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Bidirectional affinity chromatography

Westley S Bauer, Kelly A Richardson, Nicholas M Adams, Keersten M Ricks, David J Gasperino, Simon J Ghionea, Mathew Rosen, Kevin P Nichols, Bernhard H Weigl, Frederick R Haselton, David W Wright
Rapid diagnostic tests (RDTs) designed to function at the point of care are becoming more prevalent in malaria diagnostics because of their low cost and simplicity. While many of these tests function effectively with high parasite density samples, their poor sensitivity can often lead to misdiagnosis when parasitemia falls below 100 parasites/μl. In this study, a flow-through pipette-based column was explored as a cost-effective means to capture and elute more Plasmodium falciparum histidine-rich protein II (HRPII) antigen, concentrating the biomarker available in large-volume lysed whole blood samples into volumes compatible with Plasmodium falciparum-specific RDTs...
May 2017: Biomicrofluidics
Ashok Kumar Shettihalli, Sathyanarayana N Gummadi
Human phospholipid scramblase 1(hPLSCR1) is a transmembrane protein involved in bidirectional scrambling of plasma membrane phospholipids during cell activation, blood coagulation, and apoptosis in response to elevated intracellular Ca(2+) levels. Pb(2+) and Hg(2+) are known to cause procoagulant activation via phosphatidylserine exposure to the external surface in erythrocytes, resulting in blood coagulation. To explore its role in lead and mercury poisoning, hPLSCR1 was overexpressed in Escherichia coli BL21 (DE3) and purified using affinity chromatography...
June 17, 2013: Chemical Research in Toxicology
Dan-Hua Xu, Miao Yan, Huan-De Li, Ping-Fei Fang, Yan-Wen Liu
Brucine is a central agonist that can pass through the blood-brain barrier (BBB). The goal of this study is to examine whether brucine is one of the substrates of the drug transporter P-glycoprotein (P-gp) and to examine the effects of P-gp on the brucine transport at the in vitro BBB model. The P-gp ATPase assay was utilized to investigate the in vitro affinity of P-gp to brucine. Results suggested that K(m) of brucine (11.4 μmol/l) was smaller than the positive control, verapamil (16.4 μmol/l). In this study, we developed an in vitro BBB model, comprising a co-culture of primary rat brain microvessel endothelial cells and astrocytes for the transport study...
September 5, 2012: European Journal of Pharmacology
Vincent Gerard Francis, Mohammed Abdul Majeed, Sathyanarayana N Gummadi
Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca(2+)) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them. Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni(2+)-NTA) affinity chromatography and was >98% pure...
July 2012: Journal of Industrial Microbiology & Biotechnology
Cláudia Rosa-Ferreira, Sean Munro
Lysosomes move bidirectionally on microtubules, and this motility can be stimulated by overexpression of the small GTPase Arl8. By using affinity chromatography, we find that Arl8-GTP binds to the soluble protein SKIP (SifA and kinesin-interacting protein, aka PLEKHM2). SKIP was originally identified as a target of the Salmonella effector protein SifA and found to bind the light chain of kinesin-1 to activate the motor on the bacteria's replicative vacuole. We show that in uninfected cells both Arl8 and SKIP are required for lysosomes to distribute away from the microtubule-organizing center...
December 13, 2011: Developmental Cell
Takayuki Uematsu, Chieko Konishi, Daisuke Hoshino, Xiao Han, Taizo Tomari, Nagayasu Egawa, Yoshikazu Takada, Toshiaki Isobe, Motoharu Seiki, Naohiko Koshikawa
Integrins are adhesion receptors for components of the extracellular matrix (ECMs) that regulate multiple cellular functions, such as migration, invasion, proliferation, and survival by mediating bidirectional signal transmission. Even though many proteins have been reported to associate with integrins both on and in cells, systemic analyses of the adhesome have not been carried out. In previous studies, we identified proteins associating with a membrane-type protease, MT1-MMP, using nano-flow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) of associated proteins prepared by optimized conditions for cell lysis and purification...
August 2012: Journal of Cellular Physiology
Brunilda Vera, Abimael D Rodríguez, James J La Clair
A bidirectional affinity system has been developed for the identification of cancer-related natural products and their biological targets. Aplysqualenol A is thus selectively identified as a ligand of the dynein light chain. The use of forward and reverse affinity methods suggests that both small-molecule isolation and target identification can be conducted using conventional molecular biological methods.
August 22, 2011: Angewandte Chemie
Juan F Abenza, Antonio Galindo, Areti Pantazopoulou, Concha Gil, Vivian de los Ríos, Miguel A Peñalva
Aspergillus nidulans early endosomes display characteristic long-distance bidirectional motility. Simultaneous dual-channel acquisition showed that the two Rab5 paralogues RabB and RabA colocalize in these early endosomes and also in larger, immotile mature endosomes. However, RabB-GTP is the sole recruiter to endosomes of Vps34 PI3K (phosphatidylinositol-3-kinase) and the phosphatidylinositol-3-phosphate [PI(3)P] effector AnVps19 and rabB Delta, leading to thermosensitivity prevents multivesicular body sorting of endocytic cargo...
August 1, 2010: Molecular Biology of the Cell
Clifford W Mason, Hazem E Hassan, Kang-Pil Kim, Jianjing Cao, Natalie D Eddington, Amy Hauck Newman, Pamela J Voulalas
The recent discovery of novel high-affinity and selective dopamine D3 receptor (DA D3R) antagonists and partial agonists has provided tools with which to further elucidate the role DA D3R plays in substance abuse. The present study was conducted to evaluate the transport, metabolism, pharmacokinetics, and brain uptake of the DA D3R-selective fluorenyl amides, NGB 2904 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide] fumarate) and JJC 4-077 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-9H-fluorene-2-carboxamide hydrochloride], and the 2-pyridylphenyl amides, CJB 090 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridine-2-yl)benzamide hydrochloride] and PG 01037 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-trans-but-2-enyl)-4-(pyridine-2-yl)benzamide hydrochloride], all of which have been studied in animal models of psychostimulant abuse...
June 2010: Journal of Pharmacology and Experimental Therapeutics
Frauke Germer, Ingo Zebger, Miguel Saggu, Friedhelm Lendzian, Rüdiger Schulz, Jens Appel
The bidirectional [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was purified to apparent homogeneity by a single affinity chromatography step using a Synechocystis mutant with a Strep-tag II fused to the C terminus of HoxF. To increase the yield of purified enzyme and to test its overexpression capacity in Synechocystis the psbAII promoter was inserted upstream of the hoxE gene. In addition, the accessory genes (hypF, C, D, E, A, and B) from Nostoc sp. PCC 7120 were expressed under control of the psbAII promoter...
December 25, 2009: Journal of Biological Chemistry
Shan-Hua Lin, Hui-Fen Kuo, Geneviève Canivenc, Choun-Sea Lin, Marc Lepetit, Po-Kai Hsu, Pascal Tillard, Huey-Ling Lin, Ya-Yun Wang, Chyn-Bey Tsai, Alain Gojon, Yi-Fang Tsay
Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-beta-glucuronidase analysis, as well as in situ hybridization, showed that NRT1...
September 2008: Plant Cell
Liron Boyman, Reuben Hiller, W Jonathan Lederer, Daniel Khananshvili
The Na(+)-Ca(2+) exchanger in mammalian heart muscle (NCX1) is the central transporter protein that regulates extrusion of Ca(2+) from the heart cell. However, the functional biochemistry and physiology of NCX1 have been severely hampered by the absence of any specific high-affinity inhibitor. Here we describe advanced procedures for purifying a candidate inhibitor, previously called endogenous inhibitor factor (NCX(IF)), and demonstrate its direct actions on NCX1 activities in the single-cell system. A combination of advanced HILIC (hydrophilic interaction liquid chromatography) procedures with analytical tests suggests that the properties of NCX(IF) resemble those of a small (disaccharide size) polar molecule lacking any aromatic rings, conjugated bonds, or a primary amino group...
June 24, 2008: Biochemistry
Pradip Bhattacharya
In the in vitro mitochondrial (mt) transcription initiation system with mt RNA polymerase fraction and mt lysate, the transcription initiation products were shown to be synthesized bidirectionally from the only H-strand-promoter (HSP)/L-strand-promoter region (LSP) of the mitochondrial D-loop genome segment. These transcription products ranged between >100 and >800 bp with the purified mitochondrial RNA polymerase fraction, but were larger (>2030-4000 bp) in size with the mitochondrial lysate in both human and mouse...
March 30, 2007: Biochemical and Biophysical Research Communications
Oliver I Wagner, Jennifer Ascaño, Mariko Tokito, Jean-Francois Leterrier, Paul A Janmey, Erika L F Holzbaur
Neurofilaments are synthesized in the cell body of neurons and transported outward along the axon via slow axonal transport. Direct observation of neurofilaments trafficking in live cells suggests that the slow outward rate of transport is due to the net effects of anterograde and retrograde microtubule motors pulling in opposition. Previous studies have suggested that cytoplasmic dynein is required for efficient neurofilament transport. In this study, we examine the interaction of neurofilaments with cytoplasmic dynein...
November 2004: Molecular Biology of the Cell
Andrew Crowe, Penny Wong
UNLABELLED: Loperamide is a peripherally acting antidiarrheal opioid with some affinity for P-glycoprotein (P-gp). One of the main reasons for its lack of central nervous system (CNS) activity is a combination first-pass metabolism and P-gp-mediated efflux preventing brain penetration. It was assumed that P-gp would also have a similar effect at the intestinal tract, limiting loperamide systemic absorption. However, previous in vitro studies had not determined loperamide flux using pH gradients present in the intestinal tract...
May 2004: Drug Development and Industrial Pharmacy
Cuiping Chen, Elizabeth Hanson, John W Watson, Jae S Lee
The present study evaluates the impact of P-glycoprotein (P-gp) on plasma-brain disposition and transepithelial transport of sedating versus nonsedating H1-antagonists using multidrug-resistant (mdr) gene 1a and 1b (mdr1a/b) knockout (KO) mice and human MDR1-transfected Madin-Darby canine kidney (MDCK) cells. Three nonsedating (cetirizine, loratadine, and desloratadine) and three sedating (diphenhydramine, hydroxyzine, and triprolidine) H1-antagonists were tested. Each compound was administered to KO and wild-type (WT) mice intravenously at 5 mg/kg...
March 2003: Drug Metabolism and Disposition: the Biological Fate of Chemicals
Gunther Marsche, Astrid Hammer, Olga Oskolkova, Karen F Kozarsky, Wolfgang Sattler, Ernst Malle
Hypochlorous acid/hypochlorite (HOCl/OCl(-)), a potent oxidant generated in vivo by the myeloperoxidase-H(2)O(2)-chloride system of activated phagocytes, alters the physiological properties of high density lipoprotein (HDL) by generating a proatherogenic lipoprotein particle. On endothelial cells lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) and scavenger receptor class B, type I (SR-BI), act in concert by mediating the holoparticle of and selective cholesteryl ester uptake from HOCl-HDL. We therefore investigated the ligand specificity of HOCl-HDL to SR-BI-overexpressing Chinese hamster ovary cells...
August 30, 2002: Journal of Biological Chemistry
Carmen W Dessauer, Misty Chen-Goodspeed, Jun Chen
The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C1 and C2). The stimulatory G protein, Galpha(s), binds within a cleft in the C2 domain of adenylyl cyclase while Galpha(i) binds within the opposite cleft in the C1 domain. The mechanism of these two regulators also appears to be in opposition. Activation of adenylyl cyclase by Galpha(s) or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another...
August 9, 2002: Journal of Biological Chemistry
Eric Soupene, Tony Chu, Rebecca W Corbin, Donald F Hunt, Sydney Kustu
Ammonium transport (Amt) proteins appear to be bidirectional channels for NH(3). The amt genes of the hyperthermophiles Aquifex aeolicus and Methanococcus jannaschii complement enteric amtB mutants for growth at 25 nM NH(3) at 37 degrees C. To our knowledge, Amt proteins are the first hyperthermophilic membrane transport proteins shown to be active in a mesophilic bacterium. Despite low expression levels, His-tagged Aquifex Amt could be purified by heating and nickel chelate affinity chromatography. It could be studied genetically in Escherichia coli...
June 2002: Journal of Bacteriology
Irene Santamarta, Antonio Rodríguez-García, Rosario Pérez-Redondo, Juan F Martín, Paloma Liras
The putative regulatory CcaR protein, which is encoded in the beta-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region...
June 2002: Journal of Bacteriology
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