when specificity depends on reagents.

PubMed

Rup, Bonita; O'Hara, Denise

2007-05-11

Throughout the life cycle of biopharmaceutical products, bioanalytical support is provided using ligand binding assays to measure the drug product for pharmacokinetic, pharmacodynamic, and immunogenicity studies. The specificity and selectivity of these ligand binding assays are highly dependent on the ligand binding reagents. Thus the selection, characterization, and management processes for ligand binding reagents are crucial to successful assay development and application. This report describes process considerations for selection and characterization of ligand binding reagents that are integral parts of the different phases of assay development. Changes in expression, purification, modification, and storage of the ligand binding reagents may have a profound effect on the ligand binding assay performance. Thus long-term management of the critical ligand binding assay reagents is addressed including suggested characterization criteria that allow ligand binding reagents to be used in as consistent a manner as possible. Examples of challenges related to the selection, modification, and characterization of ligand binding reagents are included.

Analysis of Ethylene Receptors: Ethylene-Binding Assays.

PubMed

Binder, Brad M; Schaller, G Eric

2017-01-01

Plant ethylene receptors bind ethylene with high affinity. Most of the characterization of ethylene binding to the receptors has been carried out using a radioligand-binding assay on functional receptors expressed in yeast. In this chapter, we describe methods for expressing ethylene receptors in yeast and conducting ethylene-binding assays on intact yeast and yeast membranes. The ethylene-binding assays can be modified to analyze ethylene binding to intact plants and other organisms as well as membranes isolated from any biological source.

Monoclonal antibodies to human vitamin D-binding protein.

PubMed Central

Pierce, E A; Dame, M C; Bouillon, R; Van Baelen, H; DeLuca, H F

1985-01-01

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays. Images PMID:3936035

Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na+/K+-ATPase

PubMed Central

2013-01-01

Background Surface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK. Methods The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. Results Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. Conclusions Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth. PMID:23621895

Actomyosin kinetics and in vitro motility of wild-type Drosophila actin and the effects of two mutations in the Act88F gene.

PubMed Central

Anson, M; Drummond, D R; Geeves, M A; Hennessey, E S; Ritchie, M D; Sparrow, J C

1995-01-01

Two missense mutations of the flight muscle-specific actin gene of Drosophila melanogaster, Act88F, assemble into normally structured myofibrils but affect the flight ability of flies and the mechanical kinetics of isolated muscle fibers. We describe the isolation of actin from different homozygous Act88F strains, including wild-type, an Act88F null mutant (KM88), and two Act88F single point mutations (E316K and G368E), their biochemical interactions with rabbit myosin subfragment 1 (S1), and behavior with rabbit myosin and heavy meromyosin in in vitro motility assays. The rabbit and wild-type Drosophila actins have different association rate constants with S1 (2.64 and 1.77 microM-1 s-1, respectively) and in vitro motilities (2.51, 1.60 microns s-1) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increasing from 0.11 to 0.17 microM) and a reduced velocity in vitro (reduced by 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with respect to the recently published atomic models for the actomyosin structure and our findings that G368E fibers show a reduced rate constant for delayed tension development and increased fiber stiffness. We interpret these results as possibly caused either by effects on A1 myosin light chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed with respect to its likely position within the tropomyosin binding site of actin. Images FIGURE 1 FIGURE 9 PMID:7612841

Determining antibody-binding site of streptococcal pyrogenic exotoxin B to protect mice from group a streptococcus infection.

PubMed

Tsao, Nina; Cheng, Miao-Hui; Yang, Hsiu-Chen; Wang, Yu-Chieh; Liu, Yi-Ling; Kuo, Chih-Feng

2013-01-01

Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcal (GAS) infection. SPE B binds and cleaves antibody isotypes and further impairs the immune system by inhibiting complement activation. In this study, we examined the antibody-binding site of SPE B and used it to block SPE B actions during GAS infection. We constructed different segments of the spe B gene and induced them to express different recombinant fragments of SPE B. Using an enzyme-linked immunosorbent assay (ELISA), we found that residues 345-398 of the C-terminal domain of SPE B (rSPE B(345-398)), but not the N-terminal domain, was the major binding site for antibody isotypes. Using a competitive ELISA, we also found that rSPE B(345-398) bound to the Fc portion of IgG. The in vitro functional assays indicate that rSPE B(345-398) not only interfered with cleavage of antibody isotypes but also interfered with SPE B-induced inhibition of complement activation. Immunization of BALB/c mice using rSPE B(345-398) was able to induce production of a high titer of anti-rSPE B(345-398) antibodies and efficiently protected mice from GAS-induced death. These findings suggest that SPE B uses its C-terminal domain to bind the Fc portion of IgG and that immunization of mice with this binding domain (rSPE B(345-398)) could protect mice from GAS infection.

Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

PubMed Central

Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

2004-01-01

In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and β proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with β in vitro. A new β-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified β-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind β. A 10-amino-acid peptide containing the E. coli Hda β-binding motif was shown to compete with Hda for binding to β in an Hda-β interaction assay. These results establish that the interaction of Hda with β is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication. PMID:15150238

Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein.

PubMed

Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

2004-06-01

In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

The impact of vaccination on the breadth and magnitude of the antibody response to influenza A viruses in HIV-infected individuals.

PubMed

Kohler, Ines; Kouyos, Roger; Bianchi, Matteo; Grube, Christina; Wyrzucki, Arkadiusz; Günthard, Huldrych F; Hangartner, Lars

2015-09-10

HIV-positive individuals have lower antibody titers to influenza viruses than HIV-negative individuals, and the benefits of the annual vaccinations are controversially discussed. Also, there is no information about the breadth of the antibody response in HIV-infected individuals. The binding and neutralizing antibody titers to various human and nonhuman influenza A virus strain were determined in sera from 146 HIV-infected volunteers: They were compared with those found in 305 randomly selected HIV-negative donors, and put in relation to HIV-specific parameters. Univariable and multivariable regression was used to identify HIV-specific parameters associated with the measured binding and neutralizing activity. Enzyme-linked immunosorbent assays and in-vitro neutralization assays were used to determine the binding and neutralizing antibodiy titers to homo and heterosubtypic influenza A subtypes. We found that both homo and heterosubtypic antibody titers are lower in HIV-positive individuals. Vaccination promoted higher binding and neutralizing antibody titers to human but not to nonhuman isolates. HIV-induced immune damage (high viral load, low CD4 T-cell counts, and long untreated disease progression) is associated with impaired homosubtypic responses, but can have beneficial effects on the development of heterosubtypic antibodies, and an improved ratio of binding to neutralizing antibody titers to homosubtypic isolates. Our results indicate that repetitive vaccinations in HIV-positive individuals enhance antibody titers to human isolates. Interestingly, development of antibody titers to conserved heterosubtypic epitopes paradoxically appeared to profit from HIV-induced immune damage, as did the ratio of binding to neutralizing antibodies.

Chitosan Stabilized Gold-Folate-Poly(lactide-co-glycolide) Nanoplexes Facilitate Efficient Gene Delivery in Hepatic and Breast Cancer Cells.

PubMed

Akinyelu, Jude; Singh, Moganavelli

2018-07-01

The biodegradable polymer, poly(lactide-co-glycolide) is a popular polymer of choice in many nanotherapeutic studies. Herein, we report on the synthesis and evaluation of four chitosan stabilized poly(lactide-co-glycolide) nanoparticles with and without coating with gold, and the targeting ligand, folic acid, as potential non-viral gene delivery vectors. The poly(lactide-co-glycolide) nanoparticles were synthesized via nanoprecipitation/solvent evaporation method in conjunction with the surface functionalizing folic acid and chitosan. The physiochemical properties (morphology, particle size, zeta potential, folic acid/chitosan presence, DNA binding), and biological properties (nuclease protection, in vitro cytotoxicity and transfection potential in human kidney, hepatocellular carcinoma and breast adenocarcinoma cells), of all four gene bound nanoparticles were evaluated. Gel retardation assays confirmed that all the nanoparticles were able to successfully bind the reporter plasmid, pCMV-luc DNA at varying weight ratios. The gold-folate-poly(lactide-co-glycolide) nanoplexes with the highest binding efficiency (w/w ratio 4:1), best protected the plasmid DNA as evidenced from the nuclease protection assays. Furthermore, these nanoplexes presented as spherical particles with an average particle size of 199.4 nm and zeta potential of 35.7 mV. Folic acid and chitosan functionalization of the nanoparticles was confirmed by attenuated total reflection-Fourier transform infrared spectroscopy. All nanoplexes maintained over 90% cell viability in all cell lines investigated. Interestingly, the gold-folate-poly(lactide-co-glycolide) nanoplexes showed a greater transgene activity in the hepatic and breast cancer cells compared to the other nanocomplexes in the same cell lines. The favorable size, colloidal stability, low cytotoxicity, significant transgene expression, and nuclease protection ability in vitro, all provide support for the use of gold-folate-poly(lactide-co-glycolide) nanoplexes in future gene therapy applications.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsao, Y.-P.; Lin, J.-Y.; Jan, J.-T.

The immunogenicity of HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) peptide in severe acute respiratory syndrome coronavirus (SARS-CoV) nuclear capsid (N) and spike (S) proteins was determined by testing the proteins' ability to elicit a specific cellular immune response after immunization of HLA-A2.1 transgenic mice and in vitro vaccination of HLA-A2.1 positive human peripheral blood mononuclearcytes (PBMCs). First, we screened SARS N and S amino acid sequences for allele-specific motif matching those in human HLA-A2.1 MHC-I molecules. From HLA peptide binding predictions (http://thr.cit.nih.gov/molbio/hla{sub b}ind/), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated bymore » T2-cell stabilization assays, with immunogenicity assays revealing peptides N223-231, N227-235, and N317-325 to be First identified HLA-A*0201-restricted CTL epitopes of SARS-CoV N protein. In addition, previous reports identified three HLA-A*0201-restricted CTL epitopes of S protein (S978-986, S1203-1211, and S1167-1175), here we found two novel peptides S787-795 and S1042-1050 as S-specific CTL epitopes. Moreover, our identified N317-325 and S1042-1050 CTL epitopes could induce recall responses when IFN-{gamma} stimulation of blood CD8{sup +} T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with their cognate antigen. Our results would provide a new insight into the development of therapeutic vaccine in SARS.« less

U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

PubMed

Somarelli, Jason A; Mesa, Annia; Rodriguez, Carol E; Sharma, Shalini; Herrera, Rene J

2014-04-25

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs. Copyright © 2014 Elsevier B.V. All rights reserved.

Differential effects of clinically used derivatives and metabolites of artemisinin in the activation of constitutive androstane receptor isoforms

PubMed Central

Burk, O; Piedade, R; Ghebreghiorghis, L; Fait, JT; Nussler, AK; Gil, JP; Windshügel, B; Schwab, M

2012-01-01

BACKGROUND AND PURPOSE Widespread resistance to antimalarial drugs requires combination therapies with increasing risk of pharmacokinetic drug–drug interactions. Here, we explore the capacity of antimalarial drugs to induce drug metabolism via activation of constitutive androstane receptors (CAR) by ligand binding. EXPERIMENTAL APPROACH A total of 21 selected antimalarials and 11 major metabolites were screened for binding to CAR isoforms using cellular and in vitro CAR-coactivator interaction assays, combined with in silico molecular docking. Identified ligands were further characterized by cell-based assays and primary human hepatocytes were used to elucidate induction of gene expression. KEY RESULTS Only two artemisinin derivatives arteether and artemether, the metabolite deoxyartemisinin and artemisinin itself demonstrated agonist binding to the major isoforms CAR1 and CAR3, while arteether and artemether were also inverse agonists of CAR2. Dihydroartemisinin and artesunate acted as weak inverse agonists of CAR1. While arteether showed the highest activities in vitro, it was less active than artemisinin in inducing hepatic CYP3A4 gene expression in hepatocytes. CONCLUSIONS AND IMPLICATIONS Artemisinin derivatives and metabolites differentially affect the activities of CAR isoforms and of the pregnane X receptor (PXR). This negates a common effect of these drugs on CAR/PXR-dependent induction of drug metabolism and further provides an explanation for artemisinin consistently inducing cytochrome P450 genes in vivo, whereas arteether and artemether do not. All these drugs are metabolized very rapidly, but only artemisinin is converted to an enzyme-inducing metabolite. For better understanding of pharmacokinetic drug–drug interaction possibilities, the inducing properties of artemisinin metabolites should be considered. PMID:22577882

Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway.

PubMed

Mittal, Yash; Pavlova, Yelena; Garcia-Marcos, Mikel; Ghosh, Pradipta

2011-09-16

GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

PubMed

Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

1997-06-05

Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

Blonanserin extensively occupies rat dopamine D3 receptors at antipsychotic dose range.

PubMed

Baba, Satoko; Enomoto, Takeshi; Horisawa, Tomoko; Hashimoto, Takashi; Ono, Michiko

2015-03-01

Antagonism of the dopamine D3 receptor has been hypothesized to be beneficial for schizophrenia cognitive deficits, negative symptoms and extrapyramidal symptoms. However, recent animal and human studies have shown that most antipsychotics do not occupy D3 receptors in vivo, despite their considerable binding affinity for this receptor in vitro. In the present study, we investigated the D3 receptor binding of blonanserin, a dopamine D2/D3 and serotonin 5-HT2A receptors antagonist, in vitro and in vivo. Blonanserin showed the most potent binding affinity for human D3 receptors among the tested atypical antipsychotics (risperidone, olanzapine and aripiprazole). Our GTPγS-binding assay demonstrated that blonanserin acts as a potent full antagonist for human D3 receptors. All test-drugs exhibited antipsychotic-like efficacy in methamphetamine-induced hyperactivity in rats. Treatment with blonanserin at its effective dose blocked the binding of [(3)H]-(+)-PHNO, a D2/D3 receptor radiotracer, both in the D2 receptor-rich region (striatum) and the D3 receptor-rich region (cerebellum lobes 9 and 10). On the other hand, the occupancies of other test-drugs for D3 receptors were relatively low. In conclusion, we have shown that blonanserin, but not other tested antipsychotics, extensively occupies D3 receptors in vivo in rats. Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

Drug search for leishmaniasis: a virtual screening approach by grid computing

NASA Astrophysics Data System (ADS)

Ochoa, Rodrigo; Watowich, Stanley J.; Flórez, Andrés; Mesa, Carol V.; Robledo, Sara M.; Muskus, Carlos

2016-07-01

The trypanosomatid protozoa Leishmania is endemic in 100 countries, with infections causing 2 million new cases of leishmaniasis annually. Disease symptoms can include severe skin and mucosal ulcers, fever, anemia, splenomegaly, and death. Unfortunately, therapeutics approved to treat leishmaniasis are associated with potentially severe side effects, including death. Furthermore, drug-resistant Leishmania parasites have developed in most endemic countries. To address an urgent need for new, safe and inexpensive anti-leishmanial drugs, we utilized the IBM World Community Grid to complete computer-based drug discovery screens (Drug Search for Leishmaniasis) using unique leishmanial proteins and a database of 600,000 drug-like small molecules. Protein structures from different Leishmania species were selected for molecular dynamics (MD) simulations, and a series of conformational "snapshots" were chosen from each MD trajectory to simulate the protein's flexibility. A Relaxed Complex Scheme methodology was used to screen 2000 MD conformations against the small molecule database, producing >1 billion protein-ligand structures. For each protein target, a binding spectrum was calculated to identify compounds predicted to bind with highest average affinity to all protein conformations. Significantly, four different Leishmania protein targets were predicted to strongly bind small molecules, with the strongest binding interactions predicted to occur for dihydroorotate dehydrogenase (LmDHODH; PDB:3MJY). A number of predicted tight-binding LmDHODH inhibitors were tested in vitro and potent selective inhibitors of Leishmania panamensis were identified. These promising small molecules are suitable for further development using iterative structure-based optimization and in vitro/in vivo validation assays.

Drug search for leishmaniasis: a virtual screening approach by grid computing.

PubMed

Ochoa, Rodrigo; Watowich, Stanley J; Flórez, Andrés; Mesa, Carol V; Robledo, Sara M; Muskus, Carlos

2016-07-01

The trypanosomatid protozoa Leishmania is endemic in ~100 countries, with infections causing ~2 million new cases of leishmaniasis annually. Disease symptoms can include severe skin and mucosal ulcers, fever, anemia, splenomegaly, and death. Unfortunately, therapeutics approved to treat leishmaniasis are associated with potentially severe side effects, including death. Furthermore, drug-resistant Leishmania parasites have developed in most endemic countries. To address an urgent need for new, safe and inexpensive anti-leishmanial drugs, we utilized the IBM World Community Grid to complete computer-based drug discovery screens (Drug Search for Leishmaniasis) using unique leishmanial proteins and a database of 600,000 drug-like small molecules. Protein structures from different Leishmania species were selected for molecular dynamics (MD) simulations, and a series of conformational "snapshots" were chosen from each MD trajectory to simulate the protein's flexibility. A Relaxed Complex Scheme methodology was used to screen ~2000 MD conformations against the small molecule database, producing >1 billion protein-ligand structures. For each protein target, a binding spectrum was calculated to identify compounds predicted to bind with highest average affinity to all protein conformations. Significantly, four different Leishmania protein targets were predicted to strongly bind small molecules, with the strongest binding interactions predicted to occur for dihydroorotate dehydrogenase (LmDHODH; PDB:3MJY). A number of predicted tight-binding LmDHODH inhibitors were tested in vitro and potent selective inhibitors of Leishmania panamensis were identified. These promising small molecules are suitable for further development using iterative structure-based optimization and in vitro/in vivo validation assays.

Intrinsic mineralocorticoid agonist activity of some nonsteroidal anti-inflammatory drugs. A postulated mechanism for sodium retention.

PubMed Central

Feldman, D; Couropmitree, C

1976-01-01

Because some nonsteroidal anti-inflammatory drugs (NSAID) induce salt and water retention and exhibit other steroid-like actions, studies were performed to ascertain whether these drugs possess intrinsic mineralocorticoid agonist activity. In vitro competitive binding assays utilizing tissue from adrenalectomized rats demonstrated that some NSAID can displace [3H]-aldosterone from renal cytoplasmic mineralocorticoid receptors. Displacement potency for these sites was in the sequence: aldosterone greater than spironolactone greater than phenylbutazone (PBZ) greater than aspirin (ASA) greater than indomethacin (IDM). Concentration ratios required to obtain significant displacement of [3H]aldosterone were high but clearly within the therapeutic range for PBZ and ASA but not IDM. The analogues oxyphenbutazone (OBZ) and sodium salicylate (SS) were similar in binding activity to PBZ and ASA, respectively. Lineweaver-Burk analysis revealed that the inhibition of [3H]aldosterone binding was competitive in nature. In addition, PBZ was shown to prevent the nuclear binding of [3H]aldosterone. In vivo injection of PBZ and ASA resulted in competition for [3H]aldosterone renal binding comparable to the in vitro studies. Administration of PBZ and OBZ to adrenalectomized rats resulted in significant salt retention whereas ASA and SS did not differ significantly from controls. Salt retention elicited by PBZ and OBZ was inhibited by spironolactone, a competitive mineralocorticoid antagonist. These data suggest that, despite nonsteroidal structures, PBZ and OBZ induce salt retention via a receptor-mediated mineralocorticoid pathway analogous to aldosterone action. PMID:173739

Inverted repeats in the promoter as an autoregulatory sequence for TcrX in Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Monolekha; Das, Amit Kumar, E-mail: amitk@hijli.iitkgp.ernet.in

Highlights: Black-Right-Pointing-Pointer The regulatory sequences recognized by TcrX have been identified. Black-Right-Pointing-Pointer The regulatory region comprises of inverted repeats segregated by 30 bp region. Black-Right-Pointing-Pointer The mode of binding of TcrX with regulatory sequence is unique. Black-Right-Pointing-Pointer In silico TcrX-DNA docked model binds one of the inverted repeats. Black-Right-Pointing-Pointer Both phosphorylated and unphosphorylated TcrX binds regulatory sequence in vitro. -- Abstract: TcrY, a histidine kinase, and TcrX, a response regulator, constitute a two-component system in Mycobacterium tuberculosis. tcrX, which is expressed during iron scarcity, is instrumental in the survival of iron-dependent M. tuberculosis. However, the regulator of tcrX/Y has notmore » been fully characterized. Crosslinking studies of TcrX reveal that it can form oligomers in vitro. Electrophoretic mobility shift assays (EMSAs) show that TcrX recognizes two regions in the promoter that are comprised of inverted repeats separated by {approx}30 bp. The dimeric in silico model of TcrX predicts binding to one of these inverted repeat regions. Site-directed mutagenesis and radioactive phosphorylation indicate that D54 of TcrX is phosphorylated by H256 of TcrY. However, phosphorylated and unphosphorylated TcrX bind the regulatory sequence with equal efficiency, which was shown with an EMSA using the D54A TcrX mutant.« less

Anti-angiogenic and vascular disrupting effects of C9, a new microtubule-depolymerizing agent

PubMed Central

Ren, Xuan; Dai, Mei; Lin, Li-Ping; Li, Pui-Kai; Ding, Jian

2009-01-01

Background and purpose: The critical role of blood supply in the growth of solid tumours makes blood vessels an ideal target for anti-tumour drug discovery. The anti-angiogenic and vascular disrupting activities of C9, a newly synthesized microtubule-depolymerizing agent, were investigated with several in vitro and in vivo models. Possible mechanisms involved in its activity were also assessed. Experimental approach: Microtubule-depolymerizing actions were assessed by surface plasmon resonance binding, competitive inhibition and cytoskeleton immunofluorescence. Anti-angiogenic and vascular disrupting activities were tested on proliferation, migration, tube formation with human umbilical vein endothelial cells, and in rat aortic ring, chick chorioallantoic membrane and Matrigel plug assays. Western blots and Rho activation assays were employed to examine the role of Raf-MEK-ERK (mitogen-activated ERK kinase, extracellular signal-regulated kinase) and Rho/Rho kinase signalling. Key results: C9 inhibited proliferation, migration and tube formation of endothelial cells and inhibited angiogenesis in aortic ring and chick chorioallantoic membrane assays. C9 induced disassembly of microtubules in endothelial cells and down-regulated Raf-MEK-ERK signalling activated by pro-angiogenic factors. In addition, C9 disrupted capillary-like networks and newly formed vessels in vitro and rapidly decreased perfusion of neovasculature in vivo. Endothelial cell contraction and membrane blebbing induced by C9 in neovasculature was dependent on the Rho/Rho kinase pathway. Conclusions and implications: Anti-angiogenic and vascular disruption by C9 was associated with changes in morphology and function of endothelial cells, involving the Raf-MEK-ERK and Rho/Rho kinase signalling pathways. These findings strongly suggest that C9 is a new microtubule-binding agent that could effectively target tumour vasculature. PMID:19302593

In vitro and in vivo characterization of a reversible synthetic heparin analog.

PubMed

Whelihan, Matthew F; Cooley, Brian; Xu, Yongmei; Pawlinski, Rafal; Liu, Jian; Key, Nigel S

2016-02-01

The global supply of unfractionated heparin (UFH) and all commercially available low molecular weight heparins (LMWH) remain dependent on animal sources, such as porcine intestine or bovine lung. Recent experience has shown that contamination of the supply chain (with over-sulfated chondroitin sulfates) can result in lethal toxicity. Fondaparinux is currently the only commercially available synthetic analog of heparin. We recently described a new class of chemoenzymatically synthesized heparin analogs. One of these compounds (S12-mer) is a dodecasaccharide consisting of an antithrombin-binding moiety with repeating units of IdoA2S-GlcNS6S and two 3-O-sulfate groups that confer the ability to bind protamine. We sought to further characterize this new compound in vitro using biochemical and global coagulation assays and in vivo using thrombosis and hemostasis assays. The anticoagulant activities of the Super 12-mer (S12-mer) and Enoxaparin in anti-factor Xa and plasma-based thrombin generation assays were roughly equivalent with a 50% reduction in peak thrombin generation occurring at approximately 325nM. When protamine was titrated against a fixed concentration of S12-mer in plasma or blood, the S12-mer displayed a significant restitution of thrombin generation and clot formation. In vivo, S12-mer inhibited venous thrombosis to a similar extent as Enoxaparin, with similar bleeding profiles. These data show that the S12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the ability to ensure purity and homogeneity from batch to batch, the S12-mer is a promising new synthetic heparin analog with a potentially enhanced safety profile. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of a novel prophage-like gene cluster actively expressed in both virulent and avirulent strains of Leptospira interrogans serovar Lai.

PubMed

Qin, Jin-Hong; Zhang, Qing; Zhang, Zhi-Ming; Zhong, Yi; Yang, Yang; Hu, Bao-Yu; Zhao, Guo-Ping; Guo, Xiao-Kui

2008-06-01

DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains.

Transcriptional activation of the Escherichia coli adaptive response gene aidB is mediated by binding of methylated Ada protein. Evidence for a new consensus sequence for Ada-binding sites.

PubMed

Landini, P; Volkert, M R

1995-04-07

The Escherichia coli aidB gene is part of the adaptive response to DNA methylation damage. Genes belonging to the adaptive response are positively regulated by the ada gene; the Ada protein acts as a transcriptional activator when methylated in one of its cysteine residues at position 69. Through DNaseI protection assays, we show that methylated Ada (meAda) is able to bind a DNA sequence between 40 and 60 base pairs upstream of the aidB transcriptional startpoint. Binding of meAda is necessary to activate transcription of the adaptive response genes; accordingly, in vitro transcription of aidB is dependent on the presence of meAda. Unmethylated Ada protein shows no protection against DNaseI digestion in the aidB promoter region nor does it promote aidB in vitro transcription. The aidB Ada-binding site shows only weak homology to the proposed consensus sequences for Ada-binding sites in E. coli (AAANNAA and AAAGCGCA) but shares a higher degree of similarity with the Ada-binding regions from other bacterial species, such as Salmonella typhimurium and Bacillus subtilis. Based on the comparison of five different Ada-dependent promoter regions, we suggest that a possible recognition sequence for meAda might be AATnnnnnnG-CAA. Higher concentrations of Ada are required for the binding of aidB than for the ada promoter, suggesting lower affinity of the protein for the aidB Ada-binding site. Common features in the Ada-binding regions of ada and aidB are a high A/T content, the presence of an inverted repeat structure, and their position relative to the transcriptional start site. We propose that these elements, in addition to the proposed recognition sequence, are important for binding of the Ada protein.

SHCBP1 is over-expressed in breast cancer and is important in the proliferation and apoptosis of the human malignant breast cancer cell line.

PubMed

Feng, Wen; Li, Hong-Chang; Xu, Ke; Chen, Ya-Feng; Pan, Li-Yun; Mei, Yi; Cai, Han; Jiang, Yi-Ming; Chen, Teng; Feng, Dian-Xu

2016-08-01

SHC SH2-binding protein 1, a member of Src homolog and collagen homolog (Shc) family, has been recently identified in different contexts in unbiased screening assays. It has been reported to be over-expressed in several malignant cancers. Immunohistochemistry of SHCBP1 on 128 breast cancer tissues and adjacent normal tissues were used to evaluate the prognostic significance of SHCBP1. Survival analyses were performed by Kaplan-Meier method. CRISPR/CAS9 method was used to knockout SHCBP1 expression. CRISPR/CAS9 technology was used to knockout SHCBP1 in 2 breast cancer cell lines. MTT assay, BrdU assay, colony formation assay, cell cycle assay and apoptosis analysis in MCF-7 and MDA-MB-231 cell lines were carried out to evaluate the effects of SHCBP1 on breast cancer in vitro. Immunohistochemical analysis revealed SHCBP1 was significantly up-regulated in breast cancer tissues compared with adjacent normal tissues (82 of 128, 64%). Over-expressed SHCBP1 was correlated with advanced clinical stage and poorer survival. Ablation of SHCBP1 inhibited the proliferation in vitro. SHCBP1 knockout increased cyclin-dependent kinase inhibitor p21, and decreased the Cyclin B1 and CDK1. Our study suggests SHCBP1 is dysregulated expressed in breast cancer and plays a critical role in cancer progression, which can be a potential prognosis predictor of breast cancer. Copyright © 2016. Published by Elsevier B.V.

Target identification, lead optimization and antitumor evaluation of some new 1,2,4-triazines as c-Met kinase inhibitors.

PubMed

El-Wakil, Marwa H; Ashour, Hayam M; Saudi, Manal N; Hassan, Ahmed M; Labouta, Ibrahim M

2017-08-01

In silico target fishing approach using PharmMapper server identified c-Met kinase as the selective target for our previously synthesized compound NCI 748494/1. This approach was validated by in vitro kinase assay which showed that NCI 748494/1 possessed promising inhibitory activity against c-Met kinase (IC 50 =31.70μM). Assessment of ADMET profiling, drug-likeness, drug score as well as docking simulation for the binding pose of that compound in the active site of c-Met kinase domain revealed that NCI 748494/1 could be considered as a promising drug lead. Based on target identification and validation, it was observed that there is structure similarity between NCI 748494/1 and the reported type II c-Met kinase inhibitor BMS-777607. Optimization of our lead NCI 748494/1 furnished newly synthesized 1,2,4-triazine derivatives based on well-established structure-activity relationships, whereas three compounds namely; 4d, 7a and 8c displayed excellent in vitro cytotoxicity against three c-Met addicted cancer cell lines; A549 (lung adenocarcinoma), HT-29 (colon cancer) and MKN-45 (gastric carcinoma); with IC 50 values in the range 0.01-1.86µM. In vitro c-Met kinase assay showed 8c to possess the highest c-Met kinase inhibition profile (IC 50 =4.31µM). Docking of the active compounds in c-Met kinase active site revealed strong binding interactions comparable to the lead NCI 748494/1 and BMS-777607, suggesting that c-Met inhibition is very likely to be the mechanism of the antitumor effect of these derivatives. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition of pneumococcal choline-binding proteins and cell growth by esters of bicyclic amines.

PubMed

Maestro, Beatriz; González, Ana; García, Pedro; Sanz, Jesús M

2007-01-01

Streptococcus pneumoniae is one of the major pathogens worldwide. The use of currently available antibiotics to treat pneumococcal diseases is hampered by increasing resistance levels; also, capsular polysaccharide-based vaccination is of limited efficacy. Therefore, it is desirable to find targets for the development of new antimicrobial drugs specifically designed to fight pneumococcal infections. Choline-binding proteins are a family of polypeptides, found in all S. pneumoniae strains, that take part in important physiologic processes of this bacterium. Among them are several murein hydrolases whose enzymatic activity is usually inhibited by an excess of choline. Using a simple chromatographic procedure, we have identified several choline analogs able to strongly interact with the choline-binding module (C-LytA) of the major autolysin of S. pneumoniae. Two of these compounds (atropine and ipratropium) display a higher binding affinity to C-LytA than choline, and also increase the stability of the protein. CD and fluorescence spectroscopy analyses revealed that the conformational changes of C-LytA upon binding of these alkaloids are different to those induced by choline, suggesting a different mode of binding. In vitro inhibition assays of three pneumococcal, choline-dependent cell wall lytic enzymes also demonstrated a greater inhibitory efficiency of those molecules. Moreover, atropine and ipratropium strongly inhibited in vitro pneumococcal growth, altering cell morphology and reducing cell viability, a very different response than that observed upon addition of an excess of choline. These results may open up the possibility of the development of bicyclic amines as new antimicrobials for use against pneumococcal pathologies.

The peroxisomal multifunctional protein interacts with cortical microtubules in plant cells

PubMed Central

2005-01-01

Background The plant peroxisomal multifunctional protein (MFP) possesses up to four enzymatic activities that are involved in catalyzing different reactions of fatty acid β-oxidation in the peroxisome matrix. In addition to these peroxisomal activities, in vitro assays revealed that rice MFP possesses microtubule- and RNA-binding activities suggesting that this protein also has important functions in the cytosol. Results We demonstrate that MFP is an authentic microtubule-binding protein, as it localized to the cortical microtubule array in vivo, in addition to its expected targeting to the peroxisome matrix. MFP does not, however, interact with the three mitotic microtubule arrays. Microtubule co-sedimentation assays of truncated versions of MFP revealed that multiple microtubule-binding domains are present on the MFP polypeptide. This indicates that these regions function together to achieve high-affinity binding of the full-length protein. Real-time imaging of a transiently expressed green fluorescent protein-MFP chimera in living plant cells illustrated that a dynamic, spatial interaction exits between peroxisomes and cortical microtubules as peroxisomes move along actin filaments or oscillate at fixed locations. Conclusion Plant MFP is associated with the cortical microtubule array, in addition to its expected localization in the peroxisome. This observation, coupled with apparent interactions that frequently occur between microtubules and peroxisomes in the cell cortex, supports the hypothesis that MFP is concentrated on microtubules in order to facilitate the regulated import of MFP into peroxisomes. PMID:16313672

Application of a 1.48-microm diode laser for bisecting oocytes into two identical hemizonae for the hemizona assay.

PubMed

Edenfeld, J; Schöpper, B; Sturm, R; Diedrich, K; Al-Hasani, S

2002-04-01

Laser systems are very promising new technical tools in assisted reproduction. It was investigated if laser radiation can replace the mechanical cutting procedure via micromanipulator in the hemizona assay (HZA), a commonly used bioassay to determine the sperm-zona pellucida binding capacity. An oocyte was bisected precisely into two identical hemizonae with approximately 20 laser pulses (pulse length 30 msec) using a 1.48-microm diode laser. Compared with the conventional method using microscalpels for zona bisection, laser treated hemizonae showed equivalent sperm-binding and within the two groups there was no detectable difference between matching hemizonae in their capacity for tight sperm-binding. To evaluate whether laser radiation affects the outcome of the HZA when effects of certain substances are investigated, the spermatozoa were preincubated with human follicular fluid (hFF), which inhibits the binding of spermatozoa to zona pellucida in vitro. Supplementation with follicular fluid exerted an inhibitory effect in both groups. The hemizona index (HZI) showed no statistical differences between the two methods. Therefore, the 1.48-microm diode laser is a suitable new instrument for generating equally sized hemizonae. There is no use for holding pipettes and microscalpels, on the contrary, for performing the HZA the laser is a precise, very quick and easy to use new working tool.

MBD2 is a critical component of a methyl cytosine-binding protein complex isolated from primary erythroid cells

PubMed Central

Kransdorf, Evan P.; Wang, Shou Zhen; Zhu, Sheng Zu; Langston, Timothy B.; Rupon, Jeremy W.; Ginder, Gordon D.

2006-01-01

The chicken embryonic β-type globin gene, ρ, is a member of a small group of vertebrate genes whose developmentally regulated expression is mediated by DNA methylation. Previously, we have shown that a methyl cytosine-binding complex binds to the methylated ρ-globin gene in vitro. We have now chromatographically purified and characterized this complex from adult chicken primary erythroid cells. Four components of the MeCP1 transcriptional repression complex were identified: MBD2, RBAP48, HDAC2, and MTA1. These 4 proteins, as well as the zinc-finger protein p66 and the chromatin remodeling factor Mi2, were found to coelute by gel-filtration analysis and pull-down assays. We conclude that these 6 proteins are components of the MeCPC. In adult erythrocytes, significant enrichment for MBD2 is seen at the inactive ρ-globin gene by chromatin immunoprecipitation assay, whereas no enrichment is observed at the active βA-globin gene, demonstrating MBD2 binds to the methylated and transcriptionally silent ρ-globin gene in vivo. Knock-down of MBD2 resulted in up-regulation of a methylated ρ-gene construct in mouse erythroleukemic (MEL)-ρ cells. These results represent the first purification of a MeCP1-like complex from a primary cell source and provide support for a role for MBD2 in developmental gene regulation. PMID:16778143

Binding of human nucleotide exchange factors to heat shock protein 70 (Hsp70) generates functionally distinct complexes in vitro.

PubMed

Rauch, Jennifer N; Gestwicki, Jason E

2014-01-17

Proteins with Bcl2-associated anthanogene (BAG) domains act as nucleotide exchange factors (NEFs) for the molecular chaperone heat shock protein 70 (Hsp70). There are six BAG family NEFs in humans, and each is thought to link Hsp70 to a distinct cellular pathway. However, little is known about how the NEFs compete for binding to Hsp70 or how they might differentially shape its biochemical activities. Toward these questions, we measured the binding of human Hsp72 (HSPA1A) to BAG1, BAG2, BAG3, and the unrelated NEF Hsp105. These studies revealed a clear hierarchy of affinities: BAG3 > BAG1 > Hsp105 ≫ BAG2. All of the NEFs competed for binding to Hsp70, and their relative affinity values predicted their potency in nucleotide and peptide release assays. Finally, we combined the Hsp70-NEF pairs with cochaperones of the J protein family (DnaJA1, DnaJA2, DnaJB1, and DnaJB4) to generate 16 permutations. The activity of the combinations in ATPase and luciferase refolding assays were dependent on the identity and stoichiometry of both the J protein and NEF so that some combinations were potent chaperones, whereas others were inactive. Given the number and diversity of cochaperones in mammals, it is likely that combinatorial assembly could generate a large number of distinct permutations.

Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1

PubMed Central

McDonald, Christopher; Jovanovic, Goran; Ces, Oscar

2015-01-01

ABSTRACT Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins’ differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell’s inner membrane experiences increased SCE stress. PMID:26330516

Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing.

PubMed

Panja, Subrata; Santiago-Frangos, Andrew; Schu, Daniel J; Gottesman, Susan; Woodson, Sarah A

2015-11-06

Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Novel Atypical PKC-Iota Inhibitor, Echinochrome A, Enhances Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells.

PubMed

Kim, Hyoung Kyu; Cho, Sung Woo; Heo, Hye Jin; Jeong, Seung Hun; Kim, Min; Ko, Kyung Soo; Rhee, Byoung Doo; Mishchenko, Natalia P; Vasileva, Elena A; Fedoreyev, Sergey A; Stonik, Valentin A; Han, Jin

2018-06-02

Echinochrome A (EchA) is a marine bioproduct extracted from sea urchins having antioxidant, antimicrobial, anti-inflammatory, and chelating effects, and is the active component of the clinical drug histochrome. We investigated the potential use of Ech A for inducing cardiomyocyte differentiation from mouse embryonic stem cells (mESCs). We also assessed the effects of Ech A on mitochondrial mass, inner membrane potential (Δψm), reactive oxygen species generation, and levels of Ca 2+ . To identify the direct target of Ech A, we performed in vitro kinase activity and surface plasmon resonance binding assays. Ech A dose-dependently enhanced cardiomyocyte differentiation with higher beating rates. Ech A (50 μM) increased the mitochondrial mass and membrane potential but did not alter the mitochondrial superoxide and Ca 2+ levels. The in vitro kinase activity of the atypical protein kinase C-iota (PKCι) was significantly decreased by 50 μM of Ech A with an IC 50 for PKCι activity of 107 μM. Computational protein-ligand docking simulation results suggested the direct binding of Ech A to PKCι, and surface plasmon resonance confirmed the direct binding with a low K D of 6.3 nM. Therefore, Ech A is a potential drug for enhancing cardiomyocyte differentiation from mESCs through direct binding to PKCι and inhibition of its activity.

Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giulliani, S. E.; Frank, A. E.; Collart, F. R.

2008-12-08

We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. The assay also identified additional candidate binding ligands for several of the amino acid-binding proteins in the validation set. We extended this approach to additional targets and demonstrated the ability of the FTS assay to unambiguously identify preferential binding for several homologues of amino acid-binding proteins with known specificity andmore » to functionally annotate proteins of unknown binding specificity. The assay is implemented in a microwell plate format and provides a rapid approach to validate an anticipated function or to screen proteins of unknown function. The ABC-type transporter family is ubiquitous and transports a variety of biological compounds, but the current annotation of the ligand-binding proteins is limited to mostly generic descriptions of function. The results illustrate the feasibility of the FTS assay to improve the functional annotation of binding proteins associated with ABC-type transporters and suggest this approach that can also be extended to other protein families.« less

S-adenosylmethionine directly inhibits binding of 30S ribosomal subunits to the SMK box translational riboswitch RNA

PubMed Central

Fuchs, Ryan T.; Grundy, Frank J.; Henkin, Tina M.

2007-01-01

The SMK box is a conserved riboswitch motif found in the 5′ untranslated region of metK genes [encoding S-adenosylmethionine (SAM) synthetase] in lactic acid bacteria, including Enterococcus, Streptococcus, and Lactococcus sp. Previous studies showed that this RNA element binds SAM in vitro, and SAM binding causes a structural rearrangement that sequesters the Shine–Dalgarno (SD) sequence by pairing with an anti-SD (ASD) element. A model was proposed in which SAM binding inhibits metK translation by preventing binding of the ribosome to the SD region of the mRNA. In the current work, the addition of SAM was shown to inhibit binding of 30S ribosomal subunits to SMK box RNA; in contrast, the addition of S-adenosylhomocysteine (SAH) had no effect. A mutant RNA, which has a disrupted SD-ASD pairing, was defective in SAM binding and showed no reduction of ribosome binding in the presence of SAM, whereas a compensatory mutation that restored SD-ASD pairing restored the response to SAM. Primer extension inhibition assays provided further evidence for SD-ASD pairing in the presence of SAM. These results strongly support the model that SMK box translational repression operates through occlusion of the ribosome binding site and that SAM binding requires the SD-ASD pairing. PMID:17360376

Simultaneous Multiple MS Binding Assays Addressing D1 and D2 Dopamine Receptors.

PubMed

Schuller, Marion; Höfner, Georg; Wanner, Klaus T

2017-10-09

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D 1 and D 2 dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D 1 and D 2 receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D 1 and D 2 receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

PubMed

Pinne, Marija; Matsunaga, James; Haake, David A

2012-11-01

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

DIRECT BINDING OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE TO TELOMERIC DNA PROTECTS TELOMERES AGAINST CHEMOTHERAPY-INDUCED RAPID DEGRADATION

PubMed Central

Demarse, Neil A.; Ponnusamy, Suriyan; Spicer, Eleanor K.; Apohan, Elif; Baatz, John E.; Ogretmen, Besim; Davies, Christopher

2009-01-01

GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel shift assays, we show that recombinant GAPDH binds directly with high affinity (Kd = 45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats and that nucleotides T1, G5 and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA: GAPDH), and GAPDH appears to form a high-molecular weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition, and this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells. PMID:19800890

Operator design and mechanism for CarA repressor-mediated down-regulation of the photoinducible carB operon in Myxococcus xanthus.

PubMed

López-Rubio, José Juan; Padmanabhan, S; Lázaro, Jose María; Salas, Margarita; Murillo, Francisco José; Elías-Arnanz, Montserrat

2004-07-09

The carB operon encodes all except one of the enzymes involved in light-induced carotenogenesis in Myxococcus xanthus. Expression of its promoter (P(B)) is repressed in the dark by sequence-specific DNA binding of CarA to a palindrome (pI) located between positions -47 and -64 relative to the transcription start site. This promotes subsequent binding of CarA to additional sites that remain to be defined. CarS, produced in the light, interacts physically with CarA, abrogates CarA-DNA binding, and thereby derepresses P(B). In this study, we delineate the operator design that exists for CarA by precisely mapping out the second operator element. For this, we examined how stepwise deletions and site-directed mutagenesis in the region between the palindrome and the transcription start site affect CarA binding around P(B) in vitro and expression of P(B) in vivo. These revealed the second operator element to be an imperfect interrupted palindrome (pII) spanning positions -26 to -40. In vitro assays using purified M. xanthus RNA polymerase showed that CarA abolishes P(B)-RNA polymerase binding and runoff transcription and that both were restored by CarS, thus rationalizing the observations in vivo. CarA binding to pII (after association with pI) effectively occludes RNA polymerase from P(B) and so provides the operative mechanism for the repression of the carB operon by CarA. The bipartite operator design, whereby transcription is blocked by the low affinity CarA-pII binding and is readily restored by CarS, may have evolved to match the needs for a rapid and an effective response to light.

The tau positron-emission tomography tracer AV-1451 binds with similar affinities to tau fibrils and monoamine oxidases.

PubMed

Vermeiren, Céline; Motte, Philippe; Viot, Delphine; Mairet-Coello, Georges; Courade, Jean-Philippe; Citron, Martin; Mercier, Joël; Hannestad, Jonas; Gillard, Michel

2018-02-01

Lilly/Avid's AV-1451 is one of the most advanced tau PET tracers in the clinic. Although results obtained in Alzheimer's disease patients are compelling, discrimination of tracer uptake in healthy individuals and patients with supranuclear palsy (PSP) is less clear as there is substantial overlap of signal in multiple brain regions. Moreover, accurate quantification of [ 18 F]AV-1451 uptake in Alzheimer's disease may not be possible. The aim of the present study was to characterize the in vitro binding of AV-1451 to understand and identify potential off-target binding that could explain the poor discrimination observed in PSP patients. [ 3 H]AV-1451 and AV-1451 were characterized in in vitro binding assays using recombinant and native proteins/tissues from postmortem samples of controls and Alzheimer's disease and PSP patients. [ 3 H]AV-1451 binds to multiple sites with nanomolar affinities in brain homogenates and to tau fibrils isolated from Alzheimer's disease or PSP patients. [ 3 H]AV-1451 also binds with similarly high affinities in brain homogenates devoid of tau pathology. This unexpected binding was demonstrated to be because of nanomolar affinities of [ 3 H]AV-1451 for monoamine oxidase A and B enzymes. High affinity of AV-1451 for monoamine oxidase proteins may limit its utility as a tau PET tracer in PSP and Alzheimer's disease because of high levels of monoamine oxidase expression in brain regions also affected by tau deposition, especially if monoamine oxidase levels change over time or with a treatment intervention. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

PDZ binding motif of HTLV-1 Tax promotes virus-mediated T-cell proliferation in vitro and persistence in vivo.

PubMed

Xie, Li; Yamamoto, Brenda; Haoudi, Abdelali; Semmes, O John; Green, Patrick L

2006-03-01

HTLV-1 cellular transformation and disease induction is dependent on expression of the viral Tax oncoprotein. PDZ is a modular protein interaction domain used in organizing signaling complexes in eukaryotic cells through recognition of a specific binding motif in partner proteins. Tax-1, but not Tax-2, contains a PDZ-binding domain motif (PBM) that promotes the interaction with several cellular PDZ proteins. Herein, we investigate the contribution of the Tax-1 PBM in HTLV-induced proliferation and immortalization of primary T cells in vitro and viral survival in an infectious rabbit animal model. We generated several HTLV-1 and HTLV-2 Tax viral mutants, including HTLV-1deltaPBM, HTLV-2+C22(+PBM), and HTLV-2+ C18(deltaPBM). All Tax mutants maintained the ability to significantly activate the CREB/ATF or NFkappaB signaling pathways. Microtiter proliferation assays revealed that the Tax-1 PBM significantly increases both HTLV-1- and HTLV-2-induced primary T-cell proliferation. In addition, Tax-1 PBM was responsible for the micronuclei induction activity of Tax-1 relative to that of Tax-2. Viral infection and persistence were severely attenuated in rabbits inoculated with HTLV-1deltaPBM. Our results provide the first direct evidence suggesting that PBM-mediated associations between Tax-1 and cellular proteins play a key role in HTLV-induced cell proliferation and genetic instability in vitro and facilitate viral persistence in vivo.

Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways

PubMed Central

Franchini, Don-Marc; Incorvaia, Elisabetta; Rangam, Gopinath; Coker, Heather A.; Petersen-Mahrt, Svend K.

2013-01-01

During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes. PMID:24349193

The g.763G>C SNP of the bovine FASN gene affects its promoter activity via Sp-mediated regulation: implications for the bovine lactating mammary gland.

PubMed

Ordovás, Laura; Roy, Rosa; Pampín, Sandra; Zaragoza, Pilar; Osta, Rosario; Rodríguez-Rey, Jose Carlos; Rodellar, Clementina

2008-07-15

Fatty acid synthase (FASN) is an enzyme that catalyzes de novo synthesis of fatty acids in cells. The bovine FASN gene maps to BTA 19, where several quantitative trait loci for fat-related traits have been described. Our group recently reported the identification of a single nucleotide polymorphism (SNP), g.763G>C, in the bovine FASN 5' flanking region that was significantly associated with milk fat content in dairy cattle. The g.763G>C SNP was part of a GC-rich region that may constitute a cis element for members of the Sp transcription factor family. Thus the SNP could alter the transcription factor binding ability of the FASN promoter and consequently affect the promoter activity of the gene. However, the functional consequences of the SNP on FASN gene expression are unknown. The present study was therefore directed at elucidating the underlying molecular mechanism that could explain the association of the SNP with milk fat content. Three cellular lines (3T3L1, HepG2, and MCF-7) were used to test the promoter and the transcription factor binding activities by luciferase reporter assays and electrophoretic mobility shift assays, respectively. Band shift assays were also carried out with nuclear extracts from lactating mammary gland (LMG) to further investigate the role of the SNP in this tissue. Our results demonstrate that the SNP alters the bovine FASN promoter activity in vitro and the Sp1/Sp3 binding ability of the sequence. In bovine LMG, the specific binding of Sp3 may account for the association with milk fat content.

Cardiac muscle activation blunted by a mutation to the regulatory component, troponin T.

PubMed

Kobayashi, Minae; Debold, Edward P; Turner, Matthew A; Kobayashi, Tomoyoshi

2013-09-06

The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca(2+) sensor (TnC) and the actin filament. In the short helix preceding the IT-arm region, H1(T2), there are known dilated cardiomyopathy-linked mutations (among them R205L). Thus we hypothesized that there is an element in this short helix that plays an important role in regulating the muscle contraction, especially in Ca(2+) activation. We mutated Arg-205 and several other amino acid residues within and near the H1(T2) helix. Utilizing an alanine replacement method to compare the effects of the mutations, the biochemical and mechanical impact on the actomyosin interaction was assessed by solution ATPase activity assay, an in vitro motility assay, and Ca(2+) binding measurements. Ca(2+) activation was markedly impaired by a point mutation of the highly conserved basic residue R205A, residing in the short helix H1(T2) of cTnT, whereas the mutations to nearby residues exhibited little effect on function. Interestingly, rigor activation was unchanged between the wild type and R205A TnT. In addition to the reduction in Ca(2+) sensitivity observed in Ca(2+) binding to the thin filament, myosin S1-ADP binding to the thin filament was significantly affected by the same mutation, which was also supported by a series of S1 concentration-dependent ATPase assays. These suggest that the R205A mutation alters function through reduction in the nature of cooperative binding of S1.

Structural basis for LeishIF4E-1 modulation by an interacting protein in the human parasite Leishmania major.

PubMed

Meleppattu, Shimi; Arthanari, Haribabu; Zinoviev, Alexandra; Boeszoermenyi, Andras; Wagner, Gerhard; Shapira, Michal; Léger-Abraham, Mélissa

2018-03-19

Leishmania parasites are unicellular pathogens that are transmitted to humans through the bite of infected sandflies. Most of the regulation of their gene expression occurs post-transcriptionally, and the different patterns of gene expression required throughout the parasites' life cycle are regulated at the level of translation. Here, we report the X-ray crystal structure of the Leishmania cap-binding isoform 1, LeishIF4E-1, bound to a protein fragment of previously unknown function, Leish4E-IP1, that binds tightly to LeishIF4E-1. The molecular structure, coupled to NMR spectroscopy experiments and in vitro cap-binding assays, reveal that Leish4E-IP1 allosterically destabilizes the binding of LeishIF4E-1 to the 5' mRNA cap. We propose mechanisms through which Leish4E-IP1-mediated LeishIF4E-1 inhibition could regulate translation initiation in the human parasite.

Genome-wide identification and characterization of Notch transcription complex-binding sequence paired sites in leukemia cells

PubMed Central

Severson, Eric; Arnett, Kelly L.; Wang, Hongfang; Zang, Chongzhi; Taing, Len; Liu, Hudan; Pear, Warren S.; Liu, X. Shirley; Blacklow, Stephen C.; Aster, Jon C.

2018-01-01

Notch transcription complexes (NTCs) drive target gene expression by binding to two distinct types of genomic response elements, NTC monomer-binding sites and sequence-paired sites (SPSs) that bind NTC dimers. SPSs are conserved and are linked to the Notch-responsiveness of a few genes, but their overall contribution to Notch-dependent gene regulation is unknown. To address this issue, we determined the DNA sequence requirements for NTC dimerization using a fluorescence resonance energy transfer (FRET) assay, and applied insights from these in vitro studies to Notch-“addicted” leukemia cells. We find that SPSs contribute to the regulation of approximately a third of direct Notch target genes. While originally described in promoters, SPSs are present mainly in long-range enhancers, including an enhancer containing a newly described SPS that regulates HES5. Our work provides a general method for identifying sequence-paired sites in genome-wide data sets and highlights the widespread role of NTC dimerization in Notch-transformed leukemia cells. PMID:28465412

Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors.

PubMed

Kast, David J; Yang, Changsong; Disanza, Andrea; Boczkowska, Malgorzata; Madasu, Yadaiah; Scita, Giorgio; Svitkina, Tatyana; Dominguez, Roberto

2014-04-01

The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.

The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces

PubMed Central

Wong, Edmond; Vaaje-Kolstad, Gustav; Ghosh, Avishek; Hurtado-Guerrero, Ramon; Konarev, Peter V.; Ibrahim, Adel F. M.; Svergun, Dmitri I.; Eijsink, Vincent G. H.; Chatterjee, Nabendu S.; van Aalten, Daan M. F.

2012-01-01

Vibrio cholerae is a bacterial pathogen that colonizes the chitinous exoskeleton of zooplankton as well as the human gastrointestinal tract. Colonization of these different niches involves an N-acetylglucosamine binding protein (GbpA) that has been reported to mediate bacterial attachment to both marine chitin and mammalian intestinal mucin through an unknown molecular mechanism. We report structural studies that reveal that GbpA possesses an unusual, elongated, four-domain structure, with domains 1 and 4 showing structural homology to chitin binding domains. A glycan screen revealed that GbpA binds to GlcNAc oligosaccharides. Structure-guided GbpA truncation mutants show that domains 1 and 4 of GbpA interact with chitin in vitro, whereas in vivo complementation studies reveal that domain 1 is also crucial for mucin binding and intestinal colonization. Bacterial binding studies show that domains 2 and 3 bind to the V. cholerae surface. Finally, mouse virulence assays show that only the first three domains of GbpA are required for colonization. These results explain how GbpA provides structural/functional modular interactions between V. cholerae, intestinal epithelium and chitinous exoskeletons. PMID:22253590

Adherence of oral streptococci: evidence for nonspecific adsorption to saliva-coated hydroxylapatite surfaces.

PubMed Central

Staat, R H; Peyton, J C

1984-01-01

It is proposed that binding of oral streptococci to saliva-coated hydroxylapatite (SHA) surfaces is a multifactorial process involving both specific and nonspecific receptors. In this context, specific binding is described as a high-affinity, saturable interaction between the cell and binding surface. Conversely, nonspecific binding is considered to be a nonsaturable, generalized, low-affinity reaction. Experimental differentiation of specific binding from nonspecific binding was achieved with a competition assay which utilized a large excess of nonradiolabeled bacteria to compete with the 3H-labeled cells for attachment to receptors on 1.5 mg of SHA crystals. Competition assays of Streptococcus sanguis and Streptococcus mitis adhesion clearly demonstrated that the total binding isotherm was composed of a saturable specific binding reaction and a minor nonspecific binding component. This was further substantiated by analysis of nonlinear Scatchard plots of the total binding data. The competition data for Streptococcus mutans binding indicated that ca. 50% of the S. mutans binding appeared to be specific, although saturation of the SHA surfaces with bacterial cells could not be demonstrated. Experiments measuring desorption of radiolabeled cells from SHA crystals into buffer showed that ca. 50% of the bound S. mutans cells were removed after 4 h, whereas less than 5% of the S. sanguis cells were eluted from the SHA surfaces. The kinetics of attachment were studied by using an extract of Persea americana as a noncompetitive inhibitor of adherence. The total cell binding data for these experiments suggested a very rapid binding reaction followed by a slower rate of attachment. It was concluded from these three different experimental approaches that adherence of selected oral streptococci to SHA surfaces involves specific, high-affinity and nonspecific, low-affinity binding reactions. The concept is developed that in vitro streptococcal attachment to SHA can be described as a two-reaction process in which the low-affinity interaction of the cell with the SHA surface precedes the establishment of the stronger, specific bonds needed for the maintenance of streptococci in the oral cavity. PMID:6327530

Analysis of in vitro interactions of protein tyrosine phosphatase 1B with insulin receptors.

PubMed

Wang, X Y; Bergdahl, K; Heijbel, A; Liljebris, C; Bleasdale, J E

2001-02-28

One strategy to treat the insulin resistance that is central to type II diabetes mellitus may be to maintain insulin receptors (IR) in the active (tyrosine phosphorylated) form. Because protein tyrosine phosphatase 1B (PTP1B) binds and subsequently dephosphorylates IR, inhibitors of PTP1B-IR binding are potential insulin 'sensitizers.' A Scintillation Proximity Assay (SPA) was developed to characterize and quantitate PTP1B-IR binding. Human IR were solubilized and captured on wheat germ agglutinin (WGA)-coated SPA beads. Subsequent binding of human, catalytically inactive [35S] PTP1B Cys(215)/Ser (PTP1B(C215S)) to the lectin-anchored IR results in scintillation from the SPA beads that can be quantitated. Binding of PTP1B to IR was pH- and divalent cation-sensitive. Ca(2+) and Mn(2+), but not Mg(2+), dramatically attenuated the loss of PTP1B-IR binding observed when pH was raised from 6.2 to 7.8. PTP1B binding to IR from insulin-stimulated cells was much greater than to IR from unstimulated cells and was inhibited by either an antiphosphotyrosine antibody or treatment of IR with alkaline phosphatase, suggesting that tyrosine phosphorylation of IR is required for PTP1B binding. Phosphopeptides modeled after various IR phosphotyrosine domains each only partially inhibited PTP1B-IR binding, indicating that multiple domains of IR are likely involved in binding PTP1B. However, competitive displacement of [35S]PTP1B(C215S) by PTP1B(C215S) fitted best to a single binding site with a K(d) in the range 100-1000 nM, depending upon pH and divalent cations. PNU-200898, a potent and selective inhibitor of PTP1B whose orientation in the active site of PTP1B has been solved, competitively inhibited catalysis and PTP1B-IR binding with equal potency. The results of this novel assay for PTP1B-IR binding suggest that PTP1B binds preferentially to tyrosine phosphorylated IR through its active site and that binding may be susceptible to therapeutic disruption by small molecules.

Retro-inverso forms of gastrin5-12 are as biologically active as glycine-extended gastrin in vitro but not in vivo.

PubMed

Marshall, Kathryn M; Laval, Marie; Sims, Ioulia; Shulkes, Arthur; Baldwin, Graham S

2015-12-01

Non-amidated gastrin peptides such as glycine-extended gastrin (Ggly) are biologically active in vitro and in vivo and have been implicated in the development of gastric and colonic cancers. Previous studies have shown that the truncated form of Ggly, the octapeptide LE5AY, was still biologically active in vitro, and that activity was dependent on ferric ion binding but independent of binding to the cholecystokinin 2 (CCK2) receptor. The present work was aimed at creating more stable gastrin-derived 'super agonists' using retro-inverso technology. The truncated LE5AY peptide was synthesized using end protecting groups in three forms with l-amino acids (GL), d-amino acids (GD) or retro-inverso (reverse order with d-amino acids; GRI). All of these peptides bound ferric ions with a 2:1 (Fe: peptide) ratio. As predicted, Ggly, GL and GRI were biologically active in vitro and increased cell proliferation in mouse gastric epithelial (IMGE-5) and human colorectal cancer (DLD-1) cell lines, and increased cell migration in DLD-1 cells. These activities were likely via the same mechanism as Ggly since no CCK1 or CCK2 binding was identified, and GD remained inactive in all assays. Surprisingly, unlike Ggly, GL and GRI were not active in vivo. While Ggly stimulated colonic crypt height and proliferation rates in gastrin knockout mice, GL and GRI did not. The apparent lack of activity may be due to rapid clearance of these smaller peptides. Nevertheless further work designing and testing retro-inverso gastrins is warranted, as it may lead to the generation of super agonists that could potentially be used to treat patients with gastrointestinal disorders with reduced mucosal function. Copyright © 2015 Elsevier Inc. All rights reserved.

In vitro degradation and antitumor activity of oxime bond-linked daunorubicin-GnRH-III bioconjugates and DNA-binding properties of daunorubicin-amino acid metabolites.

PubMed

Orbán, Erika; Mezo, Gábor; Schlage, Pascal; Csík, Gabriella; Kulić, Zarko; Ansorge, Philipp; Fellinger, Erzsébet; Möller, Heiko Michael; Manea, Marilena

2011-07-01

Bioconjugates with receptor-mediated tumor-targeting functions and carrying cytotoxic agents should enable the specific delivery of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting the peripheral toxicity. In the present study, gonadotropin-releasing hormone III (GnRH-III; Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was employed as a targeting moiety to which daunorubicin was attached via oxime bond, either directly or by insertion of a GFLG or YRRL tetrapeptide spacer. The in vitro antitumor activity of the bioconjugates was determined on MCF-7 human breast and HT-29 human colon cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their degradation/stability (1) in human serum, (2) in the presence of cathepsin B and (3) in rat liver lysosomal homogenate was analyzed by liquid chromatography in combination with mass spectrometry. The results show that (1) all synthesized bioconjugates have in vitro antitumor effect, (2) they are stable in human serum at least for 24 h, except for the compound containing an YRRL spacer and (3) they are hydrolyzed by cathepsin B and in the lysosomal homogenate. To investigate the relationship between the in vitro antitumor activity and the structure of the bioconjugates, the smallest metabolites produced in the lysosomal homogenate were synthesized and their binding to DNA was assessed by fluorescence spectroscopy. Our data indicate that the incorporation of a peptide spacer in the structure of oxime bond-linked daunorubicin-GnRH-III bioconjugates is not required for their antitumor activity. Moreover, the antitumor activity is influenced by the structure of the metabolites (daunorubicin-amino acid derivatives) and their DNA-binding properties.

Eldecalcitol (ED-71), an analog of 1α,25(OH)2D3, inhibits the growth of squamous cell carcinoma (SCC) cells in vitro and in vivo by down-regulating expression of heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) and FGF-2.

PubMed

Shintani, T; Takatsu, F; Rosli, S N Z; Usui, E; Hamada, A; Sumi, K; Hayashido, Y; Toratani, S; Okamoto, Tetsuji

2017-10-01

Heparin-binding protein 17 (HBp17)/fibroblast growth factor-binding protein-1 (FGFBP-1) was first purified from medium conditioned by A431 cells for its capacity to bind to fibroblast growth factors 1 and 2 (FGF-1 and -2). Among FGF family members, FGF-2 is a potent mitogen for various cell types, including vascular endothelial cells, fibroblasts, and cancer cells such as oral squamous cell carcinoma (OSCC) cells. Besides being well known in bone metabolism, the active form of vitamin D 3 , i.e., 1α,25(OH) 2 D 3 (1,25D 3 ), was reported to have protective effects for heart disease and cancer. Previously, we reported that 1,25D 3 inhibited HBp17/FGFBP-1 expression in OSCC cell lines through NF-κB inhibition (IκBα activation) and resulted in the inactivation of FGF-2. In this study, we examined the potential anti-tumor effect of ED-71, an analog of 1α,25(OH) 2 D 3 , for squamous cell carcinoma cells in vitro and in vivo. The cell lines used were OSCC cell lines (NA-HO-1-n-1 and UE-HO-1-u-1), established from oral cancer patients in our laboratory, and an epidermoid carcinoma/SCC cell line (A431). The growth assay in serum-free culture revealed that ED-71 inhibited the growth of the cancer cell lines in a dose-dependent manner. In addition, ED-71 suppressed HBp17/FGFBP-1 expression by inhibiting the NF-κB pathway as did 1,25D 3 . Furthermore, a luciferase reporter assay revealed that the promoter activity of HBp17/FGFBP-1 (region between -217 and +61) was down-regulated by ED-71. Oral administration of ED-71 significantly inhibited the growth of A431-derived tumors in athymic nude mice. Immunohistochemical analysis revealed that the expression of HBp17/FGFBP-1, FGF-2, CD31, and Ki-67 in the tumors of ED71-treated group was down-regulated in comparison to control. These results suggest that ED-71 possesses potential anti-tumor activity for SCCs both in vitro and in vivo. This compound may act directly on the tumor cells or on endothelial cells by modulating the tumor microenvironment.

A recombined fusion protein PTD-Grb2-SH2 inhibits the proliferation of breast cancer cells in vitro.

PubMed

Yin, Jikai; Cai, Zhongliang; Zhang, Li; Zhang, Jian; He, Xianli; Du, Xilin; Wang, Qing; Lu, Jianguo

2013-03-01

The growth factor receptor bound protein 2 (Grb2) is one of the affirmative targets for cancer therapy, especially for breast cancer. In this study, we hypothesized the Src-homology 2 (SH2) domain in Grb2 may serve as a competitive protein-binding agent to interfere with the proliferation of breast cancer cells in vitro. We designed, constructed, expressed and purified a novel fusion protein containing the protein transduction domain (PTD) and Grb2-SH2 domain (we named it after PTD-Grb2-SH2). An immunofluorescence assay was used to investigate the location of PTD-Grb2-SH2 in cells. MTT assay and EdU experiments were applied to detect the proliferation of breast cancer cells. The ultra-structure was observed using transmission electron microscopy. Flow cytometry was used to determine the cytotoxicity of PTD-Grb2-SH2 on cell proliferation. We successfully obtained the PTD-Grb2-SH2 fusion protein in soluble form using a prokaryotic expression system. The new fusion protein successfully passed through both the cellular and nuclear membranes of breast cancer cells. The MTT assay showed that PTD-Grb2-SH2 exhibited significant toxicity to breast cancer cells in a dose- and time-dependent manner in vitro. EdU identified the decreased proliferation rates in treated MDA-MB-231 and SK-BR-3 cells. Observation by transmission electron microscopy and flow cytometry further confirmed the cytotoxicity as apoptosis. Our results show that the HIV1-TAT domain is a useful tool for transporting a low molecular weight protein across the cell membrane in vitro. The PTD-Grb2-SH2 may be a novel agent for breast cancer therapy.

Novel Broad Spectrum Inhibitors Targeting the Flavivirus Methyltransferase

PubMed Central

Liu, Binbin; Banavali, Nilesh K.; Jones, Susan A.; Zhang, Jing; Li, Zhong; Kramer, Laura D.; Li, Hongmin

2015-01-01

The flavivirus methyltransferase (MTase) is an essential enzyme that sequentially methylates the N7 and 2’-O positions of the viral RNA cap, using S-adenosyl-L-methionine (SAM) as a methyl donor. We report here that small molecule compounds, which putatively bind to the SAM-binding site of flavivirus MTase and inhibit its function, were identified by using virtual screening. In vitro methylation experiments demonstrated significant MTase inhibition by 13 of these compounds, with the most potent compound displaying sub-micromolar inhibitory activity. The most active compounds showed broad spectrum activity against the MTase proteins of multiple flaviviruses. Two of these compounds also exhibited low cytotoxicity and effectively inhibited viral replication in cell-based assays, providing further structural insight into flavivirus MTase inhibition. PMID:26098995

Towards novel therapeutics for HIV through fragment-based screening and drug design.

PubMed

Tiefendbrunn, Theresa; Stout, C David

2014-01-01

Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-μM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins.

In vitro DNA binding studies of therapeutic and prophylactic drug citral.

PubMed

Alam, Md Fazle; Varshney, Supriya; Khan, Masood Alam; Laskar, Amaj Ahmed; Younus, Hina

2018-07-01

The study of drug-DNA interactions is of great importance, as it paves the way towards the design of better therapeutic agents. Here, the interaction of DNA with a therapeutic and prophylactic drug citral has been studied. We have attempted to ascertain the mode of binding of citral with calf thymus DNA (Ct-DNA) through various biophysical techniques. Analysis of the UV-visible absorbance spectra and fluorescence spectra indicated the formation of a complex between citral and Ct-DNA. Competitive binding assays with ethidium bromide (EB), acridine orange (AO) and Hoechst 33258 reflected that citral possibly intercalates within the Ct-DNA. These observations were further confirmed by circular dichroism (CD) spectral analysis, viscosity measurements, DNA melting and molecular docking studies. This study is expected to contribute to a better understanding of molecular mechanisms of citral, and design of new drugs in the future. Copyright © 2018 Elsevier B.V. All rights reserved.

Functional specificity of a Hox protein mediated by the recognition of minor groove structure.

PubMed

Joshi, Rohit; Passner, Jonathan M; Rohs, Remo; Jain, Rinku; Sosinsky, Alona; Crickmore, Michael A; Jacob, Vinitha; Aggarwal, Aneel K; Honig, Barry; Mann, Richard S

2007-11-02

The recognition of specific DNA-binding sites by transcription factors is a critical yet poorly understood step in the control of gene expression. Members of the Hox family of transcription factors bind DNA by making nearly identical major groove contacts via the recognition helices of their homeodomains. In vivo specificity, however, often depends on extended and unstructured regions that link Hox homeodomains to a DNA-bound cofactor, Extradenticle (Exd). Using a combination of structure determination, computational analysis, and in vitro and in vivo assays, we show that Hox proteins recognize specific Hox-Exd binding sites via residues located in these extended regions that insert into the minor groove but only when presented with the correct DNA sequence. Our results suggest that these residues, which are conserved in a paralog-specific manner, confer specificity by recognizing a sequence-dependent DNA structure instead of directly reading a specific DNA sequence.

Antimalarial, antimicrobial, cytotoxic, DNA interaction and SOD like activities of tetrahedral copper(II) complexes

NASA Astrophysics Data System (ADS)

Mehta, Jugal V.; Gajera, Sanjay B.; Patel, Mohan N.

2015-02-01

The mononuclear copper(II) complexes with P, O-donor ligand and different fluoroquinolones have been synthesized and characterized by elemental analysis, electronic spectra, TGA, EPR, FT-IR and LC-MS spectroscopy. An antimicrobial efficiency of the complexes has been tested against five different microorganisms in terms of minimum inhibitory concentration (MIC) and displays very good antimicrobial activity. The binding strength and binding mode of the complexes with Herring Sperm DNA (HS DNA) have been investigated by absorption titration and viscosity measurement studies. The studies suggest the classical intercalative mode of DNA binding. Gel electrophoresis assay determines the ability of the complexes to cleave the supercoiled form of pUC19 DNA. Synthesized complexes have been tested for their SOD mimic activity using nonenzymatic NBT/NADH/PMS system and found to have good antioxidant activity. All the complexes show good cytotoxic and in vitro antimalarial activities.

4-Aminoquinoline-pyrimidine hybrids: synthesis, antimalarial activity, heme binding and docking studies.

PubMed

Kumar, Deepak; Khan, Shabana I; Tekwani, Babu L; Ponnan, Prija; Rawat, Diwan S

2015-01-07

A series of novel 4-aminoquinoline-pyrimidine hybrids has been synthesized and evaluated for their antimalarial activity. Several compounds showed promising in vitro antimalarial activity against both CQ-sensitive and CQ-resistant strains with high selectivity index. All the compounds were found to be non-toxic to the mammalian cell lines. Selected compound 7g exhibited significant suppression of parasitemia in the in vivo assay. The heme binding studies were conducted to determine the mode of action of these hybrid molecules. These compounds form a stable 1:1 complex with hematin suggesting that heme may be one of the possible targets of these hybrids. The interaction of these conjugate hybrids was also investigated by the molecular docking studies in the binding site of PfDHFR. The pharmacokinetic property analysis of best active compounds was also studied using ADMET prediction. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Structural insight into TPX2-stimulated microtubule assembly

PubMed Central

2017-01-01

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements (’ridge’ and ‘wedge’) in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation. PMID:29120325

Synthesis, characterization of α-amino acid Schiff base derived Ru/Pt complexes: Induces cytotoxicity in HepG2 cell via protein binding and ROS generation

NASA Astrophysics Data System (ADS)

Alsalme, Ali; Laeeq, Sameen; Dwivedi, Sourabh; Khan, Mohd. Shahnawaz; Al Farhan, Khalid; Musarrat, Javed; Khan, Rais Ahmad

2016-06-01

We have synthesized two new complexes of platinum (1) and ruthenium (2) with α-amino acid, L-alanine, and 2,3-dihydroxybenzaldehyde derived Schiff base (L). The ligand and both complexes were characterized by using elemental analysis and several other spectroscopic techniques viz; IR, 1H, 13C NMR, EPR, and ESI-MS. Furthermore, the protein-binding ability of synthesized complexes was monitored by UV-visible, fluorescence and circular dichroism techniques with a model protein, human serum albumin (HSA). Both the PtL2 and RuL2 complexes displayed significant binding towards HSA. Also, in vitro cytotoxicity assay for both complexes was carried out on human hepatocellular carcinoma cancer (HepG2) cell line. The results showed concentration-dependent inhibition of cell viability. Moreover, the generation of reactive oxygen species was also evaluated, and results exhibited substantial role in cytotoxicity.

Fluorescence anisotropy microplate assay to investigate the interaction of full-length steroid receptor coactivator-1a with steroid receptors

PubMed Central

Zhang, Chen; Nordeen, Steven K.; Shapiro, David J.

2013-01-01

Estrogens, acting via estrogen receptor (ER) play key roles in growth, differentiation and gene regulation in the reproductive, central nervous and skeletal systems. ER-mediated gene transcription contributes to the development and spread of breast, uterine, and liver cancer. Steroid receptor coactivator-1a (SRC1a) belongs to the P160 family of coactivators, which is the best known of the many coactivators implicated in ER-mediated transactivation. Binding of full-length P160 coactivators to steroid receptors has been difficult to investigate in vitro. This chapter details how to investigate the interaction of SRC1a with ER using the fluorescence anisotropy/polarization microplate assay (FAMA). PMID:23436375

Assays to Study the Interaction of Campylobacter jejuni with the Mucosal Surface.

PubMed

Clyne, Marguerite; Duggan, Gina; Dunne, Ciara; Dolan, Brendan; Alvarez, Luis; Bourke, Billy

2017-01-01

Mucosal colonization and overcoming the mucosal barrier are essential steps in the establishment of infection by Campylobacter jejuni. The interaction between C. jejuni and host cells, including binding and invasion, is thought to be the key virulence factor important for pathogenesis of C. jejuni infections in animals or humans. The intestinal mucosal barrier is composed of a polarized epithelium covered by a thick adherent mucus gel layer. There is a requirement for cell culture assays of infection to accurately represent the in vivo mucosal surface. In this chapter, we describe the use of a number of cell culture models and the use of polarized in vitro organ culture to examine the interaction of C. jejuni with mucosal surfaces.

Sulfonylureas and Glinides as New PPARγ Agonists:. Virtual Screening and Biological Assays

NASA Astrophysics Data System (ADS)

Scarsi, Marco; Podvinec, Michael; Roth, Adrian; Hug, Hubert; Kersten, Sander; Albrecht, Hugo; Schwede, Torsten; Meyer, Urs A.; Rücker, Christoph

2007-12-01

This work combines the predictive power of computational drug discovery with experimental validation by means of biological assays. In this way, a new mode of action for type 2 diabetes drugs has been unvealed. Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target PPARγ improving insulin resistance (thiazolidinediones). Our work shows that sulfonylureas and glinides bind to PPARγ and exhibit PPARγ agonistic activity. This result was predicted in silico by virtual screening and confirmed in vitro by three biological assays. This dual mode of action of sulfonylureas and glinides may open new perspectives for the molecular pharmacology of antidiabetic drugs, since it provides evidence that drugs can be designed which target both the sulfonylurea receptor and PPARγ. Targeting both receptors could in principle allow to increase pancreatic insulin secretion, as well as to improve insulin resistance.

Synthesis and characterization of PEG-conjugated quaternized chitosan and its application as a gene vector.

PubMed

Zhang, Xi; Yao, Juan; Zhang, Lihong; Fang, Jianguo; Bian, Fengling

2014-03-15

Poly(ethylene glycol)-conjugated N-(2-hydroxy) propyl-3-trimethyl ammonium chitosan chloride (PHTAC) derivatives were prepared by incorporating PEG molecules onto quaternized chitosan backbone. The copolymers were characterized by FTIR, (1)H NMR and XRD. Agarose gel retardation assay indicated that PHTAC had good plasmid DNA (pDNA) binding capability and the particle sizes of PHTAC/pDNA complexes determined by DLS were about 200 nm. Cytotoxicity assays in HeLa and 293T cells showed that PHTAC had low cytotoxicity. In vitro luciferase assay showed that PHTAC with PEGylation degree of 9% (PHTAC-1) had good transfection efficiency about 5.3-fold higher than quaternized chitosan, which was comparable with PEI (25 kDa). These results suggest that PHTAC-1 is a promising candidate as an efficient nonviral gene vector. Copyright © 2013 Elsevier Ltd. All rights reserved.

Indole cytosolic phospholipase A2 alpha inhibitors: discovery and in vitro and in vivo characterization of 4-{3-[5-chloro-2-(2-{[(3,4-dichlorobenzyl)sulfonyl]amino}ethyl)-1-(diphenylmethyl)-1H-indol-3-yl]propyl}benzoic acid, efipladib.

PubMed

McKew, John C; Lee, Katherine L; Shen, Marina W H; Thakker, Paresh; Foley, Megan A; Behnke, Mark L; Hu, Baihua; Sum, Fuk-Wah; Tam, Steve; Hu, Yonghan; Chen, Lihren; Kirincich, Steven J; Michalak, Ronald; Thomason, Jennifer; Ipek, Manus; Wu, Kun; Wooder, Lane; Ramarao, Manjunath K; Murphy, Elizabeth A; Goodwin, Debra G; Albert, Leo; Xu, Xin; Donahue, Frances; Ku, M Sherry; Keith, James; Nickerson-Nutter, Cheryl L; Abraham, William M; Williams, Cara; Hegen, Martin; Clark, James D

2008-06-26

The optimization of a class of indole cPLA 2 alpha inhibitors is described herein. The importance of the substituent at C3 and the substitution pattern of the phenylmethane sulfonamide region are highlighted. Optimization of these regions led to the discovery of 111 (efipladib) and 121 (WAY-196025), which are shown to be potent, selective inhibitors of cPLA 2 alpha in a variety of isolated enzyme assays, cell based assays, and rat and human whole blood assays. The binding of these compounds has been further examined using isothermal titration calorimetry. Finally, these compounds have shown efficacy when dosed orally in multiple acute and chronic prostaglandin and leukotriene dependent in vivo models.

A binuclear complex constituted by diethyldithiocarbamate and copper(I) functions as a proteasome activity inhibitor in pancreatic cancer cultures and xenografts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Jinbin, E-mail: hanjinbin@gmail.com; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032; Shanghai Clinical Center, Chinese Academy of Sciences/Xuhui Central Hospital, Shanghai 200031

2013-12-15

It is a therapeutic strategy for cancers including pancreatic to inhibit proteasome activity. Disulfiram (DSF) may bind copper (Cu) to form a DSF–Cu complex. DSF–Cu is capable of inducing apoptosis in cancer cells by inhibiting proteasome activity. DSF is rapidly converted to diethyldithiocarbamate (DDTC) within bodies. Copper(II) absorbed by bodies is reduced to copper(I) when it enters cells. We found that DDTC and copper(I) could form a binuclear complex which might be entitled DDTC–Cu(I), and it had been synthesized by us in the laboratory. This study is to investigate the anticancer potential of this complex on pancreatic cancer and themore » possible mechanism. Pancreatic cancer cell lines, SW1990, PANC-1 and BXPC-3 were used for in vitro assays. Female athymic nude mice grown SW1990 xenografts were used as animal models. Cell counting kit-8 (cck-8) assay and flow cytometry were used for analyzing apoptosis in cells. A 20S proteasome assay kit was used in proteasome activity analysis. Western blot (WB) and immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used in tumor sample analysis. The results suggest that DDTC–Cu(I) inhibit pancreatic cancer cell proliferation and proteasome activity in vitro and in vivo. Accumulation of ubiquitinated proteins, and increased p27 as well as decreased NF-κB expression were detected in tumor tissues of DDTC–Cu(I)-treated group. Our data indicates that DDTC–Cu(I) is an effective proteasome activity inhibitor with the potential to be explored as a drug for pancreatic cancer. - Highlights: • A new structure of DDTC–Cu(I) was reported for the first time. • DDTC–Cu(I) dissolved directly in water was for in vitro and in vivo uses. • DDTC–Cu(I) demonstrated significant anticancer effect in vitro and in vivo. • DDTC–Cu(I) is capable of inhibiting proteasome activity in vitro and in vivo.« less

Inhibition of p53 Mutant Peptide Aggregation In Vitro by Cationic Osmolyte Acetylcholine Chloride.

PubMed

Chen, Zhaolin; Kanapathipillai, Mathumai

2017-01-01

Mutations of tumor suppressor protein p53 are present in almost about 50% of all cancers. It has been reported that the p53 mutations cause aggregation and subsequent loss of p53 function, leading to cancer progression. Here in this study we focus on the inhibitory effects of cationic osmolyte molecules acetylcholine chloride, and choline on an aggregation prone 10 amino acid p53 mutant peptide WRPILTIITL, and the corresponding wildtype peptide RRPILTIITL in vitro. The characterization tools used for this study include Thioflavin- T (ThT) induced fluorescence, transmission electron microscopy (TEM), congo red binding, turbidity, dynamic light scattering (DLS), and cell viability assays. The results show that acetylcholine chloride in micromolar concentrations significantly inhibit p53 mutant peptide aggregation in vitro, and could be promising candidate for p53 mutant/ misfolded protein aggregation inhibition. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Glutathione S-Transferases Interact with AMP-Activated Protein Kinase: Evidence for S-Glutathionylation and Activation In Vitro

PubMed Central

Polge, Cécile; Ramirez, Sacnicte; Michelland, Sylvie; Sève, Michel; Vertommen, Didier; Rider, Mark; Lentze, Nicolas; Auerbach, Daniel; Schlattner, Uwe

2013-01-01

AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions. PMID:23741294

Antioxidant and Antiplasmodial Activities of Bergenin and 11-O-Galloylbergenin Isolated from Mallotus philippensis

PubMed Central

Khan, Hamayun; Amin, Hazrat; Ullah, Asad; Saba, Sumbal; Rafique, Jamal; Khan, Khalid; Ahmad, Nasir; Badshah, Syed Lal

2016-01-01

Two important biologically active compounds were isolated from Mallotus philippensis. The isolated compounds were characterized using spectroanalytical techniques and found to be bergenin (1) and 11-O-galloylbergenin (2). The in vitro antioxidant and antiplasmodial activities of the isolated compounds were determined. For the antioxidant potential, three standard analytical protocols, namely, DPPH radical scavenging activity (RSA), reducing power assay (RPA), and total antioxidant capacity (TAC) assay, were adopted. The results showed that compound 2 was found to be more potent antioxidant as compared to 1. Fascinatingly, compound 2 displayed better EC50 results as compared to α-tocopherol while being comparable with ascorbic acid. The antiplasmodial assay data showed that both the compound exhibited good activity against chloroquine sensitive strain of Plasmodium falciparum (D10) and IC50 values were found to be less than 8 μM. The in silico molecular docking analyses were also performed for the determination of binding affinity of the isolated compounds using P. falciparum proteins PfLDH and Pfg27. The results showed that compound 2 has high docking score and binding affinity to both protein receptors as compared to compound 1. The demonstrated biological potentials declared that compound 2 could be the better natural antioxidant and antiplasmodial candidate. PMID:26998192

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease

PubMed Central

Liu, Binbin; Zhang, Jing; Koetzner, Cheri A.; Jones, Susan A.; Lin, Qishan

2017-01-01

The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618. PMID:28542603

Effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro.

PubMed

Lee, J S; Kim, J M; Hong, E K; Kim, S-O; Yoo, Y-J; Cha, J-H

2009-02-01

A growing amount of attention has been placed on periodontal regeneration and wound healing for periodontal therapy. This study was conducted in an effort to determine the effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro. Human periodontal ligament cells were acquired from explant tissue of human healthy periodontal ligament. After the wounding of periodontal ligament cells, the change in expression of heparin-binding epidermal growth factor-like growth factor and epidermal growth factor receptors 1-4 mRNA was assessed. The effects of heparin-binding epidermal growth factor-like growth factor on periodontal ligament cell proliferation and repopulation were assessed in vitro via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and by photographing the injuries, respectively. Extracellular signal-regulated kinase (Erk)1/2, p38 and Akt phosphorylation was characterized via western blotting. Scratch wounding resulted in a significant up-regulation of heparin-binding epidermal growth factor-like growth factor mRNA expression, whereas wounding had no effect on the expression levels of epidermal growth factor receptors 1-4. Interestingly, no expression of epidermal growth factor receptors 2 and 4 was detectable prior to or after wounding. Heparin-binding epidermal growth factor-like growth factor treatment promoted the proliferation and repopulation of periodontal ligament cells. The scratch wounding also stimulated the phosphorylation of Erk1/2 and p38, but not of Akt, in periodontal ligament cells, and heparin-binding epidermal growth factor-like growth factor treatment applied after wounding amplified and extended the activations of Erk1/2 and p38, but not of Akt. Furthermore, Erk1/2 inhibition blocked the process of cell repopulation induced by heparin-binding epidermal growth factor-like growth factor, whereas the inhibition of p38 delayed the process. These results indicate that heparin-binding epidermal growth factor-like growth factor may constitute a critical factor in the wound healing of human periodontal ligament cells by a mechanism that requires the activation of Erk1/2 via specific interaction with epidermal growth factor receptor 1.

In Vitro Metabolism and Stability of the Actinide Chelating Agent 3,4,3-LI(1,2-HOPO)

PubMed Central

Choi, Taylor A.; Furimsky, Anna M.; Swezey, Robert; Bunin, Deborah I.; Byrge, Patricia; Iyer, Lalitha V.; Chang, Polly Y.; Abergel, Rebecca J.

2015-01-01

The hydroxypyridinonate ligand 3,4,3-LI(1,2-HOPO) is currently under development for radionuclide chelation therapy. The preclinical characterization of this highly promising ligand comprised the evaluation of its in vitro properties, including microsomal, plasma, and gastrointestinal fluid stability, cytochrome P450 inhibition, plasma protein binding, and intestinal absorption using the Caco-2 cell line. When mixed with active human liver microsomes, no loss of parent compound was observed after 60 minutes, indicating compound stability in the presence of liver microsomal P450. At the tested concentrations, 3,4,3-LI(1,2-HOPO) did not significantly influence the activities of any of the cytochromal isoforms screened. Thus, 3,4,3-LI(1,2-HOPO) is unlikely to cause drug-drug interactions by inhibiting the metabolic clearance of co-administered drugs metabolized by these enzymes. Plasma protein binding assays revealed that the compound is protein-bound in dogs and less extensively in rats and humans. In the plasma stability study, the compound was stable after 1 h at 37°C in mouse, rat, dog, and human plasma samples. Finally, a bi-directional permeability assay demonstrated that 3,4,3-LI(1,2-HOPO) is not permeable across the Caco-2 monolayer, highlighting the need to further evaluate the effects of various compounds with known permeability enhancement properties on the permeability of the ligand in future studies. PMID:25727482

In vitro metabolism and stability of the actinide chelating agent 3,4,3-LI(1,2-HOPO).

PubMed

Choi, Taylor A; Furimsky, Anna M; Swezey, Robert; Bunin, Deborah I; Byrge, Patricia; Iyer, Lalitha V; Chang, Polly Y; Abergel, Rebecca J

2015-05-01

The hydroxypyridinonate ligand 3,4,3-LI(1,2-HOPO) is currently under development for radionuclide chelation therapy. The preclinical characterization of this highly promising ligand comprised the evaluation of its in vitro properties, including microsomal, plasma, and gastrointestinal fluid stability, cytochrome P450 inhibition, plasma protein binding, and intestinal absorption using the Caco-2 cell line. When mixed with active human liver microsomes, no loss of parent compound was observed after 60 min, indicating compound stability in the presence of liver microsomal P450. At the tested concentrations, 3,4,3-LI(1,2-HOPO) did not significantly influence the activities of any of the cytochromal isoforms screened. Thus, 3,4,3-LI(1,2-HOPO) is unlikely to cause drug-drug interactions by inhibiting the metabolic clearance of coadministered drugs metabolized by these enzymes. Plasma protein-binding assays revealed that the compound is protein-bound in dogs and less extensively in rats and humans. In the plasma stability study, the compound was stable after 1 h at 37°C in mouse, rat, dog, and human plasma samples. Finally, a bidirectional permeability assay demonstrated that 3,4,3-LI(1,2-HOPO) is not permeable across the Caco-2 monolayer, highlighting the need to further evaluate the effects of various compounds with known permeability enhancement properties on the permeability of the ligand in future studies. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Effects of oversulfated and fucosylated chondroitin sulfates on coagulation. Challenges for the study of anticoagulant polysaccharides.

PubMed

Fonseca, Roberto J C; Oliveira, Stephan-Nicollas M C G; Pomin, Vitor H; Mecawi, André S; Araujo, Iracema G; Mourão, Paulo A S

2010-05-01

We report the effects of a chemically oversulfated chondroitin sulfate and a naturally fucosylated chondroitin sulfate on the coagulation system. The former has been recently identified as a contaminant of heparin preparations and the latter has been proposed as an alternative anticoagulant. The mechanism of action of these polymers on coagulation is complex and target different components of the coagulation system. They have serpin-independent anticoagulant activity, which preponderates in plasma. They also have serpin-dependent anticoagulant activity but differ significantly in the target coagulation protease and preferential serpin. Their anticoagulant effects differ even more markedly when tested as inhibitors of coagulation proteases using plasma as a source of serpins. It is possible that the difference is due to the high availability of fucosylated chondroitin sulfate whereas oversulfated chondroitin sulfate has strong unspecific binding to plasma protein and low availability for the binding to serpins. When tested using a venous thrombosis experimental model, oversulfated chondroitin sulfate is less potent as an antithrombotic agent than fucosylated chondroitin sulfate. These highly sulfated chondroitin sulfates activate factor XII in in vitro assays, based on kallikrein release. However, only fucosylated chondroitin sulfate induces hypotension when intravenously injected into rats. In conclusion, the complexity of the regulatory mechanisms involved in the action of highly sulfated polysaccharides in coagulation requires their analysis by a combination of in vitro and in vivo assays. Our results are relevant due to the urgent need for new anticoagulant drugs or alternative sources of heparin.

DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells

PubMed Central

Jain, Aklank; Bacolla, Albino; del Mundo, Imee M.; Zhao, Junhua; Wang, Guliang; Vasquez, Karen M.

2013-01-01

Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA. PMID:24049074

DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells.

PubMed

Jain, Aklank; Bacolla, Albino; Del Mundo, Imee M; Zhao, Junhua; Wang, Guliang; Vasquez, Karen M

2013-12-01

Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA.

In Vitro Metabolism and Stability of the Actinide Chelating Agent 3,4,3-LI(1,2-HOPO)

DOE PAGES

Choi, Taylor A.; Furimsky, Anna M.; Swezey, Robert; ...

2015-02-27

We report that the hydroxypyridinonate ligand 3,4,3-LI(1,2-HOPO) is currently under development for radionuclide chelation therapy. The preclinical characterization of this highly promising ligand comprised the evaluation of its in vitro properties, including microsomal, plasma, and gastrointestinal fluid stability, cytochrome P450 inhibition, plasma protein binding, and intestinal absorption using the Caco-2 cell line. When mixed with active human liver microsomes, no loss of parent compound was observed after 60 minutes, indicating compound stability in the presence of liver microsomal P450. At the tested concentrations, 3,4,3-LI(1,2-HOPO) did not significantly influence the activities of any of the cytochromal isoforms screened. Thus, 3,4,3-LI(1,2-HOPO) ismore » unlikely to cause drug-drug interactions by inhibiting the metabolic clearance of co-administered drugs metabolized by these enzymes. Plasma protein binding assays revealed that the compound is protein-bound in dogs and less extensively in rats and humans. In the plasma stability study, the compound was stable after 1 h at 37°C in mouse, rat, dog, and human plasma samples. Finally, a bi-directional permeability assay demonstrated that 3,4,3-LI(1,2-HOPO) is not permeable across the Caco-2 monolayer, highlighting the need to further evaluate the effects of various compounds with known permeability enhancement properties on the permeability of the ligand in future studies.« less

In Vitro Metabolism and Stability of the Actinide Chelating Agent 3,4,3-LI(1,2-HOPO)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Taylor A.; Furimsky, Anna M.; Swezey, Robert

We report that the hydroxypyridinonate ligand 3,4,3-LI(1,2-HOPO) is currently under development for radionuclide chelation therapy. The preclinical characterization of this highly promising ligand comprised the evaluation of its in vitro properties, including microsomal, plasma, and gastrointestinal fluid stability, cytochrome P450 inhibition, plasma protein binding, and intestinal absorption using the Caco-2 cell line. When mixed with active human liver microsomes, no loss of parent compound was observed after 60 minutes, indicating compound stability in the presence of liver microsomal P450. At the tested concentrations, 3,4,3-LI(1,2-HOPO) did not significantly influence the activities of any of the cytochromal isoforms screened. Thus, 3,4,3-LI(1,2-HOPO) ismore » unlikely to cause drug-drug interactions by inhibiting the metabolic clearance of co-administered drugs metabolized by these enzymes. Plasma protein binding assays revealed that the compound is protein-bound in dogs and less extensively in rats and humans. In the plasma stability study, the compound was stable after 1 h at 37°C in mouse, rat, dog, and human plasma samples. Finally, a bi-directional permeability assay demonstrated that 3,4,3-LI(1,2-HOPO) is not permeable across the Caco-2 monolayer, highlighting the need to further evaluate the effects of various compounds with known permeability enhancement properties on the permeability of the ligand in future studies.« less

Control of cell division and the spatial localization of assembled gene products in Caulobacter crescentus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nathan, P.D.

Experiments are described that examine the role of penicillin-binding proteins (PBPs) in the regulation of cell division in Caulobacter crescentus; and the spatial localization of methyl-accepting chemotaxis proteins (MCPs) in C. crescentus swarmer and predivisional cells. In the analysis of PBP function, in vivo and in vitro assays are used to directly label C. crescentus PBPs with (/sup 3/H) penicillin G in wild type strain CB15, in a series of conditional cell division mutants and in new temperature sensitive cephalosporin C resistant mutants PC8002 and PC8003. 14 PBPs are characterized and a high molecular weight PBP (PBP 1B) that ismore » required for cell division is identified. PBP 1B competes for ..beta..-lactams that induce filament formation and may be a high affinity binding protein. A second high molecular weight PBP (PBP 1C) is also associated with defective cell division. The examination of PBP patterns in synchronous swarmer cells reveals that the in vivo activity of PBP 1B and PBP 1C increases at the time that the cell division pathway is initiated. None of the PBPs, however, appear to be differentially localized in the C. crescentus cell. In the analysis of MCP localization, in vivo and in vitro assays are used to directly label C. crescentus MCPs with methyl-/sup 3/H. MCPs are examined in flagellated and non-flagellated vesicles prepared from cells by immunoaffinity chromatography.« less

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

The role of enzyme and substrate concentration in the evaluation of serum angiotensin converting enzyme (ACE) inhibition by enalaprilat in vitro.

PubMed

Weisser, K; Schloos, J

1991-10-09

The relationship between serum angiotensin converting enzyme (ACE) activity and concentration of the ACE inhibitor enalaprilat was determined in vitro in the presence of different concentrations (S = 4-200 mM) of the substrate Hip-Gly-Gly. From Henderson plots, a competitive tight-binding relationship between enalaprilat and serum ACE was found yielding a value of approximately 5 nM for serum ACE concentration (Et) and an inhibition constant (Ki) for enalaprilat of approximately 0.1 nM. A plot of reaction velocity (Vi) versus total inhibitor concentration (It) exhibited a non-parallel shift of the inhibition curve to the right with increasing S. This was reflected by apparent Hill coefficients greater than 1 when the commonly used inhibitory sigmoid concentration-effect model (Emax model) was applied to the data. Slopes greater than 1 were obviously due to discrepancies between the free inhibitor concentration (If) present in the assay and It plotted on the abscissa and could, therefore, be indicators of tight-binding conditions. Thus, the sigmoid Emax model leads to an overestimation of Ki. Therefore, a modification of the inhibitory sigmoid Emax model (called "Emax tight model") was applied, which accounts for the depletion of If by binding, refers to It and allows estimation of the parameters Et and IC50f (free concentration of inhibitor when 50% inhibition occurs) using non-linear regression analysis. This model could describe the non-symmetrical shape of the inhibition curves and the results for Ki and Et correlated very well with those derived from the Henderson plots. The latter findings confirm that the degree of ACE inhibition measured in vitro is, in fact, dependent on the concentration of substrate and enzyme present in the assay. This is of importance not only for the correct evaluation of Ki but also for the interpretation of the time course of serum ACE inhibition measured ex vivo. The non-linear model has some advantages over the linear Henderson equation: it is directly applicable without conversion of the data and avoids the stochastic dependency of the variables, allowing non-linear regression of all data points contributing with the same weight.

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

PubMed Central

Chiaraviglio, Lucius; Kang, Yoon-Suk; Kirby, James E.

2016-01-01

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila. PMID:27911388

Role of the Pepino mosaic virus 3'-untranslated region elements in negative-strand RNA synthesis in vitro.

PubMed

Osman, Toba A M; Olsthoorn, René C L; Livieratos, Ioannis C

2014-09-22

Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410 nt long single-stranded (ss) RNA genome flanked by a 5'-methylguanosine cap and a 3' poly-A tail. Computer-assisted folding of the 64 nt long PepMV 3'-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3'-5' direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay. Hp1, which is highly variable among different PepMV isolates, appeared dispensable for negative-strand synthesis. Hp2, which is characterized by a large U-rich loop, tolerated base-pair changes in its stem as long as they maintained the stem integrity but was very sensitive to changes in the U-rich loop. Hp3, which harbours the conserved potexvirus ACUUAA hexamer motif, was essential for template activity. Template-RNA polymerase binding competition experiments showed that the ACUUAA sequence represents a high-affinity RdRp binding element. Copyright © 2014 Elsevier B.V. All rights reserved.

Coactivator-associated arginine methyltransferase 1 enhances transcriptional activity of the human T-cell lymphotropic virus type 1 long terminal repeat through direct interaction with Tax.

PubMed

Jeong, Soo-Jin; Lu, Hanxin; Cho, Won-Kyung; Park, Hyeon Ung; Pise-Masison, Cynthia; Brady, John N

2006-10-01

In this study, we demonstrate that the coactivator-associated arginine methyltransferase 1 (CARM1), which methylates histone H3 and other proteins such as p300/CBP, is positively involved in the regulation of Tax transactivation. First, transfection studies demonstrated that overexpression of CARM1 wild-type protein resulted in increased Tax transactivation of the human T-cell lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR). In contrast, transfection of a catalytically inactive CARM1 methyltransferase mutant did not enhance Tax transactivation. CARM1 facilitated Tax transactivation of the CREB-dependent cellular GEM promoter. A direct physical interaction between HTLV-1 Tax and CARM1 was demonstrated using in vitro glutathione S-transferase-Tax binding assays, in vivo coimmunoprecipitation, and confocal microscopy experiments. Finally, chromatin immunoprecipitation analysis of the activated HTLV-1 LTR promoter showed the association of CARM1 and methylated histone H3 with the template DNA. In vitro, Tax facilitates the binding of CARM1 to the transcription complex. Together, our data provide evidence that CARM1 enhances Tax transactivation of the HTLV-1 LTR through a direct interaction between CARM1 and Tax and this binding promotes methylation of histone H3 (R2, R17, and R26).

Physical and Functional Interaction between the Eukaryotic Orthologs of Prokaryotic Translation Initiation Factors IF1 and IF2

PubMed Central

Choi, Sang Ki; Olsen, DeAnne S.; Roll-Mecak, Antonina; Martung, Agnes; Remo, Keith L.; Burley, Stephen K.; Hinnebusch, Alan G.; Dever, Thomas E.

2000-01-01

To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site. PMID:10982835

The cathelicidin-BF Lys16 mutant Cbf-K16 selectively inhibits non-small cell lung cancer proliferation in vitro.

PubMed

Tian, Yuwei; Wang, Hui; Li, Bing; Ke, Mengyun; Wang, Jing; Dou, Jie; Zhou, Changlin

2013-11-01

The 30-amino acid antimicrobial peptide Cbf-K16 is a cathelicidin-BF (BF-30) Lys16 mutant derived from the snake venom of Bungarus fasciatus. Our previous study found that BF-30 selectively inhibited the proliferation of the metastatic melanoma cell line B16F10 in vitro and in vivo, but had a negligible effect on human lung cells. In the present study, it was demonstrated for the first time that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells in vitro, with low toxicity to normal cells. The half-maximal inhibitory concentrations (IC50) of Cbf-K16 against H460 human non-small cell lung carcinoma cells and mouse Lewis lung cancer cells were only 16.5 and 10.5 µM, respectively, which were much less compared to that of BF-30 (45 and 40.3 µM). Data using a transmission electron microscope (TEM) assay showed that, at 20 and 40 µM, Cbf-K16 induced the rupture of the cytoplasmic membrane, which was consistent with data obtained from lactate dehydrogenase (LDH) release assays. The LDH release increased from 17.8 to 52.9% as the duration and dosage of Cbf-K16 increased. Annexin V-fluorescein and propidium iodide staining assays indicated that there were no obvious apoptotic effects at the different dosages and times tested. In H460 cells, the rate of genomic DNA binding increased from 51.9 to 86.8% as the concentration of Cbf-K16 increased from 5 to 10 µM. These data indicate that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells via cytoplasmic membrane permeabilization and DNA binding, rather than apoptosis. Although Cbf-K16 displayed significant cytotoxic activity (40 µM) against tumor cells, in splenocytes no significant inhibitory effect was observed and hemolysis was only 5.6%. These results suggest that Cbf-K16 is a low-toxicity anti-lung cancer drug candidate.

In silico-designed novel non-peptidic ABAD LD hot spot mimetics reverse Aβ-induced mitochondrial impairments in vitro.

PubMed

Viswanath, Ambily Nath Indu; Kim, TaeHun; Jung, Seo Yun; Lim, Sang Min; Pae, Ae Nim

2017-12-01

Present work aimed to introduce non-peptidic ABAD loop D (L D ) hot spot mimetics as ABAD-Aβ inhibitors. A full-length atomistic model of ABAD-Aβ complex was built as a scaffold to launch the lead design and its topology later verified by cross-checking the computational mutagenesis results with that of in vitro data. Thereafter, the interactions of prime Aβ-binding L D residues-Tyr101, Thr108, and Thr110-were translated into specific pharmacophore features and this hypothesis subsequently used as a virtual screen query. ELISA-based screening of 20 hits identified two promising lead candidates, VC15 and VC19 with an IC 50 of 4.4 ± 0.3 and 9.6 ± 0.1 μm, respectively. They productively reversed Aβ-induced mitochondrial dysfunctions such as mitochondrial membrane potential loss (JC-1 assay), toxicity (MTT assay), and ATP reduction (ATP assay) in addition to increased cell viabilities. This is the first reporting of L D hot spot-centric in silico scheme to discover novel compounds with promising ABAD-Aβ inhibitory potential. These chemotypes are proposed for further structural optimization to derive novel Alzheimer's disease (AD) therapeutics. © 2017 John Wiley & Sons A/S.

Schiff base-Poloxamer P85 combination demonstrates chemotherapeutic effect on prostate cancer cells in vitro.

PubMed

Demirci, Selami; Doğan, Ayşegül; Türkmen, Neşe Başak; Telci, Dilek; Rizvanov, Albert A; Şahin, Fikrettin

2017-02-01

Prostate cancer is a multistep and complicated cancer type that is regulated by androgens at the cellular level and remains the second commonest cause of death among men. Discovery and development of novel chemotherapeutic agents enabling rapid tumor cell death with minimal toxic effects to healthy tissues might greatly improve the safety of chemotherapy. The present study evaluates the anti-cancer activity of a novel heterodinuclear copper(II)Mn(II) complex (Schiff base) in combination with poly(ethylene oxide) and poly(propylene oxide) block copolymer (Pluronic) P85. We used assays for cell proliferation, apoptosis, cell migration and invasion, DNA binding and cleavage to elucidate the molecular mechanisms of action, in addition to the anti-inflammatory potency of the new combination. The combined treatment of Schiff base and P85 lead to a remarkable anti-cancer effect on prostate cancer cell lines. Cell proliferation was inhibited in Schiff base-P85 treatment. The activity of this formulation is on DNA binding and cleavage and prevents inflammation in in vitro conditions. This is the first study presenting the anti-cancer activity of the present Schiff base derivative and its combination with P85 to treat prostate cancer in vitro. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Characterization of protein--DNA interactions using surface plasmon resonance spectroscopy with various assay schemes.

PubMed

Teh, Huey Fang; Peh, Wendy Y X; Su, Xiaodi; Thomsen, Jane S

2007-02-27

Specific protein-DNA interactions play a central role in transcription and other biological processes. A comprehensive characterization of protein-DNA interactions should include information about binding affinity, kinetics, sequence specificity, and binding stoichiometry. In this study, we have used surface plasmon resonance spectroscopy (SPR) to study the interactions between human estrogen receptors (ER, alpha and beta subtypes) and estrogen response elements (ERE), with four assay schemes. First, we determined the sequence-dependent receptors' binding capacity by monitoring the binding of ER to various ERE sequences immobilized on a sensor surface (assay format denoted as the direct assay). Second, we screened the relative affinity of ER for various ERE sequences using a competition assay, in which the receptors bind to an ERE-immobilized surface in the presence of competitor ERE sequences. Third, we monitored the assembly of ER-ERE complexes on a SPR surface and thereafter the removal and/or dissociation of the ER (assay scheme denoted as the dissociation assay) to determine the binding stoichiometry. Last, a sandwich assay (ER binding to ERE followed by anti-ER recognition of a specific ER subtype) was performed in an effort to understand how ERalpha and ERbeta may associate and compete when binding to the DNA. With these assay schemes, we reaffirmed that (1) ERalpha is more sensitive than ERbeta to base pair change(s) in the consensus ERE, (2) ERalpha and ERbeta form a heterodimer when they bind to the consensus ERE, and (3) the binding stoichiometry of both ERalpha- and ERbeta-ERE complexes is dependent on salt concentration. With this study, we demonstrate the versatility of the SPR analysis. With the involvement of various assay arrangements, the SPR analysis can be further extended to more than kinetics and affinity study.

Fluorescence lifetime FRET imaging of receptor-ligand complexes in tumor cells in vitro and in vivo

NASA Astrophysics Data System (ADS)

Rudkouskaya, Alena; Sinsuebphon, Nattawut; Intes, Xavier; Mazurkiewicz, Joseph E.; Barroso, Margarida

2017-02-01

To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.

Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening.

PubMed

Wagle, Neil; Xian, Jun; Shishova, Ekaterina Y; Wei, Jie; Glicksman, Marcie A; Cuny, Gregory D; Stein, Ross L; Cohen, David E

2008-12-01

Phosphatidylcholine transfer protein (PC-TP, also referred to as StarD2) is a highly specific intracellular lipid-binding protein that catalyzes the transfer of phosphatidylcholines between membranes in vitro. Recent studies have suggested that PC-TP in vivo functions to regulate fatty acid and glucose metabolism, possibly via interactions with selected other proteins. To begin to address the relationship between activity in vitro and biological function, we undertook a high-throughput screen to identify small-molecule inhibitors of the phosphatidylcholine transfer activity of PC-TP. After adapting a fluorescence quench assay to measure phosphatidylcholine transfer activity, we screened 114,752 compounds of a small-molecule library. The high-throughput screen identified 14 potential PC-TP inhibitors. Of these, 6 compounds exhibited characteristics consistent with specific inhibition of PC-TP activity, with IC(50) values that ranged from 4.1 to 95.0muM under conditions of the in vitro assay. These compounds should serve as valuable reagents to elucidate the biological function of PC-TP. Because mice with homozygous disruption of the PC-TP gene (Pctp) are sensitized to insulin action and relatively resistant to the development of atherosclerosis, these inhibitors may also prove to be of value in the management of diabetes and atherosclerotic cardiovascular diseases.

Purification, characterization and anticancer activity of a polysaccharide from Panax ginseng.

PubMed

Li, Cong; Cai, Jianping; Geng, Jingshu; Li, Yinghong; Wang, Zhenyu; Li, Rui

2012-12-01

In this study, we purified a homogeneous polysaccharide (PGPW1) from the root of Panax ginseng. Its molecular weight was estimated to be 3.5×10(5) Da by high performance liquid chromatography (HPLC) and Gas chromatography (GC) analysis identified that PGPW1 contained Glc, Gal, Man and Ara in the molar ratio of 3.3:1.2:0.5:1.1. Furthermore the antitumor potential of PGPW1 on human bladder T24 cells was evaluated in vitro by MTT, lactate dehydrogenase (LDH), wound scratch and transwell motility assays. PGPW1 dose-dependently displayed potent anti-proliferation and anti-metastatic activities. Moreover the modulating effect of PGPW1 on the binding of (3)H-NMS to M3 muscarinic receptors on the surface of T24 cells was evaluated. In muscarinic receptor binding assay, the attenuated expression of M3 muscarinic receptor on the surface of T24 cells by PGPW1 would contribute to its antitumor functions. All the data indicated the potential of its clinical application for the prevention and treatment of bladder cancer metastasis. Copyright © 2012 Elsevier B.V. All rights reserved.

Functional characterization of p53 pathway components in the ancient metazoan Trichoplax adhaerens

NASA Astrophysics Data System (ADS)

Siau, Jia Wei; Coffill, Cynthia R.; Zhang, Weiyun Villien; Tan, Yaw Sing; Hundt, Juliane; Lane, David; Verma, Chandra; Ghadessy, Farid

2016-09-01

The identification of genes encoding a p53 family member and an Mdm2 ortholog in the ancient placozoan Trichoplax adhaerens advocates for the evolutionary conservation of a pivotal stress-response pathway observed in all higher eukaryotes. Here, we recapitulate several key functionalities ascribed to this known interacting protein pair by analysis of the placozoan proteins (Tap53 and TaMdm2) using both in vitro and cellular assays. In addition to interacting with each other, the Tap53 and TaMdm2 proteins are also able to respectively bind human Mdm2 and p53, providing strong evidence for functional conservation. The key p53-degrading function of Mdm2 is also conserved in TaMdm2. Tap53 retained DNA binding associated with p53 transcription activation function. However, it lacked transactivation function in reporter genes assays using a heterologous cell line, suggesting a cofactor incompatibility. Overall, the data supports functional roles for TaMdm2 and Tap53, and further defines the p53 pathway as an evolutionary conserved fulcrum mediating cellular response to stress.

Preparation of arginine modified PEI-conjugated chitosan copolymer for DNA delivery.

PubMed

Zhang, Xi; Duan, Yajing; Wang, Dongfang; Bian, Fengling

2015-05-20

Polyethylenimine-conjugated chitosan (CS-PEI) and arginine modified polyethylenimine-conjugated chitosan (CS-PEI-Arg) were prepared, and the copolymers were characterized by FTIR, (1)H NMR, and XRD. The properties of these copolymers like plasmid DNA (pDNA) binding capacity, complexes' size and zeta potential, cytotoxicity and transfection efficiency were also evaluated. The results show that CS-PEI-Arg derivatives can bind pDNA thoroughly, and form complexes with sizes about 170 nm. Cytotoxicity assay in HepG2 and 293T cells show that CS-PEI-Arg has lower cytotoxicity compared with CS-PEI, which is similar to CS and far below that of PEI. In vitro luciferase assay show that CS-PEI-Arg has better transfection efficiency than CS-PEI, which is superior to that of PEI. The best transfection efficiency of CS-PEI-Arg (N/P = 50) is 2.3-fold, 4.2-fold of those of CS-PEI (N/P = 20) and PEI's (N/P = 10) efficiency respectively. These results display that CS-PEI-Arg is a promising candidate as an efficient gene vector. Copyright © 2014 Elsevier Ltd. All rights reserved.

Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis.

PubMed

Kaur, Upninder; Khurana, Sumeeta; Saikia, Uma Nahar; Dubey, M L

2013-11-01

Entamoeba histolytica infection is associated with considerable morbidity and mortality in the form of intestinal and extraintestinal amoebiasis. No vaccine is yet available for amoebiasis. Heparan Sulphate Binding Proteins (HSBPs) from E. histolytica were evaluated for immunogenicity and protective efficacy in a Guinea pig model. Animals were immunized subcutaneously with 30μg of HSBP by three weekly inoculations. The immunogenicity of HSBP was determined by antibody response (IgG, IgM and IgA), splenocyte proliferation assay and in vitro direct amoebicidal assay with splenic lymphocytes and monocytes from vaccinated and control animals. The efficacy of the vaccine was evaluated by challenge infection to vaccinated and control animals by intra-caecal inoculation of E. histolytica trophozoites and comparing gross and histopathological findings in caeca of these animals. HSBP was found to induce specific anti-amoebic response as seen by specific antibody production and direct amoebicidal activity of splenocytes. The vaccine also showed partial protection against challenge infection in vaccinated animals as shown by mild/absent lesions and histopathological findings. Copyright © 2013 Elsevier Inc. All rights reserved.

Discovery of HIV Type 1 Aspartic Protease Hit Compounds through Combined Computational Approaches.

PubMed

Xanthopoulos, Dimitrios; Kritsi, Eftichia; Supuran, Claudiu T; Papadopoulos, Manthos G; Leonis, Georgios; Zoumpoulakis, Panagiotis

2016-08-05

A combination of computational techniques and inhibition assay experiments was employed to identify hit compounds from commercial libraries with enhanced inhibitory potency against HIV type 1 aspartic protease (HIV PR). Extensive virtual screening with the aid of reliable pharmacophore models yielded five candidate protease inhibitors. Subsequent molecular dynamics and molecular mechanics Poisson-Boltzmann surface area free-energy calculations for the five ligand-HIV PR complexes suggested a high stability of the systems through hydrogen-bond interactions between the ligands and the protease's flaps (Ile50/50'), as well as interactions with residues of the active site (Asp25/25'/29/29'/30/30'). Binding-energy calculations for the three most promising compounds yielded values between -5 and -10 kcal mol(-1) and suggested that van der Waals interactions contribute most favorably to the total energy. The predicted binding-energy values were verified by in vitro inhibition assays, which showed promising results in the high nanomolar range. These results provide structural considerations that may guide further hit-to-lead optimization toward improved anti-HIV drugs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of C3b-Binding Small-Molecule Complement Inhibitors Using Cheminformatics.

PubMed

Garcia, Brandon L; Skaff, D Andrew; Chatterjee, Arindam; Hanning, Anders; Walker, John K; Wyckoff, Gerald J; Geisbrecht, Brian V

2017-05-01

The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of more than two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology, such as acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases, which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small-molecule inhibitors, small-molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study, we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies, we identified 45 small molecules that putatively bind C3b near ligand-guided functional hot spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand that guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small-molecule complement inhibitors and, to our knowledge, provides the first demonstration of cheminformatics-based, complement-directed drug discovery. Copyright © 2017 by The American Association of Immunologists, Inc.

Identification of C3b-binding Small Molecule Complement Inhibitors Using Cheminformatics

PubMed Central

Garcia, Brandon L.; Skaff, D. Andrew; Chatterjee, Arindam; Hanning, Anders; Walker, John K.; Wyckoff, Gerald J.; Geisbrecht, Brian V.

2017-01-01

The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of over two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology which include acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small molecule inhibitors, small molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies we identified 45 small molecules which putatively bind C3b near ligand-guided functional hot-spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand which guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small molecule complement inhibitors, and to our knowledge, provides the first demonstration of cheminformatics-based complement-directed drug discovery. PMID:28298523

Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays

PubMed Central

Daniels, V; Wood, M; Leclercq, K; Kaminski, R M; Gillard, M

2013-01-01

Background and Purpose Synaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889. Experimental Approach UCB1244283 was characterized in vitro using radioligand binding assays with [3H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice. Key Results Saturation binding experiments in the presence of UCB1244283 demonstrated a fivefold increase in the affinity of [3H]UCB30889 for human recombinant SV2A, combined with a twofold increase of the total number of binding sites. Similar results were obtained on rat cortex. In competition binding experiments, UCB1244283 potentiated the affinity of UCB30889 while the affinity of LEV remained unchanged. UCB1244283 significantly slowed down both the association and dissociation kinetics of [3H]UCB30889. Following i.c.v. administration in sound-sensitive mice, UCB1244283 showed a clear protective effect against both tonic and clonic convulsions. Conclusions and Implications These results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies. PMID:23530581

F4+ enterotoxigenic Escherichia coli (ETEC) adhesion mediated by the major fimbrial subunit FaeG.

PubMed

Xia, Pengpeng; Song, Yujie; Zou, Yajie; Yang, Ying; Zhu, Guoqiang

2015-09-01

The FaeG subunit is the major constituent of F4(+) fimbriae, associated with glycoprotein and/or glycolipid receptor recognition and majorly contributes to the pathogen attachment to the host cells. To investigate the key factor involved in the fimbrial binding of F4(+) Escherichia coli, both the recombinant E. coli SE5000 strains carrying the fae operon gene clusters that express the different types of fimbriae in vitro, named as rF4ab, rF4ac, and rF4ad, respectively, corresponding to the fimbrial types F4ab, F4ac, and F4ad, and the three isogenic in-frame faeG gene deletion mutants were constructed. The adhesion assays and adhesion inhibition assays showed that ΔfaeG mutants had a significant reduction in the binding to porcine brush border as well as the intestinal epithelial cell lines, while the complemented strain ΔfaeG/pfaeG restored the adhesion function. The recombinant bacterial strains rF4ab, rF4ac, and rF4ad have the same binding property as wild-type F4(+) E. coli strains do and improvement in terms of binding to porcine brush border and the intestinal epithelial cells, and the adherence was blocked by the monoclonal antibody anti-F4 fimbriae. These data demonstrate that the fimbrial binding of F4(+) E. coli is directly mediated by the major FaeG subunit. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural and functional analysis of the YAP-binding domain of human TEAD2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Wei; Yu, Jianzhong; Tomchick, Diana R.

2010-06-15

The Hippo pathway controls organ size and suppresses tumorigenesis in metazoans by blocking cell proliferation and promoting apoptosis. The TEAD1-4 proteins (which contain a DNA-binding domain but lack an activation domain) interact with YAP (which lacks a DNA-binding domain but contains an activation domain) to form functional heterodimeric transcription factors that activate proliferative and prosurvival gene expression programs. The Hippo pathway inhibits the YAP-TEAD hybrid transcription factors by phosphorylating and promoting cytoplasmic retention of YAP. Here we report the crystal structure of the YAP-binding domain (YBD) of human TEAD2. TEAD2 YBD adopts an immunoglobulin-like {beta}-sandwich fold with two extra helix-turn-helixmore » inserts. NMR studies reveal that the TEAD-binding domain of YAP is natively unfolded and that TEAD binding causes localized conformational changes in YAP. In vitro binding and in vivo functional assays define an extensive conserved surface of TEAD2 YBD as the YAP-binding site. Therefore, our studies suggest that a short segment of YAP adopts an extended conformation and forms extensive contacts with a rigid surface of TEAD. Targeting a surface-exposed pocket of TEAD might be an effective strategy to disrupt the YAP-TEAD interaction and to reduce the oncogenic potential of YAP.« less

International Validation of Two Human Recombinant Estrogen ...

EPA Pesticide Factsheets

An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay using a ligand-binding domain of the human ER. Twenty three compounds were tested in 6 laboratories for the FW assay and 5 for the CERJ assay, which included three controls (used with every run), 9 uncoded, and 14 coded chemicals across 3 subtasks. The overall goal of this validation study was to demonstrate the ability of each of the two assays to reliably classify the test chemicals as binders or non-binders. Laboratories had little trouble with the ER binders that produced a full binding curve when using either the CERI or FW assays. As is typical with all ER competitive binding assays, the weak binders proved to be more challenging. However, overall results from both the FW and CERI assays were consistent and in agreement with expected classifications regardless of the form of the hrER (i.e., full length ER versus an ER ligand binding domain) or the subtle differences in the protocols for conducting each assay. The reproducibility and accuracy for classification of chemicals as potential ER binders and non- binders using the FW and CERI hrER binding assays were comparable to that of the U.S.EPA’s existing ER binding test guideline OPPTS 890.1250, while providing an improved, highe

Novel pyrrole derivatives bearing sulfonamide groups: Synthesis in vitro cytotoxicity evaluation, molecular docking and DFT study

NASA Astrophysics Data System (ADS)

Bavadi, Masoumeh; Niknam, Khodabakhsh; Shahraki, Omolbanin

2017-10-01

The synthesis of new derivatives of pyrrole substituted sulfonamide groups is described. The in vitro anticancer activity of these pyrroles was evaluated against MCF7, MOLT-4 and HL-60 cells using MTT assay. The target compounds showed inhibitory activity against tested cell lines. Among the compounds, compound 1a exhibited good cytotoxic activity. The potential of this analog to induce apoptosis was confirmed in a nuclear morphological assay by Hoechst 33258 staining in the PC-12 cells. Finally, molecular docking was performed to determine the probable binding mode of the designed pyrrole derivatives into the active site of FGFR1 protein. DFT calculations were carried out at the B3LYP levels of theory with 6-31+G (d,p) basis set for compound 1a. The point group (C1) of it was obtained based on the optimized structures; the calculation of the FT-IR vibrational frequencies, 1H NMR and 13C NMR chemical shifts of the compound were carried out and compared with those obtained experimentally.

Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery.

PubMed

Zhou, Yangbo; Tang, Zhaomin; Shi, Chunli; Shi, Shuai; Qian, Zhiyong; Zhou, Shaobing

2012-11-01

Polyethylenimine (PEI) functionalized magnetic nanoparticles were synthesized as a potential non-viral vector for gene delivery. The nanoparticles could provide the magnetic-targeting, and the cationic polymer PEI could condense DNA and avoid in vitro barriers. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, dynamic light scattering measurements, transmission electron microscopy, vibrating sample magnetometer and atomic force microscopy. Agarose gel electrophoresis was used to asses DNA binding and perform a DNase I protection assay. The Alamar blue assay was used to evaluate negative effects on the metabolic activity of cells incubated with PEI modified magnetic nanoparticles and their complexes with DNA both in the presence or absence of an external magnetic field. Flow cytometry and fluorescent microscopy were also performed to investigate the transfection efficiency of the DNA-loaded magnetic nanoparticles in A549 and B16-F10 tumor cells with (+M) or without (-M) the magnetic field. The in vitro transfection efficiency of magnetic nanoparticles was improved obviously in a permanent magnetic field. Therefore, the magnetic nanoparticles show considerable potential as nanocarriers for gene delivery.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Y.S.

(9, 10-/sup 3/H/sub 2/)Z9-14:Ac was synthesized at high specific activity (/sup 3/H, 58 Ci/mmole) by partial tritiation of the corresponding alkyne and was converted to the labeled Z9-14:OH and Z9-14:Al to study tissue specificity of acetate esterase (E), alcohol oxidase (OX), and aldehyde dehydrogenase (ALDH) in male and female Heliothis virescens. Soluble and membrane-associated enzyme activities were determined by radio-TLC assays. Compounds of the tritium-labeled Z11-16 series were synthesized and their in vitro fates examined as well. In order to achieve an alternative approach in which (1) pheromone receptor proteins would be stoichiometrically and irreversibly modified, or (2) pheromone-catabolizing enzymesmore » are inactivated by tight-binding or irreversible inhibitors, we have designed analogues of pheromones of lepidopterous insect pests and assayed their biological activity in vitro and in vivo. Various fluorinated molecules such as acyl fluorides, fluoroolefins, 2-fluoro aldehydes, 2,2-difluoro aldehydes and trifluoromethyl ketones were synthesized. The synthesis of some other functional groups such as cyclopropanones, cyclopropanols, cyclopropyl carbinols, cyclopropyl aldehydes and Michael acceptors will also be discussed.« less

Parallel Synthesis of Hexahydrodiimidazodiazepines Heterocyclic Peptidomimetics and Their in Vitro and in Vivo Activities at μ (MOR), δ (DOR), and κ (KOR) Opioid Receptors.

PubMed

Eans, Shainnel O; Ganno, Michelle L; Mizrachi, Elisa; Houghten, Richard A; Dooley, Colette T; McLaughlin, Jay P; Nefzi, Adel

2015-06-25

In the development of analgesics with mixed-opioid agonist activity, peripherally selective activity is expected to decrease side effects, minimizing respiratory depression and reinforcing properties generating significantly safer analgesic therapeutics. We synthesized diazaheterocyclics from reduced tripeptides. In vitro screening with radioligand competition binding assays demonstrated variable affinity for μ (MOR), δ (DOR), and κ (KOR) opioid receptors across the series, with the diimidazodiazepine 14 (2065-14) displaying good affinity for DOR and KOR. Central (icv), intraperitoneal (ip), or oral (po) administration of 14 produced dose-dependent, opioid-receptor mediated antinociception in the mouse, as determined from a 55 °C warm-water tail-withdrawal assay. Only trace amounts of compound 14 was found in brain up to 90 min later, suggesting poor BBB penetration and possible peripherally restricted activity. Central administration of 14 did not produce locomotor effects, acute antinociceptive tolerance, or conditioned-place preference or aversion. The data suggest these diazaheterocyclic mixed activity opioid receptor agonists may hold potential as new analgesics with fewer liabilities of use.

Investigating the pharmacodynamic and magnetic properties of pyrophosphate-bridged coordination complexes

NASA Astrophysics Data System (ADS)

Ikotun, Oluwatayo (Tayo) F.

The multidentate nature of pyrophosphate makes it an attractive ligand for complexation of metal cations. The participation of pyrophosphate in a variety of biological pathways and its metal catalyzed hydrolysis has driven our investigation into its coordination chemistry. We have successfully synthesized a library of binuclear pyrophosphate bridge coordination complexes. The problem of pyrophosphate hydrolysis to phosphate in the presence of divalent metal ions was overcome by incorporating capping ligands such as 1,10-phenanthroline and 2,2'-bipyridine prior to the addition of the pyrophosphate. The magnetic properties of these complexes was investigated and magneto-structural analysis was conducted. The biological abundance of pyrophosphate and the success of metal based drugs such as cisplatin, prompted our investigation of the cytotoxic properties of M(II) pyrophosphate dimeric complexes (where M(II) is CoII, CuII, and NiII) in adriamycin resistant human ovarian cancer cells. Thess compounds were found to exhibit toxicity in the nanomolar to picomolar range. We conducted in vitro stability studies and the mechanism of cytoxicity was elucidated by performing DNA mobility and binding assays, enzyme inhibition assays, and in vitro oxidative stress studies.

Hypotonic activation of short ClC3 isoform is modulated by direct interaction between its cytosolic C-terminal tail and subcortical actin filaments.

PubMed

McCloskey, Diana T; Doherty, Lynda; Dai, Yan-Ping; Miller, Lisa; Hume, Joseph R; Yamboliev, Ilia A

2007-06-08

Short ClC3 isoform (sClC3) functions as a volume-sensitive outwardly rectifying anion channel (VSOAC) in some cell types. In previous studies, we have shown that the hypotonic activation of sClC3 is linked to cell swelling-mediated remodeling of the actin cytoskeleton. In the present study, we have tested the hypothesis that the cytosolic tails of sClC3 bind to actin directly and that binding modulates the hypotonic activation of the channel. Co-sedimentation assays in vitro demonstrated a strong binding between the glutathione S-transferase-fused cytosolic C terminus of sClC3 (GST-sClC3-CT) to filamentous actin (F-actin) but not to globular monomeric actin (G-actin). The GST-fused N terminus (GST-sClC3-NT) exhibited low binding affinity to both G- and F-actin. Co-sedimentation experiments with progressively truncated GST-sClC3-CT indicated that the F-actin binding region is located between amino acids 690 and 760 of sClC3. Two synthetic peptides mapping basic clusters of the cytosolic sClC3-CT (CTP2, isoleucine 716 to leucine 734; and CTP3, proline 688 to proline 709) prevented binding of GST-sClC3-CT to F-actin in vitro. Dialysis into NIH/3T3 cells of these two peptides (but not of synthetic peptide CTP1 (isoleucine 737 to glutamine 748)) reduced the maximal current density by 60 and 38%, respectively. Based on these results, we have concluded that, by direct interaction with subcortical actin filaments, sClC3 contributes to the hypotonic stress-induced VSOACs in NIH/3T3 cells.

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2.

PubMed

Bubenik, Jodi L; Miniard, Angela C; Driscoll, Donna M

2014-01-01

Selenium, a micronutrient, is primarily incorporated into human physiology as selenocysteine (Sec). The 25 Sec-containing proteins in humans are known as selenoproteins. Their synthesis depends on the translational recoding of the UGA stop codon to allow Sec insertion. This requires a stem-loop structure in the 3' untranslated region of eukaryotic mRNAs known as the Selenocysteine Insertion Sequence (SECIS). The SECIS is recognized by SECIS-binding protein 2 (SBP2) and this RNA:protein interaction is essential for UGA recoding to occur. Genetic mutations cause SBP2 deficiency in humans, resulting in a broad set of symptoms due to differential effects on individual selenoproteins. Progress on understanding the different phenotypes requires developing robust tools to investigate SBP2 structure and function. In this study we demonstrate that SBP2 protein produced by in vitro translation discriminates among SECIS elements in a competitive UGA recoding assay and has a much higher specific activity than bacterially expressed protein. We also show that a purified recombinant protein encompassing amino acids 517-777 of SBP2 binds to SECIS elements with high affinity and selectivity. The affinity of the SBP2:SECIS interaction correlated with the ability of a SECIS to compete for UGA recoding activity in vitro. The identification of a 250 amino acid sequence that mediates specific, selective SECIS-binding will facilitate future structural studies of the SBP2:SECIS complex. Finally, we identify an evolutionarily conserved core cysteine signature in SBP2 sequences from the vertebrate lineage. Mutation of multiple, but not single, cysteines impaired SECIS-binding but did not affect protein localization in cells.

Identification and Characterization of Strychnine-Binding Peptides Using Phage-Display Screening.

PubMed

Zhang, Fang; Wang, Min; Qiu, Zheng; Wang, Xiao-Meng; Xu, Chun-Lei; Zhang, Xia

2017-01-01

In drug development, phage display is a high-throughput method for identifying the specific cellular targets of drugs. However, insoluble small chemicals remain intractable to this technique because of the difficulty of presenting molecules to phages without occupying or destroying the limited functional groups. In the present study, we selected Strychnine (Stry) as a model compounda and sought to develope an alternative in vitro biopanning strategy against insoluble suspension. A phage library displaying random sequences of fifteen peptides was employed to screen for interactions between Stry and its cellular selective binding peptides, which are of great value to have a complete understanding of the mechanism of Stry for its antitumor activity. After four rounds of biopanning, a selection of 100 binding clones was randomly picked and subjected to modified proliferation and diffusion assays to evaluate the binding affinity of the clones. Finally, eleven clones were identified as positive binders. The corresponding peptides were synthesized and detected for their binding activities using surface plasmon resonance imaging (SPRi). Our study provides a feasible scheme for confirming the interaction of chemical compounds and cellular binding peptides. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

IgE binding to peanut allergens is inhibited by combined D-aspartic and D-glutamic acids.

PubMed

Chung, Si-Yin; Reed, Shawndrika

2015-01-01

The objective of this study was to determine if D-amino acids (D-aas) bind and inhibit immunoglobulin E (IgE) binding to peanut allergens. D-aas such as D-Asp (aspartic acid), D-Glu (glutamic acid), combined D-[Asp/Glu] and others were each prepared in a cocktail of 9 other D-aas, along with L-amino acids (L-aas) and controls. Each sample was mixed with a pooled plasma from peanut-allergic donors, and tested by ELISA (enzyme-linked immunosorbent assay) and Western blots for IgE binding to peanut allergens. Results showed that D-[Asp/Glu] (4 mg/ml) inhibited IgE binding (75%) while D-Glu, D-Asp and other D-aas had no inhibitory effect. A higher inhibition was seen with D-[Asp/Glu] than with L-[Asp/Glu]. We concluded that IgE was specific for D-[Asp/Glu], not D-Asp or D-Glu, and that D-[Asp/Glu] was more reactive than was L-[Asp/Glu] in IgE inhibition. The finding indicates that D-[Asp/Glu] may have the potential for removing IgE or reducing IgE binding to peanut allergens in vitro. Published by Elsevier Ltd.

Spermine Attenuates the Action of the DNA Intercalator, Actinomycin D, on DNA Binding and the Inhibition of Transcription and DNA Replication

PubMed Central

Chen, Jeremy J. W.; Wu, Wen-Lin; Yuann, Jeu-Ming P.; Su, Wang-Lin; Chuang, Show-Mei; Hou, Ming-Hon

2012-01-01

The anticancer activity of DNA intercalators is related to their ability to intercalate into the DNA duplex with high affinity, thereby interfering with DNA replication and transcription. Polyamines (spermine in particular) are almost exclusively bound to nucleic acids and are involved in many cellular processes that require nucleic acids. Until now, the effects of polyamines on DNA intercalator activities have remained unclear because intercalation is the most important mechanism employed by DNA-binding drugs. Herein, using actinomycin D (ACTD) as a model, we have attempted to elucidate the effects of spermine on the action of ACTD, including its DNA-binding ability, RNA and DNA polymerase interference, and its role in the transcription and replication inhibition of ACTD within cells. We found that spermine interfered with the binding and stabilization of ACTD to DNA. The presence of increasing concentrations of spermine enhanced the transcriptional and replication activities of RNA and DNA polymerases, respectively, in vitro treated with ActD. Moreover, a decrease in intracellular polyamine concentrations stimulated by methylglyoxal-bis(guanylhydrazone) (MGBG) enhanced the ACTD-induced inhibition of c-myc transcription and DNA replication in several cancer cell lines. The results indicated that spermine attenuates ACTD binding to DNA and its inhibition of transcription and DNA replication both in vitro and within cells. Finally, a synergistic antiproliferative effect of MGBG and ACTD was observed in a cell viability assay. Our findings will be of significant relevance to future developments in combination with cancer therapy by enhancing the anticancer activity of DNA interactors through polyamine depletion. PMID:23144800

Spermine attenuates the action of the DNA intercalator, actinomycin D, on DNA binding and the inhibition of transcription and DNA replication.

PubMed

Wang, Sheng-Yu; Lee, Alan Yueh-Luen; Lee, Yueh-Luen; Lai, Yi-Hua; Chen, Jeremy J W; Wu, Wen-Lin; Yuann, Jeu-Ming P; Su, Wang-Lin; Chuang, Show-Mei; Hou, Ming-Hon

2012-01-01

The anticancer activity of DNA intercalators is related to their ability to intercalate into the DNA duplex with high affinity, thereby interfering with DNA replication and transcription. Polyamines (spermine in particular) are almost exclusively bound to nucleic acids and are involved in many cellular processes that require nucleic acids. Until now, the effects of polyamines on DNA intercalator activities have remained unclear because intercalation is the most important mechanism employed by DNA-binding drugs. Herein, using actinomycin D (ACTD) as a model, we have attempted to elucidate the effects of spermine on the action of ACTD, including its DNA-binding ability, RNA and DNA polymerase interference, and its role in the transcription and replication inhibition of ACTD within cells. We found that spermine interfered with the binding and stabilization of ACTD to DNA. The presence of increasing concentrations of spermine enhanced the transcriptional and replication activities of RNA and DNA polymerases, respectively, in vitro treated with ActD. Moreover, a decrease in intracellular polyamine concentrations stimulated by methylglyoxal-bis(guanylhydrazone) (MGBG) enhanced the ACTD-induced inhibition of c-myc transcription and DNA replication in several cancer cell lines. The results indicated that spermine attenuates ACTD binding to DNA and its inhibition of transcription and DNA replication both in vitro and within cells. Finally, a synergistic antiproliferative effect of MGBG and ACTD was observed in a cell viability assay. Our findings will be of significant relevance to future developments in combination with cancer therapy by enhancing the anticancer activity of DNA interactors through polyamine depletion.

A Single-Stranded DNA Aptamer That Selectively Binds to Staphylococcus aureus Enterotoxin B

PubMed Central

DeGrasse, Jeffrey A.

2012-01-01

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APTSEB1, successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide. PMID:22438927

A single-stranded DNA aptamer that selectively binds to Staphylococcus aureus enterotoxin B.

PubMed

DeGrasse, Jeffrey A

2012-01-01

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APT(SEB1), successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.

An electrochemiluminescence assay for analysis of rabies virus glycoprotein content in rabies vaccines

PubMed Central

Smith, Todd G.; Ellison, James A.; Ma, Xiaoyue; Kuzmina, Natalia; Carson, William C.; Rupprecht, Charles E.

2015-01-01

Vaccine potency testing is necessary to evaluate the immunogenicity of inactivated rabies virus (RABV) vaccine preparations before human or veterinary application. Currently, the NIH test is recommended by the WHO expert committee to evaluate RABV vaccine potency. However, numerous disadvantages are inherent concerning cost, number of animals and biosafety requirements. As such, several in vitro methods have been proposed for the evaluation of vaccines based on RABV glycoprotein (G) quality and quantity, which is expected to correlate with vaccine potency. In this study an antigen-capture electrochemiluminescent (ECL) assay was developed utilizing anti-RABV G monoclonal antibodies (MAb) to quantify RABV G. One MAb 2-21-14 was specific for a conformational epitope so that only immunogenic, natively-folded G was captured in the assay. A second MAb (62-80-6) that binds a linear epitope or MAb 2-21-14 was used for detection of RABV G. Vaccine efficacy was also assessed in vivo using pre-exposure vaccination of mice. Purified native RABV G induced a RABV neutralizing antibody (rVNA) response with a geometric mean titer of 4.2 IU/ml and protected 100% of immunized mice against RABV challenge, while an experimental vaccine with a lower quality and quantity of G induced a rVNA titer <0.05 IU/ml and protected <50% of immunized mice. These preliminary results support the hypothesis that in vivo immunogenicity may be predicted from the in vitro measurement of RABV G using an ECL assay. Based upon these results, the ECL assay may have utility in replacement of the NIH test. PMID:23742991

Characterization of binding affinity of CJ-023,423 for human prostanoid EP4 receptor.

PubMed

Murase, Akio; Nakao, Kazunari; Takada, Junji

2008-01-01

In order to characterize the receptor binding pharmacology of CJ-023,423, a potent and selective EP4 antagonist, we performed a radioligand receptor binding assay under various assay conditions. An acidic (pH 6) and hypotonic buffer is a conventional, well-known buffer for prostaglandin E2 receptor binding assays. CJ-023,423 showed moderate binding affinity for human EP4 receptor under conventional buffer conditions. However, its binding affinity was greatly increased under neutral (pH 7.4) and isotonic buffer conditions. In this report, the binding mechanism between CJ-023,423 and human EP4 receptor is discussed based on the binding affinities determined under various assay conditions. Copyright 2008 S. Karger AG, Basel.

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

PubMed Central

1984-01-01

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron- glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells. PMID:6725397

Parallel Force Assay for Protein-Protein Interactions

PubMed Central

Aschenbrenner, Daniela; Pippig, Diana A.; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E.

2014-01-01

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay. PMID:25546146

Parallel force assay for protein-protein interactions.

PubMed

Aschenbrenner, Daniela; Pippig, Diana A; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E

2014-01-01

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

RNA Helicase Associated with AU-rich Element (RHAU/DHX36) Interacts with the 3′-Tail of the Long Non-coding RNA BC200 (BCYRN1)*

PubMed Central

Booy, Evan P.; McRae, Ewan K. S.; Howard, Ryan; Deo, Soumya R.; Ariyo, Emmanuel O.; Dzananovic, Edis; Meier, Markus; Stetefeld, Jörg; McKenna, Sean A.

2016-01-01

RNA helicase associated with AU-rich element (RHAU) is an ATP-dependent RNA helicase that demonstrates high affinity for quadruplex structures in DNA and RNA. To elucidate the significance of these quadruplex-RHAU interactions, we have performed RNA co-immunoprecipitation screens to identify novel RNAs bound to RHAU and characterize their function. In the course of this study, we have identified the non-coding RNA BC200 (BCYRN1) as specifically enriched upon RHAU immunoprecipitation. Although BC200 does not adopt a quadruplex structure and does not bind the quadruplex-interacting motif of RHAU, it has direct affinity for RHAU in vitro. Specifically designed BC200 truncations and RNase footprinting assays demonstrate that RHAU binds to an adenosine-rich region near the 3′-end of the RNA. RHAU truncations support binding that is dependent upon a region within the C terminus and is specific to RHAU isoform 1. Tests performed to assess whether BC200 interferes with RHAU helicase activity have demonstrated the ability of BC200 to act as an acceptor of unwound quadruplexes via a cytosine-rich region near the 3′-end of the RNA. Furthermore, an interaction between BC200 and the quadruplex-containing telomerase RNA was confirmed by pull-down assays of the endogenous RNAs. This leads to the possibility that RHAU may direct BC200 to bind and exert regulatory functions at quadruplex-containing RNA or DNA sequences. PMID:26740632

Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation*

PubMed Central

Chotewutmontri, Prakitchai; Bruce, Barry D.

2015-01-01

Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences. PMID:25645915

Insights into transcription termination of Hfq-binding sRNAs of Escherichia coli and characterization of readthrough products

PubMed Central

Morita, Teppei; Ueda, Masaki; Kubo, Kento; Aiba, Hiroji

2015-01-01

The genes encoding Hfq-dependent sRNAs possess a typical Rho-independent transcription terminator. Here, we have studied the molecular events occurring at Rho-independent terminators of sRNA genes, focusing on two well-characterized Hfq-binding sRNAs, SgrS and RyhB. We constructed several hybrid genes in which the DNA sequence corresponding to a strong Rho-independent terminator was placed just downstream from the Rho-independent terminators of sRNA genes. By using this system, we demonstrate that transcripts frequently read through the Rho-independent terminators of sgrS and ryhB in normally growing cells. We show that Hfq does not affect the transcriptional readthrough event itself. We also find that the readthrough products no longer bind to Hfq in vivo. We have developed a competition assay based on a biotin–streptavidin system to analyze the interaction of Hfq and a particular RNA molecule in vitro. By using this method, we verify that the 3′-extended form of SgrS does not bind to Hfq in vitro. Finally, we demonstrate that transcription termination is significantly enhanced under stress conditions where transcription initiation of sRNA genes on the chromosome is induced. We conclude that the production of sRNAs is regulated not only at the step of transcription initiation but also at the step of transcription termination. The mechanism by which transcription termination is enhanced under stress conditions remains to be understood. PMID:26106215

Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe

PubMed Central

Zhu, Lei; Guo, Ning; Li, Quanzheng; Ma, Ying; Jacboson, Orit; Lee, Seulki; Choi, Hak Soo; Mansfield, James R.; Niu, Gang; Chen, Xiaoyuan

2012-01-01

Purpose: The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/64Cu dual-labeled cyclic RGD peptide. Methods: The integrin αvβ3 binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA)-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD) method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data. Results: The dual-labeled probe 64Cu-RGD-C(DOTA)-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp) derived from dynamic optical imaging (1.762 ± 0.020) is comparable to that from dynamic PET (1.752 ± 0.026). Conclusion: The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models. PMID:22916074

Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe.

PubMed

Zhu, Lei; Guo, Ning; Li, Quanzheng; Ma, Ying; Jacboson, Orit; Lee, Seulki; Choi, Hak Soo; Mansfield, James R; Niu, Gang; Chen, Xiaoyuan

2012-01-01

The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/(64)Cu dual-labeled cyclic RGD peptide. The integrin α(v)β(3) binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA)-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD) method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data. The dual-labeled probe (64)Cu-RGD-C(DOTA)-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp) derived from dynamic optical imaging (1.762 ± 0.020) is comparable to that from dynamic PET (1.752 ± 0.026). The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models.

Spectro Analytical, Computational and In Vitro Biological Studies of Novel Substituted Quinolone Hydrazone and it's Metal Complexes.

PubMed

Nagula, Narsimha; Kunche, Sudeepa; Jaheer, Mohmed; Mudavath, Ravi; Sivan, Sreekanth; Ch, Sarala Devi

2018-01-01

Some novel transition metal [Cu (II), Ni (II) and Co (II)] complexes of nalidixic acid hydrazone have been prepared and characterized by employing spectro-analytical techniques viz: elemental analysis, 1 H-NMR, Mass, UV-Vis, IR, TGA-DTA, SEM-EDX, ESR and Spectrophotometry studies. The HyperChem 7.5 software was used for geometry optimization of title compound in its molecular and ionic forms. Quantum mechanical parameters, contour maps of highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) and corresponding binding energy values were computed using semi empirical single point PM3 method. The stoichiometric equilibrium studies of metal complexes carried out spectrophotometrically using Job's continuous variation and mole ratio methods inferred formation of 1:2 (ML 2 ) metal complexes in respective systems. The title compound and its metal complexes screened for antibacterial and antifungal properties, exemplified improved activity in metal complexes. The studies of nuclease activity for the cleavage of CT- DNA and MTT assay for in vitro cytotoxic properties involving metal complexes exhibited high activity. In addition, the DNA binding properties of Cu (II), Ni (II) and Co (II) complexes investigated by electronic absorption and fluorescence measurements revealed their good binding ability and commended agreement of K b values obtained from both the techniques. Molecular docking studies were also performed to find the binding affinity of synthesized compounds with DNA (PDB ID: 1N37) and "Thymidine phosphorylase from E.coli" (PDB ID: 4EAF) protein targets.

Herpes simplex virus type 1 tegument protein VP22 interacts with TAF-I proteins and inhibits nucleosome assembly but not regulation of histone acetylation by INHAT.

PubMed

van Leeuwen, Hans; Okuwaki, Mitsuru; Hong, Rui; Chakravarti, Debabrata; Nagata, Kyosuke; O'Hare, Peter

2003-09-01

Affinity chromatography was used to identify cellular proteins that interact with the herpes simplex virus (HSV) tegument protein VP22. Among a small set of proteins that bind specifically to VP22, we identified TAF-I (template-activating factor I), a chromatin remodelling protein and close homologue of the histone chaperone protein NAP-1. TAF-I has been shown previously to promote more ordered transfer of histones to naked DNA through a direct interaction with histones. TAF-I, as a subunit of the INHAT (inhibitor of acetyltransferases) protein complex, also binds to histones and masks them from being substrates for the acetyltransferases p300 and PCAF. Using in vitro assays for TAF-I activity in chromatin assembly, we show that VP22 inhibits nucleosome deposition on DNA by binding to TAF-I. We also observed that VP22 binds non-specifically to DNA, an activity that is abolished by TAF-I. However, the presence of VP22 does not affect the property of INHAT in inhibiting the histone acetyltransferase activity of p300 or PCAF in vitro. We speculate that this interaction could be relevant to HSV DNA organization early in infection, for example, by interfering with nucleosomal deposition on the genome. Consistent with this possibility was the observation that overexpression of TAF-I in transfected cells interferes with the progression of HSV-1 infection.

CDB-4124 and its putative monodemethylated metabolite, CDB-4453, are potent antiprogestins with reduced antiglucocorticoid activity: in vitro comparison to mifepristone and CDB-2914.

PubMed

Attardi, Barbara J; Burgenson, Janet; Hild, Sheri A; Reel, Jerry R; Blye, Richard P

2002-02-25

To obtain selective antiprogestins, we have examined the in vitro antiprogestational/antiglucocorticoid properties of two novel compounds, CDB-4124 and the putative monodemethylated metabolite, CDB-4453, in transcription and receptor binding assays and compared them to CDB-2914 and mifepristone. All four antiprogestins bound with high affinity to rabbit uterine progestin receptors (PR) and recombinant human PR-A and PR-B (rhPR-A, rhPR-B) and were potent inhibitors of R5020-induced transactivation of the PRE2-tk-luciferase (PRE2-tk-LUC) reporter plasmid and endogenous alkaline phosphatase production in T47D-CO human breast cancer cells. None of these compounds exhibited agonist activity in these cells. Induction of luciferase activity was potentiated about five-fold by 8-Br-cAMP under basal conditions and to the same extent in the presence of the PR antagonists. Mifepristone bound to rabbit thymic glucocorticoid receptors (GR) with approximately twice the avidity of the CDB antiprogestins. Inhibition of GR-mediated transcription of PRE2-tk-LUC was assessed in HepG2 human hepatoblastoma cells. Mifepristone exhibited greater antiglucocorticoid activity than CDB-2914, 4124, and 4453, about 12-, 22-, and 185-fold, respectively. Thus, while there was a good correlation between binding to PR and functional activity of these antiprogestins, GR binding was not predictive of their glucocorticoid antagonist activity. In agreement with our in vivo results, CDB-4124 and CDB-4453, as well as CDB-2914, are potent antiprogestins in vitro, but show considerably less antiglucocorticoid activity than mifepristone.

Azetidinones as zinc-binding groups to design selective HDAC8 inhibitors.

PubMed

Galletti, Paola; Quintavalla, Arianna; Ventrici, Caterina; Giannini, Giuseppe; Cabri, Walter; Penco, Sergio; Gallo, Grazia; Vincenti, Silvia; Giacomini, Daria

2009-12-01

2-Azetidinones, commonly known as beta-lactams, are well-known heterocyclic compounds. Herein we described the synthesis and biological evaluation of a series of novel beta-lactams. In vitro inhibition assays against HDAC isoforms showed an interesting isoform-selectivity of these compounds towards HDAC6 and HDAC8. The isoform selectivity changed in response to modification of the azetidinone-ring nitrogen atom substituent. The presence of an N-thiomethyl group is a prerequisite for the activity of these compounds in the micromolar range towards HDAC8.

Iron(II) supramolecular helicates interfere with the HIV-1 Tat-TAR RNA interaction critical for viral replication.

PubMed

Malina, Jaroslav; Hannon, Michael J; Brabec, Viktor

2016-07-12

The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat-TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

Increased targeting of adenine-rich sequences by (2-amino-2-methyl-3-butanone oxime)dichloroplatinum(II) and investigations into its low cytotoxicity.

PubMed

Hambley, T W; Ling, E C; O'Mara, S; McKeage, M J; Russell, P J

2000-12-01

Using assays based on the inhibition of restriction enzyme cleavage of plasmid and synthetic DNA, the complex (2-amino-2-methyl-3-butanone oxime)dichloroplatinum(II), [PtCl2(ambo)], has been shown to have an increased tendency for binding to adenine-rich sequences when compared to cis[PtCl2(NH3)2] (cisplatin). [PtCl2(ambo)] was found to form substantially fewer interstrand adducts than does cisplatin. The in vitro cytotoxicity of [PtCl2(ambo)] against a human bladder cancer cell line was determined and found to be more than two orders of magnitude lower than that of cisplatin, yet it was also found to be equally effective at passing into cells and binding to isolated DNA.

A demonstration of the uncertainty in predicting the estrogenic activity of individual chemicals and mixtures from an in vitro estrogen receptor transcriptional activation assay (T47D-KBluc) to the in vivo uterotrophic assay using oral exposure

EPA Science Inventory

In vitro estrogen receptor assays are valuable screening tools for identifying environmental samples and chemicals that display estrogenic activity. However, in vitro potency cannot necessarily be extrapolated to estimates of in vivo potency because in vitro assays are currently...

Development of a functional cell-based assay that probes the specific interaction between influenza A virus NP and its packaging signal sequence RNA.

PubMed

Woo, Jiwon; Yu, Kyung Lee; Lee, Sun Hee; You, Ji Chang

2015-02-06

Although cis-acting packaging signal RNA sequences for the influenza virus NP encoding vRNA have been identified recently though genetic studies, little is known about the interaction between NP and the vRNA packaging signals either in vivo or in vitro. Here, we provide evidence that NP is able to interact specifically with the vRNA packaging sequence RNA within living cells and that the specific RNA binding activity of NP in vivo requires both the N-terminal and central region of the protein. This assay established would be a valuable tool for further detailed studies of the NP-packaging signal RNA interaction in living cells. Copyright © 2014 Elsevier Inc. All rights reserved.

A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.

PubMed

Corthésy, B; Cardinaux, J R; Claret, F X; Wahli, W

1989-12-01

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and, as demonstrated by DNA-binding assays, interacts with a liver-specific transcription factor. The second is required in association with the estrogen-responsive element to mediate hormonal induction and is recognized by the Xenopus liver homolog of nuclear factor I.

An integrated in vitro and in vivo high throughput screen identifies treatment leads for ependymoma

PubMed Central

Atkinson, Jennifer M.; Shelat, Anang A.; Carcaboso, Angel Montero; Kranenburg, Tanya A.; Arnold, Alexander; Boulos, Nidal; Wright, Karen; Johnson, Robert A.; Poppleton, Helen; Mohankumar, Kumarasamypet M.; Feau, Clementine; Phoenix, Timothy; Gibson, Paul; Zhu, Liqin; Tong, Yiai; Eden, Chris; Ellison, David W.; Priebe, Waldemar; Koul, Dimpy; Yung, W. K. Alfred; Gajjar, Amar; Stewart, Clinton F.; Guy, R. Kip; Gilbertson, Richard J.

2011-01-01

Summary Using a mouse model of ependymoma—a chemoresistant brain tumor—we combined multi-cell high-throughput screening (HTS), kinome-wide binding assays, and in vivo efficacy studies, to identify potential treatments with predicted toxicity against neural stem cells (NSC). We identified kinases within the insulin signaling pathway and centrosome cycle as regulators of ependymoma cell proliferation, and their corresponding inhibitors as potential therapies. FDA approved drugs not currently used to treat ependymoma were also identified that posses selective toxicity against ependymoma cells relative to normal NSCs both in vitro and in vivo e.g., 5-fluoruracil. Our comprehensive approach advances understanding of the biology and treatment of ependymoma including the discovery of several treatment leads for immediate clinical translation. PMID:21907928