Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface.

PubMed

Gorelik, Maryna; Davidson, Alan R

2012-03-16

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.

Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

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

Stanfield, R.L.; Dooley, H.; Verdino, P.

2007-07-13

Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts withmore » antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.« less

A Lectin from Platypodium elegans with Unusual Specificity and Affinity for Asymmetric Complex N-Glycans*

PubMed Central

Benevides, Raquel Guimarães; Ganne, Géraldine; Simões, Rafael da Conceição; Schubert, Volker; Niemietz, Mathäus; Unverzagt, Carlo; Chazalet, Valérie; Breton, Christelle; Varrot, Annabelle; Cavada, Benildo Sousa; Imberty, Anne

2012-01-01

Lectin activity with specificity for mannose and glucose has been detected in the seed of Platypodium elegans, a legume plant from the Dalbergieae tribe. The gene of Platypodium elegans lectin A has been cloned, and the resulting 261-amino acid protein belongs to the legume lectin family with similarity with Pterocarpus angolensis agglutinin from the same tribe. The recombinant lectin has been expressed in Escherichia coli and refolded from inclusion bodies. Analysis of specificity by glycan array evidenced a very unusual preference for complex type N-glycans with asymmetrical branches. A short branch consisting of one mannose residue is preferred on the 6-arm of the N-glycan, whereas extensions by GlcNAc, Gal, and NeuAc are favorable on the 3-arm. Affinities have been obtained by microcalorimetry using symmetrical and asymmetrical Asn-linked heptasaccharides prepared by the semi-synthetic method. Strong affinity with Kd of 4.5 μm was obtained for both ligands. Crystal structures of Platypodium elegans lectin A complexed with branched trimannose and symmetrical complex-type Asn-linked heptasaccharide have been solved at 2.1 and 1.65 Å resolution, respectively. The lectin adopts the canonical dimeric organization of legume lectins. The trimannose bridges the binding sites of two neighboring dimers, resulting in the formation of infinite chains in the crystal. The Asn-linked heptasaccharide binds with the 6-arm in the primary binding site with extensive additional contacts on both arms. The GlcNAc on the 6-arm is bound in a constrained conformation that may rationalize the higher affinity observed on the glycan array for N-glycans with only a mannose on the 6-arm. PMID:22692206

Significant relaxation of residual negative carrier in polar Alq3 film directly detected by high-sensitivity photoemission

NASA Astrophysics Data System (ADS)

Kinjo, Hiroumi; Lim, Hyunsoo; Sato, Tomoya; Noguchi, Yutaka; Nakayama, Yasuo; Ishii, Hisao

2016-02-01

Tris(8-hydroxyquinoline)aluminum (Alq3) has been widely applied as a good electron-injecting layer (EIL) in organic light-emitting diodes. High-sensitivity photoemission measurement revealed a clear photoemission by visible light, although its ionization energy is 5.7 eV. This unusual photoemission is ascribed to Alq3 anions captured by positive polarization charges. The observed electron detachment energy of the anion was about 1 eV larger than the electron affinity reported by inverse photoemission. This difference suggests that the injected electron in the Alq3 layer is energetically relaxed, leading to the reduction in injection barrier. This nature is one of the reasons why Alq3 worked well as the EIL.

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives.

PubMed

Plesniak, Leigh; Horiuchi, Yuki; Sem, Daniel; Meinenger, David; Stiles, Linda; Shaffer, Jennifer; Jennings, Patricia A; Adams, Joseph A

2002-11-26

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.

Kelvin probe force microscopy studies of the charge effects upon adsorption of carbon nanotubes and C60 fullerenes on hydrogen-terminated diamond

NASA Astrophysics Data System (ADS)

Kölsch, S.; Fritz, F.; Fenner, M. A.; Kurch, S.; Wöhrl, N.; Mayne, A. J.; Dujardin, G.; Meyer, C.

2018-01-01

Hydrogen-terminated diamond is known for its unusually high surface conductivity that is ascribed to its negative electron affinity. In the presence of acceptor molecules, electrons are expected to transfer from the surface to the acceptor, resulting in p-type surface conductivity. Here, we present Kelvin probe force microscopy (KPFM) measurements on carbon nanotubes and C60 adsorbed onto a hydrogen-terminated diamond(001) surface. A clear reduction in the Kelvin signal is observed at the position of the carbon nanotubes and C60 molecules as compared with the bare, air-exposed surface. This result can be explained by the high positive electron affinity of carbon nanotubes and C60, resulting in electron transfer from the surface to the adsorbates. When an oxygen-terminated diamond(001) is used instead, no reduction in the Kelvin signal is obtained. While the presence of a charged adsorbate or a difference in work function could induce a change in the KPFM signal, a charge transfer effect of the hydrogen-terminated diamond surface, by the adsorption of the carbon nanotubes and the C60 fullerenes, is consistent with previous theoretical studies.

Trappeindia himalayensis gen. et sp. nov., a sequestrate fungus with potential affinity to Strobilomyces (Basidiomycotina, Boletales)

Treesearch

M.A. Castellano; S.L. Miller; L. Singh; T.N. Lakhanpal

2012-01-01

An unusual sequestrate fungus forming ectomycorrhizae with Cedrus deodora (Roxb.) Laud. forms sporocarps in the northwestern Himalayas of India during spring. It has a dark brown to black peridium with a solid, white to brown, loculate gleba containing spherical, reticulate spores. It resembles no described genus and is described here as ...

Two new genera of Lumbriculidae (Annelida, Clitellata) from North Carolina, USA

USGS Publications Warehouse

Fend, S.V.; Lenat, D.R.

2007-01-01

Recent benthic macroinvertebrate collections from North Carolina contained many undescribed oligochaete taxa, mostly belonging to the family Lumbriculidae. Three of the new species had arrangements of spermathecae and atria previously unreported for the family, and were assigned to new two genera. Pilaridrilus is distinguished by the location of spermathecal pores five segments behind the male pores. The single species, Pilaridrilus uliginosus, also has unusually complex penes and spermathecae. Martinidrilus is distinguished by spermathecae beginning more than two segments anterior to the atrial segment, and also by the the vasa deferentia, which join a common duct before joining the atria. The two Martinidrilus species also have unusual digitiform blood vessels in posterior segments. Martinidrilus carolinensis has lateral spermathecae in VI, and Martinidrilus arenosus has dorsolateral spermathecae in VII and VIII. Because arrangement and morphology of reproductive organs do not resemble those of described lumbriculids, the phylogenetic affinities of the new species are not clear. These new genera and species were generally collected from areas of high water quality, suggesting that lumbriculids can be useful in water quality monitoring and conservation evaluation. Copyright ?? 2007 Magnolia Press.

A sulfhydryl-rich IgM protein with multiple serological specificities.

PubMed Central

Merlini, G; Zettervall, O; Forsgren, A; Galliano, M; Lindberg, A A; Svenson, S B; Pavesi, F; Turesson, I

1987-01-01

A monoclonal IgM lambda protein from a patient (E.T.) suffering from a lymphocytic lymphoma agglutinated Salmonella typhi bacteria and uncoated acryl particles. The antigenic determinant on Salmonella typhi bacteria was found to be 0-12 (alpha-D-Galp-(1-2)-alpha-D-Manp) while the structure on acryl particles recognized by IgM ET has not been defined. Both binding sites for bacteria and acryl particle determinants are localized on the same IgM molecule. The uncommon affinity of this IgM protein for some divalent heavy metal ions led to the finding of an unusually high content of sulfhydryl groups in the Fab portion of the molecule. PMID:2443287

Microgravity

NASA Image and Video Library

1998-12-01

As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site.

PubMed

Claveria-Gimeno, Rafael; Lanuza, Pilar M; Morales-Chueca, Ignacio; Jorge-Torres, Olga C; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

2017-01-31

Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities.

The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site

PubMed Central

Claveria-Gimeno, Rafael; Lanuza, Pilar M.; Morales-Chueca, Ignacio; Jorge-Torres, Olga C.; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

2017-01-01

Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities. PMID:28139759

Adsorption of polycyclic aromatic hydrocarbons by graphene and graphene oxide nanosheets.

PubMed

Wang, Jun; Chen, Zaiming; Chen, Baoliang

2014-05-06

The adsorption of naphthalene, phenanthrene, and pyrene onto graphene (GNS) and graphene oxide (GO) nanosheets was investigated to probe the potential adsorptive sites and molecular mechanisms. The microstructure and morphology of GNS and GO were characterized by elemental analysis, XPS, FTIR, Raman, SEM, and TEM. Graphene displayed high affinity to the polycyclic aromatic hydrocarbons (PAHs), whereas GO adsorption was significantly reduced after oxygen-containing groups were attached to GNS surfaces. An unexpected peak was found in the curve of adsorption coefficients (Kd) with the PAH equilibrium concentrations. The hydrophobic properties and molecular sizes of the PAHs affected the adsorption of G and GO. The high affinities of the PAHs to GNS are dominated by π-π interactions to the flat surface and the sieving effect of the powerful groove regions formed by wrinkles on GNS surfaces. In contrast, the adsorptive sites of GO changed to the carboxyl groups attaching to the edges of GO because the groove regions disappeared and the polar nanosheet surfaces limited the π-π interactions. The TEM and SEM images initially revealed that after loading with PAH, the conformation and aggregation of GNS and GO nanosheets dramatically changed, which explained the observations that the potential adsorption sites of GNS and GO were unusually altered during the adsorption process.

Pandoraviruses are highly derived phycodnaviruses

PubMed Central

2013-01-01

The recently discovered Pandoraviruses are by far the largest viruses known, with their 2 megabase genomes exceeding in size the genomes of numerous bacteria and archaea. Pandoraviruses show a distant relationship with other nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes, lack some of the NCLDV core genes and in particular do not appear to be specifically related to the other, better characterized family of giant viruses, the Mimiviridae. Here we report phylogenetic analysis of 6 core NCLDV genes that confidently places Pandoraviruses within the family Phycodnaviridae, with an apparent specific affinity with Coccolithoviruses. We conclude that, despite their many unusual characteristics, Pandoraviruses are highly derived phycodnaviruses. These findings imply that giant viruses have independently evolved from smaller NCLDV on at least two occasions. This article was reviewed by Patrick Forterre and Lakshminarayan Iyer. For the full reviews, see the Reviewers’ reports section. PMID:24148757

Immunological Control of Viral Infections in Bats and the Emergence of Viruses Highly Pathogenic to Humans

PubMed Central

Schountz, Tony; Baker, Michelle L.; Butler, John; Munster, Vincent

2017-01-01

Bats are reservoir hosts of many important viruses that cause substantial disease in humans, including coronaviruses, filoviruses, lyssaviruses, and henipaviruses. Other than the lyssaviruses, they do not appear to cause disease in the reservoir bats, thus an explanation for the dichotomous outcomes of infections of humans and bat reservoirs remains to be determined. Bats appear to have a few unusual features that may account for these differences, including evidence of constitutive interferon (IFN) activation and greater combinatorial diversity in immunoglobulin genes that do not undergo substantial affinity maturation. We propose these features may, in part, account for why bats can host these viruses without disease and how they may contribute to the highly pathogenic nature of bat-borne viruses after spillover into humans. Because of the constitutive IFN activity, bat-borne viruses may be shed at low levels from bat cells. With large naive antibody repertoires, bats may control the limited virus replication without the need for rapid affinity maturation, and this may explain why bats typically have low antibody titers to viruses. However, because bat viruses have evolved in high IFN environments, they have enhanced countermeasures against the IFN response. Thus, upon infection of human cells, where the IFN response is not constitutive, the viruses overwhelm the IFN response, leading to abundant virus replication and pathology. PMID:28959255

Purification of charybdotoxine, a specific inhibitor of the high-conductance Ca/sup 2 +/-activated K/sup +/ channel

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

Smith, C.; Phillips, M.; Miller, C.

1986-11-05

Charybdotoxim is a high-affinity specific inhibitor of the high-conductance Ca/sup 2 +/-activated K/sup +/ channel found in the plasma membranes of many vertebrate cell types. Using Ca/sup 2 +/-activated K/sup +/ channels reconstituted into planar lipid bilayer membranes as an assay, the authors have purified the toxin from the venom of the scorpion Leiurus quinquestriatus by a two-step procedure involving chromatofocusing on SP-Sephadex, followed by reversed-phase high-performance liquid chromatography. Charybdotoxin is shown to be a highly basic protein with a mass of 10 kDa. Under the standard assay conditions, the purified toxin inhibits the Ca/sup 2 +/-activated K/sup +/ channelmore » with an apparent dissociation constant of 3.5 nM. The protein is unusually stable, with inhibitory potency being insensitive to boiling or exposure to organic solvents. The toxin's activity is sensitive to chymotrypsin treatment and to acylation of lysine groups. The protein may be radioiodinated without loss of activity.« less

Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans*

PubMed Central

Handali, Melody; Neupane, Durga P.; Roychowdhury, Hridindu; Yukl, Erik T.

2015-01-01

ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn2+ and Mn2+ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn2+ or Mn2+ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn2+ over Mn2+ specificity, the Zn2+ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn2+-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn2+-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn2+-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn2+ specificity. PMID:25787075

Transcriptional regulation, metal binding properties and structure of Pden1597, an unusual zinc transport protein from Paracoccus denitrificans

DOE PAGES

Handali, Melody; Neupane, Durga P.; Roychowdhury, Hridindu; ...

2015-03-18

Here, ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn 2+ and Mn 2+ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn 2+ or Mn 2+ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties,more » and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn 2+ over Mn 2+ specificity, the Zn 2+ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn 2+-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn 2+-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn 2+-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn 2+ specificity.« less

Copper tolerance mediated by polyphosphate degradation and low-affinity inorganic phosphate transport system in Escherichia coli.

PubMed

Grillo-Puertas, Mariana; Schurig-Briccio, Lici Ariane; Rodríguez-Montelongo, Luisa; Rintoul, María Regina; Rapisarda, Viviana Andrea

2014-03-19

Metal tolerance in bacteria has been related to polyP in a model in which heavy metals stimulate the polymer hydrolysis, forming metal-phosphate complexes that are exported. As previously described in our laboratory, Escherichia coli cells grown in media containing a phosphate concentration >37 mM maintained an unusually high polyphosphate (polyP) level in stationary phase. The aim of the present work was to evaluate the influence of polyP levels as the involvement of low-affinity inorganic phosphate transport (Pit) system in E. coli copper tolerance. PolyP levels were modulated by the media phosphate concentration and/or using mutants in polyP metabolism. Stationary phase wild-type cells grown in high phosphate medium were significantly more tolerant to copper than those grown in sufficient phosphate medium. Copper addition to tolerant cells induced polyP degradation by PPX (an exopolyphosphatase), phosphate efflux and membrane polarization. ppk-ppx- (unable to synthesize/degrade polyP), ppx- (unable to degrade polyP) and Pit system mutants were highly sensitive to metal even in high phosphate media. In exponential phase, CopA and polyP-Pit system would act simultaneously to detoxify the metal or one could be sufficient to safeguard the absence of the other. Our results support a mechanism for copper detoxification in exponential and stationary phases of E. coli, involving Pit system and degradation of polyP. Data reflect the importance of the environmental phosphate concentration in the regulation of the microbial physiological state.

Unusual Genomic Traits Suggest Methylocystis bryophila S285 to Be Well Adapted for Life in Peatlands

PubMed Central

Han, Dongfei; Dedysh, Svetlana N

2018-01-01

Abstract The genus Methylocystis belongs to the class Alphaproteobacteria, the family Methylocystaceae, and encompasses aerobic methanotrophic bacteria with the serine pathway of carbon assimilation. All Methylocystis species are able to fix dinitrogen and several members of this genus are also capable of using acetate or ethanol in the absence of methane, which explains their wide distribution in various habitats. One additional trait that enables their survival in the environment is possession of two methane-oxidizing isozymes, the conventional particulate methane monooxygenase (pMMO) with low-affinity to substrate (pMMO1) and the high-affinity enzyme (pMMO2). Here, we report the finished genome sequence of Methylocystis bryophila S285, a pMMO2-possessing methanotroph from a Sphagnum-dominated wetland, and compare it to the genome of Methylocystis sp. strain SC2, which is the first methanotroph with confirmed high-affinity methane oxidation potential. The complete genome of Methylocystis bryophila S285 consists of a 4.53 Mb chromosome and one plasmid, 175 kb in size. The genome encodes two types of particulate MMO (pMMO1 and pMMO2), soluble MMO and, in addition, contains a pxmABC-like gene cluster similar to that present in some gammaproteobacterial methanotrophs. The full set of genes related to the serine pathway, the tricarboxylic acid cycle as well as the ethylmalonyl-CoA pathway is present. In contrast to most described methanotrophs including Methylocystis sp. strain SC2, two different types of nitrogenases, that is, molybdenum–iron and vanadium–iron types, are encoded in the genome of strain S285. This unique combination of genome-based traits makes Methylocystis bryophila well adapted to the fluctuation of carbon and nitrogen sources in wetlands. PMID:29390143

Stable coordination of the inhibitory Ca2+ ion at MIDAS in integrin CD11b/CD18 by an antibody-derived ligand aspartate: Implications for integrin regulation and structure-based drug design

PubMed Central

Mahalingam, Bhuvaneshwari; Ajroud, Kaouther; Alonso, Jose Luis; Anand, Saurabh; Adair, Brian; Horenstein, Alberto L; Malavasi, Fabio; Xiong, Jian-Ping; Arnaout, M. Amin

2011-01-01

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal-ion-dependent-adhesion site (MIDAS) in the integrin A-domain. This interaction stabilizes the A-domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A-domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking monoclonal antibody (mAb) 107 binds MIDAS of integrin CD11b/CD18 A-domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over Mg2+ at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of Ca2+ at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+. Binding of Fab 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that denticity of the ligand Asp/Glu can modify divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists. PMID:22095715

Rapid dehalogenation of pesticides and organics at the interface of reduced graphene oxide-silver nanocomposite.

PubMed

Koushik, Dibyashree; Sen Gupta, Soujit; Maliyekkal, Shihabudheen M; Pradeep, T

2016-05-05

This paper reports dehalogenation of various organohalides, especially aliphatic halocarbons and pesticides at reduced graphene oxide-silver nanocomposite (RGO@Ag). Several pesticides as well as chlorinated and fluorinated alkyl halides were chosen for this purpose. The composite and the products of degradation were characterized thoroughly by means of various microscopic and spectroscopic techniques. A sequential two-step mechanism involving dehalogenation of the target pollutants by silver nanoparticles followed by adsorption of the degraded compounds onto RGO was revealed. The composite showed unusual adsorption capacity, as high as 1534 mg/g, which facilitated the complete removal of the pollutants. Irrespective of the pollutants tested, a pseudo-second-order rate equation best described the adsorption kinetics. The affinity of the composite manifested chemical differences. The high adsorption capacity and re-usability makes the composite an excellent substrate for purification of water. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural modifications due to interface chemistry at metal-nitride interfaces

DOE PAGES

Yadav, S. K.; Shao, S.; Wang, J.; ...

2015-11-27

Based on accurate first principles density functional theory (DFT) calculations, an unusual phenomenon of interfacial structural modifications, due to the interface chemistry influence is identified at two metal-nitride interfaces with strong metal-nitrogen affinity, Al/TiN {111} and Al/VN {111} interfaces. It is shown that at such interfaces, a faulted stacking structure is energetically preferred on the Al side of the interface. And both intrinsic and extrinsic stacking fault energies in the vicinity Al layers are negligibly small. However, such phenomenon does not occur in Pt/TiN and Pt/VN interfaces because of the weak Pt-N affinity. As a result, corresponding to structural energiesmore » of metal-nitride interfaces, the linear elasticity analysis predicts characteristics of interfacial misfit dislocations at metal-nitride interfaces.« less

Structural modifications due to interface chemistry at metal-nitride interfaces

PubMed Central

Yadav, S. K.; Shao, S.; Wang, J.; Liu, X.-Y.

2015-01-01

Based on accurate first principles density functional theory (DFT) calculations, an unusual phenomenon of interfacial structural modifications, due to the interface chemistry influence is identified at two metal-nitride interfaces with strong metal-nitrogen affinity, Al/TiN {111} and Al/VN {111} interfaces. It is shown that at such interfaces, a faulted stacking structure is energetically preferred on the Al side of the interface. And both intrinsic and extrinsic stacking fault energies in the vicinity Al layers are negligibly small. However, such phenomenon does not occur in Pt/TiN and Pt/VN interfaces because of the weak Pt-N affinity. Corresponding to structural energies of metal-nitride interfaces, the linear elasticity analysis predicts characteristics of interfacial misfit dislocations at metal-nitride interfaces. PMID:26611639

Cytological studies of lunar treated tissue cultures

NASA Technical Reports Server (NTRS)

Halliwell, R. S.

1972-01-01

An electron microscopic study was made of botanical materials, particularly pine tissues, treated with lunar materials collected by Apollo 12 quarantine mission. Results show unusual structural changes within several of the treated tissues. The bodies, as yet unidentified, resemble virus particles observed within infected plant cells. Although the size and shape of the structures are comparable to rod shaped virus particles such as Tobacco mosaic, the numerical distribution, affinity for stains, and intercellular location are different.

Nickel binding to NikA: an additional binding site reconciles spectroscopy, calorimetry and crystallography.

PubMed

Addy, Christine; Ohara, Masato; Kawai, Fumihiro; Kidera, Akinori; Ikeguchi, Mitsunori; Fuchigami, Sotaro; Osawa, Masanori; Shimada, Ichio; Park, Sam-Yong; Tame, Jeremy R H; Heddle, Jonathan G

2007-02-01

Intracellular nickel is required by Escherichia coli as a cofactor for a number of enzymes and is necessary for anaerobic respiration. However, high concentrations of nickel are toxic, so both import and export systems have evolved to control the cellular level of the metal. The nik operon in E. coli encodes a nickel-uptake system that includes the periplasmic nickel-binding protein NikA. The crystal structures of wild-type NikA both bound to nickel and in the apo form have been solved previously. The liganded structure appeared to show an unusual interaction between the nickel and the protein in which no direct bonds are formed. The highly unusual nickel coordination suggested by the crystal structure contrasted strongly with earlier X-ray spectroscopic studies. The known nickel-binding site has been probed by extensive mutagenesis and isothermal titration calorimetry and it has been found that even large numbers of disruptive mutations appear to have little effect on the nickel affinity. The crystal structure of a binding-site mutant with nickel bound has been solved and it is found that nickel is bound to two histidine residues at a position distant from the previously characterized binding site. This novel site immediately resolves the conflict between the crystal structures and other biophysical analyses. The physiological relevance of the two binding sites is discussed.

Structural Principles in the Development of Cyclic Peptidic Enzyme Inhibitors

PubMed Central

Xu, Peng; Andreasen, Peter A.; Huang, Mingdong

2017-01-01

This review summarizes our studies in the development of small cyclic peptides for specifically modulating enzyme activity. Serine proteases share highly similar active sites but perform diverse physiological and pathological functions. From a phage-display peptide library, we isolated two mono-cyclic peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), which inhibit the activity of human and murine urokinase-type plasminogen activators (huPA and muPA) with Ki values in the micromolar or sub-micromolar range, respectively. The following affinity maturations significantly enhanced the potencies of the two peptides, 10-fold and >250-fold for upain-1 and mupain-1, respectively. The most potent muPA inhibitor has a potency (Ki = 2 nM) and specificity comparable to mono-clonal antibodies. Furthermore, we also found an unusual feature of mupain-1 that its inhibitory potency can be enhanced by increasing the flexibility, which challenges the traditional viewpoint that higher rigidity leading to higher affinity. Moreover, by changing a few key residues, we converted mupain-1 from a uPA inhibitor to inhibitors of other serine proteases, including plasma kallikrein (PK) and coagulation factor XIa (fXIa). PK and fXIa inhibitors showed Ki values in the low nanomolar range and high specificity. Our studies demonstrate the versatility of small cyclic peptides to engineer inhibitory potency against serine proteases and to provide a new strategy for generating peptide inhibitors of serine proteases. PMID:29104489

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

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

James, W.M.; Emerick, M.C.; Agnew, W.S.

1989-07-11

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated bindingmore » and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.« less

Structural basis for IL-1α recognition by a modified DNA aptamer that specifically inhibits IL-1α signaling

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

Ren, Xiaoming; Gelinas, Amy D.; von Carlowitz, Ira

IL-1α is an essential cytokine that contributes to inflammatory responses and is implicated in various forms of pathogenesis and cancer. Here we report a naphthyl modified DNA aptamer that specifically binds IL-1α and inhibits its signaling pathway. By solving the crystal structure of the IL-1α/aptamer, we provide a high-resolution structure of this critical cytokine and we reveal its functional interaction interface with high-affinity ligands. The non-helical aptamer, which represents a highly compact nucleic acid structure, contains a wealth of new conformational features, including an unknown form of G-quadruplex. The IL-1α/aptamer interface is composed of unusual polar and hydrophobic elements, alongmore » with an elaborate hydrogen bonding network that is mediated by sodium ion. IL-1α uses the same interface to interact with both the aptamer and its cognate receptor IL-1RI, thereby suggesting a novel route to immunomodulatory therapeutics.« less

Low-basicity 5-HT7 Receptor Agonists Synthesized Using the van Leusen Multicomponent Protocol.

PubMed

Hogendorf, Adam S; Hogendorf, Agata; Kurczab, Rafał; Satała, Grzegorz; Lenda, Tomasz; Walczak, Maria; Latacz, Gniewomir; Handzlik, Jadwiga; Kieć-Kononowicz, Katarzyna; Wierońska, Joanna M; Woźniak, Monika; Cieślik, Paulina; Bugno, Ryszard; Staroń, Jakub; Bojarski, Andrzej J

2017-05-04

A series of 5-aryl-1-alkylimidazole derivatives was synthesized using the van Leusen multicomponent reaction. The chemotype is the first example of low-basicity scaffolds exhibiting high affinity for 5-HT 7 receptor together with agonist function. The chosen lead compounds 3-(1-ethyl-1H-imidazol-5-yl)-5-iodo-1H-indole (AGH-107, 1o, K i 5-HT7  = 6 nM, EC 50  = 19 nM, 176-fold selectivity over 5-HT 1A R) and 1e (5-methoxy analogue, K i 5-HT7  = 30 nM, EC 50  = 60 nM) exhibited high selectivity over related CNS targets, high metabolic stability and low toxicity in HEK-293 and HepG2 cell cultures. A rapid absorption to the blood, high blood-brain barrier permeation and a very high peak concentration in the brain (C max  = 2723 ng/g) were found for 1o after i.p. (5 mg/kg) administration in mice. The compound was found active in novel object recognition test in mice, at 0.5, 1 and 5 mg/kg. Docking to 5-HT 7 R homology models indicated a plausible binding mode which explain the unusually high selectivity over the related CNS targets. Halogen bond formation between the most potent derivatives and the receptor is consistent with both the docking results and SAR. 5-Chlorine, bromine and iodine substitution resulted in a 13, 27 and 89-fold increase in binding affinities, respectively, and in enhanced 5-HT 1A R selectivity.

A comparison of blood nitric oxide metabolites and hemoglobin functional properties among diving mammals.

PubMed

Fago, Angela; Parraga, Daniel Garcia; Petersen, Elin E; Kristensen, Niels; Giouri, Lea; Jensen, Frank B

2017-03-01

The ability of marine mammals to hunt prey at depth is known to rely on enhanced oxygen stores and on selective distribution of blood flow, but the molecular mechanisms regulating blood flow and oxygen transport remain unresolved. To investigate the molecular mechanisms that may be important in regulating blood flow, we measured concentration of nitrite and S-nitrosothiols (SNO), two metabolites of the vasodilator nitric oxide (NO), in the blood of 5 species of marine mammals differing in their dive duration: bottlenose dolphin, South American sea lion, harbor seal, walrus and beluga whale. We also examined oxygen affinity, sensitivity to 2,3-diphosphoglycerate (DPG) and nitrite reductase activity of the hemoglobin (Hb) to search for possible adaptive variations in these functional properties. We found levels of plasma and red blood cells nitrite similar to those reported for terrestrial mammals, but unusually high concentrations of red blood cell SNO in bottlenose dolphin, walrus and beluga whale, suggesting enhanced SNO-dependent signaling in these species. Purified Hbs showed similar functional properties in terms of oxygen affinity and sensitivity to DPG, indicating that reported large variations in blood oxygen affinity among diving mammals likely derive from phenotypic variations in red blood cell DPG levels. The nitrite reductase activities of the Hbs were overall slightly higher than that of human Hb, with the Hb of beluga whale, capable of longest dives, having the highest activity. Taken together, these results underscore adaptive variations in circulatory NO metabolism in diving mammals but not in the oxygenation properties of the Hb. Copyright © 2016 Elsevier Inc. All rights reserved.

Aqueous alteration and brecciation in Bells, an unusual, saponite-bearing, CM chondrite

NASA Astrophysics Data System (ADS)

Brearley, Adrian J.

1995-06-01

The petrological and mineralogical characteristics of the unusual CM2 chondrite, Bells, have been investigated in detail by scanning electron microscopy (SEM), electron microprobe analysis (EPMA), and transmission electron microscopy (TEM). Bells is a highly brecciated chondrite which contains few intact chondrules, a very low abundance of refractory inclusions, and is notable in having an unusually high abundance of magnetite, which is disseminated throughout the fine-grained matrix. Fragmental olivines and pyroxenes are common and, based on compositional data, appear to have been derived from chondrules as a result of extensive brecciation. The fine-grained mineralogy of matrix in Bells differs considerably from other CM chondrites and has closer affinities to matrix in CI chondrites. The dominant phases are fine-grained saponite interlayered with serpentine, and phases such as tochilinite and cronstedtite, which are typical of CM chondrite matrices, are entirely absent. Pentlandite, pyrrhotite, magnetite, anhydrite, calcite, and rare Ti-oxides also occur as accessory phases. Based on its oxygen and noble gas isotopic compositions (Zadnik, 1985; Rowe et al., 1994), Bells can be considered to be a CM2 chondrite, although its bulk composition shows some departures from the typical range exhibited by this group. However, these variations in bulk chemistry are entirely consistent with the observed mineralogy of Bells. The unusual fine-grained mineralogy of Bells matrix can be reasonably attributed to the combined effects of aqueous alteration and advanced brecciation in a parent body environment. Extensive brecciation has assisted aqueous alteration by reducing chondrules and mineral grains into progressively smaller grains with high surface areas, which are more susceptible to dissolution reactions involving aqueous fluids. This has resulted in the preferential dissolution of Fe-rich chondrule olivines, which are now completely absent in Bells although present in other CM chondrites. The formation of saponite in Bells probably resulted from the dissolution of relatively silica-rich phases, such as pyroxene and olivine, that were derived from chondrules. The result of such dissolution reactions would be to increase the activity of silica in the fluid phase, at least on a localized scale, stabilizing saponite in preference to serpentine. An increase in aSiO 2 would also have destabilized preexisting cronstedtite which may have reacted to form magnetite and MgFe serpentine under conditions of constant ƒO 2 .

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

Wurzburg, Beth A.; Tarchevskaya, Svetlana S.; Jardetzky, Theodore S.

CD23, the low-affinity receptor for IgE (Fc{var_epsilon}RII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca{sup 2+}. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in humanmore » CD23. Conformational differences between the apo and Ca{sup 2+} bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.« less

Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin-dockerin interactions.

PubMed

Hamberg, Yuval; Ruimy-Israeli, Vered; Dassa, Bareket; Barak, Yoav; Lamed, Raphael; Cameron, Kate; Fontes, Carlos M G A; Bayer, Edward A; Fried, Daniel B

2014-01-01

Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin-dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be "purified" via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin-dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome's total cohesin and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

Exclusive nuclear location of estrogen receptors in Squalus testis.

PubMed Central

Callard, G V; Mak, P

1985-01-01

An estrogen (E)-binding molecule having both occupied and unoccupied sites is restricted to nuclear subfractions in the testis of the spiny dogfish (Squalus acanthias). We investigated the hypothesis that a species characterized by high body-fluid osmolarity (1010 mosM) has an estrogen receptor (ER) that binds to chromatin with high affinity and consequently resists redistribution during tissue processing. Although the steroid binding and sedimentation properties of the Squalus nuclear ER conformed to those of classical ER, its elution maximum from DNA-cellulose was unusually high (0.55 M NaCl). A tendency to adhere tightly to cell nuclei was reflected in the high salt concentration (0.43 M KCl) required to extract 50% of the receptors from the nuclear compartment during homogenization and in the stability of the nuclear ER population in the presence of high concentrations of a nonionic solute (urea) or increased buffer volume. Mixing and redistribution experiments showed that nuclear ER could be quantitatively and qualitatively measured in cytosolic extracts, ruling out the possibility that soluble receptors were being masked. Although Squalus oviduct ER was similar to that of testis, ER in the testis and liver of a related elasmobranch (Potamotrygon) that maintains osmotic equilibrium at 300 mosM more closely resembled mammalian ER in its elution maximum from DNA-cellulose (0.22 M NaCl) and cytosolic/nuclear ratios in low-salt buffers. We conclude that Squalus testis has a single ER pool located exclusively in the nuclear compartment. These observations support a revised concept of steroid action and further indicate that the chromatin affinity of the hormone-ER complex is an important factor in determining subfractional distribution during tissue processing. PMID:3856265

An unusual new species of paguroid (Crustacea, Anomura, Paguridae) from deep waters of the Gulf of Mexico

PubMed Central

Lemaitre, Rafael; Vázquez-Bader, Ana Rosa; Gracia, Adolfo

2014-01-01

Abstract A new hermit crab species of the family Paguridae, Tomopaguropsis ahkinpechensis sp. n., is described from deep waters (780–827 m) of the Gulf of Mexico. This is the second species of Tomopaguropsis known from the western Atlantic, and the fifth worldwide. The new species is morphologically most similar to a species from Indonesia, Tomopaguropsis crinita McLaughlin, 1997, the two having ocular peduncles that diminish in width distally, reduced corneas, dense cheliped setation, and males lacking paired pleopods 1. The calcified figs on the branchiostegite and anterodorsally on the posterior carapace, and the calcified first pleonal somite that is not fused to the last thoracic somite, are unusual paguroid characters. A discussion of the affinities and characters that define this new species is included, along with a key to all five species of Tomopaguropsis. PMID:25408613

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

PubMed

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

2015-01-01

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, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes.

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

PubMed Central

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

2015-01-01

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, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes. PMID:25961834

THE STRUCTURAL BIOLOGY OF THE FGF19 SUBFAMILY

PubMed Central

Beenken, Andrew; Mohammadi, Moosa

2013-01-01

The ability of the Fibroblast Growth Factor (FGF) 19 subfamily to signal in an endocrine fashion sets this subfamily apart from the remaining five FGF subfamilies known for their paracrine functions during embryonic development. Compared to the members of paracrine FGF subfamiles, the three members of the FGF 19 subfamily, namely FGF19, FGF21 and FGF23, have poor affinity for heparan sulfate (HS) and therefore can diffuse freely in the HS-rich extracellular matrix to enter into the bloodstream. In further contrast to paracrine FGFs, FGF 19 subfamily members have unusually poor affinity for their cognate FGF receptors (FGFRs) and therefore cannot bind and activate them in a solely HS-dependent fashion. As a result, the FGF 19 subfamily requires α/βklotho coreceptor proteins in order to bind, dimerize and activate their cognate FGFRs. This klotho-dependency also determines the tissue specificity of endocrine FGFs. Recent structural and biochemical studies have begun to shed light onto the molecular basis for the klotho-dependent endocrine mode of action of the FGF 19 subfamily. Crystal structures of FGF 19 and FGF23 show that the topology of the HS binding site (HBS) of FGF19 subfamily members deviates drastically from the common topology adopted by the paracrine FGFs. The distinct topologies of the HBS of FGF 19 and FGF23 prevent HS from direct hydrogen bonding with the backbone atoms of the HBS of these ligands and accordingly decrease the HS binding affinity of this subfamily. Recent biochemical data reveal that the αklotho ectodomain binds avidly to the ectodomain of FGFR1c, the main cognate FGFR of FGF23, creating a de novo high affinity binding site for the C-terminal tail of FGF23. The isolated FGF23 C-terminus can be used to effectively inhibit the formation of the FGF23-FGFR1c-αklotho complex and alleviate hypophosphatemia in renal phosphate disorders due to elevated levels of FGF23. PMID:22396159

Distinctive receptor binding properties of the surface glycoprotein of a natural feline leukemia virus isolate with unusual disease spectrum.

PubMed

Bolin, Lisa L; Chandhasin, Chandtip; Lobelle-Rich, Patricia A; Albritton, Lorraine M; Levy, Laura S

2011-05-13

Feline leukemia virus (FeLV)-945, a member of the FeLV-A subgroup, was previously isolated from a cohort of naturally infected cats. An unusual multicentric lymphoma of non-T-cell origin was observed in natural and experimental infection with FeLV-945. Previous studies implicated the FeLV-945 surface glycoprotein (SU) as a determinant of disease outcome by an as yet unknown mechanism. The present studies demonstrate that FeLV-945 SU confers distinctive properties of binding to the cell surface receptor. Virions bearing the FeLV-945 Env protein were observed to bind the cell surface receptor with significantly increased efficiency, as was soluble FeLV-945 SU protein, as compared to the corresponding virions or soluble protein from a prototype FeLV-A isolate. SU proteins cloned from other cohort isolates exhibited increased binding efficiency comparable to or greater than FeLV-945 SU. Mutational analysis implicated a domain containing variable region B (VRB) to be the major determinant of increased receptor binding, and identified a single residue, valine 186, to be responsible for the effect. The FeLV-945 SU protein binds its cell surface receptor, feTHTR1, with significantly greater efficiency than does that of prototype FeLV-A (FeLV-A/61E) when present on the surface of virus particles or in soluble form, demonstrating a 2-fold difference in the relative dissociation constant. The results implicate a single residue, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with the receptor-binding domain through residue glutamine 110 to effect increased binding affinity. Through its increased receptor binding affinity, FeLV-945 SU might function in pathogenesis by increasing the rate of virus entry and spread in vivo, or by facilitating entry into a novel target cell with a low receptor density.

Antarctic Meteorite Newsletter, Volume 29, Number 1

NASA Technical Reports Server (NTRS)

Satterwhite, Cecilia (Editor); Righter, Kevin (Editor)

2006-01-01

This newsletter contains classifications for 597 new meteorites from the 2003 and 2004 ANtarctic Search for METeorites (ANSMET) seasons. They include samples from the Cumulus Hills, Dominion Range, Grosvenor Mountains, LaPaz Icefield, MacAlpine Hills, and the Miller Range. Macroscopic and petrographic descriptions are given for 25 of the new meteorites: 1 acapulcoite/Iodranite, 1 howardite, 1 diogenite, 2 eucrites, 1 enstatite chondrite, four L3 and two H3 chondrites, 2 CM, 3 CK and 1 CV chondrites, three R chondrites, and four impact melt breccias (with affinities for H and L). Likely the most interesting sample announced in this newsletter is LAP04840, with affinity to R chondrites. This meteorite contains approximately 15% horneblende, and has mineral compositional ranges and oxygen isotopic values similar to those of R chondrites. The presence of an apparently hydrous phase in this petrologic grade 6 chondrite is very unusual, and should be of great interest to many meteoriticists.

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

PubMed Central

Chen, Chiliang; Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.; Beattie, Gwyn A.

2017-01-01

Summary We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (Km, 2.6 μM) and, although it also binds betaine (Km, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (Km, 24 μM) and the betaine-specific SBP BetX (Km, 0.6 μM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs. PMID:19919675

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

PubMed

Chen, Chiliang; Malek, Adel A; Wargo, Matthew J; Hogan, Deborah A; Beattie, Gwyn A

2010-01-01

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another

PubMed Central

Abdiche, Yasmina Noubia; Yeung, Andy Yik; Ni, Irene; Stone, Donna; Miles, Adam; Morishige, Winse; Rossi, Andrea; Strop, Pavel

2017-01-01

Here we describe how real-time label-free biosensors can be used to identify antibodies that compete for closely adjacent or minimally overlapping epitopes on their specific antigen via a mechanism of antibody displacement. By kinetically perturbing one another’s binding towards their antigen via the formation of a transient trimolecular complex, antibodies can displace one another in a fully reversible and dose-dependent manner. Displacements can be readily identified when epitope binning assays are performed in a classical sandwich assay format whereby a solution antibody (analyte) is tested for binding to its antigen that is first captured via an immobilized antibody (ligand) because an inverted sandwiching response is observed when an analyte displaces a ligand, signifying the antigen’s unusually rapid dissociation from its ligand. In addition to classifying antibodies within a panel in terms of their ability to block or sandwich pair with one another, displacement provides a hybrid mechanism of competition. Using high-throughput epitope binning studies we demonstrate that displacements can be observed on any target, if the antibody panel contains appropriate epitope diversity. Unidirectional displacements occurring between disparate-affinity antibodies can generate apparent asymmetries in a cross-blocking experiment, confounding their interpretation. However, examining competition across a wide enough concentration range will often reveal that these displacements are reversible. Displacement provides a gentle and efficient way of eluting antigen from an otherwise high affinity binding partner which can be leveraged in designing reagents or therapeutic antibodies with unique properties. PMID:28060885

Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another.

PubMed

Abdiche, Yasmina Noubia; Yeung, Andy Yik; Ni, Irene; Stone, Donna; Miles, Adam; Morishige, Winse; Rossi, Andrea; Strop, Pavel

2017-01-01

Here we describe how real-time label-free biosensors can be used to identify antibodies that compete for closely adjacent or minimally overlapping epitopes on their specific antigen via a mechanism of antibody displacement. By kinetically perturbing one another's binding towards their antigen via the formation of a transient trimolecular complex, antibodies can displace one another in a fully reversible and dose-dependent manner. Displacements can be readily identified when epitope binning assays are performed in a classical sandwich assay format whereby a solution antibody (analyte) is tested for binding to its antigen that is first captured via an immobilized antibody (ligand) because an inverted sandwiching response is observed when an analyte displaces a ligand, signifying the antigen's unusually rapid dissociation from its ligand. In addition to classifying antibodies within a panel in terms of their ability to block or sandwich pair with one another, displacement provides a hybrid mechanism of competition. Using high-throughput epitope binning studies we demonstrate that displacements can be observed on any target, if the antibody panel contains appropriate epitope diversity. Unidirectional displacements occurring between disparate-affinity antibodies can generate apparent asymmetries in a cross-blocking experiment, confounding their interpretation. However, examining competition across a wide enough concentration range will often reveal that these displacements are reversible. Displacement provides a gentle and efficient way of eluting antigen from an otherwise high affinity binding partner which can be leveraged in designing reagents or therapeutic antibodies with unique properties.

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

PubMed Central

Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

2013-01-01

In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

Ion-binding properties of Calnuc, Ca2+ versus Mg2+--Calnuc adopts additional and unusual Ca2+-binding sites upon interaction with G-protein.

PubMed

Kanuru, Madhavi; Samuel, Jebakumar J; Balivada, Lavanya M; Aradhyam, Gopala K

2009-05-01

Calnuc is a novel, highly modular, EF-hand containing, Ca(2+)-binding, Golgi resident protein whose functions are not clear. Using amino acid sequences, we demonstrate that Calnuc is a highly conserved protein among various organisms, from Ciona intestinalis to humans. Maximum homology among all sequences is found in the region that binds to G-proteins. In humans, it is known to be expressed in a variety of tissues, and it interacts with several important protein partners. Among other proteins, Calnuc is known to interact with heterotrimeric G-proteins, specifically with the alpha-subunit. Herein, we report the structural implications of Ca(2+) and Mg(2+) binding, and illustrate that Calnuc functions as a downstream effector for G-protein alpha-subunit. Our results show that Ca(2+) binds with an affinity of 7 mum and causes structural changes. Although Mg(2+) binds to Calnuc with very weak affinity, the structural changes that it causes are further enhanced by Ca(2+) binding. Furthermore, isothermal titration calorimetry results show that Calnuc and the G-protein bind with an affinity of 13 nm. We also predict a probable function for Calnuc, that of maintaining Ca(2+) homeostasis in the cell. Using Stains-all and terbium as Ca(2+) mimic probes, we demonstrate that the Ca(2+)-binding ability of Calnuc is governed by the activity-based conformational state of the G-protein. We propose that Calnuc adopts structural sites similar to the ones seen in proteins such as annexins, c2 domains or chromogrannin A, and therefore binds more calcium ions upon binding to Gialpha. With the number of organelle-targeted G-protein-coupled receptors increasing, intracellular communication mediated by G-proteins could become a new paradigm. In this regard, we propose that Calnuc could be involved in the downstream signaling of G-proteins.

Characterization of the three different states of the cholecystokinin (CCK) receptor in pancreatic acini.

PubMed

Talkad, V D; Patto, R J; Metz, D C; Turner, R J; Fortune, K P; Bhat, S T; Gardner, J D

1994-10-20

By measuring binding of [125I]CCK-8 and [3H]L-364,718 to rat pancreatic acini we demonstrated directly that the pancreatic CCK receptor can exist in three different affinity states with respect to CCK--high affinity, low affinity and very low affinity. Binding of [125I]CCK-8 reflects interaction of the tracer with the high and low affinity states, whereas binding of [3H]L-364,718 reflects interaction of the tracer with the low and very low affinity states. Treating acini with carbachol abolished the high affinity state of the CCK receptor and converted approximately 25% of the low affinity receptors to the very low affinity state. Carbachol treatment was particularly useful in establishing the values of Kd for the high and low affinity states for different CCK receptor agonists and antagonists. Of the various CCK receptor agonists tested, CCK-8 had the highest affinity for the high affinity state (Kd approximately 1 nM), whereas CCK-JMV-180 had the highest affinity for the low (Kd 7 nM) and very low affinity (Kd 200 nM) states. Gastrin and de(SO4)CCK-8 had affinities for the high and low affinity states of the receptor that were 100- to 400-fold less than those of CCK-8 but had affinities for the very low affinity state that were only 3- to 10-fold less than that of CCK-8. CCK receptor antagonists showed several patterns in interacting with the different states of the CCK receptor. L-364,718 had the same affinity for each state of the CCK receptor. CR1409 and Bt2cGMP each had similar affinities for the high and low affinity states and lower affinity for the very low affinity state. L-365,260 and CCK-JMV-179 had the highest affinity for the low affinity state and lower affinities for the high and very low affinity states. Different CCK receptor agonists caused the same maximal stimulation of amylase secretion but showed different degrees of amplification in terms of the relationship between their abilities to stimulate amylase secretion and their abilities to occupy the low affinity state of the CCK receptor. When amplification was expressed quantitatively as the value of Kd for the low affinity state divided by the corresponding EC50 for stimulating amylase secretion the values were CCK-8 (1000), de(SO)CCK-8 (1500), gastrin (100) and CCK-JMV-180 (Menozzi, D., Vinayek, R., Jensen, R.T. and Gardner, J.D. (1991) J. Biol. Chem. 266, 10385-1091).(ABSTRACT TRUNCATED AT 400 WORDS)

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

PubMed Central

Owczarek, C M; Layton, M J; Metcalf, D; Lock, P; Willson, T A; Gough, N M; Nicola, N A

1993-01-01

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins. Images PMID:8253075

Occurrence of fluororichterite and fluorian biotite in the In Tifar trachyte neck (Tazrouk district, Hoggar volcanic province, Sahara, Algeria)

NASA Astrophysics Data System (ADS)

Azzouni-Sekkal, Abla; Bonin, Bernard; Ben El Khaznadji, Riad

2013-09-01

The unusual occurrence in the In Tifar trachyte neck (Tazrouk district, Hoggar volcanic province, Sahara, Algeria) of the fluorian biotite-fluororichterite association is presented. The two mineral species were previously unknown in the Hoggar and their association is uncommon worldwide. Ti-rich biotite has 28-40% OH sites occupied by fluorine, hence the use of the modifier "fluorian". Sodic-calcic fluororichterite has more than 55% OH sites filled by fluorine, hence the use of the prefix "fluoro". Well-defined F-Mg affinities are documented in both cases, while Cl remains very low. Temperatures are estimated roughly at 775-700 °C at low pressures. The fluorian biotite → fluororichterite sequence of crystallisation implies increasingly high fH2F2/fH2O ratios in metaluminous H2O-dominated evolving to peralkaline F-enriched fluids.

Electronic Structure of Small Lanthanide Containing Molecules

NASA Astrophysics Data System (ADS)

Kafader, Jared O.; Ray, Manisha; Topolski, Josey E.; Chick Jarrold, Caroline

2016-06-01

Lanthanide-based materials have unusual electronic properties because of the high number of electronic degrees of freedom arising from partial occupation of 4f orbitals, which make these materials optimal for their utilization in many applications including electronics and catalysis. Electronic spectroscopy of small lanthanide molecules helps us understand the role of these 4f electrons, which are generally considered core-like because of orbital contraction, but are energetically similar to valence electrons. The spectroscopy of small lanthanide-containing molecules is relatively unexplored and to broaden this understanding we have completed the characterization of small cerium, praseodymium, and europium molecules using photoelectron spectroscopy coupled with DFT calculations. The characterization of PrO, EuH, EuO/EuOH, and CexOy molecules have allowed for the determination of their electron affinity, the assignment of numerous anion to neutral state transitions, modeling of anion/neutral structures and electron orbital occupation.

Mercury dilution by autochthonous organic matter in a fertilized mangrove wetland.

PubMed

Machado, Wilson; Sanders, Christian J; Santos, Isaac R; Sanders, Luciana M; Silva-Filho, Emmanoel V; Luiz-Silva, Wanilson

2016-06-01

A dated sediment core from a highly-fertilized mangrove wetland located in Cubatão (SE Brazil) presented a negative correlation between mercury (Hg) and organic carbon contents. This is an unusual result for a metal with well-known affinity to organic matter. A dilution of Hg concentrations by autochthonous organic matter explained this observation, as revealed by carbon stable isotopes signatures (δ(13)C). Mercury dilution by the predominant mangrove-derived organic matter counterbalanced the positive influences of algal-derived organic matter and clay contents on Hg levels, suggesting that deleterious effects of Hg may be attenuated. Considering the current paradigm on the positive effect of organic matter on Hg concentrations in coastal sediments and the expected increase in mangrove organic matter burial due to natural and anthropogenic stimulations of primary production, predictions on the influences of organic matter on Hg accumulation in mangrove wetlands deserve caution. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Handali, Melody; Neupane, Durga P.; Roychowdhury, Hridindu

Here, ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn 2+ and Mn 2+ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn 2+ or Mn 2+ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties,more » and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn 2+ over Mn 2+ specificity, the Zn 2+ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn 2+-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn 2+-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn 2+-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn 2+ specificity.« less

Manganese Binding Properties of Human Calprotectin Under Conditions of High and Low Calcium: X-ray Crystallographic and Advanced EPR Spectroscopic Analysis

PubMed Central

Gagnon, Derek M.; Brophy, Megan Brunjes; Bowman, Sarah E. J.; Stich, Troy A.; Drennan, Catherine L.; Britt, R. David; Nolan, Elizabeth M.

2015-01-01

The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Herein, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimer is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin-echo envelope modulation and electron nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed 15N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by ca. two orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His6 site of human calprotectin. PMID:25597447

Manganese binding properties of human calprotectin under conditions of high and low calcium: X-ray crystallographic and advanced electron paramagnetic resonance spectroscopic analysis.

PubMed

Gagnon, Derek M; Brophy, Megan Brunjes; Bowman, Sarah E J; Stich, Troy A; Drennan, Catherine L; Britt, R David; Nolan, Elizabeth M

2015-03-04

The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Herein, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimer is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin-echo envelope modulation and electron-nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed (15)N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by two to three orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His6 site of human calprotectin.

Manganese Binding Properties of Human Calprotectin under Conditions of High and Low Calcium: X-ray Crystallographic and Advanced Electron Paramagnetic Resonance Spectroscopic Analysis

DOE PAGES

Gagnon, Derek M.; Brophy, Megan Brunjes; Bowman, Sarah E. J.; ...

2015-01-18

The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Here in this paper, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimermore » is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin–echo envelope modulation and electron–nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed 15N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by two to three orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His 6 site of human calprotectin.« less

Manganese Binding Properties of Human Calprotectin under Conditions of High and Low Calcium: X-ray Crystallographic and Advanced Electron Paramagnetic Resonance Spectroscopic Analysis

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

Gagnon, Derek M.; Brophy, Megan Brunjes; Bowman, Sarah E. J.

The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Here in this paper, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimermore » is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin–echo envelope modulation and electron–nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed 15N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by two to three orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His 6 site of human calprotectin.« less

Structural determinants of phosphoinositide selectivity in splice variants of Grp1 family PH domains

PubMed Central

Cronin, Thomas C; DiNitto, Jonathan P; Czech, Michael P; Lambright, David G

2004-01-01

The pleckstrin homology (PH) domains of the homologous proteins Grp1 (general receptor for phosphoinositides), ARNO (Arf nucleotide binding site opener), and Cytohesin-1 bind phosphatidylinositol (PtdIns) 3,4,5-trisphosphate with unusually high selectivity. Remarkably, splice variants that differ only by the insertion of a single glycine residue in the β1/β2 loop exhibit dual specificity for PtdIns(3,4,5)P3 and PtdIns(4,5)P2. The structural basis for this dramatic specificity switch is not apparent from the known modes of phosphoinositide recognition. Here, we report crystal structures for dual specificity variants of the Grp1 and ARNO PH domains in either the unliganded form or in complex with the head groups of PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Loss of contacts with the β1/β2 loop with no significant change in head group orientation accounts for the significant decrease in PtdIns(3,4,5)P3 affinity observed for the dual specificity variants. Conversely, a small increase rather than decrease in affinity for PtdIns(4,5)P2 is explained by a novel binding mode, in which the glycine insertion alleviates unfavorable interactions with the β1/β2 loop. These observations are supported by a systematic mutational analysis of the determinants of phosphoinositide recognition. PMID:15359279

Towards elucidation of the mechanism of biological nanomotors

NASA Astrophysics Data System (ADS)

Zhao, Zhengyi

Biological functions such as cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging, and cell entry all involve biomotor-driven DNA translocation. In the past, the ubiquitous biological nanomotors were classified into two categories: linear and rotation motors. In 2013, we discovered a third type of biomotor, revolving motor without rotation. The revolving motion is further found to be widespread among many biological systems. In addition, the detailed sequential action mechanism of the ATPase ring in the phi29 dsDNA packaging motor has been elucidated: ATP binding induces a conformational entropy alternation of ATPase to a high affinity toward dsDNA; ATP hydrolysis triggers another conformational entropy change in ATPase to a low DNA affinity, by which the dsDNA substrate is pushed toward an adjacent ATPase subunit. The subunit communication is regulated by an arginine finger that extends from one ATPase subunit to the adjacent unit, resulting in an asymmetrical hexameric organization. Continuation of this process promotes the movement and revolving of the dsDNA within the hexameric ATPase ring. Coordination of all the motor components facilitate the motion direction control of the viral DNA packaging motors, and make it unusually powerful and effective. KEYWORDS: Phi29 dsDNA Packaging Motor, Bio-nanomotor, RNA Nanotechnology, DNA Translocase, One-Way Revolving, ASCE Superfamily, AAA+ Superfamily.

New permian fusulinids from conglomerate mesa, southeastern inyo Mountains, east-central california

USGS Publications Warehouse

Stevens, C.H.; Stone, P.

2009-01-01

In the Conglomerate Mesa area in the southeastern Inyo Mountains, east-central California, a series of distinctive fusulinid assemblages ranging in age from late Artinskian to Kungurian or Roadian was developed in units 7-10 of the sedimentary rocks of Santa Rosa Flat (part of the Owens Valley Group). The fauna of unit 7 shows some eastern Klamath Mountains affinity, but most of the species in unit 7 and the lower half of unit 8 are highly endemic and comprise three new genera with 12 new species, two unusual unassigned forms, and two other new species assigned to previously described genera. New taxa include: Crenulosepta new genus with five new species, C. inyoensis, C. delicata, C. fusiformis, C. rossi, and C. wahlmani; Nigribaccinus new genus with three new species, N. giganteus, N. elegans, and N. ? nestelli; and the new genus Inyoschwagerina with four new species, I. magnified, I. elayeri, I. elongata, and I.? linderae. Cuniculinella Skinner and Wilde, 1965, is represented by one new species, C. parva, and Skinnerella Coogan, 1960 by one new species, S.? mcallisteri. Faunas from the upper half of unit 8, unit 9, and unit 10 have a strong West Texas affinity. New species from these units are Skinnerella davydovi, S. hexagona, Parafusulina cerrogordoensis, P. complexa, P. halli, P. owensensis, and P. ubehebensis. Copyright ?? 2009, The Paleontological Society.

Cloning and functional characterization of the high-affinity K+ transporter HAK1 of pepper.

PubMed

Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

2004-10-01

High-affinity K+ uptake in plants plays a crucial role in K+ nutrition and different systems have been postulated to contribute to the high-affinity K+ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K+ uptake observed in roots. Pepper plants starved of K+ for 3 d showed high-affinity K+ uptake (Km of 6 microM K+) that was very sensitive to NH and their roots expressed a high-affinity K+ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast ( Saccharomyces cerevisiae ), CaHAK1 mediated high-affinity K+ and Rb+ uptake with Km values of 3.3 and 1.9 microM, respectively. Rb+ uptake was competitively inhibited by micromolar concentrations of NH and Cs+, and by millimolar concentrations of Na+.

Oxygenation properties and isoform diversity of snake hemoglobins

PubMed Central

Natarajan, Chandrasekhar; Moriyama, Hideaki; Hoffmann, Federico G.; Wang, Tobias; Fago, Angela; Malte, Hans; Overgaard, Johannes; Weber, Roy E.

2015-01-01

Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer-dimer dissociation. However, standardized comparative data are lacking for snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying α- and β-type globin genes to characterize 1) Hb isoform composition of definitive erythrocytes, and 2) the oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis platura (Elapidae). We analyzed allosteric properties of snake Hbs in terms of the Monod-Wyman-Changeux model and Adair four-step thermodynamic model. Hbs from each of the three species exhibited high intrinsic O2 affinities, low cooperativities, small Bohr factors in the absence of phosphates, and high sensitivities to ATP. Oxygenation properties of the snake Hbs could be explained entirely by allosteric transitions in the quaternary structure of intact tetramers, suggesting that ligation-dependent dissociation of Hb tetramers into αβ-dimers is not a universal feature of snake Hbs. Surprisingly, the major Hb isoform of the South American rattlesnake is homologous to the minor HbD of other amniotes and, contrary to the pattern of Hb isoform differentiation in birds and turtles, exhibits a lower O2 affinity than the HbA isoform. PMID:26354849

An Unusual Ligand Coordination Gives Rise to a New Family of Rhodium Metalloinsertors with Improved Selectivity and Potency

PubMed Central

2015-01-01

Rhodium metalloinsertors are octahedral complexes that bind DNA mismatches with high affinity and specificity and exhibit unique cell-selective cytotoxicity, targeting mismatch repair (MMR)-deficient cells over MMR-proficient cells. Here we describe a new generation of metalloinsertors with enhanced biological potency and selectivity, in which the complexes show Rh–O coordination. In particular, it has been found that both Δ- and Λ-[Rh(chrysi)(phen)(DPE)]2+ (where chrysi =5,6 chrysenequinone diimmine, phen =1,10-phenanthroline, and DPE = 1,1-di(pyridine-2-yl)ethan-1-ol) bind to DNA containing a single CC mismatch with similar affinities and without racemization. This is in direct contrast with previous metalloinsertors and suggests a possible different binding disposition for these complexes in the mismatch site. We ascribe this difference to the higher pKa of the coordinated immine of the chrysi ligand in these complexes, so that the complexes must insert into the DNA helix with the inserting ligand in a buckled orientation; spectroscopic studies in the presence and absence of DNA along with the crystal structure of the complex without DNA support this assignment. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than cisplatin and N-methyl-N′-nitro-nitrosoguanidine (MNNG, a common DNA-alkylating chemotherapeutic agent). Moreover, the activities of the new metalloinsertors are coupled with high levels of selective cytotoxicity for MMR-deficient versus proficient colorectal cancer cells. PMID:25254630

Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning

PubMed Central

Azarov, Ivan; Wang, Ling; Rose, Jason J.; Xu, Qinzi; Huang, Xueyin N.; Belanger, Andrea; Wang, Ying; Guo, Lanping; Liu, Chen; Ucer, Kamil B.; McTiernan, Charles F.; O’Donnell, Christopher P.; Shiva, Sruti; Tejero, Jesús; Kim-Shapiro, Daniel B.; Gladwin, Mark T.

2016-01-01

Carbon monoxide (CO) is a leading cause of poisoning deaths worldwide, with no available antidotal therapy. We introduce a potential treatment paradigm for CO poisoning, based on near-irreversible binding of CO by an engineered human neuroglobin (Ngb). Ngb is a six-coordinate hemoprotein, with the heme iron coordinated by two histidine residues. We mutated the distal histidine to glutamine (H64Q) and substituted three surface cysteines with less reactive amino acids to form a five-coordinate heme protein (Ngb-H64Q-CCC). This molecule exhibited an unusually high affinity for gaseous ligands, with a P50 value for oxygen of 0.015 mmHg. Ngb-H64Q-CCC bound CO about 500 times more strongly than did hemoglobin. Incubation of Ngb-H64Q-CCC with 100% CO-saturated hemoglobin, either cell-free or encapsulated in human red blood cells, reduced the half-life of carboxy-hemoglobin to 0.11 and 0.41 minutes, respectively, from a value that is ≥ 200 minutes when the hemoglobin or cells are only exposed to air. Infusions of Ngb-H64Q-CCC to CO-poisoned mice enhanced CO removal from red blood cells, restored heart rate and blood pressure, increased survival, and were followed by rapid renal elimination of CO-bound Ngb-H64Q-CCC. Heme-based scavenger molecules with very high CO binding affinity such as our mutant five-coordinate Ngb are potential antidotes for CO poisoning by virtue of their ability to bind and eliminate CO. PMID:27928027

An unusual Middle Permian flora from the Blaine Formation (Pease River Group: Leonardian-Guadalupian Series) of King County, West Texas

USGS Publications Warehouse

DiMichele, W.A.; Hook, R.W.; Nelson, W.J.; Chaney, D.S.

2004-01-01

A new Middle Permian plant assemblage from South Ash Pasture in King County, Texas, may be the youngest and is certainly the most unusual flora known from the Permian of either West Texas or adjoining north-central Texas. Found serendipitously in the evaporite-rich upper Blaine Formation (Pease River Group, Guadalupian Series), the flora is of very low diversity despite intensive collecting efforts, and the affinities of nearly all taxa are enigmatic. The most common elements are parallel-veined leaves that resemble cordaites but that could be isolated pinnules of a pinnate leaf. Gigantopterid foliage is present but not assignable to any known taxon. A single foliar conifer specimen is too incomplete for assignment. Numerous reproductive organs, however, and an abundance of axes may represent conifers. Conchostracans, palaeoniscoid fish scales, and small heteropolar coprolites also occur in the deposit, which originated as a small, claystone-dominated channel fill in a coastal plain setting.

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

DOE PAGES

Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S.; ...

2016-02-09

Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. Thismore » “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies.« less

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

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

Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S.

Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. Thismore » “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies.« less

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

PubMed Central

Liu, K H; Huang, C Y; Tsay, Y F

1999-01-01

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis. PMID:10330471

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Immunoglobulin Gene Insertions and Deletions in the Affinity Maturation of HIV-1 Broadly Reactive Neutralizing Antibodies

PubMed Central

Kepler, Thomas B.; Liao, Hua-Xin; Alam, S. Munir; Bhaskarabhatla, Rekha; Zhang, Ruijun; Stewart, Shelley; Anasti, Kara; Kelsoe, Garnett; Parks, Robert; Lloyd, Krissey E.; Stolarchuk, Christina; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Morris, Lynn; Karim, Salim S. Abdool; Cohen, Myron S.; Walter, Emmanuel; Moody, M. Anthony; Wu, Xueling; Altae-Tran, Han R.; Georgiev, Ivelin S.; Kwong, Peter D.; Boyd, Scott D.; Fire, Andrew Z.; Mascola, John R.; Haynes, Barton F.

2014-01-01

Summary Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point-mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events. PMID:25211073

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

PubMed

Asakura, M; Tsukamoto, T; Imafuku, J; Matsui, H; Ino, M; Hasegawa, K

1984-10-30

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs.

PubMed

Krall, Jacob; Jensen, Claus Hatt; Bavo, Francesco; Falk-Petersen, Christina Birkedahl; Haugaard, Anne Stæhr; Vogensen, Stine Byskov; Tian, Yongsong; Nittegaard-Nielsen, Mia; Sigurdardóttir, Sara Björk; Kehler, Jan; Kongstad, Kenneth Thermann; Gloriam, David E; Clausen, Rasmus Prætorius; Harpsøe, Kasper; Wellendorph, Petrine; Frølund, Bente

2017-11-09

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar K i ) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.

Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

PubMed Central

Orcutt, Kelly Davis; Slusarczyk, Adrian L; Cieslewicz, Maryelise; Ruiz-Yi, Benjamin; Bhushan, Kumar R; Frangioni, John V; Wittrup, K Dane

2014-01-01

Introduction In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to DOTA chelates for use in PRIT applications. Methods We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), reformatted as a single chain variable fragment (scFv). Results Modeling predicts that for high antigen density and saturating bsAb dose, a hapten binding affinity of 100 picomolar (pM) is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nanomolar (nM) to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2 ± 1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen (CEA), pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions We have engineered a versatile, high-affinity DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals. PMID:21315278

Enhanced Magnetization of Cobalt Defect Clusters Embedded in TiO2-δ Films.

PubMed

Cortie, David L; Khaydukov, Yury; Keller, Thomas; Sprouster, David J; Hughes, Jacob S; Sullivan, James P; Wang, Xiaolin L; Le Brun, Anton P; Bertinshaw, Joel; Callori, Sara J; Aughterson, Robert; James, Michael; Evans, Peter J; Triani, Gerry; Klose, Frank

2017-03-15

High magnetizations are desirable for spintronic devices that operate by manipulating electronic states using built-in magnetic fields. However, the magnetic moment in promising dilute magnetic oxide nanocomposites is very low, typically corresponding to only fractions of a Bohr magneton for each dopant atom. In this study, we report a large magnetization formed by ion implantation of Co into amorphous TiO 2-δ films, producing an inhomogeneous magnetic moment, with certain regions producing over 2.5 μ B per Co, depending on the local dopant concentration. Polarized neutron reflectometry was used to depth-profile the magnetization in the Co:TiO 2-δ nanocomposites, thus confirming the pivotal role of the cobalt dopant profile inside the titania layer. X-ray photoemission spectra demonstrate the dominant electronic state of the implanted species is Co 0 , with a minor fraction of Co 2+ . The detected magnetizations have seldom been reported before and lie near the upper limit set by Hund's rules for Co 0 , which is unusual because the transition metal's magnetic moment is usually reduced in a symmetric 3D crystal-field environment. Low-energy positron annihilation lifetime spectroscopy indicates that defect structures within the titania layer are strongly modified by the implanted Co. We propose that a clustering motif is promoted by the affinity of the positively charged implanted species to occupy microvoids native to the amorphous host. This provides a seed for subsequent doping and nucleation of nanoclusters within an unusual local environment.

Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

PubMed Central

Arunmanee, Wanatchaporn; Pathania, Monisha; Solovyova, Alexandra S.; Le Brun, Anton P.; Ridley, Helen; Baslé, Arnaud; van den Berg, Bert; Lakey, Jeremy H.

2016-01-01

The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin–LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin–LPS interactions and a bridging calcium ion. PMID:27493217

Cross-linking Chemistry of Squid Beak*

PubMed Central

Miserez, Ali; Rubin, Daniel; Waite, J. Herbert

2010-01-01

In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance (1H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed. PMID:20870720

Cross-linking chemistry of squid beak.

PubMed

Miserez, Ali; Rubin, Daniel; Waite, J Herbert

2010-12-03

In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance ((1)H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed.

Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants.

PubMed Central

Wang, R; Crawford, N M

1996-01-01

Two mutations have been found in a gene (NRT2) of Arabidopsis thaliana that specifically impair constitutive, high-affinity nitrate uptake. These mutants were selected for resistance to 0.1 mM chlorate in the absence of nitrate. Progency from one of the backcrossed mutants showed no constitutive uptake of nitrate below 0.5 mM at pH 7.0 in liquid culture (that is, within 30 min of initial exposure to nitrate). All other uptake activities measured (high-affinity phosphate and sulfate uptake, inducible high-affinity nitrate uptake, and constitutive low-affinity nitrate uptake) were present or nearly normal in the backcrossed mutant. Electrophysiological analysis of individual root cells showed that the nrt2 mutant showed little response to 0.25 mM of nitrate, whereas NRT2 wild-type cells showed an initial depolarization followed by recovery. At 10 mM of nitrate both the mutant and wild-type cells displayed similar, strong electrical responses. These results indicate that NRT2 is a critical and perhaps necessary gene for constitutive, high-affinity nitrate uptake in Arabidopsis, but not for inducible, high-affinity nor constitutive, low-affinity nitrate uptake. Thus, these systems are genetically distinct. PMID:8799195

Glutathione Utilization by Candida albicans Requires a Functional Glutathione Degradation (DUG) Pathway and OPT7, an Unusual Member of the Oligopeptide Transporter Family

PubMed Central

Desai, Prashant Ramesh; Thakur, Anil; Ganguli, Dwaipayan; Paul, Sanjoy; Morschhäuser, Joachim; Bachhawat, Anand K.

2011-01-01

Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15Δ strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG pathway). The major oligopeptide transporters OPT1–OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a Km of 205 μm, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3Δ) or transport (opt7Δ) of glutathione failed to show a defect in virulence. PMID:21994941

Pasireotide therapy in a rare and unusual case of plurihormonal pituitary macroadenoma.

PubMed

Rajendran, Rajesh; Naik, Sarita; Sandeman, Derek D; Nasruddin, Azraai B

2013-01-01

We report the use of pasireotide in a rare and unusual case of pituitary macroadenoma co-secreting GH, prolactin and ACTH. A 62-year-old Caucasian man presented with impotence. Clinically, he appeared acromegalic and subsequent investigations confirmed GH excess and hyperprolactinaemia. Magnetic resonance imaging (MRI) of pituitary revealed a large pituitary macroadenoma. He underwent trans-sphenoidal surgery and histology confirmed an adenoma with immunohistochemistry positive for ACTH, GH and prolactin. Acromegaly was not cured following surgery and inadequately controlled despite subsequent octreotide therapy. He underwent further debulking pituitary surgery, following which IGF1 levels improved but still high. This time adenoma cells showed immunohistochemistry positivity for ACTH only, following which subsequent investigations confirmed intermittent hypercortisolaemia compatible with pituitary Cushing's disease. We recommended radiotherapy, but in view of the pluripotential nature of the tumour, we proceeded with a trial of s.c. pasireotide therapy on the basis that it may control both his acromegaly and Cushing's disease. After 3 months of pasireotide therapy, his mean GH and IGF1 levels improved significantly, with improvement in his symptoms but intermittent hypercortisolaemia persists. His glycaemic control deteriorated requiring addition of new anti-diabetic medication. MRI imaging showed loss of contrast uptake within the tumour following pasireotide therapy but no change in size. We conclude that our patient has had a partial response to pasireotide therapy. Long-term follow-up studies are needed to establish its safety and efficacy in patients with acromegaly and/or Cushing's disease. Plurihormonal pituitary adenomas are rare and unusual.Patients with pituitary adenomas co-secreting ACTH and GH are more likely to present with acromegaly because GH excess can mask hypercortisolaemia.Pasireotide holds potential where conventional somatostatin analogues are not effective in acromegaly due to higher affinity for somatostatin receptor subtypes 1, 2, 3 and 5.Significant deterioration in glycaemic control remains a concern in the use of pasireotide.Currently, long-term safety and efficacy of pasireotide in patients with acromegaly and/or Cushing's disease are not fully clear.

Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

PubMed Central

Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

1998-01-01

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514

Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα.

PubMed

Fang, Changming; Filipp, Fabian V; Smith, Jeffrey W

2012-04-01

Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA.

IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding

PubMed Central

Hymowitz, Sarah G.; Filvaroff, Ellen H.; Yin, JianPing; Lee, James; Cai, Liping; Risser, Philip; Maruoka, Miko; Mao, Weiguang; Foster, Jessica; Kelley, Robert F.; Pan, Guohua; Gurney, Austin L.; de Vos, Abraham M.; Starovasnik, Melissa A.

2001-01-01

The proinflammatory cytokine interleukin 17 (IL-17) is the founding member of a family of secreted proteins that elicit potent cellular responses. We report a novel human IL-17 homolog, IL-17F, and show that it is expressed by activated T cells, can stimulate production of other cytokines such as IL-6, IL-8 and granulocyte colony-stimulating factor, and can regulate cartilage matrix turnover. Unexpectedly, the crystal structure of IL-17F reveals that IL-17 family members adopt a monomer fold typical of cystine knot growth factors, despite lacking the disulfide responsible for defining the canonical ‘knot’ structure. IL-17F dimerizes in a parallel manner like neurotrophins, and features an unusually large cavity on its surface. Remarkably, this cavity is located in precisely the same position where nerve growth factor binds its high affinity receptor, TrkA, suggesting further parallels between IL-17s and neurotrophins with respect to receptor recognition. PMID:11574464

Kinetic mechanism of non-muscle myosin IIB: functional adaptations for tension generation and maintenance.

PubMed

Wang, Fei; Kovacs, Mihaly; Hu, Aihua; Limouze, John; Harvey, Estelle V; Sellers, James R

2003-07-25

Besides driving contraction of various types of muscle tissue, conventional (class II) myosins serve essential cellular functions and are ubiquitously expressed in eukaryotic cells. Three different isoforms in the human myosin complement have been identified as non-muscle class II myosins. Here we report the kinetic characterization of a human non-muscle myosin IIB subfragment-1 construct produced in the baculovirus expression system. Transient kinetic data show that most steps of the actomyosin ATPase cycle are slowed down compared with other class II myosins. The ADP affinity of subfragment-1 is unusually high even in the presence of actin filaments, and the rate of ADP release is close to the steady-state ATPase rate. Thus, non-muscle myosin IIB subfragment-1 spends a significantly higher proportion of its kinetic cycle strongly attached to actin than do the muscle myosins. This feature is even more pronounced at slightly elevated ADP levels, and it may be important in carrying out the cellular functions of this isoform working in small filamentous assemblies.

An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence

PubMed Central

Jo, Jeanyoung; Cortez, Krista L; Cornell, William Cole; Price-Whelan, Alexa

2017-01-01

Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome c oxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e. ‘orphan’) operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen Pseudomonas aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets. PMID:29160206

Ecophysiology of "halarsenatibacter silvermanii" strain SLAS-1T, gen. nov., sp. nov., a facultative chemoautotrophic arsenate respirer from salt-saturated Searles Lake, California

USGS Publications Warehouse

Blum, J.S.; Han, S.; Lanoil, B.; Saltikov, C.; Witte, B.; Tabita, F.R.; Langley, S.; Beveridge, T.J.; Jahnke, L.; Oremland, R.S.

2009-01-01

Searles Lake occupies a closed basin harboring salt-saturated, alkaline brines that have exceptionally high concentrations of arsenic oxyanions. Strain SLAS-1T was previously isolated from Searles Lake (R. S. Oremland, T. R. Kulp, J. Switzer Blum, S. E. Hoeft, S. Baesman, L. G. Miller, and J. F. Stolz, Science 308:1305-1308, 2005). We now describe this extremophile with regard to its substrate affinities, its unusual mode of motility, sequenced arrABD gene cluster, cell envelope lipids, and its phylogenetic alignment within the order Halanaero-bacteriales, assigning it the name "Halarsenatibacter silvermanii" strain SLAS-1T. We also report on the substrate dynamics of an anaerobic enrichment culture obtained from Searles Lake that grows under conditions of salt saturation and whose members include a novel sulfate reducer of the order Desulfovibriales, the archaeon Halorhabdus utahensis, as well as a close homolog of strain SLAS-1T. Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

Ecophysiology of "Halarsenatibacter silvermanii" strain SLAS-1T, gen. nov., sp. nov., a facultative chemoautotrophic arsenate respirer from salt-saturated Searles Lake, California.

PubMed

Blum, Jodi Switzer; Han, Sukkyun; Lanoil, Brian; Saltikov, Chad; Witte, Brian; Tabita, F Robert; Langley, Sean; Beveridge, Terry J; Jahnke, Linda; Oremland, Ronald S

2009-04-01

Searles Lake occupies a closed basin harboring salt-saturated, alkaline brines that have exceptionally high concentrations of arsenic oxyanions. Strain SLAS-1(T) was previously isolated from Searles Lake (R. S. Oremland, T. R. Kulp, J. Switzer Blum, S. E. Hoeft, S. Baesman, L. G. Miller, and J. F. Stolz, Science 308:1305-1308, 2005). We now describe this extremophile with regard to its substrate affinities, its unusual mode of motility, sequenced arrABD gene cluster, cell envelope lipids, and its phylogenetic alignment within the order Halanaerobacteriales, assigning it the name "Halarsenatibacter silvermanii" strain SLAS-1(T). We also report on the substrate dynamics of an anaerobic enrichment culture obtained from Searles Lake that grows under conditions of salt saturation and whose members include a novel sulfate reducer of the order Desulfovibriales, the archaeon Halorhabdus utahensis, as well as a close homolog of strain SLAS-1(T).

Human Calprotectin Is an Iron-Sequestering Host-Defense Protein

PubMed Central

Nakashige, Toshiki G.; Zhang, Bo; Krebs, Carsten; Nolan, Elizabeth M.

2015-01-01

Human calprotectin (CP) is a metal-chelating antimicrobial protein of the innate immune response. The current working model states that CP sequesters manganese and zinc from pathogens. We report the discovery that CP chelates iron and deprives bacteria of this essential nutrient. Elemental analysis of CP-treated growth medium establishes that CP reduces the concentrations of manganese, iron, and zinc. Microbial growth studies reveal that iron depletion by CP contributes to the growth inhibition of bacterial pathogens. Biochemical investigations demonstrate that CP coordinates Fe(II) at an unusual hexahistidine motif, and the Mössbauer spectrum of 57Fe(II)-bound CP is consistent with coordination of high-spin Fe(II) at this site (δ = 1.20 mm/s, ΔEQ = 1.78 mm/s). In the presence of Ca(II), CP turns on its iron-sequestering function and exhibits sub-picomolar affinity for Fe(II). Our findings expand the biological coordination chemistry of iron and support a previously unappreciated role for CP in mammalian iron homeostasis. PMID:26302479

Allele-specific DNA methylation and its interplay with repressive histone marks at promoter-mutant TERT genes

PubMed Central

Stern, Josh Lewis; Paucek, Richard D.; Huang, Franklin W.; Ghandi, Mahmoud; Nwumeh, Ronald; Costello, James C.; Cech, Thomas R.

2017-01-01

SUMMARY A mutation in the promoter of the Telomerase Reverse Transcriptase (TERT) gene is the most frequent noncoding mutation in cancer. The mutation drives unusual monoallelic expression of TERT, allowing immortalization. Here we find that DNA methylation of the TERT CpG Island (CGI) is also allele-specific in multiple cancers. The expressed allele is hypomethylated, which is opposite to cancers without TERT promoter mutations. The continued presence of Polycomb repressive complex 2 (PRC2) on the inactive allele suggests that histone marks of repressed chromatin may be causally linked to high DNA methylation. Consistent with this hypothesis, TERT promoter DNA containing 5-methyl-CpG has much increased affinity for PRC2 in vitro. Thus, CpG methylation and histone marks appear to collaborate to maintain the two TERT alleles in different epigenetic states in TERT promoter-mutant cancers. Finally, in several cancers DNA methylation levels at the TERT CGI correlate with altered patient survival. PMID:29281820

Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα[S

PubMed Central

Fang, Changming; Filipp, Fabian V.; Smith, Jeffrey W.

2012-01-01

Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA. PMID:22223860

Oxygenation properties and isoform diversity of snake hemoglobins.

PubMed

Storz, Jay F; Natarajan, Chandrasekhar; Moriyama, Hideaki; Hoffmann, Federico G; Wang, Tobias; Fago, Angela; Malte, Hans; Overgaard, Johannes; Weber, Roy E

2015-11-01

Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer-dimer dissociation. However, standardized comparative data are lacking for snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying α- and β-type globin genes to characterize 1) Hb isoform composition of definitive erythrocytes, and 2) the oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis platura (Elapidae). We analyzed allosteric properties of snake Hbs in terms of the Monod-Wyman-Changeux model and Adair four-step thermodynamic model. Hbs from each of the three species exhibited high intrinsic O2 affinities, low cooperativities, small Bohr factors in the absence of phosphates, and high sensitivities to ATP. Oxygenation properties of the snake Hbs could be explained entirely by allosteric transitions in the quaternary structure of intact tetramers, suggesting that ligation-dependent dissociation of Hb tetramers into αβ-dimers is not a universal feature of snake Hbs. Surprisingly, the major Hb isoform of the South American rattlesnake is homologous to the minor HbD of other amniotes and, contrary to the pattern of Hb isoform differentiation in birds and turtles, exhibits a lower O2 affinity than the HbA isoform. Copyright © 2015 the American Physiological Society.

CO Binding and Ligand Discrimination in Human Myeloperoxidase†

PubMed Central

Murphy, Emma J.; Maréchal, Amandine; Segal, Anthony W.; Rich, Peter R.

2015-01-01

Despite the fact that ferrous myeloperoxidase (MPO) can bind both O2 and NO, its ability to bind CO has been questioned. UV/visible spectroscopy was used to confirm that CO induces small spectral shifts in ferrous MPO, and Fourier transform infrared difference spectroscopy showed definitively that these arose from formation of a heme ferrous–CO compound. Recombination rates after CO photolysis were monitored at 618 and 645 nm as a function of CO concentration and pH. At pH 6.3, kon and koff were 0.14 mM−1·s−1 and 0.23 s−1, respectively, yielding an unusually high KD of 1.6 mM. This affinity of MPO for CO is 10 times weaker than its affinity for O2. The observed rate constant for CO binding increased with increasing pH and was governed by a single protonatable group with a pKa of 7.8. Fourier transform infrared spectroscopy revealed two different conformations of bound CO with frequencies at 1927 and 1942 cm−1. Their recombination rate constants were identical, indicative of two forms of bound CO that are in rapid thermal equilibrium rather than two distinct protein populations with different binding sites. The ratio of bound states was pH-dependent (pKa ≈ 7.4) with the 1927 cm−1 form favored at high pH. Structural factors that account for the ligand-binding properties of MPO are identified by comparisons with published data on a range of other ligand-binding heme proteins, and support is given to the recent suggestion that the proximal His336 in MPO is in a true imidazolate state. PMID:20146436

Recognition of DNA bulges by dinuclear iron(II) metallosupramolecular helicates.

PubMed

Malina, Jaroslav; Hannon, Michael J; Brabec, Viktor

2014-02-01

Bulged DNA structures are of general biological significance because of their important roles in a number of biochemical processes. Compounds capable of targeting bulged DNA sequences can be used as probes for studying their role in nucleic acid function, or could even have significant therapeutic potential. The interaction of [Fe(2)L(3)](4+) metallosupramolecular helicates (L = C(25)H(20)N(4)) with DNA duplexes containing bulges has been studied by measurement of the DNA melting temperature and gel electrophoresis. This study was aimed at exploring binding affinities of the helicates for DNA bulges of various sizes and nucleotide sequences. The studies reported herein reveal that both enantiomers of [Fe(2)L(3)](4+) bind to DNA bulges containing at least two unpaired nucleotides. In addition, these helicates show considerably enhanced affinity for duplexes containing unpaired pyrimidines in the bulge and/or pyrimidines flanking the bulge on both sides. We suggest that the bulge creates the structural motif, such as the triangular prismatic pocket formed by the unpaired bulge bases, to accommodate the [Fe(2)L(3)](4+) helicate molecule, and is probably responsible for the affinity for duplexes with a varying number of bulge bases. Our results reveal that DNA bulges represent another example of unusual DNA structures recognized by dinuclear iron(II) ([Fe(2)L(3)](4+)) supramolecular helicates. © 2013 FEBS.

Paratesticular desmoplastic small round cell tumour: an unusual tumour with an unusual fusion; cytogenetic and molecular genetic analysis combining RT-PCR and COBRA-FISH.

PubMed

Cliteur, Vincent Pm; Szuhai, Károly; Baelde, Hans J; van Dam, Jurriaan; Gelderblom, Hans; Hogendoorn, Pancras Cw

2012-01-25

Desmoplastic small round cell tumour is a rare malignant tumour with a male to female ratio of 4:1. It manifests mostly at serosal sites. Here we present a case of a 28-year-old male patient, who presented with a fast growing paratesticular mass. On biopsy nests and cords of small round cells, without a clear morphological lineage of differentiation were seen. Occasionally desmoplatic small round cell tumour shows different lines of differentiation. An unequivocal histological diagnosis might be difficult in such cases. Here we demonstrate by a combination of methods the characteristic immunohistochemical profile and - albeit unusual - molecular background and discuss the eventual link with Ewing sarcoma.Immunohistochemical studies showed a membranous staining of Keratine AE1/3 and a dot-like staining of Desmine, confirming its diagnosis. Using COBRA-FISH following a metaphase approach we demonstrated a balanced translocation, t(11;22)(p13;q12) and in RT-PCR formation of the EWSR1-WT1 fusion product, a specific translocation of desmoplastic round cell tumour. The fusion involves exon 9 of EWSR1 to exon 8 of WT1, an unusual fusion product, though earlier described in a case of a desmoplastic small round cell tumour of the hand. The EWSR1-WT1 chimera seems to function as an oncogenic transcription factor. Here the zinc finger domain of the WT1 acts with affinity with certain promoter domains influencing the expression of various downstream proteins such as: PDGFA, PAX2, insulin-like growth factor 1 receptor, epidermal growth factor receptor, IL2 receptor beta, BAIAP3, MLF1, TALLA-1, LRRC15 and ENT. We discuss their potential oncogenic roles and potential therapeutic consequences.

Paratesticular desmoplastic small round cell tumour: an unusual tumour with an unusual fusion; cytogenetic and molecular genetic analysis combining RT-PCR and COBRA-FISH

PubMed Central

2012-01-01

Desmoplastic small round cell tumour is a rare malignant tumour with a male to female ratio of 4:1. It manifests mostly at serosal sites. Here we present a case of a 28-year-old male patient, who presented with a fast growing paratesticular mass. On biopsy nests and cords of small round cells, without a clear morphological lineage of differentiation were seen. Occasionally desmoplatic small round cell tumour shows different lines of differentiation. An unequivocal histological diagnosis might be difficult in such cases. Here we demonstrate by a combination of methods the characteristic immunohistochemical profile and - albeit unusual - molecular background and discuss the eventual link with Ewing sarcoma. Immunohistochemical studies showed a membranous staining of Keratine AE1/3 and a dot-like staining of Desmine, confirming its diagnosis. Using COBRA-FISH following a metaphase approach we demonstrated a balanced translocation, t(11;22)(p13;q12) and in RT-PCR formation of the EWSR1-WT1 fusion product, a specific translocation of desmoplastic round cell tumour. The fusion involves exon 9 of EWSR1 to exon 8 of WT1, an unusual fusion product, though earlier described in a case of a desmoplastic small round cell tumour of the hand. The EWSR1-WT1 chimera seems to function as an oncogenic transcription factor. Here the zinc finger domain of the WT1 acts with affinity with certain promoter domains influencing the expression of various downstream proteins such as: PDGFA, PAX2, insulin-like growth factor 1 receptor, epidermal growth factor receptor, IL2 receptor beta, BAIAP3, MLF1, TALLA-1, LRRC15 and ENT. We discuss their potential oncogenic roles and potential therapeutic consequences. PMID:22587803

Electrostatic repulsion, compensatory mutations, and long-range non-additive effects at the dimerization interface of the HIV capsid protein.

PubMed

del Alamo, Marta; Mateu, Mauricio G

2005-01-28

In previous studies, thermodynamic dissection of the dimerization interface in CA-C, the C-terminal domain of the capsid protein of human immunodeficiency virus type 1, revealed that individual mutation to alanine of Ser178, Glu180, Glu187 or Gln192 led to significant increases in dimerization affinity. Four related aspects derived from this observation have been now addressed, and the results can be summarized as follows: (i) thermodynamic analyses indicate the presence of an intersubunit electrostatic repulsion between both Glu180 residues. (ii) The mutation Glu180 to Ala was detected in nearly all type 2 human immunodeficiency virus variants, and in several simian immunodeficiency viruses analyzed. However, this mutation was strictly co-variant with mutations Ser178Asp in a neighboring residue, and Glu187Gln. Thermodynamic analysis of multiple mutants showed that Ser178Asp compensated, alone or together with Glu187Gln, the increase in affinity caused by the mutation Glu180Ala, and restored a lower dimerization affinity. (iii) The increase in the affinity constant caused by the multiple mutation to Ala of Ser178, Glu180, Glu187 and Gln192 was more than one order of magnitude lower than predicted if additivity were present, despite the fact that the 178/180 pair and the two other residues were located more than 10A apart. (iv) Mutations in CA-C that caused non-additive increases in dimerization affinity also caused a non-additive increase in the capacity of the isolated CA-C domain to inhibit the assembly of capsid-like HIV-1 particles in kinetic assays. In summary, the study of a protein-protein interface involved in the building of a viral capsid has revealed unusual features, including intersubunit electrostatic repulsions, co-variant, compensatory mutations that may evolutionarily preserve a low association constant, and long-range, large magnitude non-additive effects on association.

IgG1 memory B cells keep the memory of IgE responses.

PubMed

He, Jin-Shu; Subramaniam, Sharrada; Narang, Vipin; Srinivasan, Kandhadayar; Saunders, Sean P; Carbajo, Daniel; Wen-Shan, Tsao; Hidayah Hamadee, Nur; Lum, Josephine; Lee, Andrea; Chen, Jinmiao; Poidinger, Michael; Zolezzi, Francesca; Lafaille, Juan J; Curotto de Lafaille, Maria A

2017-09-21

The unique differentiation of IgE cells suggests unconventional mechanisms of IgE memory. IgE germinal centre cells are transient, most IgE cells are plasma cells, and high affinity IgE is produced by the switching of IgG1 cells to IgE. Here we investigate the function of subsets of IgG1 memory B cells in IgE production and find that two subsets of IgG1 memory B cells, CD80 + CD73 + and CD80 - CD73 - , contribute distinctively to the repertoires of high affinity pathogenic IgE and low affinity non-pathogenic IgE. Furthermore, repertoire analysis indicates that high affinity IgE and IgG1 plasma cells differentiate from rare CD80 + CD73 + high affinity memory clones without undergoing further mutagenesis. By identifying the cellular origin of high affinity IgE and the clonal selection of high affinity memory B cells into the plasma cell fate, our findings provide fundamental insights into the pathogenesis of allergies, and on the mechanisms of antibody production in memory B cell responses.IgE is an important mediator of protective immunity as well as allergic reaction, but how high affinity IgE antibodies are produced in memory responses is not clear. Here the authors show that IgE can be generated via class-switch recombination in IgG1 memory B cells without additional somatic hypermutation.

Semiconductor Nanomaterials-Based Fluorescence Spectroscopic and Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometric Approaches to Proteome Analysis

PubMed Central

Kailasa, Suresh Kumar; Cheng, Kuang-Hung; Wu, Hui-Fen

2013-01-01

Semiconductor quantum dots (QDs) or nanoparticles (NPs) exhibit very unusual physico-chemcial and optical properties. This review article introduces the applications of semiconductor nanomaterials (NMs) in fluorescence spectroscopy and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for biomolecule analysis. Due to their unique physico-chemical and optical properties, semiconductors NMs have created many new platforms for investigating biomolecular structures and information in modern biology. These semiconductor NMs served as effective fluorescent probes for sensing proteins and cells and acted as affinity or concentrating probes for enriching peptides, proteins and bacteria proteins prior to MALDI-MS analysis. PMID:28788422

Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

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

Sine, Steven M.; Huang, Sun; Li, Shu-Xing

2013-09-01

The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr 184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr 184 depends on local residues, we generated mutations in an α7/5HT 3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr 184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurementsmore » show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr 184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr 184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr 184 and local residues contributes to high-affinity subtype-selective α-btx binding.« less

[Interaction of human factor X with thromboplastin].

PubMed

Kiselev, S V; Zubairov, D M; Timarbaev, V N

2003-01-01

The binding of 125I-labeled human factor X to native and papaine-treated tissue tromboplastin in the presence of CaCl2 or EDTA was studied. The Scatchard analysis suggests the existence of high (Kd=l,8 x10(-9) M) and low affinity binding sites on the thromboplastin surface. The removal of Ca2+ reduced affinity of factor X to the high affinity sites. This was accompanied by some increase of their number. Proteolysis by papaine decreased affinity of high affinity sites and caused the increase of their number in the presence of Ca2+. In the absence of Ca2+ the affinity remained unchanged, but the number of sites decreased. At low concentrations of factor X positive cooperativity for high affinity binding sites was observed. It did not depend on the presence of Ca2+. The results indirectly confirm the role of hydrophobic interactons in Ca2+ dependent binding of factor X to thromboplastin and the fact that heterogeneity of this binding is determined by mesophase structure of the thromboplastin phospholipids.

Enhanced Requirement for TNFR2 in Graft Rejection Mediated by Low Affinity Memory CD8+ T Cells During Heterologous Immunity

PubMed Central

Krummey, Scott M.; Chen, Ching-Wen; Guasch, Sara A.; Liu, Danya; Wagener, Maylene; Larsen, Christian P; Ford, Mandy L.

2016-01-01

The affinity of a T cell receptor (TCR) binding to peptide:MHC profoundly impacts the phenotype and function of effector and memory cell differentiation. Little is known about the effect of low affinity priming on memory cell generation and function, which is particularly important in heterologous immunity, when microbe-specific T cells cross-react with allogeneic antigen and mediate graft rejection. We found that low affinity primed memory CD8+ T cells produced high levels of TNF ex vivo in response to heterologous rechallenge compared to high affinity primed memory T cells. Low affinity secondary effectors significantly upregulated TNFR2 on the cell surface and contained a higher frequency of TNFR2hi proliferating cells. Low affinity primed secondary effectors concurrently downregulated TNF production. Importantly, blockade of TNFR2 attenuated graft rejection in low but not high affinity primed animals. These data establish a functional connection between TNF signaling and TCR priming affinity and have implications for the immunomodulation of pathogenic T cell responses during transplantation. PMID:27481849

Aptamer loaded MoS2 nanoplates as nanoprobes for detection of intracellular ATP and controllable photodynamic therapy

NASA Astrophysics Data System (ADS)

Jia, Li; Ding, Lin; Tian, Jiangwei; Bao, Lei; Hu, Yaoping; Ju, Huangxian; Yu, Jun-Sheng

2015-09-01

In this work we designed a MoS2 nanoplate-based nanoprobe for fluorescence imaging of intracellular ATP and photodynamic therapy (PDT) via ATP-mediated controllable release of 1O2. The nanoprobe was prepared by simply assembling a chlorine e6 (Ce6) labelled ATP aptamer on MoS2 nanoplates, which have favorable biocompatibility, unusual surface-area-to-mass ratio, strong affinity to single-stranded DNA, and can quench the fluorescence of Ce6. After the nanoprobe was internalized into the cells and entered ATP-abundant lysosomes, its recognition to ATP led to the release of the single-stranded aptamer from MoS2 nanoplates and thus recovered the fluorescence of Ce6 at an excitation wavelength of 633 nm, which produced a highly sensitive and selective method for imaging of intracellular ATP. Meanwhile, the ATP-mediated release led to the generation of 1O2 under 660 nm laser irradiation, which could induce tumor cell death with a lysosomal pathway. The controllable PDT provided a model approach for design of multifunctional theranostic nanoprobes. These results also promoted the development and application of MoS2 nanoplate-based platforms in biomedicine.In this work we designed a MoS2 nanoplate-based nanoprobe for fluorescence imaging of intracellular ATP and photodynamic therapy (PDT) via ATP-mediated controllable release of 1O2. The nanoprobe was prepared by simply assembling a chlorine e6 (Ce6) labelled ATP aptamer on MoS2 nanoplates, which have favorable biocompatibility, unusual surface-area-to-mass ratio, strong affinity to single-stranded DNA, and can quench the fluorescence of Ce6. After the nanoprobe was internalized into the cells and entered ATP-abundant lysosomes, its recognition to ATP led to the release of the single-stranded aptamer from MoS2 nanoplates and thus recovered the fluorescence of Ce6 at an excitation wavelength of 633 nm, which produced a highly sensitive and selective method for imaging of intracellular ATP. Meanwhile, the ATP-mediated release led to the generation of 1O2 under 660 nm laser irradiation, which could induce tumor cell death with a lysosomal pathway. The controllable PDT provided a model approach for design of multifunctional theranostic nanoprobes. These results also promoted the development and application of MoS2 nanoplate-based platforms in biomedicine. Electronic supplementary information (ESI) available: Supplementary figures. See DOI: 10.1039/c5nr02224j

Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels.

PubMed

Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L

2013-03-05

AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl(-) lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

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

Ghadbane, Hemza; Brown, Alistair K.; Kremer, Laurent

2007-10-01

Binding of Ni{sup 2+} ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution. Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosismore » mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni{sup 2+} ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.« less

Structural and Functional Analyses of the Severe Acute Respiratory Syndrome Coronavirus Endoribonuclease Nsp15

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

Bhardwaj, Kanchan; Palaninathan, Satheesh; Alcantara, Joanna Maria Ortiz

2008-03-31

The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3' of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involvedmore » in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.« less

Thermodynamic Bounds on the Ultra- and Infra-affinity of Hsp70 for Its Substrates

NASA Astrophysics Data System (ADS)

Nguyen, Basile; Hartich, David; Seifert, Udo; Rios, Paolo De Los

2017-07-01

The 70 kDa Heat Shock Proteins Hsp70 have several essential functions in living systems, such as protecting cells against protein aggregation, assisting protein folding, remodeling protein complexes and driving the translocation into organelles. These functions require high affinity for non-specific amino-acid sequences that are ubiquitous in proteins. It has been recently shown that this high affinity, called ultra-affinity, depends on a process driven out of equilibrium by ATP hydrolysis. Here we establish the thermodynamic bounds for ultra-affinity, and further show that the same reaction scheme can in principle be used both to strengthen and to weaken affinities (leading in this case to infra-affinity). We show that cofactors are essential to achieve affinity beyond the equilibrium range. Finally, biological implications are discussed.

Affine.m—Mathematica package for computations in representation theory of finite-dimensional and affine Lie algebras

NASA Astrophysics Data System (ADS)

Nazarov, Anton

2012-11-01

In this paper we present Affine.m-a program for computations in representation theory of finite-dimensional and affine Lie algebras and describe implemented algorithms. The algorithms are based on the properties of weights and Weyl symmetry. Computation of weight multiplicities in irreducible and Verma modules, branching of representations and tensor product decomposition are the most important problems for us. These problems have numerous applications in physics and we provide some examples of these applications. The program is implemented in the popular computer algebra system Mathematica and works with finite-dimensional and affine Lie algebras. Catalogue identifier: AENA_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AENB_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, UK Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 24 844 No. of bytes in distributed program, including test data, etc.: 1 045 908 Distribution format: tar.gz Programming language: Mathematica. Computer: i386-i686, x86_64. Operating system: Linux, Windows, Mac OS, Solaris. RAM: 5-500 Mb Classification: 4.2, 5. Nature of problem: Representation theory of finite-dimensional Lie algebras has many applications in different branches of physics, including elementary particle physics, molecular physics, nuclear physics. Representations of affine Lie algebras appear in string theories and two-dimensional conformal field theory used for the description of critical phenomena in two-dimensional systems. Also Lie symmetries play a major role in a study of quantum integrable systems. Solution method: We work with weights and roots of finite-dimensional and affine Lie algebras and use Weyl symmetry extensively. Central problems which are the computations of weight multiplicities, branching and fusion coefficients are solved using one general recurrent algorithm based on generalization of Weyl character formula. We also offer alternative implementation based on the Freudenthal multiplicity formula which can be faster in some cases. Restrictions: Computational complexity grows fast with the rank of an algebra, so computations for algebras of ranks greater than 8 are not practical. Unusual features: We offer the possibility of using a traditional mathematical notation for the objects in representation theory of Lie algebras in computations if Affine.m is used in the Mathematica notebook interface. Running time: From seconds to days depending on the rank of the algebra and the complexity of the representation.

A study of the uptake of chloroquine in malaria-infected erythrocytes. High and low affinity uptake and the influence of glucose and its analogues.

PubMed

Diribe, C O; Warhurst, D C

1985-09-01

A study of concentration- and substrate-dependence of chloroquine uptake has been carried out on mouse erythrocytes infected with the chloroquine-sensitive NK65 and the chloroquine-resistant RC strains of Plasmodium berghei. The presence of drug binding sites of high and low affinity in such strains of P. berghei was confirmed. High affinity uptake sites in cells parasitized with chloroquine-sensitive and chloroquine-resistant parasites have similar characteristics, but in the sensitive strain the major component of chloroquine-uptake is at high affinity and dependent on the availability of ATP whilst in the resistant strain the major component of uptake is at low affinity and independent of energy. An absolute increase in the quantity of the low affinity site in erythrocytes parasitized with chloroquine-resistant P. berghei was noted, which may be related to an increase in quantity of parasite membrane.

Thyrotropin-Blocking Autoantibodies and Thyroid-Stimulating Autoantibodies: Potential Mechanisms Involved in the Pendulum Swinging from Hypothyroidism to Hyperthyroidism or Vice Versa

PubMed Central

Rapoport, Basil

2013-01-01

Background Thyrotropin receptor (TSHR) antibodies that stimulate the thyroid (TSAb) cause Graves' hyperthyroidism and TSHR antibodies which block thyrotropin action (TBAb) are occasionally responsible for hypothyroidism. Unusual patients switch from TSAb to TBAb (or vice versa) with concomitant thyroid function changes. We have examined case reports to obtain insight into the basis for “switching.” Summary TBAb to TSAb switching occurs in patients treated with levothyroxine (LT4); the reverse switch (TBAb to TSAb) occurs after anti-thyroid drug therapy; TSAb/TBAb alterations may occur during pregnancy and are well recognized in transient neonatal thyroid dysfunction. Factors that may impact the shift include: (i) LT4 treatment, usually associated with decreased thyroid autoantibodies, in unusual patients induces or enhances thyroid autoantibody levels; (ii) antithyroid drug treatment decreases thyroid autoantibody levels; (iii) hyperthyroidism can polarize antigen-presenting cells, leading to impaired development of regulatory T cells, thereby compromising control of autoimmunity; (iv) immune-suppression/hemodilution reduces thyroid autoantibodies during pregnancy and rebounds postpartum; (v) maternally transferred IgG transiently impacts thyroid function in neonates until metabolized; (vi) a Graves' disease model involving immunizing TSHR-knockout mice with mouse TSHR-adenovirus and transfer of TSHR antibody-secreting splenocytes to athymic mice demonstrates the TSAb to TBAb shift, paralleling the outcome of maternally transferred “term limited” TSHR antibodies in neonates. Finally, perhaps most important, as illustrated by dilution analyses of patients' sera in vitro, TSHR antibody concentrations and affinities play a critical role in switching TSAb and TBAb functional activities in vivo. Conclusions Switching between TBAb and TSAb (or vice versa) occurs in unusual patients after LT4 therapy for hypothyroidism or anti-thyroid drug treatment for Graves' disease. These changes involve differences in TSAb versus TBAb concentrations, affinities and/or potencies in individual patients. Thus, anti-thyroid drugs or suppression/hemodilution in pregnancy reduce initially low TSAb levels even further, leading to TBAb dominance. In contrast, TSAb emergence after LT4 administration may be sufficient to counteract TBAb inhibition. The occurrence of “switching” emphasizes the need for careful patient monitoring and management. Finally, whole genome screening of relatively rare “switch” patients and appropriate Graves' and Hashimoto's controls could provide unexpected and valuable information regarding the basis for thyroid autoimmunity. PMID:23025526

The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

PubMed

Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

2017-08-01

The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

Norovirus translation requires an interaction between the C Terminus of the genome-linked viral protein VPg and eukaryotic translation initiation factor 4G.

PubMed

Chung, Liliane; Bailey, Dalan; Leen, Eoin N; Emmott, Edward P; Chaudhry, Yasmin; Roberts, Lisa O; Curry, Stephen; Locker, Nicolas; Goodfellow, Ian G

2014-08-01

Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5' end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Atypical hematological response to combined calorie restriction and chronic hypoxia in Biosphere 2 crew: a possible link to latent features of hibernation capacity.

PubMed

Paglia, Donald E; Walford, Roy L

2005-01-01

Eight humans were isolated for 2 years in Biosphere 2, a sealed airtight habitat with recycled air, food, water, and wastes. A combination of conditions led to selective decline of oxygen (O2) in the internal atmosphere from 21% to 14%, inducing symptoms of high-altitude sickness but with little or no compensatory increase in red cell production. All crew members exhibited significant decreases in both erythrocyte 2,3-bisphosphoglycerate (2,3-BPG) concentrations and P50 [partial pressure of O2 for 50% hemoglobin (Hb) saturation] values, changes opposite those expected in adaptation to high-altitude hypoxia. Lower P50 with increased Hb-O2 affinity induced by low 2,3-BPG is a characteristic of hibernating species and could be advantageous in O2-impoverished environments. The mechanisms underlying these changes in the Biosphere 2 crew remain obscure but could be related to low-calorie diet (1750-2100 kcal/day). Because the combination of hypoxia and limited caloric intake is also characteristic of hibernation, this unusual response may represent a cross-adaptation phenomenon in which certain features of hibernation capability are expressed in humans.

Overproduction of alpha chains provides a proton-insensitive component to the bluefish hemoglobin system.

PubMed

Bonaventura, Celia; Godette, Gerald; Stevens, Robert; Brenowitz, Michael; Henkens, Robert

2005-12-09

Expression of alpha and beta chains and their post-translational assembly into alpha(2)beta(2) tetramers is fundamental to the formation and function of most vertebrate hemoglobins. There is a strong evolutionary bias that favors expression of equal amounts of the two types of chains, because cooperativity, pH sensitivity, and anionic control of function occurs only for the alpha(2)beta(2) tetramers. Remarkably, an over-production of alpha chains, as in the pathological condition known as beta thalassemia in humans, is adaptive rather than pathological in the bluefish hemoglobin system. The thalassemia of the bluefish is a novel means of providing for oxygen uptake and delivery when low pH conditions incapacitate the highly pH-sensitive Root effect hemoglobins of the fish. Although fish often have pH-insensitive along with highly pH-sensitive hemoglobins, having pH-insensitive alpha chain monomers in circulation is an unusual structural variation. The role of bluefish alpha chains in oxygen transport is enabled by their remarkably lower oxygen affinity relative to human alpha chains. This is the first reported case of a thalassemic condition that is maintained in a species as an adaptive advantage.

Derivatives of Ergot-alkaloids: Molecular structure, physical properties, and structure-activity relationships

NASA Astrophysics Data System (ADS)

Ivanova, Bojidarka B.; Spiteller, Michael

2012-09-01

A comprehensive screening of fifteen functionalized Ergot-alkaloids, containing bulk aliphatic cyclic substituents at D-ring of the ergoline molecular skeleton was performed, studying their structure-active relationships and model interactions with α2A-adreno-, serotonin (5HT2A) and dopamine D3 (D3A) receptors. The accounted high affinity to the receptors binding loops and unusual bonding situations, joined with the molecular flexibility of the substituents and the presence of proton accepting/donating functional groups in the studied alkaloids, may contribute to further understanding the mechanisms of biological activity in vivo and in predicting their therapeutic potential in central nervous system (CNS), including those related the Schizophrenia. Since the presented correlation between the molecular structure and properties, was based on the comprehensively theoretical computational and experimental physical study on the successfully isolated derivatives, through using routine synthetic pathways in a relatively high yields, marked these derivatives as 'treasure' for further experimental and theoretical studied in areas such as: (a) pharmacological and clinical testing; (b) molecular-drugs design of novel psychoactive substances; (c) development of the analytical protocols for determination of Ergot-alkaloids through a functionalization of the ergoline-skeleton, and more.

The Structural Basis for Recognition of the PreQ0 Metabolite by an Unusually Small Riboswitch Aptamer Domain*S⃞♦

PubMed Central

Spitale, Robert C.; Torelli, Andrew T.; Krucinska, Jolanta; Bandarian, Vahe; Wedekind, Joseph E.

2009-01-01

Riboswitches are RNA elements that control gene expression through metabolite binding. The preQ1 riboswitch exhibits the smallest known ligand-binding domain and is of interest for its economical organization and high affinity interactions with guanine-derived metabolites required to confer tRNA wobbling. Here we present the crystal structure of a preQ1 aptamer domain in complex with its precursor metabolite preQ0. The structure is highly compact with a core that features a stem capped by a well organized decaloop. The metabolite is recognized within a deep pocket via Watson-Crick pairing with C15. Additional hydrogen bonds are made to invariant bases U6 and A29. The ligand-bound state confers continuous helical stacking throughout the core fold, thus providing a platform to promote Watson-Crick base pairing between C9 of the decaloop and the first base of the ribosome-binding site, G33. The structure offers insight into the mode of ribosome-binding site sequestration by a minimal RNA fold stabilized by metabolite binding and has implications for understanding the molecular basis by which bacterial genes are regulated. PMID:19261617

A metagenome-derived thermostable β-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148.

PubMed

Angelov, Angel; Pham, Vu Thuy Trang; Übelacker, Maria; Brady, Silja; Leis, Benedikt; Pill, Nicole; Brolle, Judith; Mechelke, Matthias; Moerch, Matthias; Henrissat, Bernard; Liebl, Wolfgang

2017-12-11

The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-β-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.

[Role of hemoglobin affinity to oxygen in adaptation to hypoxemia].

PubMed

Kwasiborski, Przemysław Jerzy; Kowalczyk, Paweł; Zieliński, Jakub; Przybylski, Jacek; Cwetsch, Andrzej

2010-04-01

One of the basic mechanisms of adapting to hypoxemia is a decrease in the affinity of hemoglobin for oxygen. This process occurs mainly due to the increased synthesis of 2,3-diphosphoglycerate (2,3-DPG) in the erythrocytes, as well as through the Bohr effect. Hemoglobin with decreased affinity for oxygen increases the oxygenation of tissues, because it gives up oxygen more easily during microcirculation. In foetal circulation, however, at a partial oxygen pressure (pO2) of 25 mmHg in the umbilical vein, the oxygen carrier is type F hemoglobin which has a high oxygen affinity. The commonly accepted role for hemoglobin F is limited to facilitating diffusion through the placenta. Is fetal life the only moment when haemoglobin F is useful? THE AIM OF STUDY was to create a mathematical model, which would answer the question at what conditions an increase, rather than a decrease, in haemoglobin oxygen affinity is of benefit to the body. Using the kinetics of dissociation of oxygen from hemoglobin described by the Hill equation as the basis for further discussion, we created a mathematical model describing the pO2 value in the microcirculatory system and its dependence on arterial blood pO2. The calculations were performed for hemoglobin with low oxygen affinity (adult type) and high-affinity hemoglobin (fetal type). The modelling took into account both physiological and pathological ranges of acid-base equilibrium and tissue oxygen extraction parameters. It was shown that for the physiological range of acid-base equilibrium and the resting level of tissue oxygen extraction parameters, with an arterial blood pO2 of 26.8 mmHg, the higher-affinity hemoglobin becomes the more effective oxygen carrier. It was also demonstrated that the arterial blood pO2, below which the high-affinity hemoglobin becomes the more effective carrier, is dependent on blood pH and the difference between the arterial and venous oxygen saturation levels. Simulations performed for the pathological states showed that acidosis and increased tissue oxygen demand lead to a broadened arterial blood pO2 range, in which the high-affinity hemoglobin is more efficient. Contrary to the widely held view that the only response to hypoxemia is a decrease in haemoglobin oxygen affinity, it was shown that under extreme hypoxemic conditions, an increased haemoglobin oxygen affinity improves the oxygenation of tissues. It was also shown that the dominance of hemoglobin with a high oxygen affinity rapidly exceeds hemoglobin with low oxygen affinity in the case of acidosis with its accompanying high tissue oxygen extraction. In cases of extreme disruptions of the acid-base equilibrium, the dominance of high-oxygen-affinity hemoglobin spans over the entire possible range of pO2 in arterial blood.

Enhanced Magnetization of Cobalt Defect Clusters Embedded in TiO 2-δ Films

DOE PAGES

Cortie, David L.; Khaydukov, Yury; Keller, Thomas; ...

2017-02-23

High magnetizations are desirable for spintronic devices that operate by manipulating electronic states using built-in magnetic fields. However, the magnetic moment in promising dilute magnetic oxide nanocomposites is very low, typically corresponding to only fractions of a Bohr magneton for each dopant atom. In this study, we report a large magnetization formed by ion implantation of Co into amorphous TiO 2-δ films, producing an inhomogeneous magnetic moment, with certain regions producing over 2.5 μ B per Co, depending on the local dopant concentration. Polarized neutron reflectometry was used to depth-profile the magnetization in the Co:TiO 2-δ nanocomposites, thus confirming themore » pivotal role of the cobalt dopant profile inside the titania layer. X-ray photoemission spectra demonstrate the dominant electronic state of the implanted species is Co 0, with a minor fraction of Co 2+. The detected magnetizations have seldom been reported before and lie near the upper limit set by Hund’s rules for Co 0, which is unusual because the transition metal’s magnetic moment is usually reduced in a symmetric 3D crystal-field environment. Low-energy positron annihilation lifetime spectroscopy indicates that defect structures within the titania layer are strongly modified by the implanted Co. We propose that a clustering motif is promoted by the affinity of the positively charged implanted species to occupy microvoids native to the amorphous host. This provides a seed for subsequent doping and nucleation of nanoclusters within an unusual local environment.« less

Exploring the biochemical properties and remediation applications of the unusual explosive-degrading P450 system XplA/B

PubMed Central

Jackson, Rosamond G.; Rylott, Elizabeth L.; Fournier, Diane; Hawari, Jalal; Bruce, Neil C.

2007-01-01

Widespread contamination of land and groundwater has resulted from the use, manufacture, and storage of the military explosive hexa-hydro-1,3,5-trinitro-1,3,5-triazine (RDX). This contamination has led to a requirement for a sustainable, low-cost method to remediate this problem. Here, we present the characterization of an unusual microbial P450 system able to degrade RDX, consisting of flavodoxin reductase XplB and fused flavodoxin-cytochrome P450 XplA. The affinity of XplA for the xenobiotic compound RDX is high (Kd = 58 μM) and comparable with the Km of other P450s toward their natural substrates (ranging from 1 to 500 μM). The maximum turnover (kcat) is 4.44 per s, only 10-fold less than the fastest self-sufficient P450 reported, BM3. Interestingly, the presence of oxygen determines the final products of RDX degradation, demonstrating that the degradation chemistry is flexible, but both pathways result in ring cleavage and release of nitrite. Carbon monoxide inhibition is weak and yet the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a potent inhibitor. To test the efficacy of this system for the remediation of groundwater, transgenic Arabidopsis plants expressing both xplA and xplB were generated. They are able to remove saturating levels of RDX from liquid culture and soil leachate at rates significantly faster than those of untransformed plants and xplA-only transgenic lines, demonstrating the applicability of this system for the phytoremediation of RDX-contaminated sites. PMID:17940033

Affinity proteomics to study endogenous protein complexes: Pointers, pitfalls, preferences and perspectives

PubMed Central

LaCava, John; Molloy, Kelly R.; Taylor, Martin S.; Domanski, Michal; Chait, Brian T.; Rout, Michael P.

2015-01-01

Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls. PMID:25757543

HIGH-AFFINITY T CELL RECEPTOR DIFFERENTIATES COGNATE PEPTIDE-MHC AND ALTERED PEPTIDE LIGANDS WITH DISTINCT KINETICS AND THERMODYNAMICS

PubMed Central

Persaud, Stephen P.; Donermeyer, David L.; Weber, K. Scott; Kranz, David M.; Allen, Paul M.

2010-01-01

Interactions between the T cell receptor and cognate peptide-MHC are crucial initiating events in the adaptive immune response. These binding events are highly specific yet occur with micromolar affinity. Even weaker interactions between TCR and self-pMHC complexes play critical regulatory roles in T cell development, maintenance and coagonist activity. Due to their low affinity, the kinetics and thermodynamics of such weak interactions are difficult to study. In this work, we used M15, a high-affinity TCR engineered from the 3.L2 TCR system, to study the binding properties, thermodynamics, and specificity of two altered peptide ligands (APLs). Our affinity measurements of the high-affinity TCR support the view that the wild type TCR binds these APLs in the millimolar affinity range, and hence very low affinities can still elicit biological functions. Finally, single methylene differences among the APLs gave rise to strikingly different binding thermodynamics. These minor changes in the pMHC antigen were associated with significant and unpredictable changes in both the entropy and enthalpy of the reaction. As the identical TCR was analyzed with several structurally similar ligands, the distinct thermodynamic binding profiles provide a mechanistic perspective on how exquisite antigen specificity is achieved by the T cell receptor. PMID:20334923

Let's get specific: the relationship between specificity and affinity.

PubMed

Eaton, B E; Gold, L; Zichi, D A

1995-10-01

The factors that lead to high-affinity binding are a good fit between the surfaces of the two molecules in their ground state and charge complementarity. Exactly the same factors give high specificity for a target. We argue that selection for high-affinity binding automatically leads to highly specific binding. This principle can be used to simplify screening approaches aimed at generating useful drugs.

49 CFR 236.1021 - Discontinuances, material modifications, and amendments.

Code of Federal Regulations, 2011 CFR

2011-10-01

... such as landslide, burned bridge, high water, high and wide load, or tunnel protection when the unusual... to provide protection against unusual contingencies such as landslide, burned bridges, high water...

49 CFR 236.1021 - Discontinuances, material modifications, and amendments.

Code of Federal Regulations, 2010 CFR

2010-10-01

... such as landslide, burned bridge, high water, high and wide load, or tunnel protection when the unusual... to provide protection against unusual contingencies such as landslide, burned bridges, high water...

49 CFR 235.7 - Changes not requiring filing of application.

Code of Federal Regulations, 2013 CFR

2013-10-01

... unusual contingencies such as landslide, burned bridge, high water, high and wide load, or tunnel... used to provide protection against unusual contingencies such as landslide, burned bridges, high water...

49 CFR 235.7 - Changes not requiring filing of application.

Code of Federal Regulations, 2014 CFR

2014-10-01

... unusual contingencies such as landslide, burned bridge, high water, high and wide load, or tunnel... used to provide protection against unusual contingencies such as landslide, burned bridges, high water...

49 CFR 236.1021 - Discontinuances, material modifications, and amendments.

Code of Federal Regulations, 2013 CFR

2013-10-01

... such as landslide, burned bridge, high water, high and wide load, or tunnel protection when the unusual... to provide protection against unusual contingencies such as landslide, burned bridges, high water...

49 CFR 236.1021 - Discontinuances, material modifications, and amendments.

Code of Federal Regulations, 2014 CFR

2014-10-01

... such as landslide, burned bridge, high water, high and wide load, or tunnel protection when the unusual... to provide protection against unusual contingencies such as landslide, burned bridges, high water...

49 CFR 235.7 - Changes not requiring filing of application.

Code of Federal Regulations, 2012 CFR

2012-10-01

... unusual contingencies such as landslide, burned bridge, high water, high and wide load, or tunnel... used to provide protection against unusual contingencies such as landslide, burned bridges, high water...

49 CFR 236.1021 - Discontinuances, material modifications, and amendments.

Code of Federal Regulations, 2012 CFR

2012-10-01

... such as landslide, burned bridge, high water, high and wide load, or tunnel protection when the unusual... to provide protection against unusual contingencies such as landslide, burned bridges, high water...

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man*

PubMed Central

Macauley, Matthew S.; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M.; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C.

2015-01-01

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. PMID:26507663

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man.

PubMed

Macauley, Matthew S; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C

2015-12-11

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2-6Galβ1-4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential effects of ethanol and other inducers of drug metabolism on the two forms of hamster liver microsomal aniline hydroxylase.

PubMed

McCoy, G D

1980-03-01

The aniline hydroxylase activity of microsomes isolated from hamster liver can be differentiated kinetically into high affinity (low K(m), form I) and low affinity (high K(m), form II) forms. Microsomes isolated from uninduced animals contain slightly more form I activity. The activity of the low affinity form (form II) is preferentially enhanced by Aroclor or 3-methylcholanthrene treatment, while phenobarbital treatment increases the activity of both forms. Chronic ethanol consumption results in enhancement of only the high affinity form (form I).

The eyes of Tullimonstrum reveal a vertebrate affinity.

PubMed

Clements, Thomas; Dolocan, Andrei; Martin, Peter; Purnell, Mark A; Vinther, Jakob; Gabbott, Sarah E

2016-04-28

Tullimonstrum gregarium is an iconic soft-bodied fossil from the Carboniferous Mazon Creek Lagerstätte (Illinois, USA). Despite a large number of specimens and distinct anatomy, various analyses over the past five decades have failed to determine the phylogenetic affinities of the 'Tully monster', and although it has been allied to such disparate phyla as the Mollusca, Annelida or Chordata, it remains enigmatic. The nature and phylogenetic affinities of Tullimonstrum have defied confident systematic placement because none of its preserved anatomy provides unequivocal evidence of homology, without which comparative analysis fails. Here we show that the eyes of Tullimonstrum possess ultrastructural details indicating homology with vertebrate eyes. Anatomical analysis using scanning electron microscopy reveals that the eyes of Tullimonstrum preserve a retina defined by a thick sheet comprising distinct layers of spheroidal and cylindrical melanosomes. Time-of-flight secondary ion mass spectrometry and multivariate statistics provide further evidence that these microbodies are melanosomes. A range of animals have melanin in their eyes, but the possession of melanosomes of two distinct morphologies arranged in layers, forming retinal pigment epithelium, is a synapomorphy of vertebrates. Our analysis indicates that in addition to evidence of colour patterning, ecology and thermoregulation, fossil melanosomes can also carry a phylogenetic signal. Identification in Tullimonstrum of spheroidal and cylindrical melanosomes forming the remains of retinal pigment epithelium indicates that it is a vertebrate; considering its body parts in this new light suggests it was an anatomically unusual member of total group Vertebrata.

Purine uptake in Plasmodium: transport versus metabolism.

PubMed

Kirk, Kiaran; Howitt, Susan M; Bröer, Stefan; Saliba, Kevin J; Downie, Megan J

2009-06-01

In a recent paper, Quashie et al. have proposed that purine uptake into the intraerythrocytic malaria parasite involves four different plasma membrane transporters - two high affinity and two low affinity. They equate one of the two high-affinity transporters with PfNT1, a transporter reported previously to be a low-affinity system. Here, we offer an alternative interpretation of their data, suggesting that the conclusions drawn by Quashie et al. take insufficient account of metabolism.

Host-Guest Complexes with Protein-Ligand-Like Affinities: Computational Analysis and Design

PubMed Central

Moghaddam, Sarvin; Inoue, Yoshihisa

2009-01-01

It has recently been discovered that guests combining a nonpolar core with cationic substituents bind cucurbit[7]uril (CB[7]) in water with ultra-high affinities. The present study uses the Mining Minima algorithm to study the physics of these extraordinary associations and to computationally test a new series of CB[7] ligands designed to bind with similarly high affinity. The calculations reproduce key experimental observations regarding the affinities of ferrocene-based guests with CB[7] and β-cyclodextrin and provide a coherent view of the roles of electrostatics and configurational entropy as determinants of affinity in these systems. The newly designed series of compounds is based on a bicyclo[2.2.2]octane core, which is similar in size and polarity to the ferrocene core of the existing series. Mining Minima predicts that these new compounds will, like the ferrocenes, bind CB[7] with extremely high affinities. PMID:19133781

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*

PubMed Central

Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.

2015-01-01

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638

High-affinity K+ uptake in pepper plants.

PubMed

Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

2005-06-01

High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

High l-Trp affinity of indoleamine 2,3-dioxygenase 1 is attributed to two residues located in the distal heme pocket.

PubMed

Yuasa, Hajime J

2016-10-01

Indoleamine 2, 3-dioxygenase (IDO) catalyzes the oxidative cleavage of the pyrrole ring of l-Trp to generate N-formyl-kynurenine. Two IDO genes, IDO1 and IDO2, are found in vertebrates. Mammalian IDO1s are high-affinity, l-Trp-degrading enzymes, whereas IDO2s generally have a relatively low affinity. It has been suggested that the distal-Ser (corresponding to Ser167 of human IDO1) was crucial for improvement in the affinity for l-Trp but this idea was insufficient to explain the high affinity shown by mammalian IDO1. In this study, the amino acid sequences of vertebrate ancestral IDO1 and ancestral IDO2 were inferred, and bacterially expressed ancestral IDOs were characterized. Although the amino acid sequences of the enzymes shared high identity (86%) with each other, they showed distinct enzymatic properties. In analyses of a series of ancestral IDO1/IDO2 chimeric enzymes and their variants, the distal-Tyr (corresponding to Tyr126 of human IDO1) was detected as another and was probably the most crucial residue for high l-Trp affinity. The two amino acid substitutions (distal-Ser to Thr and distal-Tyr to His) drastically decreased the l-Trp affinity and catalytic efficiency of IDO1s. Conversely, two substitutions (distal-Thr to Ser and distal-His to Tyr) were sufficient to bestow IDO1-like high affinity on ancestral and chicken IDO2. © 2016 Federation of European Biochemical Societies.

Pertussis toxin modifies the characteristics of both the inhibitory GTP binding proteins and the somatostatin receptor in anterior pituitary tumor cells

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

Mahy, N.; Woolkalis, M.; Thermos, K.

1988-08-01

The effects of pertussis toxin treatment on the characteristics of somatostatin receptors in the anterior pituitary tumor cell line AtT-20 were examined. Pertussis toxin selectively catalyzed the ADP ribosylation of the alpha subunits of the inhibitory GTP binding proteins in AtT-20 cells. Toxin treatment abolished somatostatin inhibition of forskolin-stimulated adenylyl cyclase activity and somatostatin stimulation of GTPase activity. To examine the effects of pertussis toxin treatment on the characteristics of the somatostatin receptor, the receptor was labeled by the somatostatin analog (125I)CGP 23996. (125I)CGP 23996 binding to AtT-20 cell membranes was saturable and within a limited concentration range was tomore » a single high affinity site. Pertussis toxin treatment reduced the apparent density of the high affinity (125I)CGP 23996 binding sites in AtT-20 cell membranes. Inhibition of (125I)CGP 23996 binding by a wide concentration range of CGP 23996 revealed the presence of two binding sites. GTP predominantly reduced the level of high affinity sites in control membranes. Pertussis toxin treatment also diminished the amount of high affinity sites. GTP did not affect (125I)CGP 23996 binding in the pertussis toxin-treated membranes. The high affinity somatostatin receptors were covalently labeled with (125I) CGP 23996 and the photoactivated crosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate. No high affinity somatostatin receptors, covalently bound to (125I)CGP 23996, were detected in the pertussis toxin-treated membranes. These results are most consistent with pertussis toxin uncoupling the inhibitory G proteins from the somatostatin receptor thereby converting the receptor from a mixed population of high and low affinity sites to only low affinity receptors.« less

Cytomegalovirus-Specific CD8+ T-Cells With Different T-Cell Receptor Affinities Segregate T-Cell Phenotypes and Correlate With Chronic Graft-Versus-Host Disease in Patients Post-Hematopoietic Stem Cell Transplantation

PubMed Central

Poiret, Thomas; Axelsson-Robertson, Rebecca; Remberger, Mats; Luo, Xiao-Hua; Rao, Martin; Nagchowdhury, Anurupa; Von Landenberg, Anna; Ernberg, Ingemar; Ringden, Olle; Maeurer, Markus

2018-01-01

Virus-specific T-cell responses are crucial to control cytomegalovirus (CMV) infections/reactivation in immunocompromised individuals. Adoptive cellular therapy with CMV-specific T-cells has become a viable treatment option. High-affinity anti-viral cellular immune responses are associated with improved long-term immune protection against CMV infection. To date, the characterization of high-affinity T-cell responses against CMV has not been achieved in blood from patients after allogeneic hematopoietic stem cell transplantation (HSCT). Therefore, the purpose of this study was to describe and analyze the phenotype and clinical impact of different CMV-specific CD8+ cytotoxic T-lymphocytes (CMV-CTL) classes based on their T-cell receptor (TCR) affinity. T-cells isolated from 23 patients during the first year following HSCT were tested for the expression of memory markers, programmed cell death 1 (PD-1), as well as TCR affinity, using three different HLA-A*02:01 CMVNLVPMVATV-Pp65 tetramers (wild-type, a245v and q226a mutants). High-affinity CMV-CTL defined by q226a tetramer binding, exhibited a higher frequency in CD8+ T-cells in the first month post-HSCT and exhibited an effector memory phenotype associated with strong PD-1 expression as compared to the medium- and low-affinity CMV-CTLs. High-affinity CMV-CTL was found at higher proportion in patients with chronic graft-versus-host disease (p < 0.001). This study provides a first insight into the detailed TCR affinities of CMV-CTL. This may be useful in order to improve current immunotherapy protocols using isolation of viral-specific T-cell populations based on their TCR affinity. PMID:29692783

pMHC affinity controls duration of CD8+ T cell–DC interactions and imprints timing of effector differentiation versus expansion

PubMed Central

Sharpe, James; Zehn, Dietmar; Kreutzfeldt, Mario

2016-01-01

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity–primed T cells acquired cytotoxic activity earlier than high affinity–primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity–stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. PMID:27799622

Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics.

PubMed

Seeman, P; Ko, F; Tallerico, T

2005-09-01

Although phencyclidine and ketamine are used to model a hypoglutamate theory of schizophrenia, their selectivity for NMDA receptors has been questioned. To determine the affinities of phencyclidine, ketamine, dizocilpine and LSD for the functional high-affinity state of the dopamine D2 receptor, D2High, their dissociation constants (Ki) were obtained on [3H]domperidone binding to human cloned dopamine D2 receptors. Phencyclidine had a high affinity for D2High with a Ki of 2.7 nM, in contrast to its low affinity for the NMDA receptor, with a Ki of 313 nM, as labeled by [3H]dizocilpine on rat striatal tissue. Ketamine also had a high affinity for D2High with a Ki of 55 nM, an affinity higher than its 3100 nM Ki for the NMDA sites. Dizocilpine had a Ki of 0.3 nM at D2High, but a Kd of 1.8 nM at the NMDA receptor. LSD had a Ki of 2 nM at D2High. Because the psychotomimetics had higher potency at D2High than at the NMDA site, the psychotomimetic action of these drugs must have a major contribution from D2 agonism. Because these drugs have a combined action on both dopamine receptors and NMDA receptors, these drugs, when given in vivo, test a combined hyperdopamine and hypoglutamate theory of psychosis.

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma.

PubMed

Ikonomopoulou, M P; Bradley, A J; Whittier, J M; Ibrahim, K

2006-11-01

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4 degrees C with high affinity (K (a) = 1.49 +/- 0.09 x 10(9) M(-1); 0.17 +/- 0.02 x 10(7) M(-1)) and low binding capacity (B (max) = 3.24 +/- 0.84 x 10(-5) M; 0.33 +/- 0.06 x 10(-4) M). The binding affinity and capacity of testosterone at 23 and 36 degrees C, respectively were similar to those determined at 4 degrees C. However, oestradiol showed no binding activity at 36 degrees C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36 degrees C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.

Classical Swine Fever Virus Glycoprotein Erns Is an Endoribonuclease with an Unusual Base Specificity

PubMed Central

Hausmann, Yvonne; Roman-Sosa, Gleyder; Thiel, Heinz-Jürgen; Rümenapf, Till

2004-01-01

The glycoprotein Erns of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for Erns. Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5′ of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that Erns is a highly active endoribonuclease (kcat/Km = 2 × 106 to 10 × 106 M−1 s−1) with a strong affinity to NpU containing single-stranded RNA substrates (Km = 85 to 260 nM). PMID:15113930

Protection of Arabidopsis Blunt-Ended Telomeres Is Mediated by a Physical Association with the Ku Heterodimer[OPEN

PubMed Central

Valuchova, Sona; Prokop, Zbynek; Hofr, Ctirad

2017-01-01

Telomeres form specialized chromatin that protects natural chromosome termini from being recognized as DNA double-strand breaks. Plants possess unusual blunt-ended telomeres that are unable to form t-loops or complex with single-strand DNA binding proteins, raising the question of the mechanism behind their protection. We have previously suggested that blunt-ended telomeres in Arabidopsis thaliana are protected by Ku, a DNA repair factor with a high affinity for DNA ends. In nonhomologous end joining, Ku loads onto broken DNA via a channel consisting of positively charged amino acids. Here, we demonstrate that while association of Ku with plant telomeres also depends on this channel, Ku’s requirements for DNA binding differ between DNA repair and telomere protection. We show that a Ku complex proficient in DNA loading but impaired in translocation along DNA is able to protect blunt-ended telomeres but is deficient in DNA repair. This suggests that Ku physically sequesters blunt-ended telomeres within its DNA binding channel, shielding them from other DNA repair machineries. PMID:28584163

An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

PubMed

Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

2015-05-01

An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity. Copyright © 2015 Elsevier Inc. All rights reserved.

Allele-Specific DNA Methylation and Its Interplay with Repressive Histone Marks at Promoter-Mutant TERT Genes.

PubMed

Stern, Josh Lewis; Paucek, Richard D; Huang, Franklin W; Ghandi, Mahmoud; Nwumeh, Ronald; Costello, James C; Cech, Thomas R

2017-12-26

A mutation in the promoter of the Telomerase Reverse Transcriptase (TERT) gene is the most frequent noncoding mutation in cancer. The mutation drives unusual monoallelic expression of TERT, allowing immortalization. Here, we find that DNA methylation of the TERT CpG island (CGI) is also allele-specific in multiple cancers. The expressed allele is hypomethylated, which is opposite to cancers without TERT promoter mutations. The continued presence of Polycomb repressive complex 2 (PRC2) on the inactive allele suggests that histone marks of repressed chromatin may be causally linked to high DNA methylation. Consistent with this hypothesis, TERT promoter DNA containing 5-methyl-CpG has much increased affinity for PRC2 in vitro. Thus, CpG methylation and histone marks appear to collaborate to maintain the two TERT alleles in different epigenetic states in TERT promoter mutant cancers. Finally, in several cancers, DNA methylation levels at the TERT CGI correlate with altered patient survival. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms.

PubMed

Wu, Mingxuan; Chong, Lucy S; Perlman, David H; Resnick, Adam C; Fiedler, Dorothea

2016-11-01

Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action.

The competitive advantage of a dual-transporter system.

PubMed

Levy, Sagi; Kafri, Moshe; Carmi, Miri; Barkai, Naama

2011-12-09

Cells use transporters of different affinities to regulate nutrient influx. When nutrients are depleted, low-affinity transporters are replaced by high-affinity ones. High-affinity transporters are helpful when concentrations of nutrients are low, but the advantage of reducing their abundance when nutrients are abundant is less clear. When we eliminated such reduced production of the Saccharomyces cerevisiae high-affinity transporters for phosphate and zinc, the elapsed time from the initiation of the starvation program until the lack of nutrients limited growth was shortened, and recovery from starvation was delayed. The latter phenotype was rescued by constitutive activation of the starvation program. Dual-transporter systems appear to prolong preparation for starvation and to facilitate subsequent recovery, which may optimize sensing of nutrient depletion by integrating internal and external information about nutrient availability.

A HIGH-LEVEL CALCULATION OF THE PROTON AFFINITY OF DIBORANE

EPA Science Inventory

The experimental proton affinity of diborane (B2H6) is based on an unstable species, B2H,+, 4 which has been observed only at low temperatures. The present work calculates the proton 5 affinity of diborane using the Gaussian-3 method and other high-level compound ab initio 6 met...

Detection of ovomucoid-specific low-affinity IgE in infants and its relationship to eczema.

PubMed

Kawamoto, Norio; Kamemura, Norio; Kido, Hiroshi; Fukao, Toshiyuki

2017-06-01

Allergen-specific low-affinity IgE was previously detected in cord blood by a highly sensitive densely carboxylated protein (DCP) chip, but not by ImmunoCAP. Here, we investigated the presence of low-affinity IgE during the early life of infants and observed its relationship with eczema. We conducted a birth cohort study, collecting sera at birth and 6 and 14 months of age (n = 110). We monitored the ovomucoid (OM)- and egg white (EW)-specific IgE (sIgE) by ImmunoCAP or DCP chip and analyzed the antigen affinity of sIgE by binding inhibition assays in the presence or absence of a mild chaotropic agent, diethyl amine (DEA). The low- and high-affinity OM-sIgEs and sensitization risk factors were analyzed by a multivariate logistic analysis. The OM-sIgE measured by DCP chip significantly correlated with that measured by ImmunoCAP, but some samples assessed as OM-sIgE positive by DCP chip were considered OM-sIgE negative by ImmunoCAP. Binding inhibition analysis after DEA treatment was performed for participants judged as OM-sIgE positive by DCP chip at 14 M. The group assessed as negative for OM- and EW-sIgE by ImmunoCAP at 6 and 14 months showed a larger binding inhibition curve shift after DEA treatment than did the group assessed as positive at these times, indicating the presence of low-affinity sIgE antibodies at 14 months. The logistic regression analysis found that persistent eczema from 6 to 14 months is a significant risk factor for developing high-affinity, but not low-affinity, sIgE. Human infant peripheral blood contains allergen-specific low-affinity sIgE. Persistent eczema is related to the development of high-affinity, but not low-affinity, IgE. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multitarget-directed tricyclic pyridazinones as G protein-coupled receptor ligands and cholinesterase inhibitors.

PubMed

Pau, Amedeo; Catto, Marco; Pinna, Giovanni; Frau, Simona; Murineddu, Gabriele; Asproni, Battistina; Curzu, Maria M; Pisani, Leonardo; Leonetti, Francesco; Loza, Maria Isabel; Brea, José; Pinna, Gérard A; Carotti, Angelo

2015-06-01

By following a multitarget ligand design approach, a library of 47 compounds was prepared, and they were tested as binders of selected G protein-coupled receptors (GPCRs) and inhibitors of acetyl and/or butyryl cholinesterase. The newly designed ligands feature pyridazinone-based tricyclic scaffolds connected through alkyl chains of variable length to proper amine moieties (e.g., substituted piperazines or piperidines) for GPCR and cholinesterase (ChE) molecular recognition. The compounds were tested at three different GPCRs, namely serotoninergic 5-HT1A, adrenergic α1A, and dopaminergic D2 receptors. Our main goal was the discovery of compounds that exhibit, in addition to ChE inhibition, antagonist activity at 5-HT1A because of its involvement in neuronal deficits typical of Alzheimer's and other neurodegenerative diseases. Ligands with nanomolar affinity for the tested GPCRs were discovered, but most of them behaved as dual antagonists of α1A and 5-HT1A receptors. Nevertheless, several compounds displaying this GPCR affinity profile also showed moderate to good inhibition of AChE and BChE, thus deserving further investigations to exploit the therapeutic potential of such unusual biological profiles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of two ammonia-oxidizing bacteria isolated from reactors operated with low dissolved oxygen concentrations.

PubMed

Park, H-D; Noguera, D R

2007-05-01

To obtain ammonia-oxidizing bacterial (AOB) strains inhabiting low dissolved oxygen (DO) environments and to characterize them to better understand their function and ecology. Using a serial dilution method, two AOB strains (ML1 and NL7) were isolated from chemostat reactors operated with low DO concentrations (0.12-0.24 mg l(-1)). Phylogenetically, strains ML1 and NL7 are affiliated to AOB within the Nitrosomonas europaea and Nitrosomonas oligotropha lineages, respectively. Kinetically, strain ML1 had high affinity for oxygen (0.24 +/- 0.13 mg l(-1)) and low affinity for ammonia (1.62 +/- 0.97 mg N l(-1)), while strain NL7 had high affinity for ammonia (0.48 +/- 0.35 mg l(-1)), but a surprisingly low affinity for oxygen (1.22 +/- 0.43 mg l(-1)). A co-culture experiment was used to iteratively estimate decay constants for both strains. The results indicated that AOB without high affinity for oxygen may have other mechanisms to persist in low DO environments, with high affinity for ammonia being important. This study provides a method to determine AOB growth kinetic parameters without assuming or neglecting decay constant. And, this is the first report on oxygen affinity constant of a N. oligotropha strain.

Pharmacological and gene regulation properties point to the SlHAK5 K+ transporter as a system for high-affinity Cs+ uptake in tomato plants.

PubMed

Ródenas, Reyes; Nieves-Cordones, Manuel; Rivero, Rosa M; Martinez, Vicente; Rubio, Francisco

2018-04-01

Potassium (K + ) and cesium (Cs + ) are chemically similar but while K + is an essential nutrient, Cs + can be toxic for living organisms, plants included. Two different situations could lead to problems derived from the presence of Cs + in agricultural systems: (1) presence of Cs + at high concentrations that could produce toxic effects on plants, (2) presence of micromolar concentrations of radiocesium, which can be accumulated in the plant and affect animal and human health through the food chain. While K + uptake has been well described in tomato plants, information on molecular mechanisms involved in Cs + accumulation in this species is absent. Here, we show that in tomato plants, high concentrations of Cs + produce deficiency of K + but do not induce high-affinity K + uptake or the gene encoding the high-affinity K + transporter SlHAK5. At these concentrations, Cs + uptake takes place through a Ca 2+ -sensitive pathway, probably a non-selective cation channel. At micromolar concentrations, Cs + is accumulated by a high-affinity uptake system upregulated in K + -starved plants. This high-affinity Cs + uptake shares features with high-affinity K + uptake. It is sensitive to NH 4 + and insensitive to Ba 2+ and Ca 2+ and its presence parallels the pattern of SlHAK5 expression. Moreover, blockers of reactive oxygen species and ethylene action repress SlHAK5 and negatively regulate both high-affinity K + and Cs + uptake. Thus, we propose that SlHAK5 contributes to Cs + uptake from micromolar concentrations in tomato plants and can constitute a pathway for radiocesium transfer from contaminated areas to the food chain. © 2017 Scandinavian Plant Physiology Society.

Pasireotide therapy in a rare and unusual case of plurihormonal pituitary macroadenoma

PubMed Central

Rajendran, Rajesh; Naik, Sarita; Sandeman, Derek D; Nasruddin, Azraai B

2013-01-01

Summary We report the use of pasireotide in a rare and unusual case of pituitary macroadenoma co-secreting GH, prolactin and ACTH. A 62-year-old Caucasian man presented with impotence. Clinically, he appeared acromegalic and subsequent investigations confirmed GH excess and hyperprolactinaemia. Magnetic resonance imaging (MRI) of pituitary revealed a large pituitary macroadenoma. He underwent trans-sphenoidal surgery and histology confirmed an adenoma with immunohistochemistry positive for ACTH, GH and prolactin. Acromegaly was not cured following surgery and inadequately controlled despite subsequent octreotide therapy. He underwent further debulking pituitary surgery, following which IGF1 levels improved but still high. This time adenoma cells showed immunohistochemistry positivity for ACTH only, following which subsequent investigations confirmed intermittent hypercortisolaemia compatible with pituitary Cushing's disease. We recommended radiotherapy, but in view of the pluripotential nature of the tumour, we proceeded with a trial of s.c. pasireotide therapy on the basis that it may control both his acromegaly and Cushing's disease. After 3 months of pasireotide therapy, his mean GH and IGF1 levels improved significantly, with improvement in his symptoms but intermittent hypercortisolaemia persists. His glycaemic control deteriorated requiring addition of new anti-diabetic medication. MRI imaging showed loss of contrast uptake within the tumour following pasireotide therapy but no change in size. We conclude that our patient has had a partial response to pasireotide therapy. Long-term follow-up studies are needed to establish its safety and efficacy in patients with acromegaly and/or Cushing's disease. Learning points Plurihormonal pituitary adenomas are rare and unusual.Patients with pituitary adenomas co-secreting ACTH and GH are more likely to present with acromegaly because GH excess can mask hypercortisolaemia.Pasireotide holds potential where conventional somatostatin analogues are not effective in acromegaly due to higher affinity for somatostatin receptor subtypes 1, 2, 3 and 5.Significant deterioration in glycaemic control remains a concern in the use of pasireotide.Currently, long-term safety and efficacy of pasireotide in patients with acromegaly and/or Cushing's disease are not fully clear. PMID:24616766

Comparative geochemistry of West African kimberlites: Evidence for a micaceous kimberlite endmember of sublithospheric origin

NASA Astrophysics Data System (ADS)

Taylor, Wayne R.; Tompkins, Linda A.; Haggerty, Stephen E.

1994-10-01

A suite of largely unaltered, aphanitic, mica-bearing hypabyssal kimberlites from the Koidu kimberlite complex of the West African Craton have been investigated to determine their geochemical affinity relative to Group I (nonmicaceous) and Group II (micaceous) kimberlites of southern Africa. Comparison is made with altered kimberlites from Liberia, other West African and global kimberlites. Based on major element oxides, the Koidu kimberlites, though mica-bearing, show closest compositional similarity with the Group IA kimberlites of southern Africa. Based on major and trace elements, the Koidu kimberlites show an unusual geochemical signature. This signature is similar to that of the distinctive, micaceous Aries kimberlite of northwest Australia, and includes high Nb/U (most samples > 46), Ce/Sr(>0.4), Ta/Hf(>2), and Nb/Zr(>1) ratios and low P 2O 5/Ce ∗10 4(<27), Ba/Rb(<32), and U/Th(<0.2) ratios compared with Group I kimberlites. Koidu kimberlites can be readily discriminated from Group II kimberlites by their higher Ti/K(>0.4) and Mb/La(>1) ratios and lower Ba/Nb(<10) and Pb/Ce(<0.06) ratios. The compositions of Liberian kimberlites are leached of mobile incompatible elements, but least affected samples show affinity to Group I. Guinea kimberlites appear to be of two types: one having affinity with Group IA and the other, micaceous variety, having affinity with the Aries kimberlite. Kimberlites with an Aries geochemical signature appear to exist on some other cratons, e.g., the Kundelungu kimberlites (Zaire) and two mica-bearing Group I kimberlites (S. Africa). The Koidu kimberlites exhibit compositionally-dependent isotopic heterogeneity though initial ɛNd and ɛSr values are broadly asthenospheric (i.e., near bulk earth) similar to Group I and Aries. A compositional spectrum appears to exist between nonmicaceous Group I kimberlites through mica-bearing Koidu kimberlites to extreme endmembers of the Aries type. This spectrum can be modelled as partial melts of heterogeneous peridotite sources which incorporate a potassic, high-Nb source component. The component may represent a fluoro-K-richterite-bearing peridotite residue derived by melt extraction from subduction-zone metasomatized peridotites. Such materials may have been trapped, together with former oceanic lithosphere, in the Transition Zone of the mantle. In response to lower mantle upwelling, diapiric uprise accompanying reduced volatile degassing of the kimberlite source may occur. Because of differences in oxidation potential across the 400 km discontinuity, reactions in the ascending diapir will lead to redox melting and ultimately segregation of the kimberlite melt at the base of the thermal boundary layer (P ~ 13 GPa) separating the subcratonic lithosphere from the convective mantle.

Computational modeling and molecular imprinting for the development of acrylic polymers with high affinity for bile salts.

PubMed

Yañez, Fernando; Chianella, Iva; Piletsky, Sergey A; Concheiro, Angel; Alvarez-Lorenzo, Carmen

2010-02-05

This work has focused on the rational development of polymers capable of acting as traps of bile salts. Computational modeling was combined with molecular imprinting technology to obtain networks with high affinity for cholate salts in aqueous medium. The screening of a virtual library of 18 monomers, which are commonly used for imprinted networks, identified N-(3-aminopropyl)-methacrylate hydrochloride (APMA.HCl), N,N-diethylamino ethyl methacrylate (DEAEM) and ethyleneglycol methacrylate phosphate (EGMP) as suitable functional monomers with medium-to-high affinity for cholic acid. The polymers were prepared with a fix cholic acid:functional monomer mole ratio of 1:4, but with various cross-linking densities. Compared to polymers prepared without functional monomer, both imprinted and non-imprinted microparticles showed a high capability to remove sodium cholate from aqueous medium. High affinity APMA-based particles even resembled the performance of commercially available cholesterol-lowering granules. The imprinting effect was evident in most of the networks prepared, showing that computational modeling and molecular imprinting can act synergistically to improve the performance of certain polymers. Nevertheless, both the imprinted and non-imprinted networks prepared with the best monomer (APMA.HCl) identified by the modeling demonstrated such high affinity for the template that the imprinting effect was less important. The fitting of adsorption isotherms to the Freundlich model indicated that, in general, imprinting increases the population of high affinity binding sites, except when the affinity of the functional monomer for the target molecule is already very high. The cross-linking density was confirmed as a key parameter that determines the accessibility of the binding points to sodium cholate. Materials prepared with 9% mol APMA and 91% mol cross-linker showed enough affinity to achieve binding levels of up to 0.4 mmol g(-1) (i.e., 170 mg g(-1)) under flow (1 mL min(-1)) of 0.2 mM sodium cholate solution. Copyright 2009 Elsevier B.V. All rights reserved.

Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

PubMed

Vinayek, R; Patto, R J; Menozzi, D; Gregory, J; Mrozinski, J E; Jensen, R T; Gardner, J D

1993-03-10

Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

PubMed Central

2015-01-01

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200

High-affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays.

PubMed

Whiteaker, Jeffrey R; Zhao, Lei; Frisch, Christian; Ylera, Francisco; Harth, Stefan; Knappik, Achim; Paulovich, Amanda G

2014-04-04

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.

A Fluorescent Protein Scaffold for Presenting Structurally Constrained Peptides Provides an Effective Screening System to Identify High Affinity Target-Binding Peptides

PubMed Central

Kadonosono, Tetsuya; Yabe, Etsuri; Furuta, Tadaomi; Yamano, Akihiro; Tsubaki, Takuya; Sekine, Takuya; Kuchimaru, Takahiro; Sakurai, Minoru; Kizaka-Kondoh, Shinae

2014-01-01

Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131–L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides. PMID:25084350

Bohr effect and temperature sensitivity of hemoglobins from highland and lowland deer mice.

PubMed

Jensen, Birgitte; Storz, Jay F; Fago, Angela

2016-05-01

An important means of physiological adaptation to environmental hypoxia is an increased oxygen (O2) affinity of the hemoglobin (Hb) that can help secure high O2 saturation of arterial blood. However, the trade-off associated with a high Hb-O2 affinity is that it can compromise O2 unloading in the systemic capillaries. High-altitude deer mice (Peromyscus maniculatus) have evolved an increased Hb-O2 affinity relative to lowland conspecifics, but it is not known whether they have also evolved compensatory mechanisms to facilitate O2 unloading to respiring tissues. Here we investigate the effects of pH (Bohr effect) and temperature on the O2-affinity of high- and low-altitude deer mouse Hb variants, as these properties can potentially facilitate O2 unloading to metabolizing tissues. Our experiments revealed that Bohr factors for the high- and low-altitude Hb variants are very similar in spite of the differences in O2-affinity. The Bohr factors of deer mouse Hbs are also comparable to those of other mammalian Hbs. In contrast, the high- and low-altitude variants of deer mouse Hb exhibited similarly low temperature sensitivities that were independent of red blood cell anionic cofactors, suggesting an appreciable endothermic allosteric transition upon oxygenation. In conclusion, high-altitude deer mice have evolved an adaptive increase in Hb-O2 affinity, but this is not associated with compensatory changes in sensitivity to changes in pH or temperature. Instead, it appears that the elevated Hb-O2 affinity in high-altitude deer mice is compensated by an associated increase in the tissue diffusion capacity of O2 (via increased muscle capillarization), which promotes O2 unloading. Copyright © 2016 Elsevier Inc. All rights reserved.

New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

PubMed Central

Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

2013-01-01

3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

Direct Measurement of Equilibrium Constants for High-Affinity Hemoglobins

PubMed Central

Kundu, Suman; Premer, Scott A.; Hoy, Julie A.; Trent, James T.; Hargrove, Mark S.

2003-01-01

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (KD ≪ 1 μM) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and ∼100 μM−1 respectively, indicate that they are not capable of facilitating oxygen transport. PMID:12770899

Direct Measurement of T Cell Receptor Affinity and Sequence from Naïve Anti-Viral T Cells

PubMed Central

Zhang, Shuqi; Parker, Patricia; Ma, Keyue; He, Chenfeng; Shi, Qian; Cui, Zhonghao; Williams, Chad; Wendel, Ben S.; Meriwether, Amanda; Salazar, Mary A.; Jiang, Ning

2016-01-01

T cells recognize and kill a myriad of pathogen-infected or cancer cells using a diverse set of T cell receptors (TCR). The affinity of TCR to cognate antigen is of high interest in adoptive T cell transfer immunotherapy and antigen-specific T cell repertoire immune profiling because it is widely known to correlate with downstream T cell responses. Here, we introduce the in situ TCR affinity and sequence test (iTAST) for simultaneous measurement of TCR affinity and sequence from single primary CD8+ T cells in human blood. We demonstrate that the repertoire of primary antigen-specific T cells from pathogen inexperienced individuals has a surprisingly broad affinity range of 1000-fold composed of diverse TCR sequences. Within this range, samples from older individuals contained a reduced frequency of high affinity T cells compared to young individuals, demonstrating an age-related effect of T cell attrition that could cause holes in the repertoire. iTAST should enable the rapid selection of high affinity TCRs ex vivo for adoptive immunotherapy and measurement of T cell response for immune monitoring applications. PMID:27252176

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

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

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE PAGES

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

2016-03-09

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

Adverse Reactions to Zolpidem: Case Reports and a Review of the Literature

PubMed Central

Miyaoka, Tsuyoshi; Tsuji, Seiichi; Inami, Yasushi; Nishida, Akira; Horiguchi, Jun

2010-01-01

Objective: Zolpidem, a nonbenzodiazepine hypnotic, is very effective and widely prescribed in clinical practice for the treatment of insomnia and is thought to have few adverse effects. However, zolpidem-induced adverse effects have begun to be reported in the literature, but few systemic descriptions of the adverse effects (especially for psychotic reactions) of zolpidem have been undertaken. In light of the accumulating reports of adverse reactions to zolpidem, we present 2 case reports of zolpidem-induced adverse effects and review the literature on this subject. Data Sources: Articles were selected by the authors on the basis of our experience and by a PubMed search using the terms zolpidem or side effects or adverse effects or adverse reactions. Study Selection and Data Extraction: Publications relevant to the objective of this article were obtained (1992–2010), and some adverse neuropsychiatric reactions were summarized. Data Synthesis: Zolpidem has been associated with the development of adverse neuropsychiatric reactions, such as hallucinations/sensory distortion, amnesia, sleepwalking/somnambulism, and nocturnal eating. The following 4 variables should be considered when prescribing zolpidem: (1) gender: women have been found to have a significantly higher serum zolpidem concentration than men; (2) zolpidem dose: the adverse reactions that develop are dose dependent; (3) protein binding affinity: a high proportion of zolpidem is protein bound; therefore, low serum albumin results in a higher level of free zolpidem leading to adverse psychiatric reactions; and (4) cytochrome P450 (CYP) isoenzyme inhibition: concomitant administration of zolpidem and other drugs may cause interactions that lead to increased concentrations of zolpidem. Conclusions: Zolpidem is clinically very effective in treating insomnia. However, while rare, zolpidem-induced unusual complex behavior may develop. Primary care physicians should be alert to the possible unusual complex adverse effects of zolpidem. PMID:21494350

Nickel binding and [NiFe]-hydrogenase maturation by the metallochaperone SlyD with a single metal-binding site in Escherichia coli.

PubMed

Kaluarachchi, Harini; Altenstein, Matthias; Sugumar, Sonia R; Balbach, Jochen; Zamble, Deborah B; Haupt, Caroline

2012-03-16

SlyD (sensitive to lysis D) is a nickel metallochaperone involved in the maturation of [NiFe]-hydrogenases in Escherichia coli (E. coli) and specifically contributes to the nickel delivery step during enzyme biosynthesis. This protein contains a C-terminal metal-binding domain that is rich in potential metal-binding residues that enable SlyD to bind multiple nickel ions with high affinity. The SlyD homolog from Thermus thermophilus does not contain the extended cysteine- and histidine-rich C-terminal tail of the E. coli protein, yet it binds a single Ni(II) ion tightly. To investigate whether a single metal-binding motif can functionally replace the full-length domain, we generated a truncation of E. coli SlyD, SlyD155. Ni(II) binding to SlyD155 was investigated by using isothermal titration calorimetry, NMR and electrospray ionization mass spectrometry measurements. This in vitro characterization revealed that SlyD155 contains a single metal-binding motif with high affinity for nickel. Structural characterization by X-ray absorption spectroscopy and NMR indicated that nickel was coordinated in an octahedral geometry with at least two histidines as ligands. Heterodimerization between SlyD and another hydrogenase accessory protein, HypB, is essential for optimal hydrogenase maturation and was confirmed for SlyD155 via cross-linking experiments and NMR titrations, as were conserved chaperone and peptidyl-prolyl isomerase activities. Although these properties of SlyD are preserved in the truncated version, it does not modulate nickel binding to HypB in vitro or contribute to the maturation of [NiFe]-hydrogenases in vivo, unlike the full-length protein. This study highlights the importance of the unusual metal-binding domain of E. coli SlyD in hydrogenase biogenesis. Copyright © 2012 Elsevier Ltd. All rights reserved.

Existence of three subtypes of bradykinin B2 receptors in guinea pig.

PubMed

Seguin, L; Widdowson, P S; Giesen-Crouse, E

1992-12-01

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity-tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice

PubMed Central

Liu, Xiaojun; Jiang, Shuguang; Fang, Chongyun; Yang, Shiyu; Olalere, Devvora; Pequignot, Edward C.; Cogdill, Alexandria P.; Li, Na; Ramones, Melissa; Granda, Brian; Zhou, Li; Loew, Andreas; Young, Regina M.; June, Carl H.; Zhao, Yangbing

2015-01-01

Target-mediated toxicity is a major limitation in the development of chimeric antigen T cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues which express it at physiologic levels. A CAR-expressing T cell panel was generated with target antigen affinities varying over three orders of magnitude. High-affinity cells recognized target expressed at any level, including at levels in normal cells that were undetectable by flow cytometry. Affinity-tuned cells exhibited robust antitumor efficacy similar to high-affinity cells, but spared normal cells expressing physiologic target levels. The use of affinity-tuned scFvs offers a strategy to empower wider use of CAR T cells against validated targets widely overexpressed on solid tumors, including those considered undruggable by this approach. PMID:26330166

[Progresses in screening active compounds from herbal medicine by affinity chromatography].

PubMed

Feng, Ying-shu; Tong, Shan-shan; Xu, Xi-ming; Yu, Jiang-nan

2015-03-01

Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening.

Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74.

PubMed

Miller, Philip S; Barwell, James; Poyner, David R; Wigglesworth, Mark J; Garland, Stephen L; Donnelly, Dan

2010-01-01

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1. Copyright 2009 Elsevier Inc. All rights reserved.

Cloning, heterologous expression, and in situ characterization of the first high affinity nucleobase transporter from a protozoan.

PubMed

Burchmore, Richard J S; Wallace, Lynsey J M; Candlish, Denise; Al-Salabi, Mohammed I; Beal, Paul R; Barrett, Michael P; Baldwin, Stephen A; de Koning, Harry P

2003-06-27

While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 +/- 0.6 and 0.66 +/- 0.22 microm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine. A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by approximately 80% and H4 transport activity by approximately 90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.

Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1.

PubMed

Real Hernandez, Luis M; Gonzalez de Mejia, Elvira

2017-04-01

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (-7.2 to -7.0kcal/mol) and the cowpea bean dipeptide Lys-Asp (-7.0kcal/mol) had higher binding affinities than ezetimibe (-6.6kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (-7.2kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption. Copyright © 2017 Elsevier Inc. All rights reserved.

Selection of High-Affinity Peptidic Serine Protease Inhibitors with Increased Binding Entropy from a Back-Flip Library of Peptide-Protease Fusions.

PubMed

Sørensen, Hans Peter; Xu, Peng; Jiang, Longguang; Kromann-Hansen, Tobias; Jensen, Knud J; Huang, Mingdong; Andreasen, Peter A

2015-09-25

We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the concept by fusing a 10-residue-long, disulfide-bond-constrained inhibitory peptide, randomized in selected positions, to the catalytic domain of the serine protease murine urokinase-type plasminogen activator. High-affinity inhibitory peptide variants were identified as those that conferred to the fusion protease the lowest activity for substrate hydrolysis. The usefulness of the strategy was demonstrated by the selection of peptidic inhibitors of murine urokinase-type plasminogen activator with a low nanomolar affinity. The high affinity could not have been predicted by rational considerations, as the high affinity was associated with a loss of polar interactions and an increased binding entropy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human llamas: adaptation to altitude in subjects with high hemoglobin oxygen affinity.

PubMed Central

Hebbel, R P; Eaton, J W; Kronenberg, R S; Zanjani, E D; Moore, L G; Berger, E M

1978-01-01

To assess the adaptive value of the right-shift of the oxyhemoglobin dissociation curve (decreased affinity for oxygen) observed in humans upon altitude exposure, the short-term physiologic responses to altitude-induced hypoxia were evaluated in two subjects with a high oxygen affinity hemoglobin (Hb Andrew-Minneapolis) and in two of their normal siblings. In striking contrast to normal subjects, at moderately high altitude (3,100 m) the high affinity subjects manifested: (a) lesser increments in resting heart rate; (b) minimal increases in plasma and urinary erythropoietin; (c) no decrement in maximal oxygen consumption; and (d) no thrombocytopenia. There was no difference between subject pairs in 2,3-diphosphoglycerate response to altitude exposure. These results tend to contradict the belief that a decrease in hemoglobin oxygen affinity is of adaptive value to humans at moderate altitudes. Rather, they support the hypothesis that, despite disadvantages at low altitude, a left-shifted oxyhemoglobin dissociation curve may confer a degree of preadaptation to altitude. PMID:29054

Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H2-Oxidizing Bacteria ▿†

PubMed Central

Constant, Philippe; Chowdhury, Soumitra Paul; Hesse, Laura; Pratscher, Jennifer; Conrad, Ralf

2011-01-01

Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H2 possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H2-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H2 oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 106 to 108 hhyL gene copies g (dry weight)−1. Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H2-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H2 oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H2 by soil, because high-affinity H2 oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur. PMID:21742924

Identification of the SRC pyrimidine-binding protein (SPy) as hnRNP K: implications in the regulation of SRC1A transcription

PubMed Central

Ritchie, Shawn A.; Pasha, Mohammed K.; Batten, Danielle J. P.; Sharma, Rajendra K.; Olson, Douglas J. H.; Ross, Andrew R. S.; Bonham, Keith

2003-01-01

The human SRC gene encodes pp60c–src, a non-receptor tyrosine kinase involved in numerous signaling pathways. Activation or overexpression of c-Src has also been linked to a number of important human cancers. Transcription of the SRC gene is complex and regulated by two closely linked but highly dissimilar promoters, each associated with its own distinct non-coding exon. In many tissues SRC expression is regulated by the housekeeping-like SRC1A promoter. In addition to other regulatory elements, three substantial polypurine:polypyrimidine (TC) tracts within this promoter are required for full transcriptional activity. Previously, we described an unusual factor called SRC pyrimidine-binding protein (SPy) that could bind to two of these TC tracts in their double-stranded form, but was also capable of interacting with higher affinity to all three pyrimidine tracts in their single-stranded form. Mutations in the TC tracts, which abolished the ability of SPy to interact with its double-stranded DNA target, significantly reduced SRC1A promoter activity, especially in concert with mutations in critical Sp1 binding sites. Here we expand upon our characterization of this interesting factor and describe the purification of SPy from human SW620 colon cancer cells using a DNA affinity-based approach. Subsequent in-gel tryptic digestion of purified SPy followed by MALDI-TOF mass spectrometric analysis identified SPy as heterogeneous nuclear ribonucleoprotein K (hnRNP K), a known nucleic-acid binding protein implicated in various aspects of gene expression including transcription. These data provide new insights into the double- and single-stranded DNA-binding specificity, as well as functional properties of hnRNP K, and suggest that hnRNP K is a critical component of SRC1A transcriptional processes. PMID:12595559

Structure-based Analysis to Hu-DNA Binding

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

Swinger,K.; Rice, P.

2007-01-01

HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently publishedmore » Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from 10-14.5 kcal/mol, representing K{sub d} values in the nanomolar to low picomolar range, and a maximum stabilization of at least 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.« less

Evolution of substrate specificity for the bile salt transporter ASBT (SLC10A2)[S

PubMed Central

Lionarons, Daniël A.; Boyer, James L.; Cai, Shi-Ying

2012-01-01

The apical Na+-dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5α-bile alcohols and the skate that utilizes structurally different 5β-bile alcohols, and compared substrate specificity with ASBT from humans who utilize modern 5β-bile acids. Everted gut sacs of skate but not the more primitive lamprey transported 3H-taurocholic acid (TCA), a modern 5β-bile acid. However, molecular cloning identified ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbt's indicate that lamprey Asbt has high affinity for 5α-bile alcohols, low affinity for 5β-bile alcohols, and lacks affinity for TCA, whereas skate Asbt showed high affinity for 5α- and 5β-bile alcohols but low affinity for TCA. In contrast, human ASBT demonstrated high affinity for all three bile salt types. These findings suggest that ASBT evolved from the earliest vertebrates by gaining affinity for modern bile salts while retaining affinity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution. PMID:22669917

Hemoglobin–oxygen affinity in high-altitude vertebrates: is there evidence for an adaptive trend?

PubMed Central

2016-01-01

ABSTRACT In air-breathing vertebrates at high altitude, fine-tuned adjustments in hemoglobin (Hb)–O2 affinity provide an energetically efficient means of mitigating the effects of arterial hypoxemia. However, it is not always clear whether an increased or decreased Hb–O2 affinity should be expected to improve tissue O2 delivery under different degrees of hypoxia, due to the inherent trade-off between arterial O2 loading and peripheral O2 unloading. Theoretical results indicate that the optimal Hb–O2 affinity varies as a non-linear function of environmental O2 availability, and the threshold elevation at which an increased Hb–O2 affinity becomes advantageous depends on the magnitude of diffusion limitation (the extent to which O2 equilibration at the blood–gas interface is limited by the kinetics of O2 exchange). This body of theory provides a framework for interpreting the possible adaptive significance of evolved changes in Hb–O2 affinity in vertebrates that have colonized high-altitude environments. To evaluate the evidence for an empirical generalization and to test theoretical predictions, I synthesized comparative data in a phylogenetic framework to assess the strength of the relationship between Hb–O2 affinity and native elevation in mammals and birds. Evidence for a general trend in mammals is equivocal, but there is a remarkably strong positive relationship between Hb–O2 affinity and native elevation in birds. Evolved changes in Hb function in high-altitude birds provide one of the most compelling examples of convergent biochemical adaptation in vertebrates. PMID:27802149

Hemoglobin-oxygen affinity in high-altitude vertebrates: is there evidence for an adaptive trend?

PubMed

Storz, Jay F

2016-10-15

In air-breathing vertebrates at high altitude, fine-tuned adjustments in hemoglobin (Hb)-O 2 affinity provide an energetically efficient means of mitigating the effects of arterial hypoxemia. However, it is not always clear whether an increased or decreased Hb-O 2 affinity should be expected to improve tissue O 2 delivery under different degrees of hypoxia, due to the inherent trade-off between arterial O 2 loading and peripheral O 2 unloading. Theoretical results indicate that the optimal Hb-O 2 affinity varies as a non-linear function of environmental O 2 availability, and the threshold elevation at which an increased Hb-O 2 affinity becomes advantageous depends on the magnitude of diffusion limitation (the extent to which O 2 equilibration at the blood-gas interface is limited by the kinetics of O 2 exchange). This body of theory provides a framework for interpreting the possible adaptive significance of evolved changes in Hb-O 2 affinity in vertebrates that have colonized high-altitude environments. To evaluate the evidence for an empirical generalization and to test theoretical predictions, I synthesized comparative data in a phylogenetic framework to assess the strength of the relationship between Hb-O 2 affinity and native elevation in mammals and birds. Evidence for a general trend in mammals is equivocal, but there is a remarkably strong positive relationship between Hb-O 2 affinity and native elevation in birds. Evolved changes in Hb function in high-altitude birds provide one of the most compelling examples of convergent biochemical adaptation in vertebrates. © 2016. Published by The Company of Biologists Ltd.

Specificity of cell–cell adhesion by classical cadherins: Critical role for low-affinity dimerization through β-strand swapping

PubMed Central

Chen, Chien Peter; Posy, Shoshana; Ben-Shaul, Avinoam; Shapiro, Lawrence; Honig, Barry H.

2005-01-01

Cadherins constitute a family of cell-surface proteins that mediate intercellular adhesion through the association of protomers presented from juxtaposed cells. Differential cadherin expression leads to highly specific intercellular interactions in vivo. This cell–cell specificity is difficult to understand at the molecular level because individual cadherins within a given subfamily are highly similar to each other both in sequence and structure, and they dimerize with remarkably low binding affinities. Here, we provide a molecular model that accounts for these apparently contradictory observations. The model is based in part on the fact that cadherins bind to one another by “swapping” the N-terminal β-strands of their adhesive domains. An inherent feature of strand swapping (or, more generally, the domain swapping phenomenon) is that “closed” monomeric conformations act as competitive inhibitors of dimer formation, thus lowering affinities even when the dimer interface has the characteristics of high-affinity complexes. The model describes quantitatively how small affinity differences between low-affinity cadherin dimers are amplified by multiple cadherin interactions to establish large specificity effects at the cellular level. It is shown that cellular specificity would not be observed if cadherins bound with high affinities, thus emphasizing the crucial role of strand swapping in cell–cell adhesion. Numerical estimates demonstrate that the strength of cellular adhesion is extremely sensitive to the concentration of cadherins expressed at the cell surface. We suggest that the domain swapping mechanism is used by a variety of cell-adhesion proteins and that related mechanisms to control affinity and specificity are exploited in other systems. PMID:15937105

DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX.

PubMed

Bitaraf, F S; Rasooli, I; Mousavi Gargari, S L

2016-03-01

Haemophilus influenzae type b (Hib) causes acute bacterial meningitis (ABM) in children, with a mortality rate of about 3-6 % of the affected patients. ABM can lead to death during a period of hours to several days and, hence, rapid and early detection of the infection is crucial. Aptamers, the short single-stranded DNA or RNA with high affinity to target molecules, are selected by a high-flux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology (SELEX). In this study, whole-cell SELEX was applied for the selection of target-specific aptamers with high affinity to Hib. ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells (Hib). The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry. The aptamers with higher binding affinity were cloned. Four of 68 aptamer clones were selected for sequencing. The dissociation constant (Kd) of the high-affinity aptamer clones 45 and 63 were 47.10 and 28.46 pM, respectively. These aptamers did not bind to other bacterial species, including the seven meningitis-causing bacteria. They showed distinct affinity to various H. influenzae strains only. These aptamers showed the highest affinity to Hib and the lowest affinity to H. influenzae type c and to other meningitis-causing bacteria. Clone 63 could detect Hib in patients' cerebrospinal fluid (CSF) samples at 60 colony-forming units (CFU)/mL. The results indicate applicability of the aptamers for rapid and early detection of infections brought about by Hib.

Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

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

Poat, J.A.; Cripps, H.E.; Iversen, L.L.

1988-05-01

Forskolin labelled with (/sup 3/H) bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg/sup 2 +/ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no furthermore » increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.« less

Selectivity and permeation of alkali metal ions in K+-channels.

PubMed

Furini, Simone; Domene, Carmen

2011-06-24

Ion conduction in K(+)-channels is usually described in terms of concerted movements of K(+) progressing in a single file through a narrow pore. Permeation is driven by an incoming ion knocking on those ions already inside the protein. A fine-tuned balance between high-affinity binding and electrostatic repulsive forces between permeant ions is needed to achieve efficient conduction. While K(+)-channels are known to be highly selective for K(+) over Na(+), some K(+) channels conduct Na(+) in the absence of K(+). Other ions are known to permeate K(+)-channels with a more moderate preference and unusual conduction features. We describe an extensive computational study on ion conduction in K(+)-channels rendering free energy profiles for the translocation of three different alkali ions and some of their mixtures. The free energy maps for Rb(+) translocation show at atomic level why experimental Rb(+) conductance is slightly lower than that of K(+). In contrast to K(+) or Rb(+), external Na(+) block K(+) currents, and the sites where Na(+) transport is hindered are characterized. Translocation of K(+)/Na(+) mixtures is energetically unfavorable owing to the absence of equally spaced ion-binding sites for Na(+), excluding Na(+) from a channel already loaded with K(+). Copyright © 2011 Elsevier Ltd. All rights reserved.

Solution structure of a highly stable DNA duplex conjugated to a minor groove binder.

PubMed Central

Kumar, S; Reed, M W; Gamper, H B; Gorn, V V; Lukhtanov, E A; Foti, M; West, J; Meyer, R B; Schweitzer, B I

1998-01-01

The tripeptide 1,2-dihydro-(3 H )-pyrrolo[3,2- e ]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder is conjugated to the 5'-end of short oligonucleotides the conjugates form unusually stable hybrids with complementary DNA and thus may have useful diagnostic and/or therapeutic applications. In order to gain an understanding of the structural interactions between the CDPI3minor groove binding moiety and the DNA, we have determined and compared the solution structure of a duplex consisting of oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 and its complementary strand to an unmodified control duplex of the same sequence using nuclear magnetic resonance techniques. Thermal denaturation studies indicated that the hybrid of this conjugate with its complementary strand had a melting temperature that was 30 degrees C higher compared with the unmodified control duplex. Following restrained molecular dynamics and relaxation matrix refinement, the solution structure of the CDPI3-conjugated DNA duplex demonstrated that the overall shape of the duplex was that of a straight B-type helix and that the CDPI3moiety was bound snugly in the minor groove, where it was stabilized by extensive van der Waal's interactions. PMID:9443977

Solution structure of a highly stable DNA duplex conjugated to a minor groove binder.

PubMed

Kumar, S; Reed, M W; Gamper, H B; Gorn, V V; Lukhtanov, E A; Foti, M; West, J; Meyer, R B; Schweitzer, B I

1998-02-01

The tripeptide 1,2-dihydro-(3 H )-pyrrolo[3,2- e ]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder is conjugated to the 5'-end of short oligonucleotides the conjugates form unusually stable hybrids with complementary DNA and thus may have useful diagnostic and/or therapeutic applications. In order to gain an understanding of the structural interactions between the CDPI3minor groove binding moiety and the DNA, we have determined and compared the solution structure of a duplex consisting of oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 and its complementary strand to an unmodified control duplex of the same sequence using nuclear magnetic resonance techniques. Thermal denaturation studies indicated that the hybrid of this conjugate with its complementary strand had a melting temperature that was 30 degrees C higher compared with the unmodified control duplex. Following restrained molecular dynamics and relaxation matrix refinement, the solution structure of the CDPI3-conjugated DNA duplex demonstrated that the overall shape of the duplex was that of a straight B-type helix and that the CDPI3moiety was bound snugly in the minor groove, where it was stabilized by extensive van der Waal's interactions.

Synthesis and superconductivity of highly underdoped HgBa2CuO4+δ

NASA Astrophysics Data System (ADS)

Edwards, P. P.; Gameson, I.; Fletcher, A.; Peacock, G. B.

1998-05-01

The highest transition temperature superconductors are found within the complex homologous series HgBa2Can-1CunO2n+2+δ (n=1-7), with the third member, HgBa2Ca2Cu3O8+δ possessing the record-high transition temperature (Tc) of 135 K at room pressure. The first member of this family, HgBa2CuO4+δ having a Tc of up to 97 K, displays the highest transition temperature for any analogous compounds with a single copper-layer. The chemical reaction for the formation of this material is intrinsically complex due to the natural high volatility of mercury-bearing compounds; chemical synthesis has been postulated to proceed via a solid-vapour reaction. With this in mind, we have developed a mixed solid/vapour phase synthesis for HgBa2CuO4+δ using what one might term a `remote' source of mercury, in this case elemental Hg itself. Interestingly, because of the zero oxidation state of elemental mercury in the reagent mixture, the synthesis reaction proceeds under reducing conditions. By this route, a highly underdoped state (Tc<=35 K) of the superconducting phase HgBa2CuO4+δ is readily obtained. This level of underdoping is extremely difficult to achieve by more conventional synthetic routes. We comment on the unusually high oxygen affinity of the resulting underdoped compound, in relation to other cuprate superconductors, and the implied mobility of oxygen defects within the crystal structure.

SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

PubMed Central

Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

2016-01-01

Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

Emergence of the acute-phase protein hemopexin in jawed vertebrates.

PubMed

Dooley, Helen; Buckingham, E Bryan; Criscitiello, Michael F; Flajnik, Martin F

2010-01-01

When released from damaged erythrocytes free heme not only provides a source of iron for invading bacteria but also highly toxic due to its ability to catalyze free radical formation. Hemopexin (Hx) binds free heme with very high-affinity and thus protects against heme toxicity, sequesters heme from pathogens, and helps conserve valuable iron. Hx is also an acute-phase serum protein (APP), whose expression is induced by inflammation. To date Hx has been identified as far back in phylogeny as bony fish where it is called warm-temperature acclimation-related 65 kDa protein (WAP65), as serum protein levels are increased at elevated environmental temperatures as well as by infection. During analysis of nurse shark (Ginglymostoma cirratum) plasma we isolated a Ni(2+)-binding serum glycoprotein and characterized it as the APP Hx. We subsequently cloned Hx from nurse shark and another cartilaginous fish species, the little skate Leucoraja erinacea. Functional analysis showed shark Hx, like that of mammals, binds heme but is found at unusually high levels in normal shark serum. As an Hx orthologue could not be found in the genomes of jawless vertebrates or lower deuterostomes it appears to have arisen just prior to the emergence of jawed vertebrates, coincident with the second round of genome-wide duplication and the appearance of tetrameric hemoglobin (Hb). Copyright © 2010 Elsevier Ltd. All rights reserved.

Characterization of nicotine binding to the rat brain P/sub 2/ preparation: the identification of multiple binding sites which include specific up-regulatory site(s)

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

Sloan, J.W.

1984-01-01

These studies show that nicotine binds to the rat brain P/sub 2/ preparation by saturable and reversible processes. Multiple binding sites were revealed by the configuration of saturation, kinetic and Scatchard plots. A least squares best fit of Scatchard data using nonlinear curve fitting programs confirmed the presence of a very high affinity site, an up-regulatory site, a high affinity site and one or two low affinity sites. Stereospecificity was demonstrated for the up-regulatory site where (+)-nicotine was more effective and for the high affinity site where (-)-nicotine had a higher affinity. Drugs which selectively up-regulate nicotine binding site(s) havemore » been identified. Further, separate very high and high affinity sites were identified for (-)- and (+)-(/sup 3/H)nicotine, based on evidence that the site density for the (-)-isomer is 10 times greater than that for the (+)-isomer at these sites. Enhanced nicotine binding has been shown to be a statistically significant phenomenon which appears to be a consequence of drugs binding to specific site(s) which up-regulate binding at other site(s). Although Scatchard and Hill plots indicate positive cooperatively, up-regulation more adequately describes the function of these site(s). A separate up-regulatory site is suggested by the following: (1) Drugs vary markedly in their ability to up-regulate binding. (2) Both the affinity and the degree of up-regulation can be altered by structural changes in ligands. (3) Drugs with specificity for up-regulation have been identified. (4) Some drugs enhance binding in a dose-related manner. (5) Competition studies employing cold (-)- and (+)-nicotine against (-)- and (+)-(/sup 3/H)nicotine show that the isomers bind to separate sites which up-regulate binding at the (-)- and (+)-nicotine high affinity sites and in this regard (+)-nicotine is more specific and efficacious than (-)-nicotine.« less

A molecular recognizing system of serotonin in rat fetal axonal growth cones: uptake and high affinity binding.

PubMed

Mercado, R; Hernández, J

1992-09-18

Axonal growth cone particles (AGCP) isolated from prenatal and postnatal rat brain had different high-affinity 5-HT uptake characteristics. In postnatal AGCP the uptake behaves as in the adult rat brain, while in the prenatal AGCP the uptake characteristics seem to be in a transitional stage. Also in prenatal AGCP we observed specific, high-affinity 5-HT binding sites. These results support the idea of an important role for 5-HT during axogenesis.

Surfactant-free Colloidal Particles with Specific Binding Affinity

PubMed Central

2017-01-01

Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles. PMID:28847149

An ERP study of passive creative conceptual expansion using a modified alternate uses task.

PubMed

Kröger, Sören; Rutter, Barbara; Hill, Holger; Windmann, Sabine; Hermann, Christiane; Abraham, Anna

2013-08-21

A novel ERP paradigm was employed to investigate conceptual expansion, a central component of creative thinking. Participants were presented with word pairs, consisting of everyday objects and uses for these objects, which had to be judged based on the two defining criteria of creative products: unusualness and appropriateness. Three subject-determined trial types resulted from this judgement: high unusual and low appropriate (nonsensical uses), low unusual and high appropriate (common uses), and high unusual and high appropriate (creative uses). Word pairs of the creative uses type are held to passively induce conceptual expansion. The N400 component was not specifically modulated by conceptual expansion but was, instead, generally responsive as a function of unusualness or novelty of the stimuli (nonsense=creative>common). Explorative analyses in a later time window (500-900 ms) revealed that ERP activity in this phase indexes appropriateness (nonsense>creative=common). In the discussion of these findings with reference to the literature on semantic cognition, both components are proposed as indexing processes relevant to conceptual expansion as they are selectively involved in the encoding and integration of a newly established semantic connection between two previously unrelated concepts. Copyright © 2013 Elsevier B.V. All rights reserved.

Interaction of Ochratoxin A and Its Thermal Degradation Product 2'R-Ochratoxin A with Human Serum Albumin.

PubMed

Sueck, Franziska; Poór, Miklós; Faisal, Zelma; Gertzen, Christoph G W; Cramer, Benedikt; Lemli, Beáta; Kunsági-Máté, Sándor; Gohlke, Holger; Humpf, Hans-Ulrich

2018-06-22

Ochratoxin A (OTA) is a toxic secondary metabolite produced by several fungal species of the genus Penicillium and Aspergillus . 2′ R -Ochratoxin A (2′ R -OTA) is a thermal isomerization product of OTA formed during food processing at high temperatures. Both compounds are detectable in human blood in concentrations between 0.02 and 0.41 µg/L with 2′ R -OTA being only detectable in the blood of coffee drinkers. Humans have approximately a fifty-fold higher exposure through food consumption to OTA than to 2′ R -OTA. In human blood, however, the differences between the concentrations of the two compounds is, on average, only a factor of two. To understand these unexpectedly high 2′ R -OTA concentrations found in human blood, the affinity of this compound to the most abundant protein in human blood the human serum albumin (HSA) was studied and compared to that of OTA, which has a well-known high binding affinity. Using fluorescence spectroscopy, equilibrium dialysis, circular dichroism (CD), high performance affinity chromatography (HPAC), and molecular modelling experiments, the affinities of OTA and 2′ R -OTA to HSA were determined and compared with each other. For the affinity of HSA towards OTA, a log K of 7.0⁻7.6 was calculated, while for its thermally produced isomer 2′ R -OTA, a lower, but still high, log K of 6.2⁻6.4 was determined. The data of all experiments showed consistently that OTA has a higher affinity to HSA than 2′ R -OTA. Thus, differences in the affinity to HSA cannot explain the relatively high levels of 2′ R -OTA found in human blood samples.

Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

PubMed

Sams, Craig; Loizou, George D; Cocker, John; Lennard, Martin S

2004-03-07

The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

High-Speed Lateral Flow Strategy for a Fast Biosensing with an Improved Selectivity and Binding Affinity.

PubMed

Cho, Dong Guk; Yoo, Haneul; Lee, Haein; Choi, Yeol Kyo; Lee, Minju; Ahn, Dong June; Hong, Seunghun

2018-05-10

We report a high-speed lateral flow strategy for a fast biosensing with an improved selectivity and binding affinity even under harsh conditions. In this strategy, biosensors were fixed at a location away from the center of a round shape disk, and the disk was rotated to create the lateral flow of a target solution on the biosensors during the sensing measurements. Experimental results using the strategy showed high reaction speeds, high binding affinity, and low nonspecific adsorptions of target molecules to biosensors. Furthermore, binding affinity between target molecules and sensing molecules was enhanced even in harsh conditions such as low pH and low ionic strength conditions. These results show that the strategy can improve the performance of conventional biosensors by generating high-speed lateral flows on a biosensor surface. Therefore, our strategy can be utilized as a simple but powerful tool for versatile bio and medical applications.

High affinity ligands from in vitro selection: Complex targets

PubMed Central

Morris, Kevin N.; Jensen, Kirk B.; Julin, Carol M.; Weil, Michael; Gold, Larry

1998-01-01

Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems. PMID:9501188

Haemoglobin Pierre-Benite--a high affinity variant associated with relative polycythaemia.

PubMed

Beard, M E; Potter, H C; Spearing, R L; Brennan, S O

2001-12-01

This is the second reported example of Hb Pierre--Benite (beta90 Glu-->Asp). This mutation is associated with increased oxygen affinity and polycythaemia. No instability was found and there was no charge shift detected by cellulose acetate electrophoresis at pH 8.3. The mutation was however, clearly indicated by electrospray ionization mass spectrometry (ESI MS), which showed an abnormal beta chain with a 14 Da decrease in mass. Blood volume studies documented a relative rather than a true polycythaemia and this finding has been reported in at least two other high affinity haemoglobin variants--Hb Heathrow and Hb Rahere. This finding led to delay in diagnosis because high oxygen affinity variants are conventionally considered to cause a true polycythaemia.

Specificity and Affinity Quantification of Flexible Recognition from Underlying Energy Landscape Topography

PubMed Central

Chu, Xiakun; Wang, Jin

2014-01-01

Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less) flexibility leads to weaker (stronger) coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition. PMID:25144525

Specificity and affinity quantification of flexible recognition from underlying energy landscape topography.

PubMed

Chu, Xiakun; Wang, Jin

2014-08-01

Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less) flexibility leads to weaker (stronger) coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition.

Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

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

Senogles, S.E.; Caron, M.G.

1986-05-01

The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with /sup 3/H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..Smore » binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D/sub 2/-dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.« less

Molecular Basis of Autophagic Cell Death in Prostate Cancer

DTIC Science & Technology

2009-03-01

lipid-binding proteinwith high affinity for phosphatidic acid (PA) and cardiolipin (CL). Previously, it has been shown that PA directly interacted...lyceride), PI (phosphatidylinositol), DAG (diacylglycerol), PI4P (PtdIns(4)P), PA ( phosphatidic acid ), PI4,5P2 (PtdIns(4,5)P2), PS (phosphatidylserine...with high affinity for phosphatidic acid and cardiolipin and less affinity for various phosphoinositides. Functional genomics analysis identified 5

Efficient T-cell receptor signaling requires a high-affinity interaction between the Gads C-SH3 domain and the SLP-76 RxxK motif.

PubMed

Seet, Bruce T; Berry, Donna M; Maltzman, Jonathan S; Shabason, Jacob; Raina, Monica; Koretzky, Gary A; McGlade, C Jane; Pawson, Tony

2007-02-07

The relationship between the binding affinity and specificity of modular interaction domains is potentially important in determining biological signaling responses. In signaling from the T-cell receptor (TCR), the Gads C-terminal SH3 domain binds a core RxxK sequence motif in the SLP-76 scaffold. We show that residues surrounding this motif are largely optimized for binding the Gads C-SH3 domain resulting in a high-affinity interaction (K(D)=8-20 nM) that is essential for efficient TCR signaling in Jurkat T cells, since Gads-mediated signaling declines with decreasing affinity. Furthermore, the SLP-76 RxxK motif has evolved a very high specificity for the Gads C-SH3 domain. However, TCR signaling in Jurkat cells is tolerant of potential SLP-76 crossreactivity, provided that very high-affinity binding to the Gads C-SH3 domain is maintained. These data provide a quantitative argument that the affinity of the Gads C-SH3 domain for SLP-76 is physiologically important and suggest that the integrity of TCR signaling in vivo is sustained both by strong selection of SLP-76 for the Gads C-SH3 domain and by a capacity to buffer intrinsic crossreactivity.

Radioiodinated nondegradable gonadotropin-releasing hormone analogs: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors.

PubMed

Clayton, R N; Shakespear, R A; Duncan, J A; Marshall, J C; Munson, P J; Rodbard, D

1979-12-01

Studies of pituitary plasma membrane gonadotropin-releasing hormone (GnRH) receptors using [125I]-iodo-GnRH suffer major disadvantages. Only a small (less than 25%) proportion of specific tracer binding is to high affinity sites, with more than 70% bound to low affinity sites (Ka = 1 x 10(6) M-1). [125I]Iodo-GnRH is also inactivated during incubation with pituitary plasma membrane preparations. Two superactive analongs of GnRH, substituted in positions 6 and 10, were used as the labeled ligand to overcome these problems. Both analogs bound to the same high affinity sites as GnRH on bovine pituitary plasma membranes, though the affinity of the analogs was higher than that of the natural decapeptide (Ka = 2.0 x 10(9), 6.0 x 10(9), and 3.0 x 10(8) M-1 for [D-Ser(TBu)6]des-Gly10-GnRH ethylamide, [D-Ala6]des-Gly10-GnRH ethylamide, and GnRH, respectively. The labeled analogs bound to a single class of high affinity sites with less than 15% of the specific binding being to low affinity sites (Ka approximately equal to 1 x 10(6) M-1). The labeled analogs were not inactivated during incubation with the pituitary membrane preparations. Using the analogs as tracer, a single class of high affinity sites (K1 = 4.0 x 10(9) M-1) was also demonstrated on crude 10,800 x g rat pituitary membrane preparations. Use of these analogs as both the labeled and unlabeled ligand offers substantial advantages over GnRH for investigation of GnRH receptors, allowing accurate determination of changes in their numbers and affinities under various physiological conditions.

Hb Potomac (101 Glu replaced by Asp): speculations on placental oxygen transport in carriers of high-affinity hemoglobins.

PubMed

Charache, S; Jacobson, R; Brimhall, B; Murphy, E A; Hathaway, P; Winslow, R; Jones, R; Rath, C; Simkovich, J

1978-02-01

Blood from a woman with unexplained erythrocytosis had increased oxygen affinity, but no abnormality could be detected by electrophoresis or chromatography of her hemolysate. Separation of the tryptic peptides of her beta chains disclosed two half-sized peaks in the regions of beta T-11. The faster of these was abnormal, with the structure beta 101 Glu replaced by Asp. The new hemoglobin was called "Potomac." Three of the proband's four surviving siblings and both of her children were carriers. Differences in the ratio of carrier: normal children born to male of female carriers of 23 other high-affinity hemoglobins were not significant. The high proportion of carriers in this kindred was probably due to chance alone, and not because high maternal oxygen affinity interfered with oxygen transport to fetuses with normal hemoglobin.

Biomarker Candidates of Chlamydophila pneumoniae Proteins and Protein Fragments Identified by Affinity-Proteomics Using FTICR-MS and LC-MS/MS

NASA Astrophysics Data System (ADS)

Susnea, Iuliana; Bunk, Sebastian; Wendel, Albrecht; Hermann, Corinna; Przybylski, Michael

2011-04-01

We report here an affinity-proteomics approach that combines 2D-gel electrophoresis and immunoblotting with high performance mass spectrometry to the identification of both full length protein antigens and antigenic fragments of Chlamydophila pneumoniae (C. pneumoniae). The present affinity-mass spectrometry approach effectively utilized high resolution FTICR mass spectrometry and LC-tandem-MS for protein identification, and enabled the identification of several new highly antigenic C. pneumoniae proteins that were not hitherto reported or previously detected only in other Chlamydia species, such as Chlamydia trachomatis. Moreover, high resolution affinity-MS provided the identification of several neo-antigenic protein fragments containing N- and C-terminal, and central domains such as fragments of the membrane protein Pmp21 and the secreted chlamydial proteasome-like factor (Cpaf), representing specific biomarker candidates.

Lesion-induced plasticity of high affinity choline uptake in the developing rat fascia dentata.

PubMed

Nadler, J V; Shelton, D L; Cotman, C W

1979-03-23

After removal of the perforant path input to the rat fascia dentata at the age of 11 days, cholinergic septohippocampal fibers invade the denervated area. We have examined the effect of this lesion on hemicholinium-sensitive, high affinity choline uptake and its coupling to acetylcholine synthesis, specific properties of the septohippocampal input. Removal of the ipsilateral perforant path fibers increased the velocity of high affinity choline uptake by dentate particulate preparations, usually within 1 day. Studies conducted 5--104 days after operation showed a consistent 50--65% elevation in the molecular (denervated) layer. In contrast, the choline uptake rate in the granular layer eventually decreased slightly. Calculation of choline uptake rates independently of protein (per whole region) revealed that fasciae dentatae from operated and control sides accumulated choline at approximately equal rates, but on the operated side a greater percentage was transported by structures from the molecular layer and a lesser percentage by those from the granular layer. The rate of acetylcholine synthesis from exogenous choline increased to the same extent as high affinity choline uptake from 3 days after operation onwards. The changes in high affinity choline uptake and acetylcholine synthesis coincided spatially and temporally with the reactive growth of septohippocampal fibers. Our results support the view that a perforant path lesion during development permanently alters the distribution of functional septohippocampal boutons in the fascia dentata. Acetylcholine synthesis is regulated to the same extent by high affinity choline uptake in the anomalous boutons as in normally located boutons.

A putative role for the plasma membrane potential in the control of the expression of the gene encoding the tomato high-affinity potassium transporter HAK5.

PubMed

Nieves-Cordones, Manuel; Miller, Anthony J; Alemán, Fernando; Martínez, Vicente; Rubio, Francisco

2008-12-01

A chimeric CaHAK1-LeHAK5 transporter with only 15 amino acids of CaHAK1 in the N-terminus mediates high-affinity K(+) uptake in yeast cells. Kinetic and expression analyses strongly suggest that LeHAK5 mediates a significant proportion of the high-affinity K(+) uptake shown by K(+)-starved tomato (Solanum lycopersicum) plants. The development of high-affinity K(+) uptake, putatively mediated by LeHAK5, was correlated with increased LeHAK5 mRNA levels and a more negative electrical potential difference across the plasma membrane of root epidermal and cortical cells. However, this increase in high-affinity K(+) uptake was not correlated with the root K(+) content. Thus, (i) growth conditions that result in a hyperpolarized root plasma membrane potential, such as K(+) starvation or growth in the presence of NH(4) (+), but which do not decrease the K(+) content, lead to increased LeHAK5 expression; (ii) the presence of NaCl in the growth solution, which prevents the hyperpolarization induced by K(+) starvation, also prevents LeHAK5 expression. Moreover, once the gene is induced, depolarization of the plasma membrane potential then produces a decrease in the LeHAK5 mRNA. On the basis of these results, we propose that the plant membrane electrical potential plays a role in the regulation of the expression of this gene encoding a high-affinity K(+) transporter.

Physico-chemical and biological factors influencing dinoflagellate cyst production in the Cariaco Basin

NASA Astrophysics Data System (ADS)

Bringué, Manuel; Thunell, Robert C.; Pospelova, Vera; Pinckney, James L.; Romero, Oscar E.; Tappa, Eric J.

2018-04-01

We present a 2.5-year-long sediment trap record of dinoflagellate cyst production in the Cariaco Basin, off Venezuela (southern Caribbean Sea). The site lies under the influence of wind-driven, seasonal upwelling which promotes high levels of primary productivity during boreal winter and spring. Changes in dinoflagellate cyst production is documented between November 1996 and May 1999 at ˜ 14-day intervals and interpreted in the context of in situ observations of physico-chemical and biological parameters measured at the mooring site. Dinoflagellate cyst assemblages are diverse (57 taxa) and dominated by cyst taxa of heterotrophic affinity, primarily Brigantedinium spp. (51 % of the total trap assemblage). Average cyst fluxes to the trap are high (17.1 × 103 cysts m-2 day-1) and show great seasonal and interannual variability. On seasonal timescales, dinoflagellate cyst production responds closely to variations in upwelling strength, with increases in cyst fluxes of several protoperidinioid taxa observed during active upwelling intervals, predominantly Brigantedinium spp. Cyst taxa produced by autotrophic dinoflagellates, in particular Bitectatodinium spongium, also respond positively to upwelling. Several spiny brown cysts contribute substantially to the assemblages, including Echinidinium delicatum (9.7 %) and Echinidinium granulatum (7.3 %), and show a closer affinity to weaker upwelling conditions. The strong El Niño event of 1997/98 appears to have negatively impacted cyst production in the basin with a 1-year lag, and may have contributed to the unusually high fluxes of cysts type Cp (possibly the cysts of the toxic dinoflagellate Cochlodinium polykrikoides sensu Li et al., 2015), with cyst type Cp fluxes up to 11.8 × 103 cysts m-2 day-1 observed during the weak upwelling event of February-May 1999. Possible trophic interactions between dinoflagellates and other major planktonic groups are also investigated by comparing the timing and magnitude of cyst production with proxies for phytoplanktonic communities (from photopigment data) and micro- to macrozooplankton abundance indicators (from palynological data) at the site. This work provides new, detailed insights into the ecology of cyst-producing dinoflagellates and will allow for more detailed interpretations of fossil assemblages extracted from sedimentary records in the basin and elsewhere.

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

PubMed Central

2014-01-01

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356

ECL-IAA and ECL-GADA Can Identify High-Risk Single Autoantibody-Positive Relatives in the TrialNet Pathway to Prevention Study.

PubMed

Steck, Andrea K; Fouts, Alexandra; Miao, Dongmei; Zhao, Zhiyuan; Dong, Fran; Sosenko, Jay; Gottlieb, Peter; Rewers, Marian J; Yu, Liping

2016-07-01

Relatives with single positive islet autoantibodies have a much lower risk of progression to diabetes than those with multiple autoantibodies. TrialNet subjects positive for single autoantibody to insulin (mIAA) (n = 50) or single autoantibody to glutamic acid decarboxylase (GADA) (n = 50) were analyzed using new electrochemiluminescence (ECL) assays (ECL-IAA and ECL-GADA, respectively) at their initial visit and longitudinally over time. Affinity assays were performed on a subset of single autoantibody-positive subjects at initial and most recent visits. After a mean follow-up of 5.3 years, 20 subjects developed type 1 diabetes. Among either single GADA or single mIAA subjects, those who were positive in the ECL assay showed higher affinity at the initial visit, and affinity results stayed consistent over time. No converting events from low to high or high to low affinity were seen over time. Confirmed positivity for ECL is associated with high affinity and can help staging of risk for type 1 diabetes in single autoantibody-positive subjects.

High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

NASA Astrophysics Data System (ADS)

Orito, N.; Umekage, S.; Sato, K.; Kawauchi, S.; Tanaka, H.; Sakai, E.; Tanaka, T.; Kikuchi, Y.

2012-03-01

We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25×10-9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

High affinity IgM(+) memory B cells are generated through a germinal center-dependent pathway.

PubMed

Hara, Yasushi; Tashiro, Yasuyuki; Murakami, Akikazu; Nishimura, Miyuki; Shimizu, Takeyuki; Kubo, Masato; Burrows, Peter D; Azuma, Takachika

2015-12-01

During a T cell-dependent immune response, B cells undergo clonal expansion and selection and the induction of isotype switching and somatic hypermutation (SHM). Although somatically mutated IgM(+) memory B cells have been reported, it has not been established whether they are really high affinity B cells. We tracked (4-hydroxy-3-nitrophenyl) acetyl hapten-specific GC B cells from normal immunized mice based on affinity of their B cell receptor (BCR) and performed BCR sequence analysis. SHM was evident by day 7 postimmunization and increased with time, such that high affinity IgM(+) as well as IgG(+) memory B cells continued to be generated up to day 42. In contrast, class-switch recombination (CSR) was almost completed by day 7 and then the ratio of IgG1(+)/IgM(+) GC B cells remained unchanged. Together these findings suggest that IgM(+) B cells undergo SHM in the GC to generate high affinity IgM(+) memory cells and that this process continues even after CSR is accomplished. Copyright © 2015 Elsevier Ltd. All rights reserved.

2. GORGE HIGH DAM. UNUSUALLY HIGH WATER IN GORGE LAKE ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

2. GORGE HIGH DAM. UNUSUALLY HIGH WATER IN GORGE LAKE DUE TO THE COMBINATION OF UNIT 24 BEING DOWN FOR REWINDING AND TWO UNITS COMING ON LINE UNEXPECTEDLY AT ROSS POWERHOUSE LED TO WATER FLOWING OVER THE SPILLGATES. EACH GATE IF 47 FEET WIDE AND 50 FEET HIGH, 1989. - Skagit Power Development, Gorge High Dam, On Skagit River, 2.9 miles upstream from Newhalem, Newhalem, Whatcom County, WA

Affinity Purification of Proteins in Tag-Free Form: Split Intein-Mediated Ultrarapid Purification (SIRP).

PubMed

Guan, Dongli; Chen, Zhilei

2017-01-01

Proteins purified using affinity-based chromatography often exploit a recombinant affinity tag. Existing methods for the removal of the extraneous tag, needed for many applications, suffer from poor efficiency and/or high cost. Here we describe a simple, efficient, and potentially low-cost approach-split intein-mediated ultrarapid purification (SIRP)-for both the purification of the desired tagged protein from Escherichia coli lysate and removal of the tag in less than 1 h. The N- and C-fragment of a self-cleaving variant of a naturally split DnaE intein from Nostoc punctiforme are genetically fused to the N-terminus of an affinity tag and a protein of interest (POI), respectively. The N-intein/affinity tag is used to functionalize an affinity resin. The high affinity between the N- and C-fragment of DnaE intein enables the POI to be purified from the lysate via affinity to the resin, and the intein-mediated C-terminal cleavage reaction causes tagless POI to be released into the flow-through. The intein cleavage reaction is strongly inhibited by divalent ions (e.g., Zn 2+ ) under non-reducing conditions and is significantly enhanced by reducing conditions. The POI is cleaved efficiently regardless of the identity of the N-terminal amino acid except in the cases of threonine and proline, and the N-intein-functionalized affinity resin can be regenerated for multiple cycles of use.

The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

PubMed

Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

2012-09-01

Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses. © 2012 John Wiley & Sons A/S.

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

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

Ovchinnikov, Victor; Louveau, Joy E.; Barton, John P.

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutationsmore » increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed.« less

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

DOE PAGES

Ovchinnikov, Victor; Louveau, Joy E.; Barton, John P.; ...

2018-02-14

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutationsmore » increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed.« less

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

PubMed Central

2018-01-01

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutations increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed. PMID:29442996

Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

PubMed

Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

2016-08-26

Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

Predicting the dynamics of bacterial growth inhibition by ribosome-targeting antibiotics

NASA Astrophysics Data System (ADS)

Greulich, Philip; Doležal, Jakub; Scott, Matthew; Evans, Martin R.; Allen, Rosalind J.

2017-12-01

Understanding how antibiotics inhibit bacteria can help to reduce antibiotic use and hence avoid antimicrobial resistance—yet few theoretical models exist for bacterial growth inhibition by a clinically relevant antibiotic treatment regimen. In particular, in the clinic, antibiotic treatment is time-dependent. Here, we use a theoretical model, previously applied to steady-state bacterial growth, to predict the dynamical response of a bacterial cell to a time-dependent dose of ribosome-targeting antibiotic. Our results depend strongly on whether the antibiotic shows reversible transport and/or low-affinity ribosome binding (‘low-affinity antibiotic’) or, in contrast, irreversible transport and/or high affinity ribosome binding (‘high-affinity antibiotic’). For low-affinity antibiotics, our model predicts that growth inhibition depends on the duration of the antibiotic pulse, and can show a transient period of very fast growth following removal of the antibiotic. For high-affinity antibiotics, growth inhibition depends on peak dosage rather than dose duration, and the model predicts a pronounced post-antibiotic effect, due to hysteresis, in which growth can be suppressed for long times after the antibiotic dose has ended. These predictions are experimentally testable and may be of clinical significance.

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

Beaumont, K.; Vaughn, D.A.; Fanestil, D.D.

Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. The authors report here that ({sup 3}H)metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. ({sup 3}H)Metolazone bound with high affinity to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or not binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated withmore » their clinical potency. Halide anions specifically inhibited high-affinity binding of ({sup 3}H)metolazone to this site. ({sup 3})Metolazone also bound with lower affinity to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had K{sub i} values of 0.6-10 {mu}M for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity ({sup 3}H)metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.« less

Predicting the dynamics of bacterial growth inhibition by ribosome-targeting antibiotics

PubMed Central

Greulich, Philip; Doležal, Jakub; Scott, Matthew; Evans, Martin R; Allen, Rosalind J

2017-01-01

Understanding how antibiotics inhibit bacteria can help to reduce antibiotic use and hence avoid antimicrobial resistance—yet few theoretical models exist for bacterial growth inhibition by a clinically relevant antibiotic treatment regimen. In particular, in the clinic, antibiotic treatment is time-dependent. Here, we use a theoretical model, previously applied to steady-state bacterial growth, to predict the dynamical response of a bacterial cell to a time-dependent dose of ribosome-targeting antibiotic. Our results depend strongly on whether the antibiotic shows reversible transport and/or low-affinity ribosome binding (‘low-affinity antibiotic’) or, in contrast, irreversible transport and/or high affinity ribosome binding (‘high-affinity antibiotic’). For low-affinity antibiotics, our model predicts that growth inhibition depends on the duration of the antibiotic pulse, and can show a transient period of very fast growth following removal of the antibiotic. For high-affinity antibiotics, growth inhibition depends on peak dosage rather than dose duration, and the model predicts a pronounced post-antibiotic effect, due to hysteresis, in which growth can be suppressed for long times after the antibiotic dose has ended. These predictions are experimentally testable and may be of clinical significance. PMID:28714461

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins.

PubMed

Suzuki, N; Mihashi, K

1991-01-01

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.

Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

PubMed

Shiomi, K; Yamaguchi, S; Shimakura, K; Nagashima, Y; Yamamori, K; Matsui, T

1993-12-01

A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

Ediacaran developmental biology

PubMed Central

Liu, Alexander G.; Donoghue, Philip C. J.

2017-01-01

ABSTRACT Rocks of the Ediacaran System (635–541 Ma) preserve fossil evidence of some of the earliest complex macroscopic organisms, many of which have been interpreted as animals. However, the unusual morphologies of some of these organisms have made it difficult to resolve their biological relationships to modern metazoan groups. Alternative competing phylogenetic interpretations have been proposed for Ediacaran taxa, including algae, fungi, lichens, rhizoid protists, and even an extinct higher‐order group (Vendobionta). If a metazoan affinity can be demonstrated for these organisms, as advocated by many researchers, they could prove informative in debates concerning the evolution of the metazoan body axis, the making and breaking of axial symmetries, and the appearance of a metameric body plan. Attempts to decipher members of the enigmatic Ediacaran macrobiota have largely involved study of morphology: comparative analysis of their developmental phases has received little attention. Here we present what is known of ontogeny across the three iconic Ediacaran taxa Charnia masoni, Dickinsonia costata and Pteridinium simplex, together with new ontogenetic data and insights. We use these data and interpretations to re‐evaluate the phylogenetic position of the broader Ediacaran morphogroups to which these taxa are considered to belong (rangeomorphs, dickinsoniomorphs and erniettomorphs). We conclude, based on the available evidence, that the affinities of the rangeomorphs and the dickinsoniomorphs lie within Metazoa. PMID:29105292

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

PubMed Central

Vincentelli, Renaud; Luck, Katja; Poirson, Juline; Polanowska, Jolanta; Abdat, Julie; Blémont, Marilyne; Turchetto, Jeremy; Iv, François; Ricquier, Kevin; Straub, Marie-Laure; Forster, Anne; Cassonnet, Patricia; Borg, Jean-Paul; Jacob, Yves; Masson, Murielle; Nominé, Yves; Reboul, Jérôme; Wolff, Nicolas; Charbonnier, Sebastian; Travé, Gilles

2015-01-01

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes. PMID:26053890

Concepts in receptor optimization: targeting the RGD peptide.

PubMed

Chen, Wei; Chang, Chia-en; Gilson, Michael K

2006-04-12

Synthetic receptors have a wide range of potential applications, but it has been difficult to design low molecular weight receptors that bind ligands with high, "proteinlike" affinities. This study uses novel computational methods to understand why it is hard to design a high-affinity receptor and to explore the limits of affinity, with the bioactive peptide RGD as a model ligand. The M2 modeling method is found to yield excellent agreement with experiment for a known RGD receptor and then is used to analyze a series of receptors generated in silico with a de novo design algorithm. Forces driving binding are found to be systematically opposed by proportionate repulsions due to desolvation and entropy. In particular, strong correlations are found between Coulombic attractions and the electrostatic desolvation penalty and between the mean energy change on binding and the cost in configurational entropy. These correlations help explain why it is hard to achieve high affinity. The change in surface area upon binding is found to correlate poorly with affinity within this series. Measures of receptor efficiency are formulated that summarize how effectively a receptor uses surface area, total energy, and Coulombic energy to achieve affinity. Analysis of the computed efficiencies suggests that a low molecular weight receptor can achieve proteinlike affinity. It is also found that macrocyclization of a receptor can, unexpectedly, increase the entropy cost of binding because the macrocyclic structure further restricts ligand motion.

Cholera Toxin Inhibitors Studied with High-Performance Liquid Affinity Chromatography: A Robust Method to Evaluate Receptor–Ligand Interactions

PubMed Central

Bergström, Maria; Liu, Shuang; Kiick, Kristi L.; Ohlson, Sten

2009-01-01

Anti-adhesion drugs may be an alternative to antibiotics to control infection of micro-organisms. The well-characterized interaction between cholera toxin and the cellular glycolipid GM1 makes it an attractive model for inhibition studies in general. In this report, we demonstrate a high-performance liquid affinity chromatography approach called weak affinity chromatography to evaluate cholera toxin inhibitors. The cholera toxin B-subunit was covalently coupled to porous silica and a (weak) affinity column was produced. The KD values of galactose and meta-nitrophenyl α-D-galactoside were determined with weak affinity chromatography to be 52 and 1 mM, respectively, which agree well with IC50 values previously reported. To increase inhibition potency multivalent inhibitors have been developed and the interaction with multivalent glycopolypeptides was also evaluated. The affinity of these compounds was found to correlate with the galactoside content but KD values were not obtained because of the inhomogeneous response and slow off-rate from multivalent interactions. Despite the limitations in obtaining direct KD values of the multivalent galactopolypeptides, weak affinity chromatography represents an additional and valuable tool in the evaluation of monovalent as well as multivalent cholera toxin inhibitors. It offers multiple advantages, such as a low sample consumption, high reproducibility and short analysis time, which are often not observed in other methods of analysis. PMID:19152642

Selection of imprinted nanoparticles by affinity chromatography.

PubMed

Guerreiro, António R; Chianella, Iva; Piletska, Elena; Whitcombe, Michael J; Piletsky, Sergey A

2009-04-15

Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (K(d) 6.6x10(-8)) M and were also able to discriminate between related functional analogues of the template.

Canine Comfort: Pet Affinity Buffers the Negative Impact of Ambivalence over Emotional Expression on Perceived Social Support.

PubMed

Bryan, Jennifer L; Quist, Michelle C; Young, Chelsie M; Steers, Mai-Ly N; Foster, Dawn W; Lu, Qian

2014-10-01

This study evaluated pet affinity as a buffer between ambivalence over emotional expression (AEE) and social support. AEE occurs when one desires to express emotions but is reluctant to do so and is related to negative psychological outcomes. Individuals high in AEE may have difficulty receiving social support and thus may not gain accompanying benefits. Social support has been associated with positive health outcomes, and pet support is positively associated with human social support. The present study explores the potential protective effect of pet affinity. One hundred ninety-eight undergraduate dog owners completed measures assessing perceived social support, pet affinity, and AEE. AEE was expected to be negatively associated with social support, and pet affinity was expected to buffer the negative effects of AEE on social support. We found that AEE was negatively associated with perceived social support. An interaction between pet affinity and AEE emerged such that the negative association between AEE and social support was weaker among those higher in pet affinity. Thus, at high levels of AEE, those who felt a close connection with their pets reported more perceived social support than those less connected with their pets. Overall, these findings emphasize the potential benefits of pet affinity.

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.

PubMed

Tome, Jacob M; Ozer, Abdullah; Pagano, John M; Gheba, Dan; Schroth, Gary P; Lis, John T

2014-06-01

RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

[Comparison of acetonitrile, ethanol and chromatographic column to eliminate high-abundance proteins in human serum].

PubMed

Li, Yin; Liao, Ming; He, Xiao; Zhou, Yi; Luo, Rong; Li, Hongtao; Wang, Yun; He, Min

2012-11-01

To compare the effects of acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal to eliminate high-abundance proteins in human serum. Elimination of serum high-abundance proteins performed with acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal. Bis-Tris Mini Gels electrophoresis and two-dimensional gel electrophoresis to detect the effect. Grey value analysis from 1-DE figure showed that after serum processed by acetonitrile method, multiple affinity chromatography column Human 14 removal method and ethanol method, the grey value of albumin changed into 157.2, 40.8 and 8.2 respectively from the original value of 19. 2-DE analysis results indicated that using multiple affinity chromatography column Human 14 method, the protein points noticeable increased by 137 compared to the original serum. After processed by acetonitrile method and ethanol method, the protein point reduced, but the low abundance protein point emerged. The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecular weight, ethanol precipitation could eliminate part of high abundance proteins in serum, but low abundance proteins less harvested, and multiple affinity chromatography column Human 14 method could effectively removed the high abundance proteins, and keep a large number of low abundance proteins.

Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen

PubMed Central

1995-01-01

To gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-p- azophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among the monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars that the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment. PMID:7650481

A Link No Longer Missing: New Evidence for the Cetotheriid Affinities of Caperea.

PubMed

Marx, Felix G; Fordyce, R Ewan

2016-01-01

The origins of the enigmatic pygmy right whale Caperea marginata, the only living member of its subfamily (Neobalaeninae), are an outstanding mystery of cetacean evolution. Its strikingly disparate morphology sets Caperea apart from all other whales, and has turned it into a wildcard taxon that holds the key to understanding modern baleen whale diversity. Morphological cladistics generally ally this species with right whales, whereas molecular analyses consistently cluster it with rorquals and grey whales (Balaenopteroidea). A recent study potentially resolved this conflict by proposing that Caperea belongs with the otherwise extinct Cetotheriidae, but has been strongly criticised on morphological grounds. Evidence from the neobalaenine fossil record could potentially give direct insights into morphological transitions, but is currently limited to just a single species: the Late Miocene Miocaperea pulchra, from Peru. We show that Miocaperea has a highly unusual morphology of the auditory region, resulting from a-presumably feeding-related-strengthening of the articulation of the hyoid apparatus with the skull. This distinctive arrangement is otherwise only found in the extinct Cetotheriidae, which makes Miocaperea a "missing link" that demonstrates the origin of pygmy right whales from cetotheriids, and confirms the latter's resurrection from the dead.

A Link No Longer Missing: New Evidence for the Cetotheriid Affinities of Caperea

PubMed Central

Marx, Felix G.; Fordyce, R. Ewan

2016-01-01

The origins of the enigmatic pygmy right whale Caperea marginata, the only living member of its subfamily (Neobalaeninae), are an outstanding mystery of cetacean evolution. Its strikingly disparate morphology sets Caperea apart from all other whales, and has turned it into a wildcard taxon that holds the key to understanding modern baleen whale diversity. Morphological cladistics generally ally this species with right whales, whereas molecular analyses consistently cluster it with rorquals and grey whales (Balaenopteroidea). A recent study potentially resolved this conflict by proposing that Caperea belongs with the otherwise extinct Cetotheriidae, but has been strongly criticised on morphological grounds. Evidence from the neobalaenine fossil record could potentially give direct insights into morphological transitions, but is currently limited to just a single species: the Late Miocene Miocaperea pulchra, from Peru. We show that Miocaperea has a highly unusual morphology of the auditory region, resulting from a–presumably feeding-related–strengthening of the articulation of the hyoid apparatus with the skull. This distinctive arrangement is otherwise only found in the extinct Cetotheriidae, which makes Miocaperea a “missing link” that demonstrates the origin of pygmy right whales from cetotheriids, and confirms the latter’s resurrection from the dead. PMID:27711216

Atmospheric Hydrogen Scavenging: from Enzymes to Ecosystems

PubMed Central

Constant, Philippe; Hards, Kiel; Morales, Sergio E.; Oakeshott, John G.; Russell, Robyn J.; Taylor, Matthew C.; Berney, Michael; Conrad, Ralf; Cook, Gregory M.

2014-01-01

We have known for 40 years that soils can consume the trace amounts of molecular hydrogen (H2) found in the Earth's atmosphere. This process is predicted to be the most significant term in the global hydrogen cycle. However, the organisms and enzymes responsible for this process were only recently identified. Pure culture experiments demonstrated that several species of Actinobacteria, including streptomycetes and mycobacteria, can couple the oxidation of atmospheric H2 to the reduction of ambient O2. A combination of genetic, biochemical, and phenotypic studies suggest that these organisms primarily use this fuel source to sustain electron input into the respiratory chain during energy starvation. This process is mediated by a specialized enzyme, the group 5 [NiFe]-hydrogenase, which is unusual for its high affinity, oxygen insensitivity, and thermostability. Atmospheric hydrogen scavenging is a particularly dependable mode of energy generation, given both the ubiquity of the substrate and the stress tolerance of its catalyst. This minireview summarizes the recent progress in understanding how and why certain organisms scavenge atmospheric H2. In addition, it provides insight into the wider significance of hydrogen scavenging in global H2 cycling and soil microbial ecology. PMID:25501483

Second-generation DNA-templated macrocycle libraries for the discovery of bioactive small molecules.

PubMed

Usanov, Dmitry L; Chan, Alix I; Maianti, Juan Pablo; Liu, David R

2018-07-01

DNA-encoded libraries have emerged as a widely used resource for the discovery of bioactive small molecules, and offer substantial advantages compared with conventional small-molecule libraries. Here, we have developed and streamlined multiple fundamental aspects of DNA-encoded and DNA-templated library synthesis methodology, including computational identification and experimental validation of a 20 × 20 × 20 × 80 set of orthogonal codons, chemical and computational tools for enhancing the structural diversity and drug-likeness of library members, a highly efficient polymerase-mediated template library assembly strategy, and library isolation and purification methods. We have integrated these improved methods to produce a second-generation DNA-templated library of 256,000 small-molecule macrocycles with improved drug-like physical properties. In vitro selection of this library for insulin-degrading enzyme affinity resulted in novel insulin-degrading enzyme inhibitors, including one of unusual potency and novel macrocycle stereochemistry (IC 50  = 40 nM). Collectively, these developments enable DNA-templated small-molecule libraries to serve as more powerful, accessible, streamlined and cost-effective tools for bioactive small-molecule discovery.

Purification and kinetic characterization of recombinant human mitogen-activated protein kinase kinase kinase COT and the complexes with its cellular partner NF-kappa B1 p105.

PubMed

Jia, Yong; Quinn, Christopher M; Bump, Nancy J; Clark, Kevin M; Clabbers, Anca; Hardman, Jennifer; Gagnon, Andrew; Kamens, Joanne; Tomlinson, Medha J; Wishart, Neil; Allen, Hamish

2005-09-01

Cancer osaka thyroid (COT), a human MAP 3 K, is essential for lipopolysaccharide activation of the Erk MAPK cascade in macrophages. COT 30--467 is insoluble, whereas low levels of COT 30--397 can be expressed, but this protein is unstable. However, both COT 30--467 and COT 30--397 are expressed in a soluble and stable form when produced in complex with the C-terminal half of p105. The k(cat) of COT 30--397 is reduced approximately 47--fold in the COT 30--467/p105 Delta N complex. COT prefers Mn(2+) to Mg(2+) as the ATP metal cofactor, exhibiting an unusually high ATP K(m) in the presence of Mg(2+). When using Mn(2+) as the cofactor, the ATP K(m) is reduced to a level typical of most kinases. In contrast, the binding affinity of COT for its other substrate MEK is cofactor independent. Our results using purified proteins indicate that p105 binding improves COT solubility and stability while down-regulating kinase activity, consistent with cellular data showing that p105 functions as an inhibitor of COT.

Allyl m-Trifluoromethyldiazirine Mephobarbital: An Unusually Potent Enantioselective and Photoreactive Barbiturate General Anesthetic

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

Savechenkov, Pavel Y.; Zhang, Xi; Chiara, David C.

2012-12-10

We synthesized 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (14), a trifluoromethyldiazirine-containing derivative of general anesthetic mephobarbital, separated the racemic mixture into enantiomers by chiral chromatography, and determined the configuration of the (+)-enantiomer as S by X-ray crystallography. Additionally, we obtained the {sup 3}H-labeled ligand with high specific radioactivity. R-(-)-14 is an order of magnitude more potent than the most potent clinically used barbiturate, thiopental, and its general anesthetic EC{sub 50} approaches those for propofol and etomidate, whereas S-(+)-14 is 10-fold less potent. Furthermore, at concentrations close to its anesthetic potency, R-(-)-14 both potentiated GABA-induced currents and increased the affinity for the agonist muscimol inmore » human {alpha}1{beta}2/3{gamma}2L GABA{sub A} receptors. Finally, R-(-)-14 was found to be an exceptionally efficient photolabeling reagent, incorporating into both {alpha}1 and {beta}3 subunits of human {alpha}1{beta}3 GABAA receptors. These results indicate R-(-)-14 is a functional general anesthetic that is well-suited for identifying barbiturate binding sites on Cys-loop receptors.« less

Xyloglucan mobilisation in cotyledons of developing plantlets of Hymenaea courbaril L. (Leguminosae-Caesalpinoideae).

PubMed

Tiné; Cortelazzo; Buckeridge

2000-05-29

Many seeds contain storage compounds that are used by the embryo/plantlet as a source of nutrients after germination. In seeds of Hymenaea courbaril, a leguminous tree, the main reserve consists of a structurally unusual xyloglucan stored in thickened walls of the cotyledon cells. The present work aimed to study H. courbaril xyloglucan metabolism during and after germination in order to compare its degrading system with the other known xyloglucan containing seeds. Polysaccharide degradation occurred after germination between 35 and 55 days after planting. The activities of alpha-xylosidase, beta-glucosidase, beta-galactosidase and XET rose during the period of xyloglucan disassembling but a low level of endo-beta-glucanase activity was detected, suggesting that this XET has high affinity for the oligosaccharides. The pH optimum of beta-galactosidase was different from the alpha-xylosidase, beta-glucosidase and XET optima suggesting that the former may be important in the control of the mobilisation process. A tentative model for xyloglucan disassembling in vivo is proposed, where beta-galactosidase allows the free oligosaccharides to bypass a transglycosylation cycle and be disassembled by the other exo-enzymes. Some ecophysiological comparisons among H. courbaril and other xyloglucan storing seeds are discussed.

Evidence that the novobiocin-sensitive ATP-binding site of the heat shock protein 90 (hsp90) is necessary for its autophosphorylation.

PubMed

Langer, T; Schlatter, H; Fasold, H

2002-01-01

The 90kDa heat shock protein (Hsp90) is one of the most abundant protein and essential for all eukaryotic cells. Many proteins require the interaction with Hsp90 for proper function. Upon heat stress the expression level of Hsp90 is even enhanced. It is assumed, that under these conditions Hsp90 is required to protect other proteins from aggregation. One property of Hsp90 is its ability to undergo autophosphorylation. The N-terminal domain of Hsp90 has been shown to contain an unusual ATP-binding site. A well-known inhibitor of Hsp90 function is geldanamycin binding to the N-terminal ATP-binding site with high affinity. Recently it was shown that Hsp90 possesses a second ATP-binding site in the C-terminal region, which can be competed with novobiocin. Autophosphorylation of Hsp90 was analysed by incubation with gamma(32)P-ATP. Addition of geldanamycin did not interfere with the capability for autophosphorylation, while novobiocin indeed did. These results suggest that the C-terminal ATP-binding site is required for autophosphorylation of Hsp90.

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration* ♦

PubMed Central

Koenig, Patrick; Lee, Chingwei V.; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F.; Fuh, Germaine

2015-01-01

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. PMID:26088137

Degenerate Pax2 and Senseless binding motifs improve detection of low-affinity sites required for enhancer specificity

PubMed Central

Zandvakili, Arya; Campbell, Ian; Weirauch, Matthew T.

2018-01-01

Cells use thousands of regulatory sequences to recruit transcription factors (TFs) and produce specific transcriptional outcomes. Since TFs bind degenerate DNA sequences, discriminating functional TF binding sites (TFBSs) from background sequences represents a significant challenge. Here, we show that a Drosophila regulatory element that activates Epidermal Growth Factor signaling requires overlapping, low-affinity TFBSs for competing TFs (Pax2 and Senseless) to ensure cell- and segment-specific activity. Testing available TF binding models for Pax2 and Senseless, however, revealed variable accuracy in predicting such low-affinity TFBSs. To better define parameters that increase accuracy, we developed a method that systematically selects subsets of TFBSs based on predicted affinity to generate hundreds of position-weight matrices (PWMs). Counterintuitively, we found that degenerate PWMs produced from datasets depleted of high-affinity sequences were more accurate in identifying both low- and high-affinity TFBSs for the Pax2 and Senseless TFs. Taken together, these findings reveal how TFBS arrangement can be constrained by competition rather than cooperativity and that degenerate models of TF binding preferences can improve identification of biologically relevant low affinity TFBSs. PMID:29617378

Evaluation of Drugs for Prevention of Hyperthermia in Rats Exposed to Microwave Irradiation.

DTIC Science & Technology

1987-03-04

is deficient in antidiuretic hormone (ADH) and has diabetes insipidus (DI). The latter condition causes unusually high water intake and urine...ADH) and consequently has diabetes ihsipidus (DI). A DI-rat, also known as Brattleboro rat, has both unusually high water intake and rate of

Selectivity of the uptake of glutamate and GABA in two morphologically distinct insect neuromuscular synapses.

PubMed

van Marle, J; Piek, T; Lammertse, T; Lind, A; Van Weeren-Kramer, J

1985-11-25

The common inhibitor (CI) and slow excitor tibiae (SETi) innervated slow muscles 135cd of the locust Schistocerca gregaria were incubated under high-affinity uptake conditions either in [3H]GABA or in [3H]glutamate. [3H]GABA is accumulated in the glia of the nerve endings of the CI as well as the SETi; however, it is accumulated only in the terminal axons of the CI, not in the terminal axons of the SETi. The grain densities above the glia and above the CI terminal axons are approximately 2 grains/micron2. After incubation in [3H]glutamate the grain densities above the CI terminal axons and the SETi terminal axons are approximately 4 grains/micron2; the grain densities above the glia of both types of nerve endings are approximately 17 grains/micron2. The relatively high labeling (3 grains/micron2) of the muscles after incubation in the presence of glutamate is ascribed to the high metabolic requirements of slow muscles. The conclusion is drawn that a high-affinity uptake system for GABA is present in the CI terminal axons and in the glia of both the CI and SETi nerve endings. However, while the glutamate uptake in the CI and SETi nerve endings of the slow 135cd is comparable to the high-affinity uptake of glutamate in the fast excitor tibiae (FETi) nerve endings of the fast retractor unguis muscle, a high-affinity uptake of glutamate was only demonstrated in the glia of both types of nerve endings. A high-affinity uptake in the terminal axons of the CI and SETi may be masked by an extensively low-affinity uptake of glutamate by the muscles.

Selection of a high-affinity and in vivo bioactive ssDNA aptamer against angiotensin II peptide.

PubMed

Heiat, Mohammad; Ranjbar, Reza; Latifi, Ali Mohammad; Rasaee, Mohammad Javad

2016-08-01

Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52±2.44E-10 and 5.87±1.3E-9M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33±1.15E-9 and 4.11±1.09E-9M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (P<0.05). They also significantly reduced the serum sodium level and increased the urine volume (P<0.05). The core regions of aptamers did not show high inhibitory potential against Ang II. It can be a spotlight that ssDNA aptamers have high potential for blocking Ang II. In conclusion, it appears that the researches focusing on high affinity and bioactive aptamers may lead to excellent results in blocking Ang II activity. Copyright © 2016 Elsevier Inc. All rights reserved.

ECL-IAA and ECL-GADA Can Identify High-Risk Single Autoantibody-Positive Relatives in the TrialNet Pathway to Prevention Study

PubMed Central

Fouts, Alexandra; Miao, Dongmei; Zhao, Zhiyuan; Dong, Fran; Sosenko, Jay; Gottlieb, Peter; Rewers, Marian J.

2016-01-01

Abstract Background: Relatives with single positive islet autoantibodies have a much lower risk of progression to diabetes than those with multiple autoantibodies. Materials and Methods: TrialNet subjects positive for single autoantibody to insulin (mIAA) (n = 50) or single autoantibody to glutamic acid decarboxylase (GADA) (n = 50) were analyzed using new electrochemiluminescence (ECL) assays (ECL-IAA and ECL-GADA, respectively) at their initial visit and longitudinally over time. Affinity assays were performed on a subset of single autoantibody-positive subjects at initial and most recent visits. Results: After a mean follow-up of 5.3 years, 20 subjects developed type 1 diabetes. Among either single GADA or single mIAA subjects, those who were positive in the ECL assay showed higher affinity at the initial visit, and affinity results stayed consistent over time. No converting events from low to high or high to low affinity were seen over time. Conclusions: Confirmed positivity for ECL is associated with high affinity and can help staging of risk for type 1 diabetes in single autoantibody-positive subjects. PMID:26991969

Isolation and purification of wheat germ agglutinin and analysis of its properties

NASA Astrophysics Data System (ADS)

Wang, Han

2017-12-01

In this paper, the wheat germ agglutinin was isolated and purified by affinity chromatography of chicken ovomucoid as ligand. The physicochemical properties were analyzed. The chicken ovomucoid was isolated from egg white and conjugated to affinity chromatography column agarose gel to prepare affinity adsorbent. The crude extract of wheat germ was freezedried by affinity chromatography. The physicochemical properties were analyzed by SDSpolyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. And the relative molecular mass and isoelectric point of wheat germ agglutinin were obtained, and the high efficiency of purification of wheat germ agglutinin was proved by affinity chromatography.

Pharmacological characterization of the cloned kappa opioid receptor as a kappa 1b subtype.

PubMed

Lai, J; Ma, S W; Zhu, R H; Rothman, R B; Lentes, K U; Porreca, F

1994-10-27

Substantial pharmacological evidence in vitro and in vivo has suggested the existence of subtypes of the kappa opioid receptor. Quantitative radioligand binding techniques resolved the presence of two high affinity binding sites for the kappa 1 ligand [3H]U69,593 in mouse brain membranes, termed kappa 1a and kappa 1b, respectively. Whereas the kappa 1a site has high affinity for fedotozine and oxymorphindole and low affinity for bremazocine and alpha-neoendorphin, site kappa 1b has high affinity for bremazocine and alpha-neoendorphin and low affinity for fedotozine and oxymorphindole. CI-977 and U69,593 bind equally well at both sites. To determine the relationship between these kappa 1 receptor subtypes and the recently cloned mouse kappa 1 receptor (KOR), we examined [3H]U69,593 binding to the KOR in stably transfected cells (KORCHN-8). Competition of [3H]U69,593 binding to the KOR by bremazocine, alpha-neoendorphin, fedotozine and oxymorphindole resolved a single class of binding sites at which these agents had binding affinities similar to that of the kappa 1b site present in mouse brain. These results suggest that the cloned KOR corresponds to the kappa 1 site in mouse brain defined as kappa 1b.

The protein-protein interface evolution acts in a similar way to antibody affinity maturation.

PubMed

Li, Bohua; Zhao, Lei; Wang, Chong; Guo, Huaizu; Wu, Lan; Zhang, Xunming; Qian, Weizhu; Wang, Hao; Guo, Yajun

2010-02-05

Understanding the evolutionary mechanism that acts at the interfaces of protein-protein complexes is a fundamental issue with high interest for delineating the macromolecular complexes and networks responsible for regulation and complexity in biological systems. To investigate whether the evolution of protein-protein interface acts in a similar way as antibody affinity maturation, we incorporated evolutionary information derived from antibody affinity maturation with common simulation techniques to evaluate prediction success rates of the computational method in affinity improvement in four different systems: antibody-receptor, antibody-peptide, receptor-membrane ligand, and receptor-soluble ligand. It was interesting to find that the same evolutionary information could improve the prediction success rates in all the four protein-protein complexes with an exceptional high accuracy (>57%). One of the most striking findings in our present study is that not only in the antibody-combining site but in other protein-protein interfaces almost all of the affinity-enhancing mutations are located at the germline hotspot sequences (RGYW or WA), indicating that DNA hot spot mechanisms may be widely used in the evolution of protein-protein interfaces. Our data suggest that the evolution of distinct protein-protein interfaces may use the same basic strategy under selection pressure to maintain interactions. Additionally, our data indicate that classical simulation techniques incorporating the evolutionary information derived from in vivo antibody affinity maturation can be utilized as a powerful tool to improve the binding affinity of protein-protein complex with a high accuracy.

How Structure Defines Affinity in Protein-Protein Interactions

PubMed Central

Erijman, Ariel; Rosenthal, Eran; Shifman, Julia M.

2014-01-01

Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success. The growing number of high-resolution structures, the appearance of more precise methods for measuring binding affinities and the development of new computational algorithms enable more thorough investigations in this direction. Here, we use a large dataset of PPI structures with the documented binding affinities to calculate a number of structure-based features that could potentially define binding energetics. We explore how well each calculated biophysical feature alone correlates with binding affinity and determine the features that could be used to distinguish between high-, medium- and low- affinity PPIs. Furthermore, we test how various combinations of features could be applied to predict binding affinity and observe a slow improvement in correlation as more features are incorporated into the equation. In addition, we observe a considerable improvement in predictions if we exclude from our analysis low-resolution and NMR structures, revealing the importance of capturing exact intermolecular interactions in our calculations. Our analysis should facilitate prediction of new interactions on the genome scale, better characterization of signaling networks and design of novel binding partners for various target proteins. PMID:25329579

Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

PubMed

Dagnino, Sonia; Bellet, Virginie; Grimaldi, Marina; Riu, Anne; Aït-Aïssa, Sélim; Cavaillès, Vincent; Fenet, Hélène; Balaguer, Patrick

2014-02-01

Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 μM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 μM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

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.

Can we beat the biotin-avidin pair?: cucurbit[7]uril-based ultrahigh affinity host-guest complexes and their applications.

PubMed

Shetty, Dinesh; Khedkar, Jayshree K; Park, Kyeng Min; Kim, Kimoon

2015-12-07

The design of synthetic, monovalent host-guest molecular recognition pairs is still challenging and of particular interest to inquire into the limits of the affinity that can be achieved with designed systems. In this regard, cucurbit[7]uril (CB[7]), an important member of the host family cucurbit[n]uril (CB[n], n = 5-8, 10, 14), has attracted much attention because of its ability to form ultra-stable complexes with multiple guests. The strong hydrophobic effect between the host cavity and guests, ion-dipole and dipole-dipole interactions of guests with CB portals helps in cooperative and multiple noncovalent interactions that are essential for realizing such strong complexations. These highly selective, strong yet dynamic interactions can be exploited in many applications including affinity chromatography, biomolecule immobilization, protein isolation, biological catalysis, and sensor technologies. In this review, we summarize the progress in the development of high affinity guests for CB[7], factors affecting the stability of complexes, theoretical insights, and the utility of these high affinity pairs in different challenging applications.

A 45-Amino-Acid Scaffold Mined from the PDB for High-Affinity Ligand Engineering.

PubMed

Kruziki, Max A; Bhatnagar, Sumit; Woldring, Daniel R; Duong, Vandon T; Hackel, Benjamin J

2015-07-23

Small protein ligands can provide superior physiological distribution compared with antibodies, and improved stability, production, and specific conjugation. Systematic evaluation of the PDB identified a scaffold to push the limits of small size and robust evolution of stable, high-affinity ligands: 45-residue T7 phage gene 2 protein (Gp2) contains an α helix opposite a β sheet with two adjacent loops amenable to mutation. De novo ligand discovery from 10(8) mutants and directed evolution toward four targets yielded target-specific binders with affinities as strong as 200 ± 100 pM, Tms from 65 °C ± 3 °C to 80°C ± 1 °C, and retained activity after thermal denaturation. For cancer targeting, a Gp2 domain for epidermal growth factor receptor was evolved with 18 ± 8 nM affinity, receptor-specific binding, and high thermal stability with refolding. The efficiency of evolving new binding function and the size, affinity, specificity, and stability of evolved domains render Gp2 a uniquely effective ligand scaffold. Copyright © 2015 Elsevier Ltd. All rights reserved.

Solubilization and purification of melatonin receptors from lizard brain.

PubMed

Rivkees, S A; Conron, R W; Reppert, S M

1990-09-01

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

Mobile Technology Affinity in Renal Transplant Recipients.

PubMed

Reber, S; Scheel, J; Stoessel, L; Schieber, K; Jank, S; Lüker, C; Vitinius, F; Grundmann, F; Eckardt, K-U; Prokosch, H-U; Erim, Y

Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 ± 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Zukin, R.S.; Eghbali, M.; Olive, D.

{kappa} opioid receptors ({kappa} receptors) have been characterized in homogenates of guinea pig and rat brain under in vitro binding conditions. {kappa} receptors were labeled by using the tritiated prototypic {kappa} opioid ethylketocyclazocine under conditions in which {mu} and {delta} opioid binding was suppressed. In the case of guinea pig brain membranes, a single population of high-affinity {kappa} opioid receptor sites was observed. In contrast, in the case of rat brain, two populations of {kappa} sites were observed. To test the hypothesis that the high- and low-affinity {kappa} sites represent two distinct {kappa} receptor subtypes, a series of opioids weremore » tested for their abilities to compete for binding to the two sites. U-69,593 and Cambridge 20 selectively displaced the high-affinity {kappa} site in both guinea pig and rat tissue, but were inactive at the rat-brain low-affinity site. Other {kappa} opioid drugs competed for binding to both sites, but with different rank orders of potency. Quantitative light microscopy in vitro autoradiography was used to visualize the neuroanatomical pattern of {kappa} receptors in rat and guinea pig brain. The distribution patterns of the two {kappa} receptor subtypes of rat brain were clearly different. Collectively, these data provide direct evidence for the presence of two {kappa} receptor subtypes; the U-69,593-sensitive, high-affinity {kappa}{sub 1} site predominates in guinea pig brain, and the U-69,593-insensitive, low-affinity {kappa}{sub 2} site predominates in rat brain.« less

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

PubMed

Koenig, Patrick; Lee, Chingwei V; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F; Fuh, Germaine

2015-09-04

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis

PubMed Central

Pillon, Arnaud; Boussioux, Anne-Marie; Escande, Aurélie; Aït-Aïssa, Sélim; Gomez, Elena; Fenet, Hélène; Ruff, Marc; Moras, Dino; Vignon, Françoise; Duchesne, Marie-Josèphe; Casellas, Claude; Nicolas, Jean-Claude; Balaguer, Patrick

2005-01-01

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-αligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-αand whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-αto separate ligands for ER and AhR that are present in river sediments. Immobilized ER-α, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis. PMID:15743715

Coffee Grounds to Multifunctional Quantum Dots: Extreme Nanoenhancers of Polymer Biocomposites.

PubMed

Xu, Huan; Xie, Lan; Li, Jinlai; Hakkarainen, Minna

2017-08-23

Central to the design and execution of nanocomposite strategies is the invention of polymer-affinitive and multifunctional nanoreinforcements amenable to economically viable processing. Here, a microwave-assisted approach enabled gram-scale fabrication of polymer-affinitive luminescent quantum dots (QDs) from spent coffee grounds. The ultrasmall dimensions (approaching 20 nm), coupled with richness of diverse oxygen functional groups, conferred the zero-dimensional QDs with proper exfoliation and uniform dispersion in poly(l-lactic acid) (PLLA) matrix. The unique optical properties of QDs were inherited by PLLA nanocomposites, giving intensive luminescence and high visible transparency, as well as nearly 100% UV-blocking ratio in the full-UV region at only 0.5 wt % QDs. The strong anchoring of PLLA chains at the nanoscale surfaces of QDs facilitated PLLA crystallization, which was accompanied by substantial improvements in thermomechanical and tensile properties. With 1 wt % QDs, for example, the storage modulus at 100 °C and tensile strength increased over 2500 and 69% compared to those of pure PLLA (4 and 57.3 MPa), respectively. The QD-enabled energy-dissipating and flexibility-imparting mechanisms upon tensile deformation, including the generation of numerous shear bands, crazing, and nanofibrillation, gave an unusual combination of elasticity and extensibility for PLLA nanocomposites. This paves the way to biowaste-derived nanodots with high affinity to polymer for elegant implementation of distinct light management and extreme nanoreinforcements in an ecofriendly manner.

Extensive plasma cell infiltration with crystal IgG inclusions and mutated IgV(H) gene in an osteoarthritis patient with lymphoplasmacellular synovitis. A case report.

PubMed

Magalhães, Raquel; Gehrke, Thorsten; Souto-Carneiro, Maria M; Kriegsmann, Jörg; Krenn, Veit

2002-01-01

The presence of immunoglobulin crystal inclusions in plasma cells from plasmacytomas and B-NHLs (linked to overstimulation and overproduction) has been frequently reported. Our case describes a lymphoplasmacellular synovitis in a patient with osteoarthritis (OA) showing an unusually high plasma cell infiltration and for the first time crystals in plasma cells. Using immunohistochemistry. these crystals were identified as being IgG with a balanced lambda/kappa ratio. IgV(H) gene analysis (n = 5 clones) showed that they were somatically mutated (R/S of CDR > 3): in one case, an insertion of 9 nucleotides on the CDR2 region was observed. High R/S values in the CDR indicated antigen selectivity and affinity (4/5). Since no germinal centers could be detected and the analyzed B cells showed antigen selectivity, it may be concluded that already antigenically activated B cells migrated into the synovium and locally differentiated into plasma cells, leading to the extensive infiltration observed. Rheumatoid fibroblasts were shown to support terminal B cell differentiation. Our data suggests that the ability of fibroblasts to activate B cells is not only restricted to RA, but also occurs in OA. The intense plasma cell infiltration contributed to further cartilage damage by altering the microenvironment of the nourishing synovial tissue or by the local production of pathogenic autoantibodies.

[Specific effects of Cr ions on DNA: a comparison between the interaction of CrIII with purified DNA and with DNA from cultured cells].

PubMed

Balbi, C; Vecchio, D; Russo, P; Parodi, S; Santi, L

1981-05-30

Affinity between CrIII and purified calf thymus DNA were studied by equilibrium dialysis at different pHs. Chromium was dosed by atomic spectrometry. This affinity was compared with Chromium-DNA affinity after treatment of living mammalian cells with CrVI and its intracellular reduction. Preliminary results seem to suggest that affinity is similar in both cases and not especially high (K approximately 10(5) 1/mole).

Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate.

PubMed

Maloney, Finn P; Gerwick, Lena; Gerwick, William H; Sherman, David H; Smith, Janet L

2016-09-13

Alkyl branching at the β position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a β-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (Kd = 0.5 μM) and could not be substituted by the acceptor ACP. High-resolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre- and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA.

Tryptophanyl-tRNA synthetase mediates high-affinity tryptophan uptake into human cells.

PubMed

Miyanokoshi, Miki; Yokosawa, Takumi; Wakasugi, Keisuke

2018-06-01

The tryptophan (Trp) transport system has a high affinity and selectivity toward Trp, and has been reported to exist in both human and mouse macrophages. Although this system is highly expressed in interferon-γ (IFN-γ)-treated cells and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells, its identity remains incompletely understood. Tryptophanyl-tRNA synthetase (TrpRS) is also highly expressed in IFN-γ-treated cells and also has high affinity and selectivity for Trp. Here, we investigated the effects of human TrpRS expression on Trp uptake into IFN-γ-treated human THP-1 monocytes or HeLa cells. Inhibition of human TrpRS expression by TrpRS-specific siRNAs decreased and overexpression of TrpRS increased Trp uptake into the cells. Of note, the TrpRS-mediated uptake system had more than hundred-fold higher affinity for Trp than the known System L amino acid transporter, promoted uptake of low Trp concentrations, and had very high Trp selectivity. Moreover, site-directed mutagenesis experiments indicated that Trp- and ATP-binding sites, but not tRNA-binding sites, in TrpRS are essential for TrpRS-mediated Trp uptake into the human cells. We further demonstrate that the addition of purified TrpRS to cell culture medium increases Trp uptake into cells. Taken together, our results reveal that TrpRS plays an important role in high-affinity Trp uptake into human cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Nickel(II) Inhibits Tet-Mediated 5-Methylcytosine Oxidation by High Affinity Displacement of the Cofactor Iron(II).

PubMed

Yin, Ruichuan; Mo, Jiezhen; Dai, Jiayin; Wang, Hailin

2017-06-16

Ten-eleven translocation (Tet) family proteins are Fe(II)- and 2-oxoglutarate-dependent dioxygenases that regulate the dynamics of DNA methylation by catalyzing the oxidation of DNA 5-methylcytosine (5mC). To exert physiologically important functions, redox-active iron chelated in the catalytic center of Tet proteins directly involves the oxidation of the multiple substrates. To understand the function and interaction network of Tet dioxygenases, it is interesting to obtain high affinity and a specific inhibitor. Surprisingly, here we found that natural Ni(II) ion can bind to the Fe(II)-chelating motif (HXD) with an affinity of 7.5-fold as high as Fe(II). Consistently, we further found that Ni(II) ion can displace the cofactor Fe(II) of Tet dioxygenases and inhibit Tet-mediated 5mC oxidation activity with an estimated IC 50 of 1.2 μM. Essentially, Ni(II) can be used as a high affinity and selective inhibitor to explore the function and dynamics of Tet proteins.

Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

PubMed

Lai, H; Carino, M A

1992-07-01

Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

PubMed

Duan, Nuo; Wu, Shijia; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

2012-04-25

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

PubMed Central

Itakura, Yoko; Nakamura-Tsuruta, Sachiko; Kominami, Junko; Tateno, Hiroaki; Hirabayashi, Jun

2017-01-01

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations. PMID:28556796

The FOXP2 forkhead domain binds to a variety of DNA sequences with different rates and affinities.

PubMed

Webb, Helen; Steeb, Olga; Blane, Ashleigh; Rotherham, Lia; Aron, Shaun; Machanick, Philip; Dirr, Heini; Fanucchi, Sylvia

2017-07-01

FOXP2 is a member of the P subfamily of FOX transcription factors, the DNA-binding domain of which is the winged helix forkhead domain (FHD). In this work we show that the FOXP2 FHD is able to bind to various DNA sequences, including a novel sequence identified in this work, with different affinities and rates as detected using surface plasmon resonance. Combining the experimental work with molecular docking, we show that high-affinity sequences remain bound to the protein for longer, form a greater number of interactions with the protein and induce a greater structural change in the protein than low-affinity sequences. We propose a binding model for the FOXP2 FHD that involves three types of binding sequence: low affinity sites which allow for rapid scanning of the genome by the protein in a partially unstructured state; moderate affinity sites which serve to locate the protein near target sites and high-affinity sites which secure the protein to the DNA and induce a conformational change necessary for functional binding and the possible initiation of downstream transcriptional events. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Imprinting the Fate of Antigen-Reactive B Cells through the Affinity of the B Cell Receptor

PubMed Central

O'Connor, Brian P.; Vogel, Laura A.; Zhang, Weijun; Loo, William; Shnider, Danielle; Lind, Evan F.; Ratliff, Michelle; Noelle, Randolph J.; Erickson, Loren D.

2010-01-01

Long-lived plasma cells (PCs) and memory B cells (Bmem) constitute the cellular components of enduring humoral immunity, whereas short-lived PCs that rapidly produce Ig correspond to the host's need for immediate protection against pathogens. In this study we show that the innate affinity of the BCR for Ag imprints upon naive B cells their differentiation fate to become short-or long-lived PCs and Bmem. Using BCR transgenic mice with varying affinities for Ag, naive B cells with high affinity lose their capacity to form germinal centers (GCs), develop neither Bmem nor long-lived PCs, and are destined to a short-lived PC fate. Moderate affinity interactions result in hastened GC responses, and differentiation to long-lived PCs, but Bmem remain extinct. In contrast, lower affinity interactions show tempered GCs, producing Bmem and affinity-matured, long-lived PCs. Thus, a continuum of elementary to comprehensive humoral immune responses exists that is controlled by inherent BCR affinity. PMID:17114443

Dissection of the IgNAR V domain: molecular scanning and orthologue database mining define novel IgNAR hallmarks and affinity maturation mechanisms.

PubMed

Fennell, B J; Darmanin-Sheehan, A; Hufton, S E; Calabro, V; Wu, L; Müller, M R; Cao, W; Gill, D; Cunningham, O; Finlay, W J J

2010-07-09

The shark antigen-binding V(NAR) domain has the potential to provide an attractive alternative to traditional biotherapeutics based on its small size, advantageous physiochemical properties, and unusual ability to target clefts in enzymes or cell surface molecules. The V(NAR) shares many of the properties of the well-characterised single-domain camelid V(H)H but is much less understood at the molecular level. We chose the hen-egg-lysozyme-specific archetypal Type I V(NAR) 5A7 and used ribosome display in combination with error-prone mutagenesis to interrogate the entire sequence space. We found a high level of mutational plasticity across the V(NAR) domain, particularly within the framework 2 and hypervariable region 2 regions. A number of residues important for affinity were identified, and a triple mutant combining A1D, S61R, and G62R resulted in a K(D) of 460 pM for hen egg lysozyme, a 20-fold improvement over wild-type 5A7, and the highest K(D) yet reported for V(NAR)-antigen interactions. These findings were rationalised using structural modelling and indicate the importance of residues outside the classical complementarity determining regions in making novel antigen contacts that modulate affinity. We also located two solvent-exposed residues (G15 and G42), distant from the V(NAR) paratope, which retain function upon mutation to cysteine and have the potential to be exploited as sites for targeted covalent modification. Our findings with 5A7 were extended to all known NAR structures using an in-depth bioinformatic analysis of sequence data available in the literature and a newly generated V(NAR) database. This study allowed us to identify, for the first time, both V(NAR)-specific and V(NAR)/Ig V(L)/TCR V(alpha) overlapping hallmark residues, which are critical for the structural and functional integrity of the single domain. Intriguingly, each of our designated V(NAR)-specific hallmarks align precisely with previously defined mutational 'cold spots' in natural nurse shark cDNA sequences. These findings will aid future V(NAR) engineering and optimisation studies towards the development of V(NAR) single-domain proteins as viable biotherapeutics. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Preparation and characterization of fluorophenylboronic acid-functionalized affinity monolithic columns for the selective enrichment of cis-diol-containing biomolecules.

PubMed

Li, Qianjin; Liu, Zhen

2015-01-01

Boronate affinity monolithic columns have been developed into an important means for the selective recognition and capture of cis-diol-containing biomolecules, such as glycoproteins, nucleosides and saccharides. The ligands of boronic acids are playing an important role in boronate affinity monolithic columns. Although several boronate affinity monoliths with high affinity toward cis-diol-containing biomolecules have been reported, only few publications are focused on their detailed procedures for preparation and characterization. This chapter describes in detail the preparation and characterization of a boronate affinity monolithic column applying 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) as a ligand. The DFFPBA-functionalized monolithic column not only exhibited an ultrahigh boronate affinity toward cis-diol-containing biomolecules, but also showed great potential for the selective enrichment of cis-diol-containing biomolecules in real samples.

Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

PubMed

Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

2016-01-01

Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

PubMed

Debaigt, Colin; Meunier, Annie-Claire; Goursaud, Stephanie; Montoni, Alicia; Pineau, Nicolas; Couvineau, Alain; Laburthe, Marc; Muller, Jean-Marc; Janet, Thierry

2006-07-01

High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes.

PubMed

Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying

2015-01-01

Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

PubMed Central

2015-01-01

Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes. PMID:26681483

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT).

PubMed

Ghadbane, Hemza; Brown, Alistair K; Kremer, Laurent; Besra, Gurdyal S; Fütterer, Klaus

2007-10-01

Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 A resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

The localization and concentration of the PDE2-encoded high-affinity cAMP phosphodiesterase is regulated by cAMP-dependent protein kinase A in the yeast Saccharomyces cerevisiae.

PubMed

Hu, Yun; Liu, Enkai; Bai, Xiaojia; Zhang, Aili

2010-03-01

The genome of the yeast Saccharomyces cerevisiae encodes two cyclic AMP (cAMP) phosphodiesterases, a low-affinity one, Pde1, and a high-affinity one, Pde2. Pde1 has been ascribed a function for downregulating agonist-induced cAMP accumulation in a protein kinase A (PKA)-governed negative feedback loop, whereas Pde2 controls the basal cAMP level in the cell. Here we show that PKA regulates the localization and protein concentration of Pde2. Pde2 is accumulated in the nucleus in wild-type cells growing on glucose, or in strains with hyperactive PKA. In contrast, in derepressed wild-type cells or cells with attenuated PKA activity, Pde2 is distributed over the nucleus and cytoplasm. We also show evidence indicating that the Pde2 protein level is positively correlated with PKA activity. The increase in the Pde2 protein level in high-PKA strains and in cells growing on glucose was due to its increased half-life. These results suggest that, like its low-affinity counterpart, the high-affinity phosphodiesterase may also play an important role in the PKA-controlled feedback inhibition of intracellular cAMP.

Activated tumor cell integrin αvβ3 cooperates with platelets to promote extravasation and metastasis from the blood stream.

PubMed

Weber, Martin R; Zuka, Masahiko; Lorger, Mihaela; Tschan, Mario; Torbett, Bruce E; Zijlstra, Andries; Quigley, James P; Staflin, Karin; Eliceiri, Brian P; Krueger, Joseph S; Marchese, Patrizia; Ruggeri, Zaverio M; Felding, Brunhilde H

2016-04-01

Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvβ3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvβ3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvβ3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvβ3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease. © 2016 Elsevier Ltd. All rights reserved.

Activated tumor cell integrin αvβ3 cooperates with platelets to promote extravasation and metastasis from the blood stream

PubMed Central

Weber, Martin R.; Zuka, Masahiko; Lorger, Mihaela; Tschan, Mario; Torbett, Bruce E.; Zijlstra, Andries; Quigley, James P.; Staflin, Karin; Eliceiri, Brian P.; Krueger, Joseph S.; Marchese, Patricia; Ruggeri, Zaverio M.; Felding, Brunhilde H.

2016-01-01

Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvβ3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvβ3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvβ3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvβ3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease. PMID:27067975

Computational design of an endo-1,4-[beta]-xylanase ligand binding site

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

Morin, Andrew; Kaufmann, Kristian W.; Fortenberry, Carie

2012-09-05

The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-{beta}-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterizationmore » of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 {beta}-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the {beta}-xylanase proteins.« less

Expression of the high-affinity choline transporter CHT1 in rat and human arteries.

PubMed

Lips, Katrin S; Pfeil, Uwe; Reiners, Katja; Rimasch, Christoph; Kuchelmeister, Klaus; Braun-Dullaeus, Ruediger C; Haberberger, Rainer V; Schmidt, Rupert; Kummer, Wolfgang

2003-12-01

The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibroblasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29-40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.

Regulation of calreticulin–major histocompatibility complex (MHC) class I interactions by ATP

PubMed Central

Wijeyesakere, Sanjeeva Joseph; Gagnon, Jessica K.; Arora, Karunesh; Brooks, Charles L.; Raghavan, Malini

2015-01-01

The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin–substrate interactions and as key determinants of PLC dynamics. PMID:26420867

Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

PubMed

Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

2013-12-01

Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection.

Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity

PubMed Central

Sharma, P.; Postel, S.; Sundberg, E.J.; Kranz, D.M.

2013-01-01

Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. PMID:24167300

Alternating carrier models of asymmetric glucose transport violate the energy conservation laws.

PubMed

Naftalin, Richard J

2008-11-01

Alternating access transporters with high-affinity externally facing sites and low-affinity internal sites relate substrate transit directly to the unliganded asymmetric "carrier" (Ci) distribution. When both bathing solutions contain equimolar concentrations of ligand, zero net flow of the substrate-carrier complex requires a higher proportion of unliganded low-affinity inside sites (proportional, variant 1/KD(in)) and slower unliganded "free" carrier transit from inside to outside than in the reverse direction. However, asymmetric rates of unliganded carrier movement, kij, imply that an energy source, DeltaGcarrier = RT ln (koi/kio) = RT ln (Cin/Cout) = RT ln (KD(in)/KD(out)), where R is the universal gas constant (8.314 Joules/M/K degrees), and T is the temperature, assumed here to be 300 K degrees , sustains the asymmetry. Without this invalid assumption, the constraints of carrier path cyclicity, combined with asymmetric ligand affinities and equimolarity at equilibrium, are irreconcilable, and any passive asymmetric uniporter or cotransporter model system, e.g., Na-glucose cotransporters, espousing this fundamental error is untenable. With glucose transport via GLUT1, the higher maximal rate and Km of net ligand exit compared to net ligand entry is only properly simulated if ligand transit occurs by serial dissociation-association reactions between external high-affinity and internal low-affinity immobile sites. Faster intersite transit rates occur from lower-affinity sites than from higher-affinity sites and require no other energy source to maintain equilibrium. Similar constraints must apply to cotransport.

Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

PubMed Central

Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

2012-01-01

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

Robust feature matching via support-line voting and affine-invariant ratios

NASA Astrophysics Data System (ADS)

Li, Jiayuan; Hu, Qingwu; Ai, Mingyao; Zhong, Ruofei

2017-10-01

Robust image matching is crucial for many applications of remote sensing and photogrammetry, such as image fusion, image registration, and change detection. In this paper, we propose a robust feature matching method based on support-line voting and affine-invariant ratios. We first use popular feature matching algorithms, such as SIFT, to obtain a set of initial matches. A support-line descriptor based on multiple adaptive binning gradient histograms is subsequently applied in the support-line voting stage to filter outliers. In addition, we use affine-invariant ratios computed by a two-line structure to refine the matching results and estimate the local affine transformation. The local affine model is more robust to distortions caused by elevation differences than the global affine transformation, especially for high-resolution remote sensing images and UAV images. Thus, the proposed method is suitable for both rigid and non-rigid image matching problems. Finally, we extract as many high-precision correspondences as possible based on the local affine extension and build a grid-wise affine model for remote sensing image registration. We compare the proposed method with six state-of-the-art algorithms on several data sets and show that our method significantly outperforms the other methods. The proposed method achieves 94.46% average precision on 15 challenging remote sensing image pairs, while the second-best method, RANSAC, only achieves 70.3%. In addition, the number of detected correct matches of the proposed method is approximately four times the number of initial SIFT matches.

Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications

PubMed Central

Hage, David S.

2017-01-01

BACKGROUND The interactions between biochemical and chemical agents in the body are important in many clinical processes. Affinity chromatography and high-performance affinity chromatography (HPAC), in which a column contains an immobilized biologically-related binding agent, are two methods that can be used to study these interactions. CONTENT This review looks at various approaches that can be used in affinity chromatography and HPAC to characterize the strength or rate of a biological interaction, the number and types of sites that are involved in this process, and the interactions between multiple solutes for the same binding agent. A number of applications for these methods are examined, with an emphasis on recent developments and high-performance affinity methods. These applications include the use of these techniques for fundamental studies of biological interactions, high-throughput screening of drugs, work with modified proteins, tools for personalized medicine, and studies of drug-drug competition for a common binding agent. SUMMARY The wide range of formats and detection methods that can be used with affinity chromatography and HPAC for examining biological interactions makes these tools attractive for various clinical and pharmaceutical applications. Future directions in the development of small-scale columns and the coupling of these methods with other techniques, such as mass spectrometry or other separation methods, should continue to increase the flexibility and ease with which these approaches can be used in work involving clinical or pharmaceutical samples. PMID:28396561

Rational design of peptide affinity ligands for the purification of therapeutic enzymes.

PubMed

Trasatti, John P; Woo, James; Ladiwala, Asif; Cramer, Steven; Karande, Pankaj

2018-04-25

Non-mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof-of-concept for developing affinity peptide-based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Affinity ranking of antibodies using flow cytometry: application in antibody phage display-based target discovery.

PubMed

Geuijen, Cecilia A W; Clijsters-van der Horst, Marieke; Cox, Freek; Rood, Pauline M L; Throsby, Mark; Jongeneelen, Mandy A C; Backus, Harold H J; van Deventer, Els; Kruisbeek, Ada M; Goudsmit, Jaap; de Kruif, John

2005-07-01

Application of antibody phage display to the identification of cell surface antigens with restricted expression patterns is often complicated by the inability to demonstrate specific binding to a certain cell type. The specificity of an antibody can only be properly assessed when the antibody is of sufficient high affinity to detect low-density antigens on cell surfaces. Therefore, a robust and simple assay for the prediction of relative antibody affinities was developed and compared to data obtained using surface plasmon resonance (SPR) technology. A panel of eight anti-CD46 antibody fragments with different affinities was selected from phage display libraries and reformatted into complete human IgG1 molecules. SPR was used to determine K(D) values for these antibodies. The association and dissociation of the antibodies for binding to CD46 expressed on cell surfaces were analysed using FACS-based assays. We show that ranking of the antibodies based on FACS data correlates well with ranking based on K(D) values as measured by SPR and can therefore be used to discriminate between high- and low-affinity antibodies. Finally, we show that a low-affinity antibody may only detect high expression levels of a surface marker while failing to detect lower expression levels of this molecule, which may lead to a false interpretation of antibody specificity.

The binding properties of cycloxaprid on insect native nAChRs partially explain the low cross-resistance with imidacloprid in Nilaparvata lugens.

PubMed

Zhang, Yixi; Xu, Xiaoyong; Bao, Haibo; Shao, Xusheng; Li, Zhong; Liu, Zewen

2018-06-06

Neonicotinoids, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) to control Nilaparvata lugens, a major rice insect pest. High imidacloprid resistance has been reported in N. lugens in laboratory and in fields. Cycloxaprid, an oxabridged cis-nitromethylene neonicotinoid, showed high insecticidal activity against N. lugens and low cross-resistance in the imidacloprid resistant strains and field populations. Binding studies have demonstrated that imidacloprid had two binding sites with different affinities (Kd = 3.18 ± 0.43 pM and 1.78 ± 0.19 nM) in N. lugens nAChRs. Cycloxaprid was poor at displacing [ 3 H]imidacloprid at its high-affinity binding site (Ki = 159.38±20.43 nM), but quite efficient at the low-affinity binding site (Ki = 1.27±0.35 nM). These data showed that cycloxaprid had overlapping binding sites with imidacloprid only at its low-affinity binding site. Therefore, the low displacement ability of cycloxaprid against imidacloprid binding at its high affinity site could partially explain the low cross-resistance of cycloxaprid in the imidacloprid resistant populations. The high insecticidal activity, low cross-resistance and different binding properties on insect nAChRs of cycloxaprid demonstrating it a potential insecticide to control N. lugens and related insect pests, especially the ones with high resistance to neonicotinoids. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The Phytopathogen Pseudomonas syringae pv. tomato DC3000 Has Three High-Affinity Iron-Scavenging Systems Functional under Iron Limitation Conditions but Dispensable for Pathogenesis▿¶

PubMed Central

Jones, Alexander M.; Wildermuth, Mary C.

2011-01-01

High-affinity iron scavenging through the use of siderophores is a well-established virulence determinant in mammalian pathogenesis. However, few examples have been reported for plant pathogens. Here, we use a genetic approach to investigate the role of siderophores in Pseudomonas syringae pv. tomato DC3000 (DC3000) virulence in tomato. DC3000, an agronomically important pathogen, has two known siderophores for high-affinity iron scavenging, yersiniabactin and pyoverdin, and we uncover a third siderophore, citrate, required for growth when iron is limiting. Though growth of a DC3000 triple mutant unable to either synthesize or import these siderophores is severely restricted in iron-limited culture, it is fully pathogenic. One explanation for this phenotype is that the DC3000 triple mutant is able to directly pirate plant iron compounds such as heme/hemin or iron-nicotianamine, and our data indicate that DC3000 can import iron-nicotianamine with high affinity. However, an alternative explanation, supported by data from others, is that the pathogenic environment of DC3000 (i.e., leaf apoplast) is not iron limited but is iron replete, with available iron of >1 μM. Growth of the triple mutant in culture is restored to wild-type levels by supplementation with a variety of iron chelates at >1 μM, including iron(III) dicitrate, a dominant chelate of the leaf apoplast. This suggests that lower-affinity iron import would be sufficient for DC3000 iron nutrition in planta and is in sharp contrast to the high-affinity iron-scavenging mechanisms required in mammalian pathogenesis. PMID:21441525

Immunotherapy expands and maintains the function of high affinity tumor infiltrating CD8 T cells in situ

PubMed Central

Moran, Amy E.; Polesso, Fanny; Weinberg, Andrew D.

2016-01-01

Cancer cells harbor high affinity tumor-associated antigens capable of eliciting potent anti-tumor T cell responses yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and PD-1 have been used. We report here that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor-antigen specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Co-expression of Nur77GFP and OX40 identifies a polyclonal population of high affinity tumor-associated antigen-specific CD8+ T cells, which produce more IFNγ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or PD-L1. Moreover, expansion of these high affinity CD8 T cells prolongs survival of tumor bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor resident CD8 T cells thereby increasing the frequency of cytolytic, high affinity, tumor-associated antigen-specific cells. PMID:27503208

SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES

EPA Science Inventory

Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES (ABSTRACT)

EPA Science Inventory

Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

A solid-phase combinatorial approach for indoloquinolizidine-peptides with high affinity at D(1) and D(2) dopamine receptors.

PubMed

Molero, Anabel; Vendrell, Marc; Bonaventura, Jordi; Zachmann, Julian; López, Laura; Pardo, Leonardo; Lluis, Carme; Cortés, Antoni; Albericio, Fernando; Casadó, Vicent; Royo, Miriam

2015-06-05

Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number of neurodegenerative disorders. Specifically, compounds able to bind at D1R and D2R with high affinity could restore the effects of dopamine depletion and enhance motor activation on degenerated nigrostriatal dopaminergic systems. We have directed our research towards the synthesis and characterisation of heterocycle-peptide hybrids based on the indolo[2,3-a]quinolizidine core. This privileged structure is a water-soluble and synthetically accessible scaffold with affinity for diverse GPCRs. Herein we have prepared a solid-phase combinatorial library of 80 indoloquinolizidine-peptides to identify compounds with enhanced binding affinity at D2R, a receptor that is crucial to re-establish activity on dopamine-depleted degenerated GABAergic neurons. We applied computational tools and high-throughput screening assays to identify 9a{1,3,3} as a ligand for dopamine receptors with nanomolar affinity and agonist activity at D2R. Our results validate the application of indoloquinolizidine-peptide combinatorial libraries to fine-tune the pharmacological profiles of multiple ligands at D1 and D2 dopamine receptors. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

PubMed Central

Dunn, Steven M.; Rizkallah, Pierre J.; Baston, Emma; Mahon, Tara; Cameron, Brian; Moysey, Ruth; Gao, Feng; Sami, Malkit; Boulter, Jonathan; Li, Yi; Jakobsen, Bent K.

2006-01-01

The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential. PMID:16600963

High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

PubMed Central

Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

2016-01-01

Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

IA-2 autoantibody affinity in children at risk for type 1 diabetes.

PubMed

Krause, Stephanie; Chmiel, Ruth; Bonifacio, Ezio; Scholz, Marlon; Powell, Michael; Furmaniak, Jadwiga; Rees Smith, Bernard; Ziegler, Anette-G; Achenbach, Peter

2012-12-01

Autoantibodies to insulinoma-associated protein 2 (IA-2A) are associated with increased risk for type 1 diabetes. Here we examined IA-2A affinity and epitope specificity to assess heterogeneity in response intensity in relation to pathogenesis and diabetes risk in 50 children who were prospectively followed from birth. At first IA-2A appearance, affinity ranged from 10(7) to 10(11)L/mol and was high (>1.0×10(9)L/mol) in 41 (82%) children. IA-2A affinity was not associated with epitope specificity or HLA class II haplotype. On follow-up, affinity increased or remained high, and IA-2A were commonly against epitopes within the protein tyrosine phosphatase-like IA-2 domain and the homologue protein IA-2β. IA-2A were preceded or accompanied by other islet autoantibodies in 49 (98%) children, of which 34 progressed to diabetes. IA-2A affinity did not stratify diabetes risk. In conclusion, the IA-2A response in children is intense with rapid maturation against immunogenic epitopes and a strong association with diabetes development. Copyright © 2012 Elsevier Inc. All rights reserved.

Solubilization and purification of melatonin receptors from lizard brain

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

Rivkees, S.A.; Conron, R.W. Jr.; Reppert, S.M.

Melatonin receptors in lizard brain were identified and characterized using {sup 125}I-labeled melatonin (({sup 125}I)MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resultedmore » in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.« less

Unusual dewetting of thin polymer films in liquid media containing a poor solvent and a nonsolvent.

PubMed

Xu, Lin; Sharma, Ashutosh; Joo, Sang Woo; Liu, Hui; Shi, Tongfei

2014-12-16

We investigate the control of pattern size and kinetics in spontaneous dewetting of thin polymer films (polystyrene) that are stable to thermal annealing by annealing in a poor solvent (acetone)/nonsolvent (ethanol or n-hexane) liquid mixture. Dewetting occurs by the formation and growth of circular holes that coalesce to form droplets. The influence of the nature and the volume fraction of the nonsolvents on the contact angle of polymer droplets, number density of holes, and the kinetics of holes formation and growth is studied. Addition of ethanol greatly increases the hole density and slows down the kinetics substantially, while affecting only a small change in wettability. n-Hexane addition shows an interesting nonmonotonic response in decreasing the hole density and contact angle in the volume fraction range of 0-0.3 but an opposite effect beyond that. Although the two nonsolvents chosen cannot by themselves induce dewetting, their relative affinity for the solid substrate vis-à-vis acetone can strongly influence the observed dewetting scenarios that are not understood by the existing theoretical considerations. n-Hexane, for example, has great affinity for silicon substrate. In addition to the changes in wettability, viscosity, and film interfacial tension engendered by the nonsolvents, the possibility of the formation of adsorbed liquid layers at the substrate-polymer interface, which can modify the interfacial friction and slippage, needs to be considered.

Specific DNA binding of a potential transcriptional regulator, inosine 5'-monophosphate dehydrogenase-related protein VII, to the promoter region of a methyl coenzyme m reductase I-encoding operon retrieved from Methanothermobacter thermautotrophicus strain DeltaH.

PubMed

Shinzato, Naoya; Enoki, Miho; Sato, Hiroaki; Nakamura, Kohei; Matsui, Toru; Kamagata, Yoichi

2008-10-01

Two methyl coenzyme M reductases (MCRs) encoded by the mcr and mrt operons of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus DeltaH are expressed in response to H(2) availability. In the present study, cis elements and trans-acting factors responsible for the gene expression of MCRs were investigated by using electrophoretic mobility shift assay (EMSA) and affinity particle purification. A survey of their operator regions by EMSA with protein extracts from mrt-expressing cultures restricted them to 46- and 41-bp-long mcr and mrt upstream regions, respectively. Affinity particle purification of DNA-binding proteins conjugated with putative operator regions resulted in the retrieval of a protein attributed to IMP dehydrogenase-related protein VII (IMPDH VII). IMPDH VII is predicted to have a winged helix-turn-helix DNA-binding motif and two cystathionine beta-synthase domains, and it has been suspected to be an energy-sensing module. EMSA with oligonucleotide probes with unusual sequences showed that the binding site of IMPDH VII mostly overlaps the factor B-responsible element-TATA box of the mcr operon. The results presented here suggest that IMPDH VII encoded by MTH126 is a plausible candidate for the transcriptional regulator of the mcr operon in this methanogen.

Structural basis for collagen recognition by the immune receptor OSCAR.

PubMed

Zhou, Long; Hinerman, Jennifer M; Blaszczyk, Michal; Miller, Jeanette L C; Conrady, Deborah G; Barrow, Alexander D; Chirgadze, Dimitri Y; Bihan, Dominique; Farndale, Richard W; Herr, Andrew B

2016-02-04

The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2. © 2016 by The American Society of Hematology.

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

PubMed Central

Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

2014-01-01

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

Myelin-reactive “type B” T cells and T cells specific for low-affinity MHC-binding myelin peptides escape tolerance in HLA-DR transgenic mice

PubMed Central

Kawamura, Kazuyuki; McLaughlin, Katherine A.; Weissert, Robert; Forsthuber, Thomas G.

2009-01-01

Genes of the major histocompatibility complex (MHC) show the strongest genetic association with multiple sclerosis (MS) but the underlying mechanisms have remained unresolved. Here, we asked whether the MS-associated MHC class II molecules, HLA-DRB1*1501, HLA-DRB5*0101, and HLA-DRB1*0401 contribute to autoimmune central nervous system (CNS) demyelination by promoting pathogenic T cell responses to human myelin basic protein (hMBP), using three transgenic (Tg) mouse lines expressing these MHC molecules. Unexpectedly, profound T cell tolerance to the high-affinity MHC-binding hMBP82-100 epitope was observed in all Tg mouse lines. T cell tolerance to hMBP82-100 was abolished upon backcrossing the HLA-DR Tg mice to MBP-deficient mice. In contrast, T cell tolerance was incomplete for low-affinity MHC-binding hMBP epitopes. Furthermore, hMBP82-100-specific “type B” T cells escaped tolerance in HLA-DRB5*0101 Tg mice. Importantly, T cells specific for low-affinity MHC-binding hMBP epitopes and hMBP82-100-specific “type B” T cells were highly encephalitogenic. Collectively, the results show that MS-associated MHC class II molecules are highly efficient at inducing T cell tolerance to high-affinity MHC-binding epitope, whereas autoreactive T cells specific for the low-affinity MHC-binding epitopes and “type B” T cells can escape the induction of T cell tolerance and may promote MS. PMID:18713991

ADP binding to TF1 and its subunits induces ultraviolet spectral changes.

PubMed

Hisabori, T; Yoshida, M; Sakurai, H

1986-09-01

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.

Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification.

PubMed

Fujii, Yuki; Kaneko, Mika K; Ogasawara, Satoshi; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Honma, Ryusuke; Kato, Yukinari

2017-04-01

Affinity tag systems, possessing high affinity and specificity, are useful for protein detection and purification. The most suitable tag for a particular purpose should be selected from many available affinity tag systems. In this study, we developed a novel affinity tag called the "RAP tag" system, which comprises a mouse antirat podoplanin monoclonal antibody (clone PMab-2) and the RAP tag (DMVNPGLEDRIE). This system is useful not only for protein detection in Western blotting, flow cytometry, and sandwich enzyme-linked immunosorbent assay, but also for protein purification.

Intercostal muscle twitching: An unusual manifestation of extracardiac stimulation related to right ventricular outflow tract pacing

PubMed Central

Erdogan, Okan

2007-01-01

The present case report describes a patient who underwent successful dual-chamber pacemaker implantation with active ventricular lead fixation at a high septal region in the right ventricular outflow tract. Unexpectedly, stimulation at a high output in the right ventricular outflow tract caused an unusual extracardiac stimulation, specifically, intercostal muscle twitching. PMID:17703261

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library*

PubMed Central

Kiefer, Jonathan D.; Srinivas, Raja R.; Lobner, Elisabeth; Tisdale, Alison W.; Mehta, Naveen K.; Yang, Nicole J.; Tidor, Bruce; Wittrup, K. Dane

2016-01-01

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation. PMID:27582495

Higher Nucleoporin-Importinβ Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import

PubMed Central

Azimi, Mohammad; Mofrad, Mohammad R. K.

2013-01-01

Several in vitro studies have shown the presence of an affinity gradient in nuclear pore complex proteins for the import receptor Importinβ, at least partially contributing to nucleocytoplasmic transport, while others have historically argued against the presence of such a gradient. Nonetheless, the existence of an affinity gradient has remained an uncharacterized contributing factor. To shed light on the affinity gradient theory and better characterize how the existence of such an affinity gradient between the nuclear pore and the import receptor may influence the nucleocytoplasmic traffic, we have developed a general-purpose agent based modeling (ABM) framework that features a new method for relating rate constants to molecular binding and unbinding probabilities, and used our ABM approach to quantify the effects of a wide range of forward and reverse nucleoporin-Importinβ affinity gradients. Our results indicate that transport through the nuclear pore complex is maximized with an effective macroscopic affinity gradient of 2000 µM, 200 µM and 10 µM in the cytoplasmic, central channel and nuclear basket respectively. The transport rate at this gradient is approximately 10% higher than the transport rate for a comparable pore lacking any affinity gradient, which has a peak transport rate when all nucleoporins have an affinity of 200 µM for Importinβ. Furthermore, this optimal ratio of affinity gradients is representative of the ratio of affinities reported for the yeast nuclear pore complex – suggesting that the affinity gradient seen in vitro is highly optimized. PMID:24282617

A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

PubMed

Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

2018-04-30

Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

Detachment of affinity-captured bioparticles by elastic deformation of a macroporous hydrogel

PubMed Central

Dainiak, Maria B.; Kumar, Ashok; Galaev, Igor Yu.; Mattiasson, Bo

2006-01-01

Adsorption of bioparticles to affinity surfaces involves polyvalent interactions, complicating greatly the recovery of the adsorbed material. A unique system for the efficient binding and release of different cells and particles is described. Affinity-bound bioparticles and synthetic particles are detached from the macroporous hydrogel matrix, a so-called cryogel, when the cryogel undergoes elastic deformation. The particle detachment upon elastic deformation is believed to be due to breaking of many of the multipoint attachments between the particles and the affinity matrix and the change in the distance between affinity ligands when the matrix is deformed. However, no release of affinity-bound protein occurred upon elastic deformation. The phenomenon of particle detachment upon elastic deformation is believed to be of a generic nature, because it was demonstrated for a variety of bioparticles of different sizes and for synthetic particles, for different ligand–receptor pairs (IgG–protein A, sugar–ConA, metal ion–chelating ligand), and when the deformation was caused by either external forces (mechanical deformation) or internal forces (the shrinkage of thermosensitive, macroporous hydrogel upon an increase in temperature). The elasticity of cryogel monoliths ensures high recovery of captured cells under mild conditions, with highly retained viability. This property, along with their continuous porous structure makes cryogel monoliths very attractive for applications in affinity cell separation. PMID:16418282

Kir6.2-dependent high-affinity repaglinide binding to β-cell KATP channels

PubMed Central

Hansen, Ann Maria K; Hansen, John Bondo; Carr, Richard D; Ashcroft, Frances M; Wahl, Philip

2005-01-01

The β-cell KATP channel is composed of two types of subunit – the inward rectifier K+ channel (Kir6.2) which forms the channel pore, and the sulphonylurea receptor (SUR1), which serves as a regulatory subunit. The N-terminus of Kir6.2 is involved in transduction of sulphonylurea binding into channel closure, and deletion of the N-terminus (Kir6.2ΔN14) results in functional uncoupling of the two subunits. In this study, we investigate the interaction of the hypoglycaemic agents repaglinide and glibenclamide with SUR1 and the effect of Kir6.2 on this interaction. We further explore how the binding properties of repaglinide and glibenclamide are affected by functional uncoupling of SUR1 and Kir6.2 in Kir6.2ΔN14/SUR1 channels. All binding experiments are performed on membranes in ATP-free buffer at 37°C. Repaglinide was found to bind with low affinity (KD=59±16 nM) to SUR1 alone, but with high affinity (increased ∼150-fold) when SUR1 was co-expressed with Kir6.2 (KD=0.42±0.03 nM). Glibenclamide, tolbutamide and nateglinide all bound with marginally lower affinity to SUR1 than to Kir6.2/SUR1. Repaglinide bound with low affinity (KD=51±23 nM) to SUR1 co-expressed with Kir6.2ΔN14. In contrast, the affinity for glibenclamide, tolbutamide and nateglinide was only mildly changed as compared to wild-type channels. In whole-cell patch-clamp experiments inhibition of Kir6.2ΔN14/SUR1 currents by both repaglinide and nateglinde is abolished. The results suggest that Kir6.2 causes a conformational change in SUR1 required for high-affinity repaglinide binding, or that the high-affinity repaglinide-binding site includes contributions from both SUR1 and Kir6.2. Glibenclamide, tolbutamide and nateglinide binding appear to involve only SUR1. PMID:15678092

Overcoming HERG affinity in the discovery of the CCR5 antagonist maraviroc.

PubMed

Price, David A; Armour, Duncan; de Groot, Marcel; Leishman, Derek; Napier, Carolyn; Perros, Manos; Stammen, Blanda L; Wood, Anthony

2006-09-01

The discovery of maraviroc 17 is described with particular reference to the generation of high selectivity over affinity for the HERG potassium channel. This was achieved through the use of a high throughput binding assay for the HERG channel that is known to show an excellent correlation with functional effects.

Life on the edge: O2 binding in Atlantic cod red blood cells near their southern distribution limit is not sensitive to temperature or haemoglobin genotype

PubMed Central

Barlow, Samantha L.; Metcalfe, Julian; Righton, David A.

2017-01-01

ABSTRACT Atlantic cod are a commercially important species believed to be threatened by warming seas near their southern, equatorward upper thermal edge of distribution. Limitations to circulatory O2 transport, in particular cardiac output, and the geographic distribution of functionally different haemoglobin (Hb) genotypes have separately been suggested to play a role in setting thermal tolerance in this species. The present study assessed the thermal sensitivity of O2 binding in Atlantic cod red blood cells with different Hb genotypes near their upper thermal distribution limit and modelled its consequences for the arterio-venous O2 saturation difference, Sa–vO2, another major determinant of circulatory O2 supply rate. The results showed statistically indistinguishable red blood cell O2 binding between the three HbI genotypes in wild-caught Atlantic cod from the Irish Sea (53° N). Red blood cells had an unusually low O2 affinity, with reduced or even reversed thermal sensitivity between pH 7.4 and 7.9, and 5.0 and 20.0°C. This was paired with strongly pH-dependent affinity and cooperativity of red blood cell O2 binding (Bohr and Root effects). Modelling of Sa–vO2 at physiological pH, temperature and O2 partial pressures revealed a substantial capacity for increases in Sa–vO2 to meet rising tissue O2 demands at 5.0 and 12.5°C, but not at 20°C. Furthermore, there was no evidence for an increase of maximal Sa–vO2 with temperature. It is suggested that Atlantic cod at such high temperatures may solely depend on increases in cardiac output and blood O2 capacity, or thermal acclimatisation of metabolic rate, for matching circulatory O2 supply to tissue demand. PMID:28148818

Life on the edge: O2 binding in Atlantic cod red blood cells near their southern distribution limit is not sensitive to temperature or haemoglobin genotype.

PubMed

Barlow, Samantha L; Metcalfe, Julian; Righton, David A; Berenbrink, Michael

2017-02-01

Atlantic cod are a commercially important species believed to be threatened by warming seas near their southern, equatorward upper thermal edge of distribution. Limitations to circulatory O 2 transport, in particular cardiac output, and the geographic distribution of functionally different haemoglobin (Hb) genotypes have separately been suggested to play a role in setting thermal tolerance in this species. The present study assessed the thermal sensitivity of O 2 binding in Atlantic cod red blood cells with different Hb genotypes near their upper thermal distribution limit and modelled its consequences for the arterio-venous O 2 saturation difference, Sa-v O 2 , another major determinant of circulatory O 2 supply rate. The results showed statistically indistinguishable red blood cell O 2 binding between the three HbI genotypes in wild-caught Atlantic cod from the Irish Sea (53° N). Red blood cells had an unusually low O 2 affinity, with reduced or even reversed thermal sensitivity between pH 7.4 and 7.9, and 5.0 and 20.0°C. This was paired with strongly pH-dependent affinity and cooperativity of red blood cell O 2 binding (Bohr and Root effects). Modelling of Sa-v O 2  at physiological pH, temperature and O 2 partial pressures revealed a substantial capacity for increases in Sa-v O 2  to meet rising tissue O 2 demands at 5.0 and 12.5°C, but not at 20°C. Furthermore, there was no evidence for an increase of maximal Sa-v O 2  with temperature. It is suggested that Atlantic cod at such high temperatures may solely depend on increases in cardiac output and blood O 2 capacity, or thermal acclimatisation of metabolic rate, for matching circulatory O 2 supply to tissue demand. © 2017. Published by The Company of Biologists Ltd.

Challenges and opportunities in the purification of recombinant tagged proteins.

PubMed

Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A

2014-01-01

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. Copyright © 2013 Elsevier Inc. All rights reserved.

Discovery of Tertiary Sulfonamides as Potent Liver X Receptor Antagonists

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

Zuercher, William J.; Buckholz†, Richard G.; Campobasso, Nino

2010-08-12

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

PubMed

Zuercher, William J; Buckholz, Richard G; Campobasso, Nino; Collins, Jon L; Galardi, Cristin M; Gampe, Robert T; Hyatt, Stephen M; Merrihew, Susan L; Moore, John T; Oplinger, Jeffrey A; Reid, Paul R; Spearing, Paul K; Stanley, Thomas B; Stewart, Eugene L; Willson, Timothy M

2010-04-22

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Use of receptor chimeras to identify small molecules with high affinity for the dynorphin A binding domain of the kappa opioid receptor.

PubMed

Kumar, Virendra; Guo, Deqi; Marella, Michael; Cassel, Joel A; Dehaven, Robert N; Daubert, Jeffrey D; Mansson, Erik

2008-06-15

A series of 2-substituted sulfamoyl arylacetamides of general structure 2 were prepared as potent kappa opioid receptor agonists and the affinities of these compounds for opioid and chimeric receptors were compared with those of dynorphin A. Compounds 2e and 2i were identified as non-peptide small molecules that bound to chimeras 3 and 4 with high affinities similar to dynorphin A, resulting in K(i) values of 1.5 and 1.2 nM and 1.3 and 2.2 nM, respectively.

Cyclic peptide unguisin A is an anion receptor with high affinity for phosphate and pyrophosphate.

PubMed

Daryl Ariawan, A; Webb, James E A; Howe, Ethan N W; Gale, Philip A; Thordarson, Pall; Hunter, Luke

2017-04-05

Unguisin A (1) is a marine-derived, GABA-containing cyclic heptapeptide. The biological function of this flexible macrocycle is obscure. Here we show that compound 1 lacks any detectable activity in antimicrobial growth inhibition assays, a result that runs contrary to a previous report. However, we find that 1 functions as a promiscuous host molecule in a variety of anion-binding interactions, with high affinity particularly for phosphate and pyrophosphate. We also show that a series of rigidified, backbone-fluorinated analogues of 1 displays altered affinity for chloride ions.

A Water‐Soluble Tetraazaperopyrene Dye as Strong G‐Quadruplex DNA Binder

PubMed Central

Hahn, Lena

2016-01-01

Abstract The interactions of the water‐soluble tetraazaperopyrene dye 1 with ct‐DNA, duplex‐[(dAdT)12 ⋅(dAdT)12], duplex‐[(dGdC)12 ⋅(dGdC)12] as well as with two G‐quadruplex‐forming sequences, namely the human telomeric 22AG and the promotor sequence c‐myc, were investigated by means of UV/visible and fluorescence spectroscopy, isothermal titration calorimetry (ITC) and molecular docking studies. Dye 1 exhibits a high affinity for G‐quadruplex structures over duplex DNA structures. Furthermore, the ligand shows promising G‐quadruplex discrimination, with an affinity towards c‐myc of 2×107  m −1 (i.e., K d=50 nm), which is higher than for 22AG (4×106  m −1). The ITC data reveal that compound 1 interacts with c‐myc in a stoichiometric ratio of 1:1 but also indicate the presence of two identical lower affinity secondary binding sites per quadruplex. In 22AG, there are two high affinity binding sites per quadruplex, that is, one on each side, with a further four weaker binding sites. For both quadruplex structures, the high affinity interactions between compound 1 and the quadruplex‐forming nucleic acid structures are weakly endothermic. Molecular docking studies suggest an end‐stacking binding mode for compound 1 interacting with quadruplex structures, and a higher affinity for the parallel conformation of c‐myc than for the mixed‐hybrid conformation of 22AG. In addition, docking studies also suggest that the reduced affinity for duplex DNA structures is due to the non‐viability of an intercalative binding mode. PMID:26997208

Interaction between phloretin and the red blood cell membrane

PubMed Central

1976-01-01

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane. PMID:5575

NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

PubMed

Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

2002-07-01

Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

Affinity of Tau antibodies for solubilized pathological Tau species but not their immunogen or insoluble Tau aggregates predicts in vivo and ex vivo efficacy.

PubMed

Congdon, Erin E; Lin, Yan; Rajamohamedsait, Hameetha B; Shamir, Dov B; Krishnaswamy, Senthilkumar; Rajamohamedsait, Wajitha J; Rasool, Suhail; Gonzalez, Veronica; Levenga, Josien; Gu, Jiaping; Hoeffer, Charles; Sigurdsson, Einar M

2016-08-30

A few tau immunotherapies are now in clinical trials with several more likely to be initiated in the near future. A priori, it can be anticipated that an antibody which broadly recognizes various pathological tau aggregates with high affinity would have the ideal therapeutic properties. Tau antibodies 4E6 and 6B2, raised against the same epitope region but of varying specificity and affinity, were tested for acutely improving cognition and reducing tau pathology in transgenic tauopathy mice and neuronal cultures. Surprisingly, we here show that one antibody, 4E6, which has low affinity for most forms of tau acutely improved cognition and reduced soluble phospho-tau, whereas another antibody, 6B2, which has high affinity for various tau species was ineffective. Concurrently, we confirmed and clarified these efficacy differences in an ex vivo model of tauopathy. Alzheimer's paired helical filaments (PHF) were toxic to the neurons and increased tau levels in remaining neurons. Both toxicity and tau seeding were prevented by 4E6 but not by 6B2. Furthermore, 4E6 reduced PHF spreading between neurons. Interestingly, 4E6's efficacy relates to its high affinity binding to solubilized PHF, whereas the ineffective 6B2 binds mainly to aggregated PHF. Blocking 4E6's uptake into neurons prevented its protective effects if the antibody was administered after PHF had been internalized. When 4E6 and PHF were administered at the same time, the antibody was protective extracellularly. Overall, these findings indicate that high antibody affinity for solubilized PHF predicts efficacy, and that acute antibody-mediated improvement in cognition relates to clearance of soluble phospho-tau. Importantly, both intra- and extracellular clearance pathways are in play. Together, these results have major implications for understanding the pathogenesis of tauopathies and for development of immunotherapies.

How high do ion fluxes go? A re-evaluation of the two-mechanism model of K+ 2 transport in plant roots

USDA-ARS?s Scientific Manuscript database

Potassium acquisition in roots is described by a two-mechanism model, consisting of a saturable, high-affinity transport system (HATS) operating via H+/K+ symport at low (< 1 mM) external [K+] ([K+]ext), and a linear, low-affinity system (LATS) operating via ion channels at high (> 112 mM) [K+]ext. ...

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1.

PubMed

Baruffini, Enrico; Goffrini, Paola; Donnini, Claudia; Lodi, Tiziana

2006-12-01

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.

Development of melanoma-targeted polymer micelles by conjugation of a Melanocortin 1 Receptor (MC1R) specific ligand

PubMed Central

Barkey, Natalie M.; Tafreshi, Narges K.; Josan, Jatinder S.; De Silva, Channa R.; Sill, Kevin N.; Hruby, Victor J.; Gillies, Robert J.; Morse, David L.; Vagner, Josef

2012-01-01

The incidence of malignant melanoma is rising faster than that of any other cancer in the United States. Due to its high expression on the surface of melanomas, MC1R has been investigated as a target for selective imaging and therapeutic agents against melanoma. Eight ligands were screened against cell lines engineered to over-express MC1R, MC4R or MC5R. Of these, compound 1 (4-phenylbutyryl-His-Dphe-Arg-Trp-NH2) exhibited high (0.2 nM) binding affinity for MC1R, and low (high nM) affinities for MC4R and MC5R. Subsequently functionalization of the ligand at the C-terminus with an alkyne for use in Cu-catalyzed click chemistry was shown not to affect the binding affinity. Finally, formation of the targeted-polymer, as well as the targeted micelle formulation, also resulted in constructs with low nM binding affinity. PMID:22011200

Development of melanoma-targeted polymer micelles by conjugation of a melanocortin 1 receptor (MC1R) specific ligand.

PubMed

Barkey, Natalie M; Tafreshi, Narges K; Josan, Jatinder S; De Silva, Channa R; Sill, Kevin N; Hruby, Victor J; Gillies, Robert J; Morse, David L; Vagner, Josef

2011-12-08

The incidence of malignant melanoma is rising faster than that of any other cancer in the United States. Because of its high expression on the surface of melanomas, MC1R has been investigated as a target for selective imaging and therapeutic agents against melanoma. Eight ligands were screened against cell lines engineered to overexpress MC1R, MC4R, or MC5R. Of these, compound 1 (4-phenylbutyryl-His-dPhe-Arg-Trp-NH(2)) exhibited high (0.2 nM) binding affinity for MC1R and low (high nanomolar) affinities for MC4R and MC5R. Functionalization of the ligand at the C-terminus with an alkyne for use in Cu-catalyzed click chemistry was shown not to affect the binding affinity. Finally, formation of the targeted polymer, as well as the targeted micelle formulation, also resulted in constructs with low nanomolar binding affinity.

Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

PubMed

Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

2015-09-01

Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Probing ligand recognition of the opioid pan antagonist AT-076 at nociceptin, kappa, mu, and delta opioid receptors through structure-activity relationships.

PubMed

Journigan, V Blair; Polgar, Willma E; Tuan, Edward W; Lu, James; Daga, Pankaj R; Zaveri, Nurulain T

2017-10-16

Few opioid ligands binding to the three classic opioid receptor subtypes, mu, kappa and delta, have high affinity at the fourth opioid receptor, the nociceptin/orphanin FQ receptor (NOP). We recently reported the discovery of AT-076 (1), (R)-7-hydroxy-N-((S)-1-(4-(3-hydroxyphenyl)piperidin-1-yl)-3-methylbutan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide, a pan antagonist with nanomolar affinity for all four subtypes. Since AT-076 binds with high affinity at all four subtypes, we conducted a structure-activity relationship (SAR) study to probe ligand recognition features important for pan opioid receptor activity, using chemical modifications of key pharmacophoric groups. SAR analysis of the resulting analogs suggests that for the NOP receptor, the entire AT-076 scaffold is crucial for high binding affinity, but the binding mode is likely different from that of NOP antagonists C-24 and SB-612111 bound in the NOP crystal structure. On the other hand, modifications of the 3-hydroxyphenyl pharmacophore, but not the 7-hydroxy Tic pharmacophore, are better tolerated at kappa and mu receptors and yield very high affinity multifunctional (e.g. 12) or highly selective (e.g. 16) kappa ligands. With the availability of the opioid receptor crystal structures, our SAR analysis of the common chemotype of AT-076 suggests rational approaches to modulate binding selectivity, enabling the design of multifunctional or selective opioid ligands from such scaffolds.

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

Novel antipsychotics activate recombinant human and native rat serotonin 5-HT1A receptors: affinity, efficacy and potential implications for treatment of schizophrenia.

PubMed

Newman-Tancredi, Adrian; Assié, Marie-Bernadette; Leduc, Nathalie; Ormière, Anne-Marie; Danty, Nathalie; Cosi, Cristina

2005-09-01

Serotonin 5-HT1A receptors are promising targets in the management of schizophrenia but little information exists about affinity and efficacy of novel antipsychotics at these sites. We addressed this issue by comparing binding affinity at 5-HT1A receptors with dopamine rD2 receptors, which are important targets for antipsychotic drug action. Agonist efficacy at 5-HT1A receptors was determined for G-protein activation and adenylyl cyclase activity. Whereas haloperidol, thioridazine, risperidone and olanzapine did not interact with 5-HT1A receptors, other antipsychotic agents exhibited agonist properties at these sites. E(max) values (% effect induced by 10 microM of 5-HT) for G-protein activation at rat brain 5-HT1A receptors: sarizotan (66.5), bifeprunox (35.9), SSR181507 (25.8), nemonapride (25.7), ziprasidone (20.6), SLV313 (19), aripiprazole (15), tiospirone (8.9). These data were highly correlated with results obtained at recombinant human 5-HT1A receptors in determinations of G-protein activation and inhibition of forskolin-stimulated adenylyl cyclase. In binding-affinity determinations, the antipsychotics exhibited diverse properties at r5-HT1A receptors: sarizotan (pK(i)=8.65), SLV313 (8.64), SSR181507 (8.53), nemonapride (8.35), ziprasidone (8.30), tiospirone (8.22), aripiprazole (7.42), bifeprunox (7.19) and clozapine (6.31). The affinity ratios of the ligands at 5-HT1A vs. D2 receptors also varied widely: ziprasidone, SSR181507 and SLV313 had similar affinities whereas aripiprazole, nemonapride and bifeprunox were more potent at D2 than 5-HT1A receptors. Taken together, these data indicate that aripiprazole has low efficacy and modest affinity at 5-HT1A receptors, whereas bifeprunox has low affinity but high efficacy. In contrast, SSR181507 has intermediate efficacy but high affinity, and is likely to have more prominent 5-HT1A receptor agonist properties. Thus, the contribution of 5-HT1A receptor activation to the pharmacological profile of action of the antipsychotics will depend on the relative 5-HT1A/D2 affinities and on 5-HT1A agonist efficacy of the drugs.

Low-affinity spermine block mediating outward currents through Kir2.1 and Kir2.2 inward rectifier potassium channels

PubMed Central

Ishihara, Keiko; Yan, Ding-Hong

2007-01-01

The outward component of the strong inward rectifier K+ current (IKir) plays a pivotal role in polarizing the membranes of excitable and non-excitable cells and is regulated by voltage-dependent channel block by internal cations. Using the Kir2.1 channel, we previously showed that a small fraction of the conductance susceptible only to a low-affinity mode of block likely carries a large portion of the outward current. To further examine the relevance of the low-affinity block to outward IKir and to explore its molecular mechanism, we studied the block of the Kir2.1 and Kir2.2 channels by spermine, which is the principal Kir2 channel blocker. Current–voltage relations of outward Kir2.2 currents showed a peak, a plateau and two peaks in the presence of 10, 1 and 0.1 μm spermine, respectively, which was explained by the presence of two conductances that differ in their susceptibility to spermine block. When the current–voltage relations showed one peak, like those of native IKir, outward Kir2.2 currents were mediated mostly by the conductance susceptible to the low-affinity block. They also flowed in a narrower range than the corresponding Kir2.1 currents, because of 3- to 4-fold greater susceptibility to the low-affinity block than in Kir2.1. Reducing external [K+] shifted the voltage dependences of both the high- and low-affinity block of Kir2.1 in parallel with the shift in the reversal potential, confirming the importance of the low-affinity block in mediating outward IKir. When Kir2.1 mutants known to have reduced sensitivity to internal blockers were examined, the D172N mutation in the transmembrane pore region made almost all of the conductance susceptible only to low-affinity block, while the E224G mutation in the cytoplasmic pore region reduced the sensitivity to low-affinity block without markedly altering that to the high-affinity block or the high/low conductance ratio. The effects of these mutations support the hypothesis that Kir2 channels exist in two states having different susceptibilities to internal cationic blockers. PMID:17640933

Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

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

Rothman, R.B.; Reid, A.; Mahboubi, A.

1991-02-01

Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and lowmore » affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.« less

Invert biopanning: A novel method for efficient and rapid isolation of scFvs by phage display technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Tanomand, Asghar; Akbari, Bahman

2016-11-01

Phage display is a prominent screening technique for development of novel high affinity antibodies against almost any antigen. However, removing false positive clones in screening process remains a challenge. The aim of this study was to develop an efficient and rapid method for isolation of high affinity scFvs by removing NSBs without losing rare specific clones. Therefore, a novel two rounds strategy called invert biopanning was developed for isolating high affinity scFvs against EGFRvIII antigen from human scFv library. The efficiency of invert biopanning method (procedure III) was analyzed by comparing with results of conventional biopanning methods (procedures I and II). According to the results of polyclonal ELISA, the second round of procedure III displayed highest binding affinity against EGFRvIII peptide accompanied by lowest NSB comparing to other two procedures. Several positive clones were identified among output phages of procedure III by monoclonal phage ELISA which displayed high affinity to EGFRvIII antigen. In conclusion, results of our study indicate that invert biopanning is an efficient method for avoiding NSBs and conservation of rare specific clones during screening of a scFv phage library. Novel anti EGFRvIII scFv isolated could be a promising candidate for potential use in treatment of EGFRvIII expressing cancers. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri*

PubMed Central

Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

2016-01-01

Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H+ or Na+ electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri. Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4′,6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR.

PubMed

Gater, Deborah L; Saurel, Olivier; Iordanov, Iordan; Liu, Wei; Cherezov, Vadim; Milon, Alain

2014-11-18

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

IFN-γ regulates CD8+ memory T cell differentiation and survival in response to weak, but not strong, TCR signals.

PubMed

Stoycheva, Diana; Deiser, Katrin; Stärck, Lilian; Nishanth, Gopala; Schlüter, Dirk; Uckert, Wolfgang; Schüler, Thomas

2015-01-15

In response to primary Ag contact, naive mouse CD8(+) T cells undergo clonal expansion and differentiate into effector T cells. After pathogen clearance, most effector T cells die, and only a small number of memory T cell precursors (TMPs) survive to form a pool of long-lived memory T cells (TMs). Although high- and low-affinity CD8(+) T cell clones are recruited into the primary response, the TM pool consists mainly of high-affinity clones. It remains unclear whether the more efficient expansion of high-affinity clones and/or cell-intrinsic processes exclude low-affinity T cells from the TM pool. In this article, we show that the lack of IFN-γR signaling in CD8(+) T cells promotes TM formation in response to weak, but not strong, TCR agonists. The IFN-γ-sensitive accumulation of TMs correlates with reduced mammalian target of rapamycin activation and the accumulation of long-lived CD62L(hi)Bcl-2(hi)Eomes(hi) TMPs. Reconstitution of mammalian target of rapamycin or IFN-γR signaling is sufficient to block this process. Hence, our data suggest that IFN-γR signaling actively blocks the formation of TMPs responding to weak TCR agonists, thereby promoting the accumulation of high-affinity T cells finally dominating the TM pool. Copyright © 2015 by The American Association of Immunologists, Inc.

Influence of N-ethylmaleimide on cholinoceptors and responses in longitudinal muscles from guinea-pig ileum.

PubMed Central

Aronstam, R. S.; Carrier, G. O.

1982-01-01

1 The binding of carbamylcholine to membranes prepared from the longitudinal muscle of guinea-pig ileum was determined from its inhibition of the binding of [3H]-3-quinuclidinyl benzilate. Carbamylcholine binding was resolved into high and low affinity components with apparent dissociation constants of 0.11 +/- 0.02 and 11 +/- 1 microM; 42% of the receptors displayed high affinity carbamylcholine binding. 2 Alkylation of longitudinal muscle membranes with N-ethylmaleimide increased muscarinic receptor affinity for carbamylcholine in a manner consistent with a conversion of low affinity to high affinity receptors. After exposure the muscle membrane fragments to 1 mM N-ethylmaleimide for 20 min at 35 degrees C, carbamylcholine binding was resolved into two components with apparent dissociation constants of 0.11 +/- 0.01 and 9 +/- 2 microM, with 74% of the receptors displaying the higher affinity. 3 Exposure of longitudinal membranes mounted in an organ chamber to 1 mM N-ethylmaleimide for 30s depressed isometric contractions in response to acetylcholine by 80%, while contractions induced by K+ and Ba2+ were reduced by less than 20% and 10%, respectively. Acetylcholine dose-response curves were shifted to the right while Ba2+ curves were unaffected. 4 It is suggested that N-ethylmaleimide has a selective effect on muscarinic responses in the longitudinal muscle by disrupting processes occurring after receptor occupancy but before the induction of phospholipid turnover or calcium influx in the postsynaptic membrane. PMID:7126999

4-Alkylated homoibotenic acid (HIBO) analogues: versatile pharmacological agents with diverse selectivity profiles towards metabotropic and ionotropic glutamate receptor subtypes.

PubMed

Madsen, Ulf; Pickering, Darryl S; Nielsen, Birgitte; Bräuner-Osborne, Hans

2005-01-01

4-Alkylated analogues of homoibotenic acid (HIBO) have previously shown high potency and selectivity at ionotropic and metabotropic glutamic acid receptor (iGluR and mGluR) subtypes. Compounds with different selectivity profiles are valuable pharmacological tools for neuropharmacological studies, and the series of 4-alkyl-HIBO analogues have been extended in this paper in the search for versatile agents. Pharmacological characterization of five new analogues, branched and unbranched 4-alkyl-HIBO analogues, have been carried out. The present compounds are all weak antagonists at Group I mGluRs (mGluR1 and 5) presenting only small differences in potencies (Ki values ranging from 89 to 670 microM). Affinities were studied at native and cloned iGluRs, and the compounds described show preference for the AMPA receptor subtypes GluR1 and 2 over GluR3 and 4. However, compared to previous 4-alkyl-HIBO analogues, these compounds show a remarkably high affinity for the Kain preferring subtype GluR5. The observed GluR5 affinities were either similar or higher compared to their GluR1 and 2 affinity. Isopropyl-HIBO showed the highest affinity for GluR5 (Ki=0.16 microM), and represents a unique compound with high affinity towards the three subtypes GluR1, 2 and 5. In general, these compounds represent new selectivity profiles compared to previously reported Glu receptor analogues.

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

PubMed Central

Nitsche, Andreas; Kurth, Andreas; Dunkhorst, Anna; Pänke, Oliver; Sielaff, Hendrik; Junge, Wolfgang; Muth, Doreen; Scheller, Frieder; Stöcklein, Walter; Dahmen, Claudia; Pauli, Georg; Kage, Andreas

2007-01-01

Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. PMID:17697378

High-affinity PD-1 molecules deliver improved interaction with PD-L1 and PD-L2.

PubMed

Li, Yanyan; Liang, Zhaoduan; Tian, Ye; Cai, Wenxuan; Weng, Zhiming; Chen, Lin; Zhang, Huanling; Bao, Yifeng; Zheng, Hongjun; Zeng, Sihai; Bei, Chunhua; Li, Yi

2018-06-11

The inhibitory checkpoint molecule programmed death (PD)-1 plays a vital role in maintaining immune homeostasis upon binding to its ligands, PD-L1 and PD-L2. Several recent studies have demonstrated that soluble PD-1 (sPD-1) can block the interaction between membrane PD-1 and PD-L1 to enhance the anti-tumor capability of T cells. However, the affinity of natural sPD-1 binding to PD-L1 is too low to permit therapeutic applications. Here a PD-1 variant with ~3,000-fold and ~70-fold affinity increase to bind PD-L1 and PD-L2, respectively, was generated through directed molecular evolution and phage display technology. Structural analysis showed that mutations at amino acid positions 124 and 132 of PD-1 played major roles in enhancing the affinity of PD-1 binding to its ligands. The high-affinity PD-1 mutant could compete with the binding of antibodies specific to PD-L1 or PD-L2 on cancer cells or dendritic cells (DCs), and it could enhance the proliferation and IFN-γ release of activated lymphocytes. These features potentially qualify the high-affinity PD-1 variant as a unique candidate for the development of a new class of PD-1 immune checkpoint blockade therapeutics. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Diagnostic approach to hemoglobins with high oxygen affinity: experience from France and Belgium and review of the literature.

PubMed

Orvain, Corentin; Joly, Philippe; Pissard, Serge; Badiou, Stéphanie; Badens, Catherine; Bonello-Palot, Nathalie; Couque, Nathalie; Gulbis, Béatrice; Aguilar-Martinez, Patricia

2017-02-01

Congenital causes of erythrocytosis are now more easily identified due to the improvement of the molecular characterization of many of them. Among these causes, hemoglobins with high oxygen affinity take a large place. The aim of this work was to reevaluate the diagnostic approach of these disorders. To assess the current practices, we sent a questionnaire to the expert laboratories in the diagnosis of hemoglobinopathies in France and Belgium. In parallel, we gathered the methods used for the diagnosis of the hemoglobins with high oxygen affinity indexed in the international database HbVar. Even though they remain a rare cause of erythrocytosis (1 to 5 positive diagnosis every year in each of the questioned specialized laboratories), hemoglobins with high oxygen affinity are increasingly suspected by clinicians. Phenotypic assessment by laboratory techniques remains a main step in their diagnosis as it enables the finding of 93% of them in the questioned laboratories (28 of the 30 variants diagnosed during the last 5 years). Among the 96 hemoglobin variants with high oxygen affinity indexed in the international database, 87% could be diagnosed with phenotypic techniques. A direct measure of the p50 with the Hemox-Analyzer is included in the diagnostic approach of half of the laboratories only, because of the poor availability of this apparatus. Comparatively, the estimation of p50 by blood gas analyzers on venous blood is a much more convenient and attractive method but due to the lack of proof as to its effectiveness in the diagnosis of hemoglobins with high oxygen affinity, it requires further investigations. Beta- and alphaglobin genes analysis by molecular biology techniques is essential as it either allows a quick and definite identification of the variant or definitely excludes the diagnosis. It is thus systematically performed as a first or second step method, according to the laboratory practice.

Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis.

PubMed

Pan, Yuchen; Sackmann, Eric K; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S; Herr, Amy E

2016-12-23

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality - the binding affinity - is quantified through the dissociation constant (K D ) of each recombinant antibody and the target antigen. To characterize the K D of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The K D for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization.

Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis

PubMed Central

Pan, Yuchen; Sackmann, Eric K.; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S.; Herr, Amy E.

2016-01-01

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality – the binding affinity – is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization. PMID:28008969

Bimodal imprint chips for peptide screening: integration of high-throughput sequencing by MS and affinity analyses by surface plasmon resonance imaging.

PubMed

Wang, Weizhi; Li, Menglin; Wei, Zewen; Wang, Zihua; Bu, Xiangli; Lai, Wenjia; Yang, Shu; Gong, He; Zheng, Hui; Wang, Yuqiao; Liu, Ying; Li, Qin; Fang, Qiaojun; Hu, Zhiyuan

2014-04-15

Peptide probes and drugs have widespread applications in disease diagnostics and therapy. The demand for peptides ligands with high affinity and high specificity toward various targets has surged in the biomedical field in recent years. The traditional peptide screening procedure involves selection, sequencing, and characterization steps, and each step is manual and tedious. Herein, we developed a bimodal imprint microarray system to embrace the whole peptide screening process. Silver-sputtered silicon chip fabricated with microwell array can trap and pattern the candidate peptide beads in a one-well-one-bead manner. Peptides on beads were photocleaved in situ. A portion of the peptide in each well was transferred to a gold-coated chip to print the peptide array for high-throughput affinity analyses by surface plasmon resonance imaging (SPRi), and the peptide left in the silver-sputtered chip was ready for in situ single bead sequencing by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Using the bimodal imprint chip system, affinity peptides toward AHA were efficiently screened out from the 7 × 10(4) peptide library. The method provides a solution for high efficiency peptide screening.

A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches

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

Murtaugh, Megan L.; Fanning, Sean W.; Sharma, Tressa M.

2012-09-05

There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein-protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton-linkage dictate that the presence of multiple ionizable groups, which undergo a pK{sub a} change on protein binding, are necessary to result in highly pH-dependent binding. To test this hypothesis, a novelmore » combinatorial histidine library was developed where every possible combination of histidine and wild-type residue is sampled throughout the interface of a model anti-RNase A single domain VHH antibody. Antibodies were coselected for high-affinity binding and pH-sensitivity using an in vitro, dual-function selection strategy. The resulting antibodies retained near wild-type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine 'hot-spots,' which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH-sensitive protein affinity reagents for a number of different applications.« less

Na⁺-Dependent High-Affinity Nitrate, Phosphate and Amino Acids Transport in Leaf Cells of the Seagrass Posidonia oceanica (L.) Delile.

PubMed

Rubio, Lourdes; García-Pérez, Delia; García-Sánchez, María Jesús; Fernández, José A

2018-05-24

Posidonia oceanica (L.) Delile is a seagrass, the only group of vascular plants to colonize the marine environment. Seawater is an extreme yet stable environment characterized by high salinity, alkaline pH and low availability of essential nutrients, such as nitrate and phosphate. Classical depletion experiments, membrane potential and cytosolic sodium measurements were used to characterize the high-affinity NO₃ - , Pi and amino acids uptake mechanisms in this species. Net uptake rates of both NO₃ - and Pi were reduced by more than 70% in the absence of Na⁺. Micromolar concentrations of NO₃ - depolarized mesophyll leaf cells plasma membrane. Depolarizations showed saturation kinetics ( Km = 8.7 ± 1 μM NO₃ - ), which were not observed in the absence of Na⁺. NO₃ - induced depolarizations at increasing Na⁺ also showed saturation kinetics ( Km = 7.2 ± 2 mM Na⁺). Cytosolic Na⁺ measured in P. oceanica leaf cells (17 ± 2 mM Na⁺) increased by 0.4 ± 0.2 mM Na⁺ upon the addition of 100 μM NO₃ - . Na⁺-dependence was also observed for high-affinity l-ala and l-cys uptake and high-affinity Pi transport. All together, these results strongly suggest that NO₃ - , amino acids and Pi uptake in P. oceanica leaf cells are mediated by high-affinity Na⁺-dependent transport systems. This mechanism seems to be a key step in the process of adaptation of seagrasses to the marine environment.

Synthesis and binding affinity of new 1,4-disubstituted triazoles as potential dopamine D(3) receptor ligands.

PubMed

Insua, Ignacio; Alvarado, Mario; Masaguer, Christian F; Iglesias, Alba; Brea, José; Loza, María I; Carro, Laura

2013-10-15

A series of new 1,4-disubstituted triazoles was prepared from appropriate arylacetylenes and aminoalkylazides using click chemistry methodology. These compounds were evaluated as potential ligands on several subtypes of dopamine receptors in in vitro competition assays, showing high affinity for dopamine D3 receptors, lower affinity for D2 and D4, and no affinity for the D1 receptors. Compound 18 displayed the highest affinity at the D3 receptor with a Ki value of 2.7 nM, selectivity over D2 (70-fold) and D4 (200-fold), and behaviour as a competitive antagonist in the low nanomolar range. Copyright © 2013 Elsevier Ltd. All rights reserved.

Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

PubMed Central

Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

2016-01-01

Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

[125I]2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a high-affinity radioligand selective for I1 imidazoline receptors.

PubMed

Greney, Hugues; Urosevic, Dragan; Schann, Stephan; Dupuy, Laurence; Bruban, Véronique; Ehrhardt, Jean-Daniel; Bousquet, Pascal; Dontenwill, Monique

2002-07-01

The I1 subtype of imidazoline receptors (I1R) is a plasma membrane protein that is involved in diverse physiological functions. Available radioligands used so far to characterize the I(1)R were able to bind with similar affinities to alpha2-adrenergic receptors (alpha2-ARs) and to I1R. This feature was a major drawback for an adequate characterization of this receptor subtype. New imidazoline analogs were therefore synthesized and the present study describes one of these compounds, 2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), which was of high affinity and selectivity for the I1R. LNP 911 was radioiodinated and its binding properties characterized in different membrane preparations. Saturation experiments with [125I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (K(D) = 1.4 nM; B(max) = 398 fmol/mg protein) with low nonspecific binding. [125I]LNP 911 specific binding was inhibited by various imidazolines and analogs but was insensitive to guanosine-5'-O-(3-thio)triphosphate. The rank order of potency of some competing ligands [LNP 911, PIC, rilmenidine, 4-chloro-2-(imidazolin-2-ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [125I]LNP 911 binding sites as I1R. However, other high-affinity I1R ligands (moxonidine, efaroxan, and benazoline) exhibited low affinities for these binding sites in standard binding assays. In contrast, when [125I]LNP 911 was preincubated at 4 degrees C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [125I]LNP 911 binding sites as on I1R defined with [125I]PIC. Moxonidine proved also able to accelerate the dissociation of [125I]LNP 911 from its binding sites. These results suggest the existence of an allosteric modulation at the level of the I1R, which seems to be corroborated by the dose-dependent enhancement by LNP 911 of the agonist effects on the adenylate cyclase pathway associated to I1R. Because [125I]LNP 911 was unable to bind to the I2 binding site and alpha2AR, our data indicate that [125I]LNP 911 is the first highly selective radioiodinated probe for I1R with a nanomolar affinity. This new tool should facilitate the molecular characterization of the I1 imidazoline receptor.

Structural basis for the differential effects of CaBP1 and calmodulin on Ca(V)1.2 calcium-dependent inactivation.

PubMed

Findeisen, Felix; Minor, Daniel L

2010-12-08

Calcium-binding protein 1 (CaBP1), a calmodulin (CaM) homolog, endows certain voltage-gated calcium channels (Ca(V)s) with unusual properties. CaBP1 inhibits Ca(V)1.2 calcium-dependent inactivation (CDI) and introduces calcium-dependent facilitation (CDF). Here, we show that the ability of CaBP1 to inhibit Ca(V)1.2 CDI and induce CDF arises from interaction between the CaBP1 N-lobe and interlobe linker residue Glu94. Unlike CaM, where functional EF hands are essential for channel modulation, CDI inhibition does not require functional CaBP1 EF hands. Furthermore, CaBP1-mediated CDF has different molecular requirements than CaM-mediated CDF. Overall, the data show that CaBP1 comprises two structural modules having separate functions: similar to CaM, the CaBP1 C-lobe serves as a high-affinity anchor that binds the Ca(V)1.2 IQ domain at a site that overlaps with the Ca²+/CaM C-lobe site, whereas the N-lobe/linker module houses the elements required for channel modulation. Discovery of this division provides the framework for understanding how CaBP1 regulates Ca(V)s. Copyright © 2010 Elsevier Ltd. All rights reserved.

Structural basis for the differential effects of CaBP1 and calmodulin on CaV1.2 calcium-dependent inactivation

PubMed Central

Findeisen, Felix; Minor, Daniel L.

2010-01-01

Calcium-binding protein 1 (CaBP1), a calmodulin (CaM) homolog, endows certain voltage-gated calcium channels (CaVs) with unusual properties. CaBP1 inhibits CaV1.2 calcium-dependent inactivation (CDI) and introduces calcium-dependent facilitation (CDF). Here, we show that the ability of CaBP1 to inhibit CaV1.2 CDI and induce CDF arises from interaction between the CaBP1 N-lobe and interlobe linker residue Glu94. Unlike CaM, where functional EF hands are essential for channel modulation, CDI inhibition does not require functional CaBP1 EF-hands. Furthermore, CaBP1-mediated CDF has different molecular requirements than CaM-mediated CDF. Overall, the data show that CaBP1 comprises two structural modules having separate functions: similar to CaM, the CaBP1 C-lobe serves as a high-affinity anchor that binds the CaV1.2 IQ domain at a site that overlaps with the Ca2+/CaM C-lobe site, whereas the N-lobe/linker module houses the elements required for channel modulation. Discovery of this division provides the framework for understanding how CaBP1 regulates CaVs. PMID:21134641

Structural basis for highly effective HIV-1 neutralization by CD4-mimetic miniproteins revealed by 1.5 Å co-crystal structure of gp120 and M48U1

PubMed Central

Acharya, Priyamvada; Luongo, Timothy; Louder, Mark K.; McKee, Krisha; Yang, Yongping; Kwon, Young Do; Mascola, John R.; Kessler, Pascal; Martin, Loïc; Kwong, Peter D.

2014-01-01

The interface between HIV-1 gp120 envelope glycoprotein and CD4 receptor contains an unusual interfacial cavity, the “Phe43 cavity”, which miniprotein mimetics of CD4 with non-natural extensions can potentially utilize to enhance their neutralization of HIV-1. Here we report co-crystal structures of HIV-1 gp120 with miniproteins M48U1 and M48U7, which insert cyclohexylmethoxy and 5-hydroxypentylmethoxy extensions, respectively, into the Phe43 cavity. Both inserts displayed flexibility and hydrophobic interactions, but the M48U1 insert showed better shape complementarity with the Phe43 cavity than the M48U7 insert. Subtle alteration in gp120 conformation played a substantial role in optimizing fit. With M48U1, these translated into a YU2-gp120 affinity of 0.015 nM and neutralization of all 180-circulating HIV-1 strains tested, except clade-A/E isolates with non-canonical Phe43 cavities. Ligand chemistry, shape complementary, surface burial, and gp120 conformation act in concert to modulate binding of ligands to the gp120-Phe43 cavity and, when optimized, can effect near pan-neutralization of HIV-1. PMID:23707685

Current at domain walls, roughly speaking: nanoscales studies of disorder roughening and conduction

NASA Astrophysics Data System (ADS)

Paruch, Patrycja

2013-03-01

Domain walls in (multi)ferroic materials are the thin elastic interfaces separating regions with different orientations of magnetisation, electric polarisation, or spontaneous strain. Understanding their behaviour, and controlling domain size and stability, is key for their integration into applications, while fundamentally, domain walls provide an excellent model system in which the rich physics of disordered elastic interfaces can be accesses. In addition, domain walls can present novel properties, quite different from those of their parent materials, making them potentially useful as active components in future nano-devices. Here, we present our atomic force microscopy studies of ferroelectric domain walls in epitaxial Pb(Zr0.2Ti0.8)O3 and BiFeO3 thin films, in which we use piezorespose force microscopy to show unusual domain wall roughening behaviour, with very localised disorder regions in the sample leading to a complex, multi-affine scaling of the domain wall shape. We also show the effects of temperature, environmental conditions, and defects on switching dynamics and domain wall roughness. We combine these observations with parallel conductive-tip atomic force microscopy current measurements, which also show highly localised variations in conduction, and highlight the key role played by oxygen vacancies in the observed domain wall conduction.

Differential affinities of molindone, metoclopramide and domperidone for classes of [3H]spiroperidol binding sites in rat striatum: evidence for pharmacologically distinct classes of receptors.

PubMed

Rosenfeld, M R; Dvorkin, B; Klein, P N; Makman, M H

1982-03-04

Rat striatum contains two populations of dopaminergic [3H]spiroperidol binding sites. The two populations are similar in their affinities for chlorpromazine and dopamine. Only one population, that with a somewhat higher affinity for spiroperidol itself, exhibits high affinity for the selective D2 antagonists molindone, metoclopramide and domperidone. Hence, this population may represent D2 receptor sites. The other larger population may represent either a separate class of receptor sites or a different form of D2 receptor sites.

A Virtual Screening Approach for the Identification of High Affinity Small Molecules Targeting BCR-ABL1 Inhibitors for the Treatment of Chronic Myeloid Leukemia.

PubMed

Sharda, Saphy; Sarmandal, Palash; Cherukommu, Shirisha; Dindhoria, Kiran; Yadav, Manisha; Bandaru, Srinivas; Sharma, Anudeep; Sakhi, Aditi; Vyas, Tanmay; Hussain, Tajamul; Nayarisseri, Anuraj; Singh, Sanjeev Kumar

2017-01-01

CML originates due to reciprocal translocation in Philadelphia chromosome leading to the formation of fusion product BCR-ABL which constitutively activates tyrosine kinase signaling pathways eventually leading to abnormal proliferation of granulocytic cells. As a therapeutic strategy, BCR-ABL inhibitors have been clinically approved which terminates its phosphorylation activity and retards cancer progression. However, a number of patients develop resistance to inhibitors which demand for the discovery of new inhibitors. Given the drawbacks of present inhibitors, by high throughput virtual screening approaches, present study pursues to identify high affinity compounds targeting BCR-ABL1 anticipated to have safer pharmacological profiles. Five established BCR-ABL inhibitors formed the query compounds for identification of structurally similar compounds by Tanimoto coefficient based linear fingerprint search with a threshold of 95% against PubChemdatabase. Assisted by MolDock algorithm all compounds were docked against BCR-ABL protein in order to retrieve high affinity compounds. The parents and similars were further tested for their ADMET propertiesand bioactivity. Rebastinib formed higher affinity inhibitor than rest of the four established compound investigated in the study. Interestingly, Rebastinib similar compound with Pubchem ID: 67254402 was also shown to have highest affinity than other similars including the similars of respective five parents. In terms of ADMET properties Pubchem ID: 67254402 had appreciable ADMET profile and bioactivity. However, Rebastinib still stood as the best inhibitor in terms of binding affinity and ADMET properties than Pubchem ID: 67254402. Nevertheless, owing to the similar pharmacological properties with Rebastinib, Pubchem ID: 67254402 can be expected to form potential BCR-ABL inhibitor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

PubMed Central

Honey, Denise M.; Best, Annie; Qiu, Huawei

2018-01-01

ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

Active site residues critical for flavin binding and 5,6-dimethylbenzimidazole biosynthesis in the flavin destructase enzyme BluB.

PubMed

Yu, Ta-Yi; Mok, Kenny C; Kennedy, Kristopher J; Valton, Julien; Anderson, Karen S; Walker, Graham C; Taga, Michiko E

2012-06-01

The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12). Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB. Copyright © 2012 The Protein Society.

Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution.

PubMed

Ahmad, Kareem M; Xiao, Yi; Soh, H Tom

2012-12-01

Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K(d)) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

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

Wang, Deng-Liang; Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou; Song, Yan-Ling

2014-10-31

Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the idealmore » antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.« less

Cholinergic nicotinic and muscarinic receptors in dementia of Alzheimer, Parkinson and Lewy body types.

PubMed

Perry, E K; Smith, C J; Court, J A; Perry, R H

1990-01-01

Cholinergic nicotinic and muscarinic receptor binding were measured in post mortem human brain tissue, using low (nM) concentrations of (3H)-nicotine to detect predominately the high affinity nicotinic site and (3H)-N-methylscopolamine in the presence and absence of 3 x 10(-4) M carbachol to measure both the low and high affinity agonist subtypes of the muscarinic receptor group. Consistent with most previous reports, the nicotinic but not muscarinic binding was reduced in the different forms of dementia associated with cortical cholinergic deficits, including Alzheimer's and Parkinson's disease, senile dementia of Lewy body type (SDLT) and Down's syndrome (over 50 years). Analysis of (3H)-nicotine binding displaced by a range of carbachol concentrations (10(-9)-10(-3) M) indicated 2 binding sites for nicotine and that the high affinity rather than low affinity site was reduced in Alzheimer's disease. In all 3 cortical areas investigated (temporal, parietal and occipital) there were increases in the low affinity muscarinic site in Parkinson's disease and SDLT but not Alzheimer's disease or middle-aged Down's syndrome. This observation raised the question of whether the presence of neurofibrillary tangles (evident in the latter but not former 2 disorders) is incompatible with denervation-induced muscarinic supersensitivity in cholinoceptive neurons which include cortical pyramids generally affeted by tangle formation.

Impaired activation of adenylyl cyclase in lung of the Basenji-greyhound model of airway hyperresponsiveness: decreased numbers of high affinity beta-adrenoceptors.

PubMed Central

Emala, C. W.; Aryana, A.; Hirshman, C. A.

1996-01-01

1. To evaluate mechanisms involved in the impaired beta-adrenoceptor stimulation of adenylyl cyclase in tissues from the Basenji-greyhound (BG) dog model of airway hyperresponsiveness, we compared agonist and antagonist binding affinity of beta-adrenoceptors, beta-adrenoceptor subtypes, percentage of beta-adrenoceptors sequestered, and coupling of the beta-adrenoceptor to Gs alpha in lung membranes from BG and control mongrel dogs. We found that lung membranes from the BG dog had higher total numbers of beta-adrenoceptors with a greater percentage of receptors of the beta 2 subtype as compared to mongrel lung membranes. 2. Agonist and antagonist binding affinity and the percentage of beta-adrenoceptors sequestered were not different in BG and mongrel dog lung membranes. However, the percentage of beta-adrenoceptors in the high affinity state for agonist was decreased in BG lung membranes suggesting an uncoupling of the receptor from Gs alpha. 3. Impaired coupling between the beta-adrenoceptor and G protein documented by the decreased numbers of beta-adrenoceptors in the high affinity state in BG lung membranes, is a plausible explanation for the reduced stimulation of adenylyl cyclase and the resultant reduction in airway smooth muscle relaxation in this model. PMID:8864536

SOLID PHASE MICROEXTRACTION SAMPLING OF HIGH EXPLOSIVE RESIDUES IN THE PRESENCE OF RADIONUCLIDES AND RADIONUCLIDE SURROGATE METALS

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

Duff, M; S Crump, S; Robert02 Ray, R

2007-04-13

The Federal Bureau of Investigation (FBI) Laboratory currently does not have on site facilities for handling radioactive evidentiary materials and there are no established FBI methods or procedures for decontaminating high explosive (HE) evidence while maintaining evidentiary value. One experimental method for the isolation of HE residue involves using solid phase microextraction or SPME fibers to remove residue of interest. Due to their high affinity for organics, SPME fibers should have little affinity for most metals. However, no studies have measured the affinity of radionuclides for SPME fibers. The focus of this research was to examine the affinity of dissolvedmore » radionuclide ({sup 239/240}Pu, {sup 238}U, {sup 237}Np, {sup 85}Sr, {sup 133}Ba, {sup 137}Cs, {sup 60}Co and {sup 226}Ra) and stable radionuclide surrogate metals (Sr, Co, Ir, Re, Ni, Ba, Cs, Nb, Zr, Ru, and Nd) for SPME fibers at the exposure conditions that favor the uptake of HE residues. Our results from radiochemical and mass spectrometric analyses indicate these metals have little measurable affinity for these SPME fibers during conditions that are conducive to HE residue uptake with subsequent analysis by liquid or gas phase chromatography with mass spectrometric detection.« less

Structure-kinetic relationships--an overlooked parameter in hit-to-lead optimization: a case of cyclopentylamines as chemokine receptor 2 antagonists.

PubMed

Vilums, Maris; Zweemer, Annelien J M; Yu, Zhiyi; de Vries, Henk; Hillger, Julia M; Wapenaar, Hannah; Bollen, Ilse A E; Barmare, Farhana; Gross, Raymond; Clemens, Jeremy; Krenitsky, Paul; Brussee, Johannes; Stamos, Dean; Saunders, John; Heitman, Laura H; Ijzerman, Adriaan P

2013-10-10

Preclinical models of inflammatory diseases (e.g., neuropathic pain, rheumatoid arthritis, and multiple sclerosis) have pointed to a critical role of the chemokine receptor 2 (CCR2) and chemokine ligand 2 (CCL2). However, one of the biggest problems of high-affinity inhibitors of CCR2 is their lack of efficacy in clinical trials. We report a new approach for the design of high-affinity and long-residence-time CCR2 antagonists. We developed a new competition association assay for CCR2, which allows us to investigate the relation of the structure of the ligand and its receptor residence time [i.e., structure-kinetic relationship (SKR)] next to a traditional structure-affinity relationship (SAR). By applying combined knowledge of SAR and SKR, we were able to re-evaluate the hit-to-lead process of cyclopentylamines as CCR2 antagonists. Affinity-based optimization yielded compound 1 with good binding (Ki = 6.8 nM) but very short residence time (2.4 min). However, when the optimization was also based on residence time, the hit-to-lead process yielded compound 22a, a new high-affinity CCR2 antagonist (3.6 nM), with a residence time of 135 min.

Selective high-affinity polydentate ligands and methods of making such

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

Denardo, Sally J.; Denardo, Gerald L.; Balhorn, Rodney L.

This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

Selective high-affinity polydentate ligands and methods of making such

DOEpatents

DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

2013-09-17

This invention provides polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each binds different regions on the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

Selective high affinity polydentate ligands and methods of making such

DOEpatents

DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

2010-02-16

This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

DNA aptamer affinity ligands for highly selective purification of human plasma-related proteins from multiple sources.

PubMed

Forier, Cynthia; Boschetti, Egisto; Ouhammouch, Mohamed; Cibiel, Agnès; Ducongé, Frédéric; Nogré, Michel; Tellier, Michel; Bataille, Damien; Bihoreau, Nicolas; Santambien, Patrick; Chtourou, Sami; Perret, Gérald

2017-03-17

Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Isolation and purification of glutathione S-transferases from Brachionus plicatilis and B. calyciflorus (Rotifera).

PubMed

Bowman, B P; Snell, T W; Cochrane, B J

1990-01-01

1. The enzyme glutathione S-transferase (GST), a critical element in xenobiotic metabolism, was isolated from the marine rotifer Brachionus plicatilis and its freshwater congener B. calyciflorus. 2. In B. plicatilis, GST comprised 4.2% of cytosolic protein and was present as three separate isozymes with mol. wts 30,000, 31,400 and 33,700. Specific activity of crude homogenates was 56 nmol min-1 mg-1 protein, while that of affinity chromatography purified GST was 1850. 3. In B. calyciflorus, GST was present as two isozymes with mol. wts of 26,300 and 28,500, representing 1.0% of cytosolic protein. Crude GST specific activity was 1750 nmol min-1 mg-1 protein and purified was 72,400. 4. Rotifer GSTs are unusual because they are monomers whereas all other animals thus far investigated posses dimeric GSTs.

Rifampin phosphotransferase is an unusual antibiotic resistance kinase

PubMed Central

Stogios, Peter J.; Cox, Georgina; Spanogiannopoulos, Peter; Pillon, Monica C.; Waglechner, Nicholas; Skarina, Tatiana; Koteva, Kalinka; Guarné, Alba; Savchenko, Alexei; Wright, Gerard D.

2016-01-01

Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal structure of RPH from Listeria monocytogenes (RPH-Lm), which reveals that the enzyme is comprised of three domains: two substrate-binding domains (ATP-grasp and RIF-binding domains); and a smaller phosphate-carrying His swivel domain. Using solution small-angle X-ray scattering and mutagenesis, we reveal a mechanism where the swivel domain transits between the spatially distinct substrate-binding sites during catalysis. RPHs are previously uncharacterized dikinases that are widespread in environmental and pathogenic bacteria. These enzymes are members of a large unexplored group of bacterial enzymes with substrate affinities that have yet to be fully explored. Such an enzymatically complex mechanism of antibiotic resistance augments the spectrum of strategies used by bacteria to evade antimicrobial compounds. PMID:27103605

On the pterosaur remains from the Río Belgrano Formation (Barremian), Patagonian Andes of Argentina.

PubMed

Kellner, Alexander W A; Aguirre-Urreta, María B; Ramos, Victor A

2003-12-01

Pterosaur remains from the Río Belgrano Formation, Santa Cruz Province, Argentina, were found close to the Estancia Río Roble, along with several ammonoids that indicate a Barremian age for those strata. The specimens (MACN-SC 3617) consist of one ulna and one element tentatively identified as a portion of a wing metacarpal. The ulna shows morphological affinities with the Pteranodontoidea (sensu Kellner 1996), particularly with the members of the Anhangueridae by having a well developed ventral crest close to the proximal articulation, and is tentatively referred to this pterosaur clade. The oldest record of the Anhangueridae, previously limited to the Aptian/Albian, is therefore extended to the Barremian. The Argentinean material is preserved in three dimensions, an unusual condition for pterosaur fossils from that country, indicating that the site situated near the Estancia Río Roble has a great potential for new and well preserved specimens.

Solution to the phylogenetic enigma of Tetraplatia, a worm-shaped cnidarian

PubMed Central

Collins, Allen G; Bentlage, Bastian; Matsumoto, George I; Haddock, Steven H.D; Osborn, Karen J; Schierwater, Bernd

2005-01-01

Tetraplatia is a genus containing two species of pelagic cnidarians of curious morphology. Their vermiform shape and four swimming flaps are difficult to relate to the features of other cnidarians, thus obscuring their phylogenetic affinities. Since their discovery in the mid-1800s, a number of prominent cnidarian workers have weighed in on this conundrum, some arguing that they are aberrant hydrozoans and others concluding that they are unusual scyphozoans. Current taxonomic practice conforms to the latter view. However, data presented here from the large and small subunits of the nuclear ribosome leave little doubt that Tetraplatia is in fact a hydrozoan genus. Indeed, its precise phylogenetic position is within Narcomedusae, as some authors had previously deduced based on structural characters. The distinctive body plan of Tetraplatia is remarkable because it appears to have a recent origin, in contrast to the prevailing pattern of metazoan history. PMID:17148343

Peroxisome protein import: a complex journey.

PubMed

Baker, Alison; Lanyon-Hogg, Thomas; Warriner, Stuart L

2016-06-15

The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor-cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. © 2016 The Author(s).

Two competing ionization processes in ESI-MS analysis of N-(1,3-diphenylallyl)benzenamines: formation of the unusual [M-H]+ ion versus the regular [M+H]+ ion.

PubMed

Fang, Liwen; Dong, Cheng; Guo, Cheng; Xu, Jianxing; Liu, Qiaoling; Qu, Zhirong; Jiang, Kezhi

2018-06-01

A series of N-(1,3-diphenylallyl)benzenamine derivatives (M) were investigated by electrospray ionization mass spectrometry in the positive-ion mode. Both the anomalous [M-H] + and the regular [M+H] + were observed in the ESI mass spectra. The occurrence of [M-H] + has been supported by accurate mass spectrometry, liquid chromatography mass spectrometry, and tandem mass spectrometry analysis. Calculation results indicated that formation of [M-H] + is attributed to the ion-molecule reaction of M with the protonated ESI solvent molecule (e.g. CH 3 OH 2 + ) via hydride abstraction from a tertiary C sp3 -H. The competing ionization processes leading to [M-H] + or [M+H] + were significantly affected by the concentration of formic acid in the electrospray ionization solvent and the proton affinity of the N atom.

Isolation of high-affinity, neutralizing anti-idiotype antibodies by phage and ribosome display for application in immunogenicity and pharmacokinetic analyses.

PubMed

Chin, Stacey E; Ferraro, Franco; Groves, Maria; Liang, Meina; Vaughan, Tristan J; Dobson, Claire L

2015-01-01

Anti-idiotype antibodies against a therapeutic antibody are key reagents for the development of immunogenicity and pharmacokinetic (PK) assays during pre-clinical and clinical development. Here we have used a combination of phage and ribosome display to isolate a panel of monoclonal anti-idiotype antibodies with sub-nanomolar affinity and high specificity to a human anti-IgE monoclonal antibody. Anti-idiotype antibodies were enriched from scFv libraries using phage display, and a biochemical epitope competition assay was used to identify anti-idiotypes which neutralized IgE binding, which was essential for the intended use of the anti-idiotypes as positive controls in neutralizing anti-drug antibody (Nab) assays. The phage display-derived anti-idiotype antibodies were rapidly affinity-matured using a random point mutagenesis approach in ribosome display. Ten anti-idiotype antibodies with improved neutralizing activity relative to the parent antibodies displayed sub-nanomolar affinity for the anti-IgE antibody, representing up to 20-fold improvements in affinity from just two rounds of affinity-based selection. The optimized anti-idiotype antibodies retained the specificity of the parent antibodies, and importantly, were fit for purpose for use in PK and anti-drug antibody (ADA) assays. The approach we describe here for generation of anti-idiotype antibodies to an anti-IgE antibody is generically applicable for the rapid isolation and affinity maturation of anti-idiotype antibodies to any antibody-based drug candidate. Copyright © 2014 Elsevier B.V. All rights reserved.

Physiological characterization of putative high-affinity glucose transport protein Hxt2 of Saccharomyces cerevisiae by use of anti-synthetic peptide antibodies.

PubMed Central

Wendell, D L; Bisson, L F

1993-01-01

Characterization and quantification of the Hxt2 (hexose transport) protein of Saccharomyces cerevisiae indicate that it is one of a set of differentially expressed high-affinity glucose transporters. The protein product of the HXT2 gene was specifically detected by antibodies raised against a synthetic peptide encompassing the 13 carboxyl-terminal amino acids predicted by the HXT2 gene sequence. Hxt2 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band or closely spaced doublet with an average M(r) of 47,000. Hxt2 cofractionated with the plasma membrane ATPase, Pma1, indicating that it is a plasma membrane protein. Hxt2 was not solubilized by high pH or urea but was solublized by detergents, which is characteristic of an integral membrane protein. Expression of the Hxt2 protein was measured under two different conditions that produce expression of high-affinity glucose transport: a medium shift from a high (2.0%) to a low (0.05%) glucose concentration (referred to below as high and low glucose) and growth from high to low glucose. Hxt2 as measured by immunoblotting increased 20-fold upon a shift from high-glucose to low-glucose medium, and the high-affinity glucose transport expressed had a strong HXT2-dependent component. Surprisingly, Hxt2 was not detectable when S. cerevisiae growing in high glucose approached glucose exhaustion, and the high-affinity glucose transport expressed under these conditions did not have an HXT2-dependent component. The role of Hxt2 in growth during aerobic batch culture in low-glucose medium was examined. An hxt2 null mutant grew and consumed glucose significantly more slowly than the wild type, and this phenotype correlated directly with appearance of the Hxt2 protein. Images PMID:8244939

Development of Single-Stranded DNA Aptamers for Specific Bisphenol A Detection

PubMed Central

Jo, Minjoung; Ahn, Ji-Young; Lee, Joohyung; Lee, Seram; Hong, Sun Woo; Yoo, Jae-Wook; Kang, Jeehye; Dua, Pooja

2011-01-01

The development of reagents with high affinity and specificity to small molecules is crucial for the high-throughput detection of chemical compounds, such as toxicants or pollutants. Aptamers are short and single-stranded (ss) oligonucleotides able to recognize target molecules with high affinity. Here, we report the selection of ssDNA aptamers that bind to Bisphenol A (BPA), an environmental hormone. Using SELEX process, we isolated high affinity aptamers to BPA from a 1015 random library of 60 mer ssDNAs. The selected aptamers bound specifically to BPA, but not to structurally similar molecules, such as Bisphenol B with one methyl group difference, or 4,4′-Bisphenol with 2 methyl groups difference. Using these aptamers, we developed an aptamer-based sol–gel biochip and detected BPA dissolved in water. This novel BPA aptamer-based detection can be further applied to the universal and high-specificity detection of small molecules. PMID:21413891

Fatal fat embolism in isolated vertebral compression fracture

PubMed Central

Saldanha, Vilas; Balasubramanian, Manjula; Handal, John

2010-01-01

Fat embolism after long bone and pelvic fractures as well as orthopedic interventions is a well-documented phenomenon, but it is highly unusual after isolated vertebral fractures. We report a case of fatal fat embolism in a 78-year-old man after an isolated vertebral compression fracture with no related orthopedic intervention. A high index of suspicion is necessary for early diagnosis and successfully treating this unusual complication. PMID:20229119

Fatal fat embolism in isolated vertebral compression fracture.

PubMed

Lastra, Ricardo R; Saldanha, Vilas; Balasubramanian, Manjula; Handal, John

2010-07-01

Fat embolism after long bone and pelvic fractures as well as orthopedic interventions is a well-documented phenomenon, but it is highly unusual after isolated vertebral fractures. We report a case of fatal fat embolism in a 78-year-old man after an isolated vertebral compression fracture with no related orthopedic intervention. A high index of suspicion is necessary for early diagnosis and successfully treating this unusual complication.

Multilayer affinity adsorption of albumin on polymer brushes modified membranes in a continuous-flow system.

PubMed

Hu, Meng-Xin; Li, Xiang; Li, Ji-Nian; Huang, Jing-Jing; Ren, Ge-Rui

2018-02-23

Polymer brushes modified surfaces have been widely used for protein immobilization and isolation. Modification of membranes with polymer brushes increases the surface concentration of affinity ligands used for protein binding. Albumin is one of the transporting proteins and shows a high affinity to bile acids. In this work, the modified membranes with cholic acid-containing polymer brushes can be facilely prepared by the immobilization of cholic acid on the poly(2-hydroxyethyl methacrylate) grafted microporous polypropylene membranes (MPPMs) for affinity adsorption of albumin. ATR/FT-IR and X-ray photoelectron spectroscopy were used to characterize the chemical composition of the modified membranes. Water contact angle measurements were used to analyze the hydrophilic/hydrophobic properties of the membrane surface. The modified MPPMs show a high affinity to albumin and have little non-specific adsorption of hemoglobin. The dynamic binding capacity of albumin in the continous-flow system increases with the cycle number and feed rate as the binding degree of cholic acid is moderate. The highest binding capacity of affinity membranes is about 52.49 g/m 2 membrane, which is about 24 times more than the monolayer binding capacity. These results reveal proteins could be captured in multilayers by the polymer brushes containing affinity ligands similar to the polymer brushes containing ion-exchange groups, which open up the potential of the polymer brushes containing affinity ligands in protein or another components separation. And the cholic acid containing polymer brushes modified membranes has the promising potential for albumin separation and purification rapidly from serum or fermented solution in medical diagnosis and bioseparation. Copyright © 2018 Elsevier B.V. All rights reserved.

The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

PubMed Central

Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

2011-01-01

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

Muscarinic receptor occupation and receptor activation in the guinea-pig ileum by some acetamides related to oxotremorine.

PubMed Central

Ringdahl, B.

1984-01-01

The dissociation constants (KD values) and relative efficacies of seven acetamide analogues of oxotremorine, including two enantiomeric pairs, at muscarinic receptors in the guinea-pig isolated ileum were determined. The method used involved analysis of dose-response data before and after fractional inactivation of receptors with propylbenzilylcholine mustard. All of the compounds studied had lower affinities than oxotremorine, but some had substantially higher relative efficacies. Replacement of the pyrrolidine ring of N-methyl-N-(4- pyrrolidino -2- butynyl )acetamide(I), the parent compound in the series, by a dimethylamino or a trimethylammonium group decreased the affinity 32 and 4.5 fold, respectively, whereas the relative efficacy increased 5.7-8.3 times. There was no correlation between relative efficacies and affinities of the compounds. The structural requirements for high affinity and high efficacy appeared to be quite different. PMID:6733356

Quantum-chemical calculations and IR spectra of the (F2)MF2 molecules (M = B, Al, Ga, In, Tl) in solid matrices: a new class of very high electron affinity neutral molecules.

PubMed

Wang, Xuefeng; Andrews, Lester

2011-03-23

Electron-deficient group 13 metals react with F(2) to give the compounds MF(2) (M = B, Al, Ga, In, Tl), which combine with F(2) to form a new class of very high electron affinity neutral molecules, (F(2))MF(2), in solid argon and neon. These (F(2))MF(2) fluorine metal difluoride molecules were identified through matrix IR spectra containing new antisymmetric and symmetric M-F stretching modes. The assignments were confirmed through close comparisons with frequency calculations using DFT methods, which were calibrated against the MF(3) molecules observed in all of the spectra. Electron affinities calculated at the CCSD(T) level fall between 7.0 and 7.8 eV, which are in the range of the highest known electron affinities.

3-Arylpiperazinylethyl-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione derivatives as novel, high-affinity and selective alpha(1)-adrenoceptor ligands.

PubMed

Pittalà, Valeria; Romeo, Giuseppe; Salerno, Loredana; Siracusa, Maria Angela; Modica, Maria; Materia, Luisa; Mereghetti, Ilario; Cagnotto, Alfredo; Mennini, Tiziana; Marucci, Gabriella; Angeli, Piero; Russo, Filippo

2006-01-01

The discovery of a new series of selective and high-affinity alpha(1)-adrenoceptor (alpha(1)-AR) ligands, characterized by a 1H-pyrrolo[2,3-d]-pyrimidine-2,4(3H,7H)-dione system, is described in this paper. Some synthesized compounds, including 20, 22, and 30, displayed affinity in the nanomolar range for alpha(1)-ARs and substantial selectivity with respect to 5-HT(1A) and dopaminergic D(1) and D(2) receptors. Functional assays, performed on selected derivatives, showed antagonistic properties.

ALH85085: a unique volatile-poor carbonaceous chondrite with possible implications for nebular fractionation processes

USGS Publications Warehouse

Grossman, J.N.; Rubin, A.E.; MacPherson, G.J.

1988-01-01

Allan Hills 85085 is a unique chondrite with affinities to the Al Rais-Renazzo clan of carbonaceous chondrites. Its constituents are less than 50 ??m in mean size. Chondrules and microchondrules of all textures are present; nonporphyritic chondrules are unusually abundant. The mean compositions of porphyritic, nonporphyritic and barred olivine chondrules resemble those in ordinary chondrites except that they are depleted in volatile elements. Ca-, Al-rich inclusions are abundant and largely free of nebular alteration; they comprise types similar to those in CM and CO chondrites, as well as unique types. Calcium dialuminate occurs in several inclusions. Metal, silicate and sulfide compositions are close to those in CM-CO chondrites and Al Rais and Renazzo. C1-chondrite clasts and metal-rich "reduced" clasts are present, but opaque matrix is absent. Siderophile abundances in ALH85085 are extremely high (e.g., Fe Si = 1.7 ?? solar), and volatiles are depleted (e.g., Na Si = 0.25 ?? solar, S Si = 0.03 ?? solar). Nonvolatile lithophile abundances are similar to those in Al Rais, Renazzo, and CM and CO chondrites. ALH85085 agglomerated when temperatures in the nebula were near 1000 K, in the same region where Renazzo, Al Rais and the CI chondrites formed. Agglomeration of high-temperature material may thus be a mechanism by which the fractionation of refractory lithophiles occurred in the nebula. Chondrule formation must have occurred at high temperatures when clumps of precursors were small. After agglomeration, ALH85085 was annealed and lightly shocked. C1 and other clasts were subsequently incorporated during late-stage brecciation. ?? 1988.

High-resolution mapping of heterochromatin redistribution in a Drosophila position-effect variegation model.

PubMed

Vogel, Maartje J; Pagie, Ludo; Talhout, Wendy; Nieuwland, Marja; Kerkhoven, Ron M; van Steensel, Bas

2009-01-29

Position-effect variegation (PEV) is the stochastic transcriptional silencing of a gene positioned adjacent to heterochromatin. white-mottled X-chromosomal inversions in Drosophila are classic PEV models that show variegation of the eye color gene white due to its relocation next to pericentric heterochromatin. It has been suggested that in these models the spreading of heterochromatin across the rearrangement breakpoint causes the silencing of white. However, the extent of this spreading and the precise pattern of heterochromatin redistribution have remained unclear. To obtain insight into the mechanism of PEV, we constructed high-resolution binding maps of Heterochromatin Protein 1 (HP1) on white-mottled chromosomes. We find that HP1 invades euchromatin across the inversion breakpoints over approximately 175 kb and approximately 30 kb, causing de novo association of HP1 with 20 genes. However, HP1 binding levels in these regions show substantial local variation, and white is the most strongly bound gene. Remarkably, white is also the only gene that is detectably repressed by heterochromatin. Furthermore, we find that HP1 binding to the invaded region is particularly sensitive to the dosage of the histone methyltransferase Su(var)3-9, indicating that the de novo formed heterochromatin is less stable than naturally occurring constitutive heterochromatin. Our molecular maps demonstrate that heterochromatin can invade a normally euchromatic region, yet the strength of HP1 binding and effects on gene expression are highly dependent on local context. Our data suggest that the white gene has an unusual intrinsic affinity for heterochromatin, which may cause this gene to be more sensitive to PEV than most other genes.

Peri-implant and systemic effects of high-/low-affinity bisphosphonate-hydroxyapatite composite coatings in a rabbit model with peri-implant high bone turnover

PubMed Central

2012-01-01

Background Hydroxyapatite (HA) coatings composed with bisphosphonates (BPs) which have high mineral-binding affinities have been confirmed to successfully enhance implant stability. However, few previous studies focused on HA coatings composed with low-affinity BPs or on systemic effects of locally released BPs. Methods In this long-term study, we developed two kinds of BP-HA composite coatings using either high-affinity BP (alendronate, ALN) or low-affinity BP (risedronate, RIS). Thirty-six rabbits were divided into three groups according to different coating applications (group I: HA, group II: ALN-HA, and group III: RIS-HA). Implants were inserted into the proximal region of the medullary cavity of the left tibiay. At insertion, 2 × 108 wear particles were injected around implants to induce a peri-implant high bone turnover environment. Both local (left tibias) and systemic (right tibias and lumbar vertebrae) inhibitory effect on bone resorption were compared, including bone-implant integration, bone architecture, bone mineral density (BMD), implant stability, and serum levels of bone turnover markers. Results The results indicated that ALN-HA composite coating, which could induce higher bone-implant contact (BIC) ratio, bone mass augmentation, BMD, and implant stability in the peri-implant region, was more potent on peri-implant bone, while RIS-HA composite coating, which had significant systemic effect, was more potent on non-peri-implant bone, especially lumbar vertebrae. Conclusions It is instructive and meaningful to further clinical studies that we could choose different BP-HA composite coatings according to the patient’s condition. PMID:22686414

Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

PubMed Central

Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

2016-01-01

Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

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

Hall, Justin; Brault, Amy; Vincent, Fabien

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a highmore » affinity (K D = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.« less

The Mechanism of Interaction of Oximes with the Muscarinic-Cholinergic Complex in the Central Nervous System

DTIC Science & Technology

1983-11-03

ACh binding to the remaining sites. However, the affinity of oxotremorine to the high affinity agonist binding sites was reduced. The relative...when examined in the remaining sites in the washed membranes, were similar to those in control membranes. The affinity of the agonist oxotremorine ... oxotremorine was substituted for atropine. All determinations were carriid out in quadruplicate, each one varying by < 15%. Centrifugation assays

An unusual percutaneous transmitral commissurotomy: A collection of four rare occurrences!

PubMed

Shankarappa, Ravindranath K; Agrawal, Navin; Patra, Soumya; Karur, Satish; Nanjappa, Manjunath C

2013-09-01

We are presenting an interesting case of a 30-year-old patient taken for percutaneous transvenous mitral commissurotomy (PTMC) for severe rheumatic mitral stenosis in which there was a collection of four unusual occurrences during the course of a procedure. She had recurrent generalized tonic-clonic seizures immediately after femoral sheath insertion requiring the patient to be mechanically ventilated. Subsequently, the pressure tracings recorded with catheters in the aorta and the pulmonary artery showed transient unusually high supra-systemic pulmonary artery pressure. During inflation the Accura PTMC balloon which was used to dilate the mitral valve ruptured and the procedure subsequently had to be completed using another balloon catheter. During the procedure the presence of a distended stomach due to insufflations of air during positive pressure ventilation which subsided subsequently was another unusual documentation on fluoroscopy. The final outcome of the procedure was successful. This case presents an interesting collection of unusual occurrences during a PTMC procedure which started on an unusual note but ended on a successful one. Careful assessment and appropriate management of complications can lead to successful outcome of procedures as in our case.

Two classes of binding sites for [3H]substance P in rat cerebral cortex.

PubMed

Geraghty, D P; Burcher, E

1993-01-22

The binding characteristics of [3H]substance P ([3H]SP) were investigated in membranes prepared from rat cerebral cortex. Binding of [3H]SP reached equilibrium after 50 min at 25 degrees C and was saturable at 8 nM. Saturation data could be resolved into high affinity (equilibrium dissociation constant, Kd, 0.22 nM) and low affinity sites (Kd, 2.65 nM). The low affinity sites were more numerous than the high affinity sites, with a ratio of 4:1. The non-hydrolyzable GTP analogue GppNHp had no effect on binding, indicating that the high and low affinity sites are not guanine nucleotide-regulated states of the same (NK-1) receptor. The low affinity sites are unlikely to represent NK-3 receptors since coincubation with the selective NK-3 receptor agonist senktide did not alter the biphasic nature of [3H]SP binding. The rank order of potency for inhibition of [3H]SP (2 nM) binding was SP > or = [Sar9, Met(O2)11]-SP > or = physalaemin > SP(3-11) > NP gamma = [Ala3]-SP > or = SP(4-11) > or = NPK > or = SP(5-11) > or = NKB approximately NKA > SP(1-9), compatible with binding to an NK-1 site. N-terminal fragments and non-amidated analogues were ineffective competitors for [3H]SP binding. However, competition data for several peptides including substance P (SP) and the NK-1 selective agonist [Sar9, Met(O2)11]-SP could be resolved into two components.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Fedynyshyn, J.P.

The opioid binding characteristics of the rat (PAG) and the signal transduction mechanisms of the opioid receptors were examined with in vitro radioligand binding, GTPase, adenylyl cyclase, and inositol phosphate assays. The nonselective ligand {sup 3}H-ethylketocyclazocine (EKC), the {mu} and {delta} selective ligand {sup 3}H-(D-Ala{sup 2}, D-Leu{sup 5}) enkephalin (DADLE), the {mu} selective ligand {sup 3}H-(D-Ala{sup 2}, N-methyl Phe{sup 4}, Glyol{sup 5}) enkephalin (DAGO), and the {delta} selective ligand {sup 3}H-(D-Pen{sup 2}, D-Pen{sup 5}) enkephalin (DPDPE) were separately used as tracer ligands to label opioid binding sites in rat PAG enriched P{sub 2} membrane in competition with unlabeled DADLE, DAGO,more » DPDPE, or the {kappa} selective ligand trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzeneacetamide, methane sulfonate, hydrate (U50, 488H). Only {mu} selective high affinity opioid binding was observed. No high affinity {delta} or {kappa} selective binding was detected. {sup 3}H-DAGO was used as a tracer ligand to label {mu} selective high affinity opioid binding sites in PAG enriched P{sub 2} membrane in competition with unlabeled {beta}-endorphin, dynorphin A (1-17), BAM-18, methionine enkephalin, dynorphin A (1-8), and leucine enkephalin. Of these endogenous opioid peptides only those with previously reported high affinity {mu} type opioid binding activity competed with {sup 3}H-DAGO for binding sites in rat PAG enriched P{sub 2} membrane with affinities similar to that of unlabeled DAGO.« less

[Development of antibody medicines by bio-venture: lesson from license negotiations with mega pharmacies].

PubMed

Takada, Kenzo

2013-01-01

The current method of antibody production is mainly the hybridoma method, in which mice are immunized with an excess amount of antigen for a short period to promote activation and proliferation of B-lymphocytes producing the antibodies of interest. Because of the excess antigen, those producing low-affinity antibodies are activated. In contrast, human blood B-lymphocytes are activated through natural immune reactions, such as the reaction to infection. B-lymphocytes are stimulated repeatedly with a small amount of antigen, and thus only those producing high-affinity antibodies are activated. Consequently, the lymphocytes producing the high-affinity antibodies are accumulated in human blood. Therefore, human lymphocytes are an excellent source of high-affinity antibodies. Evec, Inc. has established a unique method to produce high-affinity antibodies from human lymphocytes using Epstein-Barr virus (EBV), which induces the proliferation of B-lymphocytes. The method first induces the proliferation of B-lymphocytes from human blood using EBV, and then isolates those producing the antibodies of interest. The key features of the Evec technique are: 1) development of a lymphocyte library consisting of 150 donors' lymphocytes from which donors suited to develop the antibodies of interest can be selected in 4 days; and 2) development of a sorting method and cell microarray method for selecting lymphocyte clones producing the target antibodies. Licensing agreements have been concluded with European and Japanese pharmaceutical companies for two types of antibody. This paper describes Evec's antibody technology and experience in license negotiations with Mega Pharmacies.

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

PubMed Central

Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

2016-01-01

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

Biphasic association of T7 RNA polymerase and a nucleotide analogue, cibacron blue as a model to understand the role of initiating nucleotide in the mechanism of enzyme action.

PubMed

Pai, Sudipta; Das, Mili; Banerjee, Rahul; Dasgupta, Dipak

2011-08-01

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. Here we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, its effect on the function of the enzyme and the probable mode of binding of the dye. We have used difference absorption spectroscopy and isothermal titration calorimetry to show that the dye binds T7 RNAP in a biphasic manner. The first phase of the binding is characterized by inactivation of the enzyme. The second binding site overlaps with the common substrate-binding site of the enzyme. We have carried out docking experiment to map the binding site of the dye in the promoter bound protein. Competitive displacement of the dye from the high affinity site by labeled GTP and isothermal titration calorimetry of high affinity GTP bound enzyme with the dye suggests a strong correlation between the high affinity dye binding and the high affinity GTP binding in T7 RNAP reported earlier from our laboratory.

The use of selective adsorbents in capillary electrophoresis-mass spectrometry for analyte preconcentration and microreactions: a powerful three-dimensional tool for multiple chemical and biological applications.

PubMed

Guzman, N A; Stubbs, R J

2001-10-01

Much attention has recently been directed to the development and application of online sample preconcentration and microreactions in capillary electrophoresis using selective adsorbents based on chemical or biological specificity. The basic principle involves two interacting chemical or biological systems with high selectivity and affinity for each other. These molecular interactions in nature usually involve noncovalent and reversible chemical processes. Properly bound to a solid support, an "affinity ligand" can selectively adsorb a "target analyte" found in a simple or complex mixture at a wide range of concentrations. As a result, the isolated analyte is enriched and highly purified. When this affinity technique, allowing noncovalent chemical interactions and biochemical reactions to occur, is coupled on-line to high-resolution capillary electrophoresis and mass spectrometry, a powerful tool of chemical and biological information is created. This paper describes the concept of biological recognition and affinity interaction on-line with high-resolution separation, the fabrication of an "analyte concentrator-microreactor", optimization conditions of adsorption and desorption, the coupling to mass spectrometry, and various applications of clinical and pharmaceutical interest.

An enantioselective enzymatic desymmetrization route to hexahydro-4H-furopyranol, a high-affinity ligand for HIV-1 protease inhibitors.

PubMed

Ghosh, Arun K; Sarkar, Anindya

2017-08-16

An enantioselective synthesis of ( 3 a S , 4S , 7 a R )-hexahydro-4 H -furo[2,3- b ]pyran-4-ol, a high-affinity nonpeptide ligand for a variety of potent HIV-1 protease inhibitors is described. The key steps involved a highly enantioselective enzymatic desymmetrization of meso -diacetate, an efficient transacetalization, and a highly diastereoselective reduction of a ketone. This route is amenable to large-scale synthesis using readily available starting materials.

Magnetic vortex excitation as spin torque oscillator and its unusual trajectories

NASA Astrophysics Data System (ADS)

Natarajan, Kanimozhi; Muthuraj, Ponsudana; Rajamani, Amuda; Arumugam, Brinda

2018-05-01

We report an interesting observation of unusual trajectories of vortex core oscillations in a spin valve pillar. Micromagnetic simulation in the composite free layer spin valve nano-pillar shows magnetic vortex excitation under critical current density. When current density is slightly increased and wave vector is properly tuned, for the first time we observe a star like and square gyration. Surprisingly this star like and square gyration also leads to steady, coherent and sustained oscillations. Moreover, the frequency of gyration is also very high for this unusual trajectories. The power spectral analysis reveals that there is a marked increase in output power and frequency with less distortions. Our investigation explores the possibility of these unusual trajectories to exhibit spin torque oscillations.

Production and characterization of a high-affinity nanobody against human endoglin.

PubMed

Ahmadvand, Davoud; Rasaee, Mohammad J; Rahbarizadeh, Fatemeh; Mohammadi, Mohammad

2008-10-01

Abstract Antibodies or antibody fragments are almost exclusively applied in human therapy and diagnosis. The high affinity and specificity of antibodies makes them suitable for these applications. Nanobody, the variable domain of Camelidae heavy chain antibodies, have superior properties compared with conventional antibodies in that they are small, non-immunogenic, very stable, highly soluble, and easy to produce in large quantities. In the present study, we report the isolation and characterization of a high-affinity binder against human endoglin retrieved from camels' nanobody gene library. Endoglin (CD105), an accessory protein of the transforming growth factor beta receptor complex, has become an attractive molecule for the targeting of the tumor vasculature. Upregulation of endoglin on proliferating endothelial cells is associated with tumor neovascularization. Here, we generated two nanobody gene libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the recombinant extracellular domain of human endoglin. The other selected anti-endoglin nanobody (AR1-86) showed strong binding to human endoglin expressing endothelial cells (HUVECs), while no binding was observed with the endoglin-negative cell line (HEK293). This high-affinity single-domain antibody could be a good candidate for the generation of vascular or tumor targeting agents in cancer therapy.

Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

PubMed Central

Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

2016-01-01

The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

beta. -Adrenoceptors in human tracheal smooth muscle: characteristics of binding and relaxation

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

van Koppen, C.J.; Hermanussen, M.W.; Verrijp, K.N.

1987-06-29

Specific binding of (/sup 125/I)-(-)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (K/sub d/ = 5.3 +/- 0.9 pmol/l and R/sub T/ = 78 +/- 7 fmol/g tissue). The ..beta../sub 1/-selective antagonists atenolol and LK 203-030 inhibited specific (/sup 125/I)-(-)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous BETA/sub 2/-adrenoceptor population. In one subject using LK 203-030 a small ..beta../sub 1/-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pK/sub H/-more » and pK/sub L/-values for the high and low affinity sites were 8.0 +/- 0.2 and 5.9 +/- 0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD/sub 2/-value of 6.63 +/- 0.19. 32 references, 4 figures, 2 tables.« less

Monospecific high-affinity and complement activating anti-GM1 antibodies are determinants in experimental axonal neuropathy.

PubMed

Notturno, Francesca; Del Boccio, Piero; Luciani, Mirella; Caporale, Christina Michaela; Pieragostino, Damiana; Prencipe, Vincenza; Sacchetta, Paolo; Uncini, Antonino

2010-06-15

It has been difficult to replicate consistently the experimental model of axonal Guillain-Barré syndrome (GBS). We immunized rabbits with two lipo-oligosaccharides (LOS1 and LOS2) derived from the same C. jejuni strain and purified in a slightly different way. LOS1 did not contain proteins whereas several proteins were present in LOS2. In spite of a robust anti-GM1 antibody response in all animals the neuropathy developed only in rabbits immunized with LOS1. To explain this discrepancy we investigated fine specificity, affinity and ability to activate the complement of anti-GM1 antibodies. Only rabbits immunized with LOS1 showed monospecific high-affinity antibodies which activated more effectively the complement. Although it is not well understood how monospecific high-affinity antibodies are induced these are crucial for the induction of experimental axonal neuropathy. Only a strict adherence to the protocols demonstrated to be successful may guarantee the reproducibility and increase the confidence in the animal model as a reliable tool for the study of the human axonal GBS. Copyright 2010 Elsevier B.V. All rights reserved.

Unusual CoS2 ellipsoids with anisotropic tube-like cavities and their application in supercapacitors.

PubMed

Zhang, Lei; Wu, Hao Bin; Lou, Xiong Wen

2012-07-14

Unusual CoS(2) ellipsoids with anisotropic tube-like cavities have been synthesized from the simultaneous thermal decomposition and sulfidation of a preformed cobalt carbonate precursor. The as-prepared CoS(2) ellipsoids show interesting supercapacitive properties with high capacitance and good cycling performance.

Engineering the production of conjugated fatty acids in Arabidopsis thaliana leaves

USDA-ARS?s Scientific Manuscript database

The seeds of many non-domesticated plant species synthesize oils containing high amounts of a single unusual fatty acid, many of which have potential usage in industry. Despite the identification of enzymes for unusual oxidized fatty acid synthesis, the production of these fatty acids in engineered ...

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

PubMed Central

2011-01-01

Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates. Conclusions Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches. PMID:22112852

The effects of SB 216469, an antagonist which discriminates between the alpha 1A-adrenoceptor and the human prostatic alpha 1-adrenoceptor.

PubMed Central

Chess-Williams, R.; Chapple, C. R.; Verfurth, F.; Noble, A. J.; Couldwell, C. J.; Michel, M. C.

1996-01-01

1. The affinity of the alpha 1-adrenoceptor antagonist SB 216469 (also known as REC 15/2739) has been determined at native and cloned alpha 1-adrenoceptor subtypes by radioligand binding and at functional alpha 1-adrenoceptor subtypes in isolated tissues. 2. In radioligand binding studies with [3H]-prazosin, SB 216469 had a high affinity at the alpha 1A-adrenoceptors of the rat cerebral cortex and kidney (9.5-9.8) but a lower affinity at the alpha 1B-adrenoceptors of the rat spleen and liver (7.7-8.2). 3. At cloned rat alpha 1-adrenoceptor subtypes transiently expressed in COS-1 cells and also at cloned human alpha 1-adrenoceptor subtypes stably transfected in Rat-1 cells, SB 216469 exhibited a high affinity at the alpha 1a-adrenoceptors (9.6-10.4) with a significantly lower affinity at the alpha 1b-adrenoceptor (8.0-8.4) and an intermediate affinity at the alpha 1d-adrenoceptor (8.7-9.2). 4. At functional alpha 1-adrenoceptors, SB 216469 had a similar pharmacological profile, with a high affinity at the alpha 1A-adrenoceptors of the rat vas deferens and anococcygeus muscle (pA2 = 9.5-10.0), a low affinity at the alpha 1B-adrenoceptors of the rat spleen (6.7) and guinea-pig aorta (8.0), and an intermediate affinity at the alpha 1D-adrenoceptors of the rat aorta (8.8). 5. Several recent studies have concluded that the alpha 1-adrenoceptor present in the human prostate has the pharmacological characteristics of the alpha 1A-adrenoceptor subtype. However, the affinity of SB 216469 at human prostatic alpha 1-adrenoceptors (pA2 = 8.1) determined in isolated tissue strips, was significantly lower than the values obtained at either the cloned alpha 1a-adrenoceptors (human, rat, bovine) or the native alpha 1A-adrenoceptors in radioligand binding and functional studies in the rat. 6. Our results with SB 216469, therefore, suggest that the alpha 1-adrenoceptor mediating contractile responses of the human prostate has properties which distinguish it from the cloned alpha 1a-adrenoceptor or native alpha 1A-adrenoceptor. Since it has previously been shown that the receptor is not the alpha 1B- or alpha 1D-adrenoceptor, the functional alpha 1-adrenoceptor of the human prostate may represent a novel receptor with properties which differ from any of the alpha 1-adrenoceptors currently defined by pharmacological means. PMID:8937710

(99m)Tc-amitrole as a novel selective imaging probe for solid tumor: In silico and preclinical pharmacological study.

PubMed

Essa, B M; Sakr, T M; Khedr, Mohammed A; El-Essawy, F A; El-Mohty, A A

2015-08-30

Lactoperoxidase (LPO) inhibitors are very selective for solid tumor due to their high binding affinity to the LPO enzyme. A computational study was used to select top-ranked LPO inhibitor (alone and in complex with (99m)Tc) with high in silico affinity. The novel prepared (99m)Tc-amitrole complex demonstrated both in silico and in vivo high affinity toward solid tumors.(99m)Tc-amitrole was radio-synthesized with a high radiochemical yield (89.7±3.25). It showed in vitro stability for up to 6h. Its preclinical evaluation in solid tumor-bearing mice showed high retention and biological accumulation in solid tumor cells with a high Target/Non-Target (T/NT) ratio equal to 4.9 at 60min post-injection. The data described previously could recommend (99m)Tc-amitrole as potential targeting scintigraphic probe for solid tumor imaging. Copyright © 2015 Elsevier B.V. All rights reserved.

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

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

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

DOE PAGES

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick; ...

2017-11-24

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

One ligand, two regulators and three binding sites: How KDPG controls primary carbon metabolism in Pseudomonas

PubMed Central

Fung, Rowena K. Y.; Grenga, Lucia; Trampari, Eleftheria; Pepe, Simona

2017-01-01

Effective regulation of primary carbon metabolism is critically important for bacteria to successfully adapt to different environments. We have identified an uncharacterised transcriptional regulator; RccR, that controls this process in response to carbon source availability. Disruption of rccR in the plant-associated microbe Pseudomonas fluorescens inhibits growth in defined media, and compromises its ability to colonise the wheat rhizosphere. Structurally, RccR is almost identical to the Entner-Doudoroff (ED) pathway regulator HexR, and both proteins are controlled by the same ED-intermediate; 2-keto-3-deoxy-6-phosphogluconate (KDPG). Despite these similarities, HexR and RccR control entirely different aspects of primary metabolism, with RccR regulating pyruvate metabolism (aceEF), the glyoxylate shunt (aceA, glcB, pntAA) and gluconeogenesis (pckA, gap). RccR displays complex and unusual regulatory behaviour; switching repression between the pyruvate metabolism and glyoxylate shunt/gluconeogenesis loci depending on the available carbon source. This regulatory complexity is enabled by two distinct pseudo-palindromic binding sites, differing only in the length of their linker regions, with KDPG binding increasing affinity for the 28 bp aceA binding site but decreasing affinity for the 15 bp aceE site. Thus, RccR is able to simultaneously suppress and activate gene expression in response to carbon source availability. Together, the RccR and HexR regulators enable the rapid coordination of multiple aspects of primary carbon metabolism, in response to levels of a single key intermediate. PMID:28658302

Evidence of environmental strains on charge injection in silole-based organic light-emitting diodes

NASA Astrophysics Data System (ADS)

Huby, N.; Hirsch, L.; Aubouy, L.; Gerbier, P.; van der Lee, A.; Amy, F.; Kahn, A.

2007-03-01

Using density functional theory (DFT) computations, we have demonstrated a substantial skeletal relaxation when the structure of 2,5-[bis-(4-anthracene-9-yl-phenyl]-1,1-dimethyl-3,4-diphenyl-silole (BAS) is optimized in the gas-phase comparing with the molecular structure determined from monocrystal x-ray diffraction. The origin of such a relaxation is explained by a strong environmental strains induced by the presence of anthracene entities. Moreover, the estimation of the frontier orbital levels showed that this structural relaxation affects mainly the LUMO that is lowered of 190meV in the gas phase. To check if these theoretical findings would be confirmed for thin films of BAS, we turned to ultraviolet photoemission spectroscopy and/or inverse photoemission spectroscopy and electro-optical measurements. Interestingly, the study of the current density or voltage and luminance or voltage characteristics of an ITO/PEDOT/BAS/Au device clearly demonstrated a very unusual temperature-dependent behavior. Using a thermally assisted tunnel transfer model, we found that this behavior likely originated from the variation of the electronic affinity of the silole derivative with the temperature. The thermal agitation relaxes the molecular strains in thin films as it is shown when passing from the crystalline to the gas phase. The relaxation of the intramolecular thus induces an increase of the electronic affinity and, as a consequence, the more efficient electron injection in organic light-emitting diodes.

Receptor residence time trumps drug-likeness and oral bioavailability in determining efficacy of complement C5a antagonists

PubMed Central

Seow, Vernon; Lim, Junxian; Cotterell, Adam J.; Yau, Mei-Kwan; Xu, Weijun; Lohman, Rink-Jan; Kok, W. Mei; Stoermer, Martin J.; Sweet, Matthew J.; Reid, Robert C.; Suen, Jacky Y.; Fairlie, David P.

2016-01-01

Drug discovery and translation are normally based on optimizing efficacy by increasing receptor affinity, functional potency, drug-likeness (rule-of-five compliance) and oral bioavailability. Here we demonstrate that residence time of a compound on its receptor has an overriding influence on efficacy, exemplified for antagonists of inflammatory protein complement C5a that activates immune cells and promotes disease. Three equipotent antagonists (3D53, W54011, JJ47) of inflammatory responses to C5a (3nM) were compared for drug-likeness, receptor affinity and antagonist potency in human macrophages, and anti-inflammatory efficacy in rats. Only the least drug-like antagonist (3D53) maintained potency in cells against higher C5a concentrations and had a much longer duration of action (t1/2 ~ 20 h) than W54011 or JJ47 (t1/2 ~ 1–3 h) in inhibiting macrophage responses. The unusually long residence time of 3D53 on its receptor was mechanistically probed by molecular dynamics simulations, which revealed long-lasting interactions that trap the antagonist within the receptor. Despite negligible oral bioavailability, 3D53 was much more orally efficacious than W54011 or JJ47 in preventing repeated agonist insults to induce rat paw oedema over 24 h. Thus, residence time on a receptor can trump drug-likeness in determining efficacy, even oral efficacy, of pharmacological agents. PMID:27094554

Receptor residence time trumps drug-likeness and oral bioavailability in determining efficacy of complement C5a antagonists

NASA Astrophysics Data System (ADS)

Seow, Vernon; Lim, Junxian; Cotterell, Adam J.; Yau, Mei-Kwan; Xu, Weijun; Lohman, Rink-Jan; Kok, W. Mei; Stoermer, Martin J.; Sweet, Matthew J.; Reid, Robert C.; Suen, Jacky Y.; Fairlie, David P.

2016-04-01

Drug discovery and translation are normally based on optimizing efficacy by increasing receptor affinity, functional potency, drug-likeness (rule-of-five compliance) and oral bioavailability. Here we demonstrate that residence time of a compound on its receptor has an overriding influence on efficacy, exemplified for antagonists of inflammatory protein complement C5a that activates immune cells and promotes disease. Three equipotent antagonists (3D53, W54011, JJ47) of inflammatory responses to C5a (3nM) were compared for drug-likeness, receptor affinity and antagonist potency in human macrophages, and anti-inflammatory efficacy in rats. Only the least drug-like antagonist (3D53) maintained potency in cells against higher C5a concentrations and had a much longer duration of action (t1/2 ~ 20 h) than W54011 or JJ47 (t1/2 ~ 1-3 h) in inhibiting macrophage responses. The unusually long residence time of 3D53 on its receptor was mechanistically probed by molecular dynamics simulations, which revealed long-lasting interactions that trap the antagonist within the receptor. Despite negligible oral bioavailability, 3D53 was much more orally efficacious than W54011 or JJ47 in preventing repeated agonist insults to induce rat paw oedema over 24 h. Thus, residence time on a receptor can trump drug-likeness in determining efficacy, even oral efficacy, of pharmacological agents.

Two new carnivores from an unusual late Tertiary forest biota in eastern North America.

PubMed

Wallace, Steven C; Wang, Xiaoming

2004-09-30

Late Cenozoic terrestrial fossil records of North America are biased by a predominance of mid-latitude deposits, mostly in the western half of the continent. Consequently, the biological history of eastern North America, including the eastern deciduous forest, remains largely hidden. Unfortunately, vertebrate fossil sites from this vast region are rare, and few pertain to the critically important late Tertiary period, during which intensified global climatic changes took place. Moreover, strong phylogenetic affinities between the flora of eastern North America and eastern Asia clearly demonstrate formerly contiguous connections, but disparity among shared genera (eastern Asia-eastern North America disjunction) implies significant periods of separation since at least the Miocene epoch. Lacustrine sediments deposited within a former sinkhole in the southern Appalachian Mountains provide a rare example of a late Miocene to early Pliocene terrestrial biota from a forested ecosystem. Here we show that the vertebrate remains contained within this deposit represent a unique combination of North American and Eurasian taxa. A new genus and species of the red (lesser) panda (Pristinailurus bristoli), the earliest and most primitive so far known, was recovered. Also among the fauna are a new species of Eurasian badger (Arctomeles dimolodontus) and the largest concentration of fossil tapirs ever recorded. Cladistical analyses of the two new carnivores strongly suggest immigration events that were earlier than and distinct from previous records, and that the close faunal affinities between eastern North America and eastern Asia in the late Tertiary period are consistent with the contemporaneous botanical record.

( sup 3 H)-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and ( sup 3 H) ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

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

Branchek, T.; Adham, N.; Macchi, M.

1990-11-01

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to (3H)ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding themore » serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both (3H)DOB and (3H)ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p) to this system caused a rightward shift and steepening of agonist competition curves for (3H) ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity (3H)DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that (3H)DOB and (3H)ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein.« less

Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

PubMed

Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

2016-10-01

Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a cost-effective, rapid, and reliable avenue for the purification of recombinant proteins in heterologous hosts. Copyright © 2016 Elsevier Inc. All rights reserved.

Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts

PubMed Central

Burel, Sebastien A.; Hart, Christopher E.; Cauntay, Patrick; Hsiao, Jill; Machemer, Todd; Katz, Melanie; Watt, Andy; Bui, Huynh-hoa; Younis, Husam; Sabripour, Mahyar; Freier, Susan M.; Hung, Gene; Dan, Amy; Prakash, T.P.; Seth, Punit P.; Swayze, Eric E.; Bennett, C. Frank; Crooke, Stanley T.; Henry, Scott P.

2016-01-01

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation. PMID:26553810

Toxic metals (Ni2+, Pb2+, Hg2+) binding affinity of dissolved organic matter (DOM) derived from different ages municipal landfill leachate

NASA Astrophysics Data System (ADS)

Rikta, S. Y.; Tareq, Shafi M.; Uddin, M. Khabir

2018-03-01

Solid waste production is rapidly increasing in Bangladesh and landfill leachate is the consequence of the decomposition of this waste. These leachates contain heavy metals and significant amount of dissolved organic matter (DOM). DOM is known to have considerable role in heavy metals speciation. Hence, it is important to characterize DOM/leachate and evaluate toxic metals binding affinity of DOM. The objectives of this study were to characterize the DOM in landfill leachate through physico-chemical and optical analyses and to investigate the toxic metals (Ni2+, Pb2+ and Hg2+) binding affinity of three different ages (fresh sample L-1, young sample L-2 and mature sample L-3) DOM samples. Results suggested that leachate is a potential pollutant which contained very high organic pollutant load. Conditional stability constant (Log K) and percentages of fluorophores that correspond to metal binding (% f) values indicated that young DOM sample (L-2) had the highest binding affinity to all the three metals ions. In general, DOM samples showed the following order affinity to the metal ions; Ni2+ binding affinity: L-2 > L-3 > L-1, Pb2+ binding affinity: L-2 > L-3 > L-1 and Hg2+ binding affinity: L-2 > L-1 > L-3.

Chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozyme.

PubMed

Yamada, H; Fukumura, T; Ito, Y; Imoto, T

1985-04-01

Preparation of chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozymes and its application to separation of N-bromosuccinimide-oxidized lysozymes are described. By pH gradient elution, two diastereomers of oxindolealanine-62-lysozyme, delta 1-acetoxytryptophan-62-lysozyme (intermediate product in the reaction in acetate buffer), and native lysozyme were all separated within 40 min.

Batrachotoxin Changes the Properties of the Muscarinic Receptor in Rat Brain and Heart: Possible Interaction(s) between Muscarinic Receptors and Sodium Channels

NASA Astrophysics Data System (ADS)

Cohen-Armon, Malca; Kloog, Yoel; Henis, Yoav I.; Sokolovsky, Mordechai

1985-05-01

The effects of Na+-channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors in homogenates of rat brain and heart were studied. BTX enhanced the affinity for the binding of the agonists carbamoylcholine and acetylcholine to the muscarinic receptors in brainstem and ventricle, but not in the cerebral cortex. Analysis of the data according to a two-site model for agonist binding indicated that the effect of BTX was to increase the affinity of the agonists to the high-affinity site. Guanyl nucleotides, known to induce interconversion of high-affinity agonist binding sites to the low-affinity state, canceled the effect of BTX on carbamoylcholine and acetylcholine binding. BTX had no effect on the binding of the agonist oxotremorine or on the binding of the antagonist [3H]-N-methyl-4-piperidyl benzilate. The local anesthetics dibucaine and tetracaine antagonized the effect of BTX on the binding of muscarinic agonists at concentrations known to inhibit the activation of Na+ channels by BTX. On the basis of these findings, we propose that in specific tissues the muscarinic receptors may interact with the BTX binding site (Na+ channels).

Concentration-Dependent Multiple Binding Sites on Saliva-Treated Hydroxyapatite for Streptococcus sanguis

PubMed Central

Gibbons, R. J.; Moreno, E. C.; Etherden, I.

1983-01-01

The influence of bacterial cell concentration on estimates of the number of binding sites and the affinity for the adsorption of a strain of Streptococcus sanguis to saliva-treated hydroxyapatite was determined, and the possible presence of multiple binding sites for this organism was tested. The range of concentrations of available bacteria varied from 4.7 × 106 to 5,960 × 106 cells per ml. The numbers of adsorbed bacteria increased over the entire range tested, but a suggestion of a break in an otherwise smooth adsorption isotherm was evident. Values for the number of binding sites and the affinity varied considerably depending upon the range of available bacterial concentrations used to estimate them; high correlation coefficients were obtained in all cases. The use of low bacterial cell concentrations yielded lower values for the number of sites and much higher values for the affinity constant than did the use of high bacterial cell concentrations. When data covering the entire range of bacterial concentrations were employed, values for the number of sites and the affinity were similar to those obtained by using only high bacterial cell concentrations. The simplest explanation for these results is that there are multiple binding sites for S. sanguis on saliva-treated hydroxyapatite surfaces. When present in low concentration, the streptococci evidently attach to more specific high-affinity sites which become saturated when higher bacterial concentrations are employed. The possibility of multiple binding sites was substantiated by comparing estimates of the adsorption parameters from a computer-simulated isotherm with those derived from the experimentally generated isotherm. A mathematical model describing bacterial adsorption to binary binding sites was further evidence for the existence of at least two classes of binding sites for S. sanguis. Far fewer streptococci adsorbed to experimental pellicles prepared from saliva depleted of bacterial aggregating activity when low numbers of streptococci were used, but the magnitude of this difference was considerably less when high streptococcal concentrations were employed. This suggests an association between salivary components which possess bacterial-aggregating activity and bacterial adsorption to high-affinity specific binding sites on saliva-treated hydroxyapatite surfaces. PMID:6822416

Surface-modified multifunctional MIP nanoparticles

NASA Astrophysics Data System (ADS)

Moczko, Ewa; Poma, Alessandro; Guerreiro, Antonio; Perez de Vargas Sansalvador, Isabel; Caygill, Sarah; Canfarotta, Francesco; Whitcombe, Michael J.; Piletsky, Sergey

2013-04-01

The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as a protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinylferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors.The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as a protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinylferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors. Electronic supplementary information (ESI) available: Details of the synthesis of eosin O-acrylate monomer and 1H-NMR spectrum of MIP NPs post-derivatised with PEG shell. See DOI: 10.1039/c3nr00354j

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

2013-01-01

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

Characterization of diadenosine tetraphosphate (Ap4A) binding sites in cultured chromaffin cells: evidence for a P2y site.

PubMed Central

Pintor, J.; Torres, M.; Castro, E.; Miras-Portugal, M. T.

1991-01-01

1. Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 +/- 0.65 x 10(-11) M and Bmax value of 5420 +/- 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 +/- 0.53 x 10(-9) M and a Bmax value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2. The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [3H]-Ap4A from the two binding sites, the Ki values being 1.0 nM, 0.013 nM, 0.013 nM and 0.013 nM for the very high affinity binding site and 0.5 microM, 0.13 microM, 0.062 microM and 0.75 microM for the second binding site. 3. The ATP analogues displaced [3H]-Ap4A with the potency order of the P2y receptors, adenosine 5'-O-(2 thiodiphosphate) (ADP-beta-S) greater than 5'-adenylyl imidodiphosphate (AMP-PNP) greater than alpha, beta-methylene ATP (alpha, beta-MeATP), in both binding sites. The Ki values were respectively 0.075 nM, 0.2 nM and 0.75 nM for the very high affinity binding site and 0.125 microM, 0.5 microM and 0.9 microM for the second binding site. PMID:1912985

Examining authentic talk and student authorship of scientific ideas: Public pedagogy and affinity space

NASA Astrophysics Data System (ADS)

Slaton, Adriane McNamara

One of the great challenges to teachers both historically and contemporarily is to facilitate a classroom as a group of students, while concurrently attending to each individual student. As the profession becomes increasingly aware and sensitive to student (dis)abilities, academic giftedness, pressures of athletics and extra-curricular events, and acknowledges the racial, cultural, sexual, economic, gendered identities of students--- the complexity of each individual student becomes ever more apparent. It is a seemingly insurmountable challenge. This dissertation examines how stem cell unit guided group learning while also attending to the individual learning needs of each student. What makes classroom study unusual in research is that it is completely "usual". This study was not part of a university partnership nor did it take place in an affluent school district. Instead, this unit took place in a large, Midwestern urban high school that was/is often written off as a failing---underscoring that powerful science teaching and learning is occurring in schools society simultaneously portrays as victim and villain in the media. Using a sociocultural lens, this ethnographic study draws upon two frameworks, Wenger's communities of practice (1998) and Hayes and Gee's (2011) public pedagogy, to examine how participation in a debate individualizes and personalizes student knowledge and participation in science. The primary use of a community of practice framework allowed for analysis of the norms, rules, practices, and routines of Classroom 507---to establish the nature of the community of learners in the study. A secondary framework, public pedagogy, allowed for deeper understanding of the practices drawn upon in the classroom through consideration of the design, resources, and an emergent "affinity space". This hybridized analysis led to further understanding of how students and teacher stand to learn, participate, engage and use a classroom lesson, the debate, to serve their own, unique purposes. During my observations of biology Classroom 507, the class engaged a three-week unit on the ethics of stem cells which ended in a class-wide debate. I had the opportunity to observe, film, and interview students and their teacher. Through observation, interviews, and film, I began to understand the classroom community (its norms, practices, and routines) but also began to recognize how this unit functioned to teach content but push on student thought creation and development as individuals. In this dissertation, I construct something I call an "affinity story" for four focal students (Mismin, Kevin, Molly, and Michael) and their teacher (Theresa). These affinity stories capture how each of the individuals co-opted the debate space in various ways allowing for engagement and participation in ways that were true to themselves and their social and academic needs. Implications from this dissertation include: 1) re-considering the importance of how both students and teacher enrich science understandings when public, authentic talk is encouraged; 2) how teachers can design units and draw from diverse resources to enrich the learning experience of the individual; 3) how students and teacher appropriate learning spaces for their own individual social and academic needs; 4) to consider what it means to allow students to follow their affinities within the science classroom; and 5) to inspire teachers as they engage in the difficult task we call teaching.

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 anti-CD3/anti-CLL-1 bispecific antibody for the treatment of acute myeloid leukemia.

PubMed

Leong, Steven R; Sukumaran, Siddharth; Hristopoulos, Maria; Totpal, Klara; Stainton, Shannon; Lu, Elizabeth; Wong, Alfred; Tam, Lucinda; Newman, Robert; Vuillemenot, Brian R; Ellerman, Diego; Gu, Chen; Mathieu, Mary; Dennis, Mark S; Nguyen, Allen; Zheng, Bing; Zhang, Crystal; Lee, Genee; Chu, Yu-Waye; Prell, Rodney A; Lin, Kedan; Laing, Steven T; Polson, Andrew G

2017-02-02

Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML. © 2017 by The American Society of Hematology.

The translocator protein gene is associated with symptom severity and cerebral pain processing in fibromyalgia.

PubMed

Kosek, Eva; Martinsen, Sofia; Gerdle, Björn; Mannerkorpi, Kaisa; Löfgren, Monika; Bileviciute-Ljungar, Indre; Fransson, Peter; Schalling, Martin; Ingvar, Martin; Ernberg, Malin; Jensen, Karin B

2016-11-01

The translocator protein (TSPO) is upregulated during glia activation in chronic pain patients. TSPO constitutes the rate-limiting step in neurosteroid synthesis, thus modulating synaptic transmission. Related serotonergic mechanisms influence if pro- or anti-nociceptive neurosteroids are produced. This study investigated the effects of a functional genetic polymorphism regulating the binding affinity to the TSPO, thus affecting symptom severity and cerebral pain processing in fibromyalgia patients. Gene-to-gene interactions with a functional polymorphism of the serotonin transporter gene were assessed. Fibromyalgia patients (n=126) were genotyped regarding the polymorphisms of the TSPO (rs6971) and the serotonin transporter (5-HTTLPR/rs25531). Functional magnetic resonance imaging (n=24) was used to study brain activation during individually calibrated pressure pain. Compared to mixed/low TSPO affinity binders, the high TSPO affinity binders rated more severe pain (p=0.016) and fibromyalgia symptoms (p=0.02). A significant interaction was found between the TSPO and the serotonin transporter polymorphisms regarding pain severity (p<0.0001). Functional connectivity analyses revealed that the TSPO high affinity binding group had more pronounced pain-evoked functional connectivity in the right frontoparietal network, between the dorsolateral prefrontal area and the parietal cortex. In conclusion, fibromyalgia patients with the TSPO high affinity binding genotype reported a higher pain intensity and more severe fibromyalgia symptoms compared to mixed/low affinity binders, and this was modulated by interaction with the serotonin transporter gene. To our knowledge this is the first evidence of functional genetic polymorphisms affecting pain severity in FM and our findings are in line with proposed glia-related mechanisms. Furthermore, the functional magnetic resonance findings indicated an effect of translocator protein on the affective-motivational components of pain perception. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Channel architecture in maltoporin: dominance studies with lamB mutations influencing maltodextrin binding provide evidence for independent selectivity filters in each subunit.

PubMed Central

Ferenci, T; Lee, K S

1989-01-01

Maltoporin trimers constitute maltodextrin-selective channels in the outer membrane of Escherichia coli. To study the organization of the maltodextrin-binding site within trimers, dominance studies were undertaken with maltoporin variants of altered binding affinity. It has been established that amino acid substitutions at three dispersed regions of the maltoporin sequence (at residues 8, 82, and 360) resulted specifically in maltodextrin-binding defects and loss of maltodextrin channel selectivity; a substitution at residue 118 increased both binding affinity and maltodextrin transport. Strains heterodiploid for lamB were constructed in which these substitutions were encoded by chromosomal and plasmid-borne genes, and the relative level of maltoporin expression from these genes was estimated. Binding assays with bacteria forming maltoporin heterotrimers were performed in order to test for complementation between binding-negative alleles, negative dominance of negative over wild-type alleles, and possible dominance of negatives over the high-affinity allele. Double mutants with mutations affecting residues 8 and 118, 82 and 118, and 118 and 360 were constructed in vitro, and the dominance properties of the mutations in cis were also tested. There was no complementation between negatives and no negative dominance in heterotrimers. The high-affinity mutation was dominant over negatives in trans but not in cis. The affinity of binding sites in heterotrimer populations was characteristic of the high-affinity allele present and uninfluenced by the negative allele. These results are consistent with the presence of three discrete binding sites in a maltoporin trimer and suggest that the selectivity filter for maltodextrins is not at the interface between the three subunits. PMID:2521623

Arrestin binds to different phosphorylated regions of the thyrotropin-releasing hormone receptor with distinct functional consequences.

PubMed

Jones, Brian W; Hinkle, Patricia M

2008-07-01

Arrestin binding to agonist-occupied phosphorylated G protein-coupled receptors typically increases the affinity of agonist binding, increases resistance of receptor-bound agonist to removal with high acid/salt buffer, and leads to receptor desensitization and internalization. We tested whether thyrotropin-releasing hormone (TRH) receptors lacking phosphosites in the C-terminal tail could form stable and functional complexes with arrestin. Fibroblasts from mice lacking arrestins 2 and 3 were used to distinguish between arrestin-dependent and -independent effects. Arrestin did not promote internalization or desensitization of a receptor that had key Ser/Thr phosphosites mutated to Ala (4Ala receptor). Nevertheless, arrestin greatly increased acid/salt resistance and the affinity of 4Ala receptor for TRH. Truncation of 4Ala receptor just distal to the key phosphosites (4AlaStop receptor) abolished arrestin-dependent acid/salt resistance but not the effect of arrestin on agonist affinity. Arrestin formed stable complexes with activated wild-type and 4Ala receptors but not with 4AlaStop receptor, as measured by translocation of arrestin-green fluorescent protein to the plasma membrane or chemical cross-linking. An arrestin mutant that does not interact with clathrin and AP2 did not internalize receptor but still promoted high affinity TRH binding, acid/salt resistance, and desensitization. A sterically restricted arrestin mutant did not cause receptor internalization or desensitization but did promote acid/salt resistance and high agonist affinity. The results demonstrate that arrestin binds to proximal or distal phosphosites in the receptor tail. Arrestin binding at either site causes increased agonist affinity and acid/salt resistance, but only the proximal phosphosites evoke the necessary conformational changes in arrestin for receptor desensitization and internalization.

The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

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

Nemoto, Takayuki; Ohara-Nemoto, Yuko; Denis, M.

1990-02-20

High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, the authors decided to investigate the possible relationship between these two phenomena. The binding of ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas ({sup 3}H)TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR showed the presence of twomore » types of binding sites. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low ({sup 3}H)TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. These results indicate that the transformed GR has a reduced ({sup 3}H)TA-binding affinity as compared to the nontransformed GR. The steroid-binding domain (amino acids 477-777) and the DNA- and steroid-binding domains (amino acids 415-777) of the human GR were expressed in Escherichia coli as protein A fused proteins. Taken together, these results suggest that the component(s) associating with the nontransformed GR, possibly the heat shock protein hsp 90, play(s) an important role in stabilizing the GR in a high-affinity state for steroids.« less

Adrenergic receptors in frontal cortex in human brain.

PubMed

Cash, R; Raisman, R; Ruberg, M; Agid, Y

1985-02-05

The binding of three adrenergic ligands ([3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol) was studied in the frontal cortex of human brain. alpha 1-Receptors, labeled by [3H]prazosin, predominated. [3H]Clonidine bound to two classes of sites, one of high affinity and one of low affinity. Guanosine triphosphate appeared to lower the affinity of [3H]clonidine for its receptor. [3H]Dihydroalprenolol bound to three classes of sites: the beta 1-receptor, the beta 2-receptor and a receptor with low affinity which represented about 40% of the total binding, but which was probably a non-specific site; the beta 1/beta 2 ratio was 1/2.

Tumour necrosis factors modulate the affinity state of the leukotriene B4 receptor on human neutrophils.

PubMed Central

Brom, J; Knöller, J; Köller, M; König, W

1988-01-01

Pre-incubation of human polymorphonuclear granulocytes with recombinant human tumour necrosis factors (TNF) revealed a time- and dose-dependent reduction of the expression of leukotriene B4-receptor sites. Analysis of the binding data by Scatchard plots showed a shift from a heterologous receptor population (indicating high- and low-affinity subsets) to a homologous population. From the results it is considered that TNF can influence host defence through the modulation of leukotriene B4 receptor affinity. PMID:2851543

Smart Hydrogel Particles: Biomarker Harvesting: One-step affinity purification, size exclusion, and protection against degradation

PubMed Central

Luchini, Alessandra; Geho, David H.; Bishop, Barney; Tran, Duy; Xia, Cassandra; Dufour, Robert; Jones, Clint; Espina, Virginia; Patanarut, Alexis; Zhu, Weidong; Ross, Mark; Tessitore, Alessandra; Petricoin, Emanuel; Liotta, Lance A.

2010-01-01

Disease-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin. We have introduced an affinity bait molecule into N-isopropylacrylamide to produce a particle that will perform three independent functions within minutes, in one step, in solution: a) molecular size sieving b) affinity capture of all solution phase target molecules, and c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis. PMID:18076201

Identification and Characterization of a High-Affinity Choline Uptake System of Brucella abortus

PubMed Central

Herrmann, Claudia K.; Bukata, Lucas; Melli, Luciano; Marchesini, M. Ines; Caramelo, Julio J.

2013-01-01

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus. PMID:23161032

Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response

PubMed Central

Rosa, Sofia Tabares-da; Rossotti, Martin; Carleiza, Carmen; Carrión, Federico; Pritsch, Otto; Ahn, Ki Chang; Last, Jerold A.; Hammock, Bruce D; González-Sapienza, Gualberto

2011-01-01

Single-domain antibodies (sdAbs) found in camelids, lack a light chain and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. While this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC50 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, KD 0.98–1.37 nM (SPR) which allowed development of a competitive assay for TCC with an IC50 = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC50 attained with other anti-hapten VHHs reported thus far. Despite the modest overall anti-hapten sdAbs response in llamas, a small subpopulation of high affinity VHHs are generated that can be isolated by carefully design of the selection process. PMID:21827167

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin.

PubMed

Nielsen, A D; Borch, K; Westh, P

2000-06-15

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.

Effect of Antipsychotic Type and Dose Changes on Tardive Dyskinesia and Parkinsonism Severity in Patients With a Serious Mental Illness: The Curaçao Extrapyramidal Syndromes Study XII.

PubMed

Mentzel, Charlotte L; Bakker, P Roberto; van Os, Jim; Drukker, Marjan; Matroos, Glenn E; Hoek, Hans W; Tijssen, Marina A J; van Harten, Peter N

2017-03-01

To test the efficacy of current treatment recommendations for parkinsonism and tardive dyskinesia (TD) severity in patients with severe mental illness (SMI). We present an 18-year prospective study including all 223 patients with SMI (as defined by the 1987 US National Institute of Mental Health, which were based on DSM-III-R diagnostic criteria) receiving care from the only psychiatric hospital of the former Netherlands Antilles. Eight clinical assessments (1992-2009) focused on movement disorders and medication use. Tardive dyskinesia was measured by the Abnormal Involuntary Movement Scale and parkinsonism by the Unified Parkinson's Disease Rating Scale. Antipsychotics were classified into first-generation antipsychotic (FGA) versus second-generation antipsychotic (SGA) and high versus low dopamine 2 (D₂) affinity categories. The effect that switching has within each category on subsequent movement scores was calculated separately by using time-lagged multilevel logistic regression models. There was a significant association between reduction in TD severity and starting/switching to an FGA (B = -3.54, P < .001) and starting/switching to a high D₂ affinity antipsychotic (B = -2.49, P < .01). Adding an SGA to existing FGA treatment was associated with reduction in TD severity (B = -2.43, P < .01). For parkinsonism, stopping antipsychotics predicted symptom reduction (B = -7.76, P < .01 in FGA/SGA-switch model; B = -7.74, P < .01 in D₂ affinity switch model), while starting a high D₂ affinity antipsychotic predicted an increase in symptoms (B = 3.29, P < .05 in D₂ affinity switch model). The results show that switching from an FGA to an SGA does not necessarily result in a reduction of TD or parkinsonism. Only stopping all antipsychotics reduces the severity of parkinsonism, and starting an FGA or a high D₂ affinity antipsychotic may reduce the severity of TD. © Copyright 2017 Physicians Postgraduate Press, Inc.

An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis

DOE PAGES

Kariolis, Mihalis S.; Miao, Yu Rebecca; Jones, Douglas S.; ...

2014-09-21

Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl ‘decoy receptor’ that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high affinity Axl variant caused structural alterations in side chains across the Gas6/Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc-fusion, the engineered decoy receptor bound tomore » Gas6 with femtomolar affinity, an 80-fold improvement compared to the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Additionally, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.« less

Affinity chromatography: A versatile technique for antibody purification.

PubMed

Arora, Sushrut; Saxena, Vikas; Ayyar, B Vijayalakshmi

2017-03-01

Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup. Among a plethora of different techniques, affinity chromatography is one of the most selective, rapid and easy method for antibody purification. This review aims to provide a detailed overview on affinity chromatography and the components involved in purification. An array of support matrices along with various classes of affinity ligands detailing their underlying working principles, together with the advantages and limitations of each system in purifying different types of antibodies, accompanying recent developments and important practical methodological considerations to optimize purification procedure are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Serotonin and cholecystokinin synergistically stimulate rat vagal primary afferent neurones

PubMed Central

Li, Y; Wu, X Y; Owyang, C

2004-01-01

Recent studies indicate that cholecystokinin (CCK) and serotonin (5-hydroxytryptamine, 5-HT) act via vagal afferent fibres to mediate gastrointestinal functions. In the present study, we characterized the interaction between CCK and 5-HT in the vagal primary afferent neurones. Single neuronal discharges of vagal primary afferent neurones innervating the duodenum were recorded from rat nodose ganglia. Two groups of nodose ganglia neurones were identified: group A neurones responded to intra-arterial injection of low doses of cholecystokinin octapeptide (CCK-8; 10–60 pmol); group B neurones responded only to high doses of CCK-8 (120–240 pmol), and were also activated by duodenal distention. CCK-JMV-180, which acts as an agonist in high-affinity states and as an antagonist in low-affinity states, dose dependently stimulated group A neurones, but inhibited the effect of the high doses of CCK-8 on group B neurones. Duodenal perfusion of 5-HT evoked dose-dependent increases in nodose neuronal discharges. Some neurones that responded to 5-HT showed no response to either high or low doses of CCK-8. A separate group of nodose neurones that possessed high-affinity CCK type A (CCK-A) receptors also responded to luminal infusion of 5-HT. Further, a subthreshold dose of CCK-8 (i.e. 5 pmol) produced no measurable electrophysiological effects but it augmented the neuronal responses to 5-HT. This potentiation effect of CCK-8 was eliminated by CR 1409. From these results we concluded that the vagal nodose ganglion contains neurones that may possess only high- or low-affinity CCK-A receptors or 5-HT3 receptors. Some neurones that express high-affinity CCK-A receptors also express 5-HT3 receptors. Pre-exposure to luminal 5-HT may augment the subsequent response to a subthreshold dose of CCK. PMID:15235095

Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

Treesearch

Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

2001-01-01

BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

Imaging Neuroinflammation in Post Traumatic Stress Disorder

DTIC Science & Technology

2012-11-01

Metabolite B = 0-30%), without evidence of lipophilic metabolites which can confound the analysis. 8 Figure 2 Left graph : Mean PSTD... graph : There is similar plasma protein binding of 18-F PBR111 in healthy and PTSD participants. Individual subject are data are indicated on the... graph . TSPO Binder status Both mixed and high afffinity TSPO binders were evident in the PTSD (4 high affinity binders, 4 mixed affinity

Structure of Greyhound hemoglobin: origin of high oxygen affinity.

PubMed

Bhatt, Veer S; Zaldívar-López, Sara; Harris, David R; Couto, C Guillermo; Wang, Peng G; Palmer, Andre F

2011-05-01

This study presents the crystal structure of Greyhound hemoglobin (GrHb) determined to 1.9 Å resolution. GrHb was found to crystallize with an α₁β₁ dimer in the asymmetric unit and belongs to the R2 state. Oxygen-affinity measurements combined with the fact that GrHb crystallizes in the R2 state despite the high-salt conditions used for crystallization strongly indicate that GrHb can serve as a model high-oxygen-affinity hemoglobin (Hb) for higher mammals, especially humans. Structural analysis of GrHb and its comparison with the R2-state of human Hb revealed several regions that can potentially contribute to the high oxygen affinity of GrHb and serve to rationalize the additional stability of the R2-state of GrHb. A previously well studied hydrophobic cluster of bar-headed goose Hb near α119 was also incorporated in the comparison between GrHb and human Hb. Finally, a structural comparison with generic dog Hb and maned wolf Hb was conducted, revealing that in contrast to GrHb these structures belong to the R state of Hb and raising the intriguing possibility of an additional allosteric factor co-purifying with GrHb that can modulate its quaternary structure.

Affinity chromatography on monolithic supports for simultaneous and high-throughput isolation of immunoglobulins from human serum.

PubMed

Martinović, Tamara; Andjelković, Uroš; Klobučar, Marko; Černigoj, Urh; Vidič, Jana; Lučić, Marina; Pavelić, Krešimir; Josić, Djuro

2017-11-01

Posttranslational modifications of immunoglobulins have been a topic of great interest and have been repeatedly reported as a major factor in disease pathology. Cost-effective, reproducible, and high-throughput (HTP) isolation of immunoglobulins from human serum is vital for studying the changes in protein structure and the following understanding of disease development. Although there are many methods for the isolation of specific immunoglobulin classes, only a few of them are applicable for isolation of all subtypes and variants. Here, we present the development of a scheme for fast and simultaneous affinity purification of α (A), γ (G), and μ (M) immunoglobulins from human serum through affinity monolith chromatography. Affinity-based monolithic columns with immobilized protein A, G, or L were used for antibody isolation. Monolithic stationary phases have a high surface accessibility of binding sites, large flow-through channels, and can be operated at high flow rates, making them the ideal supports for HTP isolation of biopolymers. The presented method can be used for HTP screening of human serum in order to simultaneously isolate all three above-mentioned immunoglobulins and determine their concentration and changes in their glycosylation pattern as potential prognostic and diagnostic disease biomarkers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Cyclic Tetrapeptide (“Cyclodal”) and Its Mirror-Image Isomer Are Both High-Affinity μ Opioid Receptor Antagonists

PubMed Central

Weltrowska, Grazyna; Nguyen, Thi M.-D.; Chung, Nga N.; Wood, JodiAnne; Ma, Xiaoyu; Guo, Jason; Wilkes, Brian C.; Ge, Yang; Laferrière, André; Coderre, Terence J.; Schiller, Peter W.

2016-01-01

Head-to-tail cyclization of the μ opioid receptor (MOR) agonist [Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2 (9; Dmt = 2′,6′-dimethyltyrosine) resulted in a highly active, selective MOR antagonist, c[-d-Arg-Phe-Lys-Dmt-] (1) (“cyclodal”), with subnanomolar binding affinity. A docking study of cyclodal using the crystal structure of MOR in the inactive form showed a unique binding mode with the two basic residues of the ligand forming salt bridges with the Asp127 and Glu229 receptor residues. Cyclodal showed high plasma stability and was able to cross the blood–brain barrier to reverse morphine-induced, centrally mediated analgesia when given intravenously. Surprisingly, the mirror-image isomer (optical antipode) of cyclodal, c[-Arg-d-Phe-d-Lys-d-Dmt-] (2), also turned out to be a selective MOR antagonist with 1 nM binding affinity, and thus, these two compounds represent the first example of mirror image opioid receptor ligands with both optical antipodes having high binding affinity. Reduction of the Lys-Dmt peptide bond in cyclodal resulted in an analogue, c[-d-Arg-Phe-LysΨ[CH2NH]Dmt-] (8), with MOR agonist activity. PMID:27676089

LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

PubMed

Marshall, J C; Shakespear, R A; Odell, W D

1976-11-01

Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

PubMed Central

Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

2016-01-01

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

PubMed

Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

2016-03-03

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (k(off )< 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

Directed evolution of PDZ variants to generate high-affinity detection reagents.

PubMed

Ferrer, Marc; Maiolo, Jim; Kratz, Patricia; Jackowski, Jessica L; Murphy, Dennis J; Delagrave, Simon; Inglese, James

2005-04-01

High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta1-beta3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.

An unusual case of diffuse Merkel cell carcinoma successfully treated with low dose radiotherapy.

PubMed

Chatzinasiou, Foteini; Papadavid, Euaggelia; Korkolopoulou, Penelope; Levidou, Georgia; Panayiotides, Ioannis; Theodoropoulos, Konstadinos; Pogka, Vasiliki; Asimakopoulos, Charalampos; Rigopoulos, Dimitrios

2015-01-01

Merkel cell carcinoma (MCC) is a rare and highly aggressive neuroendocrine carcinoma of the skin. MCC should be included in the diagnosis of a rapidly growing infiltrating mass and histology as well as laboratory investigations such as Merkel cell polyoma virus (MCPyV) detection are valuable in its diagnosis. We present an unusual case of giant MCC-positive MCPyV in a Greek woman located on the lower leg. Our patient is very unusual in terms of her extensive MCC and her rapid and complete response to radiotherapy. © 2015 Wiley Periodicals, Inc.

Pre-Ediacaran to Ediacaran Radiation in the Vindhyan Supergroup, India

NASA Astrophysics Data System (ADS)

Srivastava, P.

2009-04-01

The Vindhyan Supergroup is globally acknowledged amongst the best repositories of the Proterozoic life evidences. Fossils of the Vindhyan Supergroup exhibit extensive diversity and variable biologic affinities represented by: bacteria, cyanobacteria, algae, fungi, acritarchs, metaphytes and metazoans (including members of the Ediacaran Fauna). The size of fossils ranges from less than a micron to almost a meter. As the Ediacaran fauna has already been recorded from the uppermost Vindhyans that is from the Bhander Group, strata lying beneath and above the Ediacaran fossil bearing horizons, exhibit presence of a vast range of fossils (both micro and mega fossils) inclining towards variable biologic affinities stated earlier. Besides identified fossils, a number of peculiar morphologies (due to deviation of morphologies from conventional structures), present in various stratigraphic horizons of the entire Vindhyan Supergroup, have also been observed. It is very difficult to identify and decide biologic affinities of these peculiar morphologies or bizarre fossil forms. In thin sections of Lower Vindhyan cherts (of Semri Group), microfossils resembling, a Volvox colony like structure and a vase- shaped body without an opening, Lichen- like or fungal forms in which a sac encompassing a coiled filament may possibly indicate a symbiotic relationship are unique. Megascopic branching and associated Grypania like structure is another form preserved as an impression on a micritic limestone slab. A very unusual and interesting fossil is a transparent disc of about one mm in diameter, composed of numerous chromosome-like structures or the appendages of an unidentified mesoscopic insect- like organism. In Upper Vindhyans, microscopic unidentified forms (in thin sections of chert) include acritarchs and acanthomorphs of variable morphologies and a dividing cell like structure interpreted as rhodophycean form or a cleaving embryo of an animal affinity. Among the carbonaceous fossils, peculiar morphologies are branched filaments that have attached sporangia-like vesicles, Chuaria-like body comprising cluster of very small sized spheroids resemble with scale like structure; a chrysophycean alga or a multicellular tissue of a metaphyte. Another carbonaceous fossil represents a possible metazoan exhibiting an elongate body and a mid-gut like structure or a voucheriacean alga. Although the biologic affinities of these forms can be a matter of debate, their biogenic nature is almost undoubted. The presence of such forms in the Vindhyans, well exhibits pre-Ediacaran - Ediacaran radiation, advancement in morphology and a gradual evolution of life during the Palaeoproterozoic- Neoproterozoic period that is the time of Vindhyan deposition. In addition, presence of large- sized acritarchs, especially the presence of an age marker acanthomorph; Trachystrichosphaera sp. in Bhander Group (Uppermost Vindhyans) also suggests Vendian as an upper age limit of the Vindhyan Supergroup (which is also supported by the presence of Ediacaran fauna from the uppermost Vindhyans.

A comparison of the properties of polyurethane immobilised Sphagnum moss, seaweed, sunflower waste and maize for the biosorption of Cu, Pb, Zn and Ni in continuous flow packed columns.

PubMed

Zhang, Yue; Banks, Charles

2006-02-01

The biosorption of Cu, Pb, Zn and Ni from a mixed solution of the metals was investigated in continuous flow packed columns containing polyurethane immobilised biomass. The characteristics and biosorption properties of Sphagnum moss, the brown seaweed Ascophyllum nodosum, waste biomass from the preparation of sunflower oil, and whole plant maize were compared. All the biomass types showed a preference for the sequestration of Pb followed by Cu, with Ni and Zn having roughly equal affinity. With continuous metal loading to the column there was an initial binding of all metals and then a displacement of the lower affinity metals by those with a high affinity. This led to a chromatographic effect in the column with breakthrough concentrations for low-affinity metals higher than the concentration in the feed. A similar phenomenon was found on desorption using acidic solutions where low-affinity metals were desorbed preferentially. The results also indicated that despite competitive displacement of one metal species by another the biomass appeared to succeed in retaining some low-affinity metal species indicating that there may be selective sites present with different affinity characteristics. When using a multi-metal solution with Cu, Pb, Zn and Ni at equal 10 mgl(-1) concentrations as column influent, the total quantities of metal sequestered were: seaweed, 117.3 mg g(-1); sunflower waste, 33.2 mg g(-1); Sphagnum moss, 32.5 mg g(-1); and maize, 2.3 mg g(-1). The use of an acid base potentiometric titration showed a relationship between the number of acid functional groups and biosorption capacity, although this was not proportional for the biomass types studied. It can, however, be used in conjunction with a simple classification of metals into high and low-affinity bands to make a preliminary assessment of a biosorption system.

Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

NASA Astrophysics Data System (ADS)

Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

2011-10-01

Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

Dextran as a Generally Applicable Multivalent Scaffold for Improving Immunoglobulin-Binding Affinities of Peptide and Peptidomimetic Ligands

PubMed Central

2015-01-01

Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors. PMID:25073654

Complex reassortment events of unusual G9P[4] rotavirus strains in India between 2011 and 2013.

PubMed

Doan, Yen Hai; Suzuki, Yoshiyuki; Fujii, Yoshiki; Haga, Kei; Fujimoto, Akira; Takai-Todaka, Reiko; Someya, Yuichi; Nayak, Mukti K; Mukherjee, Anupam; Imamura, Daisuke; Shinoda, Sumio; Chawla-Sarkar, Mamta; Katayama, Kazuhiko

2017-10-01

Rotavirus A (RVA) is the predominant etiological agent of acute gastroenteritis in young children worldwide. Recently, unusual G9P[4] rotavirus strains emerged with high prevalence in many countries. Such intergenogroup reassortant strains highlight the ongoing spread of unusual rotavirus strains throughout Asia. This study was undertaken to determine the whole genome of eleven unusual G9P[4] strains detected in India during 2011-2013, and to compare them with other human and animal global RVAs to understand the exact origin of unusual G9P[4] circulating in India and other countries worldwide. Of these 11 RVAs, four G9P[4] strains were double-reassortants with the G9-VP7 and E6-NSP4 genes on a DS-1-like genetic backbone (G9-P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2). The other strains showed a complex genetic constellation, likely derived from triple reassortment event with the G9-VP7, N1-NSP2 and E6-NSP4 on a DS-1-like genetic backbone (G9-P[4]-I2-R2-C2-M2-A2-N1-T2-E6-H2). Presumably, these unusual G9P[4] strains were generated after several reassortment events between the contemporary co-circulating human rotavirus strains. Moreover, the point mutation S291L at the interaction site between inner and outer capsid proteins of VP6 gene may be important in the rapid spread of this unusual strain. The complex reassortment events within the G9[4] strains may be related to the high prevalence of mixed infections in India as reported in this study and other previous studies. Copyright © 2017. Published by Elsevier B.V.

The Intrinsically X-Ray-weak Quasar PHL 1811. II. Optical and UV Spectra and Analysis

NASA Astrophysics Data System (ADS)

Leighly, Karen M.; Halpern, Jules P.; Jenkins, Edward B.; Casebeer, Darrin

2007-11-01

This is the second of two papers reporting observations and analysis of the unusually bright (mb=14.4), luminous (MB=-25.5), nearby (z=0.192) narrow-line quasar PHL 1811. The first paper reported that PHL 1811 is intrinsically X-ray-weak and presented a spectral energy distribution (SED). Here we present HST STIS optical and UV spectra, and ground-based optical spectra. The optical and UV line emission is very unusual. There is no evidence for forbidden or semiforbidden lines. The near-UV spectrum is dominated by very strong Fe II and Fe III, and unusual low-ionization lines such as Na I D and Ca II H and K are observed. High-ionization lines are very weak; C IV has an equivalent width of 6.6 Å, a factor of ~5 smaller than measured from quasar composite spectra. An unusual feature near 1200 Å can be deblended in terms of Lyα, N V, Si II, and C III* using the blueshifted C IV profile as a template. Photoionization modeling shows that the unusual line emission can be explained qualitatively by the unusually soft SED. Principally, a low gas temperature results in inefficient emission of collisionally excited lines, including the semiforbidden lines generally used as density diagnostics. The emission resembles that of high-density gas; in both cases this is a consequence of inefficient cooling. PHL 1811 is very unusual, but we note that quasar surveys may be biased against finding similar objects. Based on observations made with the NASA/ESA Hubble Space Telescope, obtained at the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS5-26555. These observations are associated with proposal 9181. Based on observations obtained at Kitt Peak National Observatory, a division of the National Optical Astronomy Observatories, which is operated by the Association of Universities for Research in Astronomy, Inc., under cooperative agreement with the National Science Foundation.

Relationship between helix stability and binding affinities: molecular dynamics simulations of Bfl-1/A1-binding pro-apoptotic BH3 peptide helices in explicit solvent.

PubMed

Modi, Vivek; Lama, Dilraj; Sankararamakrishnan, Ramasubbu

2013-01-01

The anti-apoptotic protein Bfl-1, also known as A1, belongs to the Bcl-2 family of proteins and interacts with pro-apoptotic Bcl-2 counterparts to regulate programmed cell death. As demonstrated for other anti-apoptotic Bcl-2 proteins, Bfl-1/A1 has also been shown to be overexpressed in various human cancers and hence they are attractive targets for anticancer drugs. Peptides derived from the BH3 region of pro-apoptotic Bcl-2 proteins have been shown to elicit similar biological response as that of parent proteins. BH3 peptides from different pro-apoptotic proteins have wide range of affinities for Bfl-1/A1. Experimentally determined complex structures show that the hydrophobic side of amphipathic BH3 peptides binds to the hydrophobic groove formed by the α-helical bundle of Bfl-1/A1 protein. Apart from the length and amino acid composition, a BH3 peptide's ability to form a stable helical structure has been suggested to be important for its high binding affinity. Molecular dynamics simulations of three BH3 peptides derived from the pro-apoptotic proteins Bak, Bid, and Bmf were carried out each for a period of at least 100 ns after 2 ns equilibration run. The length of simulated BH3 peptides varied from 22 to 24 residues and their binding affinities for Bfl-1/A1 varied from 1 to 180 nM. Our results show that the hydrophobic residues from the hydrophobic face of BH3 peptides tend to cluster together quickly to avoid being exposed to the solvent. This resulted in either reduction of helix length or complete loss of helical character. Bak and Bid BH3 peptides with high affinities for Bf1-1/A1 have stable helical segments in the N-terminal region. The highly conserved Leu residue lies just outside the helical region at the C-terminal end. Capping interactions arising out of N-cap residues seem to be extremely important to maintain the helical stability. Favorable hydrophilic interactions between residues also give further stability to the helix fragment and at least one of the interacting residues resides within the helical region. Bmf BH3 peptide with a weaker binding affinity for Bmf-1/A1 completely lost its helical character at the end of 100 ns production run and a further 50 ns simulation showed that the Bmf peptide continues to remain in random conformation. The present study clearly establishes a link between a BH3 peptide's ability to form a stable helical segment and its high binding affinity for an anti-apoptotic protein. To further test this hypothesis, we simulated a mutant Bmf peptide for 100 ns in which two residues R129 and H146 were substituted by Asn in silico in the wild-type peptide. Introduction of N-terminal Asn clearly enabled the formation of capping interactions at the N-terminus and resulted in a stable N-terminal helical segment. This demonstrates that the knowledge of interactions that help to maintain stable helical segments in a high-affinity BH3 peptide will help in designing highly specific peptide-based drugs/inhibitors. Such molecules will have the ability to bind a particular anti-apoptotic protein with high affinity.

A chimera encoding the fusion of an acetylcholine-binding protein to an ion channel is stabilized in a state close to the desensitized form of ligand-gated ion channels.

PubMed

Grutter, Thomas; Prado de Carvalho, Lia; Virginie, Dufresne; Taly, Antoine; Fischer, Markus; Changeux, Jean-Pierre

2005-03-01

To understand the mechanism of allosteric coupling between the ligand-binding domain and the ion channel of the Cys-loop ligand-gated ion channels (LGICs), we fused the soluble acetylcholine-binding protein (AChBP), which lacks an ion channel, to either the cationic serotonin type-3A ion channel (5HT(3A)) or the anionic glycine ion channel. Both linear chimeras expressed in HEK-293 cells display high affinity for the nicotinic agonist epibatidine (K(D) = 0.2-0.5 nM), but are not targeted to the cell surface. Only after substituting a ring of three loops located at the putative membrane side of the AChBP three-dimensional structure by the homologous residues of 5HT(3A), the resulting chimera AChBP(ring)/5HT(3A) (i) still displayed on intact cells an apparent high affinity for epibatidine, yet with a fourfold decrease (K(D) = 2.1 nM), (ii) displayed a high proportion of low affinity sites (11 +/- 7 microM) for the resting state stabilizing competitive antagonist alpha-bungarotoxin and (iii) was successfully targeted to the cell surface, as seen by immunofluorescence labelling. The AChBP(ring)/5HT(3A) chimera forms a pentameric structure, as revealed by sucrose gradient sedimentation. However, no whole-cell patch-clamp currents were detectable. Interestingly, binding assays with membrane fragments prepared from cells expressing AChBP(ring)/5HT(3A) showed a decrease in the apparent affinity for the agonists nicotine and epibatidine (5-fold), concomitant with an increase in the proportion of high-affinity sites (48 +/- 1 nM) for alpha-bungarotoxin. These results indicate that fusion of AChBP to an ion channel forms a pentameric receptor exposed to the cell surface and able to convert between discrete allosteric states, but stabilized in a high affinity state for epibatidine that likely corresponds to a desensitized form of LGICs. These artificial chimeras might offer a useful system to investigate signal transduction in LGICs.

A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity employing NK or high-affinity NK cells in combination with cetuximab.

PubMed

Fujii, Rika; Schlom, Jeffrey; Hodge, James W

2018-05-01

OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma. METHODS Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells have been shown to influence the degree of ADCC of tumor cells, with the high-affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high-affinity CD16 allele. These irradiated high-affinity (ha)NK cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles. RESULTS Here the authors demonstrate for the first time 1) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; 2) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; 3) that engineered haNK cells-that is, cells transduced to express the CD16 V158 FcγRIIIa receptor-bind cetuximab with similar affinity to normal NK cells expressing the high-affinity VV allele; and 4) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. CONCLUSIONS These studies provide rationale for the use of cetuximab in combination with irradiated haNK cells for therapy for chordoma.

Two classes of receptor specific for sperm-activating peptide III in sand-dollar spermatozoa.

PubMed

Yoshino, K; Suzuki, N

1992-06-15

We characterized receptors specific for sperm-activating peptide III (SAP-III: DSDSAQNLIQ) in spermatozoa of the sand dollar, Clypeaster japonicus, using both binding and cross-linking techniques. Analyses of the data obtained from the equilibrium binding of a radiolabeled SAP-III analogueto C. japonicus spermatozoa, using Klotz, Scatchard and Hill plots, showed the presence of two classes of receptors specific for SAP-III in the spermatozoa. One of the receptors (high-affinity) had a Kd of 3.4 nM and 3.4 x 10(4) binding sites/spermatozoon. The other receptor (low-affinity) had a Kd of 48 nM, with 6.1 x 10(4) binding sites/spermatozoon. The Kd of the high-affinity receptor was comparable to the median effective concentration of the intracellular-pH-increasing activity of SAP-III and that of the low-affinity receptor was comparable to the median effective concentration of the cellular-cGMP-elevating activity of the peptide. In addition, Scatchard and Hill plots of the data suggested the existence of positive cooperativity between the high-affinity members. Similar results were also obtained from a binding experiment using a sperm-membrane fraction prepared from C. japonicus spermatozoa. The incubation of intact spermatozoa or sperm plasma membranes with the radioiodinated SAP-III analogue and a chemical cross-linking reagent, disuccinimidyl suberate, resulted in the radiolabeling of three proteins with molecular masses of 126, 87 and 64 kDa, estimated by SDS/PAGE under reducing conditions.

High affinity binding of amyloid β-peptide to calmodulin: Structural and functional implications.

PubMed

Corbacho, Isaac; Berrocal, María; Török, Katalin; Mata, Ana M; Gutierrez-Merino, Carlos

2017-05-13

Amyloid β-peptides (Aβ) are a major hallmark of Alzheimer's disease (AD) and their neurotoxicity develop with cytosolic calcium dysregulation. On the other hand, calmodulin (CaM), a protein which plays a major multifunctional role in neuronal calcium signaling, has been shown to be involved in the regulation of non-amyloidogenic processing of amyloid β precursor protein (APP). Using fluorescent 6-bromoacetyl-2-dimethylaminonaphthalene derivatives of CaM, Badan-CaM, and human amyloid β(1-42) HiLyte™-Fluor555, we show in this work that Aβ binds with high affinity to CaM through the neurotoxic Aβ25-35 domain. In addition, the affinity of Aβ for calcium-saturated CaM conformation is approximately 20-fold higher than for CaM conformation in the absence of calcium (apo-CaM). Moreover, the value of K d of 0.98 ± 0.11 nM obtained for Aβ1-42 dissociation from CaM saturated by calcium points out that CaM is one of the cellular targets with highest affinity for neurotoxic Aβ peptides. A major functional consequence of Aβ-CaM interaction is that it slowdowns Aβ fibrillation. The novel and high affinity interaction between calmodulin and Aβ shown in this work opens a yet-unexplored gateway to further understand the neurotoxic effect of Aβ in different neural cells and also to address the potential of calmodulin and calmodulin-derived peptides as therapeutic agents in AD. Copyright © 2017 Elsevier Inc. All rights reserved.

Selectin catch-bonds mechanotransduce integrin activation and neutrophil arrest on inflamed endothelium under shear flow.

PubMed

Morikis, Vasilios A; Chase, Shannon; Wun, Ted; Chaikof, Elliot L; Magnani, John L; Simon, Scott I

2017-11-09

E-selectin extends from the plasma membrane of inflamed endothelium and serves to capture leukocytes from flowing blood via long-lived catch-bonds that support slow leukocyte rolling under shear stress. Its ligands are glycosylated with the tetrasaccharide sialyl Lewis x (sLe x ), which contributes to bond affinity and specificity. E-selectin-mediated rolling transmits signals into neutrophils that trigger activation of high-affinity β 2 -integrins necessary for transition to shear-resistant adhesion and transendothelial migration. Rivipansel is a glycomimetic drug that inhibits E-selectin-mediated vaso-occlusion induced by integrin-dependent sickle-red blood cell-leukocyte adhesion. How Rivipansel antagonizes ligand recognition by E-selectin and blocks outside-in signaling of integrin-mediated neutrophil arrest while maintaining rolling immune-surveillance is unknown. Here, we demonstrate that sLe x expressed on human L-selectin is preferentially bound by E-selectin and, on ligation, initiates secretion of MRP8/14 that binds TLR4 to elicit the extension of β 2 -integrin to an intermediate affinity state. Neutrophil rolling over E-selectin at precise shear stress transmits tension and catch-bond formation with L-selectin via sLe x , resulting in focal clusters that deliver a distinct signal to upshift β 2 -integrins to a high-affinity state. Rivipansel effectively blocked formation of selectin catch-bonds, revealing a novel mechanotransduction circuit that rapidly converts extended β 2 -integrins to high-affinity shear-resistant bond clusters with intracellular adhesion molecule 1 on inflamed endothelium.

A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

PubMed

Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

2012-12-01

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

PubMed Central

Junicke, Henrik; Hart, Jonathan R.; Kisko, Jennifer; Glebov, Oleg; Kirsch, Ilan R.; Barton, Jacqueline K.

2003-01-01

A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design. PMID:12610209

Current advances in screening for bioactive components from medicinal plants by affinity ultrafiltration mass spectrometry.

PubMed

Chen, Guilin; Huang, Bill X; Guo, Mingquan

2018-05-21

Medicinal plants have played an important role in maintaining human health for thousands of years. However, the interactions between the active components in medicinal plants and some certain biological targets during a disease are still unclear in most cases. To conduct the high-throughput screening for small active molecules that can interact with biological targets, which is of great theoretical significance and practical value. The ultrafiltration mass spectrometry (UF-LC/MS) is a powerful bio-analytical method by combining affinity ultrafiltration and liquid chromatography-mass spectrometry (LC/MS), which could rapidly screen and identify small active molecules that bind to biological targets of interest at the same time. Compared with other analytical methods, affinity UF-LC/MS has the characteristics of fast, sensitive and high throughput, and is especially suitable for the complicated extracts of medicinal plants. In this review, the basic principle, characteristics and some most recent challenges in UF-LC/MS have been demonstrated. Meanwhile, the progress and applications of affinity UF-LC/MS in the discovery of the active components from natural medicinal plants and the interactions between small molecules and biological target proteins are also briefly summarised. In addition, the future directions for UF-LC/MS are also prospected. Affinity UF-LC/MS is a powerful tool in studies on the interactions between small active molecules and biological protein targets, especially in the high-throughput screening of active components from the natural medicinal plants. Copyright © 2018 John Wiley & Sons, Ltd.

Tuning the Protein Corona of Hydrogel Nanoparticles: The Synthesis of Abiotic Protein and Peptide Affinity Reagents.

PubMed

O'Brien, Jeffrey; Shea, Kenneth J

2016-06-21

Nanomaterials, when introduced into a complex, protein-rich environment, rapidly acquire a protein corona. The type and amount of proteins that constitute the corona depend significantly on the synthetic identity of the nanomaterial. For example, hydrogel nanoparticles (NPs) such as poly(N-isopropylacrylamide) (NIPAm) have little affinity for plasma proteins; in contrast, carboxylated poly(styrene) NPs acquire a dense protein corona. This range of protein adsorption suggests that the protein corona might be "tuned" by controlling the chemical composition of the NP. In this Account, we demonstrate that small libraries of synthetic polymer NPs incorporating a diverse pool of functional monomers can be screened for candidates with high affinity and selectivity to targeted biomacromolecules. Through directed synthetic evolution of NP compositions, one can tailor the protein corona to create synthetic organic hydrogel polymer NPs with high affinity and specificity to peptide toxins, enzymes, and other functional proteins, as well as to specific domains of large proteins. In addition, many NIPAm NPs undergo a change in morphology as a function of temperature. This transformation often correlates with a significant change in NP-biomacromolecule affinity, resulting in a temperature-dependent protein corona. This temperature dependence has been used to develop NP hydrogels with autonomous affinity switching for the protection of proteins from thermal stress and as a method of biomacromolecule purification through a selective thermally induced catch and release. In addition to temperature, changes in pH or buffer can also alter a NP protein corona composition, a property that has been exploited for protein purification. Finally, synthetic polymer nanoparticles with low nanomolar affinity for a peptide toxin were shown to capture and neutralize the toxin in the bloodstream of living mice. While the development of synthetic polymer alternatives to protein affinity reagents is in its early stages, these recent successes using only small libraries of functional monomers are most encouraging. It is likely that by expanding the chemical diversity of functional hydrogels and other polymers, a much broader range of NP-biomacromolecule affinity pairs will result. Since these robust, nontoxic polymers are readily synthesized in the chemistry laboratory, we believe the results presented in this Account offer a promising future for the development of low cost alternatives to more traditional protein affinity reagents such as antibodies.

Reflecting on My Progressive Education

ERIC Educational Resources Information Center

Friend, Nina

2012-01-01

At the end of the 2011-12 version of the project course Schools Across Borders, Schools Across Time (SABSAT), a high school senior wrote a series of letters reflecting on the experience of participating in an unusual course with an unusual outcome. In the letters, the student wanted to evoke what was personal and what was critical--to herself, her…

An unusual reaction of α-alkoxyphosphonium salts with Grignard reagents under an O2 atmosphere.

PubMed

Fujioka, Hiromichi; Goto, Akihiro; Otake, Kazuki; Kubo, Ozora; Sawama, Yoshinari; Maegawa, Tomohiro

2011-09-21

An unusual and novel reaction of α-alkoxyphosphonium salts, generated from O,O-acetals and Ph(3)P, with Grignard reagents under an O(2) atmosphere afforded alcohols in moderate to high yields. It was clarified by isotopic labelling experiments that the reaction proceeded via a novel radical pathway.