Divergent Synthesis of Revised Apratoxin E, 30-epi-Apratoxin E, and 30S/30R-Oxoapratoxin E.

PubMed

Mao, Zhuo-Ya; Si, Chang-Mei; Liu, Yi-Wen; Dong, Han-Qing; Wei, Bang-Guo; Lin, Guo-Qiang

2017-10-20

In this report, originally proposed apratoxin E (30S-7), revised apratoxin E (30R-7), and (30S)/(30R)-oxoapratoxin E (30S)-38/(30R)-38 were efficiently prepared by two synthetic methods. The chiral lactone 10, recycled from the degradation of saponin glycosides, was utilized to prepare the key nonpeptide fragment 9. Our alternative convergent assembly strategy was applied to the divergent synthesis of revised apratoxin E and its three analogues. Moreover, ring-closing metathesis (RCM) was for the first time found to be an efficient strategy for the macrocyclization of apratoxins.

Binding of Y-P30 to Syndecan 2/3 Regulates the Nuclear Localization of CASK

PubMed Central

Landgraf, Peter; Mikhaylova, Marina; Macharadze, Tamar; Borutzki, Corinna; Zenclussen, Ana-Claudia; Wahle, Petra; Kreutz, Michael R.

2014-01-01

The survival promoting peptide Y-P30 has documented neuroprotective effects as well as cell survival and neurite outgrowth promoting activity in vitro and in vivo. Previous work has shown that multimerization of the peptide with pleiotrophin (PTN) and subsequent binding to syndecan (SDC) -2 and -3 is involved in its neuritogenic effects. In this study we show that Y-P30 application regulates the nuclear localization of the SDC binding partner Calcium/calmodulin-dependent serine kinase (CASK) in neuronal primary cultures during development. In early development at day in vitro (DIV) 8 when mainly SDC-3 is expressed supplementation of the culture medium with Y-P30 reduces nuclear CASK levels whereas it has the opposite effect at DIV 18 when SDC-2 is the dominant isoform. In the nucleus CASK regulates gene expression via its association with the T-box transcription factor T-brain-1 (Tbr-1) and we indeed found that gene expression of downstream targets of this complex, like the GluN2B NMDA-receptor, exhibits a corresponding down- or up-regulation at the mRNA level. The differential effect of Y-P30 on the nuclear localization of CASK correlates with its ability to induce shedding of the ectodomain of SDC-2 but not -3. shRNA knockdown of SDC-2 at DIV 18 and SDC-3 at DIV 8 completely abolished the effect of Y-P30 supplementation on nuclear CASK levels. During early development a protein knockdown of SDC-3 also attenuated the effect of Y-P30 on axon outgrowth. Taken together these data suggest that Y-P30 can control the nuclear localization of CASK in a SDC-dependent manner. PMID:24498267

Impact of disruption of secondary binding site S2 on dopamine transporter function.

PubMed

Zhen, Juan; Reith, Maarten E A

2016-09-01

The structures of the leucine transporter, drosophila dopamine transporter, and human serotonin transporter show a secondary binding site (designated S2 ) for drugs and substrate in the extracellular vestibule toward the membrane exterior in relation to the primary substrate recognition site (S1 ). The present experiments are aimed at disrupting S2 by mutating Asp476 and Ile159 to Ala. Both mutants displayed a profound decrease in [(3) H]DA uptake compared with wild-type associated with a reduced turnover rate kcat . This was not caused by a conformational bias as the mutants responded to Zn(2+) (10 μM) similarly as WT. The dopamine transporters with either the D476A or I159A mutation both displayed a higher Ki for dopamine for the inhibition of [3H](-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane binding than did the WT transporter, in accordance with an allosteric interaction between the S1 and S2 sites. The results provide evidence in favor of a general applicability of the two-site allosteric model of the Javitch/Weinstein group from LeuT to dopamine transporter and possibly other monoamine transporters. X-ray structures of transporters closely related to the dopamine (DA) transporter show a secondary binding site S2 in the extracellular vestibule proximal to the primary binding site S1 which is closely linked to one of the Na(+) binding sites. This work examines the relationship between S2 and S1 sites. We found that S2 site impairment severely reduced DA transport and allosterically reduced S1 site affinity for the cocaine analog [(3) H]CFT. Our results are the first to lend direct support for the application of the two-site allosteric model, advanced for bacterial LeuT, to the human DA transporter. The model states that, after binding of the first DA molecule (DA1 ) to the primary S1 site (along with Na(+) ), binding of a second DA (DA2 ) to the S2 site triggers, through an allosteric interaction, the release of DA1 and Na(+) into the cytoplasm. © 2016

The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

PubMed

Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

2016-08-15

Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends. © 2016 Ly, Moroz, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Pyrazole-based cathepsin S inhibitors with arylalkynes as P1 binding elements

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

Ameriks, Michael K.; Axe, Frank U.; Bembenek, Scott D.

A crystal structure of 1 bound to a Cys25Ser mutant of cathepsin S helped to elucidate the binding mode of a previously disclosed series of pyrazole-based CatS inhibitors and facilitated the design of a new class of arylalkyne analogs. Optimization of the alkyne and tetrahydropyridine portions of the pharmacophore provided potent CatS inhibitors (IC{sub 50} = 40-300 nM), and an X-ray structure of 32 revealed that the arylalkyne moiety binds in the S1 pocket of the enzyme.

An NMR-Based Structural Rationale for Contrasting Stoichiometry and Ligand Binding Site(s) in Fatty Acid-binding Proteins†

PubMed Central

He, Yan; Estephan, Rima; Yang, Xiaomin; Vela, Adriana; Wang, Hsin; Bernard, Cédric; Stark, Ruth E.

2011-01-01

Liver fatty acid-binding protein (LFABP) is a 14-kDa cytosolic polypeptide, differing from other family members in number of ligand binding sites, diversity of bound ligands, and transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional 1H-15N NMR signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without solving the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and a R122L/S124A mutant in which electrostatic interactions viewed as essential to fatty acid binding were removed. For wild-type LFABP the results compared favorably with previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, 1H/15N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family. PMID:21226535

Estimating V̄s(30) (or NEHRP site classes) from shallow velocity models (depths < 30 m)

USGS Publications Warehouse

Boore, David M.

2004-01-01

The average velocity to 30 m [V??s(30)] is a widely used parameter for classifying sites to predict their potential to amplify seismic shaking. In many cases, however, models of shallow shear-wave velocities, from which V??s(30) can be computed, do not extend to 30 m. If the data for these cases are to be used, some method of extrapolating the velocities must be devised. Four methods for doing this are described here and are illustrated using data from 135 boreholes in California for which the velocity model extends to at least 30 m. Methods using correlations between shallow velocity and V??s(30) result in significantly less bias for shallow models than the simplest method of assuming that the lowermost velocity extends to 30 m. In addition, for all methods the percent of sites misclassified is generally less than 10% and falls to negligible values for velocity models extending to at least 25 m. Although the methods using correlations do a better job on average of estimating V??s(30), the simplest method will generally result in a lower value of V??s(30) and thus yield a more conservative estimate of ground motion [which generally increases as V??s(30) decreases].

Characterisation of the selective binding of antibiotics vancomycin and teicoplanin by the VanS receptor regulating type A vancomycin resistance in the enterococci.

PubMed

Hughes, C S; Longo, E; Phillips-Jones, M K; Hussain, R

2017-08-01

A-type resistance towards "last-line" glycopeptide antibiotic vancomycin in the leading hospital acquired infectious agent, the enterococci, is the most common in the UK. Resistance is regulated by the VanR A S A two-component system, comprising the histidine sensor kinase VanS A and the partner response regulator VanR A . The nature of the activating ligand for VanS A has not been identified, therefore this work sought to identify and characterise ligand(s) for VanS A . In vitro approaches were used to screen the structural and activity effects of a range of potential ligands with purified VanS A protein. Of the screened ligands (glycopeptide antibiotics vancomycin and teicoplanin, and peptidoglycan components N-acetylmuramic acid, D-Ala-D-Ala and Ala-D-y-Glu-Lys-D-Ala-D-Ala) only glycopeptide antibiotics vancomycin and teicoplanin were found to bind VanS A with different affinities (vancomycin 70μM; teicoplanin 30 and 170μM), and were proposed to bind via exposed aromatic residues tryptophan and tyrosine. Furthermore, binding of the antibiotics induced quicker, longer-lived phosphorylation states for VanS A , proposing them as activators of type A vancomycin resistance in the enterococci. Copyright © 2017 Diamond Light Source Ltd. Published by Elsevier B.V. All rights reserved.

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.

PubMed

Mundus, D; Wollenzien, P

1998-11-01

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.

Kinetics of phloretin binding to phosphatidylcholine vesicle membranes

PubMed Central

1980-01-01

The submillisecond kinetics for phloretin binding to unilamellar phosphatidylcholine (PC) vesicles was investigated using the temperature-jump technique. Spectrophotometric studies of the equilibrium binding performed at 328 nm demonstrated that phloretin binds to a single set of independent, equivalent sites on the vesicle with a dissociation constant of 8.0 microM and a lipid/site ratio of 4.0. The temperature of the phloretin-vesicle solution was jumped by 4 degrees C within 4 microseconds producing a monoexponential, concentration-dependent relaxation process with time constants in the 30--200-microseconds time range. An analysis of the concentration dependence of relaxation time constants at pH 7.30 and 24 degrees C yielded a binding rate constant of 2.7 X 10(8) M-1 s-1 and an unbinding constant of 2,900 s-1; approximately 66 percent of total binding sites are exposed at the outer vesicle surface. The value of the binding rate constant and three additional observations suggest that the binding kinetics are diffusion limited. The phloretin analogue, naringenin, which has a diffusion coefficient similar to phloretin yet a dissociation constant equal to 24 microM, bound to PC vesicle with the same rate constant as phloretin did. In addition, the phloretin-PC system was studied in buffers made one to six times more viscous than water by addition of sucrose or glycerol to the differ. The equilibrium affinity for phloretin binding to PC vesicles is independent of viscosity, yet the binding rate constant decreases with the expected dependence (kappa binding alpha 1/viscosity) for diffusion-limited processes. Thus, the binding rate constant is not altered by differences in binding affinity, yet depends upon the diffusion coefficient in buffer. Finally, studies of the pH dependence of the binding rate constant showed a dependence (kappa binding alpha [1 + 10pH-pK]) consistent with the diffusion-limited binding of a weak acid. PMID:7391812

First measurement of 30S+α resonant elastic scattering for the 30S(α ,p ) reaction rate

NASA Astrophysics Data System (ADS)

Kahl, D.; Yamaguchi, H.; Kubono, S.; Chen, A. A.; Parikh, A.; Binh, D. N.; Chen, J.; Cherubini, S.; Duy, N. N.; Hashimoto, T.; Hayakawa, S.; Iwasa, N.; Jung, H. S.; Kato, S.; Kwon, Y. K.; Nishimura, S.; Ota, S.; Setoodehnia, K.; Teranishi, T.; Tokieda, H.; Yamada, T.; Yun, C. C.; Zhang, L. Y.

2018-01-01

Background: Type I x-ray bursts are the most frequently observed thermonuclear explosions in the galaxy, resulting from thermonuclear runaway on the surface of an accreting neutron star. The 30S(α ,p ) reaction plays a critical role in burst models, yet insufficient experimental information is available to calculate a reliable, precise rate for this reaction. Purpose: Our measurement was conducted to search for states in 34Ar and determine their quantum properties. In particular, natural-parity states with large α -decay partial widths should dominate the stellar reaction rate. Method: We performed the first measurement of 30S+α resonant elastic scattering up to a center-of-mass energy of 5.5 MeV using a radioactive ion beam. The experiment utilized a thick gaseous active target system and silicon detector array in inverse kinematics. Results: We obtained an excitation function for 30S(α ,α ) near 150∘ in the center-of-mass frame. The experimental data were analyzed with R -matrix calculations, and we observed three new resonant patterns between 11.1 and 12.1 MeV, extracting their properties of resonance energy, widths, spin, and parity. Conclusions: We calculated the resonant thermonuclear reaction rate of 30S(α ,p ) based on all available experimental data of 34Ar and found an upper limit about one order of magnitude larger than a rate determined using a statistical model. The astrophysical impact of these two rates has been investigated through one-zone postprocessing type I x-ray burst calculations. We find that our new upper limit for the 30S(α ,p )33Cl rate significantly affects the predicted nuclear energy generation rate during the burst.

Design and Synthesis of 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile (Citalopram) Analogues as Novel Probes for the Serotonin Transporter S1 and S2 Binding Sites

PubMed Central

Banala, Ashwini K.; Zhang, Peng; Plenge, Per; Cyriac, George; Kopajtic, Theresa; Katz, Jonathan L.; Loland, Claus Juul; Newman, Amy Hauck

2013-01-01

The serotonin transporter (SERT) is the primary target for antidepressant drugs. The existence of a high affinity primary orthosteric binding site (S1) and a low affinity secondary site (S2) has been described and their relation to antidepressant pharmacology has been debated. Herein, structural modifications to the N-, 4, 5, and 4’-positions of (±)citalopram (1) are reported. All of the analogues were SERT-selective and demonstrated that steric bulk was tolerated at the SERT S1 site, including two dimeric ligands (15 and 51.) In addition, 8 analogues were identified with similar potencies to S-1 for decreasing the dissociation of [3H]S-1 from the S1 site, via allosteric modulation at S2. Both dimeric compounds had similar affinities for the SERT S1 site (Ki=19.7 and 30.2 nM, respectively), whereas only the N-substituted analogue, 51, was as effective as S-1 in allosterically modulating the binding of [3H]S-1 via S2. PMID:24237160

GenProBiS: web server for mapping of sequence variants to protein binding sites.

PubMed

Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

2017-07-03

Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

CD30 on extracellular vesicles from malignant Hodgkin cells supports damaging of CD30 ligand-expressing bystander cells with Brentuximab-Vedotin, in vitro

PubMed Central

Hansen, Hinrich P.; Trad, Ahmad; Dams, Maria; Zigrino, Paola; Moss, Marcia; Tator, Maximilian; Schön, Gisela; Grenzi, Patricia C; Bachurski, Daniel; Aquino, Bruno; Dürkop, Horst; Reiners, Katrin S; von Bergwelt-Baildon, Michael; Hallek, Michael; Grötzinger, Joachim; Engert, Andreas; Leme, Adriana F Paes; von Strandmann, Elke Pogge

2016-01-01

The goal of targeted immunotherapy in cancer is to damage both malignant and tumor-supporting cells of the microenvironment but spare unaffected tissue. The malignant cells in classical Hodgkin lymphoma (cHL) selectively express CD30. They release this receptor on extracellular vesicles (EVs) for the tumor-supporting communication with CD30 ligand (CD30L)-positive bystander cells. Here, we investigated how CD30-positive EVs influence the efficacy of the CD30 antibody drug conjugate (ADC) Brentuximab Vedotin (SGN-35). The malignant cells and the EVs expressed the active sheddase ADAM10. ADAM10 cleaved and released the CD30 ectodomain (sCD30), causing a gradual depletion of SGN-35 binding sites on EVs and creating a soluble competitor of the ADC therapy. In a 3D semi-solid tumor microenvironment model, the EVs were retained in the matrix whereas sCD30 penetrated readily into the surrounding culture medium. This resulted in a lowered ratio of EV-associated CD30 (CD30EV) to sCD30 in the surrounding medium in comparison to non-embedded cultures. A low percentage of CD30EV was also detected in the plasma of cHL patients, supporting the clinical relevance of the model. The adherence of CD30EV but not sCD30 to CD30−/CD30L+ mast cells and eosinophils allowed the indirect binding of SGN-35. Moreover, SGN-35 damaged CD30-negative cells, provided they were loaded with CD30+ EVs. PMID:27105521

sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides

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

Luo, Heng; Ye, Hao; Ng, Hui Wen

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. Furthermore, this algorithmmore » can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system.« less

sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides

PubMed Central

Luo, Heng; Ye, Hao; Ng, Hui Wen; Sakkiah, Sugunadevi; Mendrick, Donna L.; Hong, Huixiao

2016-01-01

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. This algorithm can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system. PMID:27558848

sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides

DOE PAGES

Luo, Heng; Ye, Hao; Ng, Hui Wen; ...

2016-08-25

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. Furthermore, this algorithmmore » can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system.« less

Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPgammaS binding study.

PubMed

Newman-Tancredi, A; Gavaudan, S; Conte, C; Chaput, C; Touzard, M; Verrièle, L; Audinot, V; Millan, M J

1998-08-21

Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical

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

Drug Resistance Mechanism of L10F, L10F/N88S and L90M mutations in CRF01_AE HIV-1 protease: Molecular dynamics simulations and binding free energy calculations.

PubMed

Vasavi, C S; Tamizhselvi, Ramasamy; Munusami, Punnagai

2017-08-01

HIV-1 protease plays a crucial role in viral replication and maturation, which makes it one of the most attractive targets for anti-retroviral therapy. The majority of HIV infections in developing countries are due to non-B subtype. Subtype AE is spreading rapidly and infecting huge population worldwide. The mutations in the active site of subtype AE directly impair the interactions with the inhibitor. The non-active site mutations influence the binding of the inhibitor indirectly and their resistance mechanism is not well understood. It is important to design new effective inhibitors that combat drug resistance in subtype AE protease. In this work, we examined the effect of non active site mutations L10F, L10F/N88S and L90M with nelfinavir using molecular dynamics simulation and binding free energy calculations. The simulations suggested that the L10F and L10F/N88S mutants decrease the binding affinity of nelfinavir, whereas the L90M mutant increases the binding affinity. The formation of hydrogen bonds between nelfinavir and Asp30 is crucial for effective binding. The benzamide moiety of nelfinavir shows large positional deviation in L10F and L10F/N88S complexes and the L10F/N88S mutation changes the hydrogen bond between the side chain atoms of 30th residue and the 88th residue. Consequently the hydrogen bond interaction between Asp30 and nelfinavir are destroyed leading to drug resistance. Our present study shed light on the resistance mechanism of the strongly linked mutation L10F/N88S observed experimentally in AE subtype. Copyright © 2017 Elsevier Inc. All rights reserved.

Study on the binding of procaine hydrochloride to DNA/DNA bases and the effect of CdS nanoparticles on the binding behavior.

PubMed

Ping, Gang; Lv, Gang; Gutmann, Sebastian; Chen, Chen; Zhang, Renyun; Wang, Xuemei

2006-01-01

The interaction between procaine hydrochloride and DNA/DNA bases in the absence and presence of cadmium sulfide (CdS) nanoparticles has been explored in this study by using differential pulse voltammetry, atomic force microscopy (AFM) and so on, which illustrates the different binding behaviors of procaine hydrochloride with different DNA bases. The results clearly indicate that the binding of purines to procaine hydrochloride is stronger than that of pyrimidines and the binding affinity is in the order of G > A > T > C. In addition, it was observed that the presence of CdS nanoparticles could remarkably enhance the probing sensitivity for the interaction between procaine hydrochloride and DNA/DNA bases. Furthermore, AFM study illustrates that procaine hydrochloride can bind to some specific sites of DNA chains, which indicates that procaine hydrochloride may interact with some special sequences of DNA.

Crystal Structure and Theoretical Analysis of Green Gold Au 30 (S- t Bu) 18 Nanomolecules and Their Relation to Au 30 S(S- t Bu) 18

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

Dass, Amala; Jones, Tanya; Rambukwella, Milan

We report the complete X-ray crystallographic structure as determined through single crystal X-ray diffraction and a thorough theoretical analysis of the green gold Au30(S-tBu)18. While the structure of Au30S(S-tBu)18 with 19 sulfur atoms has been reported, the crystal structure of Au30(S-tBu)18 without the μ3-sulfur has remained elusive until now, though matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) data unequivocally shows its presence in abundance. The Au30(S-tBu)18 nanomolecule is not only distinct in its crystal structure but has unique temperature dependent optical properties. Structure determination allows a rigorous comparison and an excellent agreement with theoreticalmore » predictions of structure, stability, and optical response.« less

Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

PubMed

Tan, Cheng; Li, Wenfei; Wang, Wei

2013-12-19

Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

Identification of the HrpS binding site in the hrpL promoter and effect of the RpoN binding site of HrpS on the regulation of the type III secretion system in Erwinia amylovora.

PubMed

Lee, Jae Hoon; Sundin, George W; Zhao, Youfu

2016-06-01

The type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by an RpoN-HrpL sigma factor cascade, which is activated by the bacterial alarmone (p)ppGpp. In this study, the binding site of HrpS, an enhancer binding protein, was identified for the first time in plant-pathogenic bacteria. Complementation of the hrpL mutant with promoter deletion constructs of the hrpL gene and promoter activity analyses using various lengths of the hrpL promoter fused to a promoter-less green fluorescent protein (gfp) reporter gene delineated the upstream region for HrpS binding. Sequence analysis revealed a dyad symmetry sequence between -138 and -125 nucleotides (TGCAA-N4-TTGCA) as the potential HrpS binding site, which is conserved in the promoter of the hrpL gene among plant enterobacterial pathogens. Results of quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and electrophoresis mobility shift assay coupled with site-directed mutagenesis (SDM) analysis showed that the intact dyad symmetry sequence was essential for HrpS binding, full activation of T3SS gene expression and virulence. In addition, the role of the GAYTGA motif (RpoN binding site) of HrpS in the regulation of T3SS gene expression in E. amylovora was characterized by complementation of the hrpS mutant using mutant variants generated by SDM. Results showed that a Y100F substitution of HrpS complemented the hrpS mutant, whereas Y100A and Y101A substitutions did not. These results suggest that tyrosine (Y) and phenylalanine (F) function interchangeably in the conserved GAYTGA motif of HrpS in E. amylovora. © 2015 BSPP AND JOHN WILEY & SONS LTD.

An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

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

Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

2016-06-13

ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface inmore » which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.« less

ProBiS-ligands: a web server for prediction of ligands by examination of protein binding sites.

PubMed

Konc, Janez; Janežič, Dušanka

2014-07-01

The ProBiS-ligands web server predicts binding of ligands to a protein structure. Starting with a protein structure or binding site, ProBiS-ligands first identifies template proteins in the Protein Data Bank that share similar binding sites. Based on the superimpositions of the query protein and the similar binding sites found, the server then transposes the ligand structures from those sites to the query protein. Such ligand prediction supports many activities, e.g. drug repurposing. The ProBiS-ligands web server, an extension of the ProBiS web server, is open and free to all users at http://probis.cmm.ki.si/ligands. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

PubMed

Banasik, Michał; Sachadyn, Paweł

2016-09-01

A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.

Overlearned responses hinder S-R binding.

PubMed

Moeller, Birte; Frings, Christian

2017-01-01

Two mechanisms that are important for human action control are the integration of individual action plans (see Hommel, Müsseler, Aschersleben, & Prinz, 2001) and the automatization of overlearned actions to familiar stimuli (see Logan, 1988). In the present study, we analyzed the influence of automatization on action plan integration. Integration with pronunciation responses were compared for response incompatible word and nonword stimuli. Stimulus-response binding effects were observed for nonwords. In contrast, words that automatically triggered an overlearned pronunciation response were not integrated with pronunciation of a different word. That is, automatized response retrieval hindered binding effects regarding the retrieving stimulus and a new response. The results are a first indication of the way that binding and learning processes interact, and might also be a first step to understanding the more complex interdependency of the processes responsible for stimulus-response binding in action control and stimulus-response associations in learning research. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Binding of Dihydrostreptomycin to Escherichia coli Ribosomes: Characteristics and Equilibrium of the Reaction

PubMed Central

Chang, F. N.; Flaks, Joel G.

1972-01-01

The binding of dihydrostreptomycin to ribosomes and ribosomal subunits of a number of different Escherichia coli strains was studied, and the Mg2+ and pH dependence, as well as the effect of salts and polynucleotides, was determined. The only requirement for binding with ribosomes and subunits from susceptible strains was 10 mm Mg2+. Monovalent salts weakened the binding in a manner similar to the effects on ribonucleic acid secondary structure, and this was antagonized to some extent by increased amounts of Mg2+. Bound dihydrostreptomycin could be readily exchanged by streptomycin and any antibiotically active derivative, but not by fragments of the antibiotic or any other aminoglycoside. With native (run-off) 70S ribosomes from streptomycin-susceptible strains, the binding was rapid and relatively temperature independent over the range from 0 to 37 C. Polynucleotides did not stimulate the binding. With concentrations of dihydrostreptomycin up to 10−5m, greater than 95% of native 70S ribosomes bound exactly 1 molecule of the antibiotic tightly, with a Kdiss for the bound complex at 25 C of 9.4 × 10−8m. The following thermodynamic parameters were found for the binding with 70S ribosomes at 25 C:ΔG° = −9.6 kcal/mole, ΔH° = −6.2 kcal/mole, and ΔS° = +11.4 entropy units/mole. Differences in affinity for the antibiotic were found between ribosomes of K-12 strains and those of other E. coli strains. There was insignificant binding to 70S ribosomes or subunits from streptomycin-resistant or -dependent strains, and to 50S subunits from susceptible strains. The binding to 30S subunits from susceptible strains was weaker by an order of magnitude than that to the 70S particle, with a Kdiss at 25 C of 10−6m. Polyuridylic acid stimulated this binding slightly but did not influence the affinity of the bound molecule. At antibiotic concentrations above 10−5m, streptomycin-susceptible 70S and 30S particles bound additional molecules of the antibiotic, and

Posttransplant sCD30 as a predictor of kidney graft outcome.

PubMed

Süsal, Caner; Döhler, Bernd; Sadeghi, Mahmoud; Salmela, Kaija T; Weimer, Rolf; Zeier, Martin; Opelz, Gerhard

2011-06-27

Reliable markers for assessing the biological effect of immunosuppressive drugs and identification of transplant recipients at risk of developing rejection are not available. In a prospective multicenter study, we investigated whether posttransplant measurement of the T-cell activation marker soluble CD30 (sCD30) can be used for estimating the risk of graft loss in kidney transplant recipients. Pre- and posttransplant sera of 2322 adult deceased-donor kidney recipients were tested for serum sCD30 content using a commercial enzyme-linked immunosorbent assay. sCD30 decreased posttransplant and reached a nadir on day 30. Patients with a high sCD30 of more than or equal to 40 U/mL on day 30 showed a subsequent graft survival rate after 3 years of 78.3±4.1%, significantly lower than the 90.3±1.0% rate in recipients with a low sCD30 on day 30 of less than 40 U/mL (log-rank P<0.001; Cox hazard ratio 2.02, P<0.001). Although an association was found between pre- and posttransplant sCD30 levels, patients with high sCD30 on posttransplant day 30 demonstrated significantly lower 3-year graft survival irrespective of the pretransplant level. Our data suggest that posttransplant measurement of sCD30 on day 30 is a predictor of subsequent graft loss in kidney transplant recipients and that sCD30 may potentially serve as an indicator for adjustment of immunosuppressive medication.

1-3-A Resolution Structure of Human Glutathione S-Transferase With S-Hexyl Glutathione Bound Reveals Possible Extended Ligandin Binding Site

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

Trong, I.Le; Stenkamp, R.E.; Ibarra, C.

2005-08-22

Cytosolic glutathione S-transferases (GSTs) play a critical role in xenobiotic binding and metabolism, as well as in modulation of oxidative stress. Here, the high-resolution X-ray crystal structures of homodimeric human GSTA1-1 in the apo form and in complex with S-hexyl glutathione (two data sets) are reported at 1.8, 1.5, and 1.3A respectively. At this level of resolution, distinct conformations of the alkyl chain of S-hexyl glutathione are observed, reflecting the nonspecific nature of the hydrophobic substrate binding site (H-site). Also, an extensive network of ordered water, including 75 discrete solvent molecules, traverses the open subunit-subunit interface and connects the glutathionemore » binding sites in each subunit. In the highest-resolution structure, three glycerol moieties lie within this network and directly connect the amino termini of the glutathione molecules. A search for ligand binding sites with the docking program Molecular Operating Environment identified the ordered water network binding site, lined mainly with hydrophobic residues, suggesting an extended ligand binding surface for nonsubstrate ligands, the so-called ligandin site. Finally, detailed comparison of the structures reported here with previously published X-ray structures reveal a possible reaction coordinate for ligand-dependent conformational changes in the active site and the C-terminus.« less

Domain wise docking analyses of the modular chitin binding protein CBP50 from Bacillus thuringiensis serovar konkukian S4.

PubMed

Sehar, Ujala; Mehmood, Muhammad Aamer; Hussain, Khadim; Nawaz, Salman; Nadeem, Shahid; Siddique, Muhammad Hussnain; Nadeem, Habibullah; Gull, Munazza; Ahmad, Niaz; Sohail, Iqra; Gill, Saba Shahid; Majeed, Summera

2013-01-01

This paper presents an in silico characterization of the chitin binding protein CBP50 from B. thuringiensis serovar konkukian S4 through homology modeling and molecular docking. The CBP50 has shown a modular structure containing an N-terminal CBM33 domain, two consecutive fibronectin-III (Fn-III) like domains and a C-terminal CBM5 domain. The protein presented a unique modular structure which could not be modeled using ordinary procedures. So, domain wise modeling using MODELLER and docking analyses using Autodock Vina were performed. The best conformation for each domain was selected using standard procedure. It was revealed that four amino acid residues Glu-71, Ser-74, Glu-76 and Gln-90 from N-terminal domain are involved in protein-substrate interaction. Similarly, amino acid residues Trp-20, Asn-21, Ser-23 and Val-30 of Fn-III like domains and Glu-15, Ala-17, Ser-18 and Leu-35 of C-terminal domain were involved in substrate binding. Site-directed mutagenesis of these proposed amino acid residues in future will elucidate the key amino acids involved in chitin binding activity of CBP50 protein.

Regional correlations of V s30 and velocities averaged over depths less than and greater than 30 meters

USGS Publications Warehouse

Boore, D.M.; Thompson, E.M.; Cadet, H.

2011-01-01

Using velocity profiles from sites in Japan, California, Turkey, and Europe, we find that the time-averaged shear-wave velocity to 30 m (V S30), used as a proxy for site amplification in recent ground-motion prediction equations (GMPEs) and building codes, is strongly correlated with average velocities to depths less than 30 m (V Sz, with z being the averaging depth). The correlations for sites in Japan (corresponding to the KiK-net network) show that V S30 is systematically larger for a given V Sz than for profiles from the other regions. The difference largely results from the placement of the KiK-net station locations on rock and rocklike sites, whereas stations in the other regions are generally placed in urban areas underlain by sediments. Using the KiK-net velocity profiles, we provide equations relating V S30 to V Sz for z ranging from 5 to 29 m in 1-m increments. These equations (and those for California velocity profiles given in Boore, 2004b) can be used to estimate V S30 from V Sz for sites in which velocity profiles do not extend to 30 m. The scatter of the residuals decreases with depth, but, even for an averaging depth of 5 m, a variation in log V S30 of 1 standard deviation maps into less than a 20% uncertainty in ground motions given by recent GMPEs at short periods. The sensitivity of the ground motions to V S30 uncertainty is considerably larger at long periods (but is less than a factor of 1.2 for averaging depths greater than about 20 m). We also find that V S30 is correlated with V Sz for z as great as 400 m for sites of the KiK-net network, providing some justification for using V S30 as a site-response variable for predicting ground motions at periods for which the wavelengths far exceed 30 m.

Ap4A and ADP-beta-S binding to P2 purinoceptors present on rat brain synaptic terminals.

PubMed Central

Pintor, J.; Díaz-Rey, M. A.; Miras-Portugal, M. T.

1993-01-01

1. Diadenosine tetraphosphate (Ap4A) a dinucleotide stored and released from rat brain synaptic terminals presents two types of affinity binding sites in synaptosomes. When [3H]-Ap4A was used for binding studies a Kd value of 0.10 +/- 0.014 nM and a Bmax value of 16.6 +/- 1.2 fmol mg-1 protein were obtained for the high affinity binding site from the Scatchard analysis. The second binding site, obtained by displacement studies, showed a Ki value of 0.57 +/- 0.09 microM. 2. Displacement of [3H]-Ap4A by non-labelled Ap4A and P2-purinoceptor ligands showed a displacement order of Ap4A > adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S) > 5'-adenylyl-imidodiphosphate (AMP-PNP) > alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) in both sites revealed by the Ki values of 0.017 nM, 0.030 nM, 0.058 nM and 0.147 nM respectively for the high affinity binding site and values of 0.57 microM, 0.87 microM, 2.20 microM and 4.28 microM respectively for the second binding site. 3. Studies of the P2-purinoceptors present in synaptosomes were also performed with [35S]-ADP-beta-S. This radioligand showed two binding sites the first with Kd and Bmax values of 0.11 +/- 0.022 nM and 3.9 +/- 2.1 fmol mg-1 of protein respectively for the high affinity binding site obtained from the Scatchard plot. The second binding site showed a Ki of 0.018 +/- 0.0035 microM obtained from displacement curves. 4. Competition studies with diadenosine polyphosphates of [35S]-ADP-beta-S binding showed a displacement order of Ap4A > Ap5A > Ap6A in the high affinity binding site and Ki values of 0.023 nM, 0.081 nM and 5.72 nM respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8485620

The Extracellular Heme-binding Protein HbpS from the Soil Bacterium Streptomyces reticuli Is an Aquo-cobalamin Binder*

PubMed Central

Ortiz de Orué Lucana, Darío; Fedosov, Sergey N.; Wedderhoff, Ina; Che, Edith N.; Torda, Andrew E.

2014-01-01

The extracellular protein HbpS from Streptomyces reticuli interacts with iron ions and heme. It also acts in concert with the two-component sensing system SenS-SenR in response to oxidative stress. Sequence comparisons suggested that the protein may bind a cobalamin. UV-visible spectroscopy confirmed binding (Kd = 34 μm) to aquo-cobalamin (H2OCbl+) but not to other cobalamins. Competition experiments with the H2OCbl+-coordinating ligand CN− and comparison of mutants identified a histidine residue (His-156) that coordinates the cobalt ion of H2OCbl+ and substitutes for water. HbpS·Cobalamin lacks the Asp-X-His-X-X-Gly motif seen in some cobalamin binding enzymes. Preliminary tests showed that a related HbpS protein from a different species also binds H2OCbl+. Furthermore, analyses of HbpS-heme binding kinetics are consistent with the role of HbpS as a heme-sensor and suggested a role in heme transport. Given the high occurrence of HbpS-like sequences among Gram-positive and Gram-negative bacteria, our findings suggest a great functional versatility among these proteins. PMID:25342754

Muscarinic binding sites in cultured bovine pulmonary arterial endothelial cells

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

Aronstam, R.S.; Catravas, J.D.; Ryan, U.S.

The authors have previously reported a) the presence of muscarinic binding sites on cultured bovine pulmonary arterial endothelial cells (BPAE; 2,000 sites/cell) and b) that acetylcholine inhibits the release of thromboxane B/sub 2/ fro BPAE. Since the authors findings could reflect muscarinic receptors (mAChR) on BPAE, they have further investigated the nature of BPAE muscarinic binding sites and contrast them to those of known functional mAChR. Muscarinic binding sites on BPAE resembled mAChR in that a) the binding of 3 nM /sup 3/H QNB was inhibited by muscarinic agonists and antagonists; b) /sup 3/H QNB binding was 30 times moremore » sensitive to R(-)- than to S(+)-QNB; c) carbamylcholine binding was resolved into high and low affinity components (IC50's = 0.04 and 2 ..mu..M; d) 5'-guanylylimidodiphosphate (100 ..mu..M) shifted agonist binding curves to the right by a factor of 3; 4) the atropine-sensitive binding of /sup 3/H oxotremorine-M (/sup 3/H-OXO-M) was depressed by the guanine nucleotide (IC50 + 60 ..mu..M). However, although gallamine allosterically regulates mAChR binding in other tissues, it did not affect the rates of dissociation of /sup 3/H QNB, /sup 3/H methylscopolamine or /sup 3/H OXO-M from BPAE binding sites. Thus, BPAE muscarinic binding sites posses many but not all of the properties associated with functional mAChR.« less

Polymorphisms A387P in thrombospondin-4 and N700S in thrombospondin-1 perturb calcium binding sites.

PubMed

Stenina, Olga I; Ustinov, Valentin; Krukovets, Irene; Marinic, Tina; Topol, Eric J; Plow, Edward F

2005-11-01

Recent genetic studies have associated members of the thrombospondin (TSP) gene family with premature cardiovascular disease. The disease-associated polymorphisms lead to single amino acid changes in TSP-4 (A387P) and TSP-1 (N700S). These substitutions reside in adjacent domains of these highly homologous proteins. Secondary structural predictive programs and the homology of the domains harboring these amino acid substitutions to those in other proteins pointed to potential alterations of putative Ca2+ binding sites that reside in close proximity to the polymorphic amino acids. Since Ca2+ binding is critical for the structure and function of TSP family members, direct evidence for differences in Ca2+ binding by the polymorphic forms was sought. Using synthetic peptides and purified recombinant variant fragments bearing the amino acid substitutions, we measured differences in Tb3+ luminescence as an index of Ca2+ binding. The Tb3+ binding constants placed the TSP-1 region affected by N700S polymorphism among other high-affinity Ca2+ binding sites. The affinity of Ca2+ binding was lower for peptides (3.5-fold) and recombinant fragments (10-fold) containing the S700 vs. the N700 form. In TSP-4, the P387 form acquired an additional Ca2+ binding site absent in the A387 form. The results of our study suggest that both substitutions (A387P in TSP-4 and N700S in TSP-1) alter Ca2+ binding properties. Since these substitutions exert the opposite effects on Ca2+ binding, a decrease in TSP-1 and an increase in TSP-4, the two TSP variants are likely to influence cardiovascular functions in distinct but yet pathogenic ways.

ProBiS-CHARMMing: Web Interface for Prediction and Optimization of Ligands in Protein Binding Sites.

PubMed

Konc, Janez; Miller, Benjamin T; Štular, Tanja; Lešnik, Samo; Woodcock, H Lee; Brooks, Bernard R; Janežič, Dušanka

2015-11-23

Proteins often exist only as apo structures (unligated) in the Protein Data Bank, with their corresponding holo structures (with ligands) unavailable. However, apoproteins may not represent the amino-acid residue arrangement upon ligand binding well, which is especially problematic for molecular docking. We developed the ProBiS-CHARMMing web interface by connecting the ProBiS ( http://probis.cmm.ki.si ) and CHARMMing ( http://www.charmming.org ) web servers into one functional unit that enables prediction of protein-ligand complexes and allows for their geometry optimization and interaction energy calculation. The ProBiS web server predicts ligands (small compounds, proteins, nucleic acids, and single-atom ligands) that may bind to a query protein. This is achieved by comparing its surface structure against a nonredundant database of protein structures and finding those that have binding sites similar to that of the query protein. Existing ligands found in the similar binding sites are then transposed to the query according to predictions from ProBiS. The CHARMMing web server enables, among other things, minimization and potential energy calculation for a wide variety of biomolecular systems, and it is used here to optimize the geometry of the predicted protein-ligand complex structures using the CHARMM force field and to calculate their interaction energies with the corresponding query proteins. We show how ProBiS-CHARMMing can be used to predict ligands and their poses for a particular binding site, and minimize the predicted protein-ligand complexes to obtain representations of holoproteins. The ProBiS-CHARMMing web interface is freely available for academic users at http://probis.nih.gov.

The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines.

PubMed

Kim, Hyo-Jin; Lee, Kang-Lok; Kim, Kyoung-Dong; Roe, Jung-Hye

2016-09-09

Iron homeostasis is tightly regulated since iron is an essential but toxic element in the cell. The GATA-type transcription factor Fep1 and its orthologs contribute to iron homeostasis in many fungi by repressing genes for iron uptake when intracellular iron is high. Even though the function and interaction partners of Fep1 have been elucidated extensively In Schizosaccharomyces pombe, the mechanism behind iron-sensing by Fep1 remains elusive. It has been reported that Fep1 interacts with Fe-S-containing monothiol glutaredoxin Grx4 and Grx4-Fra2 complex. In this study, we demonstrate that Fep1 also binds iron, in the form of Fe-S cluster. Spectroscopic and biochemical analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. The finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparison of S. cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site

PubMed Central

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; Kenniston, Jon A.; Mendrola, Jeannine M.; Ferguson, Kathryn M.; Lemmon, Mark A.

2015-01-01

SUMMARY F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the S. cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences, and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip, and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity, and provide a basis for its prediction from sequence. PMID:25620000

Binding of DNA-bending non-histone proteins destabilizes regular 30-nm chromatin structure

PubMed Central

Bajpai, Gaurav; Jain, Ishutesh; Inamdar, Mandar M.; Das, Dibyendu; Padinhateeri, Ranjith

2017-01-01

Why most of the in vivo experiments do not find the 30-nm chromatin fiber, well studied in vitro, is a puzzle. Two basic physical inputs that are crucial for understanding the structure of the 30-nm fiber are the stiffness of the linker DNA and the relative orientations of the DNA entering/exiting nucleosomes. Based on these inputs we simulate chromatin structure and show that the presence of non-histone proteins, which bind and locally bend linker DNA, destroys any regular higher order structures (e.g., zig-zag). Accounting for the bending geometry of proteins like nhp6 and HMG-B, our theory predicts phase-diagram for the chromatin structure as a function of DNA-bending non-histone protein density and mean linker DNA length. For a wide range of linker lengths, we show that as we vary one parameter, that is, the fraction of bent linker region due to non-histone proteins, the steady-state structure will show a transition from zig-zag to an irregular structure—a structure that is reminiscent of what is observed in experiments recently. Our theory can explain the recent in vivo observation of irregular chromatin having co-existence of finite fraction of the next-neighbor (i + 2) and neighbor (i + 1) nucleosome interactions. PMID:28135276

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

PubMed Central

Cristóvão, Michele; Sisamakis, Evangelos; Hingorani, Manju M.; Marx, Andreas D.; Jung, Caroline P.; Rothwell, Paul J.; Seidel, Claus A. M.; Friedhoff, Peter

2012-01-01

Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS–mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand. PMID:22367846

NMR Chemical Exchange as a Probe for Ligand-Binding Kinetics in a Theophylline-Binding RNA Aptamer

PubMed Central

Latham, Michael P.; Zimmermann, Grant R.; Pardi, Arthur

2009-01-01

The apparent on- and off-rate constants for theophylline binding to its RNA aptamer in the absence of Mg2+ were determined here by 2D 1H-1H NMR ZZ-exchange spectroscopy. Analysis of the build-up rate of the exchange cross peaks for several base-paired imino protons in the RNA yielded an apparent kon of 600 M-1 s-1. This small apparent kon results from the free RNA existing as a dynamic equilibrium of inactive states rapidly interconverting with a low population of active species. The data here indicate that the RNA aptamer employs a conformational selection mechanism for binding theophylline in the absence of Mg2+. The kinetic data here also explain a very unusual property of this RNA-theophylline system, slow exchange on the NMR chemical shift timescale for a weak-binding complex. To our knowledge, it is unprecedented to have such a weak binding complex (Kd ≈ 3.0 mM at 15 °C) show slow exchange on the NMR chemical shift timescale, but the results clearly demonstrate that slow exchange and weak binding are readily rationalized by a small kon. Comparisons with other ligand-receptor interactions are presented. PMID:19317486

DRUGDOG 3.0: U.S. Navy Random Urinalysis Software Package

DTIC Science & Technology

1994-03-15

NAVAL PO11GRADUATE SCHOOL Monterey, California AD-A281 748 THESIS LJuEoTE DRUGDOG 3.0: U. S . NAVY RANDOM URINALYSIS SOFTWARE PACKAGE by (% Dale E...ONLY (Leave blank) 2. REPORT DATE 3. REPORT TYPE AND DATES COVERED 15 MAR 94 Master’s Thesis 4. TITLE AND SUBTITLE DRUGDOG 3.0: U. S . NAVY RANDOM 5...FUNDING NUMBERS URINALYSIS SOFTWARE PACKAGE 6. AUTHOR( S ) Dale E. Wilson 7. PERFORMING ORGANIZATION NAME( S ) AND ADDRESS(ES) 8. PERFORMING Naval

Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing.

PubMed

Panja, Subrata; Santiago-Frangos, Andrew; Schu, Daniel J; Gottesman, Susan; Woodson, Sarah A

2015-11-06

Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs. Copyright © 2015 Elsevier Ltd. All rights reserved.

New tailored substituted benzothiazole Schiff base Cu(II)/Zn(II) antitumor drug entities: effect of substituents on DNA binding profile, antimicrobial and cytotoxic activity.

PubMed

Zehra, Siffeen; Shavez Khan, Mohammad; Ahmad, Iqbal; Arjmand, Farukh

2018-05-07

New tailored Cu(II) & Zn(II) metal-based antitumor drug entities were synthesized from substituted benzothiazole o‒vanillin Schiff base ligands. The complexes were thoroughly characterized by elemental analysis, spectroscopic studies {IR, 1 H & 13 C NMR, ESI-MS, EPR} and magnetic susceptibility measurements. The structure activity relationship (SAR) studies of benzothiazole Cu(II) & Zn(II) complexes having molecular formulas [C 30 H 22 CuN 5 O 7 S 2 ], [C 30 H 20 Cl 2 CuN 5 O 7 S 2 ], [C 30 H 20 CuF 2 N 5 O 7 S 2 ], [C 30 H 22 N 4 O 4 S 2 Zn], [C 30 H 20 Cl 2 N 4 O 4 S 2 Zn], and [C 30 H 20 F 2 N 5 O 7 S 2 Zn], with CT‒DNA were performed by employing absorption, emission titrations, and hydrodynamic measurements. The DNA binding affinity was quantified by K b and K sv values which gave higher binding propensity for chloro-substituted Cu(II) [C 30 H 20 Cl 2 CuN 5 O 7 S 2 ] complex, suggestive of groove binding mode with subtle partial intercalation. Molecular properties and drug likeness profile were assessed for the ligands and all the Lipinski's rules were found to be obeyed. The antimicrobial potential of ligands and their Cu(II) & Zn(II) complexes were screened against some notably important pathogens viz., E. coli, S. aureus, P. aeruginosa, B. subtilis, and C. albicans. The cytotoxicity of the complexes [C 30 H 20 Cl 2 CuN 5 O 7 S 2 ], [C 30 H 20 CuF 2 N 5 O 7 S 2 ], [C 30 H 20 Cl 2 N 4 O 4 S 2 Zn], and [C 30 H 20 F 2 N 5 O 7 S 2 Zn] were evaluated against five human cancer cell lines viz., MCF‒7 (breast), MIA‒PA‒CA‒2 (pancreatic), HeLa (cervix) and Hep‒G2 (Hepatoma) and A498 (Kidney) by SRB assay which revealed that chloro-substituted [C 30 H 20 Cl 2 CuN 5 O 7 S 2 ] complex, exhibited pronounced specific cytotoxicity with GI 50 value of 4.8 μg/ml against HeLa cell line. Molecular docking studies were also performed to explore the binding modes and orientation of the complexes in the DNA helix.

Functional coupling between adenosine A1 receptors and G-proteins in rat and postmortem human brain membranes determined with conventional guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding or [35S]GTPγS/immunoprecipitation assay.

PubMed

Odagaki, Yuji; Kinoshita, Masakazu; Ota, Toshio; Meana, J Javier; Callado, Luis F; Matsuoka, Isao; García-Sevilla, Jesús A

2018-06-01

Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A 1 receptor and G-protein was assessed by means of two [ 35 S]GTPγS binding assays, i.e., conventional filtration method and [ 35 S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A 1 receptor-mediated Gα i-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [ 35 S]GTPγS binding determined with conventional assay derives from functional activation of Gα i/o proteins (not restricted only to Gα i-3 ) coupled to adenosine A 1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [ 35 S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %E max values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A 1 receptor/G-protein interaction in postmortem human brain tissue.

Studies on the contributions of steric and polarity effects to the H2S-binding properties of Vitreoscilla hemoglobin

NASA Astrophysics Data System (ADS)

Wang, Dandan; Wang, Hui; Li, Haichao; Liu, Li; Li, Zhengqiang

2017-01-01

We have reported recently that Vitreoscilla hemoglobin (VHb) is a potential H2S receptor and storage molecule in bacterial metabolism. In this study, molecular cloning and site-directed mutagenesis were employed to investigate the structural basis for H2S binding. Association and dissociation rate constants (kon and koff) were determined using stopped-flow rapid-scanning spectrophotometry and compared with those for wild type VHb. Several unanticipated factors were found to govern H2S binding properties, due to the distinct structure of VHb. The results presented in this paper show that: i) bulkier residues at positions E7 and E11 decrease H2S binding accessibility, while the residue located at position B10 blocks bound H2S from escaping. ii) hydroxyl sidechains within the distal heme pocket reduce H2S reactivity to VHb; iii) Pro(E8) is involved in moving the E7-E10 loop region to trigger opening of the distal heme pocket to facilitate H2S binding.

Cluster analysis of S. Cerevisiae nucleosome binding sites

NASA Astrophysics Data System (ADS)

Suvorova, Y.; Korotkov, E.

2017-12-01

It is well known that major part of a eukaryotic genome is wrapped around histone proteins forming nucleosomes. It was also demonstrated that the DNA sequence itself is playing an important role in the nucleosome positioning process. In this work, a cluster analysis of 67 517 nucleosome binding sites from the S. Cerevisiae genome was carried out. The classification method is based on the self-adjusting dinucleotides position weight matrix. As a result, 135 significant clusters were discovered that contain 43225 sequences (which constitutes 64% of the initial set). The meaning of the found classes is discussed, as well as the possibility of the further usage.

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

PubMed Central

Thorgersen, Michael P.; Lancaster, W. Andrew; Rajeev, Lara; Ge, Xiaoxuan; Vaccaro, Brian J.; Poole, Farris L.; Arkin, Adam P.; Mukhopadhyay, Aindrila

2016-01-01

ABSTRACT Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. IMPORTANCE Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Herein, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. While other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

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

Thorgersen, Michael P.; Lancaster, W. Andrew; Rajeev, Lara

Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC hadmore » an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Here in this paper, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. Finally, while other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium.

PubMed

Thorgersen, Michael P; Lancaster, W Andrew; Rajeev, Lara; Ge, Xiaoxuan; Vaccaro, Brian J; Poole, Farris L; Arkin, Adam P; Mukhopadhyay, Aindrila; Adams, Michael W W

2017-02-15

Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Herein, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. While other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an S-layer complex

Definition of a consensus DNA-binding site for PecS, a global regulator of virulence gene expression in Erwinia chrysanthemi and identification of new members of the PecS regulon.

PubMed

Rouanet, Carine; Reverchon, Sylvie; Rodionov, Dmitry A; Nasser, William

2004-07-16

In Erwinia chrysanthemi, production of pectic enzymes is modulated by a complex network involving several regulators. One of them, PecS, which belongs to the MarR family, also controls the synthesis of various other virulence factors, such as cellulases and indigoidine. Here, the PecS consensus-binding site is defined by combining a systematic evolution of ligands by an exponential enrichment approach and mutational analyses. The consensus consists of a 23-base pair palindromic-like sequence (C(-11)G(-10)A(-9)N(-8)W(-7)T(-6)C(-5)G(-4)T(-3)A(-2))T(-1)A(0)T(1)(T(2)A(3)C(4)G(5)A(6)N(7)N(8)N(9)C(10)G(11)). Mutational experiments revealed that (i) the palindromic organization is required for the binding of PecS, (ii) the very conserved part of the consensus (-6 to 6) allows for a specific interaction with PecS, but the presence of the relatively degenerated bases located apart significantly increases PecS affinity, (iii) the four bases G, A, T, and C are required for efficient binding of PecS, and (iv) the presence of several binding sites on the same promoter increases the affinity of PecS. This consensus is detected in the regions involved in PecS binding on the previously characterized target genes. This variable consensus is in agreement with the observation that the members of the MarR family are able to bind various DNA targets as dimers by means of a winged helix DNA-binding motif. Binding of PecS on a promoter region containing the defined consensus results in a repression of gene transcription in vitro. Preliminary scanning of the E. chrysanthemi genome sequence with the consensus revealed the presence of strong PecS-binding sites in the intergenic region between fliE and fliFGHIJKLMNOPQR which encode proteins involved in the biogenesis of flagellum. Accordingly, PecS directly represses fliE expression. Thus, PecS seems to control the synthesis of virulence factors required for the key steps of plant infection.

Nucleosome Translational Position, Not Histone Acetylation, Determines TFIIIA Binding to Nucleosomal Xenopus laevis 5S rRNA Genes

PubMed Central

Howe, LeAnn; Ausió, Juan

1998-01-01

We sought to study the binding constraints placed on the nine-zinc-finger protein transcription factor IIIA (TFIIIA) by a histone octamer. To this end, five overlapping fragments of the Xenopus laevis oocyte and somatic 5S rRNA genes were reconstituted into nucleosomes, and it was subsequently shown that nucleosome translational positioning is a major determinant of the binding of TFIIIA to the 5S rRNA genes. Furthermore, it was found that histone acetylation cannot override the TFIIIA binding constraints imposed by unfavorable translational positions. PMID:9488430

The Bisphenol A analogue Bisphenol S binds to K-Ras4B--implications for 'BPA-free' plastics.

PubMed

Schöpel, Miriam; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

2016-02-01

K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers. © 2016 Federation of European Biochemical Societies.

Increases thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase by fusion of cellulose binding domain derived from Trichoderma reesei.

PubMed

Thongekkaew, Jantaporn; Ikeda, Hiroko; Iefuji, Haruyuki

2012-03-30

To improve the thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase (CSLP), the cellulose-binding domain originates from Trichoderma reesei cellobiohydrolase I was engineered into C-terminal region of the CSLP (CSLP-CBD). The CSLP and CSLP-CBD were successfully expressed in the Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from Saccharomyces cerevisiae (α factor). The recombinant CSLP and CSLP-CBD were secreted into culture medium and estimated by SDS-PAGE to be 22 and 27 kDa, respectively. The fusion enzyme was stable at 80 °C and retained more than 80% of its activity after 120-min incubation at this temperature. Our results also found that the fusion of fungal exoglucanase cellulose-binding domain to CSLP is responsible for cellulose-binding capacity. This attribute should make it an attractive applicant for enzyme immobilization. Copyright © 2012 Elsevier Inc. All rights reserved.

Regulation of E2s: A Role for Additional Ubiquitin Binding Sites?

PubMed

Middleton, Adam J; Wright, Joshua D; Day, Catherine L

2017-11-10

Attachment of ubiquitin to proteins relies on a sophisticated enzyme cascade that is tightly regulated. The machinery of ubiquitylation responds to a range of signals, which remarkably includes ubiquitin itself. Thus, ubiquitin is not only the central player in the ubiquitylation cascade but also a key regulator. The ubiquitin E3 ligases provide specificity to the cascade and often bind the substrate, while the ubiquitin-conjugating enzymes (E2s) have a pivotal role in determining chain linkage and length. Interaction of ubiquitin with the E2 is important for activity, but the weak nature of these contacts has made them hard to identify and study. By reviewing available crystal structures, we identify putative ubiquitin binding sites on E2s, which may enhance E2 processivity and the assembly of chains of a defined linkage. The implications of these new sites are discussed in the context of known E2-ubiquitin interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Definition of the binding mode of phosphoinositide 3-kinase α-selective inhibitor A-66S through molecular dynamics simulation.

PubMed

Bian, Xiaoli; Dong, Wangqing; Zhao, Yang; Sun, Rui; Kong, Wanjun; Li, Yiping

2014-04-01

Activation of the phosphatidylinositol 3-kinase α (PI3Kα) is commonly observed in human cancer and is critical for tumor progression, which has made PI3Kα an attractive target for anticancer drug discovery. To systematically investigate the binding mode of A-66S, a new selective PI3Kα inhibitor for PI3Kα, molecular docking, molecular dynamics simulation and ensuing energetic analysis were performed. The binding free energy between PI3Kα and A-66S is -11.27 kcal•mol⁻¹ using MMPBSA method, while -14.67 kcal•mol⁻¹ using MMGBSA method, which is beneficial for the binding, and the van der Waals/hydrophobic and electrostatic interactions are critical for the binding. The conserved hydrophobic adenine region of PI3Kα made up of Met772, Pro778, Ile800, Tyr836, Ile848, Val850, Val851, Met922, Phe930 and Ile932 accommodates the flat 2-tert-butyl-4'-methyl-4,5'-bithiazol moiety of A-66S, and the NH of Val851 forms a hydrogen with the nitrogen atom embedded in the aminothiazole ring of A-66S. The (S)-pyrrolidine carboxamide urea moiety especially extends toward the region of the binding site wall (Ser854-Gln859) defined by the C-terminal lobe, and has three hydrogen-bond arms with the backbone of Ser854 and the side chain of Gln859. Notably the interaction between the non-conserved residue Gln859 and A-66S is responsible for the selectivity profile of A-66S. The binding mode of A-66S for PI3Kα presented in this study should aid in the design of a new highly selective PI3Kα inhibitor.

Post-synaptic Density-95 (PSD-95) Binding Capacity of G-protein-coupled Receptor 30 (GPR30), an Estrogen Receptor That Can Be Identified in Hippocampal Dendritic Spines*

PubMed Central

Akama, Keith T.; Thompson, Louisa I.; Milner, Teresa A.; McEwen, Bruce S.

2013-01-01

The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity. PMID:23300088

Post-synaptic density-95 (PSD-95) binding capacity of G-protein-coupled receptor 30 (GPR30), an estrogen receptor that can be identified in hippocampal dendritic spines.

PubMed

Akama, Keith T; Thompson, Louisa I; Milner, Teresa A; McEwen, Bruce S

2013-03-01

The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity.

Clinical significance of calcium-binding protein S100A8 and S100A9 expression in non-small cell lung cancer.

PubMed

Huang, He; Huang, Qingdong; Tang, Tingyu; Gu, Liang; Du, Jianzong; Li, Zhijun; Lu, Xiaoling; Zhou, Xiaoxi

2018-05-07

The purpose of this study was to evaluate the correlation between calcium-binding protein S100A8 and S100A9 expression in non-small cell lung cancer (NSCLC) and patients' clinical features. Fifty-two NSCLC patients who underwent surgery at Zhejiang Hospital from February 2014 to January 2016 were included in this study. Calcium-binding protein S100A8 and S100A9 expression patterns in cancer and para-cancer tissues were examined by immunohistochemistry assay. The correlation between calcium-binding protein S100A8 and S100A9 expression patterns and NSCLC patients' clinical characteristics, including age, gender, tumor node metastasis stage, and pathology type, were evaluated. S100A8 and S100A9 were generally expressed on the cytoplasm and nucleus of NSCLC cells, mainly located in the cytoplasm, stained with brown particles, and distributed evenly. The positive expression rates of S100A8 and S100A9 in cancer tissues were 71.2% and 76.9%, respectively, which were significantly higher than in para-cancer tissues at 11.5% and 19.2%, respectively, with statistical significance (P < 0.05). S100A8 and S100A9 positive expression was associated with tumor differentiation degree (P < 0.05) but were not correlated with age, gender, smoking history, tumor diameter, pathology type, tumor node metastasis stage, or pleural effusion (P all  > 0.05). S100A8 and S100A9 positive expression in cancer tissues was significantly higher than in para-cancer tissues and was correlated with tumor differentiation, which may be a potential marker for poor prognosis. © 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2.

PubMed

Saito, Motoki; Ishikawa, Fuyuki

2002-09-20

Although mammalian MBD3 contains the mCpG-binding domain (MBD) and is highly homologous with the authentic mCpG-binding protein MBD2, it was reported that the protein does not bind to mCpG specifically. Using recombinant human wild type and mutant MBD3 proteins, we demonstrated that atypical amino acids found in MBD3 MBD, namely, His-30 and Phe-34, are responsible for the inability of MBD3 to bind to mCpG. Interestingly, although H30K/F34Y MBD3 mutant protein binds to mCpG efficiently in vitro, it was not localized at the mCpG-rich pericentromeric regions in mouse cells. We also showed that Y34F MBD2b MBD, which possesses not the mCpG-specific DNA-binding activity but the nonspecific DNA-binding activity, was localized at the pericentromeric regions. These results suggested that the mCpG-specific DNA-binding activity is largely dispensable, and another factor(s) is required for the localization of MBD proteins in vivo. MBD3 was identified as a component of the NuRD/Mi2 complex that shows chromatin remodeling and histone deacetylase activities. We demonstrated that MBD3 MBD is necessary and sufficient for binding to HDAC1 and MTA2, two components of the NuRD/Mi2 complex. It was therefore suggested that mCpG-binding-defective MBD3 has evolutionarily conserved its MBD because of the secondary role played by the MBD in protein-protein interactions.

Differential regulation of serotonin-1A receptor stimulated [35S]GTPγS binding in the dorsal raphe nucleus by citalopram and escitalopram

PubMed Central

Rossi, Dania V.; Burke, Teresa F.; Hensler, Julie G.

2008-01-01

The effect of chronic citalopram or escitalopram administration on 5-HT1A receptor function in the dorsal raphe nucleus was determined by measuring [35S]GTPγS binding stimulated by the 5-HT1A receptor agonist (R)-(+)-8-OH-DPAT (1nM-10μM). Although chronic administration of citalopram or escitalopram has been shown to desensitize somatodendritic 5-HT1A autoreceptors, we found that escitalopram treatment decreased the efficacy of 5-HT1A receptors to activate G-proteins, whereas citalopram treatment did not. The binding of [3H]8-OH-DPAT to the coupled, high affinity agonist state of the receptor was not altered by either treatment. Interestingly, escitalopram administration resulted in greater occupancy of serotonin transporter sites as measured by the inhibition of [3H]cyanoimipramine binding. As the binding and action of escitalopram is limited by the inactive enantiomer R-citalopram present in racemic citalopram, we propose that the regulation of 5-HT1A receptor function in the dorsal raphe nucleus at the level of receptor-G protein interaction may be a result of greater inhibition of the serotonin transporter by escitalopram. PMID:18289523

Iron Binding at Specific Sites within the Octameric HbpS Protects Streptomycetes from Iron-Mediated Oxidative Stress

PubMed Central

Wedderhoff, Ina; Kursula, Inari; Groves, Matthew R.; Ortiz de Orué Lucana, Darío

2013-01-01

The soil bacterium Streptomyces reticuli secretes the octameric protein HbpS that acts as a sensory component of the redox-signalling pathway HbpS-SenS-SenR. This system modulates a genetic response on iron- and haem-mediated oxidative stress. Moreover, HbpS alone provides this bacterium with a defence mechanism to the presence of high concentrations of iron ions and haem. While the protection against haem has been related to its haem-binding and haem-degrading activity, the interaction with iron has not been studied in detail. In this work, we biochemically analyzed the iron-binding activity of a set of generated HbpS mutant proteins and present evidence showing the involvement of one internal and two exposed D/EXXE motifs in binding of high quantities of ferrous iron, with the internal E78XXE81 displaying the tightest binding. We additionally show that HbpS is able to oxidize ferrous to ferric iron ions. Based on the crystal structure of both the wild-type and the mutant HbpS-D78XXD81, we conclude that the local arrangement of the side chains from the glutamates in E78XXE81 within the octameric assembly is a pre-requisite for interaction with iron. The data obtained led us to propose that the exposed and the internal motif build a highly specific route that is involved in the transport of high quantities of iron ions into the core of the HbpS octamer. Furthermore, physiological studies using Streptomyces transformants secreting either wild-type or HbpS mutant proteins and different redox-cycling compounds led us to conclude that the iron-sequestering activity of HbpS protects these soil bacteria from the hazardous side effects of peroxide- and iron-based oxidative stress. PMID:24013686

Interaction of S17 Antibody with the Functional Binding Region of the Hepatitis B Virus Pre-S2 Epitope.

PubMed

Chang, Chang-Yu; Chang, Fu-Ling; Chiang, Chen-Wei; Lo, Yan-Ni; Lin, Tsai-Yu; Chen, Wang-Chuan; Tsai, Keng-Chang; Lee, Yu-Ching

2018-05-30

To understand the mechanism for inhibition of hepatitis B virus (HBV) infection is important. In this study, single-chain variable fragment (scFv) antibodies were generated and directed to the pre-S2 epitope of HBV surface antigen (HBsAg). These human scFvs were isolated from a person with history of HBV infection by phage display technology. An evaluation of panning efficiency revealed that the eluted phage titer was increased, indicating that specific clones were enriched after panning. Selected scFvs were characterized with the recombinant HBsAg through Western blotting and enzyme-linked immunosorbent assay to confirm the binding ability. Flow cytometry analysis and immunocytochemical staining revealed that one scFv, S17, could recognize endogenous HBsAg expressed on the HepG2215 cell membrane. Moreover, the binding affinity of scFv S17 to the pre-S2 epitope was determined to be 4.2 × 10 -8 M. Two ion interactions were observed as the major driving forces for scFv S17 interacting with pre-S2 by performing a rational molecular docking analysis. This study provides insights into the structural basis to understand the interactions between an antibody and the pre-S2 epitope. The functional scFv format can potentially be used in future immunotherapeutic applications.

DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1

EPA Science Inventory

DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1. R A Pegram1 and M K Ross2. 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC; 1Pharmacokinetics Branch, NHEERL, ORD, United States Environmental Protection Ag...

NetMHCpan-3.0; improved prediction of binding to MHC class I molecules integrating information from multiple receptor and peptide length datasets.

PubMed

Nielsen, Morten; Andreatta, Massimo

2016-03-30

Binding of peptides to MHC class I molecules (MHC-I) is essential for antigen presentation to cytotoxic T-cells. Here, we demonstrate how a simple alignment step allowing insertions and deletions in a pan-specific MHC-I binding machine-learning model enables combining information across both multiple MHC molecules and peptide lengths. This pan-allele/pan-length algorithm significantly outperforms state-of-the-art methods, and captures differences in the length profile of binders to different MHC molecules leading to increased accuracy for ligand identification. Using this model, we demonstrate that percentile ranks in contrast to affinity-based thresholds are optimal for ligand identification due to uniform sampling of the MHC space. We have developed a neural network-based machine-learning algorithm leveraging information across multiple receptor specificities and ligand length scales, and demonstrated how this approach significantly improves the accuracy for prediction of peptide binding and identification of MHC ligands. The method is available at www.cbs.dtu.dk/services/NetMHCpan-3.0 .

Serum sCD30 in monitoring of alloresponse in well HLA-matched cadaveric kidney transplantations.

PubMed

Matinlauri, Irma H; Kyllönen, Lauri E J; Salmela, Kaija T; Helin, Heikki; Pelzl, Steffen; Süsal, Caner

2005-12-27

In kidney transplantation, pretransplant serum sCD30 testing has been proposed in immunological risk estimation together with anti-HLA antibodies. We evaluated the risks associated with high pretransplant serum sCD30 in well HLA-matched cadaveric kidney recipients recruited in a clinical study comparing different immunosuppressive regimens. Rejection rate was similar in 37 recipients with high pretransplant serum sCD30 compared to 117 recipients with low serum sCD30 (16% vs. 15%, P=NS). Compared to pretransplant levels, the posttransplant sCD30 levels generally decreased, also in patients with rejection, although on day 21 posttransplant, rejecting patients had significantly higher relative sCD30 than nonrejecting patients (P<0.01). However, steroid-resistant rejection was associated with increasing posttransplant sCD30 levels. High pretransplant sCD30 values were associated with tubulointerstitial rejection. There was no correlation of sCD30 with delayed graft function. Good HLA matching seems to be effective in neutralizing the negative effect of a high pretransplant serum sCD30.

RNA–protein binding kinetics in an automated microfluidic reactor

PubMed Central

Ridgeway, William K.; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R.

2009-01-01

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome. PMID:19759214

Precise half-life measurement of the superallowed emitter 30S

NASA Astrophysics Data System (ADS)

Iacob, V. E.; Hardy, J. C.; Chen, L.; Horvat, V.; Bencomo, M.; Nica, N.; Park, H. I.; Roeder, B. T.; Saastamoinen, A.

2018-03-01

We have measured the half-life of 30S, the parent of a superallowed 0+→0+β transition, to a high precision using very pure sources and a 4 π proportional gas counter to detect the decay positrons. Our result for the half-life is 1.179 92(34) s. As a by-product of this measurement, we determine the half-life of its daughter, 30P, to be 2.501(2) min.

Differential regulation of serotonin-1A receptor-stimulated [35S]GTP gamma S binding in the dorsal raphe nucleus by citalopram and escitalopram.

PubMed

Rossi, Dania V; Burke, Teresa F; Hensler, Julie G

2008-03-31

The effect of chronic citalopram or escitalopram administration on 5-HT1A receptor function in the dorsal raphe nucleus was determined by measuring [35S]GTP gamma S binding stimulated by the 5-HT1A receptor agonist (R)-(+)-8-OH-DPAT (1nM-10 microM). Although chronic administration of citalopram or escitalopram has been shown to desensitize somatodendritic 5-HT1A autoreceptors, we found that escitalopram treatment decreased the efficacy of 5-HT1A receptors to activate G proteins, whereas citalopram treatment did not. The binding of [3H]8-OH-DPAT to the coupled, high affinity agonist state of the receptor was not altered by either treatment. Interestingly, escitalopram administration resulted in greater occupancy of serotonin transporter sites as measured by the inhibition of [3H]cyanoimipramine binding. As the binding and action of escitalopram is limited by the inactive enantiomer R-citalopram present in racemic citalopram, we propose that the regulation of 5-HT1A receptor function in the dorsal raphe nucleus at the level of receptor-G protein interaction may be a result of greater inhibition of the serotonin transporter by escitalopram.

Neurosteroid binding to the amino terminal and glutamate binding domains of ionotropic glutamate receptors.

PubMed

Cameron, Krasnodara; Bartle, Emily; Roark, Ryan; Fanelli, David; Pham, Melissa; Pollard, Beth; Borkowski, Brian; Rhoads, Sarah; Kim, Joon; Rocha, Monica; Kahlson, Martha; Kangala, Melinda; Gentile, Lisa

2012-06-01

The endogenous neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5β-pregnan-20-one sulfate (PREGAS), have been shown to differentially regulate the ionotropic glutamate receptor (iGluR) family of ligand-gated ion channels. Upon binding to these receptors, PREGAS decreases current flow through the channels. Upon binding to non-NMDA or NMDA receptors containing an GluN2C or GluN2D subunit, PS also decreases current flow through the channels, however, upon binding to NMDA receptors containing an GluN2A or GluN2B subunit, flow through the channels increases. To begin to understand this differential regulation, we have cloned the S1S2 and amino terminal domains (ATD) of the NMDA GluN2B and GluN2D and AMPA GluA2 subunits. Here we present results that show that PS and PREGAS bind to different sites in the ATD of the GluA2 subunit, which when combined with previous results from our lab, now identifies two binding domains for each neurosteroid. We also show both neurosteroids bind only to the ATD of the GluN2D subunit, suggesting that this binding is distinct from that of the AMPA GluA2 subunit, with both leading to iGluR inhibition. Finally, we provide evidence that both PS and PREGAS bind to the S1S2 domain of the NMDA GluN2B subunit. Neurosteroid binding to the S1S2 domain of NMDA subunits responsible for potentiation of iGluRs and to the ATD of NMDA subunits responsible for inhibition of iGluRs, provides an interesting option for therapeutic design. Copyright © 2012 Elsevier Inc. All rights reserved.

Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics

DOE PAGES

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.; ...

2017-01-17

We studied a mango glutathione S-transferase (GST) ( Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a K m, V max and k cat for CDNB of 0.792 mM, 80.58 mM min -1 and 68.49 s -1 respectively and 0.693 mM, 105.32 mM min -1 and 89.57 s -1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 mM) or GSX (7.8 mM). As a result, the crystal structure of the MiGSTU inmore » apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.« less

Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics

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

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.

We studied a mango glutathione S-transferase (GST) ( Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a K m, V max and k cat for CDNB of 0.792 mM, 80.58 mM min -1 and 68.49 s -1 respectively and 0.693 mM, 105.32 mM min -1 and 89.57 s -1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 mM) or GSX (7.8 mM). As a result, the crystal structure of the MiGSTU inmore » apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.« less

A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

PubMed Central

Sashital, Dipali G; Greeman, Candacia A; Lyumkis, Dmitry; Potter, Clinton S; Carragher, Bridget; Williamson, James R

2014-01-01

Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3′ domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3′-domain is unanchored and the 5′-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells. DOI: http://dx.doi.org/10.7554/eLife.04491.001 PMID:25313868

The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange

PubMed Central

Qian, Yufeng; Johnson, Kenneth A.

2017-01-01

The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB–ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB)30 and (SSB)60, defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB)60 and (SSB)30 complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 × 109 m−1 s−1) and that longer DNA (≥60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein. PMID:28615444

Increases thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase by fusion of cellulose binding domain derived from Trichoderma reesei

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

Thongekkaew, Jantaporn, E-mail: jantaporn_25@yahoo.com; Ikeda, Hiroko; Iefuji, Haruyuki

Highlights: Black-Right-Pointing-Pointer The CSLP and fusion enzyme were successfully expressed in the Pichia pastoris. Black-Right-Pointing-Pointer The fusion enzyme was stable at 80 Degree-Sign C for 120-min. Black-Right-Pointing-Pointer The fusion enzyme was responsible for cellulose-binding capacity. Black-Right-Pointing-Pointer The fusion enzyme has an attractive applicant for enzyme immobilization. -- Abstract: To improve the thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase (CSLP), the cellulose-binding domain originates from Trichoderma reesei cellobiohydrolase I was engineered into C-terminal region of the CSLP (CSLP-CBD). The CSLP and CSLP-CBD were successfully expressed in the Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1)more » promoter and the secretion signal sequence from Saccharomyces cerevisiae ({alpha} factor). The recombinant CSLP and CSLP-CBD were secreted into culture medium and estimated by SDS-PAGE to be 22 and 27 kDa, respectively. The fusion enzyme was stable at 80 Degree-Sign C and retained more than 80% of its activity after 120-min incubation at this temperature. Our results also found that the fusion of fungal exoglucanase cellulose-binding domain to CSLP is responsible for cellulose-binding capacity. This attribute should make it an attractive applicant for enzyme immobilization.« less

Insights into transcription termination of Hfq-binding sRNAs of Escherichia coli and characterization of readthrough products

PubMed Central

Morita, Teppei; Ueda, Masaki; Kubo, Kento; Aiba, Hiroji

2015-01-01

The genes encoding Hfq-dependent sRNAs possess a typical Rho-independent transcription terminator. Here, we have studied the molecular events occurring at Rho-independent terminators of sRNA genes, focusing on two well-characterized Hfq-binding sRNAs, SgrS and RyhB. We constructed several hybrid genes in which the DNA sequence corresponding to a strong Rho-independent terminator was placed just downstream from the Rho-independent terminators of sRNA genes. By using this system, we demonstrate that transcripts frequently read through the Rho-independent terminators of sgrS and ryhB in normally growing cells. We show that Hfq does not affect the transcriptional readthrough event itself. We also find that the readthrough products no longer bind to Hfq in vivo. We have developed a competition assay based on a biotin–streptavidin system to analyze the interaction of Hfq and a particular RNA molecule in vitro. By using this method, we verify that the 3′-extended form of SgrS does not bind to Hfq in vitro. Finally, we demonstrate that transcription termination is significantly enhanced under stress conditions where transcription initiation of sRNA genes on the chromosome is induced. We conclude that the production of sRNAs is regulated not only at the step of transcription initiation but also at the step of transcription termination. The mechanism by which transcription termination is enhanced under stress conditions remains to be understood. PMID:26106215

A biomechanical study of laparoscopic 4S-modified Roeder and Weston knot strength in 3-0 polyglactin 910 and 3-0 polydioxanone.

PubMed

Fugazzi, Russell W; Fransson, Boel A; Curran, Katie M; Davis, Howard M; Gay, John M

2013-02-01

To (1) evaluate biomechanical strength of 4 different laparoscopic knots using 2 suture types, and (2) evaluate carotid artery ligature bursting pressure of 2 knots using a single suture type. Biomechanical materials testing. Suture material (3-0 polydioxanone, 3-0 polyglactin 910). Four knot types were tested: 4S-modified Roeder (4SMR) Weston plus 3 square throws (W3S); Weston plus 3 granny throws (W3G); and a 4 square throw knot as a control (control); 24 specimens of each knot type were tied with 3-0 polyglactin 910 and 24 of 3 knot types (4SMR, W3S, control) were tied with 3-0 polydioxanone. Suture loop constructs were tested to 3 mm displacement failure and ultimate failure. Carotid artery ligation bursting pressure was tested using 10 samples each of 4SMR and W3S knots with 3-0 polyglactin 910. The W3S, W3G, and controls were similar. The 4SMR was similar to W3S using 3-0 PDS but the 4SMR had lower load to failure and greater elongation than the Weston using 3-0 polyglactin 910. The 4SMR had a higher slippage rate with 3-0 polyglactin 910. All ligatures withstood supraphysiologic pressures. Surgeons using 3-0 polyglactin 910 should consider using the Weston knot with added throws during laparoscopic procedures. © Copyright 2012 by The American College of Veterinary Surgeons.

The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the beta(2)-adrenoceptor.

PubMed

Jozwiak, Krzysztof; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W

2010-06-01

The binding thermodynamics of the stereoisomers of fenoterol, (R,R')-, (S,S')-, (R,S')-, and (S,R')-fenoterol, to the beta(2)-adrenergic receptor (beta(2)-AR) have been determined. The experiments utilized membranes obtained from HEK cells stably transfected with cDNA encoding human beta(2)-AR. Competitive displacement studies using [(3)H]CGP-12177 as the marker ligand were conducted at 4, 15, 25, 30 and 37 degrees C, the binding affinities calculated and the standard enthalpic (DeltaH degrees ) and standard entropic (DeltaS degrees ) contribution to the standard free energy change (DeltaG degrees ) associated with the binding process determined through the construction of van't Hoff plots. The results indicate that the binding of (S,S')- and (S,R')-fenoterol were predominately enthalpy-driven processes while the binding of (R,R')- and (R,S')-fenoterol were entropy-driven. All of the fenoterol stereoisomers are full agonists of the beta(2)-AR, and, therefore, the results of this study are inconsistent with the previously described "thermodynamic agonist-antagonist discrimination", in which the binding of an agonist to the beta-AR is entropy-driven and the binding of an antagonist is enthalpy-driven. In addition, the data demonstrate that the chirality of the carbon atom containing the beta-hydroxyl group of the fenoterol molecule (the beta-OH carbon) is a key factor in the determination of whether the binding process will be enthalpy-driven or entropy-driven. When the configuration at the beta-OH carbon is S the binding process is enthalpy-driven while the R configuration produces an entropy-driven process. Published by Elsevier Inc.

Role of the terminator hairpin in the biogenesis of functional Hfq-binding sRNAs

PubMed Central

Morita, Teppei; Nishino, Ryo; Aiba, Hiroji

2017-01-01

Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of seven or more T residues at the 3′ end, as noted in previous studies. Here, we have studied the role of the terminator hairpin in the biogenesis of sRNAs focusing on SgrS and RyhB in Escherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the poly(U) tail is shortened. The transcripts with shortened poly(U) tails no longer bind to Hfq and lose the ability to repress the target mRNAs. The shortened transcripts are generated in an in vitro transcription system with purified RNA polymerase, indicating that the generation of shortened transcripts is caused by premature transcription termination. We conclude that the terminator structure of sRNA genes is optimized to generate functional sRNAs. Thus, the Rho-independent terminators of sRNA genes possess two common features: a long T residue stretch that is a prerequisite for generation of functional sRNAs and a moderate strength of hairpin structure that ensures the termination at the seventh or longer position within the consecutive T stretch. The modulation of the termination position at the Rho-independent terminators is critical for biosynthesis of functional sRNAs. PMID:28606943

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp.

PubMed

Souza, Natalie M; Vieira, Monica L; Alves, Ivy J; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2012-09-01

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coli BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K(D)) of 292 ± 24 nm and 157 ± 35 nm, respectively. Moreover, the Lsa30 is a plasminogen (PLG) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. Copyright © 2012 Elsevier Ltd. All rights reserved.

Redox Behavior of the S-Adenosylmethionine (SAM)-Binding Fe-S Cluster in Methylthiotransferase RimO, toward Understanding Dual SAM Activity.

PubMed

Molle, Thibaut; Moreau, Yohann; Clemancey, Martin; Forouhar, Farhad; Ravanat, Jean-Luc; Duraffourg, Nicolas; Fourmond, Vincent; Latour, Jean-Marc; Gambarelli, Serge; Mulliez, Etienne; Atta, Mohamed

2016-10-18

RimO, a radical-S-adenosylmethionine (SAM) enzyme, catalyzes the specific C 3 methylthiolation of the D89 residue in the ribosomal S 12 protein. Two intact iron-sulfur clusters and two SAM cofactors both are required for catalysis. By using electron paramagnetic resonance, Mössbauer spectroscopies, and site-directed mutagenesis, we show how two SAM molecules sequentially bind to the unique iron site of the radical-SAM cluster for two distinct chemical reactions in RimO. Our data establish that the two SAM molecules bind the radical-SAM cluster to the unique iron site, and spectroscopic evidence obtained under strongly reducing conditions supports a mechanism in which the first molecule of SAM causes the reoxidation of the reduced radical-SAM cluster, impeding reductive cleavage of SAM to occur and allowing SAM to methylate a HS - ligand bound to the additional cluster. Furthermore, by using density functional theory-based methods, we provide a description of the reaction mechanism that predicts the attack of the carbon radical substrate on the methylthio group attached to the additional [4Fe-4S] cluster.

The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the β2-adrenoceptor

PubMed Central

Jozwiak, Krzysztof; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W.

2010-01-01

The binding thermodynamics of the stereoisomers of fenoterol, (R,R')-, (S,S')- , (R,S')-, and (S,R')-fenoterol, to the β2-adrenergic receptor (β2-AR) have been determined. The experiments utilized membranes obtained from HEK cells stably transfected with cDNA encoding human β2-AR. Competitive displacement studies using [3H]CGP-12177 as the marker ligand were conducted at 4°, 15°, 25°, 30° and 37°C, the binding affinities calculated and the standard enthalpic (ΔH°) and standard entropic (ΔS°) contribution to the standard free energy change (ΔG°) associated with the binding process determined through the construction of van't Hoff plots. The results indicate that the binding of (S,S')- and (S,R')-fenoterol were predominately enthalpy-driven processes while the binding of (R,R')- and (R,S')-fenoterol were entropy-driven. All of the fenoterol stereoisomers are full agonists of the β2-AR, and, therefore, the results of this study are inconsistent with the previously described “thermodynamic agonist-antagonist discrimination”, in which the binding of an agonist to the β-AR is entropy-driven and the binding of an antagonist is enthalpy driven. In addition, the data demonstrate that the chirality of the carbon atom containing the β-hydroxyl group of the fenoterol molecule (the β-OH carbon) is a key factor in the determination of whether the binding process will be enthalpy-driven or entropy-driven. When the configuration at the β-OH carbon is S the binding process is enthalpy-driven while the R configuration produces an entropy-driven process. PMID:20144591

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

Potential Protein Toxicity of Synthetic Pigments: Binding of Poncean S to Human Serum Albumin☆

PubMed Central

Gao, Hong-Wen; Xu, Qing; Chen, Ling; Wang, Shi-Long; Wang, Yuan; Wu, Ling-Ling; Yuan, Yuan

2008-01-01

Using various methods, e.g., spectrophotometry, circular dichroism, and isothermal titration calorimetry, the interaction of poncean S (PS) with human serum albumin (HSA) was characterized at pH 1.81, 3.56, and 7.40 using the spectral correction technique, and Langmuir and Temkin isothermal models. The consistency among results concerning, e.g., binding number, binding energy, and type of binding, showed that ion pair electrostatic attraction fixed the position of PS in HSA and subsequently induced a combination of multiple noncovalent bonds such as H-bonds, hydrophobic interactions, and van der Waals forces. Ion pair attraction and H-bonds produced a stable PS-HSA complex and led to a marked change in the secondary structure of HSA in acidic media. The PS-HSA binding pattern and the process of change in HSA conformation were also investigated. The potentially toxic effect of PS on the transport function of HSA in a normal physiological environment was analyzed. This work provides a useful experimental strategy for studying the interaction of organic substances with biomacromolecules, helping us to understand the activity or mechanism of toxicity of an organic compound. PMID:17905844

Arsenic and 17-β-estradiol bind to each other and neutralize each other’s signaling effects

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

Kumar, Sukhdeep; Mukherjee, Tapan K.; Guptasarma, Purnananda, E-mail: guptasarma@iisermohali.ac.in

We report that arsenic trioxide (ATO) and 17-beta-estradiol (E2) abolish each other’s independent cell signaling effects in respect of cell survival and proliferation/migration of breast cancer (MCF-7) cells. The possibility that this is due to binding of ATO to E2 was confirmed through difference absorption spectroscopy, chromatography-coupled voltammometry and 1-D {sup 1}H and {sup 13}C NMR spectroscopy. Binding leads to attenuation of E2’s hydroxyl {sup 1}H peaks at its C17 and C3 carbon positions. The results suggest that ATO and E2 can titrate each other’s levels, potentially explaining why sustained arsenic exposure tends to be associated with delays in agemore » of menarche, advanced age of menopause, poorer sperm quality, higher overall morbidity in men, and lower incidences of breast cancer in women in some arsenic-contaminated areas. - Highlights: • Difference absorption spectroscopy suggests that arsenic binds to estradiol. • Interaction with arsenic alters {sup 1}H and {sup 13}C NMR spectra of estradiol at positions C3 and C17. • Estradiol traps arsenic on C{sub 18} reverse-phase columns. • Estradiol and arsenic neutralize each other’s ability to stimulate scratch wound healing. • Arsenic appears to form pnictogen bonds with hydroxyls on estradiol.« less

Engineered proteins as specific binding reagents.

PubMed

Binz, H Kaspar; Plückthun, Andreas

2005-08-01

Over the past 30 years, monoclonal antibodies have become the standard binding proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.

High variation of individual soluble serum CD30 levels of pre-transplantation patients: sCD30 a feasible marker for prediction of kidney allograft rejection?

PubMed

Altermann, Wolfgang; Schlaf, Gerald; Rothhoff, Anita; Seliger, Barbara

2007-10-01

Previous studies have suggested that the pre-transplant levels of the soluble CD30 molecule (sCD30) represent a non-invasive tool which can be used as a biomarker for the prediction of kidney allograft rejections. In order to evaluate the feasibility of sCD30 for pre-transplantation monitoring the sera of potential kidney recipients (n = 652) were collected four times in a 3 months interval. Serum from healthy blood donors (n = 203) served as controls. The sCD30 concentrations of all samples were determined using a commercially available ELISA. This strategy allowed the detection of possible variations of individual sCD30 levels over time. Heterogeneous sCD30 concentrations were found in the samples obtained from individual putative kidney transplant recipients when quarterly measured over 1 year. Total 95% of serum samples obtained from healthy controls exhibited sCD30 values <30 U/ml, whereas most recipients displayed higher serum levels (>30 U/ml). Total 524 patients (80.4%) constantly exhibited serum concentrations of <100 U/ml during the period investigated, whereas 109 patients (16.7%) showed variations by exceeding the proposed 'cut off' of 100 U/ml for one to three times. The frequency of samples exhibiting sCD30 values >100 U/ml was significantly lower than that previously reported. The high degree of variation does not allow the stratification of patients into high and low immunological risk groups based on a single sCD30 value > 100 U/ml. Due to the heterogeneity of sCD30 levels during time course and the high values of SD, its implementation as a pre-transplant marker cannot be justified to generate special provisions for the organ allocation to patients with single sCD30 values > 100 U/ml.

Role of the terminator hairpin in the biogenesis of functional Hfq-binding sRNAs.

PubMed

Morita, Teppei; Nishino, Ryo; Aiba, Hiroji

2017-09-01

Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of seven or more T residues at the 3' end, as noted in previous studies. Here, we have studied the role of the terminator hairpin in the biogenesis of sRNAs focusing on SgrS and RyhB in Escherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the poly(U) tail is shortened. The transcripts with shortened poly(U) tails no longer bind to Hfq and lose the ability to repress the target mRNAs. The shortened transcripts are generated in an in vitro transcription system with purified RNA polymerase, indicating that the generation of shortened transcripts is caused by premature transcription termination. We conclude that the terminator structure of sRNA genes is optimized to generate functional sRNAs. Thus, the Rho-independent terminators of sRNA genes possess two common features: a long T residue stretch that is a prerequisite for generation of functional sRNAs and a moderate strength of hairpin structure that ensures the termination at the seventh or longer position within the consecutive T stretch. The modulation of the termination position at the Rho-independent terminators is critical for biosynthesis of functional sRNAs. © 2017 Morita et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Characterization of GTP binding and hydrolysis in plasma membranes of zucchini

NASA Technical Reports Server (NTRS)

Perdue, D. O.; Lomax, T. L.

1992-01-01

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.

Characterization of GTP binding and hydrolysis in plasma membranes of zucchini.

PubMed

Perdue, D O; Lomax, T L

1992-01-01

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.

Structure and binding energy of the H2S dimer at the CCSD(T) complete basis set limit

NASA Astrophysics Data System (ADS)

Lemke, Kono H.

2017-06-01

This study presents results for the binding energy and geometry of the H2S dimer which have been computed using Møller-Plesset perturbation theory (MP2, MP4) and coupled cluster (CCSD, CCSD(T)) calculations with basis sets up to aug-cc-pV5Z. Estimates of De, EZPE, Do, and dimer geometry have been obtained at each level of theory by taking advantage of the systematic convergence behavior toward the complete basis set (CBS) limit. The CBS limit binding energy values of De are 1.91 (MP2), 1.75 (MP4), 1.41 (CCSD), and 1.69 kcal/mol (CCSD[T]). The most accurate values for the equilibrium S-S distance rSS (without counterpoise correction) are 4.080 (MP2/aug-cc-pV5Z), 4.131 (MP4/aug-cc-pVQZ), 4.225 (CCSD/aug-cc-pVQZ), and 4.146 Å (CCSD(T)/aug-cc-pVQZ). This study also evaluates the effect of counterpoise correction on the H2S dimer geometry and binding energy. As regards the structure of (H2S)2, MPn, CCSD, and CCSD(T) level values of rSS, obtained by performing geometry optimizations on the counterpoise-corrected potential energy surface, converge systematically to CBS limit values of 4.099 (MP2), 4.146 (MP4), 4.233 (CCSD), and 4.167 Å (CCSD(T)). The corresponding CBS limit values of the equilibrium binding energy De are 1.88 (MP2), 1.76 (MP4), 1.41 (CCSD), and 1.69 kcal/mol (CCSD(T)), the latter in excellent agreement with the measured binding energy value of 1.68 ± 0.02 kcal/mol reported by Ciaffoni et al. [Appl. Phys. B 92, 627 (2008)]. Combining CBS electronic binding energies De with EZPE predicted by CCSD(T) vibrational second-order perturbation theory calculations yields Do = 1.08 kcal/mol, which is around 0.6 kcal/mol smaller than the measured value of 1.7 ± 0.3 kcal/mol. Overall, the results presented here demonstrate that the application of high level calculations, in particular CCSD(T), in combination with augmented correlation consistent basis sets provides valuable insight into the structure and energetics of the hydrogen sulfide dimer.

Calcium-Binding Proteins S100A8 and S100A9: Investigation of Their Immune Regulatory Effect in Myeloid Cells.

PubMed

Yang, Jianxin; Anholts, Jacqueline; Kolbe, Ulrike; Stegehuis-Kamp, Janine A; Claas, Frans H J; Eikmans, Michael

2018-06-21

High expression levels of the calcium-binding proteins S100A8 and S100A9 in myeloid cells in kidney transplant rejections are associated with a favorable outcome. Here we investigated the myeloid cell subset expressing these molecules, and their function in inflammatory reactions. Different monocyte subsets were sorted from buffy coats of healthy donors and investigated for S100A8 and S100A9 expression. To characterize S100A9high and S100A9low subsets within the CD14+ classical monocyte subset, intracellular S100A9 staining was combined with flow cytometry (FACS) and qPCR profiling. Furthermore, S100A8 and S100A9 were overexpressed by transfection in primary monocyte-derived macrophages and the THP-1 macrophage cell line to investigate the functional relevance. Expression of S100A8 and S100A9 was primarily found in classical monocytes and to a much lower extent in intermediate and non-classical monocytes. All S100A9+ cells expressed human leukocyte antigen—antigen D related (HLA-DR) on their surface. A small population (<3%) of CD14+ CD11b+ CD33+ HLA-DR− cells, characterized as myeloid derived suppressor cells (MDSCs), also expressed S100A9 to high extent. Overexpression of S100A8 and S00A9 in macrophages led to enhanced extracellular reactive oxygen species (ROS) production, as well as elevated mRNA expression of anti-inflammatory IL-10 . The results suggest that the calcium-binding proteins S100A8 and S100A9 in myeloid cells have an immune regulatory effect.

Molecular investigation of active binding site of isoniazid (INH) and insight into resistance mechanism of S315T-MtKatG in Mycobacterium tuberculosis.

PubMed

Srivastava, Gaurava; Tripathi, Shubhandra; Kumar, Akhil; Sharma, Ashok

2017-07-01

Multi drug resistant tuberculosis is a major threat for mankind. Resistance against Isoniazid (INH), targeting MtKatG protein, is one of the most commonly occurring resistances in MDR TB strains. S315T-MtKatG mutation is widely reported for INH resistance. Despite having knowledge about the mechanism of INH, exact binding site of INH to MtKatG is still uncertain and proposed to have three presumable binding sites (site-1, site-2, and site-3). In the current study docking, molecular dynamics simulation, binding free energy estimation, principal component analysis and free energy landscape analysis were performed to get molecular level details of INH binding site on MtKatG, and to probe the effect of S315T mutation on INH binding. Molecular docking and MD analysis suggested site-1 as active binding site of INH, where the effects of S315T mutation were observed on both access tunnel as well as molecular interaction between INH and its neighboring residues. MMPBSA also supported site-1 as potential binding site with lowest binding energy of -44.201 kJ/mol. Moreover, PCA and FEL revealed that S315T mutation not only reduces the dimension of heme access tunnel but also showed that extra methyl group at 315 position altered heme cavity, enforcing heme group distantly from INH, and thus preventing INH activation. The present study not only investigated the active binding site of INH but also provides a new insight about the conformational changes in the binding site of S315T-MtKatG. Copyright © 2017 Elsevier Ltd. All rights reserved.

Exploring stress tolerance mechanism of evolved freshwater strain Chlorella sp. S30 under 30 g/L salt.

PubMed

Li, Xuyang; Yuan, Yizhong; Cheng, Dujia; Gao, Juan; Kong, Lingzhao; Zhao, Quanyu; Wei, Wei; Sun, Yuhan

2018-02-01

Enhancement of stress tolerance to high concentration of salt and CO 2 is beneficial for CO 2 capture by microalgae. Adaptive evolution was performed for improving the tolerance of a freshwater strain, Chlorella sp. AE10, to 30 g/L salt. A resulting strain denoted as Chlorella sp. S30 was obtained after 46 cycles (138 days). The stress tolerance mechanism was analyzed by comparative transcriptomic analysis. Although the evolved strain could tolerate 30 g/L salt, high salinity caused loss to photosynthesis, oxidative phosphorylation, fatty acid biosynthesis and tyrosine metabolism. The related genes of antioxidant enzymes, CO 2 fixation, amino acid biosynthesis, central carbon metabolism and ABC transporter proteins were up-regulated. Besides the up-regulation of several genes in Calvin-Benson cycle, they were also identified in C4 photosynthetic pathway and crassulacean acid metabolism pathway. They were essential for the survival and CO 2 fixation of Chlorella sp. S30 under 30 g/L salt and 10% CO 2 . Copyright © 2017 Elsevier Ltd. All rights reserved.

Interaction of fenoterol stereoisomers with β2-adrenoceptor-G sα fusion proteins: antagonist and agonist competition binding.

PubMed

Reinartz, Michael T; Kälble, Solveig; Wainer, Irving W; Seifert, Roland

2015-05-01

The specific interaction between G-protein-coupled receptors and ligand is the starting point for downstream signaling. Fenoterol stereoisomers were successfully used to probe ligand-specific activation (functional selectivity) of the β2-adrenoceptor (β2AR) (Reinartz et al. 2015). In the present study, we extended the pharmacological profile of fenoterol stereoisomers using β2AR-Gsα fusion proteins in agonist and antagonist competition binding assays. Dissociations between binding affinities and effector potencies were found for (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol. Our data corroborate former studies on the importance of the aminoalkyl moiety of fenoterol derivatives for functional selectivity.

Mutation detection by mismatch binding protein, MutS, in amplified DNA: Application to the cystic fibrosis gene

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

Lishanski, A.; Ostrander, E.A.; Rine, J.

1994-03-29

An experimental strategy for detecting heterozygosity in genomic DNA has been developed based on preferential binding of Escherichia coli MutS protein to DNA molecules containing mismatched bases. The binding was detected by a gel mobility-shift assay. This approach was tested by using as a model the most commonly occurring mutations within the cystic fibrosis (CFTR) gene. Genomic DNA samples were amplified with 5{prime}-end-labeled primers that bracket the site of the {Delta}F508 3-bp deletion in exon 10 of the CFTR gene. The renatured PCR products from homozygotes produced homoduplexes; the PCR products from heterozygotes produced heteroduplexes and homoduplexes (1:1). MutS proteinmore » bound more strongly to heteroduplexes that correspond to heterozygous carriers of {Delta}F508 and contain a CTT or a GAA loop in one of the strands than to homoduplexes corresponding to homozygotes. The ability of MutS protein to detect heteroduplexes in PCR-amplified DNA extended to fragments {approximately} 500 bp long. The method was also able to detect carriers of the point mutations in exon 11 of the CFTR gene by a preferential binding of MutS to single-base mismatches in PCR-amplified DNA.« less

Modeling Conformational Transitions and Energetics of Ligand Binding with the Glutamate Receptor Ligand Binding Domain

NASA Astrophysics Data System (ADS)

Kurnikova, Maria

2009-03-01

Understanding of protein motion and energetics of conformational transitions is crucial to understanding protein function. The glutamate receptor ligand binding domain (GluR2 S1S2) is a two lobe protein, which binds ligand at the interface of two lobes and undergoes conformational transition. The cleft closure conformational transition of S1S2 has been implicated in gating of the ion channel formed by the transmembrane domain of the receptor. In this study we present a composite multi-faceted theoretical analysis of the detailed mechanism of this conformational transition based on rigid cluster decomposition of the protein structure [1] and identifying hydrogen bonds that are responsible for stabilizing the closed conformation [2]. Free energy of the protein reorganization upon ligand binding was calculated using combined Thermodynamic Integration (TI) and Umbrella Sampling (US) simulations [3]. Ligand -- protein interactions in the binding cleft were analyzed using Molecular Dynamics, continuum electrostatics and QM/MM models [4]. All model calculations compare well with corresponding experimental measurements. [4pt] [1] Protein Flexibility using Constraints from Molecular Dynamics Simulations T. Mamonova, B. Hespenheide, R. Straub, M. F. Thorpe, M. G. Kurnikova , Phys. Biol., 2, S137 (2005)[0pt] [2] Theoretical Study of the Glutamate Receptor Ligand Binding Domain Flexibility and Conformational Reorganization T. Mamonova, K. Speranskiy, and M. Kurnikova , Prot.: Struct., Func., Bioinf., 73,656 (2008)[0pt] [3] Energetics of the cleft closing transition and glutamate binding in the Glutamate Receptor ligand Binding Domain T. Mamonova, M. Yonkunas, and M. Kurnikova Biochemistry 47, 11077 (2008)[0pt] [4] On the Binding Determinants of the Glutamate Agonist with the Glutamate Receptor Ligand Binding Domain K. Speranskiy and M. Kurnikova Biochemistry 44, 11208 (2005)

The Era GTPase recognizes the GAUCACCUCC sequence and binds helix 45 near the 3; end of 16S rRNA

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

Tu, Chao; Zhou, Xiaomei; Tarasov, Sergey G.

2012-03-26

Era, composed of a GTPase domain and a K homology domain, is essential for bacterial cell viability. It is required for the maturation of 16S rRNA and assembly of the 30S ribosomal subunit. We showed previously that the protein recognizes nine nucleotides (1531{sup AUCACCUCC}1539) near the 3{prime} end of 16S rRNA, and that this recognition stimulates GTP-hydrolyzing activity of Era. In all three kingdoms of life, the 1530{sup GAUCA}1534 sequence and helix 45 (h45) (nucleotides 1506-1529) are highly conserved. It has been shown that the 1530{sup GA}1531 to 1530{sup AG}1531 double mutation severely affects the viability of bacteria. However, whethermore » Era interacts with G1530 and/or h45 and whether such interactions (if any) contribute to the stimulation of Era's GTPase activity were not known. Here, we report two RNA structures that contain nucleotides 1506-1542 (RNA301), one in complex with Era and GDPNP (GNP), a nonhydrolysable GTP-analogue, and the other in complex with Era, GNP, and the KsgA methyltransferase. The structures show that Era recognizes 10 nucleotides, including G1530, and that Era also binds h45. Moreover, GTPase assay experiments show that G1530 does not stimulate Era's GTPase activity. Rather, A1531 and A1534 are most important for stimulation and h45 further contributes to the stimulation. Although G1530 does not contribute to the intrinsic GTPase activity of Era, its interaction with Era is important for binding and is essential for the protein to function, leading to the discovery of a new cold-sensitive phenotype of Era.« less

Characterization of Glutaredoxin Fe-S Cluster-Binding Interactions Using Circular Dichroism Spectroscopy.

PubMed

Albetel, Angela-Nadia; Outten, Caryn E

2018-01-01

Monothiol glutaredoxins (Grxs) with a conserved Cys-Gly-Phe-Ser (CGFS) active site are iron-sulfur (Fe-S) cluster-binding proteins that interact with a variety of partner proteins and perform crucial roles in iron metabolism including Fe-S cluster transfer, Fe-S cluster repair, and iron signaling. Various analytical and spectroscopic methods are currently being used to monitor and characterize glutaredoxin Fe-S cluster-dependent interactions at the molecular level. The electronic, magnetic, and vibrational properties of the protein-bound Fe-S cluster provide a convenient handle to probe the structure, function, and coordination chemistry of Grx complexes. However, some limitations arise from sample preparation requirements, complexity of individual techniques, or the necessity for combining multiple methods in order to achieve a complete investigation. In this chapter, we focus on the use of UV-visible circular dichroism spectroscopy as a fast and simple initial approach for investigating glutaredoxin Fe-S cluster-dependent interactions. © 2018 Elsevier Inc. All rights reserved.

Mature parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum binds to the 30-kDa domain of protein 4.1 in malaria-infected red blood cells.

PubMed

Waller, Karena L; Nunomura, Wataru; An, Xiuli; Cooke, Brian M; Mohandas, Narla; Coppel, Ross L

2003-09-01

The Plasmodium falciparum mature parasite-infected erythrocyte surface antigen (MESA) is exported from the parasite to the infected red blood cell (IRBC) membrane skeleton, where it binds to protein 4.1 (4.1R) via a 19-residue MESA sequence. Using purified RBC 4.1R and recombinant 4.1R fragments, we show MESA binds the 30-kDa region of RBC 4.1R, specifically to a 51-residue region encoded by exon 10 of the 4.1R gene. The 3D structure of this region reveals that the MESA binding site overlaps the region of 4.1R involved in the p55, glycophorin C, and 4.1R ternary complex. Further binding studies using p55, 4.1R, and MESA showed competition between p55 and MESA for 4.1R, implying that MESA bound at the IRBC membrane skeleton may modulate normal 4.1R and p55 interactions in vivo. Definition of minimal binding domains involved in critical protein interactions in IRBCs may aid the development of novel therapies for falciparum malaria.

A computer model for the 30S ribosome subunit.

PubMed Central

Kuntz, I D; Crippen, G M

1980-01-01

We describe a computer-generated model for the locations of the 21 proteins of the 30S subunit of the E. coli ribosome. The model uses a new method of incorporating experimental measurements based on a mathematical technique called distance geometry. In this paper, we use data from two sources: immunoelectron microscopy and neutron-scattering studies. The data are generally self-consistent and lead to a set of relatively well-defined structures in which individual protein coordinates differ by approximately 20 A from one structure to another. Two important features of this calculation are the use of extended proteins rather than just the centers of mass, and the ability to confine the protein locations within an arbitrary boundary surface so that only solutions with an approximate 30S "shape" are permitted. PMID:7020786

Mutant protein of recombinant human granulocyte colony-stimulating factor for receptor binding assay.

PubMed

Watanabe, M; Fukamachi, H; Uzumaki, H; Kabaya, K; Tsumura, H; Ishikawa, M; Matsuki, S; Kusaka, M

1991-05-15

A new mutant protein of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was produced for the studies on receptors for human G-CSF. The mutant protein [(Tyr1, Tyr3]rhG-CSF), the biological activity of which was almost equal to that of rhG-CSF, was prepared by the replacement of threonine-1 and leucine-3 of rhG-CSF with tyrosine. The radioiodinated preparation of the mutant protein showed high specific radioactivity and retained full biological activity for at least 3 weeks. The binding capacity of the radioiodinated ligand was compared with that of [35S]rhG-CSF. Both radiolabeled ligands showed specific binding to murine bone marrow cells. Unlabeled rhG-CSF and human G-CSF purified from the culture supernatant of the human bladder carcinoma cell line 5637 equally competed for the binding of labeled rhG-CSFs in a dose-dependent manner, demonstrating that the sugar moiety of human G-CSF made no contribution to the binding of human G-CSF to target cells. In contrast, all other colony-stimulating factors and lymphokines examined did not affect the binding. Scatchard analysis of the specific binding of both labeled ligands revealed a single class of binding site with an apparent dissociation constant (Kd) of 20-30 pM and 100-200 maximal binding sites per cell. These data indicate that the radioiodinated preparation of the mutant protein binds the same specific receptor with the same affinity as [35S]rhG-CSF. The labeled mutant protein also showed specific binding to human circulating neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

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

PubMed Central

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

2015-01-01

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

Lactose carrier protein of Escherichia coli. Transport and binding of 2'-(N-dansyl)aminoethyl beta-D-thiogalactopyranoside and p-nitrophenyl alpha-d-galactopyranoside.

PubMed

Overath, P; Teather, R M; Simoni, R D; Aichele, G; Wilhelm, U

1979-01-09

The elevated level of lactose carrier protein present in cytoplasmic membranes derived from Escherichia coli strain T31RT, which carries the Y gene of the lac operon on a plasmid vector (Teather, R. M., et al. (1978) Mol. Gen. Genet. 159, 239--248), has allowed the detection of a complex between the carrier and the fluorescent substrate 2'-(N-dansyl)-aminoethyl beta-D-thiogalactopyranoside (Dns2-S-Gal). Binding is accompanied by a 50-nm blue shift in the emission maximum of the dansyl residue. The complex (dissociation constant, KD = 30 micron) rapidly dissociates upon addition of competing substrates such as beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside or upon reaction with the thiol reagent p-chloromercuribenzenesulfonate. Binding of both Dns2-S-Gal and p-nitrophenyl alpha-D-galactopyranoside (alpha-NPG) occurs spontaneously in the absence of an electrochemical potential gradient across the membrane. Comparison of equilibrium binding experiments using Dns2-S-Gal or alpha-NPG and differential labeling of the carrier with radioactive amino acids shows that the carrier binds 1 mol of substrate per mol of polypeptide (molecular weight 30 000). In addition to specific binding to the lactose carrier, Dns2-S-gal binds unspecifically to lipid vesicles or membranes, as described by a partition coefficient, K = 60, resulting in a 25-nm blue shift in the emission maximum of the dansyl group. Both Dns2-S-Gal and alpha-NPG are not only bound by the lactose carrier but also transported across the membrane by this transport protein in cells and membrane vesicles. The fluorescence changes observed with dansylated galactosides in membrane vesicles in the presence of an electrochemical gradient (Schuldiner et al. (1975) J. Biol. Chem. 250, 1361--1370)) are interpreted as an increase in unspecific binding after translocation.

Evaluation of serum sCD30 in renal transplantation patients with and without acute rejection.

PubMed

Cervelli, C; Fontecchio, G; Scimitarra, M; Azzarone, R; Famulari, A; Pisani, F; Battistoni, C; Di Iulio, B; Fracassi, D; Scarnecchia, M A; Papola, F

2009-05-01

Despite new immunosuppressive approaches, acute rejection episodes (ARE) are still a major cause of early kidney dysfunction with a negative impact on long-term allograft survival. Noninvasive markers able to identify renal ARE earlier than creatinine measurement include sCD30. We sought to establish whether circulating levels of sCD30 in pretransplantation and posttransplantation periods were of clinical relevance to avoid graft damage. Quantitative detection of serum sCD30 was performed using an enzyme-linked immunosorbent assay. Our results demonstrated that the mean concentrations of sCD30 were significantly higher in the sera of renal transplant recipients with ARE (30.04 U/mL) and in uremic patients on the waiting list (37.7 U/mL) compared with healthy controls (HC; 9.44 U/mL), but not nonrejecting patients (12.01 U/mL). Statistical analysis revealed a strong association between high sCD30 levels in posttransplantation sera and ARE risk. This study suggested that sCD30 levels were a reliable predictor of ARE among deceased-donor kidney recipients.

YC-1 BINDING TO THE BETA SUBUNIT OF SOLUBLE GUANYLYL CYCLASE OVERCOMES ALLOSTERIC INHIBITION BY THE ALPHA SUBUNIT

PubMed Central

Purohit, Rahul; Fritz, Bradley G.; The, Juliana; Issaian, Aaron; Weichsel, Andrzej; David, Cynthia L.; Campbell, Eric; Hausrath, Andrew C.; Rassouli-Taylor, Leida; Garcin, Elsa D.; Gage, Matthew J.; Montfort, William R.

2014-01-01

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity. PMID:24328155

Structure and binding energy of the H2S dimer at the CCSD(T) complete basis set limit.

PubMed

Lemke, Kono H

2017-06-21

This study presents results for the binding energy and geometry of the H 2 S dimer which have been computed using Møller-Plesset perturbation theory (MP2, MP4) and coupled cluster (CCSD, CCSD(T)) calculations with basis sets up to aug-cc-pV5Z. Estimates of D e , E ZPE , D o , and dimer geometry have been obtained at each level of theory by taking advantage of the systematic convergence behavior toward the complete basis set (CBS) limit. The CBS limit binding energy values of D e are 1.91 (MP2), 1.75 (MP4), 1.41 (CCSD), and 1.69 kcal/mol (CCSD[T]). The most accurate values for the equilibrium S-S distance r SS (without counterpoise correction) are 4.080 (MP2/aug-cc-pV5Z), 4.131 (MP4/aug-cc-pVQZ), 4.225 (CCSD/aug-cc-pVQZ), and 4.146 Å (CCSD(T)/aug-cc-pVQZ). This study also evaluates the effect of counterpoise correction on the H 2 S dimer geometry and binding energy. As regards the structure of (H 2 S) 2 , MPn, CCSD, and CCSD(T) level values of r SS , obtained by performing geometry optimizations on the counterpoise-corrected potential energy surface, converge systematically to CBS limit values of 4.099 (MP2), 4.146 (MP4), 4.233 (CCSD), and 4.167 Å (CCSD(T)). The corresponding CBS limit values of the equilibrium binding energy D e are 1.88 (MP2), 1.76 (MP4), 1.41 (CCSD), and 1.69 kcal/mol (CCSD(T)), the latter in excellent agreement with the measured binding energy value of 1.68 ± 0.02 kcal/mol reported by Ciaffoni et al. [Appl. Phys. B 92, 627 (2008)]. Combining CBS electronic binding energies D e with E ZPE predicted by CCSD(T) vibrational second-order perturbation theory calculations yields D o = 1.08 kcal/mol, which is around 0.6 kcal/mol smaller than the measured value of 1.7 ± 0.3 kcal/mol. Overall, the results presented here demonstrate that the application of high level calculations, in particular CCSD(T), in combination with augmented correlation consistent basis sets provides valuable insight into the structure and energetics of the hydrogen sulfide

Structural and thermodynamic characterization of the recognition of the S100-binding peptides TRTK12 and p53 by calmodulin

PubMed Central

Wafer, Lucas N; Tzul, Franco O; Pandharipande, Pranav P; McCallum, Scott A; Makhatadze, George I

2014-01-01

Calmodulin (CaM) is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells in a calcium-dependent manner. CaM has been proposed to be functionally distinct from the S100 proteins, a related family of eukaryotic calcium-binding proteins. Previously, it was demonstrated that peptides derived from the actin-capping protein, TRTK12, and the tumor-suppressor protein, p53, interact with multiple members of the S100 proteins. To test the specificity of these peptides, they were screened using isothermal titration calorimetry against 16 members of the human S100 protein family, as well as CaM, which served as a negative control. Interestingly, both the TRTK12 and p53 peptides were found to interact with CaM. These interactions were further confirmed by both fluorescence and nuclear magnetic resonance spectroscopies. These peptides have distinct sequences from the known CaM target sequences. The TRTK12 peptide was found to independently interact with both CaM domains and bind with a stoichiometry of 2:1 and dissociations constants Kd,C-term = 2 ± 1 µM and Kd,N-term = 14 ± 1 µM. In contrast, the p53 peptide was found to interact only with the C-terminal domain of CaM, Kd,C-term =2 ± 1 µM, 25°C. Using NMR spectroscopy, the locations of the peptide binding sites were mapped onto the structure of CaM. The binding sites for both peptides were found to overlap with the binding interface for previously identified targets on both domains of CaM. This study demonstrates the plasticity of CaM in target binding and may suggest a possible overlap in target specificity between CaM and the S100 proteins. PMID:24947426

Insights into ligand binding to a Glutathione S-transferase from mango: structure, thermodynamics and kinetics

PubMed Central

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.; Serrano-Posada, Hugo; Lopez-Zavala, Alonso A.; García-Orozco, Karina D.; Hernandez-Paredes, Javier; Rudiño-Piñera, Enrique; Stojanoff, Vivian; Sotelo-Mundo, Rogerio R.; Islas-Osuna, Maria A.

2017-01-01

We studied a mango glutathione S-transferase (GST) (Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a Km, Vmax and kcat for CDNB of 0.792 mM, 80.58 mM·min−1 and 68.49 s−1 respectively and 0.693 mM, 105.32 mM·min−1 and 89.57 s−1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 μM) or GSX (7.8 μM). The crystal structure of the MiGSTU in apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes. PMID:28104507

Mutational analysis of human RNA polymerase II subunit 5 (RPB5): the residues critical for interactions with TFIIF subunit RAP30 and hepatitis B virus X protein.

PubMed

Le, Thi Thu Thuy; Zhang, Shijun; Hayashi, Naoyuki; Yasukawa, Mami; Delgermaa, Luvsanjav; Murakami, Seishi

2005-09-01

RNA polymerase II (RNAPII) subunit 5 (RPB5) is positioned close to DNA downstream of the initiation site and is the site of interaction with several regulators. Hepatitis B virus X protein (HBx) binds the central part of RPB5 to modulate activated transcription, and TFIIF subunit RAP30 interacts with the same part of RPB5 that is critical for the association between TFIIF and RNAPII. However the residues necessary for these interactions remain unknown. Here we report systematic mutagenesis of the central part of RPB5 using two-step alanine scanning libraries to pinpoint critical residues for its binding to RAP30 in the TFIIF complex and/or to HBx, and identified these residues in both mammalian cells and in an in vitro binding assay. Four residues, F76, I104, T111 and S113, are critical for both TFIIF- and HBx-binding, indicating the overlapping nature of the sites of interaction. In addition, V74 and N98 are required for HBx-binding, and T56 and L58 are needed for RAP30-binding. Interestingly the residues exposed to solvent, T111 and S113, are very close to the DNA, implying that two factors may modulate the interaction between DNA and RPB5.

Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site

PubMed Central

Calviño, Fabiola R.; Kharde, Satyavati; Ori, Alessandro; Hendricks, Astrid; Wild, Klemens; Kressler, Dieter; Bange, Gert; Hurt, Ed; Beck, Martin; Sinning, Irmgard

2015-01-01

During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1–RpL5-N–RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1–RpL5–RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP. PMID:25849277

Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site.

PubMed

Calviño, Fabiola R; Kharde, Satyavati; Ori, Alessandro; Hendricks, Astrid; Wild, Klemens; Kressler, Dieter; Bange, Gert; Hurt, Ed; Beck, Martin; Sinning, Irmgard

2015-04-07

During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1-RpL5-N-RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1-RpL5-RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP.

Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals

DOE PAGES

Dao, E. Han; Sierra, Raymond G.; Laksmono, Hartawan; ...

2015-04-30

In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecondmore » X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.« less

Ligand-binding pocket shape differences between S1P1 and S1P3 determine efficiency of chemical probe identification by uHTS

PubMed Central

Schürer, Stephan C.; Brown, Steven J.; Cabrera, Pedro Gonzales; Schaeffer, Marie-Therese; Chapman, Jacqueline; Jo, Euijung; Chase, Peter; Spicer, Tim; Hodder, Peter; Rosen, Hugh

2008-01-01

We have studied the Sphingosine 1-phosphate (S1P) receptor system to better understand why certain molecular targets within a closely related family are much more tractable when identifying compelling chemical leads. Five medically important G protein-coupled receptors for S1P regulate heart rate, coronary artery caliber, endothelial barrier integrity, and lymphocyte trafficking. Selective S1P receptor agonist probes would be of great utility to study receptor subtype-specific function. Through systematic screening of the same libraries, we identified novel selective agonists chemotypes for each of the S1P1 and S1P3 receptors. uHTS for S1P1 was more effective than for S1P3, with many selective, low nanomolar hits of proven mechanism emerging for. Receptor structure modeling and ligand docking reveal differences between the receptor binding pockets, which are the basis for sub-type selectivity. Novel selective agonists interact primarily in the hydrophobic pocket of the receptor in the absence of head-group interactions. Chemistry-space and shape-based analysis of the screening libraries in combination with the binding models explain the observed differential hit rates and enhanced efficiency for lead discovery for S1P1 vs. S1P3 in this closely related receptor family. PMID:18590333

Sublingual immunization with the phosphate-binding-protein (PstS) reduces oral colonization by Streptococcus mutans.

PubMed

Ferreira, E L; Batista, M T; Cavalcante, R C M; Pegos, V R; Passos, H M; Silva, D A; Balan, A; Ferreira, L C S; Ferreira, R C C

2016-10-01

Bacterial ATP-binding cassette (ABC) transporters play a crucial role in the physiology and pathogenicity of different bacterial species. Components of ABC transporters have also been tested as target antigens for the development of vaccines against different bacterial species, such as those belonging to the Streptococcus genus. Streptococcus mutans is the etiological agent of dental caries, and previous studies have demonstrated that deletion of the gene encoding PstS, the substrate-binding component of the phosphate uptake system (Pst), reduced the adherence of the bacteria to abiotic surfaces. In the current study, we generated a recombinant form of the S. mutans PstS protein (rPstS) with preserved structural features, and we evaluated the induction of antibody responses in mice after sublingual mucosal immunization with a formulation containing the recombinant protein and an adjuvant derived from the heat-labile toxin from enterotoxigenic Escherichia coli strains. Mice immunized with rPstS exhibited systemic and secreted antibody responses, measured by the number of immunoglobulin A-secreting cells in draining lymph nodes. Serum antibodies raised in mice immunized with rPstS interfered with the adhesion of bacteria to the oral cavity of naive mice challenged with S. mutans. Similarly, mice actively immunized with rPstS were partially protected from oral colonization after challenge with the S. mutans NG8 strain. Therefore, our results indicate that S. mutans PstS is a potential target antigen capable of inducing specific and protective antibody responses after sublingual administration. Overall, these observations raise interesting perspectives for the development of vaccines to prevent dental caries. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Epstein-Barr virus-encoded EBNA-5 binds to Epstein-Barr virus-induced Fte1/S3a protein

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

Kashuba, Elena; Yurchenko, Mariya; Szirak, Krisztina

Epstein-Barr virus (EBV) transforms resting human B cells into immortalized immunoblasts. EBV-encoded nuclear antigens EBNA-5 (also called EBNA-LP) is one of the earliest viral proteins expressed in freshly infected B cells. We have recently shown that EBNA-5 binds p14ARF, a nucleolar protein that regulates the p53 pathway. Here, we report the identification of another protein with partially nucleolar localization, the v-fos transformation effector Fte-1 (Fte-1/S3a), as an EBNA-5 binding partner. In transfected cells, Fte-1/S3a and EBNA-5 proteins showed high levels of colocalization in extranucleolar inclusions. Fte-1/S3a has multiple biological functions. It enhances v-fos-mediated cellular transformation and is part of themore » small ribosomal subunit. It also interacts with the transcriptional factor CHOP and apoptosis regulator poly(ADP-ribose) polymerase (PARP). Fte-1/S3a is regularly expressed at high levels in both tumors and cancer cell lines. Its high expression favors the maintenance of malignant phenotype and undifferentiated state, whereas its down-regulation is associated with cellular differentiation and growth arrest. Here, we show that EBV-induced B cell transformation leads to the up-regulation of Fte-1/S3a. We suggest that EBNA-5 through binding may influence the growth promoting, differentiation inhibiting, or apoptosis regulating functions of Fte-1/S3a.« less

Binding of /sup 18/F by cell membranes and cell walls of Streptococcus mutans

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

Yotis, W.W.; Zeb, M.; McNulty, J.

1983-07-01

The binding of /sup 18/F to isolated cell membranes and cell walls of Streptococcus mutans GS-5 or other bacteria was assayed. The attachment of /sup 18/F to these cell envelopes proceeded slowly and reached equilibrium within 60 min. /sup 18/F binding was stimulated by Ca/sup 2 +/ (1 mM). The binding of /sup 18/F to cellular components was dependent upon the pH, as well as the amount of /sup 18/F and dose of the binder employed. The binding of /sup 18/F by cell walls prepared from fluoride-sensitive and fluoride-resistant cells of S. salivarius and S. mutans did not differ significantly.more » The pretreatment of cell walls or cell membranes for 60 min at 30 degrees C with 1 mg of RNase, DNase, or trypsin per ml did not influence the binding of /sup 18/F by the walls and membranes of S. mutans GS-5. However, prior exposure of cell membranes to sodium dodecyl sulfate caused a significant reduction in the number of /sup 18/F atoms bound by the membranes. In saturated assay systems, cell membranes of S. mutans GS-5 bound 10(15) to 10(16) atoms of /sup 18/F per mg (dry weight), whereas cell walls from S. mutans GS-5, FA-1, and HS-6 or Actinomyces viscosus T14V and T14AV bound 10(12) to 10(13) atoms of /sup 18/F per mg (dry weight). /sup 18/F in this quantity (10(12) to 10(13) atoms) cannot be detected with the fluoride electrode. The data provide, for the first time, a demonstration of /sup 18/F binding by cell membranes and walls of oral flora.« less

Functions of Intracellular Retinoid Binding-Proteins.

PubMed

Napoli, Joseph L

Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.

Dynamics of 28,30S i* compound nuclei formed at sub-barrier energies

NASA Astrophysics Data System (ADS)

Kaur, Manpreet; Singh, Bir Bikram; Kaur, Sarbjeet

2018-05-01

The decay of 28S i* and 30S i* compound nuclei (CN) formed at sub-barrier energies, in the reactions induced by stable projectile 16O and exotic projectile 18O, respectively, has been investigated within the quantum mechanical fragmentation theory based dynamical cluster-decay model (DCM). The collective potential energy surface shows that xα-type (x is an integer) clusters are minimized in the decay of 28S i* while in case of 30S i* in addition to xα-type clusters, np-xα (n, p are neutron and proton, respectively) type clusters are also minimized. These minimized fragments have more preformation probability P0, which is an important factor through which nuclear structure effects of decaying CN are probed, within DCM. The results show that light particles (LPs) are contributing mostly in the fusion cross-section, σfusion. In case of 30S i*, the contribution of 1n is highest and more compared to 4He in case of 28S i*, which seems to play an important role in fusion enhancement. The DCM calculated σfusion for both the CN formed with same Ec.m. = 7.0 MeV gives more value for σfusion of 30S i*, in agreement with the experimental data.

Investigation of nuclear structure of 30-44S isotopes using spherical and deformed Skyrme-Hartree-Fock method

NASA Astrophysics Data System (ADS)

Alzubadi, A. A.

2015-06-01

Nuclear many-body system is usually described by a mean-field built upon a nucleon-nucleon effective interaction. In this work, we investigate ground state properties of the sulfur isotopes covering a wide range from the line of stability up to the dripline region (30-44S). For this purpose the Hartree-Fock mean field theory in coordinate space with a Skyrme parameterization SkM* has been utilized. In particular, we calculate the nuclear charge, neutrons, protons, mass densities, the associated radii, neutron skin thickness and binding energy. The charge form factors have been also investigated using SkM*, SkO, SkE, SLy4 and Skxs15 Skyrme parameterizations and the results obtained using the theoretical approach are compared with the available experimental data. To investigate the potential energy surface as a function of the quadrupole deformation for isotopic sulfur chains, Skyrme-Hartree-Fock-Bogoliubov theory has been adopted with SLy4 parameterization.

Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain

PubMed Central

Sakata, Eri; Yamaguchi, Yoshiki; Kurimoto, Eiji; Kikuchi, Jun; Yokoyama, Shigeyuki; Yamada, Shingo; Kawahara, Hiroyuki; Yokosawa, Hideyoshi; Hattori, Nobutaka; Mizuno, Yoshikuni; Tanaka, Keiji; Kato, Koichi

2003-01-01

Parkin, a product of the causative gene of autosomal-recessive juvenile parkinsonism (AR-JP), is a RING-type E3 ubiquitin ligase and has an amino-terminal ubiquitin-like (Ubl) domain. Although a single mutation that causes an Arg to Pro substitution at position 42 of the Ubl domain (the Arg 42 mutation) has been identified in AR-JP patients, the function of this domain is not clear. In this study, we determined the three-dimensional structure of the Ubl domain of parkin by NMR, in particular by extensive use of backbone 15N-1H residual dipolar-coupling data. Inspection of chemical-shift-perturbation data showed that the parkin Ubl domain binds the Rpn10 subunit of 26S proteasomes via the region of parkin that includes position 42. Our findings suggest that the Arg 42 mutation induces a conformational change in the Rpn10-binding site of Ubl, resulting in impaired proteasomal binding of parkin, which could be the cause of AR-JP. PMID:12634850

Mechanism of Sulfide Binding by Ferric Hemeproteins.

PubMed

Boubeta, Fernando M; Bieza, Silvina A; Bringas, Mauro; Estrin, Darío A; Boechi, Leonardo; Bari, Sara E

2018-06-19

The reaction of hydrogen sulfide (H 2 S) with hemeproteins is a key physiological reaction; still, its mechanism and implications are not completely understood. In this work, we propose a combination of experimental and theoretical tools to shed light on the reaction in model system microperoxidase 11 (MP11-Fe III ) and myoglobin (Mb-Fe III ), from the estimation of the intrinsic binding constants of the species H 2 S and hydrosulfide (HS - ), and the computational description of the overall binding process. Our results show that H 2 S and HS - are the main reactive species in Mb-Fe III and MP11-Fe III , respectively, and that the magnitude of their intrinsic binding constants are similar to most of the binding constants reported so far for hemeproteins systems and model compounds. However, while the binding of HS - to Mb-Fe III was negligible, the binding of H 2 S to MP11-Fe III was significant, providing a frame for a discriminated analysis of both species and revealing differential mechanistic aspects. A joint inspection of the kinetic data and the free energy profiles of the binding processes suggests that a dissociative mechanism with the release of a coordinated water molecule as rate limiting step is operative in the binding of H 2 S to Mb-Fe III and that the binding of HS - is prevented in the access to the protein matrix. For the MP11-Fe III case, where no access restrictions for the ligands are present, an associative component in the mechanism seems to be operative. Overall, the results suggest that if accessing the active site then both H 2 S and HS - are capable of binding a ferric heme moiety.

mRNA bound to the 30S subunit is a HigB toxin substrate

PubMed Central

Schureck, Marc A.; Maehigashi, Tatsuya; Miles, Stacey J.; Marquez, Jhomar; Dunham, Christine M.

2016-01-01

Activation of bacterial toxins during stress results in cleavage of mRNAs in the context of the ribosome. These toxins are thought to function as global translational inhibitors yet recent studies suggest each may have distinct mRNA specificities that result in selective translation for bacterial survival. Here we demonstrate that mRNA in the context of a bacterial 30S subunit is sufficient for ribosome-dependent toxin HigB endonucleolytic activity, suggesting that HigB interferes with the initiation step of translation. We determined the X-ray crystal structure of HigB bound to the 30S, revealing that two solvent-exposed clusters of HigB basic residues directly interact with 30S 16S rRNA helices 18, 30, and 31. We further show that these HigB residues are essential for ribosome recognition and function. Comparison with other ribosome-dependent toxins RelE and YoeB reveals that each interacts with similar features of the 30S aminoacyl (A) site yet does so through presentation of diverse structural motifs. PMID:27307497

Activated effector and memory T cells contribute to circulating sCD30: potential marker for islet allograft rejection.

PubMed

Saini, D; Ramachandran, S; Nataraju, A; Benshoff, N; Liu, W; Desai, N; Chapman, W; Mohanakumar, T

2008-09-01

T-cell activation up-regulates CD30 resulting in an increase in serum soluble CD30 (sCD30). CD4+ T cells, a major source for sCD30, play a significant role in the pathogenesis of rejection. In this study, sCD30 was measured pre- and posttransplant in mouse islet allograft models and human islet allograft recipients. sCD30 was measured by ELISA in diabetic C57BL/6, CD4Knockout (KO) and CD8KO islet allograft recipients. sCD30 increased significantly prior to rejection (1.8 +/- 1 days) in 80% of allograft recipients. Sensitization with donor splenocytes, or a second graft, further increased sCD30 (282.5 +/- 53.5 for the rejecting first graft vs. 374.6 +/- 129 for the rejecting second graft) prior to rejection suggesting memory CD4+ T cells contribute to sCD30. CD4KO failed to reject islet allograft and did not demonstrate sCD30 increase. CD8KO showed elevated (227 +/- 107) sCD30 (1 day) prior to rejection. High pretransplant sCD30 (>20 U/ml) correlated with poor outcome in human islet allograft recipients. Further, increase in sCD30 posttransplant preceded (3-4 months) loss of islet function. We conclude that sCD30 is released from activated CD4 T cells prior to islet allograft rejection and monitoring sCD30 can be a valuable adjunct in the follow-up of islet transplant recipients.

Posttransplant sCD30 as a biomarker to predict kidney graft outcome.

PubMed

Süsal, Caner; Opelz, Gerhard

2012-09-08

In current clinical praxis, monitoring of immunosuppressive agents in organ transplantation is restricted to measurement of drug blood levels and does not consider the drug's variable effect on the individual patient's immune system. Establishment of biological markers that measure the biological effect of immunosuppressive drugs is desirable and would enable the identification of patients who are at risk of developing rejection, or patients who are suitable for minimization or weaning of immunosuppressive therapy. Several studies demonstrated that the technically simple posttransplant measurement in serum of the T cell activation marker soluble CD30 (sCD30) allows prediction of subsequent graft loss in kidney transplant recipients. sCD30 is a relatively large molecule and therefore an attractive biological marker which is resistant to repeated thawing cycles and temperature differences and easily determined using commercial ELISA. Whether sCD30-based prospective adjustment of immunosuppressive therapy can prevent irreversible graft damage and improve long-term graft outcome awaits evaluation in randomized controlled trials. Copyright © 2011 Elsevier B.V. All rights reserved.

Isospin mixing reveals 30P(p, γ) 31S resonance influencing nova nucleosynthesis

DOE PAGES

Bennett, M. B.; Wrede, C.; Brown, B. A.; ...

2016-03-08

Here, the thermonuclear 30P(p, γ) 31S reaction rate is critical for modeling the final elemental and isotopic abundances of ONe nova nucleosynthesis, which affect the calibration of proposed nova thermometers and the identification of presolar nova grains, respectively. Unfortunately, the rate of this reaction is essentially unconstrained experimentally, because the strengths of key 31S proton capture resonance states are not known, largely due to uncertainties in their spins and parities. Using the β decay of 31Cl, we have observed the β-delayed γ decay of a 31S state at E x = 6390.2(7) keV, with a 30P(p, γ) 31S resonance energymore » of E r = 259.3(8) keV, in the middle of the 30P(p, γ) 31S Gamow window for peak nova temperatures. This state exhibits isospin mixing with the nearby isobaric analog state at E x = 6279.0(6) keV, giving it an unambiguous spin and parity of 3/2 + and making it an important l = 0 resonance for proton capture on 30P.« less

Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains.

PubMed

Dogan, Jakob; Jonasson, Josefin; Andersson, Eva; Jemth, Per

2015-08-04

Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, kon, for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 × 10(6) M(-1) s(-1) at 4 °C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the kon for the interaction between NCBD and the folded PNT domain is only 7 × 10(6) M(-1) s(-1) (4 °C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.

Role of conserved nucleotides in building the 16S rRNA binding site of E. coli ribosomal protein S8.

PubMed Central

Allmang, C; Mougel, M; Westhof, E; Ehresmann, B; Ehresmann, C

1994-01-01

Ribosomal protein S8 specifically recognizes a helical and irregular region of 16S rRNA that is highly evolutionary constrained. Despite its restricted size, the precise conformation of this region remains a question of debate. Here, we used chemical probing to analyze the structural consequences of mutations in this RNA region. These data, combined with computer modelling and previously published data on protein binding were used to investigate the conformation of the RNA binding site. The experimental data confirm the model in which adenines A595, A640 and A642 bulge out in the deep groove. In addition to the already proposed non canonical U598-U641 interaction, the structure is stabilized by stacking interactions (between A595 and A640) and an array of hydrogen bonds involving bases and the sugar phosphate backbone. Mutations that alter the ability to form these interdependent interactions result in a local destabilization or reorganization. The specificity of recognition by protein S8 is provided by the irregular and distorted backbone and the two bulged adenines 640 and 642 in the deep groove. The third adenine (A595) is not a direct recognition site but must adopt a bulged position. The U598-U641 pair should not be directly in contact with the protein. Images PMID:7937081

Thermodynamic and Kinetics Analysis of Peptides Derived from CapZ, NDR, p53, HDM2, and HDM4 Binding to Human S100B

PubMed Central

Wafer, Lucas N.; Streicher, Werner W.; McCallum, Scott A.; Makhatadze, George I.

2012-01-01

S100B is a member of the S100 subfamily of EF-hand proteins that has been implicated in malignant melanoma and neurodegenerative conditions such as Alzheimer's and Parkinson's disease. Calcium-induced conformational changes expose a hydrophobic binding cleft, facilitating interactions with a wide variety of nuclear, cytoplasmic, and extracellular target proteins. Previously, peptides derived from CapZ, p53, NDR, HDM2 and HDM4 have been shown to interact with S100B in a calcium-dependent manner. However, the thermodynamic and kinetic basis of these interactions remains largely unknown. To gain further insight, these peptides were screened against the S100B protein using isothermal titration calorimetry and nuclear magnetic resonance. All peptides were found to have binding affinities in the low micromolar to nanomolar range. Binding-induced changes in the line shapes of S100B backbone 1H and 15N were monitored to obtain the dissociation constants and the kinetic binding parameters. The large microscopic Kon rate constants observed in this study, Kon ≥1×107 M-1s-1, suggest that S100B utilizes a “fly casting mechanism” in the recognition of these peptide targets. PMID:22913742

Two nucleotide binding sites modulate ( sup 3 H) glyburide binding to rat cortex membranes

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

Johnson, D.E.; Gopalakrishnan, M.; Triggle, D.J.

1991-03-11

The effects of nucleotides on the binding of the ATP-dependent K{sup +}-channel antagonist ({sup 3}H)glyburide (GLB) to rat cortex membranes were examined. Nucleotide triphosphates (NTPs) and nucleotide diphosphate (NDPs) inhibited the binding of GLB. This effect was dependent on the presence of dithiothreitol (DTT). Inhibition of binding by NTPs, with the exception of ATP{gamma}S, was dependent on the presence of Mg{sup 2+}. GLB binding showed a biphasic response to ADP: up to 3 mM, ADP inhibited binding, and above this concentration GLB binding increased rapidly, and was restored to normal levels by 10 mM ADP. In the presence of Mg{supmore » 2+}, ADP did not stimulate binding. Saturation analysis in the presence of Mg{sup 2+} and increasing concentrations of ADP showed that ADP results primarily in a change of the B{sub max} for GLB binding. The differential effects of NTPS and NDPs indicate that two nucleotide binding sites regulate GLB binding.« less

ProBiS-2012: web server and web services for detection of structurally similar binding sites in proteins.

PubMed

Konc, Janez; Janezic, Dusanka

2012-07-01

The ProBiS web server is a web server for detection of structurally similar binding sites in the PDB and for local pairwise alignment of protein structures. In this article, we present a new version of the ProBiS web server that is 10 times faster than earlier versions, due to the efficient parallelization of the ProBiS algorithm, which now allows significantly faster comparison of a protein query against the PDB and reduces the calculation time for scanning the entire PDB from hours to minutes. It also features new web services, and an improved user interface. In addition, the new web server is united with the ProBiS-Database and thus provides instant access to pre-calculated protein similarity profiles for over 29 000 non-redundant protein structures. The ProBiS web server is particularly adept at detection of secondary binding sites in proteins. It is freely available at http://probis.cmm.ki.si/old-version, and the new ProBiS web server is at http://probis.cmm.ki.si.

ProBiS-2012: web server and web services for detection of structurally similar binding sites in proteins

PubMed Central

Konc, Janez; Janežič, Dušanka

2012-01-01

The ProBiS web server is a web server for detection of structurally similar binding sites in the PDB and for local pairwise alignment of protein structures. In this article, we present a new version of the ProBiS web server that is 10 times faster than earlier versions, due to the efficient parallelization of the ProBiS algorithm, which now allows significantly faster comparison of a protein query against the PDB and reduces the calculation time for scanning the entire PDB from hours to minutes. It also features new web services, and an improved user interface. In addition, the new web server is united with the ProBiS-Database and thus provides instant access to pre-calculated protein similarity profiles for over 29 000 non-redundant protein structures. The ProBiS web server is particularly adept at detection of secondary binding sites in proteins. It is freely available at http://probis.cmm.ki.si/old-version, and the new ProBiS web server is at http://probis.cmm.ki.si. PMID:22600737

A comparison of the binding of secretory component to immunoglobulin A (IgA) in human colostral S-IgA1 and S-IgA2

PubMed Central

Almogren, Adel; Senior, Bernard W; Kerr, Michael A

2007-01-01

A detailed investigation of the binding of secretory component to immunoglobulin A (IgA) in human secretory IgA2 (S-IgA2) was made possible by the development of a new method of purifying S-IgA1, S-IgA2 and free secretory component from human colostrum using thiophilic gel chromatography and chromatography on Jacalin-agarose. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of unreduced pure S-IgA2 revealed that, unlike in S-IgA1, a significant proportion of the secretory component was bound non-covalently in S-IgA2. When S-IgA1 was incubated with a protease purified from Proteus mirabilis the secretory component, but not the α-chain, was cleaved. This is in contrast to serum IgA1, in which the α-chain was cleaved under the same conditions – direct evidence that secretory component does protect the α-chain from proteolytic cleavage in S-IgA. Comparisons between the products of cleavage with P. mirabilis protease of free secretory component and bound secretory component in S-IgA1 and S-IgA2 also indicated that, contrary to the general assumption, the binding of secretory component to IgA is different in S-IgA2 from that in S-IgA1. PMID:17156102

Anti-CD30-targeted gold nanoparticles for photothermal therapy of L-428 Hodgkin’s cell

PubMed Central

Qu, Xiaochao; Yao, Cuiping; Wang, Jing; Li, Zheng; Zhang, Zhenxi

2012-01-01

Purpose Due to the efficient bioconjugation and highly photothermal effect, gold nanoparticles can stain receptor-overexpressing cancer cells through specific targeting of ligands to receptors, strongly absorb specific light and efficiently convert it into heat based on the property of surface plasmon resonance, and then induce the localized protein denaturation and cell death. Methods Two gold nanoparticle–antibody conjugates, gold-BerH2 antibody (anti-CD30 receptor) and gold-ACT1 antibody (anti-CD25-receptor), were synthesized. Gold-BerH2 conjugates can specifically bind to the surface of L-428 Hodgkin’s cells, and gold-ACT1 conjugates were used for the control. The gold nanoparticle-induced L-428 cell-killing experiments were implemented with different experimental parameters. Results At a relatively low concentration of gold and short incubation time, the influence of cytotoxicity of gold on cell viability can be overlooked. Under laser irradiation at suitable power, the high killing efficiency of gold-targeted L-428 cells was achieved, but little damage was done to nontargeted cancer cells. Conclusion Gold nanoparticle-mediated photothermal therapy provides a relatively safe therapeutic technique for cancer treatment. PMID:23269868

Snyder-Robinson Syndrome: Rescuing the Disease-Causing Effect of G56S mutant by Small Molecule Binding

NASA Astrophysics Data System (ADS)

Zhang, Zhe; Martiny, Virginie; Lagorce, David; Alexov, Emil; Miteva, Maria; Clemson University Team; Université Paris Diderot Team

2013-03-01

Snyder-Robinson Syndrome (SRS) is an X-linked mental retardation disorder, which is caused by defects in a particular gene coding for the spermine synthase (SMS) protein. Among the missense mutations known to be disease-causing is the G56S, which is positioned at the interface of the SMS homo-dimer. Previous computational and experimental investigations have shown that G56S mutation destabilizes the homo-dimer and thus greatly reduces the SMS enzymatic activity. In this study, we explore the possibility of mitigating the effect of G56S mutation by binding small molecules to suitable pockets around the mutation site. It is done by combined efforts of molecular dynamics simulations and in silico screening. The binding of selected molecules was calculated to fully compensate the effect of the mutation and rescue the wild type dimer affinity. This work was supported by NIH, NLM grant. No. 1R03LM009748

Role of S fimbriae in Escherichia coli K1 binding to brain microvascular endothelial cells in vitro and penetration into the central nervous system in vivo.

PubMed

Wang, Ying; Wen, Zhang Guang; Kim, Kwang Sik

2004-12-01

Bacterial binding to host cell surface is considered an important initial step in the pathogenesis of many infectious diseases including meningitis. Previous studies using a laboratory Escherichia coli (E. coli) strain HB101 possessing a recombinant plasmid carrying the cloned S fimbriae gene cluster have shown that S fimbriae are the major contributor to binding to bovine brain microvascular endothelial cells (BMEC) for HB101. Our present study, however, revealed that S fimbriae did not play a major role for E. coli K1's binding to human BMEC in vitro and crossing of the blood-brain barrier in vivo. This was shown by our demonstration that E. coli K1 strain and its S fimbriae-operon deletion mutant exhibited similar rates of binding to human BMEC and similar rates of penetration into the central nervous system in the experimental hematogenous meningitis model. Studies are needed to identify major determinants of E. coli K1 contributing to BMEC binding and subsequent crossing of the blood-brain barrier in vivo.

Cation binding at the node of Ranvier: I. Localization of binding sites during development.

PubMed

Zagoren, J C; Raine, C S; Suzuki, K

1982-06-17

Cations are known to bind to the node of Ranvier and the paranodal regions of myelinated fibers. The integrity of these specialized structures is essential for normal conduction. Sites of cation binding can be microscopically identified by the electrondense histochemical reaction product formed by the precipitate of copper sulfate/potassium ferrocyanide. This technique was used to study the distribution of cation binding during normal development of myelinating fibers. Sciatic nerves of C57B1 mice, at 1, 3, 5, 6, 7, 8, 9, 13, 16, 18, 24 and 30 days of age, were prepared for electron microscopy following fixation in phosphate-buffered 2.5% glutaraldehyde and 1% osmic acid, microdissection and incubation in phosphate-buffered 0.1 M cupric sulfate followed by 0.1 M potassium ferrocyanide. Localization of reaction product was studied by light and electron microscopy. By light microscopy, no reaction product was observed prior to 9 days of age. At 13 days, a few nodes and paranodes exhibited reaction product. This increased in frequency and intensity up to 30 days when almost all nodes or paranodes exhibited reaction product. Ultrastructurally, diffuse reaction product was first observed at 3 days of age in the axoplasm of the node, in the paranodal extracellular space of the terminal loops, in the Schwann cell proper and in the terminal loops of Schwann cell cytoplasm. When myelinated axons fulfilled the criteria for mature nodes, reaction product was no longer observed in the Schwann cell cytoplasm, while the intensity of reaction product in the nodal axoplasm and paranodal extracellular space of the terminal loops increased. Reaction product in the latter site appeared to be interrupted by the transverse bands. These results suggest that cation binding accompanies nodal maturity and that the Schwann cell may play a role in production or storage of the cation binding substance during myelinogenesis and development.

Identification of AOSC-binding proteins in neurons

NASA Astrophysics Data System (ADS)

Liu, Ming; Nie, Qin; Xin, Xianliang; Geng, Meiyu

2008-11-01

Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer’s Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.

Binding and degradation of 125I-gastrin by plasma membranes from homogenized rat gastric mucosa.

PubMed

Kleveland, P M; Waldum, H L

1986-06-01

Binding of 125I-gastrin to the 270-30,000 g fraction from homogenized rat oxyntic mucosa was studied. 'Specific' binding was calculated by subtracting the binding at excess cold gastrin from the binding with labelled gastrin (250 pM) only. At 30 degrees C specific binding rose rapidly to a short-lived maximum before falling gradually, whereas at 15 degrees C and 0 degree C specific binding rose gradually to a higher plateau level. The reduced binding at 30 degrees C could be caused by degradation of either the tracer or the binding site or by a combination of these two events. Degradation of 125I-gastrin was evaluated by trichloroacetic acid (TCA) precipitation, fast protein liquid chromatography, and binding to a gastrin antibody (immunoreactivity). The effect of incubation on the binding site was evaluated by preincubation of the homogenate fraction before adding gastrin. In separate studies, the proteolytic activity of the homogenate fraction was studied by TCA precipitation of radioactive casein. Different enzyme inhibitors tested were virtually ineffective in preventing gastrin and casein degradation. Only lowering the incubation temperature to 15 degrees C or lower could prevent this degradation. The reduced and transient binding of 125I-gastrin at 30 degrees C most probably reflects tracer degradation. Accordingly, the gastrin binding experiments were performed at 15 degrees C. Only homogenates from the oxyntic area of the stomach bound 125I-gastrin specifically and with a Kd of 0.8 nM (Scatchard analysis). However, micromolar concentrations of unlabelled gastrin were required to inhibit half maximal binding of the tracer. The tracer binding was unaffected by secretin, slightly reduced by a CCK-9 analogue, and more markedly reduced by pentagastrin.

Cognitive and neuropsychological underpinnings of relational and conjunctive working memory binding across age.

PubMed

van Geldorp, Bonnie; Parra, Mario A; Kessels, Roy P C

2015-01-01

The ability to form associations (i.e., binding) is critical for memory formation. Recent studies suggest that aging specifically affects relational binding (associating separate features) but not conjunctive binding (integrating features within an object). Possibly, this dissociation may be driven by the spatial nature of the studies so far. Alternatively, relational binding may simply require more attentional resources. We assessed relational and conjunctive binding in three age groups and we included an interfering task (i.e., an articulatory suppression task). Binding was examined in a working memory (WM) task using non-spatial features: shape and colour. Thirty-one young adults (mean age = 22.35), 30 middle-aged adults (mean age = 54.80) and 30 older adults (mean age = 70.27) performed the task. Results show an effect of type of binding and an effect of age but no interaction between type of binding and age. The interaction between type of binding and interference was significant. These results indicate that aging affects relational binding and conjunctive binding similarly. However, relational binding is more susceptible to interference than conjunctive binding, which suggests that relational binding may require more attentional resources. We suggest that a general decline in WM resources associated with frontal dysfunction underlies age-related deficits in WM binding.

The Calcium-binding Proteins S100A8 and S100A9 Initiate the Early Inflammatory Program in Injured Peripheral Nerves*

PubMed Central

Chernov, Andrei V.; Dolkas, Jennifer; Hoang, Khang; Angert, Mila; Srikrishna, Geetha; Vogl, Thomas; Baranovskaya, Svetlana; Strongin, Alex Y.; Shubayev, Veronica I.

2015-01-01

To shed light on the early immune response processes in severed peripheral nerves, we performed genome-wide transcriptional profiling and bioinformatics analyses of the proximal (P, regenerating) and distal (D, degenerating) nerve stumps on day 1 in the sciatic nerve axotomy model in rats. Multiple cell death-related pathways were activated in the degenerating D stump, whereas activation of the cytoskeletal motility and gluconeogenesis/glycolysis pathways was most prominent in the P stump of the axotomized nerve. Our bioinformatics analyses also identified the specific immunomodulatory genes of the chemokine, IL, TNF, MHC, immunoglobulin-binding Fc receptor, calcium-binding S100, matrix metalloproteinase, tissue inhibitor of metalloproteinase, and ion channel families affected in both the P and D segments. S100a8 and S100a9 were the top up-regulated genes in both the P and D segments. Stimulation of cultured Schwann cells using the purified S100A8/A9 heterodimer recapitulated activation of the myeloid cell and phagocyte chemotactic genes and pathways, which we initially observed in injured nerves. S100A8/A9 heterodimer injection into the intact nerve stimulated macrophage infiltration. We conclude that, following peripheral nerve injury, an immediate acute immune response occurs both distal and proximal to the lesion site and that the rapid transcriptional activation of the S100a8 and S100a9 genes results in S100A8/A9 hetero- and homodimers, which stimulate the release of chemokines and cytokines by activated Schwann cells and generate the initial chemotactic gradient that guides the transmigration of hematogenous immune cells into the injured nerve. PMID:25792748

Crystal structure of RlmAI: Implications for understanding the 23S rRNA G745/G748-methylation at the macrolide antibiotic-binding site

PubMed Central

Das, Kalyan; Acton, Thomas; Chiang, Yiwen; Shih, Lydia; Arnold, Eddy; Montelione, Gaetano T.

2004-01-01

The RlmA class of enzymes (RlmAI and RlmAII) catalyzes N1-methylation of a guanine base (G745 in Gram-negative and G748 in Gram-positive bacteria) of hairpin 35 of 23S rRNA. We have determined the crystal structure of Escherichia coli RlmAI at 2.8-Å resolution, providing 3D structure information for the RlmA class of RNA methyltransferases. The dimeric protein structure exhibits features that provide new insights into its molecular function. Each RlmAI molecule has a Zn-binding domain, responsible for specific recognition and binding of its rRNA substrate, and a methyltransferase domain. The asymmetric RlmAI dimer observed in the crystal structure has a well defined W-shaped RNA-binding cleft. Two S-adenosyl-l-methionine substrate molecules are located at the two valleys of the W-shaped RNA-binding cleft. The unique shape of the RNA-binding cleft, different from that of known RNA-binding proteins, is highly specific and structurally complements the 3D structure of hairpin 35 of bacterial 23S rRNA. Apart from the hairpin 35, parts of hairpins 33 and 34 also interact with the RlmAI dimer. PMID:14999102

Vibrio cholerae NspS, a homologue of ABC-type periplasmic solute binding proteins, facilitates transduction of polyamine signals independent of their transport

PubMed Central

Cockerell, Steven R.; Rutkovsky, Alex C.; Zayner, Josiah P.; Cooper, Rebecca E.; Porter, Lindsay R.; Pendergraft, Sam S.; Parker, Zach M.; McGinnis, Marcus W.

2014-01-01

The polyamines norspermidine and spermidine are among the environmental signals that regulate Vibrio cholerae biofilm formation. The effects of these polyamines are mediated by NspS, a member of the bacterial periplasmic solute binding protein superfamily. Almost all members of this superfamily characterized to date are components of ATP-binding cassette-type transporters involved in nutrient uptake. Consequently, in the current annotation of the V. cholerae genome, NspS has been assigned a function in transport. The objective of this study was to further characterize NspS and investigate its potential role in transport. Our results support a role for NspS in signal transduction in response to norspermidine and spermidine, but not their transport. In addition, we provide evidence that these polyamine signals are processed by c-di-GMP signalling networks in the cell. Furthermore, we present comparative genomics analyses which reveal the presence of NspS-like proteins in a variety of bacteria, suggesting that periplasmic ligand binding proteins may be widely utilized for sensory transduction. PMID:24530989

Dynamic Nuclear Polarization Study of Inhibitor Binding to the M218–60 Proton Transporter from Influenza A

PubMed Central

Andreas, Loren B.; Barnes, Alexander B.; Corzilius, Björn; Chou, James J.; Miller, Eric A.; Caporini, Marc; Rosay, Melanie; Griffin, Robert G

2013-01-01

We demonstrate the use of dynamic nuclear polarization (DNP) to elucidate ligand binding to a membrane protein using dipolar recoupling magic angle spinning (MAS) NMR. In particular, we detect drug binding in the proton transporter M218–60 from influenza A using recoupling experiments at room temperature and with cryogenic DNP. The results indicate that the pore binding site of rimantadine is correlated with previously reported widespread chemical shift changes, suggesting functional binding in the pore. Futhermore, the 15N labeled ammonium of rimantadine was observed near A30 13Cβ and G34 13Cα suggesting a possible hydrogen bond to A30 Carbonyl. Cryogenic DNP was required to observe the weaker external binding site(s) in a ZF-TEDOR spectrum. This approach is generally applicable, particularly for weakly bound ligands, in which case the application of MAS NMR dipolar recoupling requires the low temperatures to quench dynamic exchange processes. For the fully protonated samples investigated, we observed DNP signal enhancements of ~10 at 400 MHz using only 4–6 mM of the polarizing agent TOTAPOL. At 600 MHz and with DNP, we measured a distance between the drug and the protein to a precision of 0.2 Å. PMID:23480101

14 CFR 93.30 - Assignment provisions for domestic and U.S./Canada transborder service.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Assignment provisions for domestic and U.S./Canada transborder service. 93.30 Section 93.30 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION... domestic and U.S./Canada transborder service. (a) Whenever the FAA has determined that sufficient Arrival...

Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification.

PubMed

Andreatta, Massimo; Karosiene, Edita; Rasmussen, Michael; Stryhn, Anette; Buus, Søren; Nielsen, Morten

2015-11-01

A key event in the generation of a cellular response against malicious organisms through the endocytic pathway is binding of peptidic antigens by major histocompatibility complex class II (MHC class II) molecules. The bound peptide is then presented on the cell surface where it can be recognized by T helper lymphocytes. NetMHCIIpan is a state-of-the-art method for the quantitative prediction of peptide binding to any human or mouse MHC class II molecule of known sequence. In this paper, we describe an updated version of the method with improved peptide binding register identification. Binding register prediction is concerned with determining the minimal core region of nine residues directly in contact with the MHC binding cleft, a crucial piece of information both for the identification and design of CD4(+) T cell antigens. When applied to a set of 51 crystal structures of peptide-MHC complexes with known binding registers, the new method NetMHCIIpan-3.1 significantly outperformed the earlier 3.0 version. We illustrate the impact of accurate binding core identification for the interpretation of T cell cross-reactivity using tetramer double staining with a CMV epitope and its variants mapped to the epitope binding core. NetMHCIIpan is publicly available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.1 .

Assessment of the binding performance of histamine-imprinted microspheres by frontal analysis capillary electrophoresis.

PubMed

Romano, Edwin F; Quirino, Joselito P; Holdsworth, John L; So, Regina C; Holdsworth, Clovia I

2017-05-01

Frontal analysis capillary electrophoresis was used to evaluate the binding performance of molecularly imprinted microspheres (MIM) toward its template histamine and analogs at pH 7, and compared to the high performance liquid chromatographic method. In both methods, batch binding was employed and the binding parameters were calculated from the measured concentration of unbound amine analytes and afforded comparable histamine equilibrium dissociation constants (K d ∼ 0.4 mM). FACE was easily carried out at shorter binding equilibration time (i.e. 30 min) and without the need to separate the microspheres, circumventing laborious and, in the case of the system under study, inefficient sample filtration. It also allowed for competitive binding studies by virtue of its ability to distinctly separate intact microspheres and all tested amines which could not be resolved in HPLC. K d 's for nonimprinted (control) microspheres (NIM) from FACE and HPLC were also comparable (∼ 0.6 mM) but at higher histamine concentrations, HPLC gave lower histamine binding. This discrepancy was attributed to inefficient filtration of the batch binding samples prior to HPLC analysis resulting in an over-estimation of the concentration of free histamine brought about by the presence of unfiltered histamine-bound microspheres. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Construction of a Functional S-Layer Fusion Protein Comprising an Immunoglobulin G-Binding Domain for Development of Specific Adsorbents for Extracorporeal Blood Purification

PubMed Central

Völlenkle, Christine; Weigert, Stefan; Ilk, Nicola; Egelseer, Eva; Weber, Viktoria; Loth, Fritz; Falkenhagen, Dieter; Sleytr, Uwe B.; Sára, Margit

2004-01-01

The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed in Escherichia coli HMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA31-1068/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-μm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm2, whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm2 was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system. PMID:15006773

BK Virus Load Associated with Serum Levels of sCD30 in Renal Transplant Recipients

PubMed Central

Malik, Salma N.; Al-Saffer, Jinan M.; Jawad, Rana S.

2016-01-01

Background. Rejection is the main drawback facing the renal transplant operations. Complicated and overlapping factors, mainly related to the immune system, are responsible for this rejection. Elevated serum levels of sCD30 were frequently recorded as an indicator for renal allograft rejection, while BV virus is considered as one of the most serious consequences for immunosuppressive treatment of renal transplant recipients (RTRs). Aims. This study aimed to determine the association of BK virus load with serum levels of sCD30 in RTRs suffering from nephropathy. Patients and Methods. A total of 50 RTRs with nephropathy and 30 age-matched apparently healthy individuals were recruited for this study. Serum samples were obtained from each participant. Real-time PCR was used to quantify BK virus load in RTRs serum, while ELISA technique was employed to estimate serum levels of sCD30. Results. Twenty-two percent of RTRs had detectable BKV with mean viral load of 1.094E + 06 ± 2.291E + 06. RTRs showed higher mean serum level of sCD30 (20.669 ± 18.713 U/mL) than that of controls (5.517 ± 5.304 U/mL) with significant difference. BK virus load had significant positive correlation with the serum levels of sCD30 in RTRs group. Conclusion. These results suggest that serum levels of sCD30 could be used as an indicator of BK viremia, and accordingly the immunosuppressive regime should be adjusted. PMID:27051424

Serum sCD30: A promising biomarker for predicting the risk of bacterial infection after kidney transplantation.

PubMed

Fernández-Ruiz, Mario; Parra, Patricia; López-Medrano, Francisco; Ruiz-Merlo, Tamara; González, Esther; Polanco, Natalia; Origüen, Julia; San Juan, Rafael; Andrés, Amado; Aguado, José María

2017-04-01

The transmembrane glycoprotein CD30 contributes to regulate the balance between Th 1 and Th 2 responses. Previous studies have reported conflicting results on the utility of its soluble form (sCD30) to predict post-transplant infection. Serum sCD30 was measured by a commercial ELISA assay at baseline and post-transplant months 1, 3, and 6 in 100 kidney transplant (KT) recipients (279 monitoring points). The impact of sCD30 levels on the incidence of overall, bacterial and opportunistic infection during the first 12 months after transplantation was assessed by Cox regression. There were no differences in serum sCD30 according to the occurrence of overall or opportunistic infection. However, sCD30 levels were higher in patients with bacterial infection compared to those without at baseline (P=.038) and months 1 (P<.0001), 3 (P=.043), and 6 after transplantation (P=.006). Patients with baseline sCD30 levels ≥13.5 ng/mL had lower 12-month bacterial infection-free survival (35.0% vs 80.0%; P<.0001). After adjusting for potential confounders, baseline sCD30 levels ≥13.5 ng/mL remained as an independent risk factor for bacterial infection (adjusted hazard ratio [aHR]: 4.65; 95% confidence interval [CI]: 2.05-10.53; <.001). Analogously, sCD30 levels ≥6.0 ng/mL at month 1 acted as a risk factor for subsequent bacterial infection (aHR: 5.29; 95% CI: 1.11-25.14; P=.036). Higher serum sCD30 levels were associated with an increased risk of bacterial infection after KT. We hypothesize that this biomarker reflects a Th 2 -polarized T-cell response, which exerts a detrimental effect on the immunity against bacterial pathogens. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Streptomyces coelicolor SCO4226 Is a Nickel Binding Protein

PubMed Central

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

2014-01-01

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

Effect of the PreBind Engagement Process on Scrum Timing and Stability in the 2013 to 2016 Six Nations.

PubMed

Bradley, Edward J; Hogg, Bob; Archer, David T

2017-12-28

This study examined if changes in scrum engagement laws from the "crouch-touch-set" (CTS) in 2013 to the "PreBind" engagement from 2014 onwards have led to changes in scrum characteristics, specifically timing, in international rugby union. Duration and outcomes were identified for all scrums occurring in the 2013-2016 Six Nations (n=60 games) using video analysis. Scrum duration increased after the introduction of the PreBind engagement from 59 s in 2013 to 69 s in 2016 (ρ=0.024, ES 0.93). A significant increase in mean contact duration per scrum occurred when prebinding was adopted (ρ<0.05), moving from 7.5 s under the CTS process to 8.5, 10.0 and 10.8 s with PreBind in 2014, 2015 and 2016 (ES 0.71, 2.05 and 3.0, respectively). The number of scrum resets and collapsed scrums, along with early engagement and pulling down infringements were lower under the PreBind process. Overall, the PreBind engagement resulted in longer scrums with significant increases observed in overall and contact durations, with improved stability related characteristics. The longer contact time is a consequence of increased stability with a shift from high energy impact to a sustained push phase with a lower force that is a benefit to player welfare.

Purification, crystallization and preliminary crystallographic analysis of Streptococcus pyogenes laminin-binding protein Lbp

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

Linke, Christian, E-mail: clin180@ec.auckland.ac.nz; Caradoc-Davies, Tom T.; Australian Synchrotron, Clayton, Victoria 3168

2008-02-01

The S. pyogenes laminin-binding protein Lbp, which is essential for adhesion to human laminin, has been expressed, purified and crystallized. The laminin-binding protein Lbp (Spy2007) from Streptococcus pyogenes (a group A streptococcus) mediates adhesion to the human basal lamina glycoprotein laminin. Accordingly, Lbp is essential in in vitro models of cell adhesion and invasion. However, the molecular and structural basis of laminin binding by bacteria remains unknown. Therefore, the lbp gene has been cloned for recombinant expression in Escherichia coli. Lbp has been purified and crystallized from 30%(w/v) PEG 1500 by the sitting-drop vapour-diffusion method. The crystals belonged to themore » monoclinic space group P2{sub 1}, with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 Å, β = 106.27°, and diffracted to 2.5 Å resolution.« less

Crl binds to domain 2 of σ(S) and confers a competitive advantage on a natural rpoS mutant of Salmonella enterica serovar Typhi.

PubMed

Monteil, Véronique; Kolb, Annie; Mayer, Claudine; Hoos, Sylviane; England, Patrick; Norel, Françoise

2010-12-01

The RpoS sigma factor (σ(S)) is the master regulator of the bacterial response to a variety of stresses. Mutants in rpoS arise in bacterial populations in the absence of stress, probably as a consequence of a subtle balance between self-preservation and nutritional competence. We characterized here one natural rpoS mutant of Salmonella enterica serovar Typhi (Ty19). We show that the rpoS allele of Ty19 (rpoS(Ty19)) led to the synthesis of a σ(S)(Ty19) protein carrying a single glycine-to-valine substitution at position 282 in σ(S) domain 4, which was much more dependent than the wild-type σ(S) protein on activation by Crl, a chaperone-like protein that increases the affinity of σ(S) for the RNA polymerase core enzyme (E). We used the bacterial adenylate cyclase two-hybrid system to demonstrate that Crl bound to residues 72 to 167 of σ(S) domain 2 and that G282V substitution did not directly affect Crl binding. However, this substitution drastically reduced the ability of σ(S)(Ty19) to bind E in a surface plasmon resonance assay, a defect partially rescued by Crl. The modeled structure of the Eσ(S) holoenzyme suggested that substitution G282V could directly disrupt a favorable interaction between σ(S) and E. The rpoS(Ty19) allele conferred a competitive fitness when the bacterial population was wild type for crl but was outcompeted in Δcrl populations. Thus, these results indicate that the competitive advantage of the rpoS(Ty19) mutant is dependent on Crl and suggest that crl plays a role in the appearance of rpoS mutants in bacterial populations.

[The relationship between acute rejection and expression of sCD30 for the patients after kidney transplantation].

PubMed

Yang, Jian-Lin; Hao, Hong-Jun; Qin, Bin; Bang, Ling-Qing; Zhang, Zhi-Hong; Xin, Dian-Qi; Guo, Ying-Lu; Na, Yan-Qun

2005-03-16

To study the relationship between the sCD30 and acute rejection. We tested the sCD30 level in serum for 58 cases with kidney transplantation before and the 7th day and 28th day after operation by ELISA. 31 healthy individual for control group, and simultaneously recorded the incidence of rejection after kidney transplantation. The results showed that there is an obviously relation before kidney transplantation between the sCD30 level in serum and the incidence of acute rejection (chi = 4.843, P = 0.028, P < 0.05). There is a significantly relation at the 7th day after kidney transplantation between the sCD30 level in serum and the incidence of acute rejection (chi = 7.201, P = 0.007, P < 0.01). There is no obviously relation at 28th day after kidney transplantation between the sCD30 level in serum and the incidence of acute rejection (chi = 2.095, P = 0.148, P > 0.05). The results suggested that the expressions of sCD30 are related to acute rejection. We speculated that the expressions of sCD30 could play an important role in acute rejection.

Computational investigation of cholesterol binding sites on mitochondrial VDAC.

PubMed

Weiser, Brian P; Salari, Reza; Eckenhoff, Roderic G; Brannigan, Grace

2014-08-21

The mitochondrial voltage-dependent anion channel (VDAC) allows passage of ions and metabolites across the mitochondrial outer membrane. Cholesterol binds mammalian VDAC, and we investigated the effects of binding to human VDAC1 with atomistic molecular dynamics simulations that totaled 1.4 μs. We docked cholesterol to specific sites on VDAC that were previously identified with NMR, and we tested the reliability of multiple docking results in each site with simulations. The most favorable binding modes were used to build a VDAC model with cholesterol occupying five unique sites, and during multiple 100 ns simulations, cholesterol stably and reproducibly remained bound to the protein. For comparison, VDAC was simulated in systems with identical components but with cholesterol initially unbound. The dynamics of loops that connect adjacent β-strands were most affected by bound cholesterol, with the averaged root-mean-square fluctuation (RMSF) of multiple residues altered by 20-30%. Cholesterol binding also stabilized charged residues inside the channel and localized the surrounding electrostatic potentials. Despite this, ion diffusion through the channel was not significantly affected by bound cholesterol, as evidenced by multi-ion potential of mean force measurements. Although we observed modest effects of cholesterol on the open channel, our model will be particularly useful in experiments that investigate how cholesterol affects VDAC function under applied electrochemical forces and also how other ligands and proteins interact with the channel.

Binding of Radioactive Benzylpenicillin to Sporulating Bacillus Cultures: Chemistry and Fluctuations in Specific Binding Capacity

PubMed Central

Lawrence, Paul J.; Rogolsky, Marvin; Hanh, Vo Thi

1971-01-01

The chemistry of the binding of 14C-benzylpenicillin to sporulating cultures of Bacillus megaterium and B. subtilis is similar to that in a 4-hr vegetative culture of Staphylococcus aureus. Unlabeled penicillins prevent the binding of 14C-benzylpenicillin, but benzylpenicilloic acid and benzylpenilloic acid do not. Bound antibiotic can be removed from cells with neutral hydroxylamine at 25 C. Sporulating cultures display two intervals of enhanced binding, whereas binding to stationaryphase S. aureus cells remains constant. The first period of increased binding activity occurs during formation of the spore septum or cell wall primordium development, and the second coincides with cortex biosynthesis. PMID:4942758

Yeast one-hybrid system used to identify the binding proteins for rat glutathione S-transferase P enhancer I.

PubMed

Liao, Ming-Xiang; Liu, Dong-Yuan; Zuo, Jin; Fang, Fu-De

2002-03-01

To detect the trans-factors specifically binding to the strong enhancer element (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. Yeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library to identify potential trans-factors that can interact with core sequence of GPEI(cGPEI). Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of transfactors to cGPEI. cDNA fragments coding for the C-terminal part of the transcription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated. The binding of c-Jun and ANT to GPEI core sequence were confirmed. Rat c-jun transcriptional factor and ANT may interact with cGPEI. They could play an important role in the induced expression of GST-P gene.

Activation of the novel estrogen receptor G protein-coupled receptor 30 (GPR30) at the plasma membrane.

PubMed

Filardo, E; Quinn, J; Pang, Y; Graeber, C; Shaw, S; Dong, J; Thomas, P

2007-07-01

G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17beta-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17beta-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

Differential age-related effects on conjunctive and relational visual short-term memory binding.

PubMed

Bastin, Christine

2017-12-28

An age-related associative deficit has been described in visual short-term binding memory tasks. However, separate studies have suggested that ageing disrupts relational binding (to associate distinct items or item and context) more than conjunctive binding (to integrate features within an object). The current study directly compared relational and conjunctive binding with a short-term memory task for object-colour associations in 30 young and 30 older adults. Participants studied a number of object-colour associations corresponding to their individual object span level in a relational task in which objects were associated to colour patches and a conjunctive task where colour was integrated into the object. Memory for individual items and for associations was tested with a recognition memory test. Evidence for an age-related associative deficit was observed in the relational binding task, but not in the conjunctive binding task. This differential impact of ageing on relational and conjunctive short-term binding is discussed by reference to two underlying age-related cognitive difficulties: diminished hippocampally dependent binding and attentional resources.

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies.

PubMed

Bokori-Brown, Monika; Kokkinidou, Maria C; Savva, Christos G; Fernandes da Costa, Sérgio; Naylor, Claire E; Cole, Ambrose R; Moss, David S; Basak, Ajit K; Titball, Richard W

2013-05-01

Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity. Copyright © 2013 The Protein Society.

GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein

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

Oberdisse, E.; Lapetina, E.G.

1987-05-14

Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with (/sup 3/H)inositol. Ca/sup 2 +/ has opposite effects on the formation of (/sup 3/H)inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of (/sup 3/H)inositol phosphates is inhibited by Ca/sup 2 +/, action of thrombin is stimulated by Ca/sup 2 +/. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma Smore » on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca/sup 2 +/ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca/sup 2 +/ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein.« less

Binding of perlecan to transthyretin in vitro.

PubMed Central

Smeland, S; Kolset, S O; Lyon, M; Norum, K R; Blomhoff, R

1997-01-01

Transthyretin is one of two specific proteins involved in the transport of thyroid hormones in plasma; it possesses two binding sites for serum retinol-binding protein. In the present study we demonstrate that transthyretin also interacts in vitro with [35S]sulphate-labelled material from the medium of HepG2 cells. By using the same strategy as for purifying serum retinol-binding protein, [35S]sulphate-labelled medium was specifically eluted from a transthyretin-affinity column. Ion-exchange chromatography showed that the material was highly polyanionic, and its size and alkali susceptibility suggested that it was a proteoglycan. Structural analyses with chondroitinase ABC lyase and nitrous acid revealed that approx. 20% was chondroitin sulphate and 80% heparan sulphate. Immunoprecipitation showed that the [35S]sulphate-labelled material contained perlecan. Further analysis by binding studies revealed specific and saturable binding of 125I-transthyretin to perlecan-enriched Matrigel. Because inhibition of sulphation by treating HepG2 cells with sodium chlorate increased the affinity of the perlecan for transthyretin, and [3H]heparin was not retained by the transthyretin affinity column, the binding is probably mediated by the core protein and is not a protein-glycosaminoglycan interaction. Because perlecan is released from transthyretin in water, the binding might be due to hydrophobic interactions. PMID:9307034

Validity and Reliability of the 30-s Continuous Jump for Anaerobic Power and Capacity Assessment in Combat Sport

PubMed Central

Čular, Drazen; Ivančev, Vladimir; Zagatto, Alessandro M.; Milić, Mirjana; Beslija, Tea; Sellami, Maha; Padulo, Johnny

2018-01-01

Cycling test such Wingate anaerobic test (WAnT) is used to measure anaerobic power (AP), but not anaerobic capacity (AC, i.e., the metabolic energy demand). However, in sports that do not involve cycling movements (Karate), the continuous jump for 30 s (vertical jumps for 30 s) has been extensively used to measure anaerobic performance in all young athletes. Limited information’s are available concerning its validity and reliability especially in children. As such, the current study aimed to test validity and reliability of a continuous jumps test (the CJ30s), using WAnT as a reference. Thirteen female Karate kids (age: 11.07 ± 1.32 years; mass: 41.76 ± 15.32 kg; height: 152 ± 11.52 cm; training experience: 4.38 ± 2.14 years) were tested on three separate sessions. The first and second sessions were used to assess the reliability using Intra-class correlation coefficient (ICC) of CJ30s, whereas on the third session WAnT was administered. Following CJ30s and WAnT, we assessed AP (1/CJ30s, as jump height [JH], fatigue index [FI], and blood lactate [BL]; 2/WAnT, as mechanical power [P], FI, and BL) and AC as the excess post-exercise oxygen consumption (EPOC). Large/highly significant correlations were found between CJ30s and WAnT EPOCs (r = 0.730, P = 0.003), and BLs (r = 0.713, P = 0.009). Moderate/significant correlations were found between CJ30s and WAnT FIs (r = 0.640, P = 0.014), CJ30s first four jumps mean JH and WAnT peak P (r = 0.572, P = 0.032), and CJ30s mean JH and WAnT mean P (r = 0.589, P = 0.021). CJ30s showed excellent and moderate reliability (ICC) for AP (maximal JH 0.884, mean JH 0.742, FI 0.657, BL 0.653) and AC (EPOC 0.788), respectively. Correlations observed especially in terms of AC between CJ30s and WAnT provide evidence that former may adequately assess anaerobic performance for the young combat athlete. CJ30 is a reliable test and allow an easy assessment of AP and AC in karate children. PMID:29867580

Conformational Dynamics, Ligand Binding and Effects of Mutations in NirE an S-Adenosyl-L-Methionine Dependent Methyltransferase

NASA Astrophysics Data System (ADS)

Singh, Warispreet; Karabencheva-Christova, Tatyana G.; Black, Gary W.; Ainsley, Jon; Dover, Lynn; Christov, Christo Z.

2016-01-01

Heme d1, a vital tetrapyrrol involved in the denitrification processes is synthesized from its precursor molecule precorrin-2 in a chemical reaction catalysed by an S-adenosyl-L-methionine (SAM) dependent Methyltransferase (NirE). The NirE enzyme catalyses the transfer of a methyl group from the SAM to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. An important insight into the structure-activity relationships of NirE has been revealed by elucidating its crystal structure, but there is still no understanding about how conformational flexibility influences structure, cofactor and substrate binding by the enzyme as well as the structural effects of mutations of residues involved in binding and catalysis. In order to provide this missing but very important information we performed a comprehensive atomistic molecular dynamics study which revealed that i) the binding of the substrate contributes to the stabilization of the structure of the full complex; ii) conformational changes influence the orientation of the pyrrole rings in the substrate, iii) more open conformation of enzyme active site to accommodate the substrate as an outcome of conformational motions; and iv) the mutations of binding and active site residues lead to sensitive structural changes which influence binding and catalysis.

[Evaluation of immune status of kidney transplant recipients by combined HLA-G5 and sCD30].

PubMed

JIN, Zhan-kui; TIAN, Pu-xun; XUE, Wu-jun; DING, Xiao-ming; PAN, Xiao-ming; DING, Chen-guang; JIA, Li-ning; GE, Guan-qun; HAO, Jun-jun

2010-09-28

to study the relationship between the expression of serum human leucocyte antigen-G5 (HLA-G5)/soluble CD30 (sCD30) and the function of renal graft in kidney transplant recipients and investigate the immune status of recipients with combined HLA-G5 and sCD30. from January 2002 to November 2008, a total of 66 kidney transplant recipients in our centre were selected as subjects and divided into three groups: stable function of renal graft (n = 38), acute rejection (n = 15) and chronic rejection (n = 13). The expressions of serum HLA-G5 and sCD30 were detected. There were two different immune conditions with acute/chronic allograft rejection and normal renal graft in kidney transplant recipients as evaluated by combined HLA-G5 and sCD30. The sensitivity, specificity and critical value of the method were analyzed by the curve of receiver operating characteristic. the levels of HLA-G5 and sCD30 were significantly correlated with serum creatinine (r = -0.493, 0.691, both P < 0.01). Within the first year post-transplantation, the sensitivity was 78.6% and the specificity 85.7% when HLA-G5 critical value 82 microg/L and sCD30 critical value 12.2 microg/L. After one year post-transplantation: the sensitivity was 92.3% and the specificity 84.6% when HLA-G5 critical value 141 microg/L and sCD30 critical value 10.3 microg/L. the immune state of recipients are evaluated by combine HLA-G5 and sCD30 which may be a simple and valid method.

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain.

PubMed

Speranskiy, Kirill; Kurnikova, Maria

2005-08-30

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.

Post-transplant sCD30 and neopterin as predictors of chronic allograft nephropathy: impact of different immunosuppressive regimens.

PubMed

Weimer, R; Süsal, C; Yildiz, S; Staak, A; Pelzl, S; Renner, F; Dietrich, H; Daniel, V; Kamali-Ernst, S; Ernst, W; Padberg, W; Opelz, G

2006-08-01

Immunological monitoring for chronic allograft nephropathy (CAN) is of great potential interest. We assessed serum soluble CD30 (sCD30) together with in vitro Th2-type responses (IL-4, IL-10, CD4 helper activity) and neopterin in a prospective study of 84 renal transplant recipients with 2-year follow-up. Patients were randomized to CsA/Aza, CsA/MMF and Tacr/Aza, respectively, to analyze the effect of immunosuppression on posttransplant sCD30 and neopterin. ATG induction and acute rejections did not alter sCD30 levels whereas CMV disease was associated with transient upregulation of sCD30 (p = 0.003 at 4 months) and sustained upregulation of neopterin (corrected for graft function (Neo/CR) p = 0.005 at 2 years). Tacr versus CsA treatment proved to be an independent variable associated with downregulation of 1-year sCD30, which was positively related to Neo/CR (p = 0.007 and 0.01, respectively; logistic regression). Importantly, increased 1-year sCD30 and Neo/CR were associated with decreased glomerular filtration rate at 2 years (p = 0.02 and p < 0.0005, respectively) and evidence of CAN (p < 0.0005). High 1-year sCD30 could not be attributed to enhanced Th2-type responses and was not associated with HLA antibody formation. Our data suggest that elevated sCD30 and neopterin predict graft deterioration by CAN. Tacr effectively downregulates these responses and might be of advantage in patients with elevated sCD30 or neopterin.

Characterization and localization of /sup 3/H-arginine8-vasopressin binding to rat kidney and brain tissue

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

Dorsa, D.M.; Majumdar, L.A.; Petracca, F.M.

Anatomic, behavioral and pharmacologic evidence suggests that arginine8-vasopressin (AVP) serves as a CNS neurotransmitter or neuromodulator. AVP binding to membrane and tissue slice preparations from brain and kidney was characterized, and the anatomical distribution of these binding sites was examined. Conditions for the binding assay were optimized using kidney medullary tissue. Binding of /sup 3/H-AVP (S.A. . 30-51 Ci/mmol, NEN) to brain and kidney membranes and tissue slices was saturable, temperature dependent, linearly related to protein concentration (or number of tissue slices), reversible, and specific since the ability of cold AVP to displace /sup 3/H-AVP from binding was greater thanmore » oxytocin and other related peptide fragments. Autoradiographic localization of /sup 3/H-AVP binding was restricted to kidney medullary tissue. In brain tissue, /sup 3/H-AVP binding was found to occur in concentrated foci. Brainstem areas such as the nucleus tractus solitarius (NTS) showed a high density of AVP binding sites. Since local injections of AVP into the NTS have been shown to influence blood pressure, the present study presents the first anatomical evidence for the presence of AVP specific binding sites which might mediate this effect.« less

30. SPILLWAY BUCKET SLAB: REINFORCEMENT DETAILS NO. 4. Sheet S44, ...

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

30. SPILLWAY BUCKET SLAB: REINFORCEMENT DETAILS NO. 4. Sheet S-44, December, 1939. File no. SA 342/50. - Prado Dam, Spillway, Santa Ana River near junction of State Highways 71 & 91, Corona, Riverside County, CA

Functional identification and characterization of sodium binding sites in Na symporters

PubMed Central

Loo, Donald D. F.; Jiang, Xuan; Gorraitz, Edurne; Hirayama, Bruce A.; Wright, Ernest M.

2013-01-01

Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na+ in binding sites is beyond the resolution of the structures, two Na+ binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (Kd) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na+ binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two –OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium Kd by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na+ and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na+ binding, and that Na+ binding to Na2 promotes binding to Na1 and also sugar binding. PMID:24191006

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.

Attenuation of cancer-initiating cells stemness properties by abrogating S100A4 calcium binding ability in head and neck cancers.

PubMed

Cheng, Li-Hao; Hung, Kai-Feng; Huang, Tung-Fu; Hsieh, Hsin-Pei; Wang, Shu-Ying; Huang, Chih-Yang; Lo, Jeng-Fan

2016-11-29

S100A4 is a calcium-binding protein capable of promoting epithelial-mesenchymal transition. Previously, we have demonstrated that S100A4 is required to sustain the head and neck cancer-initiating cells (HN-CICs) subpopulation. In this study, to further investigate the molecular mechanism, we established the head and neck squamous cell carcinoma (HNSCC) cell lines stably expressing mutant S100A4 proteins with defective calcium-binding sites on either N-terminal (NM) or C-terminal (CM), or a deletion of the last 15 amino-acid residues (CD). We showed that the NM, CM and CD harboring sphere cells that were enriched with HN-CICs population exhibited impaired stemness and malignant properties in vitro, as well as reduced tumor growth ability in vivo. Mechanistically, we demonstrated that mutant S100A4 proteins decreased the promoter activity of Nanog, likely through inhibition of p53. Moreover, the biophysical analyses of purified recombinant mutant S100A4 proteins suggest that both NM and CM mutant S100A4 were very similar to the WT S100A4 with subtle difference on the secondary structure, and that the CD mutant protein displayed the unexpected monomeric form in the solution phase.Taken together, our results suggest that both the calcium-binding ability and the C-terminal region of S100A4 are important for HN-CICs to sustain its stemness property and malignancy, and that the mechanism could be mediated by repressing p53 and subsequently activating the Nanog expression.

Metal binding stoichiometry and isotherm choice in biosorption

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

Schiewer, S.; Wong, M.H.

1999-11-01

Seaweeds that possess a high metal binding capacity may be used as biosorbents for the removal of toxic heavy metals from wastewater. The binding of Cu and Ni by three brown algae (Sargassum, Colpomenia, Petalonia) and one green alga (Ulva) was investigated at pH 4.0 and pH 3.0. The greater binding strength of Cu is reflected in a binding constant that is about 10 times as high as that of Ni. The extent of metal binding followed the order Petalonia {approximately} Sargassum > Colpomenia > Ulva. This was caused by a decreasing number of binding sites and by much lowermore » metal binding constants for Ulva as compared to the brown algae. Three different stoichiometric assumptions are compared for describing the metal binding, which assume either that each metal ion M binds to one binding site B forming a BM complex or that a divalent metal ion M binds to two monovalent sites B forming BM{sub 0.5} or B{sub 2}M complexes, respectively. Stoichiometry plots are proposed as tools to discern the relevant binding stoichiometry. The pH effect in metal binding and the change in proton binding were well predicted for the B{sub 2}M or BM{sub 0.5} stoichiometries with the former being better for Cu and the latter preferable for Ni. Overall, the BM{sub 0.5} model is recommended because it avoids iterations.« less

Symmetry of Fv architecture is conducive to grafting a second antibody binding site in the Fv region.

PubMed Central

Keck, P C; Huston, J S

1996-01-01

Molecular modeling studies on antibody Fv regions have been pursued to design a second antigen-binding site (chi-site) in a chimeric single-chain Fv (chi sFv) species of about 30 kDa. This analysis has uncovered an architectural basis common to many Fv regions that permits grafting a chi-site onto the Fv surface that diametrically opposes the normal combining site. By using molecular graphics analysis, chimeric complementarity-determining regions (chi CDRs) were defined that comprised most of the CDRs from an antibody binding site of interest. The chain directionality of chi CDRs was consistent with that of specific bottom loops of the sFv, which allowed for grafting of chi CDRs with an overall geometry approximating CDRs in the parent combining site. Analysis of 10 different Fv crystal structures indicates that the positions for inserting chi CDRs are very highly conserved, as are the corresponding chi CDR boundaries in the parent binding site. The results of this investigation suggest that it should be possible to generally apply this approach to the development of chimeric bispecific antibody binding site (chi BABS) proteins. Images FIGURE 2 FIGURE 3 PMID:8889174

A differential impact of mycophenolic acid, prednisolone, and tacrolimus exposure on sCD30 levels in adult kidney transplant recipients.

PubMed

Barraclough, Katherine A; Staatz, Christine E; Johnson, David W; Gillis, David; Lee, Katie J; McWhinney, Brett C; Ungerer, Jacobus P J; Campbell, Scott B; Isbel, Nicole M

2013-04-01

Soluble CD30 (sCD30) has been associated with rejection and graft loss in kidney transplantation, leading to the suggestion that sCD30 might be a useful biomarker to adjust immunosuppressant medication dosing. However, there has been minimal study of the influence of individual immunosuppressive drugs on sCD30 levels. To evaluate the influence of mycophenolic acid (MPA), prednisolone, and tacrolimus exposure on sCD30 levels in adult kidney transplant recipients. The sCD30 levels were measured pretransplant and 30 days posttransplant. Area under the concentration-time curve (AUC) for each drug was estimated on day 30 using validated, multiple regression-derived limited sampling strategies. One hundred twenty-five subjects were included. Median (interquartile range) sCD30 levels were lower on day 30 posttransplant compared with pretransplant [10.7 (3.7-20.1) pg/mL versus 66.5 (46.0-95.1) pg/mL; P < 0.0001]. On univariate analyses, day 30 sCD30 levels were negatively correlated with MPA exposure and positively correlated with tacrolimus exposure. Using multivariate logistic regression, higher tacrolimus exposure was independently associated with higher day 30 sCD30 levels (2.2 change in odds for an SD increase in tacrolimus AUC 0-12, P = 0.01; 5.5 change in odds for an SD increase in tacrolimus predose concentration, P < 0.0001). In contrast, MPA and total and free prednisolone exposures were not independently associated with sCD30 levels. The sCD30 levels are significantly reduced in the presence of combination immunosuppression but are differentially affected by different immunosuppressant agents. More research is required before introduction of sCD30 measurement into clinical practice can be considered.

Expression of calcium-binding proteins S100A8, S100A9 and S100A12 in otitis media.

PubMed

Hong, Wenzhou; Khampang, Pawjai; Samuels, Tina L; Kerschner, Joseph E; Yan, Ke; Simpson, Pippa

2017-10-01

Calgranulins (calcium-binding proteins S100A8, S100A9 and S100A12) are predominant cytoplasmic proteins of neutrophils and produced by various cells, playing multiple functions in innate immunity and the inflammatory process. Although up-regulated expression of S100A8 and S100A9 genes were observed in an animal model of otitis media (OM), their expressions have not been studied in human middle ear epithelial cells in response to the OM pathogen or in patients with recurrent or chronic OM (recurrent OM/RecOM or chronic OM with effusion/COME). Gene expressions were compared between Streptococcus pneumoniae (SP)-infected and non-infected human middle ear epithelial cells (HMEECs) as well as between chronic OM patients and control patients (CI). Gene expressions were profiled by quantitative real time PCR (qPCR). S100 proteins in OM patient and CI middle ear biopsies were detected by immunostaining. S100A8, S100A9 and S100A12 gene expressions were elevated in SP-infected HMEECs in time-dependent manner. S100A8 and S100A9 but not S100A12 gene expression was significantly elevated in the middle ear mucosa of OM patients. S100A8 and S100A9 protein were observed in middle ear mucosa of OM, but not CI patients. Minimal co-localization was observed between S100A8 and S100A9 with neutrophil elastase and cytokeratin in ME sections of OM patients. Elevated S100A8 and S100A9 gene expression in SP-infected HMEECs and in the middle ear mucosa of OM, minor co-localized with neutrophil markers suggests that middle ear epithelial cell secretion of S100A8 and S100A9 may play a role in the pathogenesis of recurrent and chronic OM. Copyright © 2017 Elsevier B.V. All rights reserved.

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

NASA Astrophysics Data System (ADS)

Min, Wookee; Bruhn, Christopher; Grigaravicius, Paulius; Zhou, Zhong-Wei; Li, Fu; Krüger, Anja; Siddeek, Bénazir; Greulich, Karl-Otto; Popp, Oliver; Meisezahl, Chris; Calkhoven, Cornelis F.; Bürkle, Alexander; Xu, Xingzhi; Wang, Zhao-Qi

2013-12-01

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

PubMed

Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

2014-08-01

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

Computational Investigation of Cholesterol Binding Sites on Mitochondrial VDAC

PubMed Central

2015-01-01

The mitochondrial voltage-dependent anion channel (VDAC) allows passage of ions and metabolites across the mitochondrial outer membrane. Cholesterol binds mammalian VDAC, and we investigated the effects of binding to human VDAC1 with atomistic molecular dynamics simulations that totaled 1.4 μs. We docked cholesterol to specific sites on VDAC that were previously identified with NMR, and we tested the reliability of multiple docking results in each site with simulations. The most favorable binding modes were used to build a VDAC model with cholesterol occupying five unique sites, and during multiple 100 ns simulations, cholesterol stably and reproducibly remained bound to the protein. For comparison, VDAC was simulated in systems with identical components but with cholesterol initially unbound. The dynamics of loops that connect adjacent β-strands were most affected by bound cholesterol, with the averaged root-mean-square fluctuation (RMSF) of multiple residues altered by 20–30%. Cholesterol binding also stabilized charged residues inside the channel and localized the surrounding electrostatic potentials. Despite this, ion diffusion through the channel was not significantly affected by bound cholesterol, as evidenced by multi-ion potential of mean force measurements. Although we observed modest effects of cholesterol on the open channel, our model will be particularly useful in experiments that investigate how cholesterol affects VDAC function under applied electrochemical forces and also how other ligands and proteins interact with the channel. PMID:25080204

Quantum mechanics/molecular mechanics modeling of photoelectron spectra: the carbon 1s core-electron binding energies of ethanol-water solutions.

PubMed

Löytynoja, T; Niskanen, J; Jänkälä, K; Vahtras, O; Rinkevicius, Z; Ågren, H

2014-11-20

Using ethanol-water solutions as illustration, we demonstrate the capability of the hybrid quantum mechanics/molecular mechanics (QM/MM) paradigm to simulate core photoelectron spectroscopy: the binding energies and the chemical shifts. An integrated approach with QM/MM binding energy calculations coupled to preceding molecular dynamics sampling is adopted to generate binding energies averaged over the solute-solvent configurations available at a particular temperature and pressure and thus allowing for a statistical assessment with confidence levels for the final binding energies. The results are analyzed in terms of the contributions in the molecular mechanics model-electrostatic, polarization, and van der Waals-with atom or bond granulation of the corresponding MM charge and polarizability force-fields. The role of extramolecular charge transfer screening of the core-hole and explicit hydrogen bonding is studied by extending the QM core to cover the first solvation shell. The results are compared to those obtained from pure electrostatic and polarizable continuum models. Particularly, the dependence of the carbon 1s binding energies with respect to the ethanol concentration is studied. Our results indicate that QM/MM can be used as an all-encompassing model to study photoelectron binding energies and chemical shifts in solvent environments.

Interactions of 2’-O-methyl oligoribonucleotides with the RNA models of the 30S subunit A-site

PubMed Central

Jasiński, Maciej; Kulik, Marta; Wojciechowska, Monika; Stolarski, Ryszard

2018-01-01

Synthetic oligonucleotides targeting functional regions of the prokaryotic rRNA could be promising antimicrobial agents. Indeed, such oligonucleotides were proven to inhibit bacterial growth. 2’-O-methylated (2’-O-Me) oligoribonucleotides with a sequence complementary to the decoding site in 16S rRNA were reported as inhibitors of bacterial translation. However, the binding mode and structures of the formed complexes, as well as the level of selectivity of the oligonucleotides between the prokaryotic and eukaryotic target, were not determined. We have analyzed three 2’-O-Me oligoribonucleotides designed to hybridize with the models of the prokaryotic rRNA containing two neighboring aminoglycoside binding pockets. One pocket is the paromomycin/kanamycin binding site corresponding to the decoding site in the small ribosomal subunit and the other one is the close-by hygromycin B binding site whose dynamics has not been previously reported. Molecular dynamics (MD) simulations, as well as isothermal titration calorimetry, gel electrophoresis and spectroscopic studies have shown that the eukaryotic rRNA model is less conformationally stable (in terms of hydrogen bonds and stacking interactions) than the corresponding prokaryotic one. In MD simulations of the eukaryotic construct, the nucleotide U1498, which plays an important role in correct positioning of mRNA during translation, is flexible and spontaneously flips out into the solvent. In solution studies, the 2’-O-Me oligoribonucleotides did not interact with the double stranded rRNA models but all formed stable complexes with the single-stranded prokaryotic target. 2’-O-Me oligoribonucleotides with one and two mismatches bound less tightly to the eukaryotic target. This shows that at least three mismatches between the 2’-O-Me oligoribonucleotide and eukaryotic rRNA are required to ensure target selectivity. The results also suggest that, in the ribosome environment, the strand invasion is the preferred

ProBiS-database: precalculated binding site similarities and local pairwise alignments of PDB structures.

PubMed

Konc, Janez; Cesnik, Tomo; Konc, Joanna Trykowska; Penca, Matej; Janežič, Dušanka

2012-02-27

ProBiS-Database is a searchable repository of precalculated local structural alignments in proteins detected by the ProBiS algorithm in the Protein Data Bank. Identification of functionally important binding regions of the protein is facilitated by structural similarity scores mapped to the query protein structure. PDB structures that have been aligned with a query protein may be rapidly retrieved from the ProBiS-Database, which is thus able to generate hypotheses concerning the roles of uncharacterized proteins. Presented with uncharacterized protein structure, ProBiS-Database can discern relationships between such a query protein and other better known proteins in the PDB. Fast access and a user-friendly graphical interface promote easy exploration of this database of over 420 million local structural alignments. The ProBiS-Database is updated weekly and is freely available online at http://probis.cmm.ki.si/database.

A 31-residue peptide induces aggregation of tau’s microtubule-binding region in cells

PubMed Central

Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Achyraya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J.S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; DeGrado, William F.

2018-01-01

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer’s disease. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behavior of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show here that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fiber diffraction, hydrogen-deuterium exchange and solids NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide. PMID:28837163

Effects of weak/non-complement-binding HLA antibodies on C1q-binding.

PubMed

Hönger, G; Amico, P; Arnold, M-L; Spriewald, B M; Schaub, S

2017-08-01

It is unknown under what conditions and to what extent weak/non-complement (C)-binding IgG subclasses (IgG2/IgG4) can block C1q-binding triggered by C-binding IgG subclasses (IgG1/IgG3). Therefore, we investigated in vitro C1q-binding induced by IgG subclass mixtures targeting the same HLA epitope. Various mixtures of HLA class II specific monoclonal antibodies of different IgG subclasses but identical V-region were incubated with HLA DRB1*07:01 beads and monitored for C1q-binding. The lowest concentration to achieve maximum C1q-binding was measured for IgG3, followed by IgG1, while IgG2 and IgG4 did not show appreciable C1q-binding. C1q-binding occurred only after a critical amount of IgG1/3 has bound and sharply increased thereafter. When both, C-binding and weak/non-C-binding IgG subclasses were mixed, C1q-binding was diminished proportionally to the fraction of IgG2/4. A 2- to 4-fold excess of IgG2/4 inhibited C1q-binding by 50%. Very high levels (10-fold excess) almost completely abrogated C1q-binding even in the presence of significant IgG1/3 levels that would usually lead to strong C1q-binding. In sensitized renal allograft recipients, IgG subclass constellations with ≥ 2-fold excess of IgG2/4 over IgG1/3 were present in 23/66 patients (34.8%) and overall revealed slightly decreased C1q signals. However, spiking of patient sera with IgG2 targeting a different epitope than the patient's IgG1/3 synergistically increased C1q-binding. In conclusion, if targeting the same epitope, an excess of IgG2/4 is repressing the extent of IgG1/3 triggered C1q-binding in vitro. Such IgG subclass constellations are present in about a third of sensitized patients and their net effect on C1q-binding is slightly inhibitory. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography.

PubMed

Konami, Y; Yamamoto, K; Osawa, T

1991-02-01

A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

Structural evidence of the species-dependent albumin binding of the modified cyclic phosphatidic acid with cytotoxic properties

PubMed Central

Sekula, Bartosz; Ciesielska, Anna; Rytczak, Przemyslaw; Koziołkiewicz, Maria; Bujacz, Anna

2016-01-01

Cyclic phosphatidic acids (cPAs) are naturally occurring, very active signalling molecules, which are involved in several pathological states, such as cancer, diabetes or obesity. As molecules of highly lipidic character found in the circulatory system, cPAs are bound and transported by the main extracellular lipid binding protein–serum albumin. Here, we present the detailed interactions between human serum albumin (HSA) and equine serum albumin (ESA) with a derivative of cPA, 1-O-myristoyl-sn-glycerol-2,3-cyclic phosphorodithioate (Myr-2S-cPA). Initial selection of the ligand used for the structural study was made by the analysis of the therapeutically promising properties of the sulfur containing analogues of cPA in respect to the unmodified lysophospholipids (LPLs). Substitution of one or two non-bridging oxygen atoms in the phosphate group with one or two sulfur atoms increases the cytotoxic effect of cPAs up to 60% on the human prostate cancer (PC) cells. Myr-2S-cPA reduces cancer cell viability in a dose-dependent manner, with IC50 value of 29.0 μM after 24 h incubation, which is almost 30% lower than IC50 of single substituted phosphorothioate cPA. Although, the structural homology between HSA and ESA is big, their crystal complexes with Myr-2S-cPA demonstrate significantly different mode of binding of this LPL analogue. HSA binds three molecules of Myr-2S-cPA, whereas ESA only one. Moreover, none of the identified Myr-2S-cPA binding sites overlap in both albumins. PMID:27129297

Recent improvements to Binding MOAD: a resource for protein-ligand binding affinities and structures.

PubMed

Ahmed, Aqeel; Smith, Richard D; Clark, Jordan J; Dunbar, James B; Carlson, Heather A

2015-01-01

For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein-ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23,269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Gadd45a Is an RNA Binding Protein and Is Localized in Nuclear Speckles

PubMed Central

Sytnikova, Yuliya A.; Kubarenko, Andriy V.; Schäfer, Andrea; Weber, Alexander N. R.; Niehrs, Christof

2011-01-01

Background The Gadd45 proteins play important roles in growth control, maintenance of genomic stability, DNA repair, and apoptosis. Recently, Gadd45 proteins have also been implicated in epigenetic gene regulation by promoting active DNA demethylation. Gadd45 proteins have sequence homology with the L7Ae/L30e/S12e RNA binding superfamily of ribosomal proteins, which raises the question if they may interact directly with nucleic acids. Principal Findings Here we show that Gadd45a binds RNA but not single- or double stranded DNA or methylated DNA in vitro. Sucrose density gradient centrifugation experiments demonstrate that Gadd45a is present in high molecular weight particles, which are RNase sensitive. Gadd45a displays RNase-sensitive colocalization in nuclear speckles with the RNA helicase p68 and the RNA binding protein SC35. A K45A point mutation defective in RNA binding was still active in DNA demethylation. This suggests that RNA binding is not absolutely essential for demethylation of an artificial substrate. A point mutation at G39 impared RNA binding, nuclear speckle localization and DNA demethylation, emphasizing its relevance for Gadd45a function. Significance The results implicate RNA in Gadd45a function and suggest that Gadd45a is associated with a ribonucleoprotein particle. PMID:21249130

Chlorhexidine binding to mineralized versus demineralized dentin powder

PubMed Central

Kim, Jongryul; Uchiyama, Toshikazu; Carrilho, Marcela; Agee, Kelli A.; Mazzoni, Annalisa; Breschi, Lorenzo; Carvalho, Ricardo M.; Tjäderhane, Leo; Looney, Stephen; Wimmer, Courtney; Tezvergil-Mutluay, Arzu; Tay, Franklin R.; Pashley, David H.

2010-01-01

Objectives The purposes of this work were to quantitate the affinity and binding capacity of chlorhexidine (CHX) digluconate to mineralized vs. demineralized dentin powder, and to determine how much debinding would result from rinsing with water, ethanol, hydroxyethylmethacrylate (HEMA) or 0.5 M NaCl in water. Methods Dentin powder was made from coronal dentin of extracted human third molars. Standard amounts of dentin powder were tumbled with increasing concentrations of CHX (0–30 mM) for 30 min at 37 C. After centrifuging the tubes, the supernatant was removed and the decrease in CHX concentration quantitated by UV-spectroscopy. CHX-treated dentin powder was resuspended in one of the four debinding solutions for 3 min. The amount of debound CHX in the solvents was also quantitated by UV-spectroscopy. Results As the CHX concentration in the medium increased, the CHX binding to mineralized dentin powder also increased up to 6.8 μmoles/g of dry dentin powder. Demineralized dentin powder took up significantly (p<0.01) more CHX, reaching 30.1 μmoles CHX/g of dry dentin powder. Debinding of CHX was in the order: HEMA < ethanol < 0.05 M NaCl < water. The highest CHX binding to demineralized dentin occurred at 30 mM (1.5 wt%). Significance As CHX is not debound by HEMA, it may remain bound to demineralized dentin during resin-dentin bonding. This may be responsible for the long-term efficacy of CHX as an MMP inhibitor in resin-dentin bonds. PMID:20472280

Escherichia coli Ribosomal Protein S1 Unfolds Structured mRNAs Onto the Ribosome for Active Translation Initiation

PubMed Central

Duval, Mélodie; Korepanov, Alexey; Fuchsbauer, Olivier; Fechter, Pierre; Haller, Andrea; Fabbretti, Attilio; Choulier, Laurence; Micura, Ronald; Klaholz, Bruno P.; Romby, Pascale; Springer, Mathias; Marzi, Stefano

2013-01-01

Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA–protein or mRNA–ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5′ ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features. PMID:24339747

Neurogranin binds α-synuclein in the human superior temporal cortex and interaction is decreased in Parkinson’s disease

PubMed Central

Koob, Andrew O.; Shaked, Gideon M.; Bender, Andreas; Bisquertt, Alejandro; Rockenstein, Edward; Masliah, Eliezer

2016-01-01

Neurogranin is a calmodulin binding protein that has been implicated in learning and memory, long-term potentiation and synaptic plasticity. Neurons expressing neurogranin in the cortex degenerate in late stages of Parkinson’s disease with widespread α-synuclein pathology. While analyzing neurogranin gene expression levels through rtPCR in brains of mouse models overexpressing human α-synuclein, we found levels were elevated 2.5 times when compared to nontransgenic animals. Immunohistochemistry in the cortex revealed colocalization between α-synuclein and neurogranin in mouse transgenics when compared to control mice. Coimmunoprecipitation studies in the superior temporal cortex in humans confirmed interaction between α-synuclein and neurogranin, and decreased interaction between α-synuclein and neurogranin was noticed in patients diagnosed with Parkinson’s disease when compared to normal control brains. Additionally, phosphorylated neurogranin levels were also decreased in the human superior temporal cortex in patients diagnosed with Parkinson’s disease and patients diagnosed with dementia with Lewy bodies. Here, we show for the first time that neurogranin binds to α-synuclein in the human cortex, and this interaction decreases in Parkinson’s disease along with the phosphorylation of neurogranin, a molecular process thought to be involved in learning and memory. PMID:25446004

Cation binding to 15-TBA quadruplex DNA is a multiple-pathway cation-dependent process.

PubMed

Reshetnikov, Roman V; Sponer, Jiri; Rassokhina, Olga I; Kopylov, Alexei M; Tsvetkov, Philipp O; Makarov, Alexander A; Golovin, Andrey V

2011-12-01

A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 µs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange. © The Author(s) 2011. Published by Oxford University Press.

7 CFR 30.15 - Cigar binder.

Code of Federal Regulations, 2014 CFR

2014-01-01

... AND STANDARDS Classification of Leaf Tobacco Covering Classes, Types and Groups of Grades § 30.15 Cigar binder. A portion of a tobacco leaf rolled around the filler of a cigar to bind or hold it...

7 CFR 30.15 - Cigar binder.

Code of Federal Regulations, 2012 CFR

2012-01-01

... AND STANDARDS Classification of Leaf Tobacco Covering Classes, Types and Groups of Grades § 30.15 Cigar binder. A portion of a tobacco leaf rolled around the filler of a cigar to bind or hold it...

7 CFR 30.15 - Cigar binder.

Code of Federal Regulations, 2010 CFR

2010-01-01

... AND STANDARDS Classification of Leaf Tobacco Covering Classes, Types and Groups of Grades § 30.15 Cigar binder. A portion of a tobacco leaf rolled around the filler of a cigar to bind or hold it...

7 CFR 30.15 - Cigar binder.

Code of Federal Regulations, 2013 CFR

2013-01-01

... AND STANDARDS Classification of Leaf Tobacco Covering Classes, Types and Groups of Grades § 30.15 Cigar binder. A portion of a tobacco leaf rolled around the filler of a cigar to bind or hold it...

7 CFR 30.15 - Cigar binder.

Code of Federal Regulations, 2011 CFR

2011-01-01

... AND STANDARDS Classification of Leaf Tobacco Covering Classes, Types and Groups of Grades § 30.15 Cigar binder. A portion of a tobacco leaf rolled around the filler of a cigar to bind or hold it...

Androgen-sensitive spermine-binding protein of rat ventral prostate. Purification of the protein and characterization of the hormonal effect.

PubMed Central

Mezzetti, G; Loor, R; Liao, S

1979-01-01

The rat ventral prostate contains a cytosol protein that can non-covalently bind spermine much more tightly than spermidine or other natural diamines. The protein has been purified to homogeneity, as judged by electrophoresis in urea- and sodium dodecyl sulphate-containing polyacrylamide gels. The protein, with or without spermine bound to it, sediments at 3 S in a sucrose gradient with or without 0.4 M-KCl. The molecular weight of the protein is about 30 000. Each molecule of the binding protein can bind one molecule of spermine. In the prostate of rats injected with cycloheximide, the protein appears to have a half-life of about 3.5 h. The spermine-binding activity of an acidic fraction obtained by DEAE-cellulose chromatography of the prostate cytosol proteins is reduced by about 40--60% within 20--40 h after castration. This effect is reversed very rapidly within 15--30 min by intraperitoneal injection of 5 alpha-dihydrotestosterone. The hormonal effect is androgen-specific and is not mimicked by dexamethasone or oestradiol-17 beta. The androgen effect was reduced significantly when rats were injected with cycloheximide or actinomycin D, suggesting that the acidic protein may be one of the earliest proteins induced by androgen in the rat ventral prostate. Images Fig. 1. PMID:534539

The RNA-binding protein Hfq is important for ribosome biogenesis and affects translation fidelity.

PubMed

Andrade, José M; Dos Santos, Ricardo F; Chelysheva, Irina; Ignatova, Zoya; Arraiano, Cecília M

2018-06-01

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA-mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA Hfq assists ribosome assembly and associates with pre-30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq-mediated regulation of ribosomes is independent of its function as sRNA-regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm-like protein Hfq beyond its function in small RNA-mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation. © 2018 The Authors.

Different kinetic pathways of the binding of two biphenyl analogues of colchicine to tubulin.

PubMed

Dumortier, C; Gorbunoff, M J; Andreu, J M; Engelborghs, Y

1996-04-09

The kinetics of the interaction of tubulin with two biphenyl analogues of colchicine were measured by fluorescence stopped flow. The ligands were 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB) and 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB). The binding of both analogues is accompanied by a fluorescence increase with monophasic kinetics, which indicates that these drugs, unlike colchicine, do not discriminate between the isoforms of tubulin. The observed pseudo-first-order rate constant increases in a nonlinear way with the drug concentration, indicating that the binding of the biphenyl analogues to tubulin occurs, like colchicine, in two steps: a fast reversible equilibrium followed by an isomerization of the initial complex. Kinetic analysis shows that TCB and TKB exhibit differences in their K1 values. At 25 degrees C, these are 114,000 +/- 15,000 M(-1) for TCB and 8,300 +/- 900 M(-1) for TKB. Both molecules show a much higher affinity than colchicine for the initial binding site. Also at 25 degrees C, the k2 value is 0.66 +/- 0.04 s(-1) for TCB and 3.0 +/- 0.2 s(-1) for TKB. From the temperature dependence, a reaction enthalpy change for the initial binding (deltaH(zero)1) of 44 +/- 9 kJ x mol(-1) (TCB) and -40 +/- 14 kJ x mol(-1) (TKB) and an activation energy for the second forward step of 64 +/- 2 kJ x mol(-1) (TCB) and 101 +/- 10 kJ x mol(-1) (TKB) were calculated. The dissociation kinetics were studied by displacement experiments, in which podophyllotoxin was used as a displacing ligand. The rate constant for the second step in the off direction (k(-2)) is 0.25 +/- 0.05 s(-1) for TCB and 0.093 +/- 0.009 s(-1) for TKB at 25 degrees C. The activation energies for the backward isomerization of the complexes were found to be 86 +/- 20 kJ x mol(-1) (TCB) and 79 +/- 5 kJ x mol(-1) (TKB). Combination of these results with the kinetic parameters for association gives a full characterization of the enthalpy pathway for the binding of TCB and TKB. The

Structural evidence of the species-dependent albumin binding of the modified cyclic phosphatidic acid with cytotoxic properties.

PubMed

Sekula, Bartosz; Ciesielska, Anna; Rytczak, Przemyslaw; Koziołkiewicz, Maria; Bujacz, Anna

2016-07-01

Cyclic phosphatidic acids (cPAs) are naturally occurring, very active signalling molecules, which are involved in several pathological states, such as cancer, diabetes or obesity. As molecules of highly lipidic character found in the circulatory system, cPAs are bound and transported by the main extracellular lipid binding protein-serum albumin. Here, we present the detailed interactions between human serum albumin (HSA) and equine serum albumin (ESA) with a derivative of cPA, 1-O-myristoyl-sn-glycerol-2,3-cyclic phosphorodithioate (Myr-2S-cPA). Initial selection of the ligand used for the structural study was made by the analysis of the therapeutically promising properties of the sulfur containing analogues of cPA in respect to the unmodified lysophospholipids (LPLs). Substitution of one or two non-bridging oxygen atoms in the phosphate group with one or two sulfur atoms increases the cytotoxic effect of cPAs up to 60% on the human prostate cancer (PC) cells. Myr-2S-cPA reduces cancer cell viability in a dose-dependent manner, with IC50 value of 29.0 μM after 24 h incubation, which is almost 30% lower than IC50 of single substituted phosphorothioate cPA. Although, the structural homology between HSA and ESA is big, their crystal complexes with Myr-2S-cPA demonstrate significantly different mode of binding of this LPL analogue. HSA binds three molecules of Myr-2S-cPA, whereas ESA only one. Moreover, none of the identified Myr-2S-cPA binding sites overlap in both albumins. © 2016 The Author(s).

[Experiences with an isolation method for retinal S-antigen and interphotoreceptor retinoid-binding protein].

PubMed

Sych, F J; Strobel, J

1996-12-01

S-antigen (AgS) and interphotoreceptor retinoid-binding protein (IRBP) are two highly potent uveitopathogenic autoantigens of the retina frequently used to trigger autoimmune uveitis in animal trials. The aim of this study was the simultaneous isolation of both proteins from bovine retina, additionally avoiding time-consuming dialysis and employing prepacked, commercially available cartridges. Retina extract, obtained in the usual manner, was precipitated with ammonium sulfate. Both the desalting and the buffer exchange of dissolved precipitate were carried out using a Bio-Gel P6 column. Subsequent separation with Econo-Pac Q gave prepurified AgS and IRBP fractions. Further purification was realized predominantly with Econo-Pac Q, -HTP and -HIC (5-ml cartridges). AgS and IRBP were isolated in high purity (sodium dodecylsulfate polyacrylamide gel electrophoresis, silver stain) from bovine retina using prepacked cartridges with yields of approximately 0.25 (for AgS) and 0.15 (for IRBP) mg/g retina, respectively. The application of ready-to-use cartridges allows simultaneous isolation of AgS and IRBP in milligram amounts under simplified conditions. This approach might be of particular interest for small samples of retina.

Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM

PubMed Central

Hymes, Jeffrey P.; Johnson, Brant R.; Barrangou, Rodolphe

2016-01-01

Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion of fbpB lost the ability to adhere to mucin and fibronectin in vitro. Homologues of fbpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside the L. acidophilus homology group. PMID:26921419

Paced Auditory Serial Addition Test (PASAT 3.0 s): Demographically corrected norms for the Portuguese population.

PubMed

Sousa, Claudia Sofia; Neves, Mariana Rigueiro; Passos, Ana Margarida; Ferreira, Aristides; Sá, Maria José

2017-05-23

The main goal of this study was to produce adjusted normative data for the Portuguese population on the Paced Auditory Serial Addition Test (PASAT 3.0 s), the version used in the Brief Repeatable Battery of Neuropsychological Tests developed by the National Multiple Sclerosis Society. The study included 326 community-dwelling individuals (199 women and 127 men) aged between 20 and 70 (mean = 40.33, SD = 14.40), who had educational backgrounds ranging from 4 to 23 years of schooling (mean = 12.28, SD = 4.39). Age, gender and qualifications revealed differences in explaining their performance on the PASAT 3.0 s. Men had significantly better performance on the PASAT 3.0 s than women, even though this represents a small effect size r = 0.18. Demographically corrected normative data was developed and important information regarding performance on the PASAT 3.0 s test is provided. Results are discussed and presented in tables and a formula is presented for computing age, gender and education adjusted T-scores for performance on the PASAT 3.0 s. These results should be considered as useful reference values for clinicians and investigators when applying the PASAT 3.0 s to assess cognitive function like information processing speed in different pathologies.

Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin

PubMed Central

Rooijakkers, Bart J. M.

2018-01-01

Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain’s sequence–function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed. PMID:29782536

Expression of calcium binding protein S100 A7 (psoriasin) in laryngeal carcinoma.

PubMed

Tiveron, Rogério Costa; de Freitas, Luiz Carlos Conti; Figueiredo, David L; Serafini, Luciano N; Mamede, Rui Celso Martins; Zago, Marco A

2012-01-01

Many studies have reported increased expression of S100 A7 (psoriasin) in neoplastic lesions. Among them are studies on breast carcinoma, bladder squamous cell carcinoma, skin tumors and oral cavity squamous cell carcinoma. The expression of S100 A7 has not been described for laryngeal cancer. This study aims to identify the expression of the calcium-binding protein S100 A7 and its correlation with squamous cell carcinomas of the larynx. Specimens from 63 patients were submitted to immunohistochemistry testing with antibody S100 A7. Results were classified and compared. The group with highly differentiated tumors had the highest treatment failure scores. Moderately differentiated tumors had higher treatment failure scores than poorly differentiated tumors. Higher scores were predominantly seen on stages I and II in moderately differentiated tumors, whereas score distribution was more homogeneous in advanced stage disease (III and IV). Regarding failure in treatment, the group scoring zero (3/4 complications: 75%) differed significantly from the remaining groups (13/59: 22%). S100 A7 marker was expressed in 93.7% of laryngeal cancer cases, with higher positive correlation rates in more differentiated tumors and significantly lower rates of treatment failure. Scores had no impact on survival rates.

Probing the binding affinity of amyloids to reduce toxicity of oligomers in diabetes

PubMed Central

Smaoui, Mohamed Raef; Orland, Henri; Waldispühl, Jérôme

2015-01-01

Motivation: Amyloids play a role in the degradation of β-cells in diabetes patients. In particular, short amyloid oligomers inject themselves into the membranes of these cells and create pores that disrupt the strictly controlled flow of ions through the membranes. This leads to cell death. Getting rid of the short oligomers either by a deconstruction process or by elongating them into longer fibrils will reduce this toxicity and allow the β-cells to live longer. Results: We develop a computational method to probe the binding affinity of amyloid structures and produce an amylin analog that binds to oligomers and extends their length. The binding and extension lower toxicity and β-cell death. The amylin analog is designed through a parsimonious selection of mutations and is to be administered with the pramlintide drug, but not to interact with it. The mutations (T9K L12K S28H T30K) produce a stable native structure, strong binding affinity to oligomers, and long fibrils. We present an extended mathematical model for the insulin–glucose relationship and demonstrate how affecting the concentration of oligomers with such analog is strictly coupled with insulin release and β-cell fitness. Availability and implementation: SEMBA, the tool to probe the binding affinity of amyloid proteins and generate the binding affinity scoring matrices and R-scores is available at: http://amyloid.cs.mcgill.ca Contact: jeromew@cs.mcgill.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25777526

Metal binding characterization and conformational studies using Raman microscopy of resin-bound poly(aspartic acid).

PubMed

Stair, Jacqueline L; Holcombe, James A

2007-03-01

The metal binding capacities, conditional stability constants, and secondary structure of immobilized polyaspartic acid (PLAsp) (n = 6, 20, and 30) on TentaGel resin were determined when binding Mg2+, Co2+, Cd2+, and Ni2+. Metal binding to the synthesized peptides was evaluated using breakthrough curves from a packed microcolumn and flame atomic absorption spectrophotometry (FAAS) detection. The metal capacities reached values of 590, 2160, and 3710 mumol of metal/g of resin for the 6-mer, 20-mer, and 30-mer, respectively, and this resulted in 2-3 residues per metal for all peptides and metals tested. Surprisingly, the concentrated environment of the resin along with the spatial distribution of attachment groups allowed for most residues to participate in metal binding regardless of the peptide length. Conditional stability constants calculated using single metal binding isotherms indicated that binding strength decreased as the chain length increased on the resin. Raman microscopy on single beads was used to determine PLAsp secondary structure, and all peptides were of a mixed conformation (i.e., beta-sheets, alpha-helices, random chain, etc.) during neutral conditioning and metal binding. Uniquely, the longer 20-mer and 30-mer peptides showed a distinct change from a mixed conformation to beta-sheets and alpha-helices during metal release with acid. This study confirms that metal release by longer immobilized peptides is often assisted by a conformational change, which easily spoils the binding cavity, while shorter peptides may release metal primarily by H+ displacement.

[Desperately seeking decentralisation: Mexican health policies in two periods of reform: the 1920s-30s and the 1980s].

PubMed

Birn, Anne-Emannuelle

2005-01-01

This article compares public health policy reforms in Mexico during the 1920s and 1930s with subsequent reforms initiated in the 1980s. The attempts at decentralization in the 1920s-30s were supported by the Rockefeller Foundation, which was interested in the formation of local cooperative health units. In the 1980s, the aim of the Mexican government and international financial agencies, such as the Inter-American Development Bank, was to reduce public spending (as part of "structural adjustment" policies). One of the hypotheses of this article is that, in the end, the public health reforms were unable to overcome the limitations imposed by Mexico's political centralization and longstanding inequities in public spending. At the same time, one of the unforeseen achievements of these reforms was an increase in local capabilities to demand a better distribution of social services.

Longitudinal pathways from unconventional personal attributes in the late 20s to cannabis use prior to sexual intercourse in the late 30s.

PubMed

Lee, Jung Yeon; Brook, Judith S; Pahl, Kerstin; Brook, David W

2017-11-01

A quarter of people living with human immunodeficiency virus (HIV) infection in the United States are women. Furthermore, African American and Hispanic/Latina women continue to be disproportionately affected by HIV, compared with women of other races/ethnicities. Cannabis use prior to intercourse may be associated with increased risky sexual behaviors which are highly related to HIV. The ultimate goal of this research is to better understand the relationships between unconventional personal attributes (e.g., risk-taking behaviors) in the late 20s, substance use (e.g., alcohol) in the mid 30s, and cannabis use prior to intercourse in the late 30s using a community sample; such an understanding may inform interventions. This study employing data from the Harlem Longitudinal Development Study includes 343 female participants (50% African Americans, 50% Puerto Ricans). Structural equation modeling indicated that unconventional personal attributes in the late 20s were associated with substance use in the mid 30s (β=0.32, p<0.001), which in turn, was associated with cannabis use prior to sexual intercourse in the late 30s (β=0.64, p<0.001). Unconventional personal attributes in the late 20s were also directly related to cannabis use prior to sexual intercourse in the late 30s (β=0.39, p<0.01). The findings of this study suggest that interventions focused on decreasing unconventional personal attributes as well as substance use may reduce sexual risk behaviors among urban African American and Puerto Rican women. Also, the implications of this study for health care providers and researchers working in HIV prevention are that these precursors may be useful as patient screening tools. Copyright © 2017 Elsevier Ltd. All rights reserved.

15 CFR 30.39 - Special exemptions for shipments to the U.S. Armed Services.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Special exemptions for shipments to the U.S. Armed Services. 30.39 Section 30.39 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade BUREAU OF THE CENSUS, DEPARTMENT OF COMMERCE FOREIGN TRADE REGULATIONS...

RP-1551s, a family of azaphilones produced by Penicillium sp., inhibit the binding of PDGF to the extracellular domain of its receptor.

PubMed

Toki, S; Tanaka, T; Uosaki, Y; Yoshida, M; Suzuki, Y; Kita, K; Mihara, A; Ando, K; Lokker, N A; Giese, N A; Matsuda, Y

1999-03-01

Nine azaphilones designated RP-1551-1, -2, -3, -4, -5, -6, -7, -M1, and -M2 were isolated from the culture broth of Penicillium sp. SPC-21609 as inhibitors of PDGF binding to its receptor. RP-1551s inhibit the binding of PDGF AA to the extracellular domain of PDGF alpha-receptor with IC50 values ranging from 0.1 to 2 microM without affecting PDGF BB binding to the extracellular domain of PDGF beta-receptor. PDGF binding was not restored after the PDGF alpha-receptor extracellular domain was washed in an attempt to remove the RP-1551-1 bound to the receptor. This result suggests that RP-1551-1 may irreversibly interact with the PDGF alpha-receptor. Since many azaphilone compounds possess high reactivity with an amino group, RP-1551-1 may prevent PDGF AA binding by reacting with amino groups on the alpha-receptor extracellular domain.

CD68 on rat macrophages binds tightly to S100A8 and S100A9 and helps to regulate the cells' immune functions.

PubMed

Okada, Kohki; Arai, Satoshi; Itoh, Hiroshi; Adachi, Souichi; Hayashida, Masahiko; Nakase, Hiroshi; Ikemoto, Masaki

2016-11-01

S100A8 and S100A9 (S100 proteins) are regulators of immune cells of myeloid origin. Whereas S100 proteins are found at high concentrations in such cells, their immunologic roles remain unclear. We focused on cluster of differentiation 68 (CD68). The aim of this study is to investigate whether CD68 binds to extracellular S100A8 and/or S100A9 and subsequently participates in the regulation of the cells' immune functions. ELISA and affinity chromatography showed that both recombinant rat S100A8 (r-S100A8) and r-S100A9 bound to r-CD68, but not to r-CD14. Flow cytometry clearly showed evidences supporting above the 2 results. As analyzed by flow cytometry, a less amount of r-S100A8 or r-S100A9 bound to the macrophages treated with some deglycosylation enzymes. In an in vitro assay, the expression levels of S100A8 and S100A9 were significantly suppressed after the macrophages had been treated with an anti-CD68 antibody (ED1). As stimulated macrophages with r-S100A9, the expression of IL-1β mRNA in macrophages, which were treated with anti-TLR4 or -RAGE antibodies, was significantly suppressed. r-S100A8 up-regulated IL-6 and IL-10 mRNAs, while r-S100A9 did TNF-α and IL-6 mRNAs, although these regulations were not statistically significant. Small interfering CD68 also significantly suppressed activation of macrophages through an autocrine pathway by r-S100A8 or r-S100A9. In macrophages stimulated with LPS, fluorescent immunologic staining showed that most CD68 colocalized with S100A8 or S100A9 and that the levels of all 3 molecules were markedly increased. In conclusion, CD68 on macrophages binds to S100A8 and S100A9 and thereby, plays a role in the regulation of the cells' immune functions. © Society for Leukocyte Biology.

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells.

PubMed

Zhang, Zhongqiang; Gao, Bingsi; Zhao, Chengjiang; Long, Cassandra; Qi, Haizhi; Ezzelarab, Mohamed; Cooper, David Kc; Hara, Hidetaka

2017-07-01

The results of the assay for measuring anti-non-Gal antibodies (which affect pig xenograft survival) in recipients are important. Serum incubation time and concentration may be important factors in the extent of antibody binding to the graft. The aim of this in vitro study was to determine the optimal incubation time and serum concentration for measuring anti-non-Gal antibody binding to porcine aortic endothelial cells (pAECs). Pooled human, naive, and sensitized baboon sera were incubated with wild-type, α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/human CD55 pAECs. IgM/IgG binding to pAECs after varying serum incubation times (0.5, 1, 2, and 3 hour) and concentrations (5, 10, 20, and 40 μL) was determined by flow cytometry. An increase in incubation time from 30 minutes to 2 hour was associated with increases in anti-non-Gal IgM/IgG binding to GTKO and GTKO/hCD55 pAECs of pooled human, naive and sensitized baboon sera (P<.05). Pooled human serum showed a significant increase in anti-non-Gal IgM (1.5 times) and a minimal increase in anti-non-Gal IgG antibody binding. IgM/IgG binding of sensitized baboon serum to GTKO pAECs after 2-hour incubation was 1.5 times and 2 times greater than after 30-minutes incubation, respectively, whereas naïve baboon sera showed minimal (non-significant) increase in anti-non-Gal IgM/IgG antibody binding. With 2-hour incubation, increasing the serum concentration from 5 μL to 20 μL significantly increased antibody binding to non-Gal antigens in pooled human and sensitized baboon serum. With naïve baboon serum, only IgG was significantly increased. Increasing the serum incubation time contributed to improve the sensitivity of detecting anti-non-Gal antibodies, without affecting cell viability in vitro. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Rational steering of insulin binding specificity by intra-chain chemical crosslinking

NASA Astrophysics Data System (ADS)

Viková, Jitka; Collinsová, Michaela; Kletvíková, Emília; Buděšínský, Miloš; Kaplan, Vojtěch; Žáková, Lenka; Veverka, Václav; Hexnerová, Rozálie; Aviñó, Roberto J. Tarazona; Straková, Jana; Selicharová, Irena; Vaněk, Václav; Wright, Daniel W.; Watson, Christopher J.; Turkenburg, Johan P.; Brzozowski, Andrzej M.; Jiráček, Jiří

2016-01-01

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the CuI-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone’s B-chain C-terminus for its IR-B specificity.

Neutrophil-derived S100 calcium-binding proteins A8/A9 promote reticulated thrombocytosis and atherogenesis in diabetes

PubMed Central

Kraakman, Michael J.; Lee, Man K.S.; Al-Sharea, Annas; Dragoljevic, Dragana; Barrett, Tessa J.; Montenont, Emilie; Basu, Debapriya; Heywood, Sarah; Kammoun, Helene L.; Flynn, Michelle; Whillas, Alexandra; Hanssen, Nordin M.J.; Febbraio, Mark A.; Westein, Erik; Chin-Dusting, Jaye; Cooper, Mark E.; Berger, Jeffrey S.; Goldberg, Ira J.; Nagareddy, Prabhakara R.; Murphy, Andrew J.

2017-01-01

Platelets play a critical role in atherogenesis and thrombosis-mediated myocardial ischemia, processes that are accelerated in diabetes. Whether hyperglycemia promotes platelet production and whether enhanced platelet production contributes to enhanced atherothrombosis remains unknown. Here we found that in response to hyperglycemia, neutrophil-derived S100 calcium-binding proteins A8/A9 (S100A8/A9) interact with the receptor for advanced glycation end products (RAGE) on hepatic Kupffer cells, resulting in increased production of IL-6, a pleiotropic cytokine that is implicated in inflammatory thrombocytosis. IL-6 acts on hepatocytes to enhance the production of thrombopoietin, which in turn interacts with its cognate receptor c-MPL on megakaryocytes and bone marrow progenitor cells to promote their expansion and proliferation, resulting in reticulated thrombocytosis. Lowering blood glucose using a sodium-glucose cotransporter 2 inhibitor (dapagliflozin), depleting neutrophils or Kupffer cells, or inhibiting S100A8/A9 binding to RAGE (using paquinimod), all reduced diabetes-induced thrombocytosis. Inhibiting S100A8/A9 also decreased atherogenesis in diabetic mice. Finally, we found that patients with type 2 diabetes have reticulated thrombocytosis that correlates with glycated hemoglobin as well as increased plasma S100A8/A9 levels. These studies provide insights into the mechanisms that regulate platelet production and may aid in the development of strategies to improve on current antiplatelet therapies and to reduce cardiovascular disease risk in diabetes. PMID:28504650

Lactoferrin-binding proteins in Shigella flexneri.

PubMed Central

Tigyi, Z; Kishore, A R; Maeland, J A; Forsgren, A; Naidu, A S

1992-01-01

The ability of Shigella flexneri to interact with lactoferrin (Lf) was examined with a 125I-labeled protein-binding assay. The percent binding of human lactoferrin (HLf) and bovine lactoferrin (BLf) to 45 S. flexneri strains was 19 +/- 3 and 21 +/- 3 (mean +/- standard error of the mean), respectively. 125I-labeled HLf and BLf binding to strain M90T reached an equilibrium within 2 h. Unlabeled HLf and BLf displaced the 125I-HLf-bacteria interaction in a dose-dependent manner. The Lf-bacterium complex was uncoupled by KSCN or urea, but not by NaCl. The interaction was specific, and approximately 4,800 HLf binding sites (affinity constant [Ka], 690 nM) or approximately 5,700 BLf binding sites (Ka, 104 nM) per cell were estimated in strain M90T by a Scatchard plot analysis. The native cell envelope (CE) and outer membrane (OM) did not reveal Lf-binding components in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, after being boiled, the CE and OM preparations showed three distinct horseradish peroxidase-Lf reactive bands of about 39, 22, and 16 kDa. The 39-kDa component was also reactive to a monoclonal antibody specific for porin (PoI) proteins of members of the family Enterobacteriaceae. The Lf-binding protein pattern was similar with BLf or HLf, for Crb+ and Crb- strains. The protein-Lf complex was dissociable by KSCN or urea and was stable after treatment with NaCl. Variation (loss) in the O chain of lipopolysaccharide (LPS) markedly enhanced the Lf-binding capacity in the isogenic rough strain SFL1070-15 compared with its smooth parent strain, SFL1070. These data establish that Lf binds to specific components in the bacterial OM; the heat-modifiable, anti-PoI-reactive, and LPS-associated properties suggested that the Lf-binding proteins are porins in S. flexneri. Images PMID:1319403

Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

PubMed

Bae, Ji-Eun; Hwang, Kwang Yeon; Nam, Ki Hyun

2018-06-16

Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition. Copyright © 2018. Published by Elsevier Inc.

The E. coli 16S rRNA binding site of ribosomal protein S15: higher-order structure in the absence and in the presence of the protein.

PubMed Central

Mougel, M; Philippe, C; Ebel, J P; Ehresmann, B; Ehresmann, C

1988-01-01

We have investigated in detail the secondary and tertiary structures of E. coli 16S rRNA binding site of protein S15 using a variety of enzymatic and chemical probes. RNase T1 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with 1-cyclohexyl-3 (2-(1-methylmorpholino)-ethyl)-carboiimide-p- toluenesulfonate (at U(N-3) and G(N-1)) and with diethylpyrocarbonate (at A(N-7)). The RNA region corresponding to nucleotides 652 to 753 was tested within: (1) the complete 16S rRNA molecule; (2) a 16S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S15-16S rRNA complex; (4) the S15-fragment complex. Cleavage and modification sites were detected by primer extension with reverse transcriptase. Our results show that: (1) The synthetized fragment folds into the same overall secondary structure as in the complete 16S rRNA, with the exception of the large asymmetrical internal loop (nucleotides 673-676/714-733) which is fully accessible in the fragment while it appears conformationally heterogeneous in the 16S rRNA; (2) the reactivity patterns of the S15-16S rRNA and S15-fragment complexes are identical; (3) the protein protects defined RNA regions, located in the large interior loop and in the 3'-end strand of helix [655-672]-[734-751]; (4) the protein also causes enhanced chemical reactivity and enzyme accessibility interpreted as resulting from a local conformational rearrangement, induced by S15 binding. Images PMID:2453025

Detergent enhances binding of a secreted HLA-A2 molecule to solid phase peptides.

PubMed

Tussey, L G; Frelinger, J A

1991-11-01

We have constructed a secreted analogue (sA2) of the human class I molecule HLA-A2. sA2 was affinity purified both in the presence and absence of detergent and the effects of detergent on the magnitude and specificity of A2 binding to solid phase peptides tested. sA2 purified in the presence of detergent and detergent-solubilized A2 are shown to function comparably in the binding of the synthetic peptide M.Y + 57-68, a known T-cell epitope derived from the influenza A matrix protein. The molecules binding to M.Y + 57-68 typically represent 8% to 10% of the added protein. In contrast, less than 1% of sA2 protein purified in the absence of detergent binds M.Y + 57-68. This reduced binding is not due to a change in the affinity of sA2 for M.Y + 57-68. Addition of detergent at various stages of the purification and iodination procedures indicates that the longer the sA2 molecules are exposed to detergent the better they bind. However, the concentration of detergent during the actual binding assay does not appear to be critical. We also find that while the sA2-detergent and the sA2-no detergent molecules differ in the extent to which they bind various peptides, they do not differ in their patterns of binding. We conclude that detergent probably does not influence the specificity of class I/peptide binding but does increase the number of sA2 molecules that can participate in the binding of peptide either by generating and stabilizing "empty" sA2 molecules or by stabilizing a structure that is more amenable to binding peptide.

Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2

PubMed Central

Kitahara, Kei; Kajiura, Akimasa; Sato, Neuza Satomi; Suzuki, Tsutomu

2007-01-01

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function. PMID:17553838

Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM.

PubMed

Hymes, Jeffrey P; Johnson, Brant R; Barrangou, Rodolphe; Klaenhammer, Todd R

2016-05-01

Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion off bpB lost the ability to adhere to mucin and fibronectin in vitro Homologues off bpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside theL. acidophilus homology group. Copyright © 2016 Hymes et al.

Ca2+-binding Motif of βγ-Crystallins*

PubMed Central

Srivastava, Shanti Swaroop; Mishra, Amita; Krishnan, Bal; Sharma, Yogendra

2014-01-01

βγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca2+ binding. A βγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca2+-binding sites. βγ-Crystallins make a separate class of Ca2+-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in βγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca2+ binding to βγ-crystallins in mediating biological processes are yet to be elucidated. PMID:24567326

Characterization of the Binding of a Potent Synthetic Androgen, Methyltrienolone, to Human Tissues

PubMed Central

Menon, Mani; Tananis, Catherine E.; Hicks, L. Louise; Hawkins, Edward F.; McLoughlin, Martin G.; Walsh, Patrick C.

1978-01-01

The potent synthetic androgen methytrienolone (R 1881), which does not bind to serum proteins, was utilized to characterize binding to receptors in human androgen responsive tissues. Cytosol extracts prepared from hypertrophic prostates (BPH) were utilized as the source of receptor for the initial studies. High affinity binding was detected in the cytosol of 29 of 30 samples of BPH (average number of binding sites, 45.8±4.7 fmol/mg of protein; dissociation constant, 0.9±0.2 nM). This binding had the characteristics of a receptor: heat lability, precipitability by 0-33% ammonium sulfate and by protamine sulfate, and 8S sedimentation coefficient. High affinity binding was also detected in cytosol prepared from seminal vesicle, epididymis, and genital skin but not in non-genital skin or muscle. However, similar binding was demonstrated in the cytosol of human uterus. The steroid specificities of binding to the cytosol of male tissues of accessory reproduction and of uterus were similar in that progestational agents were more effective competitors than natural androgens. Binding specificities in cytosol prepared from genital skin were distinctly different and were similar to those of ventral prostate from the castrated rat in that dihydrotestosterone was much more potent than progestins in competition. Thus binding of R 1881 to the cytosol of prostate, epididymis, and seminal vesicle has some characteristics of binding to a progesterone receptor. When the nuclear extract from BPH was analyzed, high affinity binding was demonstrated that conformed to the specificities of binding to an androgen receptor. Here dihydrotestosterone was a more potent competitor than progestational agents. Similar patterns of binding were detected in the crude nuclear extracts from seminal vesicle, epididymis, and genital skin but not in uterus, muscle, or non-genital skin. We conclude that the androgen receptor is not demonstrable in the cytosol of prostate, epididymis, or seminal vesicle

Behind the scenes of vitamin D binding protein: more than vitamin D binding.

PubMed

Delanghe, Joris R; Speeckaert, Reinhart; Speeckaert, Marijn M

2015-10-01

Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

The alternative complement pathway control protein H binds to immune complexes and serves their detection

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

Nydegger, U.E.; Corvetta, A.; Spaeth, P.J.

1983-01-01

During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound /sup 125/I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of /sup 125/I-H; when fresh serum was chelated with 10 mM EDTA, /sup 125/I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samplesmore » from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), /sup 125/I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while /sup 125/I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.« less

The Y-Box Binding Protein 1 Suppresses Alzheimer’s Disease Progression in Two Animal Models

PubMed Central

Bobkova, N. V.; Lyabin, D. N.; Medvinskaya, N. I.; Samokhin, A. N.; Nekrasov, P. V.; Nesterova, I. V.; Aleksandrova, I. Y.; Tatarnikova, O. G.; Bobylev, A. G.; Vikhlyantsev, I. M.; Kukharsky, M. S.; Ustyugov, A. A.; Polyakov, D. N.; Eliseeva, I. A.; Kretov, D. A.; Guryanov, S. G.; Ovchinnikov, L. P.

2015-01-01

The Y-box binding protein 1 (YB-1) is a member of the family of DNA- and RNA binding proteins. It is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation. Previously, YB-1 was detected in neurons of the neocortex and hippocampus, but its precise role in the brain remains undefined. Here we show that subchronic intranasal injections of recombinant YB-1, as well as its fragment YB-11−219, suppress impairment of spatial memory in olfactory bulbectomized (OBX) mice with Alzheimer’s type degeneration and improve learning in transgenic 5XFAD mice used as a model of cerebral amyloidosis. YB-1-treated OBX and 5XFAD mice showed a decreased level of brain β-amyloid. In OBX animals, an improved morphological state of neurons was revealed in the neocortex and hippocampus; in 5XFAD mice, a delay in amyloid plaque progression was observed. Intranasally administered YB-1 penetrated into the brain and could enter neurons. In vitro co-incubation of YB-1 with monomeric β-amyloid (1–42) inhibited formation of β-amyloid fibrils, as confirmed by electron microscopy. This suggests that YB-1 interaction with β-amyloid prevents formation of filaments that are responsible for neurotoxicity and neuronal death. Our data are the first evidence for a potential therapeutic benefit of YB-1 for treatment of Alzheimer’s disease. PMID:26394155

Binding in agrammatic aphasia: Processing to comprehension

PubMed Central

Janet Choy, Jungwon; Thompson, Cynthia K.

2010-01-01

Background Theories of comprehension deficits in Broca’s aphasia have largely been based on the pattern of deficit found with movement constructions. However, some studies have found comprehension deficits with binding constructions, which do not involve movement. Aims This study investigates online processing and offline comprehension of binding constructions, such as reflexive (e.g., himself) and pronoun (e.g., him) constructions in unimpaired and aphasic individuals in an attempt to evaluate theories of agrammatic comprehension. Methods & Procedures Participants were eight individuals with agrammatic Broca’s aphasia and eight age-matched unimpaired individuals. We used eyetracking to examine online processing of binding constructions while participants listened to stories. Offline comprehension was also tested. Outcomes & Results The eye movement data showed that individuals with Broca’s aphasia were able to automatically process the correct antecedent of reflexives and pronouns. In addition, their syntactic processing of binding was not delayed compared to normal controls. Nevertheless, offline comprehension of both pronouns and reflexives was significantly impaired compared to the control participants. This comprehension failure was reflected in the aphasic participants’ eye movements at sentence end, where fixations to the competitor increased. Conclusions These data suggest that comprehension difficulties with binding constructions seen in agrammatic aphasic patients are not due to a deficit in automatic syntactic processing or delayed processing. Rather, they point to a possible deficit in lexical integration. PMID:20535243

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation

PubMed Central

Casillas-Ituarte, Nadia N.; Cruz, Carlos H. B.; Lins, Roberto D.; DiBartola, Alex C.; Howard, Jessica; Liang, Xiaowen; Höök, Magnus; Viana, Isabelle F. T.; Sierra-Hernández, M. Roxana; Lower, Steven K.

2017-01-01

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/koff) and dissociation constants (Kd = koff/kon), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (Kdapp = 0.2–0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide (Kdapp = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections. PMID:28400484

Polymeric brushes as functional templates for immobilizing ribonuclease A: study of binding kinetics and activity.

PubMed

Cullen, Sean P; Liu, Xiaosong; Mandel, Ian C; Himpsel, Franz J; Gopalan, Padma

2008-02-05

The ability to immobilize proteins with high binding capacities on surfaces while maintaining their activity is critical for protein microarrays and other biotechnological applications. We employed poly(acrylic acid) (PAA) brushes as templates to immobilize ribonuclease A (RNase A), which is commonly used to remove RNA from plasmid DNA preparations. The brushes are grown by surface-anchored atom-transfer radical polymerization (ATRP) initiators. RNase A was immobilized by both covalent esterification and a high binding capacity metal-ion complexation method to PAA brushes. The polymer brushes immobilized 30 times more enzyme compared to self-assembled monolayers. As the thickness of the brush increases, the surface density of the RNase A increases monotonically. The immobilization was investigated by ellipsometry, X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The activity of the immobilized RNase A was determined using UV absorbance. As much as 11.0 microg/cm(2) of RNase A was bound to PAA brushes by metal-ion complexation compared to 5.8 microg/cm(2) by covalent immobilization which is 30 and 16 times the estimated mass bound in a monolayer. The calculated diffusion coefficient D was 0.63 x 10(-14) cm(2)/s for metal-ion complexation and 0.71 x 10(-14) cm(2)/s for covalent immobilization. Similar values of D indicate that the binding kinetics is similar, but the thermodynamic equilibrium coverage varies with the binding chemistry. Immobilization kinetics and thermodynamics were characterized by ellipsometry for both methods. A maximum relative activity of 0.70-0.80 was reached between five and nine monolayers of the immobilized enzyme. However, the relative activity for covalent immobilization was greater than that of metal-ion complexation. Covalent esterification resulted in similar temperature dependence as free enzyme, whereas metal-ion complexation showed no

Sex difference in κ-opioid receptor (KOPR)-mediated behaviors, brain region KOPR level and KOPR-mediated guanosine 5'-O-(3-[35S]thiotriphosphate) binding in the guinea pig.

PubMed

Wang, Yu-Jun; Rasakham, Khampaseuth; Huang, Peng; Chudnovskaya, Darina; Cowan, Alan; Liu-Chen, Lee-Yuan

2011-11-01

We examined whether sex differences in κ-opioid receptor (KOPR) pharmacology exist in guinea pigs, which are more similar to humans in the expression level and distribution of KOPR in the brain than rats and mice. The KOPR agonist trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)benzeneacetamide methanesulfonate (U50,488H) produced a dose-dependent increase in abnormal postures and immobility with more effects in males than females. Males also showed more U50,488H-induced antinociception in the paw pressure test than females. Pretreatment with the KOPR antagonist norbinaltorphimine blocked U50,488H-induced abnormal body postures and antinociception. In contrast, inhibition of cocaine-induced hyperambulation by U50,488H was more effective in females than males. Thus, sex differences in the effects of U50,488H are endpoint-dependent. We then examined whether sex differences in KOPR levels and KOPR-mediated G protein activation in brain regions may contribute to the observed differences using quantitative in vitro autoradiography of [(3)H](5a,7a,8b)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)1-oxaspiro(4,5)dec-8-yl)benzeacetamide ([(3)H]U69,593) binding to the KOPR and U50,488H-stimulated guanosine 5'-O-(3-[(35)S]thiotriphosphate ([(35)S]GTPγS) binding. Compared with females, males exhibited more [(3)H]U69,593 binding in the deep layers of somatosensory and insular cortices, claustrum, endopiriform nucleus, periaqueductal gray, and substantial nigra. Concomitantly, U50,488H-stimulated [(35)S]GTPγS binding was greater in males than females in the superficial and deep layers of somatosensory and insular cortices, caudate putamen, claustrum, medial geniculate nucleus, and cerebellum. In contrast, compared with males, females showed more U50,488H-stimulated [(35)S]GTPγS binding in the dentate gyrus and a trend of higher [(35)S]GTPγS binding in the hypothalamus. These data demonstrate that males and females differ in KOPR expression and KOPR-mediated G protein

On the structural features of hairpin triloops in rRNA: from nucleotide to global conformational change upon ligand binding.

PubMed

Mitrasinovic, Petar M

2006-03-01

RNA structure can be viewed as both a construct composed of various structural motifs and a flexible polymer that is substantially influenced by its environment. In this light, the present paper represents an attempt to reconcile the two standpoints. By using the 3D structures both of four (16S and 23S) portions of unbound 50S, H50S, and T30S ribosomal subunits and of 38 large ribonucleoligand complexes as the starting point, the behavior, which is induced by ligand binding, of 73 hairpin triloops with closing g-c and c-g base pairs was investigated using root-mean-square deviation (RMSD) approach and pseudotorsional (eta,theta) convention at the nucleotide-by-nucleotide level. Triloops were annotated in accordance with a recent proposal of geometric nomenclature. A simple measure for the determination of the strain of a triloop is introduced. It is believed that a possible classification of the interior triloops, based on the 2D eta-theta unique path, will aid to conceive their local behavior upon ligand binding. All rRNA residues in contact with ligands as well as regions of considerable conformational changes upon complex formation were identified. The analysis offers the answer to: how proximal to and how far from the actual ligand-binding sites the structural changes occur?

Structural basis for 16S ribosomal RNA cleavage by the cytotoxic domain of colicin E3.

PubMed

Ng, C Leong; Lang, Kathrin; Meenan, Nicola Ag; Sharma, Amit; Kelley, Ann C; Kleanthous, Colin; Ramakrishnan, V

2010-10-01

The toxin colicin E3 targets the 30S subunit of bacterial ribosomes and cleaves a phosphodiester bond in the decoding center. We present the crystal structure of the 70S ribosome in complex with the cytotoxic domain of colicin E3 (E3-rRNase). The structure reveals how the rRNase domain of colicin binds to the A site of the decoding center in the 70S ribosome and cleaves the 16S ribosomal RNA (rRNA) between A1493 and G1494. The cleavage mechanism involves the concerted action of conserved residues Glu62 and His58 of the cytotoxic domain of colicin E3. These residues activate the 16S rRNA for 2' OH-induced hydrolysis. Conformational changes observed for E3-rRNase, 16S rRNA and helix 69 of 23S rRNA suggest that a dynamic binding platform is required for colicin E3 binding and function.

Predicting renal graft failure by sCD30 levels and de novo HLA antibodies at 1year post-transplantation.

PubMed

Wang, Dong; Wu, Guojun; Chen, Jinhua; Yu, Ziqiang; Wu, Weizhen; Yang, Shunliang; Tan, Jianming

2012-06-01

HLA antibodies and sCD30 levels were detected in the serum sampled from 620 renal graft recipients at 1 year post-transplantation, which were followed up for 5 years. Six-year graft and patient survivals were 81.6% and 91.0%. HLA antibodies were detected in 45 recipients (7.3%), of whom there were 14 cases with class I antibodies, 26 cases with class II, and 5 cases with both class I and II. Much more graft loss was record in recipients with HLA antibodies than those without antibodies (60% vs. 15.1%, p<0.001). Significantly higher sCD30 levels were recorded in recipients suffering graft loss than the others (73.9±48.8 U/mL vs. 37.3±14.6 U/mL, p<0.001). Compared with those with high sCD30 levels, recipients with low sCD30 levels (<50 U/mL) had much better 6-year graft survival (92.4% vs. 46.6%, p<0.001). Further statistical analysis showed that detrimental effect of de novo HLA antibodies and high sCD30 on graft survival was not only independent but also additive. Therefore, post-transplantation monitoring of HLA antibodies and sCD30 levels is necessary and recipients with elevated sCD30 level and/or de novo HLA antibody should be paid more attention in order to achieve better graft survival. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization of salivary alpha-amylase binding to Streptococcus sanguis

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

Scannapieco, F.A.; Bergey, E.J.; Reddy, M.S.

1989-09-01

The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase was the prominent salivary component eluted from S. sanguis. Studies with {sup 125}I-labeled HSMSL or {sup 125}I-labeled HPS also demonstrated a componentmore » with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of ({sup 125}I)alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.« less

30S(α , p) Thermonuclear Reaction Rate from Experimental Level Structure of 34Ar

NASA Astrophysics Data System (ADS)

Kahl, D.; Chen, A. A.; Kubono, S.; Yamaguchi, H.; Binh, D. N.; Chen, J.; Cherubini, S.; Duy, N. N.; Hashimoto, T.; Hayakawa, S.; Iwasa, N.; Jung, H. S.; Kato, S.; Kwon, Y. K.; Nishimura, S.; Ota, S.; Setoodehnia, K.; Teranishi, T.; Tokieda, H.; Yamada, T.; Yun, C. C.; Zhang, L. Y.

Type I X-ray bursts are the most frequent thermonuclear explosions in the galaxy. Owing to their recurrence from known astronomical objects, burst morphology is extensively documented, and they are modeled very successfully as neutron-deficient, thermonuclear runaway on the surface of accreting neutron stars. While reaction networks include hundreds of isotopes and thousands of nuclear processes, only a small subset appear to play a pivotal role. One such reaction is the 30S(α , p) reaction, which is believed to be a crucial link in the explosive helium burning which is responsible for the large energy flux. However, very little experimental information is available concerning the cross section itself, nor the 34Ar compound nucleus at the relevant energies. We performed the first study of the entrance channel via 30S alpha resonant elastic scattering using a state-of-the-art, low-energy, 30S radioactive ion beam. The measurement was performed in inverse kinematics using a newly-developed active target. An R-matrix analysis of the excitation function reveals previously unknown resonances, including their quantum properties of spin, parity, width, and energy.

Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide.

PubMed Central

Chiaruttini, C; Expert-Bezançon, A; Hayes, D; Ehresmann, B

1982-01-01

1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E. coli 3OS ribosomal subunit. Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels. Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form. Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence. Protein S12 is crosslinked to the terminal G of this heptanucleotide. Images PMID:6760129

Dipetalodipin, a Novel Multifunctional Salivary Lipocalin That Inhibits Platelet Aggregation, Vasoconstriction, and Angiogenesis through Unique Binding Specificity for TXA2, PGF2α, and 15(S)-HETE*

PubMed Central

Assumpção, Teresa C. F.; Alvarenga, Patricia H.; Ribeiro, José M. C.; Andersen, John F.; Francischetti, Ivo M. B.

2010-01-01

Dipetalodipin (DPTL) is an 18 kDa protein cloned from salivary glands of the triatomine Dipetalogaster maxima. DPTL belongs to the lipocalin superfamily and has strong sequence similarity to pallidipin, a salivary inhibitor of collagen-induced platelet aggregation. DPTL expressed in Escherichia coli was found to inhibit platelet aggregation by collagen, U-46619, or arachidonic acid without affecting aggregation induced by ADP, convulxin, PMA, and ristocetin. An assay based on incubation of DPTL with small molecules (e.g. prostanoids, leukotrienes, lipids, biogenic amines) followed by chromatography, mass spectrometry, and isothermal titration calorimetry showed that DPTL binds with high affinity to carbocyclic TXA2, TXA2 mimetic (U-46619), TXB2, PGH2 mimetic (U-51605), PGD2, PGJ2, and PGF2α. It also interacts with 15(S)-HETE, being the first lipocalin described to date to bind to a derivative of 15-lipoxygenase. Binding was not observed to other prostaglandins (e.g. PGE1, PGE2, 8-iso-PGF2α, prostacyclin), leukotrienes (e.g,. LTB4, LTC4, LTD4, LTE4), HETEs (e.g. 5(S)-HETE, 12(S)-HETE, 20-HETE), lipids (e.g. arachidonic acid, PAF), and biogenic amines (e.g. ADP, serotonin, epinephrine, norepinephrine, histamine). Consistent with its binding specificity, DPTL prevents contraction of rat uterus stimulated by PGF2α and induces relaxation of aorta previously contracted with U-46619. Moreover, it inhibits angiogenesis mediated by 15(S)-HETE and did not enhance inhibition of collagen-induced platelet aggregation by SQ29548 (TXA2 antagonist) and indomethacin. A 3-D model for DPTL and pallidipin is presented that indicates the presence of a conserved Arg39 and Gln135 in the binding pocket of both lipocalins. Results suggest that DPTL blocks platelet aggregation, vasoconstriction, and angiogenesis through binding to distinct eicosanoids involved in inflammation. PMID:20889972

Structure-Function Analysis of Friedreich's Ataxia Mutants Reveals Determinants of Frataxin Binding and Activation of the Fe-S Assembly Complex

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

Bridwell-Rabb, Jennifer; Winn, Andrew M; Barondeau, David P

2012-08-01

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-Smore » cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k cat/K M higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k cat/K M of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.« less

Kinematic structure of the 30 Doradus giant H II region

NASA Technical Reports Server (NTRS)

Chu, You-Hua; Kennicutt, Robert C., Jr.

1994-01-01

We have used the echelle CCD spectrograph on the Cerro Tololo Inter-American Observatory (CTIO) 4 m telescope to map the nebular velocity field in the 30 Doradus giant H II region. The kinematics of 30 Dor are very complex. The outer regions are charaterized by a smooth velocity field, but its turbulent velocity, 30-40 km/s Full Width Half Maximum (FWHM), is considerably higher than those in most smaller H II regions. In the central 9 min core, multiple velocity components are observed at most positions. The velocity field is dominated by a large number of expanding structures, ranging in size from 1 to 100 pc and expansion velocities of 20-200 km/s, and often organized into large hierarchical networks. The integral of these complex expanding structures in 30 Dor produces a surprisingly simple profile with a broad Gaussian core and faint extended wings. Several fast-expanding shells, with diameters of 2-20 pc, expansion velocities of 100-300 km/s, and kinetic energies of 0.5-10 x 10(exp 50) ergs have been identified. The large fast-expanding shells and networks are coincident with extended X-ray sources and are probably associated with supernova remnants embedded in supershells produced by the combined effects of stellar winds and supernovae from OB associations. We have used the intensity-calibrated echelle spectra to determine the basic physical and dynamical properties of the kinematic features in 30 Dor. The expanding shells contain roughly half of the kinetic energy in the 30 Dor complex, and this energy is several times higher than the gravitational binding energy of the region. The energetic requirements of the gas are consistent with the observed stellar content of 30 Dor, if the gas is accelerated by a combination of stellar winds and supernovae. Extrapolating the current energy injection rate in the nebula over the lifetime of the OB complex suggest that 30 Dor and its vicinity will evolve into a supergiant shell as seen in the LMC and other nearby

A DNA-binding protein from Candida albicans that binds to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence of C. albicans.

PubMed

Ishii, N; Yamamoto, M; Lahm, H W; Iizumi, S; Yoshihara, F; Nakayama, H; Arisawa, M; Aoki, Y

1997-02-01

Electromobility shift assays with a DNA probe containing the Saccharomyces cerevisiae ENO1 RPG box identified a specific DNA-binding protein in total protein extracts of Candida albicans. The protein, named Rbf1p (RPG-box-binding protein 1), bound to other S. cerevisiae RPG boxes, although the nucleotide recognition profile was not completely the same as that of S. cerevisiae Rap 1p (repressor-activator protein 1), an RPG-box-binding protein. The repetitive sequence of the C. albicans chromosomal telomere also competed with RPG-box binding to Rbf1p. For further analysis, we purified Rbf1p 57,600-fold from C. albicans total protein extracts, raised mAbs against the purified protein and immunologically cloned the gene, whose ORF specified a protein of 527 aa. The bacterially expressed protein showed RPG-box-binding activity with the same profile as that of the purified one. The Rbf1p, containing two glutamine-rich regions that are found in many transcription factors, showed transcriptional activation capability in S. cerevisiae and was predominantly observed in nuclei. These results suggest that Rbf1p is a transcription factor with telomere-binding activity in C. albicans.

Deciphering the GPER/GPR30-agonist and antagonists interactions using molecular modeling studies, molecular dynamics, and docking simulations.

PubMed

Méndez-Luna, D; Martínez-Archundia, M; Maroun, Rachid C; Ceballos-Reyes, G; Fragoso-Vázquez, M J; González-Juárez, D E; Correa-Basurto, J

2015-01-01

The G-protein coupled estrogen receptor 1 GPER/GPR30 is a transmembrane seven-helix (7TM) receptor involved in the growth and proliferation of breast cancer. Due to the absence of a crystal structure of GPER/GPR30, in this work, molecular modeling studies have been carried out to build a three-dimensional structure, which was subsequently refined by molecular dynamics (MD) simulations (up to 120 ns). Furthermore, we explored GPER/GPR30's molecular recognition properties by using reported agonist ligands (G1, estradiol (E2), tamoxifen, and fulvestrant) and the antagonist ligands (G15 and G36) in subsequent docking studies. Our results identified the E2 binding site on GPER/GPR30, as well as other receptor cavities for accepting large volume ligands, through GPER/GPR30 π-π, hydrophobic, and hydrogen bond interactions. Snapshots of the MD trajectory at 14 and 70 ns showed almost identical binding motifs for G1 and G15. It was also observed that C107 interacts with the acetyl oxygen of G1 (at 14 ns) and that at 70 ns the residue E275 interacts with the acetyl group and with the oxygen from the other agonist whereas the isopropyl group of G36 is oriented toward Met141, suggesting that both C107 and E275 could be involved in the protein activation. This contribution suggest that GPER1 has great structural changes which explain its great capacity to accept diverse ligands, and also, the same ligand could be recognized in different binding pose according to GPER structural conformations.

Two Aromatic Rings Coupled a Sulfur-Containing Group to Favor Protein Electron Transfer by Instantaneous Formations of π∴S:π↔π:S∴π or π∴π:S↔π:π∴S Five-Electron Bindings

PubMed Central

Sun, Weichao; Ren, Haisheng; Tao, Ye; Xiao, Dong; Qin, Xin; Deng, Li; Shao, Mengyao; Gao, Jiali; Chen, Xiaohua

2015-01-01

The cooperative interactions among two aromatic rings with a S-containing group are described, which may participate in electron hole transport in proteins. Ab initio calculations reveal the possibility for the formations of the π∴S:π↔π:S∴π and π∴π:S↔π:π∴S five-electron bindings in the corresponding microsurrounding structures in proteins, both facilitating electron hole transport as efficient relay stations. The relay functionality of these two special structures comes from their low local ionization energies and proper binding energies, which varies with the different aromatic amino acids, S-containing residues, and the arrangements of the same aromatic rings according to the local microsurroundings in proteins. PMID:26120374

Characterization of Selective Exosite-Binding Inhibitors of Matrix Metalloproteinase 13 That Prevent Articular Cartilage Degradation in Vitro

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

Spicer, Timothy P.; Jiang, Jianwen; Taylor, Alexander B.

Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for degradation of articular cartilage during osteoarthritis and therefore represents a target for drug development. Here, as a result of high-throughput screening and structure$-$activity relationship studies, we identified a novel, highly selective class of MMP-13 inhibitors (compounds 1 (Q), 2 (Q1), and 3 (Q2)). Mechanistic characterization revealed a noncompetitive nature of these inhibitors with binding constants in the low micromolar range. Crystallographic analyses revealed two binding modes for compound 2 in the MMP-13 S 1' subsite and in an S 1/S 2* subsite. Type II collagen- andmore » cartilage-protective effects exhibited by compounds 1, 2, and 3 suggested that these compounds might be efficacious in future in vivo studies. Lastly, these compounds were also highly selective when tested against a panel of 30 proteases, which, in combination with a good CYP inhibition profile, suggested low off-target toxicity and drug$-$drug interactions in humans.« less

Characterization of Selective Exosite-Binding Inhibitors of Matrix Metalloproteinase 13 That Prevent Articular Cartilage Degradation in Vitro

DOE PAGES

Spicer, Timothy P.; Jiang, Jianwen; Taylor, Alexander B.; ...

2014-10-20

Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for degradation of articular cartilage during osteoarthritis and therefore represents a target for drug development. Here, as a result of high-throughput screening and structure$-$activity relationship studies, we identified a novel, highly selective class of MMP-13 inhibitors (compounds 1 (Q), 2 (Q1), and 3 (Q2)). Mechanistic characterization revealed a noncompetitive nature of these inhibitors with binding constants in the low micromolar range. Crystallographic analyses revealed two binding modes for compound 2 in the MMP-13 S 1' subsite and in an S 1/S 2* subsite. Type II collagen- andmore » cartilage-protective effects exhibited by compounds 1, 2, and 3 suggested that these compounds might be efficacious in future in vivo studies. Lastly, these compounds were also highly selective when tested against a panel of 30 proteases, which, in combination with a good CYP inhibition profile, suggested low off-target toxicity and drug$-$drug interactions in humans.« less

PSSMHCpan: a novel PSSM-based software for predicting class I peptide-HLA binding affinity

PubMed Central

Liu, Geng; Li, Dongli; Li, Zhang; Qiu, Si; Li, Wenhui; Chao, Cheng-chi; Yang, Naibo; Li, Handong; Cheng, Zhen; Song, Xin; Cheng, Le; Zhang, Xiuqing; Wang, Jian; Yang, Huanming

2017-01-01

Abstract Predicting peptide binding affinity with human leukocyte antigen (HLA) is a crucial step in developing powerful antitumor vaccine for cancer immunotherapy. Currently available methods work quite well in predicting peptide binding affinity with HLA alleles such as HLA-A*0201, HLA-A*0101, and HLA-B*0702 in terms of sensitivity and specificity. However, quite a few types of HLA alleles that are present in the majority of human populations including HLA-A*0202, HLA-A*0203, HLA-A*6802, HLA-B*5101, HLA-B*5301, HLA-B*5401, and HLA-B*5701 still cannot be predicted with satisfactory accuracy using currently available methods. Furthermore, currently the most popularly used methods for predicting peptide binding affinity are inefficient in identifying neoantigens from a large quantity of whole genome and transcriptome sequencing data. Here we present a Position Specific Scoring Matrix (PSSM)-based software called PSSMHCpan to accurately and efficiently predict peptide binding affinity with a broad coverage of HLA class I alleles. We evaluated the performance of PSSMHCpan by analyzing 10-fold cross-validation on a training database containing 87 HLA alleles and obtained an average area under receiver operating characteristic curve (AUC) of 0.94 and accuracy (ACC) of 0.85. In an independent dataset (Peptide Database of Cancer Immunity) evaluation, PSSMHCpan is substantially better than the popularly used NetMHC-4.0, NetMHCpan-3.0, PickPocket, Nebula, and SMM with a sensitivity of 0.90, as compared to 0.74, 0.81, 0.77, 0.24, and 0.79. In addition, PSSMHCpan is more than 197 times faster than NetMHC-4.0, NetMHCpan-3.0, PickPocket, sNebula, and SMM when predicting neoantigens from 661 263 peptides from a breast tumor sample. Finally, we built a neoantigen prediction pipeline and identified 117 017 neoantigens from 467 cancer samples of various cancers from TCGA. PSSMHCpan is superior to the currently available methods in predicting peptide binding affinity with a

In Your 20s it’s Quantity, in Your 30s it’s Quality: The Prognostic Value of Social Activity Across 30 Years of Adulthood

PubMed Central

Carmichael, Cheryl L.; Reis, Harry T.; Duberstein, Paul R.

2015-01-01

Social connection, a leading factor in the promotion of health, well-being, and longevity, requires social knowledge and the capacity to cultivate intimacy. Lifespan development theorists have speculated that social information-seeking goals, emphasized at the beginning of early adulthood, give way to emotional closeness goals in later stages of early adulthood. Drawing on developmental theory (Baltes, 1997; Baltes & Carstensen, 2003), this 30-year prospective study assessed social activity at age-20 and age-30 with experience sampling methods, and psychosocial outcomes (social integration, friendship quality, loneliness, depression, and psychological well-being) at age-50. Results supported the hypothesis that the quantity (but not the quality) of social interactions at age-20, and the quality (but not the quantity) of social interactions at age-30 predict midlife psychosocial outcomes. Longitudinal structural models revealed that age-20 interaction quantity had a direct, unmediated effect on age-50 social and psychological outcomes. The effects of age-20 interaction quality on midlife outcomes, on the other hand, were mediated by age-30 interaction quality. Our findings are consistent with the idea that selection and optimization serve important functions in early adulthood, and that engaging in developmentally appropriate social activity contributes to psychosocial adjustment in the decades that follow. PMID:25774426

Carbon-11 and radioiodinated derivatives of lysergic acid diethylamide: Ligands for the study of serotonin S2 receptors in vivo

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

Lever, J.R.; Hartig, P.R.; Wong, D.F.

1985-05-01

2-(/sup 125/1)-LSD binds selectively and with high affinity to serotonin S2 receptors in vitro. In the present study, the authors prepared 2-(/sup 123/1)-LSD as well as a carbon-11 labeled analog. They also characterized the in vivo binding of these tracers to receptor sites in mouse brain to assess their potential for tomographic imaging of S2 receptors in man. The temporal distribution of 2-(/sup 125/1)-LSD paralleled the density of S2 receptors. Regional selectivity was maximal after 15 minutes when tissue to cerebellum ratios were: frontal cortex (2.6), olfactory tubercles (2.4), striatum (2.3), and cortex (2.0). Preinjection of ketanserin, a potent S2more » antagonist, inhibited binding. 2-(/sup 123/1)-LSD, prepared in 20% yield from LSD and electrophilic I-123, gave similar results in vivo and may be useful for SPECT studies. The authors then synthesized N1-((/sup 11/C)-Me)-2-Br-LSD (/sup 11/C-MBL) from (/sup 11/C)-methyl iodide and 2-Br-LSD for PET imaging trials. /sup 11/C-MBL was isolated by HPLC in high chemical and radiochemical purity within 30 minutes from E.O.B. The average radiochemical yield was 20% and the specific activity was determined by U.V. spectroscopy to be up to 1300Ci/mMol (E.O.S.). 11C-MBL showed greater regional selectivity in vivo in mouse brain than 2-(/sup 125/1)-LSD. After 30 minutes, peak tissue to cerebellum ratios were: frontal cortex (5.4), olfactory tubercles (4.2), striatum (3.0), and cortex (2.8). Preinjection of ketanserin markedly inhibited /sup 11/C-MBL binding. /sup 11/C-MBL is a promising candidate for PET studies of S2 receptors.« less

Pseudoknot and translational control in the expression of the S15 ribosomal protein.

PubMed

Bénard, L; Philippe, C; Ehresmann, B; Ehresmann, C; Portier, C

1996-01-01

Translational autocontrol of the expression of the ribosomal protein S15 proceeds through the transitory formation of a pseudoknot. A synopsis of the known data is used to propose a molecular model of the mechanism involved and for the role of the pseudoknot. This latter structure is able to recruit 30S ribosomal subunits to initiate translation, but also to bind S15 and to stop translation by trapping the ribosome on its loading site. Information on the S15 protein recognition of the messenger RNA site was deduced from mutational analyses and chemical probing. A comparison of this messenger site with the S15 ribosomal binding site was conducted by analysing hydroxyl radical footprintings of these two sites. The existence of two subsites in 16S RNA suggests that the ribosomal protein S15 might present either two different binding sites or at least one common subsite. Clues for the presence of a common site between the messenger and 16S RNA are given which cannot rule out that recognition specificity is linked to a few other determinants. Whether these determinants are different or not remains an open question.

Prognostic values of soluble CD30 and CD30 gene polymorphisms in heart transplantation.

PubMed

Frisaldi, Elisa; Conca, Raffaele; Magistroni, Paola; Fasano, Maria Edvige; Mazzola, Gina; Patanè, Francesco; Zingarelli, Edoardo; Dall'omo, Anna M; Brusco, Alfredo; Amoroso, Antonio

2006-04-27

Pretransplant soluble CD30 (sCD30) is a predictor of kidney graft outcome. Its status as a predictor of heart transplant (HT) outcome has not been established. We have studied this question by assessing sCD30 levels and the number of (CCAT)n repeats of the microsatellite in the CD30 promoter region, which is able alone to repress gene transcription, in the sera of 83 HT patients and 77 of their donors. sCD30 was non-significantly increased in the patients, whereas there were no differences in the CD30 microsatellite allele frequencies. A negative correlation between the number of (CCAT)n and sCD30 levels was evident in the donors. Patients with pretransplant sCD3030 U/ml displayed a significantly better survival. In conclusion, sCD30 levels are predictive of HT outcome.

Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

PubMed Central

Lill, R; Robertson, J M; Wintermeyer, W

1989-01-01

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA. PMID:2583120

Structure of the Reston ebolavirus VP30 C-terminal domain.

PubMed

Clifton, Matthew C; Kirchdoerfer, Robert N; Atkins, Kateri; Abendroth, Jan; Raymond, Amy; Grice, Rena; Barnes, Steve; Moen, Spencer; Lorimer, Don; Edwards, Thomas E; Myler, Peter J; Saphire, Erica Ollmann

2014-04-01

The ebolaviruses can cause severe hemorrhagic fever. Essential to the ebolavirus life cycle is the protein VP30, which serves as a transcriptional cofactor. Here, the crystal structure of the C-terminal, NP-binding domain of VP30 from Reston ebolavirus is presented. Reston VP30 and Ebola VP30 both form homodimers, but the dimeric interfaces are rotated relative to each other, suggesting subtle inherent differences or flexibility in the dimeric interface.

Domain structure, GTP-hydrolyzing activity and 7S RNA binding of Acidianus ambivalens ffh-homologous protein suggest an SRP-like complex in archaea.

PubMed

Moll, R; Schmidtke, S; Schäfer, G

1999-01-01

In this study we provide, for the first time, experimental evidence that a protein homologous to bacterial Ffh is part of an SRP-like ribonucleoprotein complex in hyperthermophilic archaea. The gene encoding the Ffh homologue in the hyperthermophilic archaeote Acidianus ambivalens has been cloned and sequenced. Recombinant Ffh protein was expressed in E. coli and subjected to biochemical and functional studies. A. ambivalens Ffh encodes a 50.4-kDa protein that is structured by three distinct regions: the N-terminal hydrophilic N-region (N), the GTP/GDP-binding domain (G) and a C-terminal located C-domain (C). The A. ambivalens Ffh sequence shares 44-46% sequence similarity with Ffh of methanogenic archaea, 34-36% similarity with eukaryal SRP54 and 30-34% similarity with bacterial Ffh. A polyclonal antiserum raised against the first two domains of A. ambivalens Ffh reacts specifically with a single protein (apparent molecular mass: 46 kDa, termed p46) present in cytosolic and in plasmamembrane cell fractions of A. ambivalens. Recombinant Ffh has a melting point of tm = 89 degreesC. Its intrinsic GTPase activity obviously depends on neutral pH and low ionic strength with a preference for chloride and acetate salts. Highest rates of GTP hydrolysis have been achieved at 81 degreesC in presence of 0.1-1 mm Mg2+. GTP hydrolysis is significantly inhibited by high glycerol concentrations, and the GTP hydrolysis rate also markedly decreases by addition of detergents. The Km for GTP is 13.7 microm at 70 degreesC and GTP hydrolysis is strongly inhibited by GDP (Ki = 8 microm). A. ambivalens Ffh, which includes an RNA-binding motif in the C-terminal domain, is shown to bind specifically to 7S RNA of the related crenarchaeote Sulfolobus solfataricus. Comparative sequence analysis reveals the presence of typical signal sequences in plasma membrane as well as extracellular proteins of hyperthermophilic crenarchaea which strongly supposes recognition events by an Ffh containing

Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins

NASA Astrophysics Data System (ADS)

Rasol, Aveen; Eassa, Souzan; Tarasenko, Olga

2010-04-01

Early recognition of Bacillus cereus group species is important since they can cause food-borne illnesses and deadly diseases in humans. Glycoconjugates (GCs) are carbohydrates covalently linked to non-sugar moieties including lipids, proteins or other entities. GCs are involved in recognition and signaling processes intrinsic to biochemical functions in cells. They also stimulate cell-cell adhesion and subsequent recognition and activation of receptors. We have demonstrated that GCs are involved in Bacillus cereus spore recognition. In the present study, we have investigated whether GCs possess the ability to bind and recognize B. cereus spores and Bacillus anthracis recombinant single toxins (sTX) and complex toxins (cTX). The affinity of GCs to spores + sTX and spores + cTX toxins was studied in the binding essay. Our results demonstrated that GC9 and GC10 were able to selectively bind to B. cereus spores and B. anthracis toxins. Different binding affinities for GCs were found toward Bacillus cereus spores + sTX and spores + cTX. Dilution of GCs does not impede the recognition and binding. Developed method provides a tool for simultaneous recognition and targeting of spores, bacteria toxins, and/or other entities.

Binding of environmental carcinogens to asbestos and mineral fibres.

PubMed Central

Harvey, G; Pagé, M; Dumas, L

1984-01-01

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

Binding of Disordered Peptides to Kelch: Insights from Enhanced Sampling Simulations.

PubMed

Do, Trang Nhu; Choy, Wing-Yiu; Karttunen, Mikko

2016-01-12

Keap1 protein plays an essential role in regulating cellular oxidative stress response and is a crucial binding hub for multiple proteins, several of which are intrinsically disordered proteins (IDP). Among Kelch's IDP binding partners, NRF2 and PTMA are the two most interesting cases. They share a highly similar binding motif; however, NRF2 binds to Kelch with a binding affinity of approximately 100-fold higher than that of PTMA. In this study, we perform an exhaustive sampling composed of 6 μs well-tempered metadynamics and 2 μs unbiased molecular dynamics (MD) simulations aiming at characterizing the binding mechanisms and structural properties of these two peptides. Our results agree with previous experimental observations that PTMA is remarkably more disordered than NRF2 in both the free and bound states. This explains PTMA's lower binding affinity. Our extensive sampling also provides valuable insights into the vast conformational ensembles of both NRF2 and PTMA, supports the hypothesis of coupled folding-binding, and confirms the essential role of linear motifs in IDP binding.

Association of elevated pretransplant sCD30 levels with graft loss in 206 patients treated with modern immunosuppressive therapies after renal transplantation.

PubMed

Heinemann, Falko M; Rebmann, Vera; Witzke, Oliver; Philipp, Thomas; Broelsch, Christoph E; Grosse-Wilde, Hans

2007-03-27

Recent reports suggest that high pretransplant serum levels of soluble CD30 (sCD30) are a risk factor for rejections after kidney transplantation. The aim of our study was to elucidate the predictive value of pretransplant sCD30 levels for kidney transplantation outcome in a single-center patient cohort that has been treated with modern immunosuppressive therapies after transplantation. We retrospectively analyzed sCD30 in multiple pretransplant sera from 206 patients, of whom 174 were transplanted with a cadaveric kidney and 32 patients received an allograft from a living donor. Renal function after transplantation was estimated by measuring serum creatinine and by rejection diagnosis. We could demonstrate a statistically significant association between increased pretransplant sCD30 values and graft failures (P=0.005). Receiver operating curve analysis revealed a cutoff value of 124 U/mL pretransplant sCD30. A multivariate analysis confirmed pretransplant sCD30 values >124 U/mL (P=0.011) and rejection episodes (P<0.0001) as independent risk factors for graft loss. Our study revealed a strong correlation between pretransplant sCD30 levels and the incidence of graft failure, but we could not confirm that the development of rejection episodes is correlated with pretransplant sCD30 values.

Access and Binding of H2S to Hemeproteins: The Case of HbI of Lucina pectinata.

PubMed

Boubeta, Fernando M; Bari, Sara E; Estrin, Dario A; Boechi, Leonardo

2016-09-15

Hydrogen sulfide (H2S) was recently discovered as a gasotransmitter, capable of coordinating to the heme iron of hemeproteins. H2S is unique for its ability to render varying concentrations of the nucleophilic conjugate bases (HS(-) or S(2-)), either as free or bound species with expected outcomes on its further reactivity. There is no direct evidence about which species (H2S, HS(-), or S(2-)) coordinates to the iron. We performed computer simulations to address the migration and binding processes of H2S species to the hemoglobin I of Lucina pectinata, which exhibits the highest affinity for the substrate measured to date. We found that H2S is the most favorable species in the migration from the bulk to the active site, through an internal pathway of the protein. After the coordination of H2S, an array of clustered water molecules modifies the active site environment, and assists in the subsequent deprotonation of the ligand, forming Fe(III)-SH(-). The feasibility of the second deprotonation of the coordinated ligand is also discussed.

Characterization of binding of N'-nitrosonornicotine to protein

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

Hughes, M.F.

1986-01-01

The NADPH-dependent activation of the carcinogenic nitrosamine, N'-nitrosonornicotine (NNN) to a reactive intermediate which binds covalently to protein was assessed using male Sprague-Dawley rat liver and lung microsomes. The NADPH-dependent covalent binding of (/sup 14/C)NNN to liver and lung microsomes was linear with time up to 90 and 45 min, respectively and was also linear with protein concentrations up to 3.0 and 2.0 mg/ml, respectively. The apparent K/sub m/ and V/sub max/ of the NADPH-dependent binding to liver microsomes were determined from the initial velocities. Addition of the thiols glutathione, cystein, N-acetylcysteine or 2-mercapthoethanol significantly decreased the non-NADPH-dependent binding tomore » liver microsomal protein, but did not affect the NADPH-dependent binding. Glutathione was required in order to observe any NADPH-dependent binding to lung microsomal protein. In lung microsomes, SKF-525A significantly decreased the NADPH-dependent binding by 79%. Replacement of an air atmosphere with N/sub 2/ or CO:O/sub 2/ (8:2) significantly decreased the NADPH-dependent binding of (/sup 14/C)NNN to liver microsomal protein by 40% or 27% respectively. Extensive covalent binding of (/sup 14/C)NNN to liver and muscle microsomal protein occurred in the absence of an NADPH-generating system, in the presence of 50% methanol and also to bovine serum albumin, indicating a nonenzymatic reaction. These data indicate that cytochrome P-450 is at least in part responsible for the metabolic activation of the carcinogen NNN, but also suggest additional mechanisms of activation.« less

Influence of binding pH and protein solubility on the dynamic binding capacity in hydrophobic interaction chromatography.

PubMed

Baumann, Pascal; Baumgartner, Kai; Hubbuch, Jürgen

2015-05-29

Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in biopharmaceutical industry. A major drawback of HIC, however, is the rather low dynamic binding capacity (DBC) obtained when compared to e.g. ion exchange chromatography (IEX). The typical purification procedure for HIC includes binding at neutral pH, independently of the proteins nature and isoelectric point. Most approaches to process intensification are based on resin and salt screenings. In this paper a combination of protein solubility data and varying binding pH leads to a clear enhancement of dynamic binding capacity. This is shown for three proteins of acidic, neutral, and alkaline isoelectric points. High-throughput solubility screenings as well as miniaturized and parallelized breakthrough curves on Media Scout RoboColumns (Atoll, Germany) were conducted at pH 3-10 on a fully automated robotic workstation. The screening results show a correlation between the DBC and the operational pH, the protein's isoelectric point and the overall solubility. Also, an inverse relationship of DBC in HIC and the binding kinetics was observed. By changing the operational pH, the DBC could be increased up to 30% compared to the standard purification procedure performed at neutral pH. As structural changes of the protein are reported during HIC processes, the applied samples and the elution fractions were proven not to be irreversibly unfolded. Copyright © 2015 Elsevier B.V. All rights reserved.

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation.

PubMed

Casillas-Ituarte, Nadia N; Cruz, Carlos H B; Lins, Roberto D; DiBartola, Alex C; Howard, Jessica; Liang, Xiaowen; Höök, Magnus; Viana, Isabelle F T; Sierra-Hernández, M Roxana; Lower, Steven K

2017-05-26

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands ( e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/ k off ) and dissociation constants ( K d = k off / k on ), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I ( K d app = 0.2-0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide ( K d app = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Sleep Deprivation Decreases [11C]Raclopride’s Binding to Dopamine D2/D3 Receptors in the Human Brain

PubMed Central

Volkow, Nora D.; Wang, Gene-Jack; Telang, Frank; Fowler, Joanna S.; Logan, Jean; Wong, Christopher; Ma, Jim; Pradhan, Kith; Tomasi, Dardo; Thanos, Peter K.; Ferré, Sergi; Jayne, Millard

2009-01-01

Sleep deprivation can markedly impair human performance contributing to accidents and poor productivity. The mechanisms underlying this impairment are not well understood but brain dopamine systems have been implicated. Here we test whether one night of sleep deprivation changes dopamine brain activity. We studied fifteen healthy subjects using positron emission tomography and [11C]raclopride (dopamine D2/3 receptor radioligand) and [11C]cocaine (dopamine transporter radioligand). Subjects were tested twice; after one night of rested sleep and after on night of sleep deprivation. [11C]Raclopride’s specific binding in striatum and thalamus were significantly reduced after sleep deprivation and the magnitude of this reduction correlated with increases in fatigue (tiredness and sleepiness) and with deterioration in cognitive performance (visual attention and working memory). In contrast sleep deprivation did not affect the specific binding of [11C]cocaine in striatum. Since [11C]raclopride competes with endogenous dopamine for binding to D2/D3 receptors, we interpret the decreases in binding to reflect dopamine increases with sleep deprivation. However, we can not rule out the possibility that decreased [11C]raclopride binding reflects decreases in receptor levels or affinity. Sleep deprivation did not affect dopamine transporters (target for most wake-promoting medications) and thus dopamine increases are likely to reflect increases in dopamine cell firing and/or release rather than decreases in dopamine reuptake. Inasmuch as dopamine-enhancing drugs increase wakefulness we postulate that dopamine increases after sleep deprivation is a mechanism by which the brain maintains arousal as the drive to sleep increases but one that is insufficient to counteract behavioral and cognitive impairment. PMID:18716203

Post-translational modifications of transthyretin affect the triiodonine-binding potential

PubMed Central

Henze, Andrea; Homann, Thomas; Serteser, Mustafa; Can, Ozge; Sezgin, Ozlem; Coskun, Abdurrahman; Unsal, Ibrahim; Schweigert, Florian J; Ozpinar, Aysel

2015-01-01

Transthyretin (TTR) is a visceral protein, which facilitates the transport of thyroid hormones in blood and cerebrospinal fluid. The homotetrameric structure of TTR enables the simultaneous binding of two thyroid hormones per molecule. Each TTR subunit provides a single cysteine residue (Cys10), which is frequently affected by oxidative post-translational modifications. As Cys10 is part of the thyroid hormone-binding channel within the TTR molecule, PTM of Cys10 may influence the binding of thyroid hormones. Therefore, we analysed the effects of Cys10 modification with sulphonic acid, cysteine, cysteinylglycine and glutathione on binding of triiodothyronine (T3) by molecular modelling. Furthermore, we determined the PTM pattern of TTR in serum of patients with thyroid disease by immunoprecipitation and mass spectrometry to evaluate this association in vivo. The in silico assays demonstrated that oxidative PTM of TTR resulted in substantial reorganization of the intramolecular interactions and also affected the binding of T3 in a chemotype- and site-specific manner with S-glutathionylation as the most potent modulator of T3 binding. These findings were supported by the in vivo results, which indicated thyroid function-specific patterns of TTR with a substantial decrease in S-sulphonated, S-cysteinylglycinated and S-glutathionylated TTR in hypothyroid patients. In conclusion, this study provides evidence that oxidative modifications of Cys10 seem to affect binding of T3 to TTR probably because of the introduction of a sterical hindrance and induction of conformational changes. As oxidative modifications can be dynamically regulated, this may represent a sensitive mechanism to adjust thyroid hormone availability. PMID:25311081

The 5HT(1A) receptor ligand, S15535, antagonises G-protein activation: a [35S]GTPgammaS and [3H]S15535 autoradiography study.

PubMed

Newman-Tancredi, A; Rivet, J; Chaput, C; Touzard, M; Verrièle, L; Millan, M J

1999-11-19

4-(Benzodioxan-5-yl)1-(indan-2-yl)piperazine (S15535) is a highly selective ligand at 5-HT(1A) receptors. The present study compared its autoradiographic labelling of rat brain sections with its functional actions, visualised by guanylyl-5'-[gamma-thio]-triphosphate ([35S]GTPgammaS) autoradiography, which affords a measure of G-protein activation. [3H]S15535 binding was highest in hippocampus, frontal cortex, entorhinal cortex, lateral septum, interpeduncular nucleus and dorsal raphe, consistent with specific labelling of 5-HT(1A) receptors. In functional studies, S15535 (10 microM) did not markedly stimulate G-protein activation in any brain region, but abolished the activation induced by the selective 5-HT(1A) agonist, (+)-8-hydroxy-dipropyl-aminotetralin ((+)-8-OH-DPAT, 1 microM), in structures enriched in [3H]S15535 labelling. S15535 did not block 5-HT-stimulated activation in caudate nucleus or substantia nigra, regions where (+)-8-OH-DPAT was ineffective and [3H]S15535 binding was absent. Interestingly, S15535 attenuated (+)-8-OH-DPAT and 5-HT-stimulated G-protein activation in dorsal raphe, a region in which S15535 is known to exhibit agonist properties in vivo [Lejeune, F., Millan, M.J., 1998. Induction of burst firing in ventral tegmental area dopaminergic neurons by activation of serotonin (5-HT)(1A) receptors: WAY100,635-reversible actions of the highly selective ligands, flesinoxan and S15535. Synapse 30, 172-180.]. The present data show that (i) [3H]S15535 labels pre- and post-synaptic populations of 5-HT(1A) sites in rat brain sections, (ii) S15535 exhibits antagonist properties at post-synaptic 5-HT(1A) receptors in corticolimbic regions, and (iii) S15535 also attenuates agonist-stimulated G-protein activation at raphe-localised 5-HT(1A) receptors.

Mapping Polymerization and Allostery of Hemoglobin S Using Point Mutations

PubMed Central

Weinkam, Patrick; Sali, Andrej

2014-01-01

Hemoglobin is a complex system that undergoes conformational changes in response to oxygen, allosteric effectors, mutations, and environmental changes. Here, we study allostery and polymerization of hemoglobin and its variants by application of two previously described methods: (i) AllosMod for simulating allostery dynamics given two allosterically related input structures and (ii) a machine-learning method for dynamics- and structure-based prediction of the mutation impact on allostery (Weinkam et al. J. Mol. Biol. 2013), now applicable to systems with multiple coupled binding sites such as hemoglobin. First, we predict the relative stabilities of substates and microstates of hemoglobin, which are determined primarily by entropy within our model. Next, we predict the impact of 866 annotated mutations on hemoglobin’s oxygen binding equilibrium. We then discuss a subset of 30 mutations that occur in the presence of the sickle cell mutation and whose effects on polymerization have been measured. Seven of these HbS mutations occur in three predicted druggable binding pockets that might be exploited to directly inhibit polymerization; one of these binding pockets is not apparent in the crystal structure but only in structures generated by AllosMod. For the 30 mutations, we predict that mutation-induced conformational changes within a single tetramer tend not to significantly impact polymerization; instead, these mutations more likely impact polymerization by directly perturbing a polymerization interface. Finally, our analysis of allostery allows us to hypothesize why hemoglobin evolved to have multiple subunits and a persistent low frequency sickle cell mutation. PMID:23957820

Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

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

Nuemket, Nipawan; Tanaka, Yoshikazu; Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810

2011-07-29

Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinummore » neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.« less

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

Growth, Persistence, and Desistance of Alcohol Use for At-Risk Men in Their 30s

PubMed Central

Capaldi, Deborah M.; Tiberio, Stacey S.; Washburn, Isaac J.; Yoerger, Karen; Feingold, Alan

2015-01-01

Background Little is known about heterogeneity in men's drinking behaviors and their related consequences across midadulthood, and moreover, whether individual or social factors may predict such differences. The present study examined 3 indicators of alcohol use; namely, alcohol volume, heavy episodic drinking (HED), and drinking-related problems for men in their 30s. Methods Participants were 197 at-risk men from the Oregon Youth Study assessed 5 times across ages 29–38 years. Growth mixture modeling with count outcomes was used to examine unobserved heterogeneity in alcohol trajectories. Associations of latent classes of alcohol users with (i) classes for the other alcohol indicators, (ii) alcohol use by peers and romantic partners, (iii) alcohol classes previously extracted from ages 18–29 years, and (iv) past year alcohol use disorder (AUD) diagnostic status at ages 35–36 years was examined. Results A 3-class solution afforded the best fit for each alcohol indicator. Alcohol problems were relatively established in the 30s, with an ascending use class found only for volume. Although relatively few men were in higher classes for all 3 indicators, 45% of the sample was in the highest class on at least 2 indicators of use. Peer drunkenness was a robust predictor of the alcohol classes. Concordance among classes of alcohol users was seen from the 20s to the 30s, with prior desistance likely to be maintained for alcohol volume and HED. AUD diagnoses at ages 35–36 years were more common in the higher classes obtained for alcohol volume and alcohol problems. Conclusions Many men in their 30s engaged in high volume of alcohol without frequent engagement in HED, likely relating to continuing alcohol problems. The convergence of men's alcohol use with that of their peers found at younger ages was maintained into early midadulthood. PMID:26010338

Human GIP(3-30)NH2 inhibits G protein-dependent as well as G protein-independent signaling and is selective for the GIP receptor with high-affinity binding to primate but not rodent GIP receptors.

PubMed

Gabe, Maria Buur Nordskov; Sparre-Ulrich, Alexander Hovard; Pedersen, Mie Fabricius; Gasbjerg, Lærke Smidt; Inoue, Asuka; Bräuner-Osborne, Hans; Hartmann, Bolette; Rosenkilde, Mette Marie

2018-04-01

GIP(3-30)NH 2 is a high affinity antagonist of the GIP receptor (GIPR) in humans inhibiting insulin secretion via G protein-dependent pathways. However, its ability to inhibit G protein-independent signaling is unknown. Here we determine its action on arrestin-recruitment and receptor internalization in recombinant cells. As GIP is adipogenic, we evaluate the inhibitory actions of GIP(3-30)NH 2 in human adipocytes. Finally, we determine the receptor selectivity of GIP(3-30)NH 2 among other human and animal GPCRs. cAMP accumulation and β-arrestin 1 and 2 recruitment were studied in transiently transfected HEK293 cells and real-time internalization in transiently transfected HEK293A and in HEK293A β-arrestin 1 and 2 knockout cells. Furthermore, human subcutaneous adipocytes were assessed for cAMP accumulation following ligand stimulation. Competition binding was examined in transiently transfected COS-7 cells using human 125 I-GIP(3-30)NH 2 . The selectivity of human GIP(3-30)NH 2 was examined by testing for agonistic and antagonistic properties on 62 human GPCRs. Human GIP(3-30)NH 2 inhibited GIP(1-42)-induced cAMP and β-arrestin 1 and 2 recruitment on the human GIPR and Schild plot analysis showed competitive antagonism with a pA 2 and Hill slope of 16.8 nM and 1.11 ± 0.02 in cAMP, 10.6 nM and 1.15 ± 0.05 in β-arrestin 1 recruitment, and 10.2 nM and 1.06 ± 0.05 in β-arrestin 2 recruitment. Efficient internalization of the GIPR was dependent on the presence of either β-arrestin 1 or 2. Moreover, GIP(3-30)NH 2 inhibited GIP(1-42)-induced internalization in a concentration-dependent manner and notably also inhibited GIP-mediated signaling in human subcutaneous adipocytes. Finally, the antagonist was established as GIPR selective among 62 human GPCRs being species-specific with high affinity binding to the human and non-human primate (Macaca fascicularis) GIPRs, and low affinity binding to the rat and mouse GIPRs (K d values of 2.0, 2.5, 31

Specific strychnine binding sites on acrosome-associated membranes of golden hamster spermatozoa.

PubMed

Llanos, Miguel N; Ronco, Ana M; Aguirre, María C

2003-06-27

This study demonstrates for the first time, that membrane vesicles originated from the hamster sperm head after the occurrence of the acrosome reaction, possess specific strychnine binding sites. [3H]Strychnine binding was saturable and reversible, being displaced by unlabeled strychnine (IC(50)=26.7+/-2.3 microM). Kinetic analysis revealed one binding site with K(d)=120nM and B(max)=142fmol/10(6) spermatozoa. Glycine receptor agonists beta-alanine and taurine inhibited strychnine binding by 20-30%. Surprisingly, glycine stimulated binding by about 40-50%. Results obtained in this study strongly suggest the presence of glycine receptors-with distinctive kinetic properties on the periacrosomal plasma membrane of hamster spermatozoa. Localization of this receptor fits well with its previously proposed role in acrosomal exocytosis during mammalian fertilization.

Cortical serotonin-S2 receptor binding in Lewy body dementia, Alzheimer's and Parkinson's diseases.

PubMed

Cheng, A V; Ferrier, I N; Morris, C M; Jabeen, S; Sahgal, A; McKeith, I G; Edwardson, J A; Perry, R H; Perry, E K

1991-11-01

The binding of the selective 5-HT2 antagonist [3H]ketanserin has been investigated in the temporal cortex of patients with Alzheimer's disease (SDAT), Parkinson's disease (PD), senile dementia of Lewy body type (SDLT) and neuropathologically normal subjects (control). 5-HT2 binding was reduced in SDAT, PD with dementia and SDLT. SDAT showed a 5-HT2 receptor deficit across most of the cortical layers. A significant decrease in 5-HT2 binding in the deep cortical layers was found in those SDLT cases without hallucinations. SDLT cases with hallucinations only showed a deficit in one upper layer. There was a significant difference in cortical layers III and V between SDLT without hallucinations and SDLT with hallucinations. The results confirm an abnormality of serotonin binding in various forms of dementia and suggest that preservation of 5-HT2 receptor in the temporal cortex may differentiate hallucinating from non-hallucinating cases of SDLT.

The study of zinc ions binding to casein.

PubMed

Pomastowski, P; Sprynskyy, M; Buszewski, B

2014-08-01

The presented research was focused on physicochemical study of casein properties and the kinetics of zinc ions binding to the protein. Moreover, a fast and simple method of casein extraction from cow's milk has been proposed. Casein isoforms, zeta potential (ζ) and particle size of the separated caseins were characterized with the use of capillary electrophoresis, zeta potential analysis and field flow fractionation (FFF) technique, respectively. The kinetics of the metal-binding process was investigated in batch adsorption experiments. Intraparticle diffusion model, first-order and zero-order kinetic models were applied to test the kinetic experimental data. Analysis of changes in infrared bands registered for casein before and after zinc binding was also performed. The obtained results showed that the kinetic process of zinc binding to casein is not homogeneous but is expressed with an initial rapid stage with about 70% of zinc ions immobilized by casein and with a much slower second step. Maximum amount of bound zinc in the experimental conditions was 30.04mgZn/g casein. Copyright © 2014 Elsevier B.V. All rights reserved.

U.S. EPA, Pesticide Product Label, BIOMIST 30+30 ULV, 05/03/1994

EPA Pesticide Factsheets

2011-04-21

... m~~I~~~~~3~.5~~~~0~.4~~~~IUD~~.F. 30-30 ULYIIOLYENT DILUTtON RATtO FlIIIglcldo, '" 3O>3D WIth lIght II) plrts lol,ont Ind apply at tho al .... ...

GPR30 Signaling and Regulation in Breast Cancer

DTIC Science & Technology

2011-04-01

binds to and activates the orphan G protein-coupled receptor, GPR30, which has been renamed G Protein Coupled Estrogen Receptor ( GPER ). Since the...that inhibition of GPER reduces estrogen-mediated tumor growth. 15. SUBJECT TERMS Breast Cancer, GPR30, Proliferation, Metastasis 16. SECURITY...receptor ( GPER ) [5,  6]. E2 plays a central role in the progression of breast cancer (BrCa), and enhances the proliferation, migration, and invasion of

Neutron Scattering and the 30 S Ribosomal Subunit of E. Coli

DOE R&D Accomplishments Database

Moore, P. B.; Engelman, D. M.; Langer, J. A.; Ramakrishnan, V. R.; Schindler, D. G.; Schoenborn, B. P.; Sillers, I. Y.; Yabuki, S.

1982-06-01

This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today.

CXCL4 is a novel nickel-binding protein and augments nickel allergy.

PubMed

Kuroishi, T; Bando, K; Tanaka, Y; Shishido, K; Kinbara, M; Ogawa, T; Muramoto, K; Endo, Y; Sugawara, S

2017-08-01

Nickel (Ni) is the most frequent metal allergen and induces a TH 1 -dependent type-IV allergy. Although Ni 2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. BALB/cA mice were sensitized with an i.p. injection of NiCl 2 and LPS. Ten days after sensitization, mice were challenged with NiCl 2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni 2+ . Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo. © 2017 John Wiley & Sons Ltd.

42 CFR 137.30 - What is a self-governance compact?

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false What is a self-governance compact? 137.30 Section... SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES TRIBAL SELF-GOVERNANCE Self-Governance compact § 137.30 What is a self-governance compact? A self-governance compact is a legally binding and mutually...

Virtual and biomolecular screening converge on a selective agonist for GPR30.

PubMed

Bologa, Cristian G; Revankar, Chetana M; Young, Susan M; Edwards, Bruce S; Arterburn, Jeffrey B; Kiselyov, Alexander S; Parker, Matthew A; Tkachenko, Sergey E; Savchuck, Nikolay P; Sklar, Larry A; Oprea, Tudor I; Prossnitz, Eric R

2006-04-01

Estrogen is a hormone critical in the development, normal physiology and pathophysiology of numerous human tissues. The effects of estrogen have traditionally been solely ascribed to estrogen receptor alpha (ERalpha) and more recently ERbeta, members of the soluble, nuclear ligand-activated family of transcription factors. We have recently shown that the seven-transmembrane G protein-coupled receptor GPR30 binds estrogen with high affinity and resides in the endoplasmic reticulum, where it activates multiple intracellular signaling pathways. To differentiate between the functions of ERalpha or ERbeta and GPR30, we used a combination of virtual and biomolecular screening to isolate compounds that selectively bind to GPR30. Here we describe the identification of the first GPR30-specific agonist, G-1 (1), capable of activating GPR30 in a complex environment of classical and new estrogen receptors. The development of compounds specific to estrogen receptor family members provides the opportunity to increase our understanding of these receptors and their contribution to estrogen biology.

The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).

PubMed

Yamaguchi, K; von Knoblauch, K; Subramanian, A R

2000-09-15

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid

SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G

PubMed Central

Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P.; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, Manfred; Fulde, Marcus

2017-01-01

The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm-positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis, respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis. The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis. PMID:28401063

Protective Immunity to Ricin Toxin Conferred by Antibodies against the Toxin’s Binding Subunit (RTB)

PubMed Central

Yermakova, Anastasiya; Mantis, Nicholas J.

2011-01-01

The B subunit (RTB) of ricin toxin is a galactose-/N-acetyl galactosamine-specific lectin that promotes attachment and entry of ricin into host cells. RTB is also the archetype of the so-called R-type lectin family, whose members include haemagglutinins of botulinum neurotoxin (BoNT) progenitor toxins, as well as the binding subunits of cytolethal distending toxins. Although RTB is an appealing subunit vaccine candidate, as well as a potential target for immunotherapeutics, the degree to which RTB immunization elicits protective antibodies against ricin toxin remains unresolved. To address this issue, groups of mice were immunized with RTB and then challenged with 5xLD50s of ricin administered intraperitoneally. Despite high RTB-specific serum antibody titers, groups of RTB immunized mice were only partially immune to ricin challenge. Analysis of a collection of RTB-specific B cell hybridomas suggested that only a small fraction of antibodies against RTB have demonstrable neutralizing activity. Two RTB-specific neutralizing monoclonal IgG1 antibodies, 24B11 and SylH3, when passively administered to mice, were sufficient to protect the animals against a 5xLD50 dose of ricin. Both 24B11 and SylH3 blocked ricin attachment to terminal galactose residues and prevented toxin binding to the surfaces of bone marrow-derived macrophages (BMM), suggesting that they function by steric hindrance and recognize epitopes located on RTB’s carbohydrate recognition sub-domains (1α or 2γ). These data raise the possibility of using specific RTB sub-domains, rather than RTB itself, as antigens to more efficiently elicit neutralizing antibodies and protective immunity against ricin. PMID:21872634

Melting relations in the Fe-rich portion of the system FeFeS at 30 kb pressure

USGS Publications Warehouse

Brett, R.; Bell, P.M.

1969-01-01

The melting relations of FeFeS mixtures covering the composition range from Fe to Fe67S33 have been determined at 30 kb pressure. The phase relations are similar to those at low pressure. The eutectic has a composition of Fe72.9S27.1 and a temperature of 990??C. Solubility of S in Fe at elevated temperatures at 30 kb is of the same order of magnitude as at low pressure. Sulfur may have significantly lowered the melting point of iron in the upper mantle during the period of coalescence of metal prior to core formation in the primitive earth. ?? 1969.

AtSPX1 affects the AtPHR1-DNA-binding equilibrium by binding monomeric AtPHR1 in solution.

PubMed

Qi, Wanjun; Manfield, Iain W; Muench, Stephen P; Baker, Alison

2017-10-23

Phosphorus is an essential macronutrient for plant growth and is deficient in ∼50% of agricultural soils. The transcription factor phosphate starvation response 1 (PHR1) plays a central role in regulating the expression of a subset of phosphate starvation-induced (PSI) genes through binding to a cis -acting DNA element termed P1BS (PHR1-binding sequences). In Arabidopsis and rice, activity of AtPHR1/OsPHR2 is regulated in part by their downstream target SPX ( S yg1, P ho81, X pr1) proteins through protein-protein interaction. Here, we provide kinetic and affinity data for interaction between AtPHR1 and P1BS sites. Using surface plasmon resonance, a tandem P1BS sequence showed ∼50-fold higher affinity for MBPAtdPHR1 (a fusion protein comprising the DNA-binding domain and coiled-coil domain of AtPHR1 fused to maltose-binding protein) than a single site. The affinity difference was largely reflected in a much slower dissociation rate from the 2× P1BS-binding site, suggesting an important role for protein co-operativity. Injection of AtSPX1 in the presence of phosphate or inositol hexakisphosphate (InsP6) failed to alter the MBPAtdPHR1-P1BS dissociation rate, while pre-mixing of these two proteins in the presence of either 5 mM Pi or 500 µM InsP6 resulted in a much lower DNA-binding signal from MBPAtdPHR1. These data suggest that, in the Pi-restored condition, AtSPX1 can bind to monomeric AtPHR1 in solution and therefore regulate PSI gene expression by tuning the AtPHR1-DNA-binding equilibrium. This Pi-dependent regulation of AtPHR1-DNA-binding equilibrium also generates a negative feedback loop on the expression of AtSPX1 itself, providing a tight control of PSI gene expression. © 2017 The Author(s).

GABAergic control of neostriatal dopamine D2 receptor binding and behaviors in the rat.

PubMed

Nikolaus, Susanne; Beu, Markus; de Souza Silva, Maria Angelica; Huston, Joseph P; Antke, Christina; Müller, Hans-Wilhelm; Hautzel, Hubertus

2017-02-01

The present study assessed the influence of the GABA A receptor agonist muscimol and the GABA A receptor antagonist bicuculline on neostriatal dopamine D 2 receptor binding in relation to motor and exploratory behaviors in the rat. D 2 receptor binding was measured in baseline and after challenge with either 1mg/kg muscimol or 1mg/kg bicuculline. In additional rats, D 2 receptor binding was measured after injection of saline. After treatment with muscimol, bicuculline and saline, motor and exploratory behaviors were assessed for 30min in an open field prior to administration of [ 123 I]S-3-iodo-N-(1-ethyl-2-pyrrolidinyl)methyl-2-hydroxy-6-methoxybenzamide ([ 123 I]IBZM). For baseline and challenges, striatal equilibrium ratios (V 3 ″) were computed as estimation of the binding potential. Muscimol but not bicuculline reduced D 2 receptor binding relative to baseline and to saline. Travelled distance, duration of rearing and frequency of rearing and of head-shoulder motility were lower after muscimol compared to saline. In contrast, duration of rearing and grooming and frequency of rearing, head-shoulder motility and grooming were elevated after bicuculline relative to saline. Moreover, bicuculline decreased duration of sitting and head-shoulder motility. The muscimol-induced decrease of motor/exploratory behaviors can be related to an elevation of striatal dopamine levels. In contrast, bicuculline is likely to elicit a decline of synaptic dopamine, which, however, is compensated by the time of D 2 receptor imaging studies. The results indicate direct GABAergic control over D 2 receptor binding in the neostriatum in relation to behavioral action, and, thus, complement earlier pharmacological studies. Copyright © 2016. Published by Elsevier Inc.

Identification of a Second Substrate-binding Site in Solute-Sodium Symporters*

PubMed Central

Li, Zheng; Lee, Ashley S. E.; Bracher, Susanne; Jung, Heinrich; Paz, Aviv; Kumar, Jay P.; Abramson, Jeff; Quick, Matthias; Shi, Lei

2015-01-01

The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ∼1. In addition, the related and more experimentally tractable SSS member PutP (the Na+/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport. PMID:25398883

Sequence characterization of S100A8 gene reveals structural differences of protein and transcriptional factor binding sites in water buffalo and yak.

PubMed

Kathiravan, P; Goyal, S; Kataria, R S; Mishra, B P; Jayakumar, S; Joshi, B K

2011-01-01

The present study was undertaken to characterize the structure of S100A8 gene and its promoter in water buffalo and yak. Sequence data of 2.067 kb, 2.071 kb, and 2.052 kb with respect to complete S100A8 gene including 5' flanking region was generated in river buffalo, swamp buffalo, and yak, respectively. BLAST analysis of coding DNA sequences (CDS) of S100A8 gene revealed 95% homology of buffalo sequence with cattle, 85% with pig and horse, 83% with dog, 72-73% with murines, and around 79% with primates and humans. Phylogenetic analysis of predicted CDS revealed distinct clustering of murines, primates, and domestic animals with bovines and bubalines forming a subcluster among farm animals. In silico translation of predicted CDS revealed a sequence of 89 amino acids with 7 amino acid changes between cattle and buffalo and 2 changes between cattle and yak. The search for Pfam family revealed the N-terminal calcium binding domain and the noncanonical EF hand domain in the carboxy terminus, with more variations being observed in the N-terminal domain among different species. Two amino acid changes observed in carboxy terminal EF hand domain resulted in altered secondary structure of yak S100A8 protein. Analysis of S100A8 gene promoter revealed 14 putative motifs for transcriptional factor binding sites. Two putative motifs viz. C/EBP and v-Myb were found to be absent in swamp buffalo as compared to river buffalo and cattle. Differences in the structure of S100A8 protein and the transcriptional factor binding sites identified in the present study need to be analyzed further for their functional significance in yak and swamp buffalo respectively. Copyright © Taylor & Francis Group, LLC

Direct Binding of the Corrector VX-809 to Human CFTR NBD1: Evidence of an Allosteric Coupling between the Binding Site and the NBD1:CL4 Interface.

PubMed

Hudson, Rhea P; Dawson, Jennifer E; Chong, P Andrew; Yang, Zhengrong; Millen, Linda; Thomas, Philip J; Brouillette, Christie G; Forman-Kay, Julie D

2017-08-01

Understanding the mechanism of action of modulator compounds for the cystic fibrosis transmembrane conductance regulator (CFTR) is key for the optimization of therapeutics as well as obtaining insights into the molecular mechanisms of CFTR function. We demonstrate the direct binding of VX-809 to the first nucleotide-binding domain (NBD1) of human CFTR. Disruption of the interaction between C-terminal helices and the NBD1 core upon VX-809 binding is observed from chemical shift changes in the NMR spectra of residues in the helices and on the surface of β -strands S3, S9, and S10. Binding to VX-809 leads to a significant negative shift in NBD1 thermal melting temperature (T m ), pointing to direct VX-809 interaction shifting the NBD1 conformational equilibrium. An inter-residue correlation analysis of the chemical shift changes provides evidence of allosteric coupling between the direct binding site and the NBD1:CL4 interface, thus enabling effects on the interface in the absence of direct binding in that location. These NMR binding data and the negative T m shifts are very similar to those previously reported by us for binding of the dual corrector-potentiator CFFT-001 to NBD1 (Hudson et al., 2012), suggesting that the two compounds may share some aspects of their mechanisms of action. Although previous studies have shown an important role for VX-809 in modulating the conformation of the first membrane spanning domain (Aleksandrov et al., 2012; Ren et al., 2013), this additional mode of VX-809 binding provides insight into conformational dynamics and allostery within CFTR. Copyright © 2017 by The Author(s).

Plasma sex-steroid binding protein in a seasonally breeding reptile, Alligator mississippiensis.

PubMed

Ho, S M; Lance, V; Megaloudis, M

1987-01-01

The properties of a sex-steroid binding protein (SSBP) in the plasma of the American alligator, Alligator mississippiensis, were partially characterized. Alligator SSBP has a sedimentation coefficient of 4S in a 5-20% sucrose gradient. It binds to estradiol-17 beta (E2) and testosterone (T) with limited capacities and moderate affinities (association constant for [3H]E2 is 4.70 +/- 0.09 X 10(8) M-1 and for [3H]T is 1.05 +/- 0.07 X 10(8) M-1, mean +/- SEM of six determinations). Plasma SSBP level, as measured by plasma [3H]E2 binding capacity, varies from 30 to 140 nmol per liter plasma (nM) and was found to be dependent on the gender, sexual maturity, and reproductive state of the animal. Distinct annual fluctuations in plasma SSBP level were observed in female alligators. In adult females, plasma SSBP levels were high (122 +/- 6 nM) in the fall during the nonbreeding season and low (30-60 nM) in spring and early summer during the breeding season. A minimum (33 +/- 6 nM) was reached in mid-June coinciding with the time of oviposition and rapid decline in circulating estrogen levels. This decline in adult female plasma SSBP levels during the breeding season was not observed in immature females. On the contrary, plasma SSBP levels in immature females increased from 81 +/- 14 nM in April to 134 +/- 9 nM in June. Plasma SSBP levels in male alligators showed little changes throughout the entire breeding season; they remained within the range of 80-100 nM from March to June. We believe that seasonal fluctuations in plasma SSBP levels constitute part of the mechanism involved in the regulation of free steroid delivered to target organs in female alligators and that such a mechanism does not exist in male animals.

Monomeric Yeast Frataxin is an Iron-Binding Protein

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

Cook,J.; Bencze, K.; Jankovic, A.

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly)more » share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.« less

Monomeric Yeast Frataxin is an Iron Binding Protein†

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

Cook, J.; Bencze, K; Jankovic, A

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) sharemore » requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.« less

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains

PubMed Central

Walker, Sarah E.; Zhou, Fujun; Mitchell, Sarah F.; Larson, Victoria S.; Valasek, Leos; Hinnebusch, Alan G.; Lorsch, Jon R.

2013-01-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B’s domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome’s mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment. PMID:23236192

The Collagen-Binding Adhesin Is a Virulence Factor in Staphylococcus aureus Keratitis

PubMed Central

Rhem, Marcus N.; Lech, Elizabeth M.; Patti, Joseph M.; McDevitt, Damien; Höök, Magnus; Jones, Dan B.; Wilhelmus, Kirk R.

2000-01-01

A collagen-binding strain of Staphylococcus aureus produced suppurative inflammation in a rabbit model of soft contact lens-associated bacterial keratitis more often than its collagen-binding-negative isogenic mutant. Reintroduction of the cna gene on a multicopy plasmid into the mutant helped it regain its corneal adherence and infectivity. The topical application of a collagen-binding peptide before bacterial challenge decreased S. aureus adherence to deepithelialized corneas. These data suggest that the collagen-binding adhesin is involved in the pathogenesis of S. aureus infection of the cornea. PMID:10816547

Inactivation by Phenylglyoxal of the Specific Binding of 1-Naphthyl Acetic Acid with Membrane-Bound Auxin Binding Sites from Maize Coleoptiles

PubMed Central

Navé, Jean-François; Benveniste, Pierre

1984-01-01

The specific binding of 1-[3H]naphthyl acetic acid (NAA) to membrane-bound binding sites from maize (Zea mays cv INRA 258) coleoptiles is inactivated by phenylglyoxal. The inactivation obeys pseudo first-order kinetics. The rate of inactivation is proportional to phenylglyoxal concentration. Under conditions at which significant binding occurs, NAA, R and S-1-naphthyl 2-propionic acids protect the auxin binding site against inactivation by phenylglyoxal. Scatchard analysis shows that the inhibition of binding corresponds to a decrease in the concentration of sites but not in the affinity. The results of the present chemical modification study indicate that at least one arginyl residue is involved in the positively charged recognition site of the carboxylate anion of NAA. PMID:16663499

Phosphorylation and calcium antagonistically tune myosin-binding protein C’s structure and function

PubMed Central

Previs, Michael J.; Mun, Ji Young; Michalek, Arthur J.; Previs, Samantha Beck; Gulick, James; Robbins, Jeffrey; Warshaw, David M.; Craig, Roger

2016-01-01

During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C’s N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain’s extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C’s inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C’s calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C’s phosphorylation state. PMID:26908872

Estimation of the Binding Free Energy of AC1NX476 to HIV-1 Protease Wild Type and Mutations Using Free Energy Perturbation Method.

PubMed

Ngo, Son Tung; Mai, Binh Khanh; Hiep, Dinh Minh; Li, Mai Suan

2015-10-01

The binding mechanism of AC1NX476 to HIV-1 protease wild type and mutations was studied by the docking and molecular dynamics simulations. The binding free energy was calculated using the double-annihilation binding free energy method. It is shown that the binding affinity of AC1NX476 to wild type is higher than not only ritonavir but also darunavir, making AC1NX476 become attractive candidate for HIV treatment. Our theoretical results are in excellent agreement with the experimental data as the correlation coefficient between calculated and experimentally measured binding free energies R = 0.993. Residues Asp25-A, Asp29-A, Asp30-A, Ile47-A, Gly48-A, and Val50-A from chain A, and Asp25-B from chain B play a crucial role in the ligand binding. The mutations were found to reduce the receptor-ligand interaction by widening the binding cavity, and the binding propensity is mainly driven by the van der Waals interaction. Our finding may be useful for designing potential drugs to combat with HIV. © 2015 John Wiley & Sons A/S.

Roadmap to developing a recombinant coronavirus S protein receptor-binding domain vaccine for severe acute respiratory syndrome

PubMed Central

Jiang, Shibo; Bottazzi, Maria Elena; Du, Lanying; Lustigman, Sara; Tseng, Chien-Te Kent; Curti, Elena; Jones, Kathryn; Zhan, Bin; Hotez, Peter J

2013-01-01

A subunit vaccine, RBD-S, is under development to prevent severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV), which is classified by the US NIH as a category C pathogen. This vaccine is comprised of a recombinant receptor-binding domain (RBD) of the SARS-CoV spike (S) protein and formulated on alum, together with a synthetic glucopyranosyl lipid A. The vaccine would induce neutralizing antibodies without causing Th2-type immunopathology. Vaccine development is being led by the nonprofit product development partnership; Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development in collaboration with two academic partners (the New York Blood Center and University of Texas Medical Branch); an industrial partner (Immune Design Corporation); and Walter Reed Army Institute of Research. A roadmap for the product development of the RBD-S SARS vaccine is outlined with a goal to manufacture the vaccine for clinical testing within the next 5 years. PMID:23252385

THE EFFECTS OF TYPE II BINDING ON METABOLIC STABILITY AND BINDING AFFINITY IN CYTOCHROME P450 CYP3A4

PubMed Central

Peng, Chi-Chi; Pearson, Josh T.; Rock, Dan A.; Joswig-Jones, Carolyn A.; Jones, Jeffrey P.

2010-01-01

One goal in drug design is to decrease clearance due to metabolism. It has been suggested that a compound’s metabolic stability can be increased by incorporation of a sp2 nitrogen into an aromatic ring. Nitrogen incorporation is hypothesized to increase metabolic stability by coordination of nitrogen to the heme iron (termed type II binding). However, questions regarding binding affinity, metabolic stability, and how metabolism of type II binders occurs remain unanswered. Herein, we use pyridinyl quinoline-4-carboxamide analogs to answer these questions. We show that type II binding can have a profound influence on binding affinity for CYP3A4, and the difference in binding affinity can be as high as 1,200 fold. We also find that type II binding compounds can be extensively metabolized, which is not consistent with the dead-end complex kinetic model assumed for type II binders. Two alternate kinetic mechanisms are presented to explain the results. The first involves a rapid equilibrium between the type II bound substrate and a metabolically oriented binding mode. The second involves direct reduction of the nitrogen-coordinated heme followed by oxygen binding. PMID:20346909

Thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli.

PubMed

Fukada, H; Sturtevant, J M; Quiocho, F A

1983-11-10

The thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli have been studied by isothermal and scanning calorimetry. The binding reaction with arabinose is characterized by an enthalpy change of -15.3 +/- 0.5 kcal mol-1 at 25 degrees C, and a large decrease in apparent heat capacity, amounting to -0.44 +/- 0.05 kcal K-1 mol-1, which is constant over the temperature range 8 to 30 degrees C. Very similar results were obtained with D-galactose. These calorimetric results have been combined with binding constants determined by equilibrium dialysis (Clark, A. F., Gerken, T. A., and Hogg, R. W. (1982) Biochemistry 21, 2227-2233) to obtain free energy and entropy changes over the range 5 to 30 degrees C, and by extrapolation to 60 degrees C. The protein undergoes reversible unfolding on being heated with an increase in enthalpy at 53.5 degrees C of 151.8 +/- 1.1 kcal mol-1 (169.2 +/- 1.2 kcal mol-1 at 59.0 degrees C) and in apparent heat capacity of 3.16 +/- 0.07 kcal K-1 mol-1. In the presence of arabinose, the unfolding enthalpy is increased to 200.7 +/- 1.8 kcal mol-1 at 59.0 degrees C, the increase being due to the enthalpy of dissociation of the ligand which amounts to 31 kcal mol-1 at the unfolding temperature. The unfolding temperature is increased by the presence of excess arabinose or galactose, an effect which is due solely to displacement by the added ligand of the unfolding-dissociation equilibrium. The thermodynamic data are discussed in connection with the detailed structural information available for this system from x-ray crystallography (Newcomer, M. E., Gilliland, G. L. and Quiocho, F. A. (1981) J. Biol. Chem. 256, 13213-13217, and references cited therein).

Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

NASA Astrophysics Data System (ADS)

Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

2016-03-01

not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions. Electronic supplementary information (ESI) available: DNA sequences and nomenclature (Table 1S); SDS-PAGE assay of IHF stock solution (Fig. 1S); determination of the concentration of IHF stock solution by Bradford assay (Fig. 2S); equilibrium binding isotherm fitting

A Bridging [4Fe-4S] Cluster and Nucleotide Binding Are Essential for Function of the Cfd1-Nbp35 Complex as a Scaffold in Iron-Sulfur Protein Maturation*

PubMed Central

Netz, Daili J. A.; Pierik, Antonio J.; Stümpfig, Martin; Bill, Eckhard; Sharma, Anil K.; Pallesen, Leif J.; Walden, William E.; Lill, Roland

2012-01-01

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo. PMID:22362766

Kinetics of Binding of Caldesmon to Actin*

PubMed Central

Chalovich, Joseph M.; Chen, Yi-der; Dudek, Ronald; Luo, Hai

2005-01-01

The time course of interaction of caldesmon with actin may be monitored by fluorescence changes that occur upon the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or to acrylodan actin. The concentration dependence of the observed rate of caldesmon-actin binding was analyzed to a first approximation as a single-step reaction using a Monte Carlo simulation. The derived association and dissociation rates were 107 m−1 s−1 and 18.2 s−1, respectively. Smooth muscle tropomyosin enhances the binding of caldesmon to actin, and this was found to be due to a reduction in the rate of dissociation to 6.3 s −1. There is no evidence from this study for a different mechanism of binding in the presence of tropomyosin. The fluorescence changes that occurred with the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or actin-tropomyosin were reversed by the addition of myosin subfragment 1 as predicted by a competitive binding mechanism. PMID:7730374

Influence of freeze-thawing on hyaluronic acid binding of human spermatozoa.

PubMed

Nijs, Martine; Creemers, Eva; Cox, Annemie; Janssen, Mia; Vanheusden, Elke; Castro-Sanchez, Yovanna; Thijs, Herbert; Ombelet, Willem

2009-08-01

Mature human spermatozoa have at least three specific hyaluronic acid (HA) binding proteins present on their sperm membrane. These receptors play a role in the acrosome reaction, hyaluronidase activity, hyaluronan-mediated motility and sperm-zona and sperm-oolemmal binding. Cryopreservation of spermatozoa can cause ultrastructural and even molecular damage. The aim of this study was to investigate if HA binding receptors of human spermatozoa remain functional after freeze-thawing. Forty patients were enrolled in the study. Semen samples were analysed before and after cryopreservation. Parameters analysed included concentration, motility, morphology and hyaluronan binding. Samples were frozen in CBS straws using a glycerol-glucose-based cryoprotectant. HA binding was studied using the sperm-hyaluronan binding assay. Freeze-thawing resulted in a significant decline in motility: the percentage of motile spermatozoa reduced from 50.6 to 30.3% (P < 0.001). HA binding properties of frozen-thawed spermatozoa remained unchanged after the freeze-thawing process: 68.5 +/- 17.1% spermatozoa of the neat sample were bound to HA, as were 71.3 +/- 20.4 of the frozen-thawed sample. This study indicates that freeze-thawing did not alter the functional hyaluronan binding sites of mature motile spermatozoa, and therefore will not alter their fertilizing potential.

Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode

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

Stanek, Kimberly A.; Patterson-West, Jennifer; Randolph, Peter S.

The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homologmore » has been identified in the phylogenetically deep-branching thermophileAquifex aeolicus(Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore,AaeHfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures ofAaeHfq were determined in space groupsP1 andP6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U 6RNA reveals that the outer rim of theAaeHfq hexamer features a well defined binding pocket that is selective for uracil. ThisAaeHfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.« less

Thermochronologic Evidence for Late Eocene Andean Mountain Building at 30°S

NASA Astrophysics Data System (ADS)

Lossada, Ana C.; Giambiagi, Laura; Hoke, Gregory D.; Fitzgerald, Paul G.; Creixell, Christian; Murillo, Ismael; Mardonez, Diego; Velásquez, Ricardo; Suriano, Julieta

2017-11-01

The Andes between 28° and 30°S represent a transition between the Puna-Altiplano Plateau and the Frontal/Principal Cordillera fold-and-thrust belts to the south. While significant early Cenozoic deformation documented in the Andean Plateau, deciphering the early episodes of deformation during Andean mountain building in the transition area is largely unstudied. Apatite fission track (AFT) and (U-Th-Sm)/He (AHe) thermochronology from a vertical and a horizontal transect reveal the exhumation history of the High Andes at 30°S, an area at the heart of this major transition. Interpretation of the age-elevation profile, combined with inverse thermal modeling, indicates that the onset of rapid cooling was underway by 35 Ma, followed by a significant decrease in cooling rate at 30-25 Ma. AFT thermal models also reveal a second episode of rapid cooling in the early Miocene ( 18 Ma) related to rock exhumation to its present position. Low exhumation between the rapid cooling events allowed for the development of a partial annealing zone. We interpret the observed Eocene rapid exhumation as the product of a previously unrecognized compressive event in this part of the Andes that reflects a southern extension of Eocene orogenesis recognized in the Puna/Altiplano. Renewed early-Miocene exhumation indicates that the late Cenozoic compressional stresses responsible for the main phase of uplift of the South Central Andes also impacted the core of the range in this transitional sector. The major episode of Eocene exhumation suggests the creation of significant topographic relief in the High Andes earlier than previously thought.

Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

NASA Technical Reports Server (NTRS)

Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

1993-01-01

Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

20 CFR 220.30 - Special period required for eligibility of widow(er)s.

Code of Federal Regulations, 2013 CFR

2013-04-01

... RAILROAD RETIREMENT ACT DETERMINING DISABILITY Disability Under the Railroad Retirement Act for Any Regular Employment § 220.30 Special period required for eligibility of widow(er)s. In order to be found disabled for any regular employment, a widow(er) must have a permanent physical or mental impairment which...

Binding and activation of major histocompatibility complex class II-deficient macrophages by staphylococcal exotoxins

NASA Technical Reports Server (NTRS)

Beharka, A. A.; Armstrong, J. W.; Iandolo, J. J.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

Macrophages from C2D transgenic mice deficient in the expression of major histocompatibility complex (MHC) class II proteins were used to identify binding sites for superantigens distinct from the MHC class II molecule. Iodinated staphylococcal enterotoxins A and B (SEA and SEB) and exfoliative toxins A and B (ETA and ETB) bound to C2D macrophages in a concentration-dependent and competitive manner. All four toxins increased F-actin concentration within 30 s of their addition to C2D macrophages, indicating that signal transduction occurred in response to toxin in the absence of class II MHC. Furthermore, ETA, ETB, SEA, and, to a lesser extent, SEB induced C2D macrophages to produce interleukin 6. Several molecular species on C2D macrophages with molecular masses of 140, 97, 61, 52, 43, and 37 kDa bound SEA in immunoprecipitation experiments. These data indicate the presence of novel, functionally active toxin binding sites on murine macrophages distinct from MHC class II molecules.

Mo-CBP3, an Antifungal Chitin-Binding Protein from Moringa oleifera Seeds, Is a Member of the 2S Albumin Family

PubMed Central

Freire, José E. C.; Vasconcelos, Ilka M.; Moreno, Frederico B. M. B.; Batista, Adelina B.; Lobo, Marina D. P.; Pereira, Mirella L.; Lima, João P. M. S.; Almeida, Ricardo V. M.; Sousa, Antônio J. S.; Monteiro-Moreira, Ana C. O.; Oliveira, José T. A.; Grangeiro, Thalles B.

2015-01-01

Mo-CBP3 is a chitin-binding protein from M. oleifera seeds that inhibits the germination and mycelial growth of phytopathogenic fungi. This protein is highly thermostable and resistant to pH changes, and therefore may be useful in the development of new antifungal drugs. However, the relationship of MoCBP3 with the known families of carbohydrate-binding domains has not been established. In the present study, full-length cDNAs encoding 4 isoforms of Mo-CBP3 (Mo-CBP3-1, Mo-CBP3-2, Mo-CBP3-3 and Mo-CBP3-4) were cloned from developing seeds. The polypeptides encoded by the Mo-CBP3 cDNAs were predicted to contain 160 (Mo-CBP3-3) and 163 amino acid residues (Mo-CBP3-1, Mo-CBP3-2 and Mo-CBP3-4) with a signal peptide of 20-residues at the N-terminal region. A comparative analysis of the deduced amino acid sequences revealed that Mo-CBP3 is a typical member of the 2S albumin family, as shown by the presence of an eight-cysteine motif, which is a characteristic feature of the prolamin superfamily. Furthermore, mass spectrometry analysis demonstrated that Mo-CBP3 is a mixture of isoforms that correspond to different mRNA products. The identification of Mo-CBP3 as a genuine member of the 2S albumin family reinforces the hypothesis that these seed storage proteins are involved in plant defense. Moreover, the chitin-binding ability of Mo-CBP3 reveals a novel functionality for a typical 2S albumin. PMID:25789746

SAM-VI RNAs selectively bind S-adenosylmethionine and exhibit similarities to SAM-III riboswitches.

PubMed

Mirihana Arachchilage, Gayan; Sherlock, Madeline E; Weinberg, Zasha; Breaker, Ronald R

2018-03-04

Five distinct riboswitch classes that regulate gene expression in response to the cofactor S-adenosylmethionine (SAM) or its metabolic breakdown product S-adenosylhomocysteine (SAH) have been reported previously. Collectively, these SAM- or SAH-sensing RNAs constitute the most abundant collection of riboswitches, and are found in nearly every major bacterial lineage. Here, we report a potential sixth member of this pervasive riboswitch family, called SAM-VI, which is predominantly found in Bifidobacterium species. SAM-VI aptamers selectively bind the cofactor SAM and strongly discriminate against SAH. The consensus sequence and structural model for SAM-VI share some features with the consensus model for the SAM-III riboswitch class, whose members are mainly found in lactic acid bacteria. However, there are sufficient differences between the two classes such that current bioinformatics methods separately cluster representatives of the two motifs. These findings highlight the abundance of RNA structures that can form to selectively recognize SAM, and showcase the ability of RNA to utilize diverse strategies to perform similar biological functions.

31Cl beta decay and the 30P31S reaction rate in nova nucleosynthesis

NASA Astrophysics Data System (ADS)

Bennett, Michael; Wrede, C.; Brown, B. A.; Liddick, S. N.; Pérez-Loureiro, D.; NSCL e12028 Collaboration

2016-03-01

The 30P31S reaction rate is critical for modeling the final isotopic abundances of ONe nova nucleosynthesis, identifying the origin of presolar nova grains, and calibrating proposed nova thermometers. Unfortunately, this rate is essentially experimentally unconstrained because the strengths of key 31S proton capture resonances are not known, due to uncertainties in their spins and parities. Using a 31Cl beam produced at the National Superconducting Cyclotron Laboratory, we have populated several 31S states for study via beta decay and devised a new decay scheme which includes updated beta feedings and gamma branchings as well as multiple states previously unobserved in 31Cl beta decay. Results of this study, including the unambiguous identification due to isospin mixing of a new l = 0 , Jπ = 3 /2+ 31S resonance directly in the middle of the Gamow Window, will be presented, and significance to the evaluation of the 30P31S reaction rate will be discussed. Work supported by U.S. Natl. Sci. Foundation (Grants No. PHY-1102511, PHY-1404442, PHY-1419765, and PHY-1431052); U.S. Dept. of Energy, Natl. Nucl. Security Administration (Award No. DE-NA0000979); Nat. Sci. and Eng. Research Council of Canada.

Synthesis and characterization of iodinated tetrahydroquinolines targeting the G protein-coupled estrogen receptor GPR30.

PubMed

Ramesh, Chinnasamy; Nayak, Tapan K; Burai, Ritwik; Dennis, Megan K; Hathaway, Helen J; Sklar, Larry A; Prossnitz, Eric R; Arterburn, Jeffrey B

2010-02-11

A series of iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines was synthesized as potential targeted imaging agents for the G protein-coupled estrogen receptor GPR30. The affinity and specificity of binding to GPR30 versus the classical estrogen receptors ER alpha/beta and functional responses associated with ligand-binding were determined. Selected iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines exhibited IC(50) values lower than 20 nM in competitive binding studies with GPR30-expressing human endometrial cancer cells. These compounds functioned as antagonists of GPR30 and blocked estrogen-induced PI3K activation and calcium mobilization. The tributylstannyl precursors of selected compounds were radiolabeled with (125)I using the iodogen method. In vivo biodistribution studies in female ovariectomized athymic (NCr) nu/nu mice bearing GPR30-expressing human endometrial tumors revealed GPR30-mediated uptake of the radiotracer ligands in tumor, adrenal, and reproductive organs. Biodistribution and quantitative SPECT/CT studies revealed structurally related differences in the pharmacokinetic profiles, target tissue uptake, and metabolism of the radiolabeled compounds as well as differences in susceptibility to deiodination. The high lipophilicity of the compounds adversely affects the in vivo biodistribution and clearance of these radioligands and suggests that further optimization of this parameter may lead to improved targeting characteristics.

Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein

NASA Technical Reports Server (NTRS)

Yang, T.; Poovaiah, B. W.

2000-01-01

Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis. Copyright 2000 Academic Press.

In silico analysis of AHJD-like viruses, Staphylococcus aureus phages S24-1 and S13′, and study of phage S24-1 adsorption

PubMed Central

Uchiyama, Jumpei; Takemura-Uchiyama, Iyo; Kato, Shin-ichiro; Sato, Miho; Ujihara, Takako; Matsui, Hidehito; Hanaki, Hideaki; Daibata, Masanori; Matsuzaki, Shigenobu

2014-01-01

Staphylococcus aureus is a clinically important bacterium that is commensal in both humans and animals. Bacteriophage (phage) attachment to the host bacterial surface is an important process during phage infection, which involves interactions between phage receptor-binding proteins and host receptor molecules. However, little information is available on the receptor-binding protein of S. aureus phages. S. aureus virulent phages S24-1 and S13′ (family Podoviridae, genus AHJD-like viruses) were isolated from sewage. In the present study, we investigated the receptor-binding protein of AHJD-like viruses using phage S24-1. First, based on a comparative genomic analysis of phages S24-1 and S13′, open reading frame 16 (ORF16) of phage S24-1 was speculated to be the receptor-binding protein, which possibly determines the host range. Second, we demonstrated that this was the receptor-binding protein of phage S24-1. Third, our study suggested that wall teichoic acids in the cell walls of S. aureus are the main receptor molecules for ORF16 and phage S24-1. Finally, the C-terminal region of ORF16 may be essential for binding to S. aureus. These results strongly suggest that ORF16 of phage S24-1 and its homologs may be the receptor-binding proteins of AHJD-like viruses. PMID:24591378

Crystallization and preliminary X-ray diffraction analysis of the phosphate-binding protein PhoX from Xanthomonas citri

PubMed Central

Pegos, Vanessa R.; Medrano, Francisco Javier; Balan, Andrea

2014-01-01

Xanthomonas axonopodis pv. citri (X. citri) is an important bacterium that causes citrus canker disease in plants in Brazil and around the world, leading to significant economic losses. Determination of the physiology and mechanisms of pathogenesis of this bacterium is an important step in the development of strategies for its containment. Phosphate is an essential ion in all microrganisms owing its importance during the synthesis of macromolecules and in gene and protein regulation. Interestingly, X. citri has been identified to present two periplasmic binding proteins that have not been further characterized: PstS, from an ATP-binding cassette for high-affinity uptake and transport of phosphate, and PhoX, which is encoded by an operon that also contains a putative porin for the transport of phosphate. Here, the expression, purification and crystallization of the phosphate-binding protein PhoX and X-ray data collection at 3.0 Å resolution are described. Biochemical, biophysical and structural data for this protein will be helpful in the elucidation of its function in phosphate uptake and the physiology of the bacterium. PMID:25484207

Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor

PubMed Central

Burvenich, Ingrid J. G.; Farrugia, William; Lee, Fook T.; Catimel, Bruno; Liu, Zhanqi; Makris, Dahna; Cao, Diana; O'Keefe, Graeme J.; Brechbiel, Martin W.; King, Dylan; Spirkoska, Violeta; Allan, Laura C.; Ramsland, Paul A.; Scott, Andrew M.

2016-01-01

ABSTRACT IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic. PMID:27030023

Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

DOE PAGES

Chen, Qi; Luan, Zheng-Jiao; Cheng, Xiaolin; ...

2015-02-25

A recombinant carboxylesterase, cloned from Pseudomonas putida and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2 -chlorophenyl)acetate (rac-AcO-CPA) with excellent (S)-enantioselectivity. Semi-rational design of the enzyme showed that the W187H variant could increase the activity by ~100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (S)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Moreover, our results show differential binding of (S)-AcO-CPA in the WT and W187H enzymes, especially the interactions of themore » substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. Based on these computational results, furthermore, we predicted that the mutations W187Y and D287G might also be able to increase the substrate binding, thus improve the enzyme s catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100 fold increase in turn-over rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turn-over rate. Finally, our work provides important molecular-level insights into the binding of the (S)-AcO-CPA substrate to carboxylesterase r

Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

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

Chen, Qi; Luan, Zheng-Jiao; Cheng, Xiaolin

A recombinant carboxylesterase, cloned from Pseudomonas putida and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2 -chlorophenyl)acetate (rac-AcO-CPA) with excellent (S)-enantioselectivity. Semi-rational design of the enzyme showed that the W187H variant could increase the activity by ~100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (S)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Moreover, our results show differential binding of (S)-AcO-CPA in the WT and W187H enzymes, especially the interactions of themore » substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. Based on these computational results, furthermore, we predicted that the mutations W187Y and D287G might also be able to increase the substrate binding, thus improve the enzyme s catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100 fold increase in turn-over rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turn-over rate. Finally, our work provides important molecular-level insights into the binding of the (S)-AcO-CPA substrate to carboxylesterase r

ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR-573 binding.

PubMed

Wang, Qiong; Hui, Haipeng; Guo, Zhendong; Zhang, Weina; Hu, Yaou; He, Tao; Tai, Yanhong; Peng, Peng; Wang, Li

2013-11-01

Rho GTPase activating protein 26 (ARHGAP26) is a negative regulator of the Rho family that converts the small G proteins RhoA and Cdc42 to their inactive GDP-bound forms. It is essential for the CLIC/GEEC endocytic pathway, cell spreading, and muscle development. The present study shows that ARHGAP26 mRNA undergoes extensive A-to-I RNA editing in the 3' UTR that is specifically catalyzed by ADAR1. Furthermore, the mRNA and protein levels of ARHGAP26 were decreased in cells in which ADAR1 was knocked down. Conversely, ADAR1 overexpression increased the abundance of ARHGAP26 mRNA and protein. In addition, we found that both miR-30b-3p and miR-573 target the ARHGAP26 gene and that RNA editing of ARHGAP26 mediated by ADAR1 abolished the repression of its expression by miR-30b-3p or miR-573. When ADAR1 was overexpressed, the reduced abundance of ARHGAP26 protein mediated by miR-30b-3p or miR-573 was rescued. Importantly, we also found that knocking down ADAR1 elevated RhoA activity, which was consistent with the reduced level of ARHGAP26. Conversely, when ADAR1 was overexpressed, the amount of RhoA-GTP decreased. The similar expression patterns of ARHGAP26 and ADAR1 in human tissue samples further confirmed our findings. Taken together, our results suggest that ADAR1 regulates the expression of ARHGAP26 through A-to-I RNA editing by disrupting the binding of miR-30b-3p and miR-573 within the 3' UTR of ARHGAP26. This study provides a novel insight into the mechanism by which ADAR1 and its RNA editing function regulate microRNA-mediated modulation of target genes.

30 CFR 550.215 - What hydrogen sulfide (H2S) information must accompany the EP?

Code of Federal Regulations, 2013 CFR

2013-07-01

... 30 Mineral Resources 2 2013-07-01 2013-07-01 false What hydrogen sulfide (H2S) information must accompany the EP? 550.215 Section 550.215 Mineral Resources BUREAU OF OCEAN ENERGY MANAGEMENT, DEPARTMENT OF... Information Contents of Exploration Plans (ep) § 550.215 What hydrogen sulfide (H2S) information must...

30 CFR 550.215 - What hydrogen sulfide (H2S) information must accompany the EP?

Code of Federal Regulations, 2012 CFR

2012-07-01

... 30 Mineral Resources 2 2012-07-01 2012-07-01 false What hydrogen sulfide (H2S) information must accompany the EP? 550.215 Section 550.215 Mineral Resources BUREAU OF OCEAN ENERGY MANAGEMENT, DEPARTMENT OF... Information Contents of Exploration Plans (ep) § 550.215 What hydrogen sulfide (H2S) information must...

30 CFR 550.215 - What hydrogen sulfide (H2S) information must accompany the EP?

Code of Federal Regulations, 2014 CFR

2014-07-01

... 30 Mineral Resources 2 2014-07-01 2014-07-01 false What hydrogen sulfide (H2S) information must accompany the EP? 550.215 Section 550.215 Mineral Resources BUREAU OF OCEAN ENERGY MANAGEMENT, DEPARTMENT OF... Information Contents of Exploration Plans (ep) § 550.215 What hydrogen sulfide (H2S) information must...

Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

USDA-ARS?s Scientific Manuscript database

Background: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to better u...

γ-Secretase binding sites in aged and Alzheimer’s disease human cerebrum: The choroid plexus as a putative origin of CSF Aβ

PubMed Central

Liu, Fei; Xue, Zhi-Qin; Deng, Si-Hao; Kun, Xiong; Luo, Xue-Gang; Patrylo, Peter R.; Rose, Gregory M.; Cai, Huaibin; Struble, Robert G.; Cai, Yan; Yan, Xiao-Xin

2013-01-01

Deposition of β-amyloid (Aβ) peptides, cleavage products of β-amyloid precursor protein (APP) by β-secretase-1 (BACE1) and γ-secretase, is a neuropathological hallmark of Alzheimer’s disease (AD). γ-Secretase inhibition is a therapeutical anti-Aβ approach, although less is clear about the change of the enzyme’s activity in AD brain. Cerebrospinal fluid (CSF) Aβ peptides are considered to derive from brain parenchyma, thus may serve as biomarkers for assessing cerebral amyloidosis and anti-Aβ efficacy. The present study compared active γ-secretase binding sites with Aβ deposition in aged and AD human cerebrum, and explored a possibility of Aβ production and secretion by the choroid plexus (CP). Specific binding density of [3H]-L-685,458, a radiolabeled high affinity γ-secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with comparable ages and postmortem delays. The CP in postmortem samples exhibited exceptionally high [3H]-L-685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin-1 immunoreactivity, and β-site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins but released Aβ40 and Aβ42 into the medium. These results suggest that γ-secretase activity appears not altered in the cerebrum in AD related to aged control, nor correlated with regional amyloid plaque pathology. The choroid plexus appears to represent a novel non-neuronal source in the brain that may contribute Aβ into cerebrospinal fluid, probably at reduced levels in AD. PMID:23432732

Molecular interactions between single-stranded DNA-binding proteins associated with an essential MCAT element in the mouse smooth muscle alpha-actin promoter.

PubMed

Kelm, R J; Cogan, J G; Elder, P K; Strauch, A R; Getz, M J

1999-05-14

Transcriptional activity of the mouse vascular smooth muscle alpha-actin gene in fibroblasts is regulated, in part, by a 30-base pair asymmetric polypurine-polypyrimidine tract containing an essential MCAT enhancer motif. The double-stranded form of this sequence serves as a binding site for a transcription enhancer factor 1-related protein while the separated single strands interact with two distinct DNA binding activities termed VACssBF1 and 2 (Cogan, J. G., Sun, S., Stoflet, E. S., Schmidt, L. J., Getz, M. J., and Strauch, A. R. (1995) J. Biol. Chem. 270, 11310-11321; Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2936). VACssBF2 has been recently cloned and shown to consist of two closely related proteins, Puralpha and Purbeta (Kelm, R. J., Elder, P. K., Strauch, A. R., and Getz, M. J. (1997) J. Biol. Chem. 272, 26727-26733). In this study, we demonstrate that Puralpha and Purbeta interact with each other via highly specific protein-protein interactions and bind to the purine-rich strand of the MCAT enhancer in the form of both homo- and heteromeric complexes. Moreover, both Pur proteins interact with MSY1, a VACssBF1-like protein cloned by virtue of its affinity for the pyrimidine-rich strand of the enhancer. Interactions between Puralpha, Purbeta, and MSY1 do not require the participation of DNA. Combinatorial interactions between these three single-stranded DNA-binding proteins may be important in regulating activity of the smooth muscle alpha-actin MCAT enhancer in fibroblasts.

Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

PubMed

Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

2015-09-11

Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

Color-motion feature-binding errors are mediated by a higher-order chromatic representation

PubMed Central

Shevell, Steven K.; Wang, Wei

2017-01-01

Peripheral and central moving objects of the same color may be perceived to move in the same direction even though peripheral objects have a different true direction of motion [Nature 429, 262 (2004)]. The perceived, illusory direction of peripheral motion is a color-motion feature-binding error. Recent work shows that such binding errors occur even without an exact color match between central and peripheral objects, and, moreover, the frequency of the binding errors in the periphery declines as the chromatic difference increases between the central and peripheral objects [J. Opt. Soc. Am. A 31, A60 (2014)]. This change in the frequency of binding errors with the chromatic difference raises the general question of the chromatic representation from which the difference is determined. Here, basic properties of the chromatic representation are tested to discover whether it depends on independent chromatic differences on the l and the s cardinal axes or, alternatively, on a more specific higher-order chromatic representation. Experimental tests compared the rate of feature-binding errors when the central and peripheral colors had the identical s chromaticity (so zero difference in s) and a fixed magnitude of l difference, while varying the identical s level in center and periphery (thus always keeping the s difference at zero). A chromatic representation based on independent l and s differences would result in the same frequency of color-motion binding errors at every s level. The results are contrary to this prediction, thus showing that the chromatic representation at the level of color-motion feature binding depends on a higherorder chromatic mechanism. PMID:26974945

The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.

PubMed

Kerkhoff, Claus; Nacken, Wolfgang; Benedyk, Malgorzata; Dagher, Marie Claire; Sopalla, Claudia; Doussiere, Jacques

2005-03-01

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

30 CFR 250.215 - What hydrogen sulfide (H2S) information must accompany the EP?

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 2 2010-07-01 2010-07-01 false What hydrogen sulfide (H2S) information must accompany the EP? 250.215 Section 250.215 Mineral Resources MINERALS MANAGEMENT SERVICE, DEPARTMENT OF THE... Contents of Exploration Plans (ep) § 250.215 What hydrogen sulfide (H2S) information must accompany the EP...

30. BUILDING NO.S 271K AND 271L, VIEW LOOKING SOUTH AT ...