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Modern directions for potentiometric sensors

Bakker,Eric; Chumbimuni-Torres,Karin
Fonte: Sociedade Brasileira de Química Publicador: Sociedade Brasileira de Química
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2008 EN
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This paper gives an overview of the newest developments of polymeric membrane ion-selective electrodes. A short essence of the underlying theory is given, emphasizing how the electromotive force may be used to assess binding constants of the ionophore, and how the selectivity and detection limit are related to the basic membrane processes. The recent developments in lowering the detection limits of ISEs are described, including recent approaches of developing all solid state ISEs, and breakthroughs in detecting ultra-small quantities of ions at low concentrations. These developments have paved the way to use potentiometric sensors as in ultra-sensitive affinity bioanalysis in conjunction with nanoparticle labels. Recent results establish that potentiometry compares favorably to electrochemical stripping analysis. Other new developments with ion-selective electrodes are also described, including the concept of backside calibration potentiometry, controlled current coulometry, pulsed chronopotentiometry, and localized flash titration with ion-selective membranes to design sensors for the direct detection of total acidity without net sample perturbation. These developments have further opened the field for exciting new possibilities and applications.

Nanomolar Levels of Dimethylsulfoniopropionate, Dimethylsulfonioacetate, and Glycine Betaine Are Sufficient To Confer Osmoprotection to Escherichia coli

Cosquer, Anne; Pichereau, Vianney; Pocard, Jean-Alain; Minet, Jacques; Cormier, Michel; Bernard, Théophile
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/1999 EN
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We combined the use of low inoculation titers (300 ± 100 CFU/ml) and enumeration of culturable cells to measure the osmoprotective potentialities of dimethylsulfoniopropionate (DMSP), dimethylsulfonioacetate (DMSA), and glycine betaine (GB) for salt-stressed cultures of Escherichia coli. Dilute bacterial cultures were grown with osmoprotectant concentrations that encompassed the nanomolar levels of GB and DMSP found in nature and the millimolar levels of osmoprotectants used in standard laboratory osmoprotection bioassays. Nanomolar concentrations of DMSA, DMSP, and GB were sufficient to enhance the salinity tolerance of E. coli cells expressing only the ProU high-affinity general osmoporter. In contrast, nanomolar levels of osmoprotectants were ineffective with a mutant strain (GM50) that expressed only the low-affinity ProP osmoporter. Transport studies showed that DMSA and DMSP, like GB, were taken up via both ProU and ProP. Moreover, ProU displayed higher affinities for the three osmoprotectants than ProP displayed, and ProP, like ProU, displayed much higher affinities for GB and DMSA than for DMSP. Interestingly, ProP did not operate at substrate concentrations of 200 nM or less, whereas ProU operated at concentrations ranging from 1 nM to millimolar levels. Consequently...

Identification of High-Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells.

Schroeder, JI; Schmidt, C; Sheaffer, J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1993 EN
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Slow anion channels in the plasma membrane of guard cells have been suggested to constitute an important control mechanism for long-term ion efflux, which produces stomatal closing. Identification of pharmacological blockers of these slow anion channels is instrumental for understanding plant anion channel function and structure. Patch clamp studies were performed on guard cell protoplasts to identify specific extracellular inhibitors of slow anion channels. Extracellular application of the anion channel blockers NPPB and IAA-94 produced a strong inhibition of slow anion channels in the physiological voltage range with half inhibition constants (K1/2) of 7 and 10 [mu]M, respectively. Single slow anion channels that had a high open probability at depolarized potentials were identified. Anion channels had a main conductance state of 33 [plus or minus] 8 pS and were inhibited by IAA-94. DIDS, which has been shown to be a potent blocker of rapid anion channels in guard cells (K1/2 = 0.2 [mu]M), blocked less than 20% of peak slow anion currents at extracellular or cytosolic concentrations of 100 [mu]M. The pharmacological properties of slow anion channels described here differ from those recently described for rapid anion channels in guard cells...

Glycylcyclines bind to the high-affinity tetracycline ribosomal binding site and evade Tet(M)- and Tet(O)-mediated ribosomal protection.

Bergeron, J; Ammirati, M; Danley, D; James, L; Norcia, M; Retsema, J; Strick, C A; Su, W G; Sutcliffe, J; Wondrack, L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1996 EN
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N,N-dimethylglycylamido (DMG) derivatives of 6-demethyl-6-deoxytetracycline and doxycycline bind 5-fold more effectively than tetracycline to the tetracycline high-affinity binding site on the Escherichia coli 70S ribosome, which correlates with a 10-fold increase in potency for inhibition of E. coli cell-free translation. The potencies of DMG-doxycycline and DMG-6-demethyl-6-deoxytetracycline were unaffected by the ribosomal tetracycline resistance factors Tet(M) and Tet(O) in cell-free translation assays and whole-cell bioassays with a conditional Tet(M)-producing E. coli strain.

Identification and characterization of an iron-regulated hemopexin receptor in Haemophilus influenzae type b.

Wong, J C; Holland, J; Parsons, T; Smith, A; Williams, P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1994 EN
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Heme can serve Haemophilus influenzae as a source of both essential porphyrin and iron. In extracellular mammalian body fluids neither free heme nor free iron is available, since they are tightly bound to hemopexin and transferrin, respectively. Since H. influenzae grows in the presence of iron-transferrin and heme-hemopexin and is known to express a saturable receptor for transferrin, we investigated the process by which this pathogen acquired heme from hemopexin for use as an iron source. The ability of human and rabbit hemopexin to donate heme as a source of iron to H. influenzae type b strains was demonstrated by plate bioassays. With a dot enzyme assay with biotinylated hemopexin as ligand, H. influenzae bound heme-hemopexin and apo-hemopexin following growth in iron-restricted, but not in iron-sufficient, medium. Competitive binding studies with heme-hemopexin and apo-hemopexin demonstrated saturability of binding. Neither heme, protoporphyrin IX, hemoglobin, nor transferrin blocked the binding of hemopexin to whole cells, demonstrating the specificity of binding. Treatment of whole H. influenzae cells with trypsin abolished binding. Taken together, these observations suggest that H. influenzae type b expresses an outer membrane protein(s) which acts as a receptor for hemopexin and which is regulated by the availability of iron in the growth medium. In iron-restricted media...

MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist

Catalioto, R-M; Criscuoli, M; Cucchi, P; Giachetti, A; Giannotti, D; Giuliani, S; Lecci, A; Lippi, A; Patacchini, R; Quartara, L; Renzetti, A R; Tramontana, M; Arcamone, F; Maggi, C A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1998 EN
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The pharmacological profile was studied of MEN 11420, or cyclo{[Asn(β-D-GlcNAc)-Asp-Trp-Phe-Dap-Leu]cyclo(2β-5β)}, a glycosylated derivative of the potent, selective, conformationally-constrained tachykinin NK2 receptor antagonist MEN 10627 (cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2β-5β)).MEN 11420 competitively bound with high affinity to the human NK2 receptor stably transfected in CHO cells, displacing radiolabelled [125I]-neurokinin A and [3H]-SR 48968 with Ki values of 2.5±0.7 nM (n=6) and 2.6±0.4 nM (n=3), respectively.MEN 11420 showed negligible binding affinity (pIC50<6) at 50 different receptors (including tachykinin NK1 and NK3 receptors) and ion channels.In the rabbit isolated pulmonary artery and rat urinary bladder MEN 11420 potently and competitively antagonized tachykinin NK2 receptor-mediated contractions (pKB=8.6±0.07, n=10, and 9.0±0.04, n=12; Schild plot slope=−1.06 (95% c.l.=−1.3; −0.8) and −1.17 (95% c.l.=−1.3; −1.0), respectively). MEN 11420 produced an insurmountable antagonism at NK2 receptors in the hamster trachea and mouse urinary bladder. However, in both preparations, the effect of MEN 11420 was reverted by washout and an apparent pKB of 10.2±0.14, n= 9, and 9.8±0.15...

Novel strategies for the design of receptor-selective vasopressin analogues: Aib-substitution and retro-inverso transformation

Howl, John; Prochazka, Zdenko; Wheatley, Mark; Slaninová, Jirina
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1999 EN
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We determined the pharmacological profile of novel backbone-modified peptides designed as protease-resistant, selective analogues of AVP. Binding affinities of peptides were determined at both V1A and V2 subtypes of vasopressin receptor (VPR). Biological potencies of selected peptides were tested in pressor and antidiuretic bioassays.Substitution of the achiral α-aminoisobutyric acid (Aib) at position 4 or 7 of AVP produced peptides that selectively bound the V2 VPR. Both [Aib4]AVP (140 IU mg−1) and [Aib7]AVP (36 IU mg−1) are selective antidiuretic agonists with little or no activity in uterotonic and pressor assays.[Aib4] and [Aib7] derivatives of the linear V1A-selective antagonist [PhaaDTyr(Et)2Arg6Tyr(NH2)9]AVP bound selectively and with high affinity (Kd 0.51 and 4.1 nM respectively) to the V1A VPR. Bioassays confirmed that these peptides were potent antivasopressor agents (pA2 8.10 and 8.36 respectively).A total retro-inverso strategy was used to prepare protease-resistant mimetics of both AVP and linear V1A-selective antagonists. Cyclic retro-inverso mimetics of AVP did not bind either V1A or V2 VPRs. In contrast, rationally designed retro-inverso mimetics of linear V1A-selective antagonists selectively bound the V1A VPR.Our findings indicate novel methods to improve the pharmacodynamic and pharmacokinetic parameters of neurohypophysial hormone analogues which could be equally applicable to other peptide-receptor systems.

BIIE0246, a potent and highly selective non-peptide neuropeptide Y Y2 receptor antagonist

Dumont, Yvan; Cadieux, Alain; Doods, Henri; Pheng, Leng Hong; Abounader, Roger; Hamel, Edith; Jacques, Danielle; Regoli, Domenico; Quirion, Rémi
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/2000 EN
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BIIE0246, a newly synthesized non-peptide neuropeptide Y (NPY) Y2 receptor antagonist, was able to compete with high affinity (8 to 15 nM) for specific [125I]PYY3–36 binding sites in HEK293 cells transfected with the rat Y2 receptor cDNA, and in rat brain and human frontal cortex membrane homogenates.Interestingly, in rat brain homogenates while NPY, C2-NPY and PYY3–36 inhibited all specific [125I]PYY3–36 labelling, BIIE0246 failed to compete for all specific binding suggesting that [125I]PYY3–36 recognized, in addition to the Y2 subtype, another population of specific NPY binding sites, most likely the Y5 receptor.Quantitative receptor autoradiographic data confirmed the presence of [125I]PYY3–36/BIIE0246-sensitive (Y2) and-insensitive (Y5) binding sites in the rat brain as well as in the marmoset monkey and human hippocampal formation.In the rat vas deferens and dog saphenous vein (two prototypical Y2 bioassays), BIIE0246 induced parallel shifts to the right of NPY concentration-response curves with pA2 values of 8.1 and 8.6, respectively. In the rat colon (a Y2/Y4 bioassay), BIIE0246 (1 μM) completely blocked the contraction induced by PYY3–36, but not that of [Leu31,Pro34]NPY (a Y1, Y4 and Y5 agonist) and hPP (a Y4 and Y5 agonist). Additionally...

GW627368X ((N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetyl} benzene sulphonamide): a novel, potent and selective prostanoid EP4 receptor antagonist

Wilson, Richard J; Giblin, Gerard M P; Roomans, Susan; Rhodes, Sharron A; Cartwright, Kerri-Ann; Shield, Vanessa J; Brown, Jason; Wise, Alan; Chowdhury, Jannatara; Pritchard, Sara; Coote, Jim; Noel, Lloyd S; Kenakin, Terry; Burns-Kurtis, Cynthia L; Morris
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
EN
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N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetyl}benzene sulphonamide (GW627368X) is a novel, potent and selective competitive antagonist of prostanoid EP4 receptors with additional human TP receptor affinity.At recombinant human prostanoid EP4 receptors expressed in HEK293 cells, GW627368X produced parallel rightward shifts of PGE2 concentration–effect (E/[A]) curves resulting in an affinity (pKb) estimate of 7.9±0.4 and a Schild slpoe not significantly different from unity. The affinity was independent of the agonist used.In rings of phenylephrine precontracted piglet saphenous vein, GW627368X (30–300 nM) produced parallel rightward displacement of PGE2 E/[A] curves (pKb=9.2±0.2; slope=1).GW627368X appears to bind to human prostanoid TP receptors but not the TP receptors of other species. In human washed platelets, GW627368X (10 μM) produced 100% inhibition of U-46619 (EC100)-induced aggregation (approximate pA2 ∼7.0). However, in rings of rabbit and piglet saphenous vein and of guinea-pig aorta GW627368X (10 μM) did not displace U-46619 E/[A] curves indicating an affinity of <5.0 for rabbit and guinea-pig prostanoid TP receptors.In functional assays GW627368X is devoid of both agonism and antagonist affinity for prostanoid CRTH2...

Pre-incubation of guinea-pig myenteric plexus with beta-funaltrexamine: discrepancy between binding assays and bioassays.

Corbett, A. D.; Kostelitz, H. W.; McKnight, A. T.; Paterson, S. J.; Robson, L. E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1985 EN
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The acute effects of beta-funaltrexamine and the effects of pre-incubation with this compound were examined in five in vitro assay tissues and in selective binding assays in homogenates of guinea-pig brain and myenteric plexus. In competitive displacement assays with selective ligands, beta-funaltrexamine had highest affinity for the mu-binding site in the myenteric plexus and brain of guinea-pig. Its affinity for the kappa-site was about 15% of that for the mu-site. Pre-incubation of the assay tissues with beta-funaltrexamine caused an increase in the IC50 values of mu- and delta-receptor agonists but not of kappa-agonists. Although in bioassays on the myenteric plexus-longitudinal muscle preparation of the guinea-pig, the IC50 value of the mu-receptor ligand [D-Ala2, MePhe4, Gly-ol5] enkephalin was increased up to 124 fold, its binding at the mu-site in homogenates of the preparation was not affected by this treatment. These findings indicate that the effects of pre-incubation with beta-funaltrexamine on agonist potency of the mu-receptor ligand are due to an interference with the coupling mechanism between the mu-binding site and the effector system.

New, long-acting, potent bradykinin antagonists.

Lembeck, F.; Griesbacher, T.; Eckhardt, M.; Henke, S.; Breipohl, G.; Knolle, J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1991 EN
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1. Three new bradykinin (BK) antagonists, D-Arg0-Hyp3-Thi5-D-Tic7-Oic8-BK (compound I), D-Arg0-Hyp3-D-Tic7-Oic8-BK (compound II), and Arg(Tos)1-Hyp3-Thi5-D-Tic7-Oic8-BK (compound III), were tested against the effects of BK in 9 bioassay preparations including visceral smooth muscles, vasoconstriction, plasma protein extravasation, release of prostaglandin E2, bronchoconstriction, and stimulation of afferent C-fibre nociceptors. In some of these tests the effects of the new compounds were compared with those of the antagonist D-Arg0-Hyp2-Thi5,8-D-Phe7-BK (compound IV), described by Stewart & Vavrek (1987). 2. For all bioassays the general rank order of potency of the compounds was found to be I greater than II greater than III much greater than IV. The new antagonists were long-acting; in some bioassays their effects outlasted the duration of the experiment. 3. The inhibitory effects of the new BK antagonists were specific for BK; actions of noradrenaline, angiotensin II, acetylcholine or histamine were unaffected by the antagonists. They did not stimulate the release of histamine or prostaglandins. An agonistic effect was observed only with very high concentrations of compounds I and II in the plasma protein extravasation test. 4. The long duration of action of the new BK antagonists is probably due to a high and long-lasting affinity to the BK receptors. A high resistance of the antagonists to enzymatic destruction may be another reason. 5. The new BK antagonists will be valuable tools for the investigation of the pathophysiological role of BK. In addition they may offer a potential for therapeutic applications.

Highly sensitive, quantitative cell-based assay for prions adsorbed to solid surfaces

Edgeworth, Julie Ann; Jackson, Graham S.; Clarke, Anthony R.; Weissmann, Charles; Collinge, John
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
EN
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Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of humans and animals, such as variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Prions pose significant public health concerns, including contamination of blood products and surgical instruments; require laborious and often insensitive animal bioassay to detect; and resist conventional hospital sterilization methods. A major experimental advance was the cell culture-based scrapie cell assay, allowing prion titres to be estimated more precisely and an order of magnitude faster than by animal bioassays. Here we describe a bioassay method that exploits the marked binding affinity of prions to steel surfaces. Using steel wires as a concentrating and sensitization tool and combining with an adapted scrapie cell endpoint assay we can achieve, for mouse prions, a sensitivity 100× higher than that achieved in standard mouse bioassays. The rapidity and sensitivity of this assay offers a major advance over small animal bioassay in many aspects of prion research. In addition, its specific application in assay of metal-bound prions allows evaluation of novel prion decontamination methods.

Reactivation of the vanchrobactin siderophore system of Vibrio anguillarum by removal of a chromosomal insertion sequence originated in plasmid pJM1 encoding the anguibactin siderophore system

Naka, Hiroaki; López, Claudia S.; Crosa, Jorge H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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A chromosomal gene cluster encoding vanchrobactin biosynthesis and transport genes was identified in the Vibrio anguillarum serotype O1 strain, 775(pJM1), harbouring the anguibactin biosynthetic genes in the pJM1 plasmid. In this strain only anguibactin is produced as the vanchrobactin chromosome cluster has a RS1 transposition insertion into vabF, one of the vanchrobactin biosynthesis genes. Removal of this RS1 generating 775(pJM1)Δtnp, still resulted in the detection of only anguibactin in specific bioassays. Surprisingly, when the pJM1 plasmid was not present as in the plasmidless strain H775-3, removal of the RS1 resulted in the detection of only vanchrobactin. These results thus can be interpreted as if presence of the pJM1 plasmid or of anguibactin itself is associated with the lack of detection of the vanchrobactin siderophore in bioassays. As high-performance liquid chromatography (HPLC) and mass spectrometry analysis demonstrated that both vanchrobactin and anguibactin were indeed produced in 775(pJM1)Δtnp, it is clear that the pJM1-encoded anguibactin siderophore has higher affinity for iron than the vanchrobactin system in strains in which both systems are expressed at the same time. Our results underscore the importance of the anguibactin system in the survival of V. anguillarum 775 under conditions of iron limitation.

Luminescent trimethoprim-polyaminocarboxylate lanthanide complex conjugates for selective protein labeling and time-resolved bioassays

Reddy, D. Rajasekhar; Pedró Rosa, Laura E.; Miller, Lawrence W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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Labeling proteins with long-lifetime emitting lanthanide (III) chelate reporters enables sensitive, time-resolved luminescence bioaffinity assays. Heterodimers of trimethoprim (TMP) covalently linked to various cs124-sensitized, polyaminocarboxylate chelates stably retain lanthanide ions and exhibit quantum yields of europium emission up to 20% in water. A time-resolved, luminescence resonance energy transfer (LRET) assay showed that TMP-polyaminocarboxylates bind to Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins with nanomolar affinity in purified solutions and in bacterial lysates. The ability to selectively impart terbium or europium luminescence to fusion proteins in complex physiological mixtures bypasses the need for specific antibodies and simplifies sample preparation.

Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab

Papadopoulos, Nicholas; Martin, Joel; Ruan, Qin; Rafique, Ashique; Rosconi, Michael P.; Shi, Ergang; Pyles, Erica A.; Yancopoulos, George D.; Stahl, Neil; Wiegand, Stanley J.
Fonte: Springer Netherlands Publicador: Springer Netherlands
Tipo: Artigo de Revista Científica
EN
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Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A...

Recent Progress in Nucleic Acid Aptamer-Based Biosensors and Bioassays

Mok, Wendy; Li, Yingfu
Fonte: Molecular Diversity Preservation International (MDPI) Publicador: Molecular Diversity Preservation International (MDPI)
Tipo: Artigo de Revista Científica
Publicado em 07/11/2008 EN
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As the key constituents of the genetic code, the importance of nucleic acids to life has long been appreciated. Despite being composed of only four structurally similar nucleotides, single-stranded nucleic acids, as in single-stranded DNAs and RNAs, can fold into distinct three-dimensional shapes due to specific intramolecular interactions and carry out functions beyond serving as templates for protein synthesis. These functional nucleic acids (FNAs) can catalyze chemical reactions, regulate gene expression, and recognize target molecules. Aptamers, whose name is derived from the Latin word aptus meaning “to fit”, are oligonucleotides that can bind their target ligands with high affinity and specificity. Since aptamers exist in nature but can also be artificially isolated from pools of random nucleic acids through a process called in vitro selection, they can potentially bind a diverse array of compounds. In this review, we will discuss the research that is being done to develop aptamers against various biomolecules, the progress in engineering biosensors by coupling aptamers to signal transducers, and the prospect of employing these sensors for a range of chemical and biological applications. Advances in aptamer technology emphasizes that nucleic acids are not only the fundamental molecules of life...

The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases

Abd-Ella, Aly; Stankiewicz, Maria; Mikulska, Karolina; Nowak, Wieslaw; Pennetier, Cédric; Goulu, Mathilde; Fruchart-Gaillard, Carole; Licznar, Patricia; Apaire-Marchais, Véronique; List, Olivier; Corbel, Vincent; Servent, Denis; Lapied, Bruno
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 11/05/2015 EN
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Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally...

Interleukin-2 Engineering for improved therapeutic effectiveness

Rao, Balaji Madhav, 1978-
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 106 leaves; 4783503 bytes; 4796102 bytes; application/pdf; application/pdf
EN_US
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(cont.) (K[d] [approximately] 10pM) for its private alpha receptor subunit, unlike wild-type IL-2 (K[d] [approximately] 10 nM). IL-2 mutants with picomolar affinity for IL-2Rα stimulate T cell growth responses quantitatively equivalent to those mediated by IL-15. Our results suggest that the contrasting effects of IL-2 and IL-15 on T cells in vivo are largely due to the 1,000-fold different affinities of wild-type IL-2 and IL-15 for their respective private alpha receptor subunits.; Interleukin-2 (IL-2) is an immunomodulatory cytokine that is clinically relevant for the treatment of metastatic renal cell carcinoma and melanoma. The primary objective of the research presented in this thesis was to generate IL-2 mutants with potentially improved therapeutic effectiveness. Based on qualitative considerations and simple mathematical modeling, we hypothesized that IL-2 mutants with increased affinity for the alpha subunit of the IL-2 receptor (IL-2Rα) would have increased potency for proliferation of activated T cells and hence potentially improved therapeutic value. Yeast surface display and directed evolution were used to generate a class of IL-2 mutants with enhanced IL-2Rα affinity. In a novel pulsed bioassay designed to approximate the rapid systemic clearance pharmacokinetics of IL-2...

Entwicklung und Miniaturisierung heterogener Fluoreszenz-Bioassays basierend auf FRET in Nanoliterkavitäten aus Kunststoff; Development and miniaturisation of heterogeneous fluorescence bioassays based on FRET in nanoliter cavities produced in platic

Seidel, Michael
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
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In dieser Arbeit wurden zwei heterogene Assays basierend auf FRET entwickelt und auf dem Nanotiterplattenformat miniaturisiert. Im Phasenseparation Fluoreszenz-immunoassay (PSFIA) wurde gezeigt, dass ein kompetitiver heterogener Fluoreszenzimmunoassay in Nanotiterplatten (NTP) mit einem Fassungsvolumen von 75 nL pro Kavität durchführbar war, indem man den strahlungslosen Energietransfer an der heterogenen Phase als waschschrittfreies Trennprinzip einsetzte. Ein wichtiger Punkt des heterogenen Nachweisformates bestand darin, Protokolle für die Oberflächenchemie auf mikrostrukturierten Kunststoffträgern zu etablieren. Die Oberfläche musste sowohl affnitätsbindende als auch fluoreszenzlöschende Eigenschaften haben. Die einfachste Methode war, die NTP mit einem Protein adsorptiv zu belegen. Dazu wurde beispielhaft BSA immobilisiert, an welches sowohl das Analytderivat Atrazincapronsäure als auch der Akzeptorfarbstoff Cy5.5 gekoppelt waren. Im PSFIA wurde der Cy5-markierte Antikörper Anti-Atrazin eingesetzt, um dessen Bindungstaschen der Analyt in Lösung und das immobilisierte Analytderivat konkurrierten. Mit dieser Immobilisierungsstrategie konnte im PSFIA Atrazin mit einer Nachweisgrenze (LOD) von 0,03 µg/L detektiert werden. Die LOD konnte noch weiter herabgesetzt werden...

Characterization of a high-affinity iron transport system in Acinetobacter baumannii.

Echenique, J R; Arienti, H; Tolmasky, M E; Read, R R; Staneloni, R J; Crosa, J H; Actis, L A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1992 EN
Relevância na Pesquisa
26.879424%
Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes.