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Sample records for binding modulate ataxin-1

  1. FOX-2 dependent splicing of ataxin-2 transcript is affected by ataxin-1 overexpression.

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    Franziska Welzel

    Full Text Available Alternative splicing is a fundamental posttranscriptional mechanism for controlling gene expression, and splicing defects have been linked to various human disorders. The splicing factor FOX-2 is part of a main protein interaction hub in a network related to human inherited ataxias, however, its impact remains to be elucidated. Here, we focused on the reported interaction between FOX-2 and ataxin-1, the disease-causing protein in spinocerebellar ataxia type 1. In this line, we further evaluated this interaction by yeast-2-hybrid analyses and co-immunoprecipitation experiments in mammalian cells. Interestingly, we discovered that FOX-2 localization and splicing activity is affected in the presence of nuclear ataxin-1 inclusions. Moreover, we observed that FOX-2 directly interacts with ataxin-2, a protein modulating spinocerebellar ataxia type 1 pathogenesis. Finally, we provide evidence that splicing of pre-mRNA of ataxin-2 depends on FOX-2 activity, since reduction of FOX-2 levels led to increased skipping of exon 18 in ataxin-2 transcripts. Most striking, we observed that ataxin-1 overexpression has an effect on this splicing event as well. Thus, our results demonstrate that FOX-2 is involved in splicing of ataxin-2 transcripts and that this splicing event is altered by overexpression of ataxin-1.

  2. Polyglutamine expansion accelerates the dynamics of ataxin-1 and does not result in aggregate formation.

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    Hilde A Krol

    Full Text Available BACKGROUND: Polyglutamine expansion disorders are caused by an expansion of the polyglutamine (polyQ tract in the disease related protein, leading to severe neurodegeneration. All polyQ disorders are hallmarked by the presence of intracellular aggregates containing the expanded protein in affected neurons. The polyQ disorder SpinoCerebellar Ataxia 1 (SCA1 is caused by a polyQ-expansion in the ataxin-1 protein, which is thought to lead to nuclear aggregates. METHODOLOGY/PRINCIPAL FINDINGS: Using advanced live cell fluorescence microscopy and a filter retardation assay we show that nuclear accumulations formed by polyQ-expanded ataxin-1 do not resemble aggregates of other polyQ-expanded proteins. Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1. During mitosis, ataxin-1 accumulations redistributed equally among daughter cells, in contrast to polyQ aggregates. Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before. CONCLUSIONS/SIGNIFICANCE: These results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1. The unexpected findings for a polyQ-expanded protein and their consequences for ongoing SCA1 research are discussed.

  3. Identification of human proteins that modify misfolding and proteotoxicity of pathogenic ataxin-1.

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    Spyros Petrakis

    Full Text Available Proteins with long, pathogenic polyglutamine (polyQ sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1-interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.

  4. Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures

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    Skinner, P J; Koshy, B T; Cummings, C J

    1997-01-01

    Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative disorders caused by an expansion of a polyglutamine tract. It is characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells. To understand the pathogenesis of SCA1, we examined.......5 microm across, whereas the expanded ataxin-1 localizes to a single approximately 2-microm structure, before the onset of ataxia. Mutant ataxin-1 localizes to a single nuclear structure in affected neurons of SCA1 patients. Similarly, COS-1 cells transfected with wild-type or mutant ataxin-1 show...

  5. dAtaxin-2 mediates expanded Ataxin-1-induced neurodegeneration in a Drosophila model of SCA1.

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    Ismael Al-Ramahi

    2007-12-01

    Full Text Available Spinocerebellar ataxias (SCAs are a genetically heterogeneous group of neurodegenerative disorders sharing atrophy of the cerebellum as a common feature. SCA1 and SCA2 are two ataxias caused by expansion of polyglutamine tracts in Ataxin-1 (ATXN1 and Ataxin-2 (ATXN2, respectively, two proteins that are otherwise unrelated. Here, we use a Drosophila model of SCA1 to unveil molecular mechanisms linking Ataxin-1 with Ataxin-2 during SCA1 pathogenesis. We show that wild-type Drosophila Ataxin-2 (dAtx2 is a major genetic modifier of human expanded Ataxin-1 (Ataxin-1[82Q] toxicity. Increased dAtx2 levels enhance, and more importantly, decreased dAtx2 levels suppress Ataxin-1[82Q]-induced neurodegeneration, thereby ruling out a pathogenic mechanism by depletion of dAtx2. Although Ataxin-2 is normally cytoplasmic and Ataxin-1 nuclear, we show that both dAtx2 and hAtaxin-2 physically interact with Ataxin-1. Furthermore, we show that expanded Ataxin-1 induces intranuclear accumulation of dAtx2/hAtaxin-2 in both Drosophila and SCA1 postmortem neurons. These observations suggest that nuclear accumulation of Ataxin-2 contributes to expanded Ataxin-1-induced toxicity. We tested this hypothesis engineering dAtx2 transgenes with nuclear localization signal (NLS and nuclear export signal (NES. We find that NLS-dAtx2, but not NES-dAtx2, mimics the neurodegenerative phenotypes caused by Ataxin-1[82Q], including repression of the proneural factor Senseless. Altogether, these findings reveal a previously unknown functional link between neurodegenerative disorders with common clinical features but different etiology.

  6. Mapping the self-association domains of ataxin-1: identification of novel non overlapping motifs

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    Rajesh P. Menon

    2014-03-01

    Full Text Available The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1 is caused by aggregation and misfolding of the ataxin-1 protein. While the pathology correlates with mutations that lead to expansion of a polyglutamine tract in the protein, other regions contribute to the aggregation process as also non-expanded ataxin-1 is intrinsically aggregation-prone and forms nuclear foci in cell. Here, we have used a combined approach based on FRET analysis, confocal microscopy and in vitro techniques to map aggregation-prone regions other than polyglutamine and to establish the importance of dimerization in self-association/foci formation. Identification of aggregation-prone regions other than polyglutamine could greatly help the development of SCA1 treatment more specific than that based on targeting the low complexity polyglutamine region.

  7. Ataxin1L is a regulator of HSC function highlighting the utility of cross-tissue comparisons for gene discovery.

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    Kahle, Juliette J; Souroullas, George P; Yu, Peng; Zohren, Fabian; Lee, Yoontae; Shaw, Chad A; Zoghbi, Huda Y; Goodell, Margaret A

    2013-03-01

    Hematopoietic stem cells (HSCs) are rare quiescent cells that continuously replenish the cellular components of the peripheral blood. Observing that the ataxia-associated gene Ataxin-1-like (Atxn1L) was highly expressed in HSCs, we examined its role in HSC function through in vitro and in vivo assays. Mice lacking Atxn1L had greater numbers of HSCs that regenerated the blood more quickly than their wild-type counterparts. Molecular analyses indicated Atxn1L null HSCs had gene expression changes that regulate a program consistent with their higher level of proliferation, suggesting that Atxn1L is a novel regulator of HSC quiescence. To determine if additional brain-associated genes were candidates for hematologic regulation, we examined genes encoding proteins from autism- and ataxia-associated protein-protein interaction networks for their representation in hematopoietic cell populations. The interactomes were found to be highly enriched for proteins encoded by genes specifically expressed in HSCs relative to their differentiated progeny. Our data suggest a heretofore unappreciated similarity between regulatory modules in the brain and HSCs, offering a new strategy for novel gene discovery in both systems.

  8. Ataxin1L is a regulator of HSC function highlighting the utility of cross-tissue comparisons for gene discovery.

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    Juliette J Kahle

    2013-03-01

    Full Text Available Hematopoietic stem cells (HSCs are rare quiescent cells that continuously replenish the cellular components of the peripheral blood. Observing that the ataxia-associated gene Ataxin-1-like (Atxn1L was highly expressed in HSCs, we examined its role in HSC function through in vitro and in vivo assays. Mice lacking Atxn1L had greater numbers of HSCs that regenerated the blood more quickly than their wild-type counterparts. Molecular analyses indicated Atxn1L null HSCs had gene expression changes that regulate a program consistent with their higher level of proliferation, suggesting that Atxn1L is a novel regulator of HSC quiescence. To determine if additional brain-associated genes were candidates for hematologic regulation, we examined genes encoding proteins from autism- and ataxia-associated protein-protein interaction networks for their representation in hematopoietic cell populations. The interactomes were found to be highly enriched for proteins encoded by genes specifically expressed in HSCs relative to their differentiated progeny. Our data suggest a heretofore unappreciated similarity between regulatory modules in the brain and HSCs, offering a new strategy for novel gene discovery in both systems.

  9. Suppression of the novel ER protein Maxer by mutant ataxin-1 in Bergman glia contributes to non-cell-autonomous toxicity

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    Shiwaku, Hiroki; Yoshimura, Natsue; Tamura, Takuya; Sone, Masaki; Ogishima, Soichi; Watase, Kei; Tagawa, Kazuhiko; Okazawa, Hitoshi

    2010-01-01

    Non-cell-autonomous effect of mutant proteins expressed in glia has been implicated in several neurodegenerative disorders, whereas molecules mediating the toxicity are currently not known. We identified a novel molecule named multiple α-helix protein located at ER (Maxer) downregulated by mutant ataxin-1 (Atx1) in Bergmann glia. Maxer is an endoplasmic reticulum (ER) membrane protein interacting with CDK5RAP3. Maxer anchors CDK5RAP3 to the ER and inhibits its function of Cyclin D1 transcript...

  10. Systematic analysis of fly models with multiple drivers reveals different effects of ataxin-1 and huntingtin in neuron subtype-specific expression.

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    Risa Shiraishi

    Full Text Available The fruit fly, Drosophila melanogaster, is a commonly used model organism for neurodegenerative diseases. Its major advantages include a short lifespan and its susceptibility to manipulation using sophisticated genetic techniques. Here, we report the systematic comparison of fly models of two polyglutamine (polyQ diseases. We induced expression of the normal and mutant forms of full-length Ataxin-1 and Huntingtin exon 1 in cholinergic, dopaminergic, and motor neurons, and glial cells using cell type-specific drivers. We systematically analyzed their effects based on multiple phenotypes: eclosion rate, lifespan, motor performance, and circadian rhythms of spontaneous activity. This systematic assay system enabled us to quantitatively evaluate and compare the functional disabilities of different genotypes. The results suggest different effects of Ataxin-1 and Huntingtin on specific types of neural cells during development and in adulthood. In addition, we confirmed the therapeutic effects of LiCl and butyrate using representative models. These results support the usefulness of this assay system for screening candidate chemical compounds that modify the pathologies of polyQ diseases.

  11. Using structure to inform carbohydrate binding module function

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    Abbott, D. Wade; Lammerts van Bueren, Alicia

    2014-01-01

    Generally, non-catalytic carbohydrate binding module (CBM) specificity has been shown to parallel the catalytic activity of the carbohydrate active enzyme (CAZyme) module it is appended to. With the rapid expansion in metagenomic sequence space for the potential discovery of new CBMs in addition to

  12. Suppression of the novel ER protein Maxer by mutant ataxin-1 in Bergman glia contributes to non-cell-autonomous toxicity.

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    Shiwaku, Hiroki; Yoshimura, Natsue; Tamura, Takuya; Sone, Masaki; Ogishima, Soichi; Watase, Kei; Tagawa, Kazuhiko; Okazawa, Hitoshi

    2010-07-21

    Non-cell-autonomous effect of mutant proteins expressed in glia has been implicated in several neurodegenerative disorders, whereas molecules mediating the toxicity are currently not known. We identified a novel molecule named multiple alpha-helix protein located at ER (Maxer) downregulated by mutant ataxin-1 (Atx1) in Bergmann glia. Maxer is an endoplasmic reticulum (ER) membrane protein interacting with CDK5RAP3. Maxer anchors CDK5RAP3 to the ER and inhibits its function of Cyclin D1 transcription repression in the nucleus. The loss of Maxer eventually induces cell accumulation at G1 phase. It was also shown that mutant Atx1 represses Maxer and inhibits proliferation of Bergmann glia in vitro. Consistently, Bergmann glia are reduced in the cerebellum of mutant Atx1 knockin mice before onset. Glutamate-aspartate transporter reduction in Bergmann glia by mutant Atx1 and vulnerability of Purkinje cell to glutamate are both strengthened by Maxer knockdown in Bergmann glia, whereas Maxer overexpression rescues them. Collectively, these results suggest that the reduction of Maxer mediates functional deficiency of Bergmann glia, and might contribute to the non-cell-autonomous pathology of SCA1.

  13. Direct and accurate measurement of CAG repeat configuration in the ataxin-1 (ATXN-1) gene by "dual-fluorescence labeled PCR-restriction fragment length analysis".

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    Lin, Jiang X; Ishikawa, Kinya; Sakamoto, Masaki; Tsunemi, Taiji; Ishiguro, Taro; Amino, Takeshi; Toru, Shuta; Kondo, Ikuko; Mizusawa, Hidehiro

    2008-01-01

    Spinocerebellar ataxia type 1 (SCA1; OMIM: #164400) is an autosomal dominant cerebellar ataxia caused by an expansion of CAG repeat, which encodes polyglutamine, in the ataxin-1 (ATXN1) gene. Length of polyglutamine in the ATXN1 protein is the critical determinant of pathogenesis of this disease. Molecular diagnosis of SCA1 is usually undertaken by assessing the length of CAG repeat configuration using primers spanning this configuration. However, this conventional method may potentially lead to misdiagnosis in assessing polyglutamine-encoding CAG repeat length, since CAT interruptions may be present within the CAG repeat configuration, not only in normal controls but also in neurologically symptomatic subjects. We developed a new method for assessing actual CAG repeat numbers not interrupted by CAT sequences. Polymerase chain reaction using a primer pair labeled with two different fluorescences followed by restriction enzyme digestion with SfaNI which recognizes the sequence "GCATC(N)(5)", lengths of actual CAG repeats that encode polyglutamine were directly detected. We named this method "dual fluorescence labeled PCR-restriction fragment length analysis". We found that numbers of actual CAG repeat encoding polyglutamine do not overlap between our cohorts of normal chromosomes (n=385) and SCA1 chromosomes (n=5). We conclude that the present method is a useful way for molecular diagnosis of SCA1.

  14. Modeling of Carbohydrate Binding Modules Complexed to Cellulose

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    Nimlos, M. R.; Beckham, G. T.; Bu, L.; Himmel, M. E.; Crowley, M. F.; Bomble, Y. J.

    2012-01-01

    Modeling results are presented for the interaction of two carbohydrate binding modules (CBMs) with cellulose. The family 1 CBM from Trichoderma reesei's Cel7A cellulase was modeled using molecular dynamics to confirm that this protein selectively binds to the hydrophobic (100) surface of cellulose fibrils and to determine the energetics and mechanisms for locating this surface. Modeling was also conducted of binding of the family 4 CBM from the CbhA complex from Clostridium thermocellum. There is a cleft in this protein, which may accommodate a cellulose chain that is detached from crystalline cellulose. This possibility is explored using molecular dynamics.

  15. Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

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    Mykol Larvie

    2012-01-01

    Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

  16. Glial cell lineage expression of mutant ataxin-1 and huntingtin induces developmental and late-onset neuronal pathologies in Drosophila models.

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    Takuya Tamura

    Full Text Available BACKGROUND: In several neurodegenerative disorders, toxic effects of glial cells on neurons are implicated. However the generality of the non-cell autonomous pathologies derived from glial cells has not been established, and the specificity among different neurodegenerative disorders remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We newly generated Drosophila models expressing human mutant huntingtin (hHtt103Q or ataxin-1 (hAtx1-82Q in the glial cell lineage at different stages of differentiation, and analyzed their morphological and behavioral phenotypes. To express hHtt103Q and hAtx1-82Q, we used 2 different Gal4 drivers, gcm-Gal4 and repo-Gal4. Gcm-Gal4 is known to be a neuroglioblast/glioblast-specific driver whose effect is limited to development. Repo-Gal4 is known to be a pan-glial driver and the expression starts at glioblasts and continues after terminal differentiation. Gcm-Gal4-induced hHtt103Q was more toxic than repo-Gal4-induced hHtt103Q from the aspects of development, locomotive activity and survival of flies. When hAtx1-82Q was expressed by gcm- or repo-Gal4 driver, no fly became adult. Interestingly, the head and brain sizes were markedly reduced in a part of pupae expressing hAtx1-82Q under the control of gcm-Gal4, and these pupae showed extreme destruction of the brain structure. The other pupae expressing hAtx1-82Q also showed brain shrinkage and abnormal connections of neurons. These results suggested that expression of polyQ proteins in neuroglioblasts provided a remarkable effect on the developmental and adult brains, and that glial cell lineage expression of hAtx1-82Q was more toxic than that of hHtt103Q in our assays. CONCLUSION/SIGNIFICANCE: All these studies suggested that the non-cell autonomous effect of glial cells might be a common pathology shared by multiple neurodegenerative disorders. In addition, the fly models would be available for analyzing molecular pathologies and developing novel therapeutics against

  17. A new zinc binding fold underlines the versatility of zinc binding modules in protein evolution.

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    Sharpe, Belinda K; Matthews, Jacqueline M; Kwan, Ann H Y; Newton, Anthea; Gell, David A; Crossley, Merlin; Mackay, Joel P

    2002-05-01

    Many different zinc binding modules have been identified. Their abundance and variety suggests that the formation of zinc binding folds might be relatively common. We have determined the structure of CH1(1), a 27-residue peptide derived from the first cysteine/histidine-rich region (CH1) of CREB binding protein (CBP). This peptide forms a highly ordered zinc-dependent fold that is distinct from known folds. The structure differs from a subsequently determined structure of a larger region from the CH3 region of CBP, and the CH1(1) fold probably represents a nonphysiologically active form. Despite this, the fold is thermostable and tolerant to both multiple alanine mutations and changes in the zinc-ligand spacing. Our data support the idea that zinc binding domains may arise frequently. Additionally, such structures may prove useful as scaffolds for protein design, given their stability and robustness.

  18. Modulation of RNase E activity by alternative RNA binding sites.

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    Daeyoung Kim

    Full Text Available Endoribonuclease E (RNase E affects the composition and balance of the RNA population in Escherichia coli via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 (24LYDLDIESPGHEQK37 of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne that was UV crosslinked with a 5'-32P-end-labeled, 13-nt oligoribonucleotide (p-BR13 containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 (427LIEEEALK433, whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (65HGFLPL*K71. Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E in vivo. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.

  19. Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

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    Shoseyov, Oded; Shani, Ziv; Levy, Ilan

    2006-01-01

    Polysaccharide-degrading microorganisms express a repertoire of hydrolytic enzymes that act in synergy on plant cell wall and other natural polysaccharides to elicit the degradation of often-recalcitrant substrates. These enzymes, particularly those that hydrolyze cellulose and hemicellulose, have a complex molecular architecture comprising discrete modules which are normally joined by relatively unstructured linker sequences. This structure is typically comprised of a catalytic module and one or more carbohydrate binding modules (CBMs) that bind to the polysaccharide. CBMs, by bringing the biocatalyst into intimate and prolonged association with its substrate, allow and promote catalysis. Based on their properties, CBMs are grouped into 43 families that display substantial variation in substrate specificity, along with other properties that make them a gold mine for biotechnologists who seek natural molecular “Velcro” for diverse and unusual applications. In this article, we review recent progress in the field of CBMs and provide an up-to-date summary of the latest developments in CBM applications. PMID:16760304

  20. Capacitance-modulated transistor detects odorant binding protein chiral interactions

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    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.

  1. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

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    Lummis, S.C.R.; Johnston, G.A.R. (Univ. of Sydney, New South Wales (Australia)); Nicoletti, G. (Royal Melbourne Inst. of Tech. (Australia)); Holan, G. (CSIRO, Melbourne (Australia))

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial ({sup 3}H)diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli ({sup 3}H)diazepam binding are those that are active in displacing ({sup 3}H)benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed.

  2. Copper at synapse: Release, binding and modulation of neurotransmission.

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    D'Ambrosi, Nadia; Rossi, Luisa

    2015-11-01

    Over the last decade, a piece of the research studying copper role in biological systems was devoted to unravelling a still elusive, but extremely intriguing, aspect that is the involvement of copper in synaptic function. These studies were prompted to provide a rationale to the finding that copper is released in the synaptic cleft upon depolarization. The copper pump ATP7A, which mutations are responsible for diseases with a prominent neurodegenerative component, seems to play a pivotal role in the release of copper at synapses. Furthermore, it was found that, when in the synaptic cleft, copper can control, directly or indirectly, the activity of the neurotransmitter receptors (NMDA, AMPA, GABA, P2X receptors), thus affecting excitability. In turn, neurotransmission can affect copper trafficking and delivery in neuronal cells. Furthermore, it was reported that copper can also modulate synaptic vesicles trafficking and the interaction between proteins of the secretory pathways. Interestingly, proteins with a still unclear role in neuronal system though associated with the pathogenesis of neurodegenerative diseases (the amyloid precursor protein, APP, the prion protein, PrP, α-synuclein, α-syn) show copper-binding domains. They may act as copper buffer at synapses and participate in the interplay between copper and the neurotransmitters receptors. Given that copper dysmetabolism occurs in several diseases affecting central and peripheral nervous system, the findings on the contribution of copper in synaptic transmission, beside its more consolidate role as a neuronal enzymes cofactor, may open new insights for therapy interventions.

  3. Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

    DEFF Research Database (Denmark)

    Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska;

    2016-01-01

    γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently...

  4. Binding Preferences, Surface Attachment, Diffusivity, and Orientation of a Family 1 Carbohydrate-Binding Module on Cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Nimlos, M. R.; Beckham, G. T.; Matthews, J. F.; Bu, L.; Himmel, M. E.; Crowley, M. F.

    2012-06-08

    Cellulase enzymes often contain carbohydrate-binding modules (CBMs) for binding to cellulose. The mechanisms by which CBMs recognize specific surfaces of cellulose and aid in deconstruction are essential to understand cellulase action. The Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase, Cel7A, is known to selectively bind to hydrophobic surfaces of native cellulose. It is most commonly suggested that three aromatic residues identify the planar binding face of this CBM, but several recent studies have challenged this hypothesis. Here, we use molecular simulation to study the CBM binding orientation and affinity on hydrophilic and hydrophobic cellulose surfaces. Roughly 43 {mu}s of molecular dynamics simulations were conducted, which enables statistically significant observations. We quantify the fractions of the CBMs that detach from crystal surfaces or diffuse to other surfaces, the diffusivity along the hydrophobic surface, and the overall orientation of the CBM on both hydrophobic and hydrophilic faces. The simulations demonstrate that there is a thermodynamic driving force for the Cel7A CBM to bind preferentially to the hydrophobic surface of cellulose relative to hydrophilic surfaces. In addition, the simulations demonstrate that the CBM can diffuse from hydrophilic surfaces to the hydrophobic surface, whereas the reverse transition is not observed. Lastly, our simulations suggest that the flat faces of Family 1 CBMs are the preferred binding surfaces. These results enhance our understanding of how Family 1 CBMs interact with and recognize specific cellulose surfaces and provide insights into the initial events of cellulase adsorption and diffusion on cellulose.

  5. Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-Binding Module

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    Taylor, C. B.; Talib, M. F.; McCabe, C.; Bu, L.; Adney, W. S.; Himmel, M. E.; Crowley, M. F.; Beckham, G. T.

    2012-01-27

    Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3-6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function.

  6. Two DNA-binding and Nick Recognition Modules in Human DNA Ligase III*

    OpenAIRE

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Tomkinson, Alan E.; Ellenberger, Tom

    2008-01-01

    Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is not required for DNA nick joining activity in vitro. In light of new structural information for mammalian ligases, we measured the DNA binding affinity and specificity of each domain of DNA ligase III. These studies identified two separate, independent DNA-binding modules in DNA ligase III that each bin...

  7. A CESA from Griffithsia monilis (Rhodophyta, Florideophyceae) has a family 48 carbohydrate-binding module.

    Science.gov (United States)

    Matthews, Peter R; Schindler, Michael; Howles, Paul; Arioli, Tony; Williamson, Richard E

    2010-10-01

    Cellulose synthases form rosette terminal complexes in the plasma membranes of Streptophyta and various linear terminal complexes in other taxa. The sequence of a putative CESA from Griffithsia monilis (Rhodophyta, Floridiophyceae) was deduced using a cloning strategy involving degenerate primers, a cDNA library screen, and 5' and 3' rapid amplification of cDNA ends (RACE). RACE identified two alternative transcriptional starts and four alternative polyadenylation sites. The first translation start codon provided an open reading frame of 2610 bp encoding 870 amino acids and was PCR amplified without introns from genomic DNA. Southern hybridization indicated one strongly hybridizing gene with possible weakly related genes or pseudogenes. Amino acid sequence analysis identified a family 48 carbohydrate-binding module (CBM) upstream of the protein's first predicted transmembrane domain. There are broad similarities in predicted 3D structures of the family 48 modules from CESA, from several glycogen- and starch-binding enzymes, and from protein kinases, but there are substitutions at some residues thought to be involved in ligand binding. The module in G. monilis CESA will be on the cytoplasmic face of the plasma membrane so that it could potentially bind either low molecular weight ligands or starch which is cytosolic rather than inside membrane-bound plastids in red algae. Possible reasons why red algal CESAs have evolved family 48 modules perhaps as part of a system to regulate cellulose synthase activity in relation to cellular carbohydrate status are briefly discussed.

  8. Sulindac-derived RXRα modulators inhibit cancer cell growth by binding to a novel site.

    Science.gov (United States)

    Chen, Liqun; Wang, Zhi-Gang; Aleshin, Alexander E; Chen, Fan; Chen, Jiebo; Jiang, Fuquan; Alitongbieke, Gulimiran; Zeng, Zhiping; Ma, Yue; Huang, Mingfeng; Zhou, Hu; Cadwell, Gregory; Zheng, Jian-Feng; Huang, Pei-Qiang; Liddington, Robert C; Zhang, Xiao-kun; Su, Ying

    2014-05-22

    Retinoid X receptor-alpha (RXRα), an intriguing and unique drug target, can serve as an intracellular target mediating the anticancer effects of certain nonsteroidal anti-inflammatory drugs (NSAIDs), including sulindac. We report the synthesis and characterization of two sulindac analogs, K-8008 and K-8012, which exert improved anticancer activities over sulindac in a RXRα-dependent manner. The analogs inhibit the interaction of the N-terminally truncated RXRα (tRXRα) with the p85α subunit of PI3K, leading to suppression of AKT activation and induction of apoptosis. Crystal structures of the RXRα ligand-binding domain (LBD) with K-8008 or K-8012 reveal that both compounds bind to tetrameric RXRα LBD at a site different from the classical ligand-binding pocket. Thus, these results identify K-8008 and K-8012 as tRXRα modulators and define a binding mechanism for regulating the nongenomic action of tRXRα.

  9. Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules

    Directory of Open Access Journals (Sweden)

    Cicortas Gunnarsson Lavinia

    2009-10-01

    Full Text Available Abstract Background Molecular evolution of carbohydrate binding modules (CBM is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.

  10. The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins.

    Science.gov (United States)

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-03-20

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (alpha, beta, and gamma), in which three laminin globular modules in the alpha chain and the Glu residue in the C-terminal tail of the gamma chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the beta chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the beta1 or beta2 chain toward a panel of laminin-binding integrins, and we found that beta2 chain-containing laminins (beta2-laminins) bound more avidly to alpha3beta1 and alpha7X2beta1 integrins than beta1 chain-containing laminins (beta1-laminins), whereas alpha6beta1, alpha6beta4, and alpha7X1beta1 integrins did not show any preference toward beta2-laminins. Because alpha3beta1 contains the "X2-type" variable region in the alpha3 subunit and alpha6beta1 and alpha6beta4 contain the "X1-type" region in the alpha6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between beta1-laminins and beta2-laminins. In support of this possibility, a putative X2-type variant of alpha6beta1 was produced and found to bind preferentially to beta2-laminins. Production of a series of swap mutants between the beta1 and beta2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by beta2-laminins. Taken together, the results provide evidence that the C-terminal region of beta chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.

  11. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin

    DEFF Research Database (Denmark)

    Eghorn, Laura Friis; Høstgaard-Jensen, Kirsten; Kongstad, Kenneth Thermann

    2014-01-01

    conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed......γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate...... whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive...

  12. Calcium modulates promoter occupancy by the Entamoeba histolytica Ca2+-binding transcription factor URE3-BP.

    Science.gov (United States)

    Gilchrist, Carol A; Leo, Megan; Line, C Genghis; Mann, Barbara J; Petri, William A

    2003-02-14

    The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) binds to the URE3 sequence of the Gal/GalNAc-inhibitable lectin hgl5 and ferredoxin 1 (fdx) gene promoters. This binding can be inhibited in vitro by addition of calcium. Two EF-hand motifs, which are associated with the ability to bind calcium, are present in the amino acid sequence of URE3-BP. Mutation of the second EF-hand motif in URE3-BP resulted in the loss of calcium inhibition of DNA binding as monitored by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays revealed that URE3-BP was physically bound to the hgl5 and fdx promoters in vivo. Parasite intracellular calcium concentrations were altered by changes in extracellular calcium. Promoter occupancy was lost when intracellular calcium levels were increased by coordinate increases in extracellular calcium. Increased intracellular calcium also resulted in decreased levels of URE3-BP mRNA. Together these results demonstrate that changes in extracellular calcium result in changes in URE3-BP mRNA and in the ability of URE3-BP to bind to URE3-containing promoters. Modulation of URE3-BP by calcium may represent an important mechanism of control of gene expression in E. histolytica.

  13. Disabled-2 modulates homotypic and heterotypic platelet interactions by binding to sulfatides.

    Science.gov (United States)

    Welsh, John D; Charonko, John J; Salmanzadeh, Alireza; Drahos, Karen E; Shafiee, Hadi; Stremler, Mark A; Davalos, Rafael V; Capelluto, Daniel G S; Vlachos, Pavlos P; Finkielstein, Carla V

    2011-07-01

    Disabled-2 (Dab2) inhibits platelet aggregation by competing with fibrinogen for binding to the α(IIb) β(3) integrin receptor, an interaction that is modulated by Dab2 binding to sulfatides at the outer leaflet of the platelet plasma membrane. The disaggregatory function of Dab2 has been mapped to its N-terminus phosphotyrosine-binding (N-PTB) domain. Our data show that the surface levels of P-selectin, a platelet transmembrane protein known to bind sulfatides and promote cell-cell interactions, are reduced by Dab2 N-PTB, an event that is reversed in the presence of a mutant form of the protein that is deficient in sulfatide but not in integrin binding. Importantly, Dab2 N-PTB, but not its sulfatide binding-deficient form, was able to prevent sulfatide-induced platelet aggregation when tested under haemodynamic conditions in microfluidic devices at flow rates with shear stress levels corresponding to those found in vein microcirculation. Moreover, the regulatory role of Dab2 N-PTB extends to platelet-leucocyte adhesion and aggregation events, suggesting a multi-target role for Dab2 in haemostasis.

  14. Glutamate-containing dipeptides do not modulate ligand binding at excitatory amino acid receptors.

    Science.gov (United States)

    Baud, J; Fagg, G E

    1986-10-08

    Dipeptides of the structure X-Glu (e.g. X = Phe, Leu) have been proposed as allosteric modulators of excitatory amino acid receptors in rat brain membranes. Here we report that these dipeptides reduce the binding of L-[3H]Glu (predominantly N-methyl-D-aspartate-sensitive sites) and of [3H]kainate to postsynaptic density preparations isolated from rat brain. However, several observations indicate that the effects of these dipeptides are mediated not by allosteric modulation, but by free L-Glu liberated by the actions of a membrane-associated aminopeptidase. The absolute and relative potencies of the dipeptides are similar at all acidic amino acid binding sites examined to date, suggesting the involvement of a factor with similar activity at each site (e.g. L-Glu). N-Acetyl-Met-Glu is a weak inhibitor of L-Glu and kainate binding, and N-blocked peptides are known to be poor substrates of aminopeptidases. Bestatin, an inhibitor of aminopeptidases, decreases or abolishes the effects of substrate dipeptides on L-Glu and kainate receptor binding, while having no effect itself.

  15. IQGAP1 Binds to Yes-associated Protein (YAP) and Modulates Its Transcriptional Activity.

    Science.gov (United States)

    Sayedyahossein, Samar; Li, Zhigang; Hedman, Andrew C; Morgan, Chase J; Sacks, David B

    2016-09-09

    During development, the Hippo signaling pathway regulates key physiological processes, such as control of organ size, regeneration, and stem cell biology. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. The scaffold protein IQGAP1 interacts with more than 100 binding partners to integrate diverse signaling pathways. In this study, we report that IQGAP1 binds to YAP and modulates its activity. IQGAP1 and YAP co-immunoprecipitated from cells. In vitro analysis with pure proteins demonstrated a direct interaction between IQGAP1 and YAP. Analysis with multiple fragments of each protein showed that the interaction occurs via the IQ domain of IQGAP1 and the TEAD-binding domain of YAP. The interaction between IQGAP1 and YAP has functional effects. Knock-out of endogenous IQGAP1 significantly increased the formation of nuclear YAP-TEAD complexes. Transcription assays were performed with IQGAP1-null mouse embryonic fibroblasts and HEK293 cells with IQGAP1 knockdown by CRISPR/Cas9. Quantification demonstrated that YAP-TEAD-mediated transcription in cells lacking IQGAP1 was significantly greater than in control cells. These data reveal that IQGAP1 binds to YAP and modulates its co-transcriptional function, suggesting that IQGAP1 participates in Hippo signaling.

  16. Cycle modulation of insulin-like growth factor-binding protein 1 in human endometrium

    Directory of Open Access Journals (Sweden)

    Corleta H.

    2000-01-01

    Full Text Available Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1 interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5% followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia.

  17. Reciprocal allosteric modulation of carbon monoxide and warfarin binding to ferrous human serum heme-albumin.

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    Alessio Bocedi

    Full Text Available Human serum albumin (HSA, the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s. As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i of carbon monoxide (CO binding to ferrous human serum heme-albumin (HSA-heme-Fe(II by warfarin (WF, and (ii of WF binding to HSA-heme-Fe(II by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II, respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands. This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II. The HSA-heme-Fe(II populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i upon CO binding a conformational change of HSA-heme-Fe(II takes place (likely reflecting the displacement of an endogenous ligand by CO, and (ii CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II.

  18. Does protein binding modulate the effect of angiotensin II receptor antagonists?

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    Marc P Maillard

    2001-03-01

    Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

  19. Insights into the modulation of dopamine transporter function by amphetamine, orphenadrine and cocaine binding

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    Mary Hongying Cheng

    2015-06-01

    Full Text Available Human dopamine transporter (hDAT regulates dopaminergic signaling in the central nervous system by maintaining the synaptic concentration of dopamine (DA at physiological levels, upon reuptake of DA into presynaptic terminals. DA translocation involves the co-transport of two sodium ions and the channeling of a chloride ion, and it is achieved via alternating access between outward-facing and inward-facing states of DAT. hDAT is a target for addictive drugs such as cocaine, amphetamine (AMPH and therapeutic antidepressants. Our recent quantitative systems pharmacology study suggested that orphenadrine (ORPH, an anticholinergic agent and anti-PD drug, might be repurposable as a DAT drug. Previous studies have shown that DAT-substrates like AMPH or -blockers like cocaine modulate the function of DAT in different ways. However, the molecular mechanisms of modulation remained elusive due to the lack of structural data on DAT. The newly resolved DAT structure from Drosophila melanogaster opens the way to a deeper understanding of the mechanism and time evolution of DAT-drug/ligand interactions. Using a combination of homology modeling, docking analysis, molecular dynamics simulations and molecular biology experiments, we performed a comparative study of the binding properties of DA, AMPH, ORPH and cocaine, and their modulation of hDAT function. Simulations demonstrate that binding DA or AMPH drives a structural transition towards a functional form predisposed to translocate the ligand. In contrast, ORPH appears to inhibit DAT function by arresting it in the outward-facing open conformation. The analysis shows that cocaine and ORPH competitively bind DAT, with the binding pose and affinity dependent on the conformational state of DAT. Further assays show that the effect of ORPH on DAT uptake and endocytosis is comparable to that of cocaine.

  20. The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

    Science.gov (United States)

    Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

    2007-01-01

    The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

  1. Binding of histone H1 to DNA is differentially modulated by redox state of HMGB1.

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    Eva Polanská

    Full Text Available HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. In this study we demonstrate that redox state of HMGB1 can significantly modulate the ability of the protein to bind and bend DNA, as well as to promote DNA end-joining. We also report a high affinity binding of histone H1 to hemicatenated DNA loops and DNA minicircles. Finally, we show that reduced HMGB1 can readily displace histone H1 from DNA, while oxidized HMGB1 has limited capacity for H1 displacement. Our results suggested a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA. Possible biological consequences of linker histones H1 replacement by HMGB1 for the functioning of chromatin are discussed.

  2. Enthalpy-Entropy Compensation in the Binding of Modulators at Ionotropic Glutamate Receptor GluA2

    DEFF Research Database (Denmark)

    Krintel, Christian; Francotte, Pierre; Pickering, Darryl S;

    2016-01-01

    The 1,2,4-benzothiadiazine 1,1-dioxide type of positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) are promising lead compounds for the treatment of cognitive disorders, e.g., Alzheimer’s disease. The modulators bind in a cleft formed by the interface of two neighboring...

  3. Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus

    DEFF Research Database (Denmark)

    Deng, Ling; Kenchappa, Chandra Shekar; Peng, Xu

    2012-01-01

    CRISPR loci are essential components of the adaptive immune system of archaea and bacteria. They consist of long arrays of repeats separated by DNA spacers encoding guide RNAs (crRNA), which target foreign genetic elements. Cbp1 (CRISPR DNA repeat binding protein) binds specifically to the multiple...... direct repeats of CRISPR loci of members of the acidothermophilic, crenarchaeal order Sulfolobales. cbp1 gene deletion from Sulfolobus islandicus REY15A produced a strong reduction in pre-crRNA yields from CRISPR loci but did not inhibit the foreign DNA targeting capacity of the CRISPR/Cas system....... Conversely, overexpression of Cbp1 in S. islandicus generated an increase in pre-crRNA yields while the level of reverse strand transcripts from CRISPR loci remained unchanged. It is proposed that Cbp1 modulates production of longer pre-crRNA transcripts from CRISPR loci. A possible mechanism...

  4. The carbohydrate-binding module family 20-diversity, structure, and function

    DEFF Research Database (Denmark)

    Christiansen, Camilla; Abou Hachem, Maher; Janecek, S.

    2009-01-01

    Starch-active enzymes often possess starch-binding domains (SBDs) mediating attachment to starch granules and other high molecular weight substrates. SBDs are divided into nine carbohydrate-binding module (CBM) families, and CBM20 is the earliest-assigned and best characterized family. High...... diversity characterizes CBM20s, which occur in starch-active glycoside hydrolase families 13, 14, 15, and 77, and enzymes involved in starch or glycogen metabolism, exemplified by the starch-phosphorylating enzyme glucan, water dikinase 3 from Arabidopsis thaliana and the mammalian glycogen phosphatases......, laforins. The clear evolutionary relatedness of CBM20s to CBM21s, CBM48s and CBM53s suggests a common clan hosting most of the known SBDs. This review surveys the diversity within the CBM20 family, and makes an evolutionary comparison with CBM21s, CBM48s and CBM53s, discussing intrafamily and interfamily...

  5. Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

    Science.gov (United States)

    Zhou, Shun-Hua; Liu, Yuan; Zhao, Yu-Juan; Chi, Zhe; Chi, Zhen-Ming; Liu, Guang-Lei

    2016-09-01

    In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

  6. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    Science.gov (United States)

    Eghorn, Laura F; Hoestgaard-Jensen, Kirsten; Kongstad, Kenneth T; Bay, Tina; Higgins, David; Frølund, Bente; Wellendorph, Petrine

    2014-10-05

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

  7. It's not my fault: postdictive modulation of intentional binding by monetary gains and losses.

    Directory of Open Access Journals (Sweden)

    Keisuke Takahata

    Full Text Available Sense of agency refers to the feeling that one's voluntary actions caused external events. Past studies have shown that compression of the subjective temporal interval between actions and external events, called intentional binding, is closely linked to the experience of agency. Current theories postulate that the experience of agency is constructed via predictive and postdictive pathways. One remaining problem is the source of human causality bias; people often make misjudgments on the causality of voluntary actions and external events depending on their rewarding or punishing outcomes. Although human causality bias implies that sense of agency can be modified by post-action information, convincing empirical findings for this issue are lacking. Here, we hypothesized that sense of agency would be modified by affective valences of action outcomes. To examine this issue, we investigated how rewarding and punishing outcomes following voluntary action modulate behavioral measures of agency using intentional binding paradigm and classical conditioning procedures. In the acquisition phase, auditory stimuli were paired with positive, neutral or negative monetary outcomes. Tone-reward associations were evaluated using reaction times and preference ratings. In the experimental session, participants performed a variant of intentional binding task, where participants made timing judgments for onsets of actions and sensory outcomes while playing simple slot games. Our results showed that temporal binding was modified by affective valences of action outcomes. Specifically, intentional binding was attenuated when negative outcome occurred, consistent with self-serving bias. Our study not only provides evidence for postdictive modification of agency, but also proposes a possible mechanism of human causality bias.

  8. Modulation of oncogenic DBL activity by phosphoinositol phosphate binding to pleckstrin homology domain.

    Science.gov (United States)

    Russo, C; Gao, Y; Mancini, P; Vanni, C; Porotto, M; Falasca, M; Torrisi, M R; Zheng, Y; Eva, A

    2001-06-01

    The Dbl family guanine nucleotide exchange factors (GEFs) contain a region of sequence similarity consisting of a catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of signaling molecules to plasma membranes by protein-protein and/or protein-lipid interactions. Here we show that Dbl PH domain binding to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to the PH domain significantly inhibits the Cdc42 interactive activity of the DH domain suggesting that the DH domain is subjected to the PH domain modulation under the influence of phosphoinositides (PIPs). We generated Dbl mutants unable to interact with PIPs. These mutants retained GEF activity on Cdc42 in the presence of PIPs and showed a markedly enhanced activating potential for both Cdc42 and RhoA in vivo while displaying decreased cellular transforming activity. Immunofluorescence analysis of NIH3T3 transfectants revealed that whereas the PH domain localizes to actin stress fibers and plasma membrane, the PH mutants are no longer detectable on the plasma membrane. These results suggest that modulation of PIPs in both the GEF catalytic activity and the targeting to plasma membrane determines the outcome of the biologic activity of Dbl.

  9. Modulating supramolecular binding of carbon dioxide in a redox-active porous metal-organic framework

    Science.gov (United States)

    Lu, Zhenzhong; Godfrey, Harry G. W.; da Silva, Ivan; Cheng, Yongqiang; Savage, Mathew; Tuna, Floriana; McInnes, Eric J. L.; Teat, Simon J.; Gagnon, Kevin J.; Frogley, Mark D.; Manuel, Pascal; Rudić, Svemir; Ramirez-Cuesta, Anibal J.; Easun, Timothy L.; Yang, Sihai; Schröder, Martin

    2017-01-01

    Hydrogen bonds dominate many chemical and biological processes, and chemical modification enables control and modulation of host–guest systems. Here we report a targeted modification of hydrogen bonding and its effect on guest binding in redox-active materials. MFM-300(VIII) {[VIII2(OH)2(L)], LH4=biphenyl-3,3′,5,5′-tetracarboxylic acid} can be oxidized to isostructural MFM-300(VIV), [VIV2O2(L)], in which deprotonation of the bridging hydroxyl groups occurs. MFM-300(VIII) shows the second highest CO2 uptake capacity in metal-organic framework materials at 298 K and 1 bar (6.0 mmol g−1) and involves hydrogen bonding between the OH group of the host and the O-donor of CO2, which binds in an end-on manner, =1.863(1) Å. In contrast, CO2-loaded MFM-300(VIV) shows CO2 bound side-on to the oxy group and sandwiched between two phenyl groups involving a unique ···c.g.phenyl interaction [3.069(2), 3.146(3) Å]. The macroscopic packing of CO2 in the pores is directly influenced by these primary binding sites. PMID:28194014

  10. Modulating supramolecular binding of carbon dioxide in a redox-active porous metal-organic framework

    Science.gov (United States)

    Lu, Zhenzhong; Godfrey, Harry G. W.; da Silva, Ivan; Cheng, Yongqiang; Savage, Mathew; Tuna, Floriana; McInnes, Eric J. L.; Teat, Simon J.; Gagnon, Kevin J.; Frogley, Mark D.; Manuel, Pascal; Rudić, Svemir; Ramirez-Cuesta, Anibal J.; Easun, Timothy L.; Yang, Sihai; Schröder, Martin

    2017-02-01

    Hydrogen bonds dominate many chemical and biological processes, and chemical modification enables control and modulation of host-guest systems. Here we report a targeted modification of hydrogen bonding and its effect on guest binding in redox-active materials. MFM-300(VIII) {[VIII2(OH)2(L)], LH4=biphenyl-3,3',5,5'-tetracarboxylic acid} can be oxidized to isostructural MFM-300(VIV), [VIV2O2(L)], in which deprotonation of the bridging hydroxyl groups occurs. MFM-300(VIII) shows the second highest CO2 uptake capacity in metal-organic framework materials at 298 K and 1 bar (6.0 mmol g-1) and involves hydrogen bonding between the OH group of the host and the O-donor of CO2, which binds in an end-on manner, =1.863(1) Å. In contrast, CO2-loaded MFM-300(VIV) shows CO2 bound side-on to the oxy group and sandwiched between two phenyl groups involving a unique ...c.g.phenyl interaction [3.069(2), 3.146(3) Å]. The macroscopic packing of CO2 in the pores is directly influenced by these primary binding sites.

  11. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    Directory of Open Access Journals (Sweden)

    Klein-Seetharaman Judith

    2008-02-01

    Full Text Available Abstract Metabotropic glutamate receptors (mGluRs are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%, the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%, the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86% and 8/9 (89% for ArgusLab and 10/14 (71% and 7/9 (78% for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by

  12. Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280).

    Science.gov (United States)

    Li, M; Bermak, J C; Wang, Z W; Zhou, Q Y

    2000-03-01

    Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.

  13. Allosteric enhancers, allosteric agonists and ago-allosteric modulators: where do they bind and how do they act?

    DEFF Research Database (Denmark)

    Schwartz, Thue W; Holst, Birgitte

    2007-01-01

    Many small-molecule agonists also display allosteric properties. Such ago-allosteric modulators act as co-agonists, providing additive efficacy--instead of partial antagonism--and they can affect--and often improve--the potency of the endogenous agonist. Surprisingly, the apparent binding sites...... different binding modes. In another, dimeric, receptor scenario, the endogenous agonist binds to one protomer while the ago-allosteric modulator binds to the other, 'allosteric' protomer. It is suggested that testing for ago-allosteric properties should be an integral part of the agonist drug discovery...... process because a compound that acts with--rather than against--the endogenous agonist could be an optimal agonist drug....

  14. A novel function for the cellulose binding module of cellobiohydrolase I

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A homogeneous cellulose-binding module(CBM)of cellobiohydrolase I(CBHI)from Trichoderma pseudokoningii S-38 was obtained by the limited proteolysis with papain and a series of chromatographs filtration.Analysis of FT-IR spectra demonstrated that the structural changes result from a weakening and splitting of the hydrogen bond network in cellulose by the action of CBMCBHI at 40℃for 24 h.The results of molecular dynamic simulations are consistent with the experimental conclusions, and provide a nanoscopic view of the mechanism that strong and medium H-bonds decreased dramatically when CBM was bound to the cellulose surface.The function of CBMCBHI is not only limited to locating intact CBHI in close proximity with cellulose fibrils,but also is involved in the structural disruption at the fibre surface.The present studies provided considerable evidence for the model of the intramolecular synergy between the catalytic domain and their CBMs.

  15. Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells1

    Science.gov (United States)

    Shpigel, Etai; Roiz, Levava; Goren, Raphael; Shoseyov, Oded

    1998-01-01

    Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL−1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL−1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control. PMID:9701575

  16. Binding of β-VLDL to heparan sulfate proteoglycans requires lipoprotein lipase, whereas apoE only modulates binding affinity

    NARCIS (Netherlands)

    Beer, F. de; Hendriks, W.L.; Vark, L.C. van; Kamerling, S.W.A.; Dijk, K.W. van; Hofker, M.H.; Smelt, A.H.M.; Havekes, L.M.

    1999-01-01

    The binding of β-VLDL to heparan sulfate proteoglycans (HSPG) has been reported to be stimulated by both apoE and lipoprotein lipase (LPL). In the present study we investigated the effect of the isoform and the amount of apoE per particle, as well as the role of LPL on the binding of β-VLDL to HSPG.

  17. γ-Secretase modulator (GSM) photoaffinity probes reveal distinct allosteric binding sites on presenilin.

    Science.gov (United States)

    Pozdnyakov, Nikolay; Murrey, Heather E; Crump, Christina J; Pettersson, Martin; Ballard, T Eric; Am Ende, Christopher W; Ahn, Kwangwook; Li, Yue-Ming; Bales, Kelly R; Johnson, Douglas S

    2013-04-05

    γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic β-amyloid peptides (i.e. Aβ42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule γ-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of Aβ42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of γ-secretase and the modulatory binding site of certain GSMs.

  18. Engineering and Comparative Characteristics of Double Carbohydrate Binding Modules as a Strength Additive for Papermaking Applications

    Directory of Open Access Journals (Sweden)

    Xiaoran Shi

    2014-04-01

    Full Text Available In this study, four engineered proteins containing two family 1 and/or family 3 carbohydrate binding modules (CBMs were constructed and expressed as soluble forms in Escherichia coli. Their binding performances and effect on paper’s mechanical properties were comprehensively studied with the aim to design suitably engineered CBMs as novel biomaterials for use in the production of new cellulose materials. The recombinant engineered double CBMs exhibited obvious differences in their adsorption to different cellulosic substrates. The CBM3-GS-CBM3 was the most effective in enhancing paper mechanical properties in terms of folding endurance (27.4% and tensile strength (15.5% among the four engineered double CBMs, but it gave rise to only a slight increase in bursting strength (3.1%. On the other hand, CBM1-NL-CBM1 achieved a significant simultaneous increase in tensile strength (12.6% and burst strength (8.8%, as well as folding endurance (16.7%. Unexpectedly, CBM3-GS-CBM1 and CBM3-NL-CBM1 had the lowest effective paper property improvement. The differences in types of CBMs and linker peptides in engineered double CBMs may contribute to the considerable differences in their cellulose binding and paper property modification. Our data suggested that CBM1-NL-CBM1 may provide a better upgrade of the secondary pulp, which makes it very suitable for fiber recycling. Meanwhile, CBM3-GS-CBM3 may have particular potential for paper manufacture requiring high folding endurance.

  19. The C-terminal Region of Laminin β Chains Modulates the Integrin Binding Affinities of Laminins*S⃞

    OpenAIRE

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-01-01

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (α, β, and γ), in which three laminin globular modules in the α chain and the Glu residue in the C-terminal tail of the γ chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the β chain is involved in laminin-integrin interactions. We compared the binding affinities of ...

  20. Changes in BQCA Allosteric Modulation of [(3)H]NMS Binding to Human Cortex within Schizophrenia and by Divalent Cations.

    Science.gov (United States)

    Dean, Brian; Hopper, Shaun; Conn, P Jeffrey; Scarr, Elizabeth

    2016-05-01

    Stimulation of the cortical muscarinic M1 receptor (CHRM1) is proposed as a treatment for schizophrenia, a hypothesis testable using CHRM1 allosteric modulators. Allosteric modulators have been shown to change the activity of CHRMs using cloned human CHRMs and CHRM knockout mice but not human CNS, a prerequisite for them working in humans. Here we show in vitro that BQCA, a positive allosteric CHRM1 modulator, brings about the expected change in affinity of the CHRM1 orthosteric site for acetylcholine in human cortex. Moreover, this effect of BQCA is reduced in the cortex of a subset of subjects with schizophrenia, separated into a discrete population because of a profound loss of cortical [(3)H]pirenzepine binding. Surprisingly, there was no change in [(3)H]NMS binding to the cortex from this subset or those with schizophrenia but without a marked loss of cortical CHRM1. Hence, we explored the nature of [(3)H]pirenzepine and [(3)H]NMS binding to human cortex and showed total [(3)H]pirenzepine and [(3)H]NMS binding was reduced by Zn(2+), acetylcholine displacement of [(3)H]NMS binding was enhanced by Mg(2+) and Zn(2+), acetylcholine displacement of [(3)H]pirenzepine was reduced by Mg(2+) and enhanced by Zn(2+), whereas BQCA effects on [(3)H]NMS, but not [(3)H]pirenzepine, binding was enhanced by Mg(2+) and Zn(2+). These data suggest the orthosteric and allosteric sites on CHRMs respond differently to divalent cations and the effects of allosteric modulation of the cortical CHRM1 is reduced in a subset of people with schizophrenia, a finding that may have ramifications for the use of CHRM1 allosteric modulators in the treatment of schizophrenia.

  1. An in silico analysis of the binding modes and binding affinities of small molecule modulators of PDZ-peptide interactions.

    Directory of Open Access Journals (Sweden)

    Garima Tiwari

    Full Text Available Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson's disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known K(i or K(d values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high K(i or K(d moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors

  2. Integrative decomposition procedure and Kappa statistics set up ATF2 ion binding module in malignant pleural mesothelioma (MPM)

    Institute of Scientific and Technical Information of China (English)

    Ying SUN; Lin WANG; Lei LIU

    2008-01-01

    Activating transcription factor 2 (ATF2) is a member of the ATF/cyclic AMP-responsive element bind-ing protein family of transcription factors. However, the information concerning ATF2 ion-mediated DNA binding module and function of ATF2 in malignant pleural mesothelioma (MPM) has never been addressed. In this study, by using GRNInfer and GVedit based on linear pro-gramming and a decomposition procedure, with integrated analysis of the function cluster using Kappa statistics and fuzzy heuristic clustering in MPM, we identified one ATF2 ion-mediated DNA binding module involved in invasive function including ATF2 inhibition to target genes FALZ, C20orf31, NME2, PLOD2, RNF10, and RNASEH1, upstream RNF10 and PLOD2 activation to ATF2, upstream RNASEH1 and FALZ inhibition to ATF2 from 40 MPM tumors and 5 normal pleural tissues. Remarkably, our results showed that the predominant effect of ATF2 occupancy is to suppress the activation of target genes on MPM. Importantly, the ATF2 ion-mediated DNA binding module reflects 'mutual' positive and negative feedback regulation mechanism of ATF2 with up-and down-stream genes. It may be useful for developing novel prognostic markers and therapeutic targets in MPM.

  3. A role for cysteine 3635 of RYR1 in redox modulation and calmodulin binding

    Science.gov (United States)

    Porter Moore, C.; Zhang, J. Z.; Hamilton, S. L.

    1999-01-01

    Oxidation of the skeletal muscle Ca(2+) release channel (RYR1) increases its activity, produces intersubunit disulfide bonds, and blocks its interaction with calmodulin. Conversely, bound calmodulin protects RYR1 from the effects of oxidants (Zhang, J.-Z., Wu, Y., Williams, B. Y., Rodney, G., Mandel, F., Strasburg, G. M., and Hamilton, S. L. (1999) Am. J. Physiol. 276, Cell Physiol. C46-C53). In addition, calmodulin protects RYR1 from trypsin cleavage at amino acids 3630 and 3637 (Moore, C. P., Rodney, G., Zhang, J.-Z., Santacruz-Toloza, L., Strasburg, G. M., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). The sequence between these two tryptic sites is AVVACFR. Alkylation of RYR1 with N-ethylmaleimide (NEM) blocks both (35)S-apocalmodulin binding and oxidation-induced intersubunit cross-linking. In the current work, we demonstrate that both cysteines needed for the oxidation-induced intersubunit cross-link are protected from alkylation with N-ethylmaleimide by bound calmodulin. We also show, using N-terminal amino acid sequencing together with analysis of the distribution of [(3)H]NEM labeling with each sequencing cycle, that cysteine 3635 of RYR1 is rapidly labeled by NEM and that this labeling is blocked by bound calmodulin. We propose that cysteine 3635 is located at an intersubunit contact site that is close to or within a calmodulin binding site. These findings suggest that calmodulin and oxidation modulate RYR1 activity by regulating intersubunit interactions in a mutually exclusive manner and that these interactions involve cysteine 3635.

  4. A novel fibronectin binding motif in MSCRAMMs targets F3 modules.

    Directory of Open Access Journals (Sweden)

    Sabitha Prabhakaran

    Full Text Available BACKGROUND: BBK32 is a surface expressed lipoprotein and fibronectin (Fn-binding microbial surface component recognizing adhesive matrix molecule (MSCRAMM of Borrelia burgdorferi, the causative agent of Lyme disease. Previous studies from our group showed that BBK32 is a virulence factor in experimental Lyme disease and located the Fn-binding region to residues 21-205 of the lipoprotein. METHODOLOGY/PRINCIPAL FINDINGS: Studies aimed at identifying interacting sites between BBK32 and Fn revealed an interaction between the MSCRAMM and the Fn F3 modules. Further analysis of this interaction showed that BBK32 can cause the aggregation of human plasma Fn in a similar concentration-dependent manner to that of anastellin, the superfibronectin (sFn inducing agent. The resulting Fn aggregates are conformationally distinct from plasma Fn as indicated by a change in available thermolysin cleavage sites. Recombinant BBK32 and anastellin affect the structure of Fn matrices formed by cultured fibroblasts and inhibit endothelial cell proliferation similarly. Within BBK32, we have located the sFn-forming activity to a region between residues 160 and 175 which contains two sequence motifs that are also found in anastellin. Synthetic peptides mimicking these motifs induce Fn aggregation, whereas a peptide with a scrambled sequence motif was inactive, suggesting that these motifs represent the sFn-inducing sequence. CONCLUSIONS/SIGNIFICANCE: We conclude that BBK32 induces the formation of Fn aggregates that are indistinguishable from those formed by anastellin. The results of this study provide evidence for how bacteria can target host proteins to manipulate host cell activities.

  5. ATP-Binding Cassette Transporters Modulate Both Coelenterazine- and D-Luciferin-Based Bioluminescence Imaging

    Directory of Open Access Journals (Sweden)

    Ruimin Huang

    2011-05-01

    Full Text Available Bioluminescence imaging (BLI of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI readout intensity from intact living cells. To investigate the effect of ATP-binding cassette (ABC transporters on BLI readout, we generated click beetle (cLuc, firefly (fLuc, Renilla (rLuc, and Gaussia (gLuc luciferase HEK-293 reporter cells that overexpressed different ABC transporters (ABCB1, ABCC1, and ABCG2. In vitro studies showed a significant BLI intensity decrease in intact cells compared to cell lysates, when ABCG2 was overexpressed in HEK-293/cLuc, fLuc, and rLuc cells. Selective ABC transporter inhibitors were also applied. Inhibition of ABCG2 activity increased the BLI intensity more than two-fold in HEK-293/cLuc, fLuc, and rLuc cells; inhibition of ABCB1 elevated the BLI intensity two-fold only in HEK-293/rLuc cells. BLI of xenografts derived from HEK-293/ABC transporter/luciferase reporter cells confirmed the results of inhibitor treatment in vivo. These findings demonstrate that coelenterazine-based rLuc-BLI intensity can be modulated by ABCB1 and ABCG2. ABCG2 modulates d-luciferin-based BLI in a luciferase type–independent manner. Little ABC transporter effect on gLuc-BLI intensity is observed because a large fraction of gLuc is secreted. The expression level of ABC transporters is one key factor affecting BLI intensity, and this may be particularly important in luciferase-based applications in stem cell research.

  6. In vitro binding of a radio-labeled positive allosteric modulator for metabotropic glutamate receptor subtype 5.

    Science.gov (United States)

    Zysk, John R; Spear, Nathan; Fieles, William; Stein, Mark M; Sygowski, Linda S; King, Megan M; Hoesch, Valerie; Hastings, Richard; Brockel, Becky; Do, Mylinh; Ström, Peter; Gadient, Reto; Chhajlani, Vijay; Elmore, Charles S; Maier, Donna L

    2013-03-01

    The positive allosteric modulator (PAM) binding site for metabotropic glutamate receptor subtype 5 (mGlu(5)) lacks a readily available radio-labeled tracer fordetailed structure-activity studies. This communication describes a selective mGlu(5) compound, 7-methyl-2-(4-(pyridin-2-yloxy)benzyl)-5-(pyridin-3-yl)isoindolin-1-one (PBPyl) that binds with high affinity to human mGlu(5) and exhibits functional PAM activity. Analysis of PBPyl by FLIPR revealed an EC(50) of 87 nM with an 89% effect in transfected HEK293 cells and an EC(50) of 81 nM with a 42% effect in rat primary neurons. PBPyl exhibited 5-fold higher functional selectivity for mGlu(5) in a full mGlu receptor panel. Unlabeled PBPyl was tested for specific binding using a liquid chromatography mass spectrometry (LC/MS/MS)-based filtration binding assay and exhibited 40% specific binding in recombinant membranes, a value higher than any candidate compound tested. In competition binding studies with [(3)H]MPEP, the mGlu(5) receptor negative allosteric modulator (NAM), PBPyl exhibited a k(i) value of 34 nM. PBPyl also displaced [(3)H]ABP688, a mGluR(5) receptor NAM, in tissue sections from mouse and rat brain using autoradiography. Areas of specific binding included the frontal cortex, striatum and nucleus accumbens. PBPyl was radiolabeled to a specific activity of 15 Ci/mmol and tested for specific binding in a filter plate format. In recombinant mGlu(5b) membranes, [(3)H] PBPyl exhibited saturable binding with a K(d) value of 18.6 nM. In competition binding experiments, [(3)H] PBPyl was displaced by high affinity mGlu(5) positive and negative modulators. Further tests showed that PBPyl displays less than optimal characteristics as an in vivo tool, including a high volume of distribution and ClogP, making it more suitable as an in vitro compound. However, as a first report of direct binding of an mGlu(5) receptor PAM, this study offers value toward the development of novel PET imaging agents for this important

  7. Macromolecular depletion modulates the binding of red blood cells to activated endothelial cells.

    Science.gov (United States)

    Yang, Yang; Koo, Stephanie; Lin, Cheryl Shuyi; Neu, Björn

    2010-09-01

    Adhesion of red blood cells (RBCs) to endothelial cells (ECs) is usually insignificant but an enhanced adhesion has been observed in various diseases associated with vascular complications. This abnormal adhesion under pathological conditions such as sickle cell disease has been correlated with increased levels of various plasma proteins but the detailed underlying mechanism(s) remains unclear. Usually it is assumed that the proadhesive effects of plasma proteins originate from ligand interactions cross-linking receptors on adjacent cells, but explicit results detailing binding sites or receptors for some proteins (e.g., fibrinogen) on either RBC or EC surfaces that would support this model are missing. In this study, the authors tested whether there is an alternative mechanism. Their results demonstrate that dextran 2 MDa promotes the adhesion of normal RBCs to thrombin-activated ECs and that this effect becomes more pronounced with increasing thrombin concentration or with prolonged thrombin incubation time. It is concluded that depletion interaction originating from nonadsorbing macromolecules (i.e., dextran) can modulate the adhesion of red blood cells to thrombin-activated EC. This study thereby suggests macromolecular depletion as an alternative mechanism for the adhesion-promoting effects of nonadsorbing plasma proteins. These findings should not only aid in getting a better understanding of diseases associated with vascular complications but should also have many potential applications in biomedical or biotechnological areas that require the control of cell-cell or cell surface interactions.

  8. Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

    Science.gov (United States)

    Kitange, Gaspar J.; Mladek, Ann C.; Schroeder, Mark A.; Pokorny, Jenny C.; Carlson, Brett L.; Zhang, Yuji; Nair, Asha A.; Lee, Jeong-Heon; Yan, Huihuang; Decker, Paul A.; Zhang, Zhiguo; Sarkaria, Jann N.

    2016-01-01

    Summary Here we provide evidence that RBBP4 modulates temozolomide (TMZ) sensitivity through coordinate regulation of 2 key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT) and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51 and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac) and increased H3K9 tri-methylation (H3K9me3). Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin modifying complex that binds within the promoter of MGMT, RAD51 and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. RBBP4/CBP/p300 complex may provide an interesting target for developing therapy sensitizing strategies for GBM and other tumors. PMID:26972001

  9. Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

    Directory of Open Access Journals (Sweden)

    Gaspar J. Kitange

    2016-03-01

    Full Text Available Here we provide evidence that RBBP4 modulates temozolomide (TMZ sensitivity through coordinate regulation of two key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition, and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51, and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac and increased H3K9 tri-methylation (H3K9me3. Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin-modifying complex that binds within the promoter of MGMT, RAD51, and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. The RBBP4/CBP/p300 complex may provide an interesting target for developing therapy-sensitizing strategies for GBM and other tumors.

  10. RETENTION AND PAPER-STRENGTH CHARACTERISTICS OF ANIONIC POLYACRYLAMIDES CONJUGATED WITH CARBOHYDRATE-BINDING MODULES

    Directory of Open Access Journals (Sweden)

    Shingo Yokota

    2009-02-01

    Full Text Available The retention behavior of polymers having the specific affinities of glyco-hydrolases for pulp fibers was investigated with regard to paper-strength enhancement in contaminated papermaking systems. Carbohydrate-binding modules (CBMs of cellulases derived from Trichoderma viride and T. reesei, and of xylanase from Thermomyces lanuginosus, were obtained by site-directed digestion with papain, then introduced into anionic polyacrylamide (A-PAM via a peptide condensation reaction. Three types of CBM-conjugated A-PAMs (CBM-A-PAMs displayed different retention behavior, depending on the kind of pulp substrates, i.e. hardwood and softwood fibers. The CBM-A-PAM from T. viride demonstrated good additive retention for hardwood pulp fibers, resulting in high tensile strength of paper sheets, even under contaminated conditions in the presence of Ca2+ ions and ligninsulfonate. The CBM-A-PAM from T. reesei showed better performance for softwood than for hardwood sheets. The xylanase CBM-A-PAM was preferentially retained on hardwood fibers in which hemicelluloses might be present. Such an additive retention system, with inherent affinities of enzymes for pulp fibers, is expected to expand the application range of CBM-polymers in practical wet-end processes.

  11. Fusion of carbohydrate binding modules from Thermotoga neapolitana with a family 10 xylanase from Bacillus halodurans S7.

    Science.gov (United States)

    Mamo, Gashaw; Hatti-Kaul, Rajni; Mattiasson, Bo

    2007-01-01

    Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)(6) tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature and pH for the activity of the xylanase did not change.

  12. Studies on the binding of modest modulators of the human enzyme, Sirtuin 6, using STD-NMR.

    Science.gov (United States)

    Bolívar, Beatriz E; Welch, John T

    2017-02-21

    Pyrazinamide (PZA), an essential constituent of short course tuberculosis chemotherapy, binds weakly but selectively to Sirtuin 6 (SIRT6). Despite structural similarities between nicotinamide (NAM), PZA, and pyrazinoic acid (POA), these inhibitors modulate SIRT6 via different mechanisms and binding sites, as suggested by Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR). Available experimental evidence, such as that derived from crystal structures and kinetic experiments, is of only limited utility in the elucidation of the mechanistic details of sirtuin inhibition by NAM or other inhibitors. For example, crystallographic structural analysis of sirtuin binding sites does not illuminate differences in binding affinities among sirtuins or capture details of the dynamic process. STD NMR led to findings that not only agreed with the binding kinetics experiments, but also yielded insights into the binding process. The data also illustrate the geometry of the ligand binding epitopes in solution for both the apo- and holoenzyme. Recognition that SIRT6 is affected selectively by PZA, an established clinical agent, suggests that the rational development of more potent and selective NAM surrogates may be possible. These derivatives may be accessible by employing the malleability of this scaffold to assist in the identification by STD-NMR of the motifs that interact with the holo- and apo-enzymes in solution.

  13. High throughput techniques for discovering new glycine receptor modulators and their binding sites

    Directory of Open Access Journals (Sweden)

    Daniel F Gilbert

    2009-10-01

    Full Text Available The inhibitory glycine receptor (GlyR is a member of the Cys-loop receptor family that mediates inhibitory neurotransmission in the central nervous system. These receptors are emerging as potential drug targets for inflammatory pain, immunomodulation, spasticity and epilepsy. Antagonists that specifically inhibit particular GlyR isoforms are also required as pharmacological probes for elucidating the roles of particular GlyR isoforms in health and disease. Although a substantial number of both positive and negative GlyR modulators have been identified, very few of these are specific for the GlyR over other receptor types. Thus, the potential of known compounds as either therapeutic leads or pharmacological probes is limited. It is therefore surprising that there have been few published studies describing attempts to discover novel GlyR isoform-specific compounds. The first aim of this review is to consider various methods for efficiently screening compounds against these receptors. We conclude that an anion sensitive yellow fluorescent protein is optimal for primary screening and that automated electrophysiology of cells stably expressing GlyRs is useful for confirming hits and quantitating the actions of identified compounds. The second aim of this review is to demonstrate how these techniques are used in our laboratory for the purpose of both discovering novel GlyR-active compounds and characterizing their binding sites. We also describe a reliable, cost effective method for transfecting HEK293 cells in single wells of a 384 well plate using nanogram quantities of cDNA.

  14. Module structure of interphotoreceptor retinoid-binding protein (IRBP may provide bases for its complex role in the visual cycle – structure/function study of Xenopus IRBP

    Directory of Open Access Journals (Sweden)

    Ghosh Debashis

    2007-08-01

    Full Text Available Abstract Background Interphotoreceptor retinoid-binding protein's (IRBP remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling. Results The full-length Xenopus IRBP cDNA codes for a polypeptide of 1,197 amino acid residues beginning with a signal peptide followed by four homologous modules each ~300 amino acid residues in length. Modules 1 and 3 are more closely related to each other than either is to modules 2 and 4. Modules 1 and 4 are most similar to the N- and C-terminal modules of the two module IRBP of teleosts. Our data are consistent with the model that vertebrate IRBPs arose through two genetic duplication events, but that the middle two modules were lost during the evolution of the ray finned fish. The sequence of the expressed full-length IRBP was confirmed by liquid chromatography-tandem mass spectrometry. The recombinant full-length Xenopus IRBP bound all-trans retinol and 11-cis retinaldehyde at 3 to 4 sites with Kd's of 0.2 to 0.3 μM, and was active in protecting all-trans retinol from degradation. Module 2 showed selectivity for all-trans retinol over 11-cis retinaldehyde. The binding data are correlated to the results of docking of all-trans-retinol to the crystal structure of Xenopus module 2 suggesting two ligand-binding sites

  15. Fluorescence Resonance Energy Transfer Imaging Reveals that Chemokine-Binding Modulates Heterodimers of CXCR4 and CCR5 Receptors

    OpenAIRE

    2008-01-01

    BACKGROUND: Dimerization has emerged as an important feature of chemokine G-protein-coupled receptors. CXCR4 and CCR5 regulate leukocyte chemotaxis and also serve as a co-receptor for HIV entry. Both receptors are recruited to the immunological synapse during T-cell activation. However, it is not clear whether they form heterodimers and whether ligand binding modulates the dimer formation. METHODOLOGY/PRINCIPAL FINDINGS: Using a sensitive Fluorescence Resonance Energy Transfer (FRET) imaging ...

  16. Sex Steroid Modulation of Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    Science.gov (United States)

    Ockner, Robert K.; Lysenko, Nina; Manning, Joan A.; Monroe, Scott E.; Burnett, David A.

    1980-01-01

    The mechanism by which sex steroids influence very low density hepatic lipoprotein triglyceride production has not been fully elucidated. In previous studies we showed that [14C]oleate utilization and incorporation into triglycerides were greater in hepatocyte suspensions from adult female rats than from males. The sex differences were not related to activities of the enzymes of triglyceride biosynthesis, whereas fatty acid binding protein (FABP) concentration in liver cytosol was greater in females. These findings suggested that sex differences in lipoprotein could reflect a sex steroid influence on the availability of fatty acids for hepatocellular triglyceride biosynthesis. In the present studies, sex steroid effects on hepatocyte [14C]oleate utilization and FABP concentration were investigated directly. Hepatocytes from immature (30-d-old) rats exhibited no sex differences in [14C]oleate utilization. With maturation, total [14C]oleate utilization and triglyceride biosynthesis increased moderately in female cells and decreased markedly in male cells; the profound sex differences in adults were maximal by age 60 d. Fatty acid oxidation was little affected. Rats were castrated at age 30 d, and received estradiol, testosterone, or no hormone until age 60 d, when hepatocyte [14C]oleate utilization was studied. Castration virtually eliminated maturational changes and blunted the sex differences in adults. Estradiol or testosterone largely reproduced the appropriate adult pattern of [14C]oleate utilization regardless of the genotypic sex of the treated animal. In immature females and males, total cytosolic FABP concentrations were similar. In 60-d-old animals, there was a striking correlation among all groups (females, males, castrates, and hormone-treated) between mean cytosolic FABP concentration on the one hand, and mean total [14C]oleate utilization (r = 0.91) and incorporation into triglycerides (r = 0.94) on the other. In 30-d-old animals rates of [14C

  17. Differential Modulation of Annexin I Binding Sites on Monocytes and Neutrophils

    Directory of Open Access Journals (Sweden)

    H. S. Euzger

    1999-01-01

    Full Text Available Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN. These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

  18. Crystal structure of an integron gene cassette-associated protein from Vibrio cholerae identifies a cationic drug-binding module.

    Directory of Open Access Journals (Sweden)

    Chandrika N Deshpande

    Full Text Available The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes.We report the 1.8 Å crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators.Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  19. Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

    Energy Technology Data Exchange (ETDEWEB)

    Deshpande, Chandrika N.; Harrop, Stephen J.; Boucher, Yan; Hassan, Karl A.; Di Leo, Rosa; Xu, Xiaohui; Cui, Hong; Savchenko, Alexei; Chang, Changsoo; Labbate, Maurizio; Paulsen, Ian T.; Stokes, H.W.; Curmi, Paul M.G.; Mabbutt, Bridget C. (MIT); (UT-Australia); (Macquarie); (Toronto); (New South)

    2012-02-15

    The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. We report the 1.8 {angstrom} crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  20. Membrane Binding and Modulation of the PDZ Domain of PICK1

    DEFF Research Database (Denmark)

    Erlendsson, Simon; Madsen, Kenneth Lindegaard

    2015-01-01

    Scaffolding proteins serve to assemble protein complexes in dynamic processes by means of specific protein-protein and protein-lipid binding domains. Many of these domains bind either proteins or lipids exclusively; however, it has become increasingly evident that certain domains are capable of b...... lipids. Moreover, we review how these PDZ-membrane interactions are regulated in the case of the synaptic scaffolding protein PICK1 and how this might affect cellular localization and function....

  1. Allosteric modulation of neurotoxin binding to voltage-sensitive sodium channels by Ptychodiscus brevis toxin 2.

    Science.gov (United States)

    Sharkey, R G; Jover, E; Couraud, F; Baden, D G; Catterall, W A

    1987-03-01

    The effects of Ptychodiscus brevis toxin 2 (PbTx-2) on the binding of neurotoxins at four different neurotoxin receptor sites on voltage-sensitive sodium channels in rat brain synaptosomes were examined. Binding of saxitoxin at neurotoxin receptor site 1 and Leiurus quinquestriatus alpha-scorpion toxin (LqTx) at neurotoxin receptor site 3 was unaffected. PbTx-2 enhanced binding of batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 and Centruroides suffusus suffusus beta-scorpion toxin (CsTx II) to site 4 on sodium channels. These results support the proposal that PbTx-2 and related toxins act at a new receptor site (site 5) that has not been previously analyzed in binding experiments. Half-maximal effects of PbTx-2 were observed in the range of 20-50 nM PbTx-2. The enhancement of BTX-B binding was reduced by depolarization. Saturating concentrations of PbTx-2 reduced KD values for binding of BTX-B and CsTx-II 2.9-fold and 2.6-fold, respectively. The effects of PbTx-2 and LqTx in enhancing BTX-B binding were synergistic. A model involving both preferential binding of BTX-B, PbTx-2, LqTx, and CsTx II to active states of sodium channels and allosteric interactions among the four receptor sites at which these toxins act accommodates these and previous results.

  2. Guanine Nucleotides Modulate Cell Surface cAMP-Binding Sites in Membranes from Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1984-01-01

    D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (Kd = 15 nM, t½ = 15 s). A second class is fast dissociating (t½ about 1 s) and is composed of high affinity binding sites H (Kd ≈ 60 nM), and low a

  3. Control of Mycosphaerella graminicola on wheat seedlings by medical drugs known to modulate the activity of ATP-binding cassette transporters

    NARCIS (Netherlands)

    Roohparvar, R.; Huser, A.; Zwiers, L.H.; Waard, de M.A.

    2007-01-01

    Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due

  4. The First Residue of the PWWP Motif Modulates HATH Domain Binding, Stability, and Protein-Protein Interaction.

    Science.gov (United States)

    Hung, Yi-Lin; Lee, Hsia-Ju; Jiang, Ingjye; Lin, Shang-Chi; Lo, Wei-Cheng; Lin, Yi-Jan; Sue, Shih-Che

    2015-07-01

    Hepatoma-derived growth factor (hHDGF) and HDGF-related proteins (HRPs) contain conserved N-terminal HATH domains with a characteristic structural motif, namely the PWWP motif. The HATH domain has attracted attention because of its ability to bind with heparin/heparan sulfate, DNA, and methylated histone peptide. Depending on the sequence of the PWWP motif, HRP HATHs are classified into P-type (Pro-His-Trp-Pro) and A-type (Ala-His-Trp-Pro) forms. A-type HATH is highly unstable and tends to precipitate in solution. We replaced the Pro residue in P-type HATHHDGF with Ala and evaluated the influence on structure, dynamics, and ligand binding. Nuclear magnetic resonance (NMR) hydrogen/deuterium exchange and circular dichroism (CD) measurements revealed reduced stability. Analysis of NMR backbone (15)N relaxations (R1, R2, and nuclear Overhauser effect) revealed additional backbone dynamics in the interface between the β-barrel and the C-terminal helix bundle. The β1-β2 loop, where the AHWP sequence is located, has great structural flexibility, which aids HATH-HATH interaction through the loop. A-type HATH, therefore, shows a stronger tendency to aggregate when binding with heparin and DNA oligomers. This study defines the role of the first residue of the PWWP motif in modulating HATH domain stability and oligomer formation in binding.

  5. Structural proof of a dimeric positive modulator bridging two identical AMPA receptor-binding sites

    DEFF Research Database (Denmark)

    Kaae, Birgitte Høiriis; Harpsøe, Kasper; Kastrup, Jette Sandholm Jensen;

    2007-01-01

    Dimeric positive allosteric modulators of ionotropic glutamate receptors were designed, synthesized, and characterized pharmacologically in electrophysiological experiments. The designed compounds are dimers of arylpropylsulfonamides and have been constructed without a linker. The monomeric...

  6. Global identification of hnRNP A1 binding sites for SSO-based splicing modulation

    DEFF Research Database (Denmark)

    Bruun, Gitte H; Doktor, Thomas K; Borch-Jensen, Jonas;

    2016-01-01

    for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. RESULTS: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing...... regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP......-identified hnRNP A1 binding site immediately downstream of the 5' splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site...

  7. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response.

    Science.gov (United States)

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; Di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D'Andrea, Luca Domenico

    2016-08-08

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor.

  8. Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding.

    Science.gov (United States)

    Reichart, Timothy M; Baksh, Michael M; Rhee, Jin-Kyu; Fiedler, Jason D; Sligar, Stephen G; Finn, M G; Zwick, Michael B; Dawson, Philip E

    2016-02-18

    The membrane-proximal external region (MPER) of HIV gp41 is an established target of antibodies that neutralize a broad range of HIV isolates. To evaluate the role of the transmembrane (TM) domain, synthetic MPER-derived peptides were incorporated into lipid nanoparticles using natural and designed TM domains, and antibody affinity was measured using immobilized and solution-based techniques. Peptides incorporating the native HIV TM domain exhibit significantly stronger interactions with neutralizing antibodies than peptides with a monomeric TM domain. Furthermore, a peptide with a trimeric, three-helix bundle TM domain recapitulates the binding profile of the native sequence. These studies suggest that neutralizing antibodies can bind the MPER when the TM domain is a three-helix bundle and this presentation could influence the binding of neutralizing antibodies to the virus. Lipid-bilayer presentation of viral antigens in Nanodiscs is a new platform for evaluating neutralizing antibodies.

  9. Ligand binding modulates the structural dynamics and compactness of the major birch pollen allergen.

    Science.gov (United States)

    Grutsch, Sarina; Fuchs, Julian E; Freier, Regina; Kofler, Stefan; Bibi, Marium; Asam, Claudia; Wallner, Michael; Ferreira, Fátima; Brandstetter, Hans; Liedl, Klaus R; Tollinger, Martin

    2014-12-16

    Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.

  10. Conformational Analysis of the Streptococcus pneumoniae Hyaluronate Lyase and Characterization of Its Hyaluronan-specific Carbohydrate-binding Module*

    Science.gov (United States)

    Suits, Michael D. L.; Pluvinage, Benjamin; Law, Adrienne; Liu, Yan; Palma, Angelina S.; Chai, Wengang; Feizi, Ten; Boraston, Alisdair B.

    2014-01-01

    For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl. PMID:25100731

  11. Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier.

    Science.gov (United States)

    Zhang, Jingjing; Ni, Chen; Yang, Zhenguo; Piontek, Anna; Chen, Huapu; Wang, Sijie; Fan, Yiming; Qin, Zhihai; Piontek, Joerg

    2015-08-01

    Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers.

  12. The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Nemčovičová, Ivana [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States); Slovak Academy of Sciences, Dúbravská cesta 9, SK 84505 Bratislava (Slovakia); Zajonc, Dirk M., E-mail: dzajonc@liai.org [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States)

    2014-03-01

    The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155 as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host

  13. Modulating the function of ATP-binding cassette subfamily G member 2 (ABCG2) with inhibitor cabozantinib.

    Science.gov (United States)

    Zhang, Guan-Nan; Zhang, Yun-Kai; Wang, Yi-Jun; Barbuti, Anna Maria; Zhu, Xi-Jun; Yu, Xin-Yue; Wen, Ai-Wen; Wurpel, John N D; Chen, Zhe-Sheng

    2017-01-25

    Cabozantinib (XL184) is a small molecule tyrosine kinase receptor inhibitor, which targets c-Met and VEGFR2. Cabozantinib has been approved by the Food and Drug Administration to treat advanced medullary thyroid cancer and renal cell carcinoma. In the present study, we evaluated the ability of cabozantinib to modulate the function of the ATP-binding cassette subfamily G member 2 (ABCG2) by sensitizing cells that are resistant to ABCG2 substrate antineoplastic drugs. We used a drug-selected resistant cell line H460/MX20 and three ABCG2 stable transfected cell lines ABCG2-482-R2, ABCG2-482-G2, and ABCG2-482-T7, which overexpress ABCG2. Cabozantinib, at non-toxic concentrations (3 or 5μM), sensitized the ABCG2-overexpressing cells to mitoxantrone, SN-38, and topotecan. Our results indicate that cabozantinib reverses ABCG2-mediated multidrug resistance by antagonizing the drug efflux function of the ABCG2 transporter instead of downregulating its expression. The molecular docking analysis indicates that cabozantinib binds to the drug-binding site of the ABCG2 transporter. Overall, our findings demonstrate that cabozantinib inhibits the ABCG2 transporter function and consequently enhances the effect of the antineoplastic agents that are substrates of ABCG2. Cabozantinib may be a useful agent in anticancer treatment regimens for patients who are resistant to ABCG2 substrate drugs.

  14. Fluorescence resonance energy transfer imaging reveals that chemokine-binding modulates heterodimers of CXCR4 and CCR5 receptors.

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    Nilgun Isik

    Full Text Available BACKGROUND: Dimerization has emerged as an important feature of chemokine G-protein-coupled receptors. CXCR4 and CCR5 regulate leukocyte chemotaxis and also serve as a co-receptor for HIV entry. Both receptors are recruited to the immunological synapse during T-cell activation. However, it is not clear whether they form heterodimers and whether ligand binding modulates the dimer formation. METHODOLOGY/PRINCIPAL FINDINGS: Using a sensitive Fluorescence Resonance Energy Transfer (FRET imaging method, we investigated the formation of CCR5 and CXCR4 heterodimers on the plasma membrane of live cells. We found that CCR5 and CXCR4 exist as constitutive heterodimers and ligands of CCR5 and CXCR4 promote different conformational changes within these preexisting heterodimers. Ligands of CCR5, in contrast to a ligand of CXCR4, induced a clear increase in FRET efficiency, indicating that selective ligands promote and stabilize a distinct conformation of the heterodimers. We also found that mutations at C-terminus of CCR5 reduced its ability to form heterodimers with CXCR4. In addition, ligands induce different conformational transitions of heterodimers of CXCR4 and CCR5 or CCR5(STA and CCR5(Delta4. CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest a model in which CXCR4 and CCR5 spontaneously form heterodimers and ligand-binding to CXCR4 or CCR5 causes different conformational changes affecting heterodimerization, indicating the complexity of regulation of dimerization/function of these chemokine receptors by ligand binding.

  15. Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

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    Romain Pardoux

    Full Text Available To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9TKE(12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d = 25±6 nM to K(d = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d = 0.25±0.06 nM. FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as(P-O and ν(s(P-O IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as(UO(2(2+ vibration (from 923 cm(-1 to 908 cm(-1 was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.

  16. Specific tracking of xylan using fluorescent-tagged carbohydrate-binding module 15 as molecular probe

    OpenAIRE

    Khatri, Vinay; Hébert-Ouellet, Yannick; Meddeb-Mouelhi, Fatma; Beauregard,Marc

    2016-01-01

    Background Xylan has been identified as a physical barrier which limits cellulose accessibility by covering the outer surface of fibers and interfibrillar space. Therefore, tracking xylan is a prerequisite for understanding and optimizing lignocellulosic biomass processes. Results In this study, we developed a novel xylan tracking approach using a two-domain probe called OC15 which consists of a fusion of Cellvibrio japonicus carbohydrate-binding domain 15 with the fluorescent protein mOrange...

  17. C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms.

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    Christopher J Brown

    Full Text Available The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD and an equivalent phage optimized peptide (12/1 were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.

  18. Ligand Binding Modulates the Structural Dynamics and Compactness of the Major Birch Pollen Allergen

    Science.gov (United States)

    Grutsch, Sarina; Fuchs, Julian E.; Freier, Regina; Kofler, Stefan; Bibi, Marium; Asam, Claudia; Wallner, Michael; Ferreira, Fátima; Brandstetter, Hans; Liedl, Klaus R.; Tollinger, Martin

    2014-01-01

    Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens. PMID:25517162

  19. Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors.

    Science.gov (United States)

    Ahmed, Ahmed H; Oswald, Robert E

    2010-03-11

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

  20. Cell Surface Binding and Internalization of Aβ Modulated by Degree of Aggregation

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    David A. Bateman

    2011-01-01

    Full Text Available The amyloid peptides, Aβ40 and Aβ42, are generated through endoproteolytic cleavage of the amyloid precursor protein. Here we have developed a model to investigate the interaction of living cells with various forms of aggregated Aβ40/42. After incubation at endosomal pH 6, we observed a variety of Aβ conformations after 3 (Aβ3, 24 (Aβ24, and 90 hours (Aβ90. Both Aβ4224 and Aβ4024 were observed to rapidly bind and internalize into differentiated PC12 cells, leading to accumulation in the lysosome. In contrast, Aβ40/4290 were both found to only weakly associate with cells, but were observed as the most aggregated using dynamic light scattering and thioflavin-T. Internalization of Aβ40/4224 was inhibited with treatment of monodansylcadaverine, an endocytosis inhibitor. These studies indicate that the ability of Aβ40/42 to bind and internalize into living cells increases with degree of aggregation until it reaches a maximum beyond which its ability to interact with cells diminishes drastically.

  1. Temperature dependence and GABA modulation of (TH)triazolam binding in the rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Earle, M.E.; Concas, A.; Wamsley, J.K.; Yamamura, H.I.

    1987-07-27

    The hypnotic triazolam (TZ), a triazolobenzodiazepine displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. The authors major objectives were the direct measurement of the temperature dependence and the gamma-aminobutyric acid (GABA) effect of (TH)TZ binding in the rat brain. Saturation studies showed a shift to lower affinity with increasing temperatures (K/sub d/ = 0.27 +/- 08 nM at 0C; K/sub d/ = 1.96 +/- 0.85 nM at 37C) while the B/sub max/ values remained unchanged (1220 +/- 176 fmoles/mg protein at 0C and 1160 +/- 383 fmoles/mg protein at 37C). Saturation studies of (TH)TZ binding in the presence or absence of GABA (100 M) showed a GABA-shift. At 0C the K/sub d/ values were (K/sub d/ = 0.24 +/- 0.03 nM/-GABA; K/sub d/ = 0.16 +/- 0.04/+GABA) and at 37C the K/sub d/ values were (K/sub d/ = 1.84 +/- 0.44 nM/-GABA; K/sub d/ = 0.95 +/- 0.29 nM/+GABA). In contrast to reported literature, the authors findings show that TZ interacts with benzodiazepine receptors with a temperature dependence and GABA-shift consistent with predicted behavior for benzodiazepine agonists. 20 references, 3 tables.

  2. Fatty-acid binding proteins modulate sleep and enhance long-term memory consolidation in Drosophila.

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    Jason R Gerstner

    Full Text Available Sleep is thought to be important for memory consolidation, since sleep deprivation has been shown to interfere with memory processing. However, the effects of augmenting sleep on memory formation are not well known, and testing the role of sleep in memory enhancement has been limited to pharmacological and behavioral approaches. Here we test the effect of overexpressing the brain-type fatty acid binding protein (Fabp7 on sleep and long-term memory (LTM formation in Drosophila melanogaster. Transgenic flies carrying the murine Fabp7 or the Drosophila homologue dFabp had reduced baseline sleep but normal LTM, while Fabp induction produced increases in both net sleep and LTM. We also define a post-training consolidation "window" that is sufficient for the observed Fabp-mediated memory enhancement. Since Fabp overexpression increases consolidated daytime sleep bouts, these data support a role for longer naps in improving memory and provide a novel role for lipid-binding proteins in regulating memory consolidation concurrently with changes in behavioral state.

  3. Cholesterol Oxidase Binds TLR2 and Modulates Functional Responses of Human Macrophages

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    Katarzyna Bednarska

    2014-01-01

    Full Text Available Cholesterol oxidase (ChoD is considered to be an important virulence factor for Mycobacterium tuberculosis (Mtb, but its influence on macrophage activity is unknown. Here we used Nocardia erythropolis ChoD, which is very similar to the Mtb enzyme (70% identity at the amino-acid level, to evaluate the impact of bacterial ChoD on the activity of THP-1-derived macrophages in vitro. We found that ChoD decreased the surface expression of Toll-like receptor type 2 (TLR2 and complement receptor 3 (CR3 on these macrophages. Flow cytometry and confocal microscopy showed that ChoD competed with lipoteichoic acid for ligand binding sites on TLR2 but not on CR3, suggesting that ChoD signaling is mediated via TLR2. Binding of ChoD to the membrane of macrophages had diverse effects on the activity of macrophages, activating p38 mitogen activated kinase and stimulating production of a large amount of interleukin-10. Moreover, ChoD primed macrophages to enhance the production of reactive oxygen species in response to the phorbol myristate acetate, which was reduced by “switching off” TLR-derived signaling through interleukin-1 receptor-associated kinases 1 and 4 inhibition. Our study revealed that ChoD interacts directly with macrophages via TLR2 and influences the biological activity of macrophages during the development of the initial response to infection.

  4. Tight-binding electrons on triangular and kagome lattices under staggered modulated magnetic fields: quantum Hall effects and Hofstadter butterflies

    Energy Technology Data Exchange (ETDEWEB)

    Li Juan; Wang Yifei; Gong Changde, E-mail: yfwang_nju@hotmail.com [Center for Statistical and Theoretical Condensed Matter Physics, and Department of Physics, Zhejiang Normal University, Jinhua 321004 (China)

    2011-04-20

    We consider the tight-binding models of electrons on a two-dimensional triangular lattice and kagome lattice under staggered modulated magnetic fields. Such fields have two components: a uniform-flux part with strength {phi}, and a staggered-flux part with strength {Delta}{phi}. Various properties of the Hall conductances and Hofstadter butterflies are studied. When {phi} is fixed, variation of {Delta}{phi} leads to the quantum Hall transitions and Chern numbers of Landau subbands being redistributed between neighboring pairs. The energy spectra with nonzero {Delta}{phi}s have similar fractal structures but quite different energy gaps compared with the original Hofstadter butterflies of {Delta}{phi} = 0. Moreover, the fan-like structure of Landau levels in the low magnetic field region is also modified appreciably by {Delta}{phi}.

  5. The carboxy-terminal domain of Dictyostelium C-module-binding factor is an independent gene regulatory entity.

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    Jörg Lucas

    Full Text Available The C-module-binding factor (CbfA is a multidomain protein that belongs to the family of jumonji-type (JmjC transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD. An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo.

  6. NITRIC OXIDE BINDS TO AND MODULATES THE ACTIVITY OF A POLLEN SPECIFIC ARABIDOPSIS DIACYLGLYCEROL KINASE

    KAUST Repository

    Wong, Aloysius Tze

    2014-06-01

    Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5\\'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered

  7. Structure of the ligand-binding domain (LBD) of human androgen receptor in complex with a selective modulator LGD2226

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng; Liu, Xiao-qin; Li, He; Liang, Kai-ni [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China); Miner, Jeffrey N.; Hong, Mei; Kallel, E. Adam; Oeveren, Arjan van; Zhi, Lin [Discovery Research, Ligand Pharmaceuticals Inc., 10275 Science Center Drive, San Diego, California 92121 (United States); Jiang, Tao, E-mail: x-ray@sun5.ibp.ac.cn [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China)

    2006-11-01

    Crystal structure of the ligand-binding domain of androgen receptor in complex with LGD2226. The androgen receptor (AR) is a ligand-inducible steroid hormone receptor that mediates androgen action, determining male sexual phenotypes and promoting spermatogenesis. As the androgens play a dominant role in male sexual development and function, steroidal androgen agonists have been used clinically for some years. However, there is a risk of potential side effects and most steroidal androgens cannot be dosed orally, which limits the use of these substances. 1,2-Dihydro-6-N,N-bis(2,2,2-trifluoroethyl) amino-4-trifluoromethyl-2-quinolinone (LGD2226) is a synthetic nonsteroidal ligand and a novel selective AR modulator. The crystal structure of the complex of LGD2226 with the androgen receptor ligand-binding domain (AR LBD) at 2.1 Å was solved and compared with the structure of the AR LBD–R1881 complex. It is hoped that this will aid in further explaining the selectivity of LGD2226 observed in in vitro and in vivo assays and in developing more selective and effective therapeutic agents.

  8. Does positive selection drive transcription factor binding site turnover? A test with Drosophila cis-regulatory modules.

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    Bin Z He

    2011-04-01

    Full Text Available Transcription factor binding site(s (TFBS gain and loss (i.e., turnover is a well-documented feature of cis-regulatory module (CRM evolution, yet little attention has been paid to the evolutionary force(s driving this turnover process. The predominant view, motivated by its widespread occurrence, emphasizes the importance of compensatory mutation and genetic drift. Positive selection, in contrast, although it has been invoked in specific instances of adaptive gene expression evolution, has not been considered as a general alternative to neutral compensatory evolution. In this study we evaluate the two hypotheses by analyzing patterns of single nucleotide polymorphism in the TFBS of well-characterized CRM in two closely related Drosophila species, Drosophila melanogaster and Drosophila simulans. An important feature of the analysis is classification of TFBS mutations according to the direction of their predicted effect on binding affinity, which allows gains and losses to be evaluated independently along the two phylogenetic lineages. The observed patterns of polymorphism and divergence are not compatible with neutral evolution for either class of mutations. Instead, multiple lines of evidence are consistent with contributions of positive selection to TFBS gain and loss as well as purifying selection in its maintenance. In discussion, we propose a model to reconcile the finding of selection driving TFBS turnover with constrained CRM function over long evolutionary time.

  9. Trichomonas vaginalis Lipophosphoglycan Exploits Binding to Galectin-1 and -3 to Modulate Epithelial Immunity.

    Science.gov (United States)

    Fichorova, Raina N; Yamamoto, Hidemi S; Fashemi, Titilayo; Foley, Evan; Ryan, Stanthia; Beatty, Noah; Dawood, Hassan; Hayes, Gary R; St-Pierre, Guillaume; Sato, Sachiko; Singh, Bibhuti N

    2016-01-08

    Trichomoniasis is the most common non-viral sexually transmitted infection caused by the vaginotropic extracellular protozoan parasite Trichomonas vaginalis. The infection is recurrent, with no lasting immunity, often asymptomatic, and linked to pregnancy complications and risk of viral infection. The molecular mechanisms of immune evasion by the parasite are poorly understood. We demonstrate that galectin-1 and -3 are expressed by the human cervical and vaginal epithelial cells and act as pathogen-recognition receptors for the ceramide phosphoinositol glycan core (CPI-GC) of the dominant surface protozoan lipophosphoglycan (LPG). We used an in vitro model with siRNA galectin knockdown epithelial clones, recombinant galectins, clinical Trichomonas isolates, and mutant protozoan derivatives to dissect the function of galectin-1 and -3 in the context of Trichomonas infection. Galectin-1 suppressed chemokines that facilitate recruitment of phagocytes, which can eliminate extracellular protozoa (IL-8) or bridge innate to adaptive immunity (MIP-3α and RANTES (regulated on activation normal T cell expressed and secreted)). Silencing galectin-1 increased and adding exogenous galectin-1 suppressed chemokine responses to Trichomonas or CPI-GC/LPG. In contrast, silencing galectin-3 reduced IL-8 response to LPG. Live Trichomonas depleted the extracellular levels of galectin-3. Clinical isolates and mutant Trichomonas CPI-GC that had reduced affinity to galectin-3 but maintained affinity to galectin-1 suppressed chemokine expression. Thus via CPI-GC binding, Trichomonas is capable of regulating galectin bioavailability and function to the benefit of its parasitic survival. These findings suggest novel approaches to control trichomoniasis and warrant further studies of galectin-binding diversity among clinical isolates as a possible source for symptom disparity in parasitic infections.

  10. Modulation of glutamate transport and receptor binding by glutamate receptor antagonists in EAE rat brain.

    Science.gov (United States)

    Sulkowski, Grzegorz; Dąbrowska-Bouta, Beata; Salińska, Elżbieta; Strużyńska, Lidia

    2014-01-01

    The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.

  11. The ATP-binding cassette transporter-2 (ABCA2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism.

    Science.gov (United States)

    Davis, Warren

    2014-01-01

    The ATP-binding cassette transporters are a large family (~48 genes divided into seven families A-G) of proteins that utilize the energy of ATP-hydrolysis to pump substrates across lipid bilayers against a concentration gradient. The ABC "A" subfamily is comprised of 13 members and transport sterols, phospholipids and bile acids. ABCA2 is the most abundant ABC transporter in human and rodent brain with highest expression in oligodendrocytes, although it is also expressed in neurons. Several groups have studied a possible connection between ABCA2 and Alzheimer's disease as well as early atherosclerosis. ABCA2 expression levels have been associated with changes in cholesterol and sphingolipid metabolism. In this paper, we hypothesized that ABCA2 expression level may regulate esterification of plasma membrane-derived cholesterol by modulation of sphingolipid metabolism. ABCA2 overexpression in N2a neuroblastoma cells was associated with an altered bilayer distribution of the sphingolipid ceramide that inhibited acylCoA:cholesterol acyltransferase (ACAT) activity and cholesterol esterification. In contrast, depletion of endogenous ABCA2 in the rat schwannoma cell line D6P2T increased esterification of plasma membrane cholesterol following treatment with exogenous bacterial sphingomyelinase. These findings suggest that control of ABCA2 expression level may be a key locus of regulation for esterification of plasma membrane-derived cholesterol through modulation of sphingolipid metabolism.

  12. Adhesive and migratory effects of phosphophoryn are modulated by flanking peptides of the integrin binding motif.

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    Shigeki Suzuki

    Full Text Available Phosphophoryn (PP is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP. Gene duplications in the ancestor dentin matrix protein-1 (DMP-1 genomic sequence created the DSPP gene in toothed animals. PP and DMP-1 are phosphorylated extracellular matrix proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs. Many SIBLING members have been shown to evoke various cell responses through the integrin-binding Arg-Gly-Asp (RGD domain; however, the RGD-dependent function of PP is not yet fully understood. We demonstrated that recombinant PP did not exhibit any obvious cell adhesion ability, whereas the simultaneously purified recombinant DMP-1 did. A cell adhesion inhibitory analysis was performed by pre-incubating human osteosarcoma MG63 cells with various PP peptides before seeding onto vitronectin. The results obtained revealed that the incorporation of more than one amino acid on both sides of the PP-RGD domain was unable to inhibit the adhesion of MG63 cells onto vitronectin. Furthermore, the inhibitory activity of a peptide containing the PP-RGD domain with an open carboxyl-terminal side (H-463SDESDTNSESANESGSRGDA482-OH was more potent than that of a peptide containing the RGD domain with an open amino-terminal side (H-478SRGDASYTSDESSDDDNDSDSH499-OH. This phenomenon was supported by the potent cell adhesion and migration abilities of the recombinant truncated PP, which terminated with Ala482. Furthermore, various point mutations in Ala482 and/or Ser483 converted recombinant PP into cell-adhesive proteins. Therefore, we concluded that the Ala482-Ser483 flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PP-RGD domain to be sequestered. The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin

  13. Regulatable and Modulable Background Expression Control in Prokaryotic Synthetic Circuits by Auxiliary Repressor Binding Sites.

    Science.gov (United States)

    Merulla, Davide; van der Meer, Jan Roelof

    2016-01-15

    Expression control in synthetic genetic circuitry, for example, for construction of sensitive biosensors, is hampered by the lack of DNA parts that maintain ultralow background yet achieve high output upon signal integration by the cells. Here, we demonstrate how placement of auxiliary transcription factor binding sites within a regulatable promoter context can yield an important gain in signal-to-noise output ratios from prokaryotic biosensor circuits. As a proof of principle, we use the arsenite-responsive ArsR repressor protein from Escherichia coli and its cognate operator. Additional ArsR operators placed downstream of its target promoter can act as a transcription roadblock in a distance-dependent manner and reduce background expression of downstream-placed reporter genes. We show that the transcription roadblock functions both in cognate and heterologous promoter contexts. Secondary ArsR operators placed upstream of their promoter can also improve signal-to-noise output while maintaining effector dependency. Importantly, background control can be released through the addition of micromolar concentrations of arsenite. The ArsR-operator system thus provides a flexible system for additional gene expression control, which, given the extreme sensitivity to micrograms per liter effector concentrations, could be applicable in more general contexts.

  14. The quaternary structure of the recombinant bovine odorant-binding protein is modulated by chemical denaturants.

    Directory of Open Access Journals (Sweden)

    Olga V Stepanenko

    Full Text Available A large group of odorant-binding proteins (OBPs has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer's β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl. Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M. This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.

  15. Roles of multiple surface sites, long substrate binding clefts, and carbohydrate binding modules in the action of amylolytic enzymes on polysaccharide substrates

    DEFF Research Database (Denmark)

    Nielsen, Morten Munch; Seo, E.S.; Dilokpimol, Adiphol

    2008-01-01

    with a characteristic subsite binding energy profile around the catalytic site. Furthermore, several amylolytic enzymes that facilitate attack on the natural substrate, i.e. the endosperm starch granules, have secondary sugar binding sites either situated on the surface of the protein domain or structural unit...... that contains the catalytic site or belonging to a separate starch binding domain. The role of surface sites in the function of barley alpha-amylase 1 has been investigated by using mutational analysis in conjunction with carbohydrate binding analyses and crystallography. The ability to bind starch depends...

  16. Glutathione selectively modulates the binding of platinum drugs to human copper chaperone Cox17.

    Science.gov (United States)

    Zhao, Linhong; Wang, Zhen; Wu, Han; Xi, Zhaoyong; Liu, Yangzhong

    2015-12-01

    The copper chaperone Cox17 (cytochrome c oxidase copper chaperone) has been shown to facilitate the delivery of cisplatin to mitochondria, which contributes to the overall cytotoxicity of the drug [Zhao et al. (2014) Chem. Commun. 50: , 2667-2669]. Kinetic data indicate that Cox17 has reactivity similar to glutathione (GSH), the most abundant thiol-rich molecule in the cytoplasm. In the present study, we found that GSH significantly modulates the reaction of platinum complexes with Cox17. GSH enhances the reactivity of three anti-cancer drugs (cisplatin, carboplatin and oxaliplatin) to Cox17, but suppresses the reaction of transplatin. Surprisingly, the pre-formed cisplatin-GSH adducts are highly reactive to Cox17; over 90% platinum transfers from GSH to Cox17. On the other hand, transplatin-GSH adducts are inert to Cox17. These different effects are consistent with the drug activity of these platinum complexes. In addition, GSH attenuates the protein aggregation of Cox17 induced by platination. These results indicate that the platinum-protein interactions could be substantially influenced by the cellular environment.

  17. Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence

    Science.gov (United States)

    Mazor, Yariv; Sachsenmeier, Kris F.; Yang, Chunning; Hansen, Anna; Filderman, Jessica; Mulgrew, Kathy; Wu, Herren; Dall’Acqua, William F.

    2017-01-01

    Bispecific antibodies are considered attractive bio-therapeutic agents owing to their ability to target two distinct disease mediators. Cross-arm avidity targeting of antigen double-positive cancer cells over single-positive normal tissue is believed to enhance the therapeutic efficacy, restrict major escape mechanisms and increase tumor-targeting selectivity, leading to reduced systemic toxicity and improved therapeutic index. However, the interplay of factors regulating target selectivity is not well understood and often overlooked when developing clinically relevant bispecific therapeutics. We show in vivo that dual targeting alone is not sufficient to endow selective tumor-targeting, and report the pivotal roles played by the affinity of the individual arms, overall avidity and format valence. Specifically, a series of monovalent and bivalent bispecific IgGs composed of the anti-HER2 trastuzumab moiety paired with affinity-modulated VH and VL regions of the anti-EGFR GA201 mAb were tested for selective targeting and eradication of double-positive human NCI-H358 non-small cell lung cancer target tumors over single-positive, non-target NCI-H358-HER2 CRISPR knock out tumors in nude mice bearing dual-flank tumor xenografts. Affinity-reduced monovalent bispecific variants, but not their bivalent bispecific counterparts, mediated a greater degree of tumor targeting selectivity, while the overall efficacy against the targeted tumor was not substantially affected. PMID:28067257

  18. Expression of the C-terminal family 22 carbohydrate-binding module of xylanase 10B of Clostridium themocellum in tobacco plant

    NARCIS (Netherlands)

    Olawole, O.

    2009-01-01

    Carbohydrate-binding modules have been shown to alter plant cell wall structural architecture. Hence, they have the potential application of being used to engineer the plant to produce tailor-made natural fibers in the cell wall. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that

  19. Bioinformatics Identification of Modules of Transcription Factor Binding Sites in Alzheimer's Disease-Related Genes by In Silico Promoter Analysis and Microarrays

    Directory of Open Access Journals (Sweden)

    Regina Augustin

    2011-01-01

    Full Text Available The molecular mechanisms and genetic risk factors underlying Alzheimer's disease (AD pathogenesis are only partly understood. To identify new factors, which may contribute to AD, different approaches are taken including proteomics, genetics, and functional genomics. Here, we used a bioinformatics approach and found that distinct AD-related genes share modules of transcription factor binding sites, suggesting a transcriptional coregulation. To detect additional coregulated genes, which may potentially contribute to AD, we established a new bioinformatics workflow with known multivariate methods like support vector machines, biclustering, and predicted transcription factor binding site modules by using in silico analysis and over 400 expression arrays from human and mouse. Two significant modules are composed of three transcription factor families: CTCF, SP1F, and EGRF/ZBPF, which are conserved between human and mouse APP promoter sequences. The specific combination of in silico promoter and multivariate analysis can identify regulation mechanisms of genes involved in multifactorial diseases.

  20. Biotin uptake in prokaryotes by solute transporters with an optional ATP-binding cassette-containing module.

    Science.gov (United States)

    Hebbeln, Peter; Rodionov, Dmitry A; Alfandega, Anja; Eitinger, Thomas

    2007-02-20

    BioMNY proteins are considered to constitute tripartite biotin transporters in prokaryotes. Recent comparative genomic and experimental analyses pointed to the similarity of BioMN to homologous modules of prokaryotic transporters mediating uptake of metals, amino acids, and vitamins. These systems resemble ATP-binding cassette-containing transporters and include typical ATPases (e.g., BioM). Absence of extracytoplasmic solute-binding proteins among the members of this group, however, is a distinctive feature. Genome context analyses uncovered that only one-third of the widespread bioY genes are linked to bioMN. Many bioY genes are located at loci encoding biotin biosynthesis, and others are unlinked to biotin metabolic or transport genes. Heterologous expression of the bioMNY operon and of the single bioY of the alpha-proteobacterium Rhodobacter capsulatus conferred biotin-transport activity on recombinant Escherichia coli cells. Kinetic analyses identified BioY as a high-capacity transporter that was converted into a high-affinity system in the presence of BioMN. BioMNY-mediated biotin uptake was severely impaired by replacement of the Walker A lysine residue in BioM, demonstrating dependency of high-affinity transport on a functional ATPase. Biochemical assays revealed that BioM, BioN, and BioY proteins form stable complexes in membranes of the heterologous host. Expression of truncated bio transport operons, each with one gene deleted, resulted in stable BioMN complexes but revealed only low amounts of BioMY and BioNY aggregates in the absence of the respective third partner. The results substantiate our earlier suggestion of a mechanistically novel group of membrane transporters.

  1. Phosphorylation of the leukemic oncoprotein EVI1 on serine 196 modulates DNA binding, transcriptional repression and transforming ability.

    Directory of Open Access Journals (Sweden)

    Daniel J White

    Full Text Available The EVI1 (ecotropic viral integration site 1 gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196 in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D, which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.

  2. ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling

    Energy Technology Data Exchange (ETDEWEB)

    Charlton, Hayley K.; Webster, Julie; Kruger, Sarah; Simpson, Fiona; Richards, Ayanthi A. [Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, QLD 4102 (Australia); Whitehead, Jonathan P., E-mail: j.whitehead1@uq.edu.au [Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, QLD 4102 (Australia)

    2010-02-05

    The pleiotropic effects of the insulin-sensitizing adipokine adiponectin are mediated, at least in part, by two seven-transmembrane domain receptors AdipoR1 and AdipoR2. Recent reports indicate a role for AdipoR-binding proteins, namely APPL1, RACK1 and CK2{beta}, in proximal signal transduction events. Here we demonstrate that endoplasmic reticulum protein 46 (ERp46) interacts specifically with AdipoR1 and provide evidence that ERp46 modulates adiponectin signalling. Co-immunoprecipitation followed by mass spectrometry identified ERp46 as an AdipoR1-, but not AdipoR2-, interacting protein. Analysis of truncated constructs and GST-fusion proteins revealed the interaction was mediated by the cytoplasmic, N-terminal residues (1-70) of AdipoR1. Indirect immunofluorescence microscopy and subcellular fractionation studies demonstrated that ERp46 was present in the ER and the plasma membrane (PM). Transient knockdown of ERp46 increased the levels of AdipoR1, and AdipoR2, at the PM and this correlated with increased adiponectin-stimulated phosphorylation of AMPK. In contrast, adiponectin-stimulated phosphorylation of p38MAPK was reduced following ERp46 knockdown. Collectively these results establish ERp46 as the first AdipoR1-specific interacting protein and suggest a role for ERp46 in adiponectin receptor biology and adiponectin signalling.

  3. Cyclic AMP response element binding protein and brain-derived neurotrophic factor: Molecules that modulate our mood?

    Indian Academy of Sciences (India)

    A Nair; V A Vaidya

    2006-09-01

    Depression is the major psychiatric ailment of our times, afflicting ∼20% of the population. Despite its prevalence, the pathophysiology of this complex disorder is not well understood. In addition, although antidepressants have been in existence for the past several decades, the mechanisms that underlie their therapeutic effects remain elusive. Building evidence implicates a role for the plasticity of specific neuro-circuitry in both the pathophysiology and treatment of depression. Damage to limbic regions is thought to contribute to the etiology of depression and antidepressants have been reported to reverse such damage and promote adaptive plasticity. The molecular pathways that contribute to the damage associated with depression and antidepressant-mediated plasticity are a major focus of scientific enquiry. The transcription factor cyclic AMP response element binding protein (CREB) and the neurotrophin brain-derived neurotrophic factor (BDNF) are targets of diverse classes of antidepressants and are known to be regulated in animal models and in patients suffering from depression. Given their role in neuronal plasticity, CREB and BDNF have emerged as molecules that may play an important role in modulating mood. The purpose of this review is to discuss the role of CREB and BDNF in depression and as targets/mediators of antidepressant action.

  4. Thyroid hormone receptors bind to the promoter of the mouse histone H10 gene and modulate its transcription.

    Science.gov (United States)

    Bauer-Hofmann, R; Alonso, A

    1995-01-01

    It has been shown that the mouse histone H10 promoter contains a DNA element, composed of a direct repeat of the sequence GGTGACC separated by 7 nt, which is able to bind retinoic acid receptors and to modulate transcription of reporter genes following treatment with retinoic acid. We have now investigated whether this DNA motif is also responsive to thyroid hormone. We co-transfected CV-1 monkey kidney cells with chloramphenicol acetyltransferase (CAT) expression plasmids containing either 740 bp of the H10 wild-type promoter or five copies of the repeat element cloned in front of the thymidine kinase promoter and expression vectors for human thyroid hormone receptors (TRs) alpha or beta and retinoid X receptor alpha (RXR alpha). Treatment of transfected cells with triiodothyronine led to a dose-dependent increase in CAT activity. Transfection experiments with increasing amounts of expression vectors for either TR alpha or RXR alpha resulted in up to 6-fold enhancement of CAT transcription. Furthermore, point mutations within the half-sites of the response element of the H10 promoter, as well as deletions within the interspace region, lowered CAT activity to 60-80% of that of the wild-type control. Electrophoretic mobility shift assays showed that the repeat element was able to form retarded complexes with TR alpha homodimers, as well as with TR alpha-RXR alpha heterodimers. Our results suggest that thyroid hormone receptors are involved in the regulation of mouse histone H10 expression. Images PMID:8559662

  5. PilN Binding Modulates the Structure and Binding Partners of the Pseudomonas aeruginosa Type IVa Pilus Protein PilM.

    Science.gov (United States)

    McCallum, Matthew; Tammam, Stephanie; Little, Dustin J; Robinson, Howard; Koo, Jason; Shah, Megha; Calmettes, Charles; Moraes, Trevor F; Burrows, Lori L; Howell, P Lynne

    2016-05-20

    Pseudomonas aeruginosa is an opportunistic bacterial pathogen that expresses type IVa pili. The pilus assembly system, which promotes surface-associated twitching motility and virulence, is composed of inner and outer membrane subcomplexes, connected by an alignment subcomplex composed of PilMNOP. PilM binds to the N terminus of PilN, and we hypothesize that this interaction causes functionally significant structural changes in PilM. To characterize this interaction, we determined the crystal structures of PilM and a PilM chimera where PilM was fused to the first 12 residues of PilN (PilM·PilN(1-12)). Structural analysis, multiangle light scattering coupled with size exclusion chromatography, and bacterial two-hybrid data revealed that PilM forms dimers mediated by the binding of a novel conserved motif in the N terminus of PilM, and binding PilN abrogates this binding interface, resulting in PilM monomerization. Structural comparison of PilM with PilM·PilN(1-12) revealed that upon PilN binding, there is a large domain closure in PilM that alters its ATP binding site. Using biolayer interferometry, we found that the association rate of PilN with PilM is higher in the presence of ATP compared with ADP. Bacterial two-hybrid data suggested the connectivity of the cytoplasmic and inner membrane components of the type IVa pilus machinery in P. aeruginosa, with PilM binding to PilB, PilT, and PilC in addition to PilN. Pull-down experiments demonstrated direct interactions of PilM with PilB and PilT. We propose a working model in which dynamic binding of PilN facilitates functionally relevant structural changes in PilM.

  6. A starch-binding domain identified in α-amylase (AmyP) represents a new family of carbohydrate-binding modules that contribute to enzymatic hydrolysis of soluble starch.

    Science.gov (United States)

    Peng, Hui; Zheng, Yunyun; Chen, Maojiao; Wang, Ying; Xiao, Yazhong; Gao, Yi

    2014-04-02

    A novel starch-binding domain (SBD) that represents a new carbohydrate-binding module family (CBM69) was identified in the α-amylase (AmyP) of the recently established alpha-amylase subfamily GH13_37. The SBD and its homologues come mostly from marine bacteria, and phylogenetic analysis indicates that they are closely related to the CBM20 and CBM48 families. The SBD exhibited a binding preference toward raw rice starch, but the truncated mutant (AmyPΔSBD) still retained similar substrate preference. Kinetic analyses revealed that the SBD plays an important role in soluble starch hydrolysis because different catalytic efficiencies have been observed in AmyP and the AmyPΔSBD.

  7. Differential modulation in binding of ketoprofen to bovine serum albumin in the presence and absence of surfactants: spectroscopic and calorimetric insights.

    Science.gov (United States)

    Misra, Pinaki P; Kishore, Nand

    2013-07-01

    Surfactants have long been implicated in the unique static and dynamic effect on the structure and function of serum albumins. However, there is very little information on the mode of interactions of drugs to serum albumins in presence of surfactants. The importance of such studies lay in the fact that apart from binding to serum albumins, surfactants are known to radically influence the solvents' micro environment and protein structure. Thus, we have studied the binding of the racemic form of ketoprofen with bovine serum albumin at pH 7.4 in the presence and absence of hexadecyl trimethyl ammonium bromide, sodium dodecyl sulfate, Triton X-100, and NaCl. The structural studies of ketoprofen with bovine serum albumin as investigated by circular dichroism spectroscopy revealed a significant stabilization of bovine serum albumin. However, the combined presence of the surfactants, NaCl and ketoprofen, demonstrated an extremely erratic behavior in terms of stabilization. Further the values of Stern-Volmer and dynamic quenching constant suggested the binding site of ketoprofen to be scattered in the region of domain I B and II A, close to Trp 134. The results of differential scanning calorimetry revealed that the binding of ketoprofen to bovine serum albumin leads to its temperature-dependent separation into two units. The binding parameters of bovine serum albumin obtained from isothermal titration calorimetry in the combined presence of ketoprofen and surfactants/NaCl correlate well with the differential scanning calorimetry studies further confirming the localization of ketoprofen in domain I B and II A. In the combined presence of surfactants, NaCl and ketoprofen, the binding of ketoprofen to bovine serum albumin exhibited altered binding parameters far different from the binding of ketoprofen alone. Overall, the experimental findings strongly indicated positive as well as negative modulation in the binding of ketoprofen to bovine serum albumin in the presence of

  8. Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

    DEFF Research Database (Denmark)

    Binti Jamek, Shariza; Nyffenegger, Christian; Muschiol, Jan

    2017-01-01

    /α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type...... A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools...

  9. Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity.

    Directory of Open Access Journals (Sweden)

    Paulo M F Cunha

    2010-07-01

    Full Text Available Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. Here, we assessed this assumption using the regulatory input of two essential transcription factors within the Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2 or the zinc-finger transcription factor lame duck (lmd lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, we obtained a global view of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation.

  10. Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity.

    Science.gov (United States)

    Cunha, Paulo M F; Sandmann, Thomas; Gustafson, E Hilary; Ciglar, Lucia; Eichenlaub, Michael P; Furlong, Eileen E M

    2010-07-01

    Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. Here, we assessed this assumption using the regulatory input of two essential transcription factors within the Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2) or the zinc-finger transcription factor lame duck (lmd) lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, we obtained a global view of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation.

  11. KIR polymorphisms modulate peptide-dependent binding to an MHC class I ligand with a Bw6 motif.

    Directory of Open Access Journals (Sweden)

    Arnaud D Colantonio

    2011-03-01

    Full Text Available Molecular interactions between killer immunoglobulin-like receptors (KIRs and their MHC class I ligands play a central role in the regulation of natural killer (NK cell responses to viral pathogens and tumors. Here we identify Mamu-A1*00201 (Mamu-A*02, a common MHC class I molecule in the rhesus macaque with a canonical Bw6 motif, as a ligand for Mamu-KIR3DL05. Mamu-A1*00201 tetramers folded with certain SIV peptides, but not others, directly stained primary NK cells and Jurkat cells expressing multiple allotypes of Mamu-KIR3DL05. Differences in binding avidity were associated with polymorphisms in the D0 and D1 domains of Mamu-KIR3DL05, whereas differences in peptide-selectivity mapped to the D1 domain. The reciprocal exchange of the third predicted MHC class I-contact loop of the D1 domain switched the specificity of two Mamu-KIR3DL05 allotypes for different Mamu-A1*00201-peptide complexes. Consistent with the function of an inhibitory KIR, incubation of lymphocytes from Mamu-KIR3DL05(+ macaques with target cells expressing Mamu-A1*00201 suppressed the degranulation of tetramer-positive NK cells. These observations reveal a previously unappreciated role for D1 polymorphisms in determining the selectivity of KIRs for MHC class I-bound peptides, and identify the first functional KIR-MHC class I interaction in the rhesus macaque. The modulation of KIR-MHC class I interactions by viral peptides has important implications to pathogenesis, since it suggests that the immunodeficiency viruses, and potentially other types of viruses and tumors, may acquire changes in epitopes that increase the affinity of certain MHC class I ligands for inhibitory KIRs to prevent the activation of specific NK cell subsets.

  12. α-Tocopherols modify the membrane dipole potential leading to modulation of ligand binding by P-glycoprotein.

    Science.gov (United States)

    Davis, Sterenn; Davis, Benjamin M; Richens, Joanna L; Vere, Kelly-Ann; Petrov, Peter G; Winlove, C Peter; O'Shea, Paul

    2015-08-01

    α-Tocopherol (vitamin E) has attracted considerable attention as a potential protective or palliative agent. In vitro, its free radical-scavenging antioxidant action has been widely demonstrated. In vivo, however, vitamin E treatment exhibits negligible benefits against oxidative stress. α-Tocopherol influences lipid ordering within biological membranes and its derivatives have been suggested to inhibit the multi-drug efflux pump, P-glycoprotein (P-gp). This study employs the fluorescent membrane probe, 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine, to investigate whether these effects are connected via influences on the membrane dipole potential (MDP), an intrinsic property of biological membranes previously demonstrated to modulate P-gp activity. α-Tocopherol and its non-free radical-scavenging succinate analog induced similar decreases in the MDP of phosphatidylcholine vesicles. α-Tocopherol succinate also reduced the MDP of T-lymphocytes, subsequently decreasing the binding affinity of saquinavir for P-gp. Additionally, α-tocopherol succinate demonstrated a preference for cholesterol-treated (membrane microdomain enriched) cells over membrane cholesterol-depleted cells. Microdomain disruption via cholesterol depletion decreased saquinavir's affinity for P-gp, potentially implicating these structures in the influence of α-tocopherol succinate on P-gp. This study provides evidence of a microdomain dipole potential-dependent mechanism by which α-tocopherol analogs influence P-gp activity. These findings have implications for the use of α-tocopherol derivatives for drug delivery across biological barriers.

  13. IGD motifs, which are required for migration stimulatory activity of fibronectin type I modules, do not mediate binding in matrix assembly.

    Directory of Open Access Journals (Sweden)

    Lisa M Maurer

    Full Text Available Picomolar concentrations of proteins comprising only the N-terminal 70-kDa region (70K of fibronectin (FN stimulate cell migration into collagen gels. The Ile-Gly-Asp (IGD motifs in four of the nine FN type 1 (FNI modules in 70K are important for such migratory stimulating activity. The 70K region mediates binding of nanomolar concentrations of intact FN to cell-surface sites where FN is assembled. Using baculovirus, we expressed wildtype 70K and 70K with Ile-to-Ala mutations in (3FNI and (5FNI; (7FNI and (9FNI; or (3FNI, (5FNI, (7FNI, and (9FNI. Wildtype 70K and 70K with Ile-to-Ala mutations were equally active in binding to assembly sites of FN-null fibroblasts. This finding indicates that IGD motifs do not mediate the interaction between 70K and the cell-surface that is important for FN assembly. Further, FN fragment N-(3FNIII, which does not stimulate migration, binds to assembly sites on FN-null fibroblast. The Ile-to-Ala mutations had effects on the structure of FNI modules as evidenced by decreases in abilities of 70K with Ile-to-Ala mutations to bind to monoclonal antibody 5C3, which recognizes an epitope in (9FNI, or to bind to FUD, a polypeptide based on the F1 adhesin of Streptococcus pyogenes that interacts with 70K by the β-zipper mechanism. These results suggest that the picomolar interactions of 70K with cells that stimulate cell migration require different conformations of FNI modules than the nanomolar interactions required for assembly.

  14. The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.

    Directory of Open Access Journals (Sweden)

    Luke J Alderwick

    2011-02-01

    Full Text Available The D-arabinan-containing polymers arabinogalactan (AG and lipoarabinomannan (LAM are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbC(CT encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM. Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985 at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT, linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis.

  15. Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

    OpenAIRE

    Gauer, Jacob W.; Knutson, Kristofer J.; Jaworski, Samantha R.; Rice, Anne M.; Rannikko, Anika M.; Lentz, Barry R.; Hinderliter, Anne

    2013-01-01

    Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturati...

  16. Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

    Energy Technology Data Exchange (ETDEWEB)

    Colson, Brett A.; Locher, Matthew R.; Bekyarova, Tanya; Patel, Jitandrakumar R.; Fitzsimons, Daniel P.; Irving, Thomas C.; Moss, Richard L. (IIT); (UW-MED)

    2010-05-25

    Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca{sup 2+} sensitivity of force (pCa{sub 50}), PKA treatment has been shown to decrease pCa{sub 50}, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca{sup 2+}-independent force and maximum Ca{sup 2+}-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I{sub 1,1}/I{sub 1,0}) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d{sub 1,0}) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I{sub 1,1}/I{sub 1,0} and, as hypothesized, treatment with MLCK also increased I{sub 1,1}/I{sub 1,0}, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by {approx}2 nm ({Delta} 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca{sup 2+} sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

  17. Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

    Science.gov (United States)

    Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B

    2013-12-06

    Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

  18. Crystal Structure of a Fibroblast Growth Factor Homologous Factor (FHF) Defines a Conserved Surface on FHFs for Binding and Modulation of Voltage-gated Sodium Channels

    Energy Technology Data Exchange (ETDEWEB)

    Goetz, R.; Dover, K; Laezza, F; Shtraizent, N; Huang, X; Tchetchik, D; Eliseenkova, A; Goldfarb, M; Mohammadi, M; et. al.

    2009-01-01

    Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav alpha subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs.

  19. A synthetic peptide derived from A1 module in CRD4 of human TNF receptor-1 inhibits binding and proinflammatory effect of human TNF-alpha.

    Science.gov (United States)

    Cao, Yingnan; Wang, Zhaohe; Bu, Xianzhang; Tang, Shu; Mei, Zhengrong; Liu, Peiqing

    2009-06-01

    Tumour necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine, which has been shown to be a causative factor in rheumatoid arthritis, inflammatory bowel disease and septic shock. Proinflammatory effect of TNF-alpha is activated mainly through human TNF receptor-1 (TNF-R1). However, the role of the fourth cystein-rich domain (CRD4) of TNF-R1 extracellular portion in the interaction of TNF-alpha with TNF-R1 is still unclear. In the present study, binding activity of TNF-alpha to TNF-R1 and protein levels of IkappaB-alpha and nuclear transcription factor kappa B (NF-kappaB) p65 subunit in HeLa cells were measured using enzyme-linked immunosorbent assay (ELISA) and western-blot analysis. Pep 3 (LRENECVS) which was derived from the hydrophilic region of A1 module in CRD4 remarkably inhibited the binding of TNF-alpha to TNF-R1, and also reversed TNF-alpha-induced degradation of IkappaB-alpha and nuclear translocation of NF-kappaB p65 subunit in HeLa cells. Our results confirmed that the hydrophilic region of A1 module in CRD4 participated in the interaction of TNF-alpha with TNF-R1, and demonstrated the potential of small-molecule TNF-alpha extracellular inhibitors targeting at A1 module in CRD4 of TNF-R1 in suppressing proinflammatory effect of TNF-alpha.

  20. Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

    Science.gov (United States)

    Gauer, Jacob W.; Knutson, Kristofer J.; Jaworski, Samantha R.; Rice, Anne M.; Rannikko, Anika M.; Lentz, Barry R.; Hinderliter, Anne

    2013-01-01

    Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response. PMID:23746516

  1. Role of fatty acid binding proteins and long chain fatty acids in modulating nuclear receptors and gene transcription.

    Science.gov (United States)

    Schroeder, Friedhelm; Petrescu, Anca D; Huang, Huan; Atshaves, Barbara P; McIntosh, Avery L; Martin, Gregory G; Hostetler, Heather A; Vespa, Aude; Landrock, Danilo; Landrock, Kerstin K; Payne, H Ross; Kier, Ann B

    2008-01-01

    Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 mircroM-mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-alpha (PPARalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha)] exhibit high affinity (low nM KdS) for LCFA (PPARalpha) and/or LCFA-CoA (PPARalpha, HNF4alpha)-in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARalpha, HNF4alpha, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that

  2. Probing the electrostatics and pharmacological modulation of sequence-specific binding by the DNA-binding domain of the ETS family transcription factor PU.1: a binding affinity and kinetics investigation.

    Science.gov (United States)

    Munde, Manoj; Poon, Gregory M K; Wilson, W David

    2013-05-27

    Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤10(5)M(-)(1)s(-)(1)), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>10(7)M(-)(1)s(-)(1)). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes.

  3. Potential energy surface and binding energy in the presence of an external electric field: modulation of anion-π interactions for graphene-based receptors.

    Science.gov (United States)

    Foroutan-Nejad, Cina; Marek, Radek

    2014-02-14

    Measuring the binding energy or scanning the potential energy surface (PES) of the charged molecular systems in the presence of an external electric field (EEF) requires a careful evaluation of the origin-dependency of the energy of the system and references. Scanning the PES for charged or purely ionic systems for obtaining the intrinsic energy barriers needs careful analysis of the electric work applied on ions by the EEF. The binding energy in the presence of an EEF is different from that in the absence of an electric field as the binding energy is an anisotropic characteristic which depends on the orientation of molecules with respect to the EEF. In this contribution we discuss various aspects of the PES and the concept of binding energy in the presence of an EEF. In addition, we demonstrate that the anion-π bonding properties can be modulated by applying a uniform EEF, which has a more pronounced effect on the larger, more polarizable π-systems. An analogous behavior is presumed for cation-π systems. We predict that understanding the phenomenon introduced in the present account has enormous potential, for example, for separating charged species on the surface of polarizable two-dimensional materials such as graphene or the surface of carbon nanotubes, in desalination of water.

  4. Expression of the C-terminal family 22 carbohydrate-binding module of xylanase 10B of Clostridium themocellum in tobacco plant

    OpenAIRE

    Olawole, O.

    2009-01-01

    Carbohydrate-binding modules have been shown to alter plant cell wall structural architecture. Hence, they have the potential application of being used to engineer the plant to produce tailor-made natural fibers in the cell wall. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22). The C-terminal CBM22-2 of the glycoside hydrolase (GH) 10 had been characterized to interact with xylan, a major hemicellulosic component in the secondary cell wall of ...

  5. Characterization of a modular enzyme of exo-1,5-alpha-L-arabinofuranosidase and arabinan binding module from Streptomyces avermitilis NBRC14893.

    Science.gov (United States)

    Ichinose, Hitomi; Yoshida, Makoto; Fujimoto, Zui; Kaneko, Satoshi

    2008-09-01

    A gene encoding an alpha-L-arabinofuranosidase, designated SaAraf43A, was cloned from Streptomyces avermitilis. The deduced amino acid sequence implies a modular structure consisting of an N-terminal glycoside hydrolase family 43 module and a C-terminal family 42 carbohydrate-binding module (CBM42). The recombinant enzyme showed optimal activity at pH 6.0 and 45 degrees C and was stable over the pH range of 5.0-6.5 at 30 degrees C. The enzyme hydrolyzed p-nitrophenol (PNP)-alpha-L-arabinofuranoside but did not hydrolyze PNP-alpha-L-arabinopyranoside, PNP-beta-D-xylopyranoside, or PNP-beta-D-galactopyranoside. Debranched 1,5-arabinan was hydrolyzed by the enzyme but arabinoxylan, arabinogalactan, gum arabic, and arabinan were not. Among the synthetic regioisomers of arabinofuranobiosides, only methyl 5-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside was hydrolyzed by the enzyme, while methyl 2-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside and methyl 3-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside were not. These data suggested that the enzyme only cleaves alpha-1,5-linked arabinofuranosyl linkages. The analysis of the hydrolysis product of arabinofuranopentaose suggested that the enzyme releases arabinose in exo-acting manner. These results indicate that the enzyme is definitely an exo-1,5-alpha-L-arabinofuranosidase. The C-terminal CBM42 did not show any affinity for arabinogalactan and debranched arabinan, although it bound arabinan and arabinoxylan, suggesting that the CBM42 bound to branched arabinofuranosyl residues. Removal of the module decreased the activity of the enzyme with regard to debranched arabinan. The CBM42 plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains.

  6. Cytoplasmic polyadenylation-element-binding protein (CPEB)1 and 2 bind to the HIF-1alpha mRNA 3'-UTR and modulate HIF-1alpha protein expression.

    Science.gov (United States)

    Hägele, Sonja; Kühn, Uwe; Böning, Melanie; Katschinski, Dörthe M

    2009-01-01

    The heterodimeric HIF (hypoxia-inducible factor)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homoeostasis. Besides the well described regulation of the HIF-1alpha subunit via hydroxylation-mediated protein stability in hypoxia, there are several indications of an additional translational control of the HIF-1alpha mRNA, especially after growth factor stimulation. We identified an interaction of CPEB (cytoplasmic polyadenylation-element-binding protein) 1 and CPEB2 with the 3'-UTR (untranslated region) of HIF-1alpha mRNA. Overexpression of CPEB1 and CPEB2 affected HIF-1alpha protein levels mediated by the 3'-UTR of HIF-1alpha mRNA. Stimulation of neuroblastoma SK-N-MC cells with insulin and thus activation of endogenous CPEBs increased the expression of a luciferase reporter gene fused to the 3'-UTR of HIF-1alpha as well as endogenous HIF-1alpha protein levels. This could be abrogated by treating the cells with CPEB1 or CPEB2 siRNAs (short interfering RNAs). Injection of HIF-1alpha cRNA into Xenopus oocytes verified the elongation of the poly(A)+ (polyadenylated) tail by cytoplasmic polyadenylation. Thus CPEB1 and CPEB2 are involved in the regulation of HIF-1alpha following insulin stimulation.

  7. The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.

    Science.gov (United States)

    Oeste, Clara L; Díez-Dacal, Beatriz; Bray, Francesca; García de Lacoba, Mario; de la Torre, Beatriz G; Andreu, David; Ruiz-Sánchez, Antonio J; Pérez-Inestrosa, Ezequiel; García-Domínguez, Carlota A; Rojas, José M; Pérez-Sala, Dolores

    2011-01-06

    Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2) selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2). Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.

  8. A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1.

    Science.gov (United States)

    García-Rodríguez, Luis J; De Piccoli, Giacomo; Marchesi, Vanessa; Jones, Richard C; Edmondson, Ricky D; Labib, Karim

    2015-10-15

    Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the 'alternative clamp loader' known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved 'Pol ϵ binding module' in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint.

  9. Acute handling disturbance modulates plasma insulin-like growth factor binding proteins in rainbow trout (Oncorhynchus mykiss)

    Science.gov (United States)

    The effects of acute stressor exposure on proximal (growth hormone; GH) and distal (insulin-like growth factor-I; IGF-I and IGF-binding proteins) components of the somatotropic axis are poorly understood in finfish. We exposed rainbow trout (Oncorhynchus mykiss) to a 5-minute handling disturbance to...

  10. Neuroplastin-65 and a mimetic peptide derived from its homophilic binding site modulate neuritogenesis and neuronal plasticity

    DEFF Research Database (Denmark)

    Owczarek, Sylwia; Soroka, Vladislav; Kiryushko, Darya;

    2011-01-01

    on fibroblast growth factor receptor, p38 mitogen-activated protein kinase, Ca(2+) /calmodulin-dependent protein kinase, and cytoplasmic Ca(2+) concentration. In addition, we show that interference with Np65 homophilic binding by enplastin has an inhibitory effect on Np65-mediated neurite outgrowth in vitro...

  11. Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module

    DEFF Research Database (Denmark)

    Salcini, A E; Confalonieri, S; Doria, M;

    1997-01-01

    EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct...

  12. Binding of a biosynthetic intermediate to AtrA modulates the production of lidamycin by Streptomyces globisporus.

    Science.gov (United States)

    Li, Xingxing; Yu, Tengfei; He, Qing; McDowall, Kenneth J; Jiang, Bingya; Jiang, Zhibo; Wu, Linzhuan; Li, Guangwei; Li, Qinglian; Wang, Songmei; Shi, Yuanyuan; Wang, Lifei; Hong, Bin

    2015-06-01

    The control of secondary production in streptomycetes involves the funneling of environmental and physiological signals to the cluster-situated (transcriptional) regulators (CSRs) of the biosynthetic genes. For some systems, the binding of biosynthetic products to the CSR has been shown to provide negative feedback. Here we show for the production of lidamycin (C-1027), a clinically relevant antitumor agent, by Streptomyces globisporus that negative feedback can extend to a point higher in the regulatory cascade. We show that the DNA-binding activity of the S. globisporus orthologue of AtrA, which was initially described as a transcriptional activator of actinorhodin biosynthesis in S. coelicolor, is inhibited by the binding of heptaene, a biosynthetic intermediate of lidamycin. Additional experiments described here show that S. globisporus AtrA binds in vivo as well as in vitro to the promoter region of the gene encoding SgcR1, one of the CSRs of lidamycin production. The feedback to the pleiotropic regulator AtrA is likely to provide a mechanism for coordinating the production of lidamycin with that of other secondary metabolites. The activity of AtrA is also regulated by actinorhodin. As AtrA is evolutionarily conserved, negative feedback of the type described here may be widespread within the streptomycetes.

  13. PACSINs bind to the TRPV4 cation channel. PACSIN 3 modulates the subcellular localization of TRPV4.

    NARCIS (Netherlands)

    Cuajungco, M.P.; Grimm, C.; Oshima, K.; D'hoedt, D.; Nilius, B.; Mensenkamp, A.R.; Bindels, R.J.M.; Plomann, M.; Heller, S.

    2006-01-01

    TRPV4 is a cation channel that responds to a variety of stimuli including mechanical forces, temperature, and ligand binding. We set out to identify TRPV4-interacting proteins by performing yeast two-hybrid screens, and we isolated with the avian TRPV4 amino terminus the chicken orthologues of mamma

  14. Humanized mAb H22 binds the human high affinity Fc receptor for IgG (FcgammaRI), blocks phagocytosis, and modulates receptor expression.

    Science.gov (United States)

    Wallace, P K; Keler, T; Coleman, K; Fisher, J; Vitale, L; Graziano, R F; Guyre, P M; Fanger, M W

    1997-10-01

    About 10-15% of patients with immune thrombocytopenic purpura (ITP) cannot be controlled by corticosteroid therapy and splenectomy. For these patients treatment with high-dose IVIgG induces partial or complete responses. The clinical benefits of IVIgG could be due to blockade of Fc receptors for IgG (FcgammaR), because several model systems clearly show that functional FcgammaR are essential for establishment of ITP and related diseases. However, the specific contributions of the three individual classes of FcgammaR remain to be more completely defined. Recently monoclonal antibody (mAb) H22, which recognizes an epitope on FcgammaRI (CD64) outside the ligand binding domain, was humanized by grafting its complementarity determining regions onto human IgG1 constant domains. Because FcgammaRI has a high affinity for human IgG1 antibodies, we predicted mAb H22 would also bind to FcgammaRI through its Fc domain and block FcgammaRI-mediated phagocytosis. These studies demonstrate that mAb H22 blocked phagocytosis of opsonized red blood cells 1000 times more effectively than an irrelevant IgG. Moreover, cross-linking FcgammaRI with mAb H22 rapidly down-modulated FcgammaRI expression on monocytes without affecting other surface antigens. We conclude that because mAb H22 is a humanized mAb that blocks the FcgammaRI ligand binding domain and down-modulates FcgammaRI expression, it is a particularly good candidate for evaluating the role of FcgammaRI in patients with ITP.

  15. Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

    Energy Technology Data Exchange (ETDEWEB)

    Weidmann, Chase A.; Qiu, Chen; Arvola, René M.; Lou, Tzu-Fang; Killingsworth, Jordan; Campbell, Zachary T.; Tanaka Hall, Traci M.; Goldstrohm, Aaron C.

    2016-08-02

    Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation byDrosophilaPumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulatedin vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

  16. Comparative biochemical characterization of peroxidases (class III) tightly bound to the maize root cell walls and modulation of the enzyme properties as a result of covalent binding.

    Science.gov (United States)

    Hadži-Tašković Šukalović, Vesna; Vuletić, Mirjana; Marković, Ksenija; Cvetić Antić, Tijana; Vučinić, Željko

    2015-01-01

    Comparative biochemical characterization of class III peroxidase activity tightly bound to the cell walls of maize roots was performed. Ionically bound proteins were solubilized from isolated walls by salt washing, and the remaining covalently bound peroxidases were released, either by enzymatic digestion or by a novel alkaline extraction procedure that released covalently bound alkali-resistant peroxidase enzyme. Solubilized fractions, as well as the salt-washed cell wall fragments containing covalently bound proteins, were analyzed for peroxidase activity. Peroxidative and oxidative activities indicated that peroxidase enzymes were predominately associated with walls by ionic interactions, and this fraction differs from the covalently bound one according to molecular weight, isozyme patterns, and biochemical parameters. The effect of covalent binding was evaluated by comparison of the catalytic properties of the enzyme bound to the salt-washed cell wall fragments with the corresponding solubilized and released enzyme. Higher thermal stability, improved resistance to KCN, increased susceptibility to H2O2, stimulated capacity of wall-bound enzyme to oxidize indole-3-acetic acid (IAA) as well as the difference in kinetic parameters between free and bound enzymes point to conformational changes due to covalent binding. Differences in biochemical properties of ionically and covalently bound peroxidases, as well as the modulation of the enzyme properties as a result of covalent binding to the walls, indicate that these two fractions of apoplastic peroxidases play different roles.

  17. Macro Domain from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Is an Efficient ADP-ribose Binding Module: CRYSTAL STRUCTURE AND BIOCHEMICAL STUDIES.

    Science.gov (United States)

    Cho, Chao-Cheng; Lin, Meng-Hsuan; Chuang, Chien-Ying; Hsu, Chun-Hua

    2016-03-01

    The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) encodes the conserved macro domain within non-structural protein 3. However, the precise biochemical function and structure of the macro domain is unclear. Using differential scanning fluorimetry and isothermal titration calorimetry, we characterized the MERS-CoV macro domain as a more efficient adenosine diphosphate (ADP)-ribose binding module than macro domains from other CoVs. Furthermore, the crystal structure of the MERS-CoV macro domain was determined at 1.43-Å resolution in complex with ADP-ribose. Comparison of macro domains from MERS-CoV and other human CoVs revealed structural differences in the α1 helix alters how the conserved Asp-20 interacts with ADP-ribose and may explain the efficient binding of the MERS-CoV macro domain to ADP-ribose. This study provides structural and biophysical bases to further evaluate the role of the MERS-CoV macro domain in the host response via ADP-ribose binding but also as a potential target for drug design.

  18. Heat Shock Protein 90 Modulates Lipid Homeostasis by Regulating the Stability and Function of Sterol Regulatory Element-binding Protein (SREBP) and SREBP Cleavage-activating Protein.

    Science.gov (United States)

    Kuan, Yen-Chou; Hashidume, Tsutomu; Shibata, Takahiro; Uchida, Koji; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2017-02-17

    Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis. SREBPs are synthesized as endoplasmic reticulum-bound precursors that require proteolytic activation in the Golgi apparatus. The stability and maturation of precursor SREBPs depend on their binding to SREBP cleavage-activating protein (SCAP), which escorts the SCAP-SREBP complex to the Golgi apparatus. In this study, we identified heat shock protein (HSP) 90 as a novel SREBP regulator that binds to and stabilizes SCAP-SREBP. In HepG2 cells, HSP90 inhibition led to proteasome-dependent degradation of SCAP-SREBP, which resulted in the down-regulation of SREBP target genes and the reduction in intracellular triglyceride and cholesterol levels. We also demonstrated in vivo that HSP90 inhibition decreased SCAP-SREBP protein, down-regulated SREBP target genes, and reduced lipids levels in mouse livers. We propose that HSP90 plays an indispensable role in SREBP regulation by stabilizing the SCAP-SREBP complex, facilitating the activation of SREBP to maintain lipids homeostasis.

  19. Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution.

    Science.gov (United States)

    Li, Shangyong; Yang, Xuemei; Bao, Mengmeng; Wu, Ying; Yu, Wengong; Han, Feng

    2015-05-01

    The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in alginate lyases has not been elucidated. A new alginate lyase gene, alyL2, was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The alginate lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide lyase (PL7) module. To better understand the function of CBM13 in alginate lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7-6.6 times those of AlyL2-CM at 30-50°C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in alginate lyase provides new insights into the degradation of alginate by marine microorganisms.

  20. Controlled Modulation of Serum Protein Binding and Biodistribution of Asymmetric Cyanine Dyes by Variation of the Number of Sulfonate Groups

    Directory of Open Access Journals (Sweden)

    Franziska M. Hamann

    2011-07-01

    Full Text Available To assess the suitability of asymmetric cyanine dyes for in vivo fluoro-optical molecular imaging, a comprehensive study on the influence of the number of negatively charged sulfonate groups governing the hydrophilicity of the DY-67x family of asymmetric cyanines was performed. Special attention was devoted to the plasma protein binding capacity and related pharmacokinetic properties. Four members of the DY-67x cyanine family composed of the same main chromophore, but substituted with a sequentially increasing number of sulfonate groups (n = 1−4; DY-675, DY-676, DY-677, DY-678, respectively, were incubated with plasma proteins dissolved in phosphate-buffered saline. Protein binding was assessed by absorption spectroscopy, gel electrophoresis, ultrafiltration, and dialysis. Distribution of dye in organs was studied by intraveneous injection of 62 nmol dye/kg body weight into mice (n = 12; up to 180 minutes postinjection using whole-body near-infrared fluorescence imaging. Spectroscopic studies, gel electrophoresis, and dialysis demonstrated reduced protein binding with increasing number of sulfonate groups. The bovine serum albumin binding constant of the most hydrophobic dye, DY-675, is 18 times higher than that of the most hydrophilic fluorophore, DY-678. In vivo biodistribution analysis underlined a considerable influence of dye hydrophilicity on biodistribution and excretion pathways, with the more hydrophobic dyes, DY-675 and DY-676, accumulating in the liver, followed by strong fluorescence signals in bile and gut owing to accumulation in feces and comparatively hydrophilic DY-678-COOH accumulating in the bladder. Our results demonstrate the possibility of selectively controlling dye-protein interactions and, thus, biodistribution and excretion pathways via proper choice of the fluorophore's substitution pattern. This underlines the importance of structure-property relationships for fluorescent labels. Moreover, our data could provide the

  1. Dynamics of starch granule biogenesis - the role of redox-regulated enzymes and low-affinity carbohydrate-binding modules

    DEFF Research Database (Denmark)

    Blennow, A.; Svensson, Birte

    2010-01-01

    The deposition and degradation of starch in plants is subject to extensive post-translational regulation. To permit degradation of B-type crystallites present in tuberous and leaf starch these starch types are phosphorylated by glucan, water dikinase (GWD). At the level of post-translational redo...... families and can enable diurnal dynamics of starch-enzyme recognition. Such diurnal changes in starch binding have been indicated for the redox-regulated GWD and SEX4....

  2. Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca(2+) channels.

    Science.gov (United States)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin; Korotchenko, Svetlana; Lievens, Patricia Marie-Jeanne; Berezin, Vladimir; Dityatev, Alexander

    2015-12-01

    Our previous studies revealed that L-type voltage-dependent Ca(2+) channels (Cav1.2 L-VDCCs) are modulated by the neural extracellular matrix backbone, polyanionic glycan hyaluronic acid. Here we used isothermal titration calorimetry and screened a set of peptides derived from the extracellular domains of Cav1.2α1 to identify putative binding sites between the channel and hyaluronic acid or another class of polyanionic glycans, such as heparin/heparan sulfates. None of the tested peptides showed detectable interaction with hyaluronic acid, but two peptides derived from the first pore-forming domain of Cav1.2α1 subunit bound to heparin. At 25 °C the binding of the peptide P7 (MGKMHKTCYN) was at ~50 μM, and that of the peptide P8 (GHGRQCQNGTVCKPGWDGPKHG) was at ~21 μM. The Cav1.2α1 first pore forming segment that contained both peptides maintained a high affinity for heparin (~23 μM), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin-agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1.2 channels revealed that enzymatic digestion of highly sulfated heparan sulfates with heparinase 1 affects neither voltage-dependence of channel activation nor the level of steady state inactivation, but did speed up channel inactivation. Treatment of hippocampal cultures with heparinase 1 reduced the firing rate and led to appearance of long-lasting bursts in the same manner as treatment with the inhibitor of L-VDCC diltiazem. Thus, heparan sulfate proteoglycans may bind to and regulate L-VDCC inactivation and network activity.

  3. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1

    Science.gov (United States)

    El-Diwany, Ramy; Mankowski, Madeleine C.; Wasilewski, Lisa N.; Brady, Jillian K.; Snider, Anna E.; Osburn, William O.; Murrell, Ben; Ray, Stuart C.

    2017-01-01

    Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes. PMID:28235087

  4. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1.

    Science.gov (United States)

    El-Diwany, Ramy; Cohen, Valerie J; Mankowski, Madeleine C; Wasilewski, Lisa N; Brady, Jillian K; Snider, Anna E; Osburn, William O; Murrell, Ben; Ray, Stuart C; Bailey, Justin R

    2017-02-01

    Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes.

  5. The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes

    DEFF Research Database (Denmark)

    Morrill, Johan; Kulcinskaja, Evelina; Sulewska, Anna Maria;

    2015-01-01

    β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here....... Surface plasmon resonance analysis confirmed the binding of the CBM10 to manno-oligosaccharides, albeit with slightly lower affinity than the catalytic module of the enzyme. This is the first example of a low-affinity mannan-specific CBM, which forms a subfamily of CBM10 together with close homologs...... by an exceptionally low Km and the presence of an atypical low affinity CBM, which increases binding to specifically to soluble mannan while causing minimal decrease in catalytic efficiency as opposed to enzymes with canonical mannan binding modules. These features highlight fine tuning of catalytic and binding...

  6. Chemokine binding protein M3 of murine gammaherpesvirus 68 modulates the host response to infection in a natural host.

    Directory of Open Access Journals (Sweden)

    David J Hughes

    2011-03-01

    Full Text Available Murine γ-herpesvirus 68 (MHV-68 infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus. Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology.

  7. Binding of Candida albicans to Human CEACAM1 and CEACAM6 Modulates the Inflammatory Response of Intestinal Epithelial Cells

    Science.gov (United States)

    Müller, Mario M.; Schäfer, Miriam R.; Clauder, Ann-Katrin; Feer, Sabina; Heyl, Kerstin A.; Stock, Magdalena; Klassert, Tilman E.; Zipfel, Peter F.; Singer, Bernhard B.

    2017-01-01

    ABSTRACT Candida albicans colonizes human mucosa, including the gastrointestinal tract, as a commensal. In immunocompromised patients, C. albicans can breach the intestinal epithelial barrier and cause fatal invasive infections. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a), CEACAM5 (CEA), and CEACAM6 (CD66c) are immunomodulatory receptors expressed on human mucosa and are recruited by bacterial and viral pathogens. Here we show for the first time that a fungal pathogen (i.e., C. albicans) also binds directly to the extracellular domain of human CEACAM1, CEACAM3, CEACAM5, and CEACAM6. Binding was specific for human CEACAMs and mediated by the N-terminal IgV-like domain. In enterocytic C2BBe1 cells, C. albicans caused a transient tyrosine phosphorylation of CEACAM1 and induced higher expression of membrane-bound CEACAM1 and soluble CEACAM6. Lack of the CEACAM1 receptor after short hairpin RNA (shRNA) knockdown abolished CXCL8 (interleukin-8) secretion by C2BBe1 cells in response to C. albicans. In CEACAM1-competent cells, the addition of recombinant soluble CEACAM6 reduced the C. albicans-induced CXCL8 secretion. PMID:28292985

  8. Chemokine Binding Protein M3 of Murine Gammaherpesvirus 68 Modulates the Host Response to Infection in a Natural Host

    Science.gov (United States)

    Hughes, David J.; Kipar, Anja; Leeming, Gail H.; Bennett, Elaine; Howarth, Deborah; Cummerson, Joanne A.; Papoula-Pereira, Rita; Flanagan, Brian F.; Sample, Jeffery T.; Stewart, James P.

    2011-01-01

    Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology. PMID:21445235

  9. Lipocalin 2 binds to membrane phosphatidylethanolamine to induce lipid raft movement in a PKA-dependent manner and modulates sperm maturation.

    Science.gov (United States)

    Watanabe, Hitomi; Takeo, Toru; Tojo, Hiromasa; Sakoh, Kazuhito; Berger, Thorsten; Nakagata, Naomi; Mak, Tak W; Kondoh, Gen

    2014-05-01

    Mammalian sperm undergo multiple maturation steps after leaving the testis in order to become competent for fertilization, but the molecular mechanisms underlying this process remain unclear. In terms of identifying factors crucial for these processes in vivo, we found that lipocalin 2 (Lcn2), which is known as an innate immune factor inhibiting bacterial and malarial growth, can modulate sperm maturation. Most sperm that migrated to the oviduct of wild-type females underwent lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding, which are signatures of sperm maturation, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine to reinforce lipid raft reorganization via a PKA-dependent mechanism and promotes sperm to acquire fertility by facilitating cholesterol efflux. These observations imply that mammals possess a mode for sperm maturation in addition to the albumin-mediated pathway.

  10. Carbohydrate Binding Module and Its Applications%碳水化合物结合结构域及其应用

    Institute of Scientific and Technical Information of China (English)

    周顺华

    2016-01-01

    碳水化合物结合结构域(Carbohydrate binding module),简称CBM.其是一种具有独立折叠结构的蛋白质.主要功能是特异性结合多糖(尤其不溶性多糖),并能通过4种机制来提高碳水化合物活性酶类的催化效率.其在生物工艺、环境的生物疗法、分析诊断工具及改善多糖水解酶酶特性上均具有广泛的应用.

  11. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Directory of Open Access Journals (Sweden)

    Qingxi Yue

    Full Text Available Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the

  12. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Science.gov (United States)

    Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

    2016-01-01

    Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K

  13. Characterization of an endo-processive-type xyloglucanase having a β-1,4-glucan-binding module and an endo-type xyloglucanase from Streptomyces avermitilis.

    Science.gov (United States)

    Ichinose, Hitomi; Araki, Yuko; Michikawa, Mari; Harazono, Koichi; Yaoi, Katsuro; Karita, Shuichi; Kaneko, Satoshi

    2012-11-01

    We cloned two glycoside hydrolase family 74 genes, the sav_1856 gene and the sav_2574 gene, from Streptomyces avermitilis NBRC14893 and characterized the resultant recombinant proteins. The sav_1856 gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while the sav_2574 gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. Both recombinant proteins were xyloglucanases. SaGH74A had optimal activity at 60°C and pH 5.5, while SaGH74B had optimal activity at 55°C and pH 6.0. SaGH74A was stable over a broad pH range (pH 4.5 to 9.0), whereas SaGH74B was stable over a relatively narrow pH range (pH 6.0 to 6.5). Analysis of the hydrolysis products of tamarind xyloglucan and xyloglucan-derived oligosaccharides indicated that SaGH74A was endo-processive, while SaGH74B was a typical endo-enzyme. The C terminus of SaGH74A, which was annotated as a carbohydrate-binding module, bound to β-1,4-linked glucan-containing soluble polysaccharides such as hydroxyethyl cellulose, barley glucan, and xyloglucan.

  14. Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation.

    Science.gov (United States)

    Feng, Jing; Yang, Weishan; Xie, Zili; Xiang, Fang; Cao, Zhijian; Li, Wenxin; Hu, Hongzhen; Chen, Zongyun; Wu, Yingliang

    2015-06-19

    Among the three extracellular domains of the tetrameric voltage-gated K(+) (Kv) channels consisting of six membrane-spanning helical segments named S1-S6, the functional role of the S1-S2 linker still remains unclear because of the lack of a peptide ligand. In this study, the Kv1.3 channel S1-S2 linker was reported as a novel receptor site for human β-defensin 2 (hBD2). hBD2 shifts the conductance-voltage relationship curve of the human Kv1.3 channel in a positive direction by nearly 10.5 mV and increases the activation time constant for the channel. Unlike classical gating modifiers of toxin peptides from animal venoms, which generally bind to the Kv channel S3-S4 linker, hBD2 only targets residues in both the N and C termini of the S1-S2 linker to influence channel gating and inhibit channel currents. The increment and decrement of the basic residue number in a positively charged S4 sensor of Kv1.3 channel yields conductance-voltage relationship curves in the positive direction by ∼31.2 mV and 2-4 mV, which suggests that positively charged hBD2 is anchored in the channel S1-S2 linker and is modulating channel activation through electrostatic repulsion with an adjacent S4 helix. Together, these findings reveal a novel peptide ligand that binds with the Kv channel S1-S2 linker to modulate channel activation. These findings also highlight the functional importance of the Kv channel S1-S2 linker in ligand recognition and modification of channel activation.

  15. Kr-pok increases FASN expression by modulating the DNA binding of SREBP-1c and Sp1 at the proximal promoter.

    Science.gov (United States)

    Jeon, Bu-Nam; Kim, Yeon-Sook; Choi, Won-Il; Koh, Dong-In; Kim, Min-Kyeong; Yoon, Jae-Hyeon; Kim, Min-Young; Hur, Benjamin; Paik, Philip Dong-Hyun; Hur, Man-Wook

    2012-04-01

    Kr-pok (kidney cancer-related POZ domain and Krüppel-like protein) is a new proto-oncogenic POZ-domain transcription factor. Fatty acid synthase gene (FASN) encodes one of the key enzymes in fatty acids synthesis and is the only enzyme that synthesizes fatty acids in cancer cells. Sp1 and SREBP-1c are the two major transcription activators of FASN. We investigated whether Kr-pok modulates transcription of the FASN. FASN expression is significantly decreased in Kr-pok knockout murine embryonic fibroblasts. Coimmunoprecipitation, GST fusion protein pull-down, and immunocytochemistry assays show that the zinc-finger domain of Kr-pok interacts directly with the bZIP DNA binding domain of SREBP-1. Electrophoretic mobility shift assay, oligonucleotide pull-down, and chromatin immunoprecipitation assays showed that Kr-pok changes the transcription factor binding dynamics of Sp1 and SREBP-1c to the SRE/E-box elements of the proximal promoter. We found that Kr-pok expression increased during 3T3-L1 preadipocyte differentiation and that FASN expression is decreased by the knockdown of Kr-pok. Kr-pok facilitates the SREBP-1c-mediated preadipocyte differentiation and/or fatty acid synthesis. Kr-pok may act as an important regulator of fatty acid synthesis and may induce rapid cancer cell proliferation by increasing palmitate synthesis.

  16. Treatment with Ginger Ameliorates Fructose-Induced Fatty Liver and Hypertriglyceridemia in Rats: Modulation of the Hepatic Carbohydrate Response Element-Binding Protein-Mediated Pathway

    Directory of Open Access Journals (Sweden)

    Huanqing Gao

    2012-01-01

    Full Text Available Ginger has been demonstrated to improve lipid derangements. However, its underlying triglyceride-lowering mechanisms remain unclear. Fructose overconsumption is associated with increase in hepatic de novo lipogenesis, thereby resulting in lipid derangements. Here we found that coadministration of the alcoholic extract of ginger (50 mg/kg/day, oral gavage, once daily over 5 weeks reversed liquid fructose-induced increase in plasma triglyceride and glucose concentrations and hepatic triglyceride content in rats. Plasma nonesterified fatty acid concentration was also decreased. Attenuation of the increased vacuolization and Oil Red O staining area was evident on histological examination of liver in ginger-treated rats. However, ginger treatment did not affect chow intake and body weight. Further, ginger treatment suppressed fructose-stimulated overexpression of carbohydrate response element-binding protein (ChREBP at the mRNA and protein levels in the liver. Consequently, hepatic expression of the ChREBP-targeted lipogenic genes responsible for fatty acid biosynthesis was also downregulated. In contrast, expression of neither peroxisome proliferator-activated receptor- (PPAR- alpha and its downstream genes, nor PPAR-gamma and sterol regulatory element-binding protein 1c was altered. Thus the present findings suggest that in rats, amelioration of fructose-induced fatty liver and hypertriglyceridemia by ginger treatment involves modulation of the hepatic ChREBP-mediated pathway.

  17. PARP1 promotes gene expression at the post-transcriptional level by modulating the RNA-binding protein HuR

    Science.gov (United States)

    Ke, Yueshuang; Han, Yanlong; Guo, Xiaolan; Wen, Jitao; Wang, Ke; Jiang, Xue; Tian, Xue; Ba, Xueqing; Boldogh, Istvan; Zeng, Xianlu

    2017-01-01

    Poly(ADP-ribosyl)ation (PARylation) is mainly catalysed by poly-ADP-ribose polymerase 1 (PARP1), whose role in gene transcription modulation has been well established. Here we show that, in response to LPS exposure, PARP1 interacts with the adenylateuridylate-rich element-binding protein embryonic lethal abnormal vision-like 1 (Elavl1)/human antigen R (HuR), resulting in its PARylation, primarily at site D226. PARP inhibition and the D226 mutation impair HuR's PARylation, nucleocytoplasmic shuttling and mRNA binding. Increases in mRNA level or stability of pro-inflammatory cytokines/chemokines are abolished by PARP1 ablation or inhibition, or blocked in D226A HuR-expressing cells. The present study demonstrates a mechanism to regulate gene expression at the post-transcriptional level, and suggests that blocking the interaction of PARP1 with HuR could be a strategy to treat inflammation-related diseases that involve increased mRNA stability. PMID:28272405

  18. White spot syndrome virus IE1 and WSV056 modulate the G1/S transition by binding to the host retinoblastoma protein.

    Science.gov (United States)

    Ran, Xiaozhuo; Bian, Xiaofang; Ji, Yongchang; Yan, Xiumin; Yang, Feng; Li, Fang

    2013-12-01

    DNA viruses often target cellular proteins to modulate host cell cycles and facilitate viral genome replication. However, whether proliferation of white spot syndrome virus (WSSV) requires regulation of the host cell cycle remains unclear. In the present study, we show that two WSSV paralogs, IE1 and WSV056, can interact with Litopenaeus vannamei retinoblastoma (Rb)-like protein (lv-RBL) through the conserved LxCxE motif. Further investigation revealed that IE1 and WSV056 could also bind to Drosophila retinoblastoma family protein 1 (RBF1) in a manner similar to how they bind to lv-RBL. Using the Drosophila RBF-E2F pathway as a model system, we demonstrated that both IE1 and WSV056 could sequester RBF1 from Drosophila E2F transcription factor 1 (E2F1) and subsequently activate E2F1 to stimulate the G1/S transition. Our findings provide the first evidence that WSSV may regulate cell cycle progression by targeting the Rb-E2F pathway.

  19. Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat

    Directory of Open Access Journals (Sweden)

    Muller Sylviane

    2008-07-01

    Full Text Available Abstract Background During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. Results To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20°C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37°C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. Conclusion Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.

  20. The structure of a Streptomyces avermitilis α-L-rhamnosidase reveals a novel carbohydrate-binding module CBM67 within the six-domain arrangement.

    Science.gov (United States)

    Fujimoto, Zui; Jackson, Adam; Michikawa, Mari; Maehara, Tomoko; Momma, Mitsuru; Henrissat, Bernard; Gilbert, Harry J; Kaneko, Satoshi

    2013-04-26

    α-L-rhamnosidases hydrolyze α-linked L-rhamnosides from oligosaccharides or polysaccharides. We determined the crystal structure of the glycoside hydrolase family 78 Streptomyces avermitilis α-L-rhamnosidase (SaRha78A) in its free and L-rhamnose complexed forms, which revealed the presence of six domains N, D, E, F, A, and C. In the ligand complex, L-rhamnose was bound in the proposed active site of the catalytic module, revealing the likely catalytic mechanism of SaRha78A. Glu(636) is predicted to donate protons to the glycosidic oxygen, and Glu(895) is the likely catalytic general base, activating the nucleophilic water, indicating that the enzyme operates through an inverting mechanism. Replacement of Glu(636) and Glu(895) resulted in significant loss of α-rhamnosidase activity. Domain D also bound L-rhamnose in a calcium-dependent manner, with a KD of 135 μm. Domain D is thus a non-catalytic carbohydrate binding module (designated SaCBM67). Mutagenesis and structural data identified the amino acids in SaCBM67 that target the features of L-rhamnose that distinguishes it from the other major sugars present in plant cell walls. Inactivation of SaCBM67 caused a substantial reduction in the activity of SaRha78A against the polysaccharide composite gum arabic, but not against aryl rhamnosides, indicating that SaCBM67 contributes to enzyme function against insoluble substrates.

  1. ERα dimerization: a key factor for the weak estrogenic activity of an ERα modulator unable to compete with estradiol in binding assays.

    Science.gov (United States)

    Leclercq, Guy; Laïos, Ioanna; Elie-Caille, Céline; Leiber, Denis; Laurent, Guy; Lesniewska, Eric; Tanfin, Zahra; Jacquot, Yves

    2017-04-01

    Estrothiazine (ESTZ) is a weak estrogen sharing structural similarities with coumestrol. ESTZ failed to compete with [(3)H]17β-estradiol ([(3)H]17β-E2) for binding to the estrogen receptor α (ERα), questioning its ability to interact with the receptor. However, detection by atomic force spectroscopy (AFS) of an ESTZ-induced ERα dimerization has eliminated any remaining doubts. The effect of the compound on the proliferation of ERα-positive and negative breast cancer cells confirmed the requirement of the receptor. The efficiency of ESTZ in MCF-7 cells was weak without any potency to modify the proliferation profile of estradiol and coumestrol. Growth enhancement was associated with a proteasomal degradation of ERα without substantial recruitment of LxxLL coactivators. This may be related to an unusual delay between the acquisition by the receptor of an ERE-binding capacity and the subsequent estrogen-dependent transcription. A complementary ability to enhance TPA-induced AP-1 transcription was observed, even at concentrations insufficient to activate the ERα, suggesting a partly independent mechanism. ESTZ also rapidly and transiently activated ERK1/2 likely through membrane estrogenic pathways provoking a reorganization of the actin network. Finally, the systematic absence of biological responses with an ESTZ derivative unable to induce ERα dimerization stresses the importance of this step in the action of the compound, as reported for conventional estrogens. In view of the existence of many other ERα modulators (endocrine disruptors such as, for example, pesticides, environmental contaminants or phytoestrogens) with extremely weak or similar apparent lack of binding ability, our work may appear as pilot investigation for assessing their mechanism of action.

  2. Estrogen modulates NFκB signaling by enhancing IκBα levels and blocking p65 binding at the promoters of inflammatory genes via estrogen receptor-β.

    Directory of Open Access Journals (Sweden)

    Dongqi Xing

    Full Text Available BACKGROUND: NFκB signaling is critical for expression of genes involved in the vascular injury response. We have shown that estrogen (17β-estradiol, E2 inhibits expression of these genes in an estrogen receptor (ER-dependent manner in injured rat carotid arteries and in tumor necrosis factor (TNF-α treated rat aortic smooth muscle cells (RASMCs. This study tested whether E2 inhibits NFκB signaling in RASMCs and defined the mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: TNF-α treated RASMCs demonstrated rapid degradation of IκBα (10-30 min, followed by dramatic increases in IκBα mRNA and protein synthesis (40-60 min. E2 enhanced TNF-α induced IκBα synthesis without affecting IκBα degradation. Chromatin immunoprecipitation (ChIP assays revealed that E2 pretreatment both enhanced TNF-α induced binding of NFκB p65 to the IκBα promoter and suppressed TNF-α induced binding of NFκB p65 to and reduced the levels of acetylated histone 3 at promoters of monocyte chemotactic protein (MCP-1 and cytokine-induced neutrophil chemoattractant (CINC-2β genes. ChIP analyses also demonstrated that ERβ can be recruited to the promoters of MCP-1 and CINC-2β during co-treatment with TNF-α and E2. CONCLUSIONS: These data demonstrate that E2 inhibits inflammation in RASMCs by two distinct mechanisms: promoting new synthesis of IκBα, thus accelerating a negative feedback loop in NFκB signaling, and directly inhibiting binding of NFκB to the promoters of inflammatory genes. This first demonstration of multifaceted modulation of NFκB signaling by E2 may represent a novel mechanism by which E2 protects the vasculature against inflammatory injury.

  3. Dissecting the functional significance of non-catalytic carbohydrate binding modules in the deconstruction of plant cell walls

    Energy Technology Data Exchange (ETDEWEB)

    Hahn, Michael G. [Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center

    2017-03-16

    The project seeks to investigate the mechanism by which CBMs potentiate the activity of glycoside hydrolases against complete plant cell walls. The project is based on the hypothesis that the wide range of CBMs present in bacterial enzymes maximize the potential target substrates by directing the cognate enzymes not only to different regions of a specific plant cell wall, but also increases the range of plant cell walls that can be degraded. In addition to maximizing substrate access, it was also proposed that CBMs can target specific subsets of hydrolases with complementary activities to the same region of the plant cell wall, thereby maximizing the synergistic interactions between these enzymes. This synergy is based on the premise that the hydrolysis of a specific polysaccharide will increase the access of closely associated polymers to enzyme attack. In addition, it is unclear whether the catalytic module and appended CBM of modular enzymes have evolved unique complementary activities.

  4. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

    Science.gov (United States)

    Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T; Ruggles, Kelly V; DeGiorgis, Joseph A; Kohlwein, Sepp D; Schon, Eric A; Sturley, Stephen L

    2015-11-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.

  5. Telomere-binding protein TPP1 modulates telomere homeostasis and confers radioresistance to human colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Lei Yang

    Full Text Available BACKGROUND: Radiotherapy is one of the major therapeutic strategies in cancer treatment. The telomere-binding protein TPP1 is an important component of the shelterin complex at mammalian telomeres. Our previous reports showed that TPP1 expression was elevated in radioresistant cells, but the exact effects and mechanisms of TPP1 on radiosensitivity is unclear. PRINCIPAL FINDINGS: In this study, we found that elevated TPP1 expression significantly correlated with radioresistance and longer telomere length in human colorectal cancer cell lines. Moreover, TPP1 overexpression showed lengthened telomere length and a significant decrease of radiosensitivity to X-rays. TPP1 mediated radioresistance was correlated with a decreased apoptosis rate after IR exposure. Furthermore, TPP1 overexpression showed prolonged G2/M arrest mediated by ATM/ATR-Chk1 signal pathway after IR exposure. Moreover, TPP1 overexpression accelerated the repair kinetics of total DNA damage and telomere dysfunction induced by ionizing radiation. CONCLUSIONS: We demonstrated that elevated expressions of TPP1 in human colorectal cancer cells could protect telomere from DNA damage and confer radioresistance. These results suggested that TPP1 may be a potential target in the radiotherapy of colorectal cancer.

  6. Binding of PAI-1 to endothelial cells stimulated by thymosin beta4 and modulation of their fibrinolytic potential.

    Science.gov (United States)

    Boncela, Joanna; Smolarczyk, Katarzyna; Wyroba, Elzbieta; Cierniewski, Czeslaw S

    2006-01-13

    Our previous studies showed that thymosin beta4 (Tbeta4) induced the synthesis of plasminogen activator inhibitor-1 (PAI-1) in cultured human umbilical vein endothelial cells (HUVECs) via the AP-1 dependent mechanism and its enhanced secretion. In this work we provide evidence that the released PAI-1 is accumulated on the surface of HUVECs, exclusively in its active form, in a complex with alpha1-acid glycoprotein (AGP) that is also up-regulated and released from the cells. This mechanism is supported by several lines of experiments, in which expression of both proteins was analyzed by flow cytometry and their colocalization supported by confocal microscopy. PAI-1 did not bind to quiescent cells but only to the Tbeta4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-alpha1-acid glycoprotein (AGP) without Tbeta4 treatment. The AGP.PAI-1 complex was accumulated essentially at the basal surface of endothelial cells, and such cells showed (a) morphology characteristic for strongly adhered and spread cells and (b) significantly reduced plasmin formation. Taken together, these results provide the evidence supporting a novel mechanism by which active PAI-1 can be bound to the Tbeta4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.

  7. Dopaminergic modulation of mitral cell activity in the frog olfactory bulb: a combined radioligand binding-electrophysiological study

    Energy Technology Data Exchange (ETDEWEB)

    Duchamp, A.; Moyse, E.; Delaleu, J.-C.; Coronas, V.; Duchamp-Viret, P. [Laboratoire de Physiologie Neurosensorielle, Universite Claude Bernard and CNRS, F69622 Villeurbanne (France)

    1997-04-28

    Dopamine content in the amphibian olfactory bulb is supplied by interneurons scattered among mitral cells in the external plexiform/mitral cell layer. In mammals, dopamine has been found to be involved in various aspects of bulbar information processing by influencing mitral cell odour responsiveness. Dopamine action in the bulb depends directly on the localization of its receptor targets, found to be mainly of the D{sub 2} type in mammals. The present study assessed, in the frog, both the anatomical localization of D{sub 2}-like, radioligand-labelled receptors of dopamine and the in vivo action of dopamine on unitary mitral cell activity in response to odours delivered over a wide range of concentrations. The [{sup 125}I]iodosulpride-labelled D{sub 2} binding sites were visualized on frozen sagittal sections of frog brains by film radioautography. The sites were found to be restricted to the external plexiform/mitral cell layer; other layers of the olfactory bulb were devoid of specific labelling. Electrophysiological recordings of mitral unit activity revealed that dopamine or its agonist apomorphine induced a drastic reduction of spontaneous firing rate of mitral cells in most cases without altering odour intensity coding properties of these cells. Moreover, pre-treatment with the D{sub 2} antagonist eticlopride blocked the dopamine-induced reduction of mitral cell spontaneous activity.In the frog olfactory bulb, both anatomical localization of D{sub 2}-like receptors and functional data on dopamine involvement in information processing differ from those reported in mammals. This suggests a phylogenetic evolution of dopamine action in the olfactory bulb. In the frog, anatomical data perfectly corroborate electrophysiological results, together strongly suggesting a direct action of dopamine on mitral cells. In a physiologically operating system, such an action would result in a global improvement of signal-to-noise ratio. (Copyright (c) 1997 Elsevier Science B

  8. Modulation of nuclear T3 binding by T3 in a human hepatocyte cell-line (Chang-liver) - T3 stimulation of cell growth but not of malic enzyme, glucose-6-phosphatdehydrogenase or 6-phosphogluconate-dehydrogenase

    DEFF Research Database (Denmark)

    Matzen, L E; Kristensen, S R; Kvetny, J

    1991-01-01

    The T3 modulation of nuclear T3 binding (NBT3), the T3 effect on cell growth, and the T3 and insulin effects on malic enzyme (ME), glucose-6-phosphat-dehydrogenase (G6PD) and 6-phosphogluconat-dehydrogenase (G6PD) were studied in a human hepatocyte cell-line (Chang-liver). T3 was bound to a high...

  9. von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII.

    Science.gov (United States)

    Sorvillo, Nicoletta; Hartholt, Robin B; Bloem, Esther; Sedek, Magdalena; ten Brinke, Anja; van der Zwaan, Carmen; van Alphen, Floris P; Meijer, Alexander B; Voorberg, Jan

    2016-03-01

    It has been proposed that von Willebrand factor might affect factor VIII immunogenicity by reducing factor VIII uptake by antigen presenting cells. Here we investigate the interaction of recombinant von Willebrand factor with immature monocyte-derived dendritic cells using flow cytometry and confocal microscopy. Surprisingly, von Willebrand factor was not internalized by immature dendritic cells, but remained bound to the cell surface. As von Willebrand factor reduces the uptake of factor VIII, we investigated the repertoire of factor VIII presented peptides when in complex with von Willebrand factor. Interestingly, factor VIII-derived peptides were still abundantly presented on major histocompatibility complex class II molecules, even though a reduction of factor VIII uptake by immature dendritic cells was observed. Inspection of peptide profiles from 5 different donors showed that different core factor VIII peptide sequences were presented upon incubation with factor VIII/von Willebrand factor complex when compared to factor VIII alone. No von Willebrand factor peptides were detected when immature dendritic cells were pulsed with different concentrations of von Willebrand factor, confirming lack of von Willebrand factor endocytosis. Several von Willebrand factor derived peptides were recovered when cells were pulsed with von Willebrand factor/factor VIII complex, suggesting that factor VIII promotes endocytosis of small amounts of von Willebrand factor by immature dendritic cells. Taken together, our results establish that von Willebrand factor is poorly internalized by immature dendritic cells. We also show that von Willebrand factor modulates the internalization and presentation of factor VIII-derived peptides on major histocompatibility complex class II.

  10. Binding and inhibition of drug transport proteins by heparin: a potential drug transporter modulator capable of reducing multidrug resistance in human cancer cells.

    Science.gov (United States)

    Chen, Yunliang; Scully, Michael; Petralia, Gloria; Kakkar, Ajay

    2014-01-01

    A major problem in cancer treatment is the development of resistance to chemotherapeutic agents, multidrug resistance (MDR), associated with increased activity of transmembrane drug transporter proteins which impair cytotoxic treatment by rapidly removing the drugs from the targeted cells. Previously, it has been shown that heparin treatment of cancer patients undergoing chemotherapy increases survival. In order to determine whether heparin is capable reducing MDR and increasing the potency of chemotherapeutic drugs, the cytoxicity of a number of agents toward four cancer cell lines (a human enriched breast cancer stem cell line, two human breast cancer cell lines, MCF-7 and MDA-MB-231, and a human lung cancer cell line A549) was tested in the presence or absence of heparin. Results demonstrated that heparin increased the cytotoxicity of a range of chemotherapeutic agents. This effect was associated with the ability of heparin to bind to several of the drug transport proteins of the ABC and non ABC transporter systems. Among the ABC system, heparin treatment caused significant inhibition of the ATPase activity of ABCG2 and ABCC1, and of the efflux function observed as enhanced intracellular accumulation of specific substrates. Doxorubicin cytoxicity, which was enhanced by heparin treatment of MCF-7 cells, was found to be under the control of one of the major non-ABC transporter proteins, lung resistance protein (LRP). LRP was also shown to be a heparin-binding protein. These findings indicate that heparin has a potential role in the clinic as a drug transporter modulator to reduce multidrug resistance in cancer patients.

  11. A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and displays altered DNA binding.

    Science.gov (United States)

    Gopalakrishnan, Suhasni; Van Emburgh, Beth O; Shan, Jixiu; Su, Zhen; Fields, C Robert; Vieweg, Johannes; Hamazaki, Takashi; Schwartz, Philip H; Terada, Naohiro; Robertson, Keith D

    2009-10-01

    DNA methylation is an epigenetic mark essential for mammalian development, genomic stability, and imprinting. DNA methylation patterns are established and maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has nearly 40 known splice variants expressed in a tissue- and disease-specific manner, but very little is known about the role of these splice variants in modulating DNMT3B function. We describe here the identification and characterization of a novel alternatively spliced form of DNMT3B lacking exon 5 within the NH(2)-terminal regulatory domain. This variant, which we term DNMT3B3Delta5 because it is closely related in structure to the ubiquitously expressed DNMT3B3 isoform, is highly expressed in pluripotent cells and brain tissue, is downregulated during differentiation, and is conserved in the mouse. Creation of pluripotent iPS cells from fibroblasts results in marked induction of DNMT3B3Delta5. DNMT3B3Delta5 expression is also altered in human disease, with tumor cell lines displaying elevated or reduced expression depending on their tissue of origin. We then compared the DNA binding and subcellular localization of DNMT3B3Delta5 versus DNMT3B3, revealing that DNMT3B3Delta5 possessed significantly enhanced DNA binding affinity and displayed an altered nuclear distribution. Finally, ectopic overexpression of DNMT3B3Delta5 resulted in repetitive element hypomethylation and enhanced cell growth in a colony formation assay. Taken together, these results show that DNMT3B3Delta5 may play an important role in stem cell maintenance or differentiation and suggest that sequences encoded by exon 5 influence the functional properties of DNMT3B.

  12. Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism.

    Science.gov (United States)

    James, P L; Stewart, C E; Rotwein, P

    1996-05-01

    The insulin-like growth factor binding proteins (IGFBPs) are a family of six secreted proteins which bind to and modulate the actions of insulin-like growth factors-I and -II (IGF-I and -II). IGFBP-5 is more conserved than other IGFBPs characterized to date, and is expressed in adult rodent muscle and in the developing myotome. We have shown previously that C2 myoblasts secrete IGFBP-5 as their sole IGFBP. Here we use these cells to study the function of IGFBP-5 during myogenesis, a process stimulated by IGFs. We stably transfected C2 cells with IGFBP-5 cDNAs under control of a constitutively active promoter. Compared with vector-transfected control cells, C2 myoblasts expressing the IGFBP-5 transgene in the sense orientation exhibit increased IGFBP-5 levels in the extracellular matrix during proliferation, and subsequently fail to differentiate normally, as assessed by both morphological and biochemical criteria. Compared to controls, IGFBP-5 sense myoblasts show enhanced survival in low serum medium, remaining viable for at least four weeks in culture. By contrast, myoblasts expressing the IGFBP-5 antisense transcript differentiate prematurely and more extensively than control cells. The inhibition of myogenic differentiation by high level expression of IGFBP-5 could be overcome by exogenous IGFs, with des (1-3) IGF-I, an analogue with decreased affinity for IGFBP-5 but normal affinity for the IGF-I receptor, showing the highest potency. These results are consistent with a model in which IGFBP-5 blocks IGF-stimulated myogenesis, and indicate that sequestration of IGFs in the extracellular matrix could be a possible mechanism of action. Our observations also suggest that IGFBP-5 normally inhibits muscle differentiation, and imply a role for IGFBP-5 in regulating IGF action during myogenic development in vivo.

  13. The expression of spinal methyl-CpG-binding protein 2, DNA methyltransferases and histone deacetylases is modulated in persistent pain states

    Directory of Open Access Journals (Sweden)

    Tochiki Keri K

    2012-02-01

    Full Text Available Abstract Background DNA CpG methylation is carried out by DNA methyltransferases and induces chromatin remodeling and gene silencing through a transcription repressor complex comprising the methyl-CpG-binding protein 2 (MeCP2 and a subset of histone deacetylases. Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA in the rat ankle joint. The aim of the present study was to analyse the changes in expression of MeCP2, DNA methyltransferases and a subset of histone deacetylases in the superficial dorsal horn during the maintenance phase of persistent pain states. In this process, the cell specific expression of MeCP2 was also investigated. Results Using immunohistochemistry, we found that neurones, oligodendrocytes and astrocytes expressed MeCP2. Microglia, oligodendrocyte precursor cells and Schwann cells never showed any positive stain for MeCP2. Quantitative analyses showed that MeCP2 expression was increased in the superficial dorsal horn 7 days following CFA injection in the ankle joint but decreased 7 days following spared nerve injury. Overall, the expression of DNA methyltransferases and a subset of histone deacetylases followed the same pattern of expression. However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint. Finally, the expression of MeCP2 was also down regulated in damaged dorsal root ganglion neurones following spared nerve injury. Conclusion Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

  14. Complement C3a binding to its receptor as a negative modulator of Th2 response in liver injury in trichloroethylene-sensitized mice.

    Science.gov (United States)

    Wang, Feng; Zha, Wan-sheng; Zhang, Jia-xiang; Li, Shu-long; Wang, Hui; Ye, Liang-ping; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

    2014-08-17

    Trichloroethylene (TCE) is a major occupational health hazard and causes occupational medicamentosa-like dermatitis (OMLDT) and liver damage. Recent evidence suggests immune response as a distinct mode of action for TCE-induced liver damage. This study aimed to explore the role of the key complement activation product C3a and its receptor C3aR in TCE-induced immune liver injury. A mouse model of skin sensitization was induced by TCE in the presence and absence of the C3aR antagonist SB 290157. Liver function was evaluated by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in conjunction with histopathological characterizations. C3a and C3aR were detected by immunohistochemistry and C5b-9 was assessed by immunofluorescence. IFN-γ and IL4 expressions were determined by flow cytometry and ELISA. The total sensitization rate was 44.1%. TCE sensitization caused liver cell necrosis and inflammatory infiltration, elevated serum ALT and AST, expression of C3a and C3aR, and deposition of C5b-9 in the liver. IFN-γ and IL-4 expressions were up-regulated in spleen mononuclear cells and their serum levels were also increased. Pretreatment with SB 290157 resulted in more inflammatory infiltration in the liver, higher levels of AST, reduced C3aR expression on Kupffer cells, and decreased IL-4 levels while IFN-γ remained unchanged. These data demonstrate that blocking of C3a binding to C3aR reduces IL4, shifts IFN-γ and IL-4 balance, and aggravates TCE-sensitization induced liver damage. These findings reveal a novel mechanism whereby modulation of Th2 response by C3a binding to C3a receptor contributes to immune-mediated liver damage by TCE exposure.

  15. A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation.

    Science.gov (United States)

    Maglinao, Maha; Eriksson, Magdalena; Schlegel, Mark K; Zimmermann, Stephanie; Johannssen, Timo; Götze, Sebastian; Seeberger, Peter H; Lepenies, Bernd

    2014-02-10

    Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties.

  16. Infiltration anesthetic lidocaine inhibits cancer cell invasion by modulating ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF).

    Science.gov (United States)

    Mammoto, Tadanori; Higashiyama, Shigeki; Mukai, Mutsuko; Mammoto, Akiko; Ayaki, Masako; Mashimo, Takashi; Hayashi, Yukio; Kishi, Yoshihiko; Nakamura, Hiroyuki; Akedo, Hitoshi

    2002-09-01

    Although the mechanism is unknown, infiltration anesthetics are believed to have membrane-stabilizing action. We report here that such a most commonly used anesthetic, lidocaine, effectively inhibited the invasive ability of human cancer (HT1080, HOS, and RPMI-7951) cells at concentrations used in surgical operations (5-20 mM). Ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) from the cell surface plays an important role in invasion by HT1080 cells. Lidocaine reduced the invasion ability of these cells by partly inhibiting the shedding of HB-EGF from the cell surface and modulation of intracellular Ca2+ concentration contributed to this action. The anesthetic action of lidocaine (sodium channel blocking ability) did not contribute to this anti-invasive action. In addition, lidocaine (5-30 mM), infiltrated around the inoculation site, inhibited pulmonary metastases of murine osteosarcoma (LM 8) cells in vivo. These data point to previously unrecognized beneficial actions of lidocaine and suggest that lidocaine might be an ideal infiltration anesthetic for surgical cancer operations.

  17. Transduction of the light signal during complementary chromatic adaptation in the cyanobacterium Calothrix sp. PCC 7601: DNA-binding proteins and modulation by phosphorylation.

    Science.gov (United States)

    Sobczyk, A; Schyns, G; Tandeau de Marsac, N; Houmard, J

    1993-03-01

    The cyanobacterium Calothrix sp. PCC 7601 can adapt its pigment content in response to changes in the incident light wavelength. It synthesizes, as major light-harvesting pigments, either phycocyanin 2 (PC2, encoded by the cpc2 operon) under red light or phycoerythrin (PE, encoded by the cpeBA operon) under green light conditions. The last step of the signal transduction pathway is characterized by a transcriptional control of the expression of these operons. Partially purified protein extracts were used in gel retardation assays and DNase I footprinting experiments to identify the factors that interact with the promoter region of the cpeBA operon. We found that two proteins, RcaA and RcaB, only detected in extracts of cells grown under green light, behave as positive transcriptional factors for the expression of the cpeBA operon. Treatment of the fractions containing RcaA and RcaB with alkaline phosphatase prevents the binding of RcaA but not of RcaB to the cpeBA promoter region. A post-translational modification of RcaA thus modulates its affinity for DNA.

  18. Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module.

    Science.gov (United States)

    Kurokawa, J; Hemjinda, E; Arai, T; Kimura, T; Sakka, K; Ohmiya, K

    2002-08-01

    The celT gene of Clostridium thermocellum strain F1 was found downstream of the mannanase gene man26B [Kurokawa J et al. (2001) Biosci Biotechnol Biochem 65:548-554] in pKS305. The open reading frame of celT consists of 1,833 nucleotides encoding a protein of 611 amino acids with a predicted molecular weight of 68,510. The mature form of CelT consists of a family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but lacks a family 3c carbohydrate-binding module (CBM) and an immunoglobulin (Ig)-like domain, which are often found with family 9 catalytic domains. CelT devoid of the dockerin domain (CelTDeltadoc) was constructed and purified from a recombinant Escherichia coli, and its enzyme properties were examined. CelTDeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan, and low activity toward xylan. The V(max) and K(m) values were 137 micro mol min(-1) mg(-1) and 16.7 mg/ml, respectively, for CMC. Immunological analysis indicated that CelT is a catalytic component of the C. thermocellum F1 cellulosome. This is the first report describing the characterization of a family 9 cellulase without an Ig-like domain or family 3c CBM.

  19. An H3K9/S10 methyl-phospho switch modulates Polycomb and Pol II binding at repressed genes during differentiation.

    Science.gov (United States)

    Sabbattini, Pierangela; Sjoberg, Marcela; Nikic, Svetlana; Frangini, Alberto; Holmqvist, Per-Henrik; Kunowska, Natalia; Carroll, Tom; Brookes, Emily; Arthur, Simon J; Pombo, Ana; Dillon, Niall

    2014-03-01

    Methylated histones H3K9 and H3K27 are canonical epigenetic silencing modifications in metazoan organisms, but the relationship between the two modifications has not been well characterized. H3K9me3 coexists with H3K27me3 in pluripotent and differentiated cells. However, we find that the functioning of H3K9me3 is altered by H3S10 phosphorylation in differentiated postmitotic osteoblasts and cycling B cells. Deposition of H3K9me3/S10ph at silent genes is partially mediated by the mitogen- and stress-activated kinases (MSK1/2) and the Aurora B kinase. Acquisition of H3K9me3/S10ph during differentiation correlates with loss of paused S5 phosphorylated RNA polymerase II, which is present on Polycomb-regulated genes in embryonic stem cells. Reduction of the levels of H3K9me3/S10ph by kinase inhibition results in increased binding of RNAPIIS5ph and the H3K27 methyltransferase Ezh1 at silent promoters. Our results provide evidence of a novel developmentally regulated methyl-phospho switch that modulates Polycomb regulation in differentiated cells and stabilizes repressed states.

  20. Analyzing radioligand binding data.

    Science.gov (United States)

    Motulsky, Harvey; Neubig, Richard

    2002-08-01

    Radioligand binding experiments are easy to perform, and provide useful data in many fields. They can be used to study receptor regulation, discover new drugs by screening for compounds that compete with high affinity for radioligand binding to a particular receptor, investigate receptor localization in different organs or regions using autoradiography, categorize receptor subtypes, and probe mechanisms of receptor signaling, via measurements of agonist binding and its regulation by ions, nucleotides, and other allosteric modulators. This unit reviews the theory of receptor binding and explains how to analyze experimental data. Since binding data are usually best analyzed using nonlinear regression, this unit also explains the principles of curve fitting with nonlinear regression.

  1. Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm

    DEFF Research Database (Denmark)

    Helledie, T; Antonius, M; Sorensen, R V;

    2000-01-01

    Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty...

  2. Galectin-3 silencing inhibits epirubicin-induced ATP binding cassette transporters and activates the mitochondrial apoptosis pathway via β-catenin/GSK-3β modulation in colorectal carcinoma.

    Directory of Open Access Journals (Sweden)

    Yung-Kuo Lee

    Full Text Available Multidrug resistance (MDR, an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp, MDR-associated protein (MRP 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.

  3. Double-stranded RNA-binding protein DRB3 negatively regulates anthocyanin biosynthesis by modulating PAP1 expression in Arabidopsis thaliana.

    Science.gov (United States)

    Sawano, Hikaru; Matsuzaki, Takuma; Usui, Tomoyuki; Tabara, Midori; Fukudome, Akihito; Kanaya, Akihiro; Tanoue, Daichi; Hiraguri, Akihiro; Horiguchi, Gorou; Ohtani, Misato; Demura, Taku; Kozaki, Toshinori; Ishii, Kazuo; Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2017-01-01

    The model plant Arabidopsis thaliana has five double-stranded RNA-binding proteins (DRB1-DRB5), two of which, DRB1 and DRB4, are well characterized. In contrast, the functions of DRB2, DRB3 and DRB5 have yet to be elucidated. In this study, we tried to uncover their functions using drb mutants and DRB-over-expressed lines. In over-expressed lines of all five DRB genes, the over-expression of DRB2 or DRB3 (DRB2ox or DRB3ox) conferred a downward-curled leaf phenotype, but the expression profiles of ten small RNAs were similar to that of the wild-type (WT) plant. Phenotypes were examined in response to abiotic stresses. Both DRB2ox and DRB3ox plants exhibited salt-tolerance. When these plants were exposed to cold stress, drb2 and drb3 over-accumulated anthocyanin but DRB2ox and DRB3ox did not. Therefore, the over-expression of DRB2 or DRB3 had pleiotropic effects on host plants. Microarray and deep-sequencing analyses indicated that several genes encoding key enzymes for anthocyanin biosynthesis, including chalcone synthase (CHS), dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS), were down-regulated in DRB3ox plants. When DRB3ox was crossed with the pap1-D line, which is an activation-tagged transgenic line that over-expresses the key transcription factor PAP1 (Production of anthocyanin pigmentation1) for anthocyanin biosynthesis, over-expression of DRB3 suppressed the expression of PAP1, CHS, DFR and ANS genes. DRB3 negatively regulates anthocyanin biosynthesis by modulating the level of PAP1 transcript. Since two different small RNAs regulate PAP1 gene expression, a possible function of DRB3 for small RNA biogenesis is discussed.

  4. MiRNA-205 modulates cellular invasion and migration via regulating zinc finger E-box binding homeobox 2 expression in esophageal squamous cell carcinoma cells

    Directory of Open Access Journals (Sweden)

    Yamashita Shunichi

    2011-03-01

    Full Text Available Abstract Background Esophageal squamous cell carcinoma (ESCC is often diagnosed at later stages until they are incurable. MicroRNA (miR is a small, non-coding RNA that negatively regulates gene expression mainly via translational repression. Accumulating evidence indicates that deregulation of miR is associated with human malignancies including ESCC. The aim of this study was to identify miR that could be specifically expressed and exert distinct biological actions in ESCC. Methods Total RNA was extracted from ESCC cell lines, OE21 and TE10, and a non-malignant human esophageal squamous cell line, Het-1A, and subjected to microarray analysis. Expression levels of miR that showed significant differences between the 2 ESCC and Het-1A cells based on the comprehensive analysis were analyzed by the quantitative reverse transcriptase (RT-PCR method. Then, functional analyses, including cellular proliferation, apoptosis and Matrigel invasion and the wound healing assay, for the specific miR were conducted. Using ESCC tumor samples and paired surrounding non-cancerous tissue obtained endoscopically, the association with histopathological differentiation was examined with quantitative RT-PCR. Results Based on the miR microarray analysis, there were 14 miRs that showed significant differences (more than 2-fold in expression between the 2 ESCC cells and non-malignant Het-1A. Among the significantly altered miRs, miR-205 expression levels were exclusively higher in 5 ESCC cell lines examined than any other types of malignant cell lines and Het-1A. Thus, miR-205 could be a specific miR in ESCC. Modulation of miR-205 expression by transfection with its precursor or anti-miR-205 inhibitor did not affect ESCC cell proliferation and apoptosis, but miR-205 was found to be involved in cell invasion and migration. Western blot revealed that knockdown of miR-205 expression in ESCC cells substantially enhanced expression of zinc finger E-box binding homeobox 2

  5. Broad distribution of ataxin 1 silencing in rhesus cerebella for spinocerebellar ataxia type 1 therapy.

    Science.gov (United States)

    Keiser, Megan S; Kordower, Jeffrey H; Gonzalez-Alegre, Pedro; Davidson, Beverly L

    2015-12-01

    Spinocerebellar ataxia type 1 is one of nine polyglutamine expansion diseases and is characterized by cerebellar ataxia and neuronal degeneration in the cerebellum and brainstem. Currently, there are no effective therapies for this disease. Previously, we have shown that RNA interference mediated silencing of ATXN1 mRNA provides therapeutic benefit in mouse models of the disease. Adeno-associated viral delivery of an engineered microRNA targeting ATXN1 to the cerebella of well-established mouse models improved motor phenotypes, neuropathy, and transcriptional changes. Here, we test the translatability of this approach in adult rhesus cerebella. Nine adult male and three adult female rhesus macaque were unilaterally injected with our therapeutic vector, a recombinant adeno-associated virus type 1 (rAAV1) expressing our RNAi trigger (miS1) and co-expressing enhanced green fluorescent protein (rAAV1.miS1eGFP) into the deep cerebellar nuclei using magnetic resonance imaging guided techniques combined with a Stealth Navigation system (Medtronics Inc.). Transduction was evident in the deep cerebellar nuclei, cerebellar Purkinje cells, the brainstem and the ventral lateral thalamus. Reduction of endogenous ATXN1 messenger RNA levels were ≥30% in the deep cerebellar nuclei, the cerebellar cortex, inferior olive, and thalamus relative to the uninjected hemisphere. There were no clinical complications, and quantitative and qualitative analyses suggest that this therapeutic intervention strategy and subsequent reduction of ATXN1 is well tolerated. Collectively the data illustrate the biodistribution and tolerability of rAAV1.miS1eGFP administration to the adult rhesus cerebellum and are supportive of clinical application for spinocerebellar ataxia type 1.

  6. Genome-Wide DNA Binding of GBF1 IsModulated by Its Heterodimerizing ProteinPartners, HY5 and HYH

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    Dear Editor, In today's post-genomic era where direct targets of manytranscription factors have been identified, it is becomingincreasingly evident that transcriptional networks are verycomplex. Heterodimerization of transcription factors is oneof the several methods by which these complex transcrip-tional networks are formed. By heterodimerization, DNA-binding specificity and affinity, transactivation properties,and ultimately cell physiology might be altered (Naar et al.,2001). The formation of heterodimers has the potential torecognize additional binding sites and increase the rangeof DNA-binding specificity (Foster et al., 1994). Further, het-erodimerization also allows the production of new proteinconfigurations. For example, the protein STF1 from soybeancan dimerize with GBF proteins and this dimerization bringstogether the acidic region from STF1 and the proline-richregion of the GBF proteins into one binding element (Cheonget al., 1998). These results highlight the importance and/orconsequences of heterodimerization of transcription factorsat particular locus. However, to understand the complex tran-scriptional networks, it is important to investigate that howheterodimerization affects the whole-genome-wide bind-ing and transcriptional properties of a transcription factor.Here in this study, we have investigated genome-wide DNAbinding of bZIP transcription factor GBF1, and analyzed theimportance of its heterodimerization with HY5 and HYH forits genome-wide binding. We have found that GBF1 bindingsites are enriched within the 1-kb regions upstream to thetranscription start sites of target genes. Moreover, the bind-ings of GBF1 to most of its targets are largely dependent onHY5, while HYH only affects the binding of GBF1 to somespecific sites.

  7. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

    Science.gov (United States)

    Freeman, N L; Lila, T; Mintzer, K A; Chen, Z; Pahk, A J; Ren, R; Drubin, D G; Field, J

    1996-02-01

    Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

  8. Binding of copper(II) ions to the polyproline II helices of PEVK modules of the giant elastic protein titin as revealed by ESI-MS, CD, and NMR.

    Science.gov (United States)

    Ma, Kan; Wang, Kuan

    2003-10-01

    Titin, a family of giant elastic proteins, constitutes an elastic sarcomere matrix in striated muscle. In the I-band region of the sarcomere, the titin PEVK segment acts as a molecular spring to generate elasticity as well as sites of adhesion with parallel thin filaments. Previously, we reported that PEVK consists of tandem repeats of 28 residue modules and that the "polyproline II-coil" motif is the fundamental conformational motif of the PEVK module. In order to characterize the factors that may affect and alter the PPII-coil conformational motifs, we have initiated a systematic study of the interaction with divalent cations (Cu2+, Ca2+, Zn2+, and Ni2+) and a conformational profile of PEVK peptides (a representative 28-mer peptide PR: PEPPKEVVPEKKAPVAPPKKPEVPPVKV and its subfragments PR1: kvPEPPKEVVPE, PR2: VPEKKAPVAPPK, PR3: KPEVPPVKV). UV-Vis absorption difference spectra and CD spectra showed that Cu2+ bound to PR1 with high affinity (20 microM), while its binding to PR2 and PR3 as well as the binding of other cations to all four peptides were of lower affinity (>100 microM). Conformational studies by CD revealed that Cu2+ binding to PR1 resulted in a polyproline II to turn transition up to a 1:2 PR1/Cu2+ ratio and a coil to turn transition at higher Cu2+ concentration. ESI-MS provided the stoichiometry of PEVK peptide-Cu2+ complexes at both low and high ion strength, confirming the specific high affinity binding of Cu2+ to PR1 and PR. Furthermore, NMR and ESI-MS/MS fragmentation analysis elucidated the binding sites of the PEVK peptide-Cu2+ complexes at (-2)KVPE2, 8VPE10, 13APV15, and 22EVP24. A potential application of Cu2+ binding in peptide sequencing by mass spectrometry was also revealed. We conclude that Cu2+ binds and bends PEVK peptides to a beta-turn-like structure at specific sites. The specific targeting of Cu2+ towards PPII is likely to be of significant value in elucidating the roles of PPII in titin elasticity as well as in interactions of

  9. Copper Binding and Subsequent Aggregation of α-Synuclein Are Modulated by N-Terminal Acetylation and Ablated by the H50Q Missense Mutation.

    Science.gov (United States)

    Mason, Rebecca J; Paskins, Aimee R; Dalton, Caroline F; Smith, David P

    2016-08-30

    The Parkinson's disease-associated protein α-synuclein exhibits significant conformational heterogeneity. Bacterially expressed α-synuclein is known to bind to copper, resulting in the formation of aggregation-prone compact conformations. However, in vivo, α-synuclein undergoes acetylation at its N-terminus. Here the effect of this modification and the pathological H50Q mutation on copper binding and subsequent conformational transitions were investigated by electrospray ionization-ion mobility spectrometry-mass spectrometry. We demonstrate that acetylation perturbs the ability of α-synuclein to bind copper and that the H50Q missense mutation in the presence of N-terminal acetylation prevents copper binding. These modifications and mutations prevent the formation of the most compact conformations and inhibit copper-induced aggregation.

  10. The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes

    DEFF Research Database (Denmark)

    Morrill, Johan; Kulcinskaja, Evelina; Sulewska, Anna Maria

    2015-01-01

    β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here......, we report the biochemical properties of the first family 5 subfamily 8 glycoside hydrolase (GH5_8) mannanase from the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 (BlMan5_8). BlMan5_8 possesses a novel low affinity carbohydrate binding module (CBM) specific for soluble mannan...

  11. The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes

    DEFF Research Database (Denmark)

    Morrill, Johan; Kulcinskaja, Evelina; Sulewska, Anna Maria;

    2015-01-01

    β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here......, we report the biochemical properties of the first family 5 subfamily 8 glycoside hydrolase (GH5_8) mannanase from the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 (BlMan5_8). BlMan5_8 possesses a novel low affinity carbohydrate binding module (CBM) specific for soluble mannan...

  12. Nicotinic stimulation modulates tyrosine hydroxylase mRNA half-life and protein binding to the 3'UTR in a manner that requires transcription.

    Science.gov (United States)

    Roe, David F; Craviso, Gale L; Waymire, Jack C

    2004-01-05

    Tyrosine hydroxylase (TH) expression increases in adrenal chromaffin cells treated with the nicotinic agonist, dimethylphenylpiperazinium (DMPP; 1 microM). We are using this response as a model of the changes in TH level that occur during increased cholinergic neural activity. Here we report a 4-fold increase in TH mRNA half-life in DMPP-treated cells chromaffin cells that is apparent when using a pulse-chase analysis to measure TH mRNA half-life. No increase is apparent using actinomycin D to measure half-life, indicating a requirement for ongoing transcription. Characterization of protein binding to the TH 3'UTR responsible for stabilization using labeled TH 3'UTR probes and electro-mobility shift assays shows the presence of two complexes both of which are increased by DMPP-treatment. The faster migrating complex (FMC) increases 2.5-fold and the slower migrating complex (SMC) increases 1.5-fold. Both changes are prevented by actinomycin D. Characterization of the protein binding to the TH UTR probes indicates SMC is disrupted by polyribonucleotides, poly (A) and poly (U), while binding to FMC is reduced by poly (CU). Separation of UV crosslinked RNA-protein complexes on SDS polyacrylamide gels shows FMC to contain a single protein whereas SMC contains three proteins. Northwesterns yielded similar results. Comparison of DMPP-induced protein binding with the poly C binding protein (PCBP) involved in hypoxia induced rat PC12 TH mRNA stability indicates none of the bovine UTR binding proteins are the PCBP. Thus, nicotinic stimulation produces a transcription-dependent increase in TH mRNA half-life that is mediated by previously unrecognized TH mRNA binding proteins.

  13. Distribution of a 69-kD laminin-binding protein in aortic and microvascular endothelial cells: modulation during cell attachment, spreading, and migration

    DEFF Research Database (Denmark)

    Yannariello-Brown, J; Wewer, U; Liotta, L;

    1988-01-01

    Affinity chromatography and immunolocalization techniques were used to investigate the mechanism(s) by which endothelial cells interact with the basement membrane component laminin. Bovine aortic endothelial cells (BAEC) membranes were solubilized and incubated with a laminin-Sepharose affinity...... column. SDS-PAGE analysis of the eluted proteins identified a 69-kD band as the major binding protein, along with minor components migrating at 125, 110, 92, 85, 75, 55, and 30 kD. Polyclonal antibodies directed against a peptide sequence of the 69-kD laminin-binding protein isolated from human tumor......, with a granular perinuclear distribution and in linear arrays throughout the cell. During migration a redistribution from diffuse to predominanately linear arrays that co-distributed with actin microfilaments was noted in double-label experiments. The 69-kD laminin-binding protein colocalized with actin filaments...

  14. ICAT Inhibits beta-Catenin Binding to Tcf/Lef-Family Transcription Factors and in the General Coactivator p300 Using Independent Structural Modules

    Energy Technology Data Exchange (ETDEWEB)

    Daniels, D. L.

    2002-01-01

    In the canonical Wnt signaling pathway, {beta}-catenin activates target genes through its interactions with Tcf/Lef-family transcription factors and additional transcriptional coactivators. The crystal structure of ICAT, an inhibitor of {beta}-catenin-mediated transcription, bound to the armadillo repeat domain of {beta}-catenin, has been determined. ICAT contains an N-terminal helilical domain that binds to repeats 11 and 12 of {beta}-catenin, and an extended C-terminal region that binds to repeats 5-10 in a manner similar that of Tcfs and other {beta}-catenin ligands. Full-length ICAT dissociates complexes of {beta}-catenin, Lef-1, and the transcriptional coactivator p300, whereas the helical domain alone selectively blocks binding to p300. The C-terminal armadillo repeats of {beta}-catenin may be an attractive target for compounds designed to disrupt aberrant {beta}-catenin-mediated transcription associated with various cancers.

  15. D1 dopamine receptor signaling is modulated by the R7 RGS protein EAT-16 and the R7 binding protein RSBP-1 in Caenoerhabditis elegans motor neurons.

    Directory of Open Access Journals (Sweden)

    Khursheed A Wani

    Full Text Available Dopamine signaling modulates voluntary movement and reward-driven behaviors by acting through G protein-coupled receptors in striatal neurons, and defects in dopamine signaling underlie Parkinson's disease and drug addiction. Despite the importance of understanding how dopamine modifies the activity of striatal neurons to control basal ganglia output, the molecular mechanisms that control dopamine signaling remain largely unclear. Dopamine signaling also controls locomotion behavior in Caenorhabditis elegans. To better understand how dopamine acts in the brain we performed a large-scale dsRNA interference screen in C. elegans for genes required for endogenous dopamine signaling and identified six genes (eat-16, rsbp-1, unc-43, flp-1, grk-1, and cat-1 required for dopamine-mediated behavior. We then used a combination of mutant analysis and cell-specific transgenic rescue experiments to investigate the functional interaction between the proteins encoded by two of these genes, eat-16 and rsbp-1, within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 act together to modulate dopamine signaling and that while they are coexpressed with both D1-like and D2-like dopamine receptors, they do not modulate D2 receptor signaling. Instead, EAT-16 and RSBP-1 act together to selectively inhibit D1 dopamine receptor signaling in cholinergic motor neurons to modulate locomotion behavior.

  16. D1 dopamine receptor signaling is modulated by the R7 RGS protein EAT-16 and the R7 binding protein RSBP-1 in Caenoerhabditis elegans motor neurons.

    Science.gov (United States)

    Wani, Khursheed A; Catanese, Mary; Normantowicz, Robyn; Herd, Muriel; Maher, Kathryn N; Chase, Daniel L

    2012-01-01

    Dopamine signaling modulates voluntary movement and reward-driven behaviors by acting through G protein-coupled receptors in striatal neurons, and defects in dopamine signaling underlie Parkinson's disease and drug addiction. Despite the importance of understanding how dopamine modifies the activity of striatal neurons to control basal ganglia output, the molecular mechanisms that control dopamine signaling remain largely unclear. Dopamine signaling also controls locomotion behavior in Caenorhabditis elegans. To better understand how dopamine acts in the brain we performed a large-scale dsRNA interference screen in C. elegans for genes required for endogenous dopamine signaling and identified six genes (eat-16, rsbp-1, unc-43, flp-1, grk-1, and cat-1) required for dopamine-mediated behavior. We then used a combination of mutant analysis and cell-specific transgenic rescue experiments to investigate the functional interaction between the proteins encoded by two of these genes, eat-16 and rsbp-1, within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 act together to modulate dopamine signaling and that while they are coexpressed with both D1-like and D2-like dopamine receptors, they do not modulate D2 receptor signaling. Instead, EAT-16 and RSBP-1 act together to selectively inhibit D1 dopamine receptor signaling in cholinergic motor neurons to modulate locomotion behavior.

  17. Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca2+ channels

    DEFF Research Database (Denmark)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin

    2015-01-01

    M), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin–agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1...... rate and led to appearance of long-lasting bursts in the same manner as treatment with the inhibitor of L-VDCC diltiazem. Thus, heparan sulfate proteoglycans may bind to and regulate L-VDCC inactivation and network activity....

  18. GTP is an allosteric modulator of the interaction between the guanylate-binding protein 1 and the prosurvival kinase PIM1.

    Science.gov (United States)

    Persico, Marco; Petrella, Lella; Orteca, Nausicaa; Di Dato, Antonio; Mariani, Marisa; Andreoli, Mirko; De Donato, Marta; Scambia, Giovanni; Novellino, Ettore; Ferlini, Cristiano; Fattorusso, Caterina

    2015-02-16

    GBP1 and PIM1 are known to interact with a molar ratio 1:1. GBP1:PIM1 binding initiates a signaling pathway that induces resistance to common chemotherapeutics such as paclitaxel. Since GBP1 is a large GTPase which undergoes conformational changes in a nucleotide-dependent manner, we investigated the effect of GTP/GDP binding on GBP1:PIM1 interaction by using computational and biological studies. It resulted that only GTP decreases the formation of the GBP1:PIM1 complex through an allosteric mechanism, putting the bases for the identification of new compounds potentially able to revert resistance to paclitaxel.

  19. Specific binding of photoaffinity-labeling peptidomimetics of Pro-Leu-Gly-NH2 to the dopamine D2L receptor: evidence for the allosteric modulation of the dopamine receptor.

    Science.gov (United States)

    Mann, Amandeep; Verma, Vaneeta; Basu, Dipannita; Skoblenick, Kevin J; Beyaert, Michael G R; Fisher, Abigail; Thomas, Nancy; Johnson, Rodney L; Mishra, Ram K

    2010-09-01

    The present study was undertaken to investigate the mechanistic role of l-prolyl-l-leucyl-glycinamide (PLG) in modulating agonist binding to the dopamine D(2L) receptor. Competition and displacement assays indicate that the photoaffinity-labeling peptidomimetics of PLG, 3(R)-[(4(S)-(4-azido-2-hydroxy-benzoyl) amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1a) and 3(R)-[(4(S)-(4-azido-2-hydroxy-5-iodo-benzoyl)amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1b) bind at the same site as PLG. Autoradiography was used to establish the covalent binding of [(125)I]-1b to an approximately 51kDa protein in bovine striatal membranes. Western blot analysis with a dopamine D(2L)-specific antibody, in combination with autoradiography, following a two-dimensional gel separation, suggested this approximately 51kDa protein to be the dopamine D(2L) receptor. Further evidence for binding of 1b to dopamine D(2L) was provided by samples immunoprecipitated with the D(2L) antibody. These samples were analyzed by western blotting in parallel with autoradiography of [(125)I]-1b labeled protein. Both methods revealed bands at approximately 51kDa. Furthermore, PLG is shown to compete with 1b for binding to the dopamine D(2L) receptor as determined by autoradiography, as well as competition experiments with PLG and 1a. Collectively, these findings suggest the successful development of a photoaffinity-labeling agent, compound 1b, that has been used to elucidate the interaction of PLG specifically with the dopamine D(2L) receptor.

  20. Study on Binding Specificity of Tt APuX21 Module for Polysaccharides%结构域Tt APuX21与碳水化合物的结合特性分析

    Institute of Scientific and Technical Information of China (English)

    谢晚彬

    2009-01-01

    This study aimed to investigate whether Tt APuX21 was a functional carbohydrate-binding module (CBM). In order to test whether Tt APuX21 possessed affinity for polysaccharides, non-denaturing affinity electrophoresis (NDAE) was carried out using different carbohydrates as ligands. NDAE displayed that Tt APuX21 had affinity for soluble starch, dextrin and pullulan. The results showed that Tt APuX21 was a functional carbohydrate-binding module.%以多种碳水化合物为底物,通过非变性亲和电泳(NDAE)来检验Tt APuX21是否能与碳水化合物结合,以证明Tt APuX21是否确为有功能的碳水化合物结合结构域.NDAE的分析结果表明,Tt A-PuX21能与水溶性淀粉、糊精和普鲁兰糖结合,Tt APuX21是一个有功能的碳水化合物结合结构域.

  1. Binding of Streptococcus pneumoniae endopeptidase O (PepO) to complement component C1q modulates the complement attack and promotes host cell adherence.

    Science.gov (United States)

    Agarwal, Vaibhav; Sroka, Magdalena; Fulde, Marcus; Bergmann, Simone; Riesbeck, Kristian; Blom, Anna M

    2014-05-30

    The Gram-positive species Streptococcus pneumoniae is a human pathogen causing severe local and life-threatening invasive diseases associated with high mortality rates and death. We demonstrated recently that pneumococcal endopeptidase O (PepO) is a ubiquitously expressed, multifunctional plasminogen and fibronectin-binding protein facilitating host cell invasion and evasion of innate immunity. In this study, we found that PepO interacts directly with the complement C1q protein, thereby attenuating the classical complement pathway and facilitating pneumococcal complement escape. PepO binds both free C1q and C1 complex in a dose-dependent manner based on ionic interactions. Our results indicate that recombinant PepO specifically inhibits the classical pathway of complement activation in both hemolytic and complement deposition assays. This inhibition is due to direct interaction of PepO with C1q, leading to a strong activation of the classical complement pathway, and results in consumption of complement components. In addition, PepO binds the classical complement pathway inhibitor C4BP, thereby regulating downstream complement activation. Importantly, pneumococcal surface-exposed PepO-C1q interaction mediates bacterial adherence to host epithelial cells. Taken together, PepO facilitates C1q-mediated bacterial adherence, whereas its localized release consumes complement as a result of its activation following binding of C1q, thus representing an additional mechanism of human complement escape by this versatile pathogen.

  2. Positive versus negative modulation of different endogenous chemokines for CC-chemokine receptor 1 by small molecule agonists through allosteric versus orthosteric binding

    DEFF Research Database (Denmark)

    Jensen, Pia C; Thiele, Stefanie; Ulven, Trond

    2008-01-01

    5 and not CCL3 activation is affected by substitutions in the main ligand binding pocket including the conserved GluVII:06 anchor point. A series of metal ion chelator complexes were found to act as full agonists on CCR1 and to be critically affected by the same substitutions in the main ligand...

  3. Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35 for gluco- and galacto- substituted mannans and its calcium induced stability.

    Directory of Open Access Journals (Sweden)

    Arabinda Ghosh

    Full Text Available This study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35 in polysaccharide recognition. CtCBM35 was cloned into pET28a (+ vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3 cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC. Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×10(4 M(-1, carob galactomannan (Ka = 12.4×10(4 M(-1 and negligible association (Ka = 12 µM(-1 with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca(2+ ion with konjac glucomannan (Ka = 41×10(4 M(-1 and carob galactomannan (Ka = 30×10(4 M(-1. The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (K a with konjac glucomannan and carob galactomannan, 14.3×10(4 M(-1 and 11.4×10(4 M(-1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG -25 kJ mole(-1 as compared to carob galactomannan (ΔG -22 kJ mole(-1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH as analysed by dynamic light scattering (DLS study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca(2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was

  4. Modulated Binding of SATB1, a Matrix Attachment Region Protein, to the AT-Rich Sequence Flanking the Major Breakpoint Region of BCL2

    Science.gov (United States)

    Ramakrishnan, Meera; Liu, Wen-Man; DiCroce, Patricia A.; Posner, Aleza; Zheng, Jian; Kohwi-Shigematsu, Terumi; Krontiris, Theodore G.

    2000-01-01

    The t(14,18) chromosomal translocation that occurs in human follicular lymphoma constitutively activates the BCL2 gene and disrupts control of apoptosis. Interestingly, 70% of the t(14,18) translocations are confined to three 15-bp clusters positioned within a 150-bp region (major breakpoint region or [MBR]) in the untranslated portion of terminal exon 3. We analyzed DNA-protein interactions in the MBR, as these may play some role in targeting the translocation to this region. An 87-bp segment (87MBR) immediately 3′ to breakpoint cluster 3 was essential for DNA-protein interaction monitored with mobility shift assays. We further delineated a core binding region within 87MBR: a 33-bp, very AT-rich sequence highly conserved between the human and mouse BCL2 gene (37MBR). We have purified and identified one of the core factors as the matrix attachment region (MAR) binding protein, SATB1, which is known to bind to AT-rich sequences with a high propensity to unwind. Additional factors in nuclear extracts, which we have not yet characterized further, increased SATB1 affinity for the 37MBR target four- to fivefold. Specific binding activity within 37MBR displayed cell cycle regulation in Jurkat T cells, while levels of SATB1 remained constant throughout the cell cycle. Finally, we demonstrated in vivo binding of SATB1 to the MBR, strongly suggesting the BCL2 major breakpoint region is a MAR. We discuss the potential consequences of our observations for both MBR fragility and regulatory function. PMID:10629043

  5. Modulation of ligand-heme reactivity by binding pocket residues demonstrated in cytochrome c' over the femtosecond-second temporal range.

    Science.gov (United States)

    Russell, Henry J; Hardman, Samantha J O; Heyes, Derren J; Hough, Michael A; Greetham, Gregory M; Towrie, Michael; Hay, Sam; Scrutton, Nigel S

    2013-12-01

    The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affinity for their chosen ligand, is fundamental to the existence of life. These processes are often controlled by precise structural arrangements in proteins, with heme pocket residues driving reactivity and specificity. One such protein is cytochrome c', which has the ability to bind nitric oxide (NO) and carbon monoxide (CO) on opposite faces of the heme, a property that is shared with soluble guanylate cycle. Like soluble guanylate cyclase, cytochrome c' also excludes O2 completely from the binding pocket. Previous studies have shown that the NO binding mechanism is regulated by a proximal arginine residue (R124) and a distal leucine residue (L16). Here, we have investigated the roles of these residues in maintaining the affinity for NO in the heme binding environment by using various time-resolved spectroscopy techniques that span the entire femtosecond-second temporal range in the UV-vis spectrum, and the femtosecond-nanosecond range by IR spectroscopy. Our findings indicate that the tightly regulated NO rebinding events following excitation in wild-type cytochrome c' are affected in the R124A variant. In the R124A variant, vibrational and electronic changes extend continuously across all time scales (from fs-s), in contrast to wild-type cytochrome c' and the L16A variant. Based on these findings, we propose a NO (re)binding mechanism for the R124A variant of cytochrome c' that is distinct from that in wild-type cytochrome c'. In the wider context, these findings emphasize the importance of heme pocket architecture in maintaining the reactivity of hemoproteins towards their chosen ligand, and demonstrate the power of spectroscopic probes spanning a wide temporal range.

  6. pRB binds to and modulates the transrepressing activity of the E1A-regulated transcription factor p120E4F

    NARCIS (Netherlands)

    Fajas, L.; Paul, C.; Zugasti, O.; Cam, L. Le; Polanowska, J.; Fabbrizio, E.; Medema, R.H.; Vignais, M.-L.; Sardet, C.

    2000-01-01

    The retinoblastoma protein pRB is involved in the transcriptional control of genes essential for cell cycle progression and differentiation. pRB interacts with different transcription factors and thereby modulates their activity by sequestration, corepression, or activation. We report that pRB, but

  7. A peptide motif from the second fibronectin module of the neural cell adhesion molecule, NCAM, NLIKQDDGGSPIRHY, is a binding site for the FGF receptor

    DEFF Research Database (Denmark)

    Jacobsen, Jacob Hedemand; Kiselyov, Vladislav; Bock, Elisabeth

    2008-01-01

    The mechanism of fibroblast growth factor receptor (FGFR) activation by the neural cell adhesion molecule (NCAM) is not well understood. A motif in the second NCAM fibronectin type III (FN3) module, termed FGL, has by means of nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) a...

  8. Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function.

    Science.gov (United States)

    Chen, D; Zhang, Z; Li, M; Wang, W; Li, Y; Rayburn, E R; Hill, D L; Wang, H; Zhang, R

    2007-08-01

    As a major negative regulator of p53, the MDM2 oncogene plays an important role in carcinogenesis and tumor progression. MDM2 promotes p53 proteasomal degradation and negatively regulates p53 function. The mechanisms by which the MDM2-p53 interaction is regulated are not fully understood, although several MDM2-interacting molecules have recently been identified. To search for novel MDM2-binding partners, we screened a human prostate cDNA library by the yeast two-hybrid assay using full-length MDM2 protein as the bait. Among the candidate proteins, ribosomal protein S7 was identified and confirmed as a novel MDM2-interacting protein. Herein, we demonstrate that S7 binds to MDM2, in vitro and in vivo, and that the interaction between MDM2 and S7 leads to modulation of MDM2-p53 binding by forming a ternary complex among MDM2, p53 and S7. This results in the stabilization of p53 protein through abrogation of MDM2-mediated p53 ubiquitination. Consequently, S7 overexpression increases p53 transactivational activities, induces apoptosis, and inhibits cell proliferation. The identification of S7 as a novel MDM2-interacting partner contributes to elucidation of the complex regulation of the MDM2-p53 interaction and has implications in cancer prevention and therapy.

  9. The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

    Directory of Open Access Journals (Sweden)

    John M Morrison

    2012-03-01

    Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

  10. DcR3 binds to ovarian cancer via heparan sulfate proteoglycans and modulates tumor cells response to platinum with corresponding alteration in the expression of BRCA1

    Directory of Open Access Journals (Sweden)

    Connor Joseph P

    2012-05-01

    Full Text Available Abstract Background Overcoming platinum resistance is a major obstacle in the treatment of Epithelial Ovarian Cancer (EOC. In our previous work Decoy Receptor 3 (DcR3 was found to be related to platinum resistance. The major objective of this work was to define the cellular interaction of DcR3 with EOC and to explore its effects on platinum responsiveness. Methods We studied cell lines and primary cultures for the expression of and the cells ability to bind DcR3. Cells were cultured with DcR3 and then exposed to platinum. Cell viability was determined by MTT assay. Finally, the cells molecular response to DcR3 was studied using real time RT-PCR based differential expression arrays, standard RT-PCR, and Western blot. Results High DcR3 in the peritoneal cavity of women with EOC is associated with significantly shorter time to first recurrence after platinum based therapy (p = 0.02. None-malignant cells contribute DcR3 in the peritoneal cavity. The cell lines studied do not secrete DcR3; however they all bind exogenous DcR3 to their surface implying that they can be effected by DcR3 from other sources. DcR3s protein binding partners are minimally expressed or negative, however, all cells expressed the DcR3 binding Heparan Sulfate Proteoglycans (HSPGs Syndecans-2, and CD44v3. DcR3 binding was inhibited by heparin and heparinase. After DcR3 exposure both SKOV-3 and OVCAR-3 became more resistant to platinum with 15% more cells surviving at high doses. On the contrary CaOV3 became more sensitive to platinum with 20–25% more cell death. PCR array analysis showed increase expression of BRCA1 mRNA in SKOV-3 and OVCAR-3 and decreased BRCA1 expression in CaOV-3 after exposure to DcR3. This was confirmed by gene specific real time PCR and Western blot analysis. Conclusions Non-malignant cells contribute to the high levels of DcR3 in ovarian cancer. DcR3 binds readily to EOC cells via HSPGs and alter their responsiveness to platinum chemotherapy. The

  11. The fibronectin-binding integrins alpha5beta1 and alphavbeta3 differentially modulate RhoA-GTP loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis

    DEFF Research Database (Denmark)

    Danen, Erik H J; Sonneveld, Petra; Brakebusch, Cord

    2002-01-01

    We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels...... receptors expressed on a cell dictates the ability of fibronectin to stimulate RhoA-mediated organization of cell matrix adhesions....

  12. sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis‐dependent release of eDNA

    DEFF Research Database (Denmark)

    Christner, Martin; Heinze, Constanze; Busch, Michael;

    2012-01-01

    Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant‐associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related...... virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed...

  13. Arabidopsis Sec1/Munc18 protein SEC11 is a competitive and dynamic modulator of SNARE binding and SYP121-dependent vesicle traffic.

    Science.gov (United States)

    Karnik, Rucha; Grefen, Christopher; Bayne, Robert; Honsbein, Annegret; Köhler, Tim; Kioumourtzoglou, Dimitrios; Williams, Mary; Bryant, Nia J; Blatt, Michael R

    2013-04-01

    The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual "handshaking" mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.

  14. A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and displays altered DNA binding

    OpenAIRE

    Gopalakrishnan, Suhasni; Van Emburgh, Beth O.; Shan, Jixiu; Su, Zhen; Fields, C. Robert; Vieweg, Johannes; Hamazaki, Takashi; Schwartz, Philip H; Terada, Naohiro; Robertson, Keith D.

    2009-01-01

    DNA methylation is an epigenetic mark essential for mammalian development, genomic stability, and imprinting. DNA methylation patterns are established and maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has nearly 40 known splice variants expressed in a tissue- and disease-specific manner, but very little is known about the role of these splice variants in modulating DNMT3B function. We describe her...

  15. Collagen and Stretch Modulate Autocrine Secretion of Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Proteins from Differentiated Skeletal Muscle Cells

    Science.gov (United States)

    Perrone, Carmen E.; Fenwick-Smith, Daniela; Vandenburgh, Herman H.

    1995-01-01

    Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micro-g of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.

  16. Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Julien

    Full Text Available New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664 reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.

  17. Feedback control of mammalian Hedgehog signaling by the Hedgehog-binding protein, Hip1, modulates Fgf signaling during branching morphogenesis of the lung

    OpenAIRE

    Chuang, Pao-Tien; Kawcak, T'Nay; McMahon, Andrew P.

    2003-01-01

    Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. A key issue is how negative regulation of Hh signaling might contribute to generating differential responses over tens of cell diameters. In cells that respond to Hh, two proteins that are up-regulated are Patched1 (Ptch1), the Hh receptor, a general target in both invertebrate and vertebrate organisms, and Hip1, a Hh-binding protein that is vertebrate specific. To address the developmental role of Hip1 i...

  18. Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene, GmDREB3, in soybean (Glycine max L.)

    OpenAIRE

    Chen, Ming; Xu, Zhaoshi; Xia, Lanqin; Li, Liancheng; Cheng, Xianguo; Dong, Jianhui; Wang, Qiaoyan; Ma, Youzhi

    2008-01-01

    DREB (dehydration-responsive element-binding protein) transcription factors have important roles in the stress-related regulation network in plants. A DREB orthologue, GmDREB3, belonging to the A-5 subgroup of the DREB subfamily, was isolated from soybean using the RACE (rapid amplification of cDNA ends) method. Northern blot analysis showed that expression of GmDREB3 in soybean seedlings was induced following cold stress treatment for 0.5 h and was not detected after 3 h. However, it was not...

  19. Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

    Science.gov (United States)

    Pal Sharma, C; Goldmann, Wolfgang H

    2004-01-01

    Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

  20. TBL2 is a novel PERK-binding protein that modulates stress-signaling and cell survival during endoplasmic reticulum stress.

    Directory of Open Access Journals (Sweden)

    Yoshinori Tsukumo

    Full Text Available Under ER stress, PKR-like ER-resident kinase (PERK phosphorylates translation initiation factor eIF2α, resulting in repression of global protein synthesis and concomitant upregulation of the translation of specific mRNAs such as activating transcription factor 4 (ATF4. This PERK function is important for cell survival under ER stress and poor nutrient conditions. However, mechanisms of the PERK signaling pathway are not thoroughly understood. Here we identify transducin (beta-like 2 (TBL2 as a novel PERK-binding protein. We found that TBL2 is an ER-localized type-I transmembrane protein and preferentially binds to the phosphorylated form of PERK, but not another eIF2α kinase GCN2 or ER-resident kinase IRE1, under ER stress. Immunoprecipitation analysis using various deletion mutants revealed that TBL2 interacts with PERK via the N-terminus proximal region and also associates with eIF2α via the WD40 domain. In addition, TBL2 knockdown can lead to impaired ATF4 induction under ER stress or poor nutrient conditions such as glucose and oxygen deprivation. Consistently, TBL2 knockdown rendered cells vulnerable to stresses similarly to PERK knockdown. Thus, TBL2 serves as a potential regulator of the PERK pathway.

  1. New modulated design and synthesis of chiral CuII/SnIV bimetallic potential anticancer drug entity: In vitro DNA binding and pBR322 DNA cleavage activity

    Science.gov (United States)

    Tabassum, Sartaj; Sharma, Girish Chandra; Arjmand, Farukh

    2012-05-01

    A new chiral ligand scaffold L derived from (R)-2-amino-2-phenyl ethanol and diethyl oxalate was isolated and thoroughly characterized by various spectroscopic methods. The ligand L was allowed to react with CuCl2·2H2O and NiCl2·6H2O to achieve monometallic complexes 1 and 2, respectively. Subsequently modulation of 1 and 2 was carried out in the presence of SnCl4·5H2O to obtain heterobimetallic potential drug candidates 3 and 4 possessing (CuII/SnIV and NiII/SnIV) metallic cores, respectively and characterized by elemental analysis and spectroscopic data including 1H, 13C and 119Sn NMR in case of 3 and 4. In vitro DNA binding studies revealed that complex 3 avidly binds to DNA as quantified by Kb and Ksv values. Complex 3 exhibits a remarkable DNA cleavage activity (concentration dependent) with pBR322 DNA and the cleavage activity of 3 was significantly enhanced in the presence of activators and follows the order H2O2 > Asc > MPA > GSH. Complex 3 cleave pBR322 DNA via hydrolytic pathway and accessible to major groove of DNA.

  2. Oleanolic Acid Diminishes Liquid Fructose-Induced Fatty Liver in Rats: Role of Modulation of Hepatic Sterol Regulatory Element-Binding Protein-1c-Mediated Expression of Genes Responsible for De Novo Fatty Acid Synthesis

    Directory of Open Access Journals (Sweden)

    Changjin Liu

    2013-01-01

    Full Text Available Oleanolic acid (OA, contained in more than 1620 plants and as an aglycone precursor for naturally occurred and synthesized triterpenoid saponins, is used in China for liver disorders in humans. However, the underlying liver-protecting mechanisms remain largely unknown. Here, we found that treatment of rats with OA (25 mg/kg/day, gavage, once daily over 10 weeks diminished liquid fructose-induced excess hepatic triglyceride accumulation without effect on total energy intake. Attenuation of the increased vacuolization and Oil Red O staining area was evident on histological examination of liver in OA-treated rats. Hepatic gene expression profile demonstrated that OA suppressed fructose-stimulated overexpression of sterol regulatory element-binding protein-(SREBP- 1/1c mRNA and nuclear protein. In accord, overexpression of SREBP-1c-responsive genes responsible for fatty acid synthesis was also downregulated. In contrast, overexpressed nuclear protein of carbohydrate response element-binding protein and its target genes liver pyruvate kinase and microsomal triglyceride transfer protein were not altered. Additionally, OA did not affect expression of peroxisome proliferator-activated receptor-gamma- and -alpha and their target genes. It is concluded that modulation of hepatic SREBP-1c-mediated expression of the genes responsible for de novo fatty acid synthesis plays a pivotal role in OA-elicited diminishment of fructose-induced fatty liver in rats.

  3. Curdlan sulphate modulates protein synthesis and enhances NF-κB and C/EBP binding activity in HepG2 cells

    Directory of Open Access Journals (Sweden)

    A. Guzdek

    1997-01-01

    Full Text Available In human hepatoma HepG2 cell line curdlan sulphate enhances basal and interleukin-6-stimulated fibrinogen and antichymotrypsin (ACT synthesis, slightly increases basal ceruloplasmin production and exerts only minor effects on alpha-1-proteinase inhibitor and transferrin. Curdlan sulphate may, at least in part, affect protein synthesis at a pretranslational level, as the expression of ACT mRNA was found to be increased, whereas intracellular enzyme, manganese superoxide dismutase mRNA level was decreased in the cell culture treated with curdlan sulphate. Gel mobility shift analysis revealed that curdlan sulphate increases the DNA binding activity of NF-κB and C/EBP, suggesting that these transcription factors may participate in the regulatory effects of curdlan sulphate in HepG2 cells.

  4. C-type lectin-like domain and fibronectin-like type II domain of phospholipase A(2) receptor 1 modulate binding and migratory responses to collagen.

    Science.gov (United States)

    Takahashi, Soichiro; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-ei; Kugiyama, Kiyotaka

    2015-03-24

    Phospholipase A2 receptor 1 (PLA2R) mediates collagen-dependent migration. The mechanisms by which PLA2R interacts with collagen remain unclear. We produced HEK293 cells expressing full-length wild-type PLA2R or a truncated PLA2R that lacks fibronectin-like type II (FNII) domains or several regions of C-type lectin-like domain (CTLD). We show that the CTLD1-2 as well as the FNII domain of PLA2R are responsible for binding to collagen and for collagen-dependent migration. Thus, multiple regions and domains of the extracellular portion of PLA2R participate in the responses to collagen. These data suggest a potentially new mechanism for PLA2R-mediated biological response beyond that of a receptor for secretory PLA2.

  5. Modulation of calcium oxalate dihydrate growth by selective crystal-face binding of phosphorylated osteopontin and polyaspartate peptide showing occlusion by sectoral (compositional) zoning.

    Science.gov (United States)

    Chien, Yung-Ching; Masica, David L; Gray, Jeffrey J; Nguyen, Sarah; Vali, Hojatollah; McKee, Marc D

    2009-08-28

    Calcium oxalate dihydrate (COD) mineral and the urinary protein osteopontin/uropontin (OPN) are commonly found in kidney stones. To investigate the effects of OPN on COD growth, COD crystals were grown with phosphorylated OPN or a polyaspartic acid-rich peptide of OPN (DDLDDDDD, poly-Asp(86-93)). Crystals grown with OPN showed increased dimensions of the {110} prismatic faces attributable to selective inhibition at this crystallographic face. At high concentrations of OPN, elongated crystals with dominant {110} faces were produced, often with intergrown, interpenetrating twin crystals. Poly-Asp(86-93) dose-dependently elongated crystal morphology along the {110} faces in a manner similar to OPN. In crystal growth studies using fluorescently tagged poly-Asp(86-93) followed by imaging of crystal interiors using confocal microscopy, sectoral (compositional) zoning in COD was observed resulting from selective binding and incorporation (occlusion) of peptide exclusively into {110} crystal sectors. Computational modeling of poly-Asp(86-93) adsorption to COD {110} and {101} surfaces also suggests increased stabilization of the COD {110} surface and negligible change to the natively stable {101} surface. Ultrastructural, colloidal-gold immunolocalization of OPN by transmission electron microscopy in human stones confirmed an intracrystalline distribution of OPN. In summary, OPN and its poly-Asp(86-93) sequence similarly affect COD mineral growth; the {110} crystallographic faces become enhanced and dominant attributable to {110} face inhibition by the protein/peptide, and peptides can incorporate into the mineral phase. We, thus, conclude that the poly-Asp(86-93) domain is central to the OPN ability to interact with the {110} faces of COD, where it binds to inhibit crystal growth with subsequent intracrystalline incorporation (occlusion).

  6. Multi-organ expression profiling uncovers a gene module in coronary artery disease involving transendothelial migration of leukocytes and LIM domain binding 2: the Stockholm Atherosclerosis Gene Expression (STAGE study.

    Directory of Open Access Journals (Sweden)

    Sara Hägg

    2009-12-01

    Full Text Available Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD. The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE study was to determine whether there are functionally associated genes (rather than individual genes important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue and atherosclerotic and unaffected arterial wall (n = 40/tissue isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes. In the second step (performed within tissue clusters, one atherosclerotic lesion (n = 49/48 and one visceral fat (n = 59 cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015. The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54 relating to carotid stenosis (P = 0.04, 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10(-27 and-30. Genes in the transendothelial migration of leukocytes (TEML pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module. In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004. The transcription co-factor LIM domain binding 2 (LDB2 was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2

  7. Carbon monoxide binding to the heme group at the dimeric interface modulates structure and copper accessibility in the Cu,Zn superoxide dismutase from Haemophilus ducreyi: in silico and in vitro evidences.

    Science.gov (United States)

    Chillemi, Giovanni; De Santis, Serena; Falconi, Mattia; Mancini, Giordano; Migliorati, Valentina; Battistoni, Andrea; Pacello, Francesca; Desideri, Alessandro; D'Angelo, Paola

    2012-01-01

    X-ray absorption near-edge structure (XANES) spectroscopy and molecular dynamics (MD) simulations have been jointly applied to the study of the Cu,Zn superoxide dismutase from Haemophilus ducreyi (HdSOD) in interaction with the carbon monoxide molecule. The configurational flexibility of the Fe(II)-heme group, intercalated between the two subunits, has been sampled by MD simulations and included in the XANES data analysis without optimization in the structural parameter space. Our results provide an interpretation of the observed discrepancy in the Fe-heme distances as detected by extended X-ray absorption fine structure (EXAFS) spectroscopy and the classical XANES analysis, in which the structural parameters are optimized in a unique structure. Moreover, binding of the CO molecule to the heme induces a long range effect on the Cu,Zn active site, as evidenced by both MD simulations and in vitro experiments. MD simulation of the CO bound system, in fact, highlighted a structural rearrangement of the protein-protein hydrogen bond network in the region of the Cu,Zn active site, correlated with an increase in water accessibility at short distance from the copper atom. In line, in vitro experiments evidenced an increase of copper accessibility to a chelating agent when the CO molecule binds to the heme group, as compared to a heme deprived HdSOD. Altogether, our results support the hypothesis that the HdSOD is a heme-sensor protein, in which binding to small gaseous molecules modulates the enzyme superoxide activity as an adaptive response to the bacterial environment.

  8. PPIP5K1 modulates ligand competition between diphosphoinositol polyphosphates and PtdIns(3,4,5)P3 for polyphosphoinositide-binding domains.

    Science.gov (United States)

    Gokhale, Nikhil A; Zaremba, Angelika; Janoshazi, Agnes K; Weaver, Jeremy D; Shears, Stephen B

    2013-08-01

    We describe new signalling consequences for PPIP5K1 (diphosphoinositol pentakisphosphate kinase type 1)-mediated phosphorylation of InsP6 and 5-InsP7 to 1-InsP7 and InsP8. In NIH 3T3 cells, either hyperosmotic stress or receptor activation by PDGF (platelet-derived growth factor) promoted translocation of PPIP5K1 from the cytoplasm to the plasma membrane. The PBD1 (polyphosphoinositide-binding domain) in PPIP5K1 recapitulated that translocation. Mutagenesis of PBD1 to reduce affinity for PtdIns(3,4,5)P3 prevented translocation. Using surface plasmon resonance, we found that PBD1 association with vesicular PtdIns(3,4,5)P3 was inhibited by InsP6 and diphosphoinositol polyphosphates. However, the inhibition by PPIP5K1 substrates (IC50: 5-InsP7=5 μM and InsP6=7 μM) was substantially more potent than that of the PPIP5K1 products (IC50: InsP8=32 μM and 1-InsP7=43 μM). This rank order of ligand competition with PtdIns(3,4,5)P3 was also exhibited by the PH (pleckstrin homology) domains of Akt (also known as protein kinase B), GRP1 (general receptor for phosphoinositides 1) and SIN1 (stress-activated protein kinase-interaction protein 1). We propose that, in vivo, PH domain binding of InsP6 and 5-InsP7 suppresses inappropriate signalling ('noise') from stochastic increases in PtdIns(3,4,5)P3. That restraint may be relieved by localized depletion of InsP6 and 5-InsP7 at the plasma membrane following PPIP5K1 recruitment. We tested this hypothesis in insulin-stimulated L6 myoblasts, using mTOR (mechanistic/mammalian target of rapamycin)-mediated phosphorylation of Akt on Ser473 as a readout for SIN1-mediated translocation of mTORC (mTOR complex) 2 to the plasma membrane [Zoncu, Efeyan and Sabatini (2011) Nat. Rev. Mol. Cell Biol. 12, 21-35]. Knockdown of PPIP5K1 expression was associated with a 40% reduction in Ser473 phosphorylation. A common feature of PtdIns(3,4,5)P3-based signalling cascades may be their regulation by PPIP5K1.

  9. Retinoids modulate expression of the endocytic partners megalin, cubilin, and disabled-2 and uptake of vitamin D-binding protein in human mammary cells.

    Science.gov (United States)

    Chlon, Timothy M; Taffany, David A; Welsh, Joellen; Rowling, Matthew J

    2008-07-01

    The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-trans-retinoic acid (RA) in T-47D human breast cancer cells and that RA-treated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells.

  10. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination.

    Science.gov (United States)

    Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R; Guerrero, Justin A; Shen, Tian; Casali, Paolo

    2017-02-08

    Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S-S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52(+/+) counterparts, which display normal CSR, Rad52(-/-) B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S-S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S-S recombination, as emphasized by the significantly greater CSR reduction in Rad52(-/-) versus Rad52(+/+) B cells on Ku86 knockdown.

  11. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

    Science.gov (United States)

    Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R.; Guerrero, Justin A.; Shen, Tian; Casali, Paolo

    2017-01-01

    Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S–S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52+/+ counterparts, which display normal CSR, Rad52−/− B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S–S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S–S recombination, as emphasized by the significantly greater CSR reduction in Rad52−/− versus Rad52+/+ B cells on Ku86 knockdown. PMID:28176781

  12. Pathways for Modulating Exosome Lipids Identified By High-Density Lipoprotein-Like Nanoparticle Binding to Scavenger Receptor Type B-1.

    Science.gov (United States)

    Angeloni, Nicholas L; McMahon, Kaylin M; Swaminathan, Suchitra; Plebanek, Michael P; Osman, Iman; Volpert, Olga V; Thaxton, C Shad

    2016-03-11

    Exosomes are produced by cells to mediate intercellular communication, and have been shown to perpetuate diseases, including cancer. New tools are needed to understand exosome biology, detect exosomes from specific cell types in complex biological media, and to modify exosomes. Our data demonstrate a cellular pathway whereby membrane-bound scavenger receptor type B-1 (SR-B1) in parent cells becomes incorporated into exosomes. We tailored synthetic HDL-like nanoparticles (HDL NP), high-affinity ligands for SR-B1, to carry a fluorescently labeled phospholipid. Data show SR-B1-dependent transfer of the fluorescent phospholipid from HDL NPs to exosomes. Modified exosomes are stable in serum and can be directly detected using flow cytometry. As proof-of-concept, human serum exosomes were found to express SR-B1, and HDL NPs can be used to label and isolate them. Ultimately, we discovered a natural cellular pathway and nanoparticle-receptor pair that enables exosome modulation, detection, and isolation.

  13. Src-Like adaptor protein (SLAP) binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

    Science.gov (United States)

    Kazi, Julhash U; Rönnstrand, Lars

    2012-01-01

    Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  14. Src-Like adaptor protein (SLAP binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

    Directory of Open Access Journals (Sweden)

    Julhash U Kazi

    Full Text Available Fms-like tyrosine kinase 3 (Flt3 is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML. Src-like adaptor protein (SLAP is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  15. Multi-organ expression profiling uncovers a gene module in coronary artery disease involving transendothelial migration of leukocytes and LIM domain binding 2: The Stockholm Atherosclerosis Gene Expression (STAGE) study

    KAUST Repository

    Hägg, Sara

    2009-12-04

    Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD). The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE) study was to determine whether there are functionally associated genes (rather than individual genes) important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n =66/tissue) and atherosclerotic and unaffected arterial wall (n =40/tissue) isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n =15,042/12,621 RefSeqs/genes) in each tissue, resulted in a total of 60 tissue clusters (n= 3958 genes). In the second step (performed within tissue clusters), one atherosclerotic lesion (n =49/48) and one visceral fat (n =59) cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P=0.008 and P=0.00015). The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n =55/54) relating to carotid stenosis (P =0.04), 27 genes in the two clusters relating to coronary stenosis were confirmed (n= 16/17, P<10 -27and-30). Genes in the transendothelial migration of leukocytes (TEML) pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module). In a second validation step, using three independent cohorts, the Amodule was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004). The transcription co-factor LIM domain binding 2 (LDB2) was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2, and by the

  16. SseK3 Is a Salmonella Effector That Binds TRIM32 and Modulates the Host's NF-κB Signalling Activity.

    Directory of Open Access Journals (Sweden)

    Zhe Yang

    Full Text Available Salmonella Typhimurium employs an array of type III secretion system effectors that facilitate intracellular survival and replication during infection. The Salmonella effector SseK3 was originally identified due to amino acid sequence similarity with NleB; an effector secreted by EPEC/EHEC that possesses N-acetylglucoasmine (GlcNAc transferase activity and modifies death domain containing proteins to block extrinsic apoptosis. In this study, immunoprecipitation of SseK3 defined a novel molecular interaction between SseK3 and the host protein, TRIM32, an E3 ubiquitin ligase. The conserved DxD motif within SseK3, which is essential for the GlcNAc transferase activity of NleB, was required for TRIM32 binding and for the capacity of SseK3 to suppress TNF-stimulated activation of NF-κB pathway. However, we did not detect GlcNAc modification of TRIM32 by SseK3, nor did the SseK3-TRIM32 interaction impact on TRIM32 ubiquitination that is associated with its activation. In addition, lack of sseK3 in Salmonella had no effect on production of the NF-κB dependent cytokine, IL-8, in HeLa cells even though TRIM32 knockdown suppressed TNF-induced NF-κB activity. Ectopically expressed SseK3 partially co-localises with TRIM32 at the trans-Golgi network, but SseK3 is not recruited to Salmonella induced vacuoles or Salmonella induced filaments during Salmonella infection. Our study has identified a novel effector-host protein interaction and suggests that SseK3 may influence NF-κB activity. However, the lack of GlcNAc modification of TRIM32 suggests that SseK3 has further, as yet unidentified, host targets.

  17. Modulation of phenytoin teratogenicity and embryonic covalent binding by acetylsalicylic acid, caffeic acid, and alpha-phenyl-N-t-butylnitrone: implications for bioactivation by prostaglandin synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Wells, P.G.; Zubovits, J.T.; Wong, S.T.; Molinari, L.M.; Ali, S.

    1989-02-01

    Teratogenicity of the anticonvulsant drug phenytoin is thought to involve its bioactivation by cytochromes P-450 to a reactive arene oxide intermediate. We hypothesized that phenytoin also may be bioactivated to a teratogenic free radical intermediate by another enzymatic system, prostaglandin synthetase. To evaluate the teratogenic contribution of this latter pathway, an irreversible inhibitor of prostaglandin synthetase, acetylsalicylic acid (ASA), 10 mg/kg intraperitoneally (ip), was administered to pregnant CD-1 mice at 9:00 AM on Gestational Days 12 and 13, 2 hr before phenytoin, 65 mg/kg ip. Other groups were pretreated 2 hr prior to phenytoin administration with either the antioxidant caffeic acid or the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). Caffeic acid and PBN were given ip in doses that respectively were up to 1.0 to 0.05 molar equivalents to the dose of phenytoin. Dams were killed on Day 19 and the fetuses were assessed for teratologic anomalies. A similar study evaluated the effect of ASA on the in vivo covalent binding of radiolabeled phenytoin administered on Day 12, in which case dams were killed 24 hr later on Day 13. ASA pretreatment produced a 50% reduction in the incidence of fetal cleft palates induced by phenytoin (p less than 0.05), without significantly altering the incidence of resorptions or mean fetal body weight. Pretreatment with either caffeic acid or PBN resulted in dose-related decreases in the incidence of fetal cleft palates produced by phenytoin, with maximal respective reductions of 71 and 82% at the highest doses of caffeic acid and PBN (p less than 0.05).

  18. Binding Procurement

    Science.gov (United States)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    2007-01-01

    This viewgraph presentation reviews the use of the binding procurement process in purchasing Aerospace Flight Battery Systems. NASA Engineering and Safety Center (NESC) requested NASA Aerospace Flight Battery Systems Working Group to develop a set of guideline requirements document for Binding Procurement Contracts.

  19. Intestinal DMBT1 expression is modulated by Crohn's disease-associated IL23R variants and by a DMBT1 variant which influences binding of the transcription factors CREB1 and ATF-2.

    Directory of Open Access Journals (Sweden)

    Julia Diegelmann

    Full Text Available OBJECTIVES: DMBT is an antibacterial pattern recognition and scavenger receptor. In this study, we analyzed the role of DMBT1 single nucleotide polymorphisms (SNPs regarding inflammatory bowel disease (IBD susceptibility and examined their functional impact on transcription factor binding and downstream gene expression. METHODS: Seven SNPs in the DMBT1 gene region were analyzed in 2073 individuals including 818 Crohn's disease (CD patients and 972 healthy controls in two independent case-control panels. Comprehensive epistasis analyses for the known CD susceptibility genes NOD2, IL23R and IL27 were performed. The influence of IL23R variants on DMBT1 expression was analyzed. Functional analysis included siRNA transfection, quantitative PCR, western blot, electrophoretic mobility shift and luciferase assays. RESULTS: IL-22 induces DMBT1 protein expression in intestinal epithelial cells dependent on STAT3, ATF-2 and CREB1. IL-22 expression-modulating, CD risk-associated IL23R variants influence DMBT1 expression in CD patients and DMBT1 levels are increased in the inflamed intestinal mucosa of CD patients. Several DMBT1 SNPs were associated with CD susceptibility. SNP rs2981804 was most strongly associated with CD in the combined panel (p = 3.0 × 10(-7, OR 1.42; 95% CI 1.24-1.63. All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18. The most strongly CD risk-associated, non-coding DMBT1 SNP rs2981804 modifies the DNA binding sites for the transcription factors CREB1 and ATF-2 and the respective genomic region comprising rs2981804 is able to act as a transcriptional regulator in vitro. Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele. CONCLUSION: We identified novel associations of DMBT1 variants with CD susceptibility and discovered a novel functional role of rs2981804 in regulating DMBT1 expression. Our data suggest an important

  20. Actin-binding protein regulation by microRNAs as a novel microbial strategy to modulate phagocytosis by host cells: the case of N-Wasp and miR-142-3p

    Directory of Open Access Journals (Sweden)

    Paulo eBettencourt

    2013-06-01

    Full Text Available Mycobacterium tuberculosis (Mtb is a successful intracellular pathogen that thrives in macrophages (Mφs. There is a need to better understand how Mtb hijacks cellular processes like phagolysosome biogenesis, a classical pathogenesis determinant. A central feature to this microbial strategy is the manipulation of actin in Mφs. Here, we examine the role of microRNAs (miRNAs as a potential mechanism in the regulation of actin-mediated events leading to phagocytosis in the context of mycobacteria infection. Given that non-virulent Mycobacterium smegmatis also controls actin filamentation to prolong its instracellular survival inside host cells, our approach was to perform a global transcriptomic approach to assess the modulation of miRNAs upon M. smegmatis infection of the murine Mφ cell line, J774A.1. This approach yielded miR-142-3p as a top candidate to be involved in the regulation of actin dynamics required in phagocytosis. We unequivocally demonstrate miR-142-3p targets N-Wasp, an actin-binding protein required during microbial challenge. A gain-of-function approach for miR-142-3p revealed a down-regulation of N-Wasp expression accompanied by a decrease of mycobacteria intake, while a loss-of-function approach yielded the reciprocal increase of the phagocytosis process. Equally important, we show Mtb induces the early expression of miR-142-3p and down-regulates N-Wasp protein levels in both the murine J774A.1 cell line and primary human Mφs. As proof of principle, the partial siRNA-mediated knock down of N-Wasp resulted in a decrease of Mtb intake by human Mφs, reflected in lower levels of colony-forming units counts over time. We therefore propose the modulation of miRNAs as a novel strategy in mycobacterial infection to control factors involved in actin filamentation and other early events of phagolysosome biogenesis, such as the case presented here for miR-142-3p influencing N-Wasp activity during the phagocytosis process.

  1. Bioluminescence of beetle luciferases with 6'-amino-D-luciferin analogues reveals excited keto-oxyluciferin as the emitter and phenolate/luciferin binding site interactions modulate bioluminescence colors.

    Science.gov (United States)

    Viviani, Vadim R; Neves, Deimison Rodrigues; Amaral, Danilo Trabuco; Prado, Rogilene A; Matsuhashi, Takuto; Hirano, Takashi

    2014-08-19

    Beetle luciferases produce different bioluminescence colors from green to red using the same d-luciferin substrate. Despite many studies of the mechanisms and structural determinants of bioluminescence colors with firefly luciferases, the identity of the emitters and the specific active site interactions responsible for bioluminescence color modulation remain elusive. To address these questions, we analyzed the bioluminescence spectra with 6'-amino-D-luciferin (aminoluciferin) and its 5,5-dimethyl analogue using a set of recombinant beetle luciferases that naturally elicit different colors and different pH sensitivities (pH-sensitive, Amydetes vivianii λmax=538 nm, Macrolampis sp2 λmax=564 nm; pH-insensitive, Phrixotrix hirtus λmax=623 nm, Phrixotrix vivianii λmax=546 nm, and Pyrearinus termitilluminans λmax=534 nm), a luciferase-like enzyme (Tenebrionidae, Zophobas morio λmax=613 nm), and mutants of C311 (S314). The green-yellow-emitting luciferases display red-shifted bioluminescence spectra with aminoluciferin in relation to those with D-luciferin, whereas the red-emitting luciferases displayed blue-shifted spectra. Bioluminescence spectra with 5,5-dimethylaminoluciferin, in which enolization is blocked, were almost identical to those of aminoluciferin. Fluorescence probing using 2-(4-toluidino)naphthalene-6-sulfonate and inference with aminoluciferin confirm that the luciferin binding site of the red-shifted luciferases is more polar than in the case of the green-yellow-emitting luciferases. Altogether, the results show that the keto form of excited oxyluciferin is the emitter in beetle bioluminescence and that bioluminescence colors are essentially modulated by interactions of the 6'-hydroxy group of oxyluciferin and basic moieties under the influence of the microenvironment polarity of the active site: a strong interaction between a base moiety and oxyluciferin phenol in a hydrophobic microenvironment promotes green-yellow emission, whereas a more polar

  2. Defining Starch Binding by Glucan Phosphatases

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2015-01-01

    phosphatases. The main objective of this study was to quantify the binding affinity of different enzymes that are involved in this cyclic process. We established a protocol to quickly, reproducibly, and quantitatively measure the binding of the enzymes to glucans utilizing Affinity Gel Electrophoresis (AGE...... glucan phosphatases showed similar affinities for the short oligosaccharide β-cyclodextrin. We performed structure-guided mutagenesis to define the mechanism of these differences. We found that the carbohydrate binding module (CBM) domain provided a stronger binding affinity compared to surface binding...

  3. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    Energy Technology Data Exchange (ETDEWEB)

    Taguchi, J.; Kuriyama, K. (Kyoto Prefectural Univ. of Medicine (Japan))

    1990-05-01

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  4. -Regular Modules

    Directory of Open Access Journals (Sweden)

    Areej M. Abduldaim

    2013-01-01

    Full Text Available We introduced and studied -regular modules as a generalization of -regular rings to modules as well as regular modules (in the sense of Fieldhouse. An -module is called -regular if for each and , there exist and a positive integer such that . The notion of -pure submodules was introduced to generalize pure submodules and proved that an -module is -regular if and only if every submodule of is -pure iff   is a -regular -module for each maximal ideal of . Many characterizations and properties of -regular modules were given. An -module is -regular iff is a -regular ring for each iff is a -regular ring for finitely generated module . If is a -regular module, then .

  5. Negative modulation of the chicken infectious anemia virus promoter by COUP-TF1 and an E box-like element at the transcription start site binding dEF1

    Science.gov (United States)

    Expression of enhanced green fluorescent protein (EGFP) under control of the promoter-enhancer of chicken infectious anemia virus (CAV) is increased in an estrogen receptor-enhanced cell line when treated with estrogen. This promoter-enhancer also binds unidentified proteins that recognize a consens...

  6. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  7. The histidine composition of the amyloid-β domain, but not the E1 copper binding domain, modulates β-secretase processing of amyloid-β protein precursor in Alzheimer's disease.

    Science.gov (United States)

    Gough, Mallory; Blanthorn-Hazell, Sophee; Parkin, Edward T

    2015-01-01

    Amyloid-β protein precursor (AβPP) proteolysis by β- and γ-secretases generates neurotoxic amyloid-β (Aβ)-peptides in Alzheimer's disease (AD). We have investigated the role of histidine residues within the extracellular E1 copper binding and Aβ domains of AβPP in its proteolysis. By stably expressing histidine to alanine AβPP mutant constructs in SH-SY5Y cells, we show that mutations in the E1 copper binding domain had no impact on α- or β-secretase processing. Mutation of histidine 14 within the Aβ-domain specifically down-regulated β-secretase processing without impacting on non-amyloidogenic proteolysis. Understanding how histidine 14 participates in AβPP proteolysis may reveal new intervention points for AD treatments.

  8. Structural and functional studies of titin's fn3 modules reveal conserved surface patterns and binding to myosin S1--a possible role in the Frank-Starling mechanism of the heart.

    Science.gov (United States)

    Muhle-Goll, C; Habeck, M; Cazorla, O; Nilges, M; Labeit, S; Granzier, H

    2001-10-19

    The A-band part of titin, a striated-muscle specific protein spanning from the Z-line to the M-line, mainly consists of a well-ordered super-repeat array of immunoglobulin-like and fibronectin-type III (fn3)-like domains. Since it has been suspected that the fn3 domains might represent titin's binding sites to myosin, we have developed structural models for all of titin's 132 fn3-like domains. A subset of eight experimentally determined fn3 structures from a range of proteins, including titin itself, was used as homology templates. After grouping the models according to their position within the super-repeat segment of the central A-band titin region, we analyzed the models with respect to side-chain conservation. This showed that conserved residues form an extensive surface pattern predominantly at one side of the domains, whereas domains outside the central C-zone super-repeat region show generally less conserved surfaces. Since the conserved surface residues may function as protein-binding sites, we experimentally studied the binding properties of expressed multi-domain fn3 fragments. This revealed that fn3 fragments specifically bind to the sub-fragment 1 of myosin. We also measured the effect of fn3 fragments on the contractile properties of single cardiac myocytes. At sub-maximal Ca(2+) concentrations, fn3 fragments significantly enhance active tension. This effect is most pronounced at short sarcomere length, and as a result the length-dependence of Ca(2+) activation is reduced. A model of how titin's fn3-like domains may influence actomyosin interaction is proposed.

  9. Analyzing binding data.

    Science.gov (United States)

    Motulsky, Harvey J; Neubig, Richard R

    2010-07-01

    Measuring the rate and extent of radioligand binding provides information on the number of binding sites, and their affinity and accessibility of these binding sites for various drugs. This unit explains how to design and analyze such experiments.

  10. Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5)

    DEFF Research Database (Denmark)

    Karlshøj, Stefanie; Amarandi, Roxana Maria; Larsen, Olav;

    2016-01-01

    The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn(2+) (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site...

  11. Dietary fiber prevents obesity-related liver lipotoxicity by modulating sterol-regulatory element binding protein pathway in C57BL/6J mice fed a high-fat/cholesterol diet.

    Science.gov (United States)

    Han, Shufen; Jiao, Jun; Zhang, Wei; Xu, Jiaying; Wan, Zhongxiao; Zhang, Weiguo; Gao, Xiaoran; Qin, Liqiang

    2015-10-29

    Adequate intake of dietary fibers has proven metabolic and cardiovascular benefits, molecular mechanisms remain still limited. This study was aimed to investigate the effects of cereal dietary fiber on obesity-related liver lipotoxicity in C57BL/6J mice fed a high-fat/cholesterol (HFC) diet and underlying mechanism. Forty-eight adult male C57BL/6J mice were randomly given a reference chow diet, or a high fat/cholesterol (HFC) diet supplemented with or without oat fiber or wheat bran fiber for 24 weeks. Our results showed mice fed oat or wheat bran fiber exhibited lower weight gain, lipid profiles and insulin resistance, compared with HFC diet. The two cereal dietary fibers potently decreased protein expressions of sterol regulatory element binding protein-1 and key factors involved in lipogenesis, including fatty acid synthase and acetyl-CoA carboxylase in target tissues. At molecular level, the two cereal dietary fibers augmented protein expressions of peroxisome proliferator-activated receptor alpha and gamma, liver X receptor alpha, and ATP-binding cassette transporter A1 in target tissues. Our findings indicated that cereal dietary fiber supplementation abrogated obesity-related liver lipotoxicity and dyslipidemia in C57BL/6J mice fed a HFC diet. In addition, the efficacy of oat fiber is greater than wheat bran fiber in normalizing these metabolic disorders and pathological profiles.

  12. Modulation of inhibitory activity of xylanase - α-amylase inhibitor protein (XAIP: binding studies and crystal structure determination of XAIP- II from Scadoxus multiflorus at 1.2 Å resolution

    Directory of Open Access Journals (Sweden)

    Dey Sharmistha

    2010-11-01

    Full Text Available Abstract Background Plants produce a wide range of proteinaceous inhibitors to protect themselves against hydrolytic enzymes. Recently a novel protein XAIP belonging to a new sub-family (GH18C was reported to inhibit two structurally unrelated enzymes xylanase GH11 and α-amylase GH13. It was shown to inhibit xylanase GH11 with greater potency than that of α-amylase GH13. A new form of XAIP (XAIP-II that inhibits α-amylase GH13 with a greater potency than that of XAIP and xylanase GH11 with a lower potency than that of XAIP, has been identified in the extracts of underground bulbs of Scadoxus multiflorus. This kind of occurrence of isoforms of inhibitor proteins is a rare observation and offers new opportunities for understanding the principles of protein engineering by nature. Results In order to determine the structural basis of the enhanced potency of XAIP-II against α-amylase GH13 and its reduced potency against xylanase GH11 as compared to that of XAIP, we have purified XAIP-II to homogeneity and obtained its complete amino acid sequence using cloning procedure. It has been crystallized with 0.1 M ammonium sulphate as the precipitating agent and the three-dimensional structure has been determined at 1.2 Å resolution. The binding studies of XAIP-II with xylanase GH11 and α-amylase GH13 have been carried out with surface plasmon resonance (SPR. Conclusion The structure determination revealed that XAIP-II adopts the well known TIM barrel fold. The xylanase GH11 binding site in XAIP-II is formed mainly with loop α3-β3 (residues, 102 - 118 which has acquired a stereochemically less favorable conformation for binding to xylanase GH11 because of the addition of an extra residue, Ala105 and due to replacements of two important residues, His106 and Asn109 by Thr107 and Ser110. On the other hand, the α-amylase binding site, which consists of α-helices α6 (residues, 193 - 206, α7 (residues, 230 - 243 and loop β6-α6 (residues, 180 - 192

  13. ANDROGEN REGULATION OF PROSTATIC STEROID BINDING PROTEIN GENE TRANSCRIPTION

    Institute of Scientific and Technical Information of China (English)

    ZHANGYong-Lian; ZHOUZong-Xun; ZHANGYou-Duan; PARKERMalcolmG

    1989-01-01

    Prostatic steroid binding protein (PSBP) is a major protein secreted in the rat ventral prostate (V.P.) and also one of the components in seminal fluid, The potential importance of this protein in male fertility emerged from its ability of binding cholesterol which might modulate the proportion of phospholipids and cholesterol in sperm making it suitable

  14. The extrinsic PsbO protein modulates the oxidation/reduction rate of the exogenous Mn cation at the high-affinity Mn-binding site of Mn-depleted PSII membranes.

    Science.gov (United States)

    Semin, Boris K; Podkovirina, Tatiana E; Davletshina, Lira N; Timofeev, Kirill N; Ivanov, Il'ya I; Rubin, Andrei B

    2015-08-01

    The oxidation of exogenous Mn(II) cations at the high-affinity (HA) Mn-binding site in Mn-depleted photosystem II (PSII) membranes with or without the presence of the extrinsic PsbO polypeptide was studied by EPR. The six-lines EPR spectrum of Mn(II) cation disappears in the absence of the PsbO protein in membranes under illumination, but there was no effect when PSII preparations bound the PsbO protein. Our study demonstrates that such effect is determined by significant influence of the PsbO protein on the ratio between the rates of Mn oxidation and reduction at the HA site when the membranes are illuminated.

  15. Microfluidic Screening of Electrophoretic Mobility Shifts Elucidates Riboswitch Binding Function

    OpenAIRE

    Karns, Kelly; Vogan, Jacob M.; Qin, Qian; Hickey, Scott F.; Wilson, Stephen C.; Hammond, Ming C.; Herr, Amy E.

    2013-01-01

    Riboswitches are RNA sensors that change conformation upon binding small molecule metabolites, in turn modulating gene expression. Our understanding of riboswitch regulatory function would be accelerated by a high throughput, quantitative screening tool capable of measuring riboswitch-ligand binding. We introduce a microfluidic mobility shift assay that enables precise and rapid quantitation of ligand binding and subsequent riboswitch conformational change. In 0.3% of the time required for be...

  16. Irreducible Specht modules are signed Young modules

    OpenAIRE

    Hemmer, David J.

    2005-01-01

    Recently Donkin defined signed Young modules as a simultaneous generalization of Young and twisted Young modules for the symmetric group. We show that in odd characteristic, if a Specht module $S^\\lambda$ is irreducible, then $S^\\lambda$ is a signed Young module. Thus the set of irreducible Specht modules coincides with the set of irreducible signed Young modules. This provides evidence for our conjecture that the signed Young modules are precisely the class of indecomposable self-dual module...

  17. *-Modules, co-*-modules and cotilting modules over Noetherian rings

    Institute of Scientific and Technical Information of China (English)

    汪明义; 许永华

    1996-01-01

    Let R be a Noetherian ring. The projectivity and injectivity of modules over R are discussed. The concept of modules is introduced and the descriptions for co-*-modules over R are given. At last, cotilting modules over R are characterized by means of co-*-modules.

  18. Thruster Module

    Science.gov (United States)

    Andersson, G.

    2015-09-01

    The thruster module described in this paper provides a low but controlled acceleration in a mission which would normally be labelled “microgravity”. The first mission was Cryofenix, where tanks containing liquid hydrogen were used in the experiment. The experiment utilizing the low acceleration is using liquids and requires a precise acceleration profile throughout the mission. Acceleration obtained by payload rotation is not feasible due to that the transversal forces required to change the acceleration will cause undesired liquid turbulence. In order to satisfy the experiment requirements a thruster module was developed by SSC for the Cryofenix mission funded by CNES. The Cryofenix mission had a payload weight of 380 kg and an apogee of about 260 km. The module produces a controlled thrust in flight direction by means of a cold gas system.

  19. Membrane binding domains

    OpenAIRE

    Hurley, James H.

    2006-01-01

    Eukaryotic signaling and trafficking proteins are rich in modular domains that bind cell membranes. These binding events are tightly regulated in space and time. The structural, biochemical, and biophysical mechanisms for targeting have been worked out for many families of membrane binding domains. This review takes a comparative view of seven major classes of membrane binding domains, the C1, C2, PH, FYVE, PX, ENTH, and BAR domains. These domains use a combination of specific headgroup inter...

  20. Signed Young Modules and Simple Specht Modules

    OpenAIRE

    Danz, Susanne; Lim, Kay Jin

    2015-01-01

    By a result of Hemmer, every simple Specht module of a finite symmetric group over a field of odd characteristic is a signed Young module. While Specht modules are parametrized by partitions, indecomposable signed Young modules are parametrized by certain pairs of partitions. The main result of this article establishes the signed Young module labels of simple Specht modules. Along the way we prove a number of results concerning indecomposable signed Young modules that are of independent inter...

  1. TNFα modulates Fibroblast Growth Factor Receptor 2 gene expression through the pRB/E2F1 pathway: identification of a non-canonical E2F binding motif.

    Directory of Open Access Journals (Sweden)

    Sirio D'Amici

    Full Text Available Interactions between epithelium and mesenchyme during wound healing are not fully understood, but Fibroblast Growth Factors (FGFs and their receptors FGFRs are recognized as key elements. FGFR2 gene encodes for two splicing transcript variants, FGFR2-IIIb or Keratinocyte Growth Factor Receptor (KGFR and FGFR2-IIIc, which differ for tissue localization and ligand specificity. Proinflammatory cytokines play an essential role in the regulation of epithelial-mesenchymal interactions, and have been indicated to stimulate FGFs production. Here we demonstrated that upregulation of FGFR2 mRNA and protein expression is induced by the proinflammatory cytokines Tumor Necrosis Factor-α, Interleukin-1β and Interleukin 2. Furthermore, we found that TNFα determines FGFR2 transcriptional induction through activation of pRb, mediated by Raf and/or p38 pathways, and subsequent release of the transcription factor E2F1. Experiments based on FGFR2 promoter serial deletions and site-directed mutagenesis allowed us to identify a minimal responsive element that retains the capacity to be activated by E2F1. Computational analysis indicated that this element is a non-canonical E2F responsive motif. Thus far, the molecular mechanisms of FGFR2 upregulation during wound healing or in pathological events are not known. Our data suggest that FGFR2 expression can be modulated by local recruitment of inflammatory cytokines. Furthermore, since alterations in FGFR2 expression have been linked to the pathogenesis of certain human cancers, these findings could also provide elements for diagnosis and potential targets for novel therapeutic approaches.

  2. Memory Modulation

    NARCIS (Netherlands)

    Roozendaal, Benno; McGaugh, James L.

    2011-01-01

    Our memories are not all created equally strong: Some experiences are well remembered while others are remembered poorly, if at all. Research on memory modulation investigates the neurobiological processes and systems that contribute to such differences in the strength of our memories. Extensive evi

  3. Modulation of the homophilic interaction between the first and second Ig modules of neural cell adhesion molecule by heparin

    DEFF Research Database (Denmark)

    Kulahin, Nikolaj; Rudenko, Olga; Kiselyov, V.;

    2005-01-01

    -binding sites (HBS and CBS, respectively) in IgII coincide, and that this site overlaps with the homophilic binding site. Using NMR and surface plasmon resonance (SPR) analyses we demonstrate that interaction between IgII and heparin indeed interferes with the homophilic interaction between IgI and Ig......The second Ig module (IgII) of the neural cell adhesion molecule (NCAM) is known to bind to the first Ig module (IgI) of NCAM (so-called homophilic binding) and to interact with heparan sulfate and chondroitin sulfate glycoconjugates. We here show by NMR that the heparin and chondroitin sulfate...

  4. Positive modulator of bone morphogenic protein-2

    Science.gov (United States)

    Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua; Takahashi, Kazuyuki

    2009-01-27

    Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

  5. Module Evaluation

    Science.gov (United States)

    2006-02-01

    MODULES IN LIFE TEST CHAMBER (LEFT SIDE) 68 MODULE TRANSMIT TEMP CA1 54.8°C CB1 65.3°C CC1 70.5°C CD1 75.2°C CE1 68.5°C CA2 72.1°C CB2 ...NO. PA (Contractor) PA (MELTS) DRV (Contractor) DRV (MELTS) CA1 058 +11.0 VOLTS +10.3 VOLTS + 7.5 VOLTS + 3.64 VOLTS CB1 085 +11.0 VOLTS +10.13...CE1 032 +11.0 VOLTS + 7.02 VOLTS + 7.5 VOLTS + 4.23 VOLTS CA2 065 +11.0 VOLTS +11.03 VOLTS + 7.5 VOLTS + 7.53 VOLTS CB2 057 +11.0 VOLTS + 9.49

  6. Ureaplasma urealyticum binds mannose-binding lectin.

    Science.gov (United States)

    Benstein, Barbara D; Ourth, Donald D; Crouse, Dennis T; Shanklin, D Radford

    2004-10-01

    Mannose-binding C-type lectin (MBL) is an important component of innate immunity in mammals. Mannose-binding lectin (MBL), an acute phase protein, acts as an opsonin for phagocytosis and also activates the mannan-binding lectin complement pathway. It may play a particularly significant role during infancy before adequate specific protection can be provided by the adaptive immune system. Ureaplasma urealyticum has been linked to several diseases including pneumonia and chronic lung disease (CLD) in premature infants. We therefore investigated the ability of U. urealyticum to bind MBL. A guinea pig IgG anti-rabbit-MBL antiserum was produced. An immunoblot (dot-blot) assay done on nitrocellulose membrane determined that the anti-MBL antibody had specificity against both rabbit and human MBL. Pure cultures of U. urealyticum, serotype 3, were used to make slide preparations. The slides containing the organisms were then incubated with nonimmune rabbit serum containing MBL. Ureaplasma was shown to bind rabbit MBL with an immunocytochemical assay using the guinea pig IgG anti-rabbit MBL antiserum. Horseradish peroxidase (HRP)-labeled anti-guinea pig IgG was used to localize the reaction. The anti-MBL antiserum was also used in an immunocytochemical assay to localize U. urealyticum in histological sections of lungs from mice specifically infected with this organism. The same method also indicated binding of MBL by ureaplasma in human lung tissue obtained at autopsy from culture positive infants. Our results demonstrate that ureaplasma has the capacity to bind MBL. The absence of MBL may play a role in the predisposition of diseases related to this organism.

  7. Ligand binding mechanics of maltose binding protein.

    Science.gov (United States)

    Bertz, Morten; Rief, Matthias

    2009-11-13

    In the past decade, single-molecule force spectroscopy has provided new insights into the key interactions stabilizing folded proteins. A few recent studies probing the effects of ligand binding on mechanical protein stability have come to quite different conclusions. While some proteins seem to be stabilized considerably by a bound ligand, others appear to be unaffected. Since force acts as a vector in space, it is conceivable that mechanical stabilization by ligand binding is dependent on the direction of force application. In this study, we vary the direction of the force to investigate the effect of ligand binding on the stability of maltose binding protein (MBP). MBP consists of two lobes connected by a hinge region that move from an open to a closed conformation when the ligand maltose binds. Previous mechanical experiments, where load was applied to the N and C termini, have demonstrated that MBP is built up of four building blocks (unfoldons) that sequentially detach from the folded structure. In this study, we design the pulling direction so that force application moves the two MBP lobes apart along the hinge axis. Mechanical unfolding in this geometry proceeds via an intermediate state whose boundaries coincide with previously reported MBP unfoldons. We find that in contrast to N-C-terminal pulling experiments, the mechanical stability of MBP is increased by ligand binding when load is applied to the two lobes and force breaks the protein-ligand interactions directly. Contour length measurements indicate that MBP is forced into an open conformation before unfolding even if ligand is bound. Using mutagenesis experiments, we demonstrate that the mechanical stabilization effect is due to only a few key interactions of the protein with its ligand. This work illustrates how varying the direction of the applied force allows revealing important details about the ligand binding mechanics of a large protein.

  8. Module descriptor

    DEFF Research Database (Denmark)

    Vincenti, Gordon; Klausen, Bodil; Kjær Jensen, Jesper

    2016-01-01

    The Module Descriptor including a Teacher’s Guide explains and describes how to work innovatively and co-creatively with wicked problems and young people. The descriptor shows how interested educators and lecturers in Europe can copy the lessons of the Erasmus+ project HIP when teaching their own...... students how to include marginalized young people and practitioners in the education of future social workers and social educators....

  9. Protein Binding Pocket Dynamics.

    Science.gov (United States)

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  10. Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals

    DEFF Research Database (Denmark)

    Janeček, Štefan; Svensson, Birte; MacGregor, E. Ann

    2011-01-01

    Starch-binding domains (SBDs) comprise distinct protein modules that bind starch, glycogen or related carbohydrates and have been classified into different families of carbohydrate-binding modules (CBMs). The present review focuses on SBDs of CBM20 and CBM48 found in amylolytic enzymes from sever...

  11. Photovoltaic module and module arrays

    Science.gov (United States)

    Botkin, Jonathan; Graves, Simon; Lenox, Carl J. S.; Culligan, Matthew; Danning, Matt

    2012-07-17

    A photovoltaic (PV) module including a PV device and a frame. The PV device has a PV laminate defining a perimeter and a major plane. The frame is assembled to and encases the laminate perimeter, and includes leading, trailing, and side frame members, and an arm that forms a support face opposite the laminate. The support face is adapted for placement against a horizontal installation surface, to support and orient the laminate in a non-parallel or tilted arrangement. Upon final assembly, the laminate and the frame combine to define a unitary structure. The frame can orient the laminate at an angle in the range of 3.degree.-7.degree. from horizontal, and can be entirely formed of a polymeric material. Optionally, the arm incorporates integral feature(s) that facilitate interconnection with corresponding features of a second, identically formed PV module.

  12. Python bindings for libcloudph++

    OpenAIRE

    Jarecka, Dorota; Arabas, Sylwester; Del Vento, Davide

    2015-01-01

    This technical note introduces the Python bindings for libcloudph++. The libcloudph++ is a C++ library of algorithms for representing atmospheric cloud microphysics in numerical models. The bindings expose the complete functionality of the library to the Python users. The bindings are implemented using the Boost.Python C++ library and use NumPy arrays. This note includes listings with Python scripts exemplifying the use of selected library components. An example solution for using the Python ...

  13. SCA1-Like Disease in Mice Expressing Wild Type Ataxin-1 with a Serine to Aspartic Acid Replacement at Residue 776

    Science.gov (United States)

    Duvick, Lisa; Barnes, Justin; Ebner, Blake; Agrawal, Smita; Andresen, Michael; Lim, Janghoo; Giesler, Glenn J.; Zoghbi, Huda Y.; Orr, Harry T.

    2010-01-01

    SUMMARY Glutamine tract expansion triggers nine neurodegenerative diseases by conferring toxic properties to the mutant protein. In SCA1, phosphorylation of ATXN1 at Ser776 is thought to be key for pathogenesis. Here we show that replacing Ser776 with a phospho-mimicking Asp converted ATXN1 with a wild type glutamine tract into a pathogenic protein. ATXN1[30Q]-D776-induced disease in Purkinje cells shared most features with disease caused by ATXN1[82Q] having an expanded polyglutamine tract. However, in contrast to disease induced by ATXN1[82Q] that progresses to cell death, ATXN1[30Q]-D776 failed to induce cell death. These results support a model where pathogenesis involves changes in regions of the protein in addition to the polyglutamine tract. In ATXN1, placing an Asp at residue 776 mimics this change. Moreover, disease initiation and progression to neuronal dysfunction are distinct from induction of cell death. Ser776 is critical for the pathway to neuronal dysfunction, while an expanded polyglutamine tract is essential for neuronal death. PMID:20869591

  14. Python bindings for libcloudph++

    CERN Document Server

    Jarecka, Dorota; Del Vento, Davide

    2015-01-01

    This technical note introduces the Python bindings for libcloudph++. The libcloudph++ is a C++ library of algorithms for representing atmospheric cloud microphysics in numerical models. The bindings expose the complete functionality of the library to the Python users. The bindings are implemented using the Boost.Python C++ library and use NumPy arrays. This note includes listings with Python scripts exemplifying the use of selected library components. An example solution for using the Python bindings to access libcloudph++ from Fortran is presented.

  15. DNS & Bind Cookbook

    CERN Document Server

    Liu, Cricket

    2011-01-01

    The DNS & BIND Cookbook presents solutions to the many problems faced by network administrators responsible for a name server. Following O'Reilly's popular problem-and-solution cookbook format, this title is an indispensable companion to DNS & BIND, 4th Edition, the definitive guide to the critical task of name server administration. The cookbook contains dozens of code recipes showing solutions to everyday problems, ranging from simple questions, like, "How do I get BIND?" to more advanced topics like providing name service for IPv6 addresses. It's full of BIND configuration files that yo

  16. On Binding Domains

    NARCIS (Netherlands)

    Everaert, M.B.H.

    2005-01-01

    In this paper I want to explore reasons for replacing Binding Theory based on the anaphor-pronoun dichotomy by a Binding Theory allowing more domains restricting/defining anaphoric dependencies. This will, thus, have consequences for the partitioning of anaphoric elements, presupposing more types of

  17. Melanin-binding radiopharmaceuticals

    Energy Technology Data Exchange (ETDEWEB)

    Packer, S; Fairchild, R G; Watts, K P; Greenberg, D; Hannon, S J

    1980-01-01

    The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed. (PSB)

  18. DNS BIND Server Configuration

    Directory of Open Access Journals (Sweden)

    Radu MARSANU

    2011-01-01

    Full Text Available After a brief presentation of the DNS and BIND standard for Unix platforms, the paper presents an application which has a principal objective, the configuring of the DNS BIND 9 server. The general objectives of the application are presented, follow by the description of the details of designing the program.

  19. Thermodynamics of fragment binding.

    Science.gov (United States)

    Ferenczy, György G; Keserű, György M

    2012-04-23

    The ligand binding pockets of proteins have preponderance of hydrophobic amino acids and are typically within the apolar interior of the protein; nevertheless, they are able to bind low complexity, polar, water-soluble fragments. In order to understand this phenomenon, we analyzed high resolution X-ray data of protein-ligand complexes from the Protein Data Bank and found that fragments bind to proteins with two near optimal geometry H-bonds on average. The linear extent of the fragment binding site was found not to be larger than 10 Å, and the H-bonding region was found to be restricted to about 5 Å on average. The number of conserved H-bonds in proteins cocrystallized with multiple different fragments is also near to 2. These fragment binding sites that are able to form limited number of strong H-bonds in a hydrophobic environment are identified as hot spots. An estimate of the free-energy gain of H-bond formation versus apolar desolvation supports that fragment sized compounds need H-bonds to achieve detectable binding. This suggests that fragment binding is mostly enthalpic that is in line with their observed binding thermodynamics documented in Isothermal Titration Calorimetry (ITC) data sets and gives a thermodynamic rationale for fragment based approaches. The binding of larger compounds tends to more rely on apolar desolvation with a corresponding increase of the entropy content of their binding free-energy. These findings explain the reported size-dependence of maximal available affinity and ligand efficiency both behaving differently in the small molecule region featured by strong H-bond formation and in the larger molecule region featured by apolar desolvation.

  20. Oxypred: Prediction and Classification of Oxygen-Binding Proteins

    Institute of Scientific and Technical Information of China (English)

    S.; Muthukrishnan; Aarti; Garg; G.P.S.; Raghava

    2007-01-01

    This study describes a method for predicting and classifying oxygen-binding pro- teins. Firstly, support vector machine (SVM) modules were developed using amino acid composition and dipeptide composition for predicting oxygen-binding pro- teins, and achieved maximum accuracy of 85.5% and 87.8%, respectively. Sec- ondly, an SVM module was developed based on amino acid composition, classify- ing the predicted oxygen-binding proteins into six classes with accuracy of 95.8%, 97.5%, 97.5%, 96.9%, 99.4%, and 96.0% for erythrocruorin, hemerythrin, hemo- cyanin, hemoglobin, leghemoglobin, and myoglobin proteins, respectively. Finally, an SVM module was developed using dipeptide composition for classifying the oxygen-binding proteins, and achieved maximum accuracy of 96.1%, 98.7%, 98.7%, 85.6%, 99.6%, and 93.3% for the above six classes, respectively. All modules were trained and tested by five-fold cross validation. Based on the above approach, a web server Oxypred was developed for predicting and classifying oxygen-binding proteins(available from http://www.imtech.res.in/raghava/oxypred/).

  1. Starch‐binding domains in the CBM45 family – low‐affinity domains from glucan, water dikinase and α‐amylase involved in plastidial starch metabolism

    DEFF Research Database (Denmark)

    Glaring, Mikkel Andreas; Baumann, Martin; Abou Hachem, Maher

    2011-01-01

    Starch‐binding domains are noncatalytic carbohydrate‐binding modules that mediate binding to granular starch. The starch‐binding domains from the carbohydrate‐binding module family 45 (CBM45, ) are found as N‐terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing...... amylolytic enzymes. This suggests that low‐affinity starch‐binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low‐affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and...

  2. SHBG (Sex Hormone Binding Globulin)

    Science.gov (United States)

    ... as: Testosterone-estrogen Binding Globulin; TeBG Formal name: Sex Hormone Binding Globulin Related tests: Testosterone , Free Testosterone, ... I should know? How is it used? The sex hormone binding globulin (SHBG) test may be used ...

  3. Sex Differences in Serotonin 1 Receptor Binding in Rat Brain

    Science.gov (United States)

    Fischette, Christine T.; Biegon, Anat; McEwen, Bruce S.

    1983-10-01

    Male and female rats exhibit sex differences in binding by serotonin 1 receptors in discrete areas of the brain, some of which have been implicated in the control of ovulation and of gonadotropin release. The sex-specific changes in binding, which occur in response to the same hormonal (estrogenic) stimulus, are due to changes in the number of binding sites. Castration alone also affects the number of binding sites in certain areas. The results lead to the conclusion that peripheral hormones modulate binding by serotonin 1 receptors. The status of the serotonin receptor system may affect the reproductive capacity of an organism and may be related to sex-linked emotional disturbances in humans.

  4. Module theory, extending modules and generalizations

    CERN Document Server

    Tercan, Adnan

    2016-01-01

    The main focus of this monograph is to offer a comprehensive presentation of known and new results on various generalizations of CS-modules and CS-rings. Extending (or CS) modules are generalizations of injective (and also semisimple or uniform) modules. While the theory of CS-modules is well documented in monographs and textbooks, results on generalized forms of the CS property as well as dual notions are far less present in the literature. With their work the authors provide a solid background to module theory, accessible to anyone familiar with basic abstract algebra. The focus of the book is on direct sums of CS-modules and classes of modules related to CS-modules, such as relative (injective) ejective modules, (quasi) continuous modules, and lifting modules. In particular, matrix CS-rings are studied and clear proofs of fundamental decomposition results on CS-modules over commutative domains are given, thus complementing existing monographs in this area. Open problems round out the work and establish the...

  5. Cofinitely weak supplemented modules

    OpenAIRE

    Alizade, Rafail; Büyükaşık, Engin

    2003-01-01

    We prove that a module M is cofinitely weak supplemented or briefly cws (i.e., every submodule N of M with M/N finitely generated, has a weak supplement) if and only if every maximal submodule has a weak supplement. If M is a cws-module then every M-generated module is a cws-module. Every module is cws if and only if the ring is semilocal. We study also modules, whose finitely generated submodules have weak supplements.

  6. On generalized extending modules

    Institute of Scientific and Technical Information of China (English)

    ZENG Qing-yi

    2007-01-01

    A module M is called generalized extending if for any submodule N of M, there is a direct summand K of M such that N≤K and K/N is singular. Any extending module and any singular module are generalized extending. Any homomorphic image of a generalized extending module is generalized extending. Any direct sum of a singular (uniform) module and a semi-simple module is generalized extending. A ring R is a right Co-H-ring ifand only ifall right R modules are generalized extending modules.

  7. CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

    DEFF Research Database (Denmark)

    1995-01-01

    Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

  8. FOXO-binding partners : it takes two to tango

    NARCIS (Netherlands)

    van der Vos, K. E.; Coffer, P. J.

    2008-01-01

    Modulation FOXO transcription factor activities can lead to a variety of cellular outputs resulting in changes in proliferation, apoptosis, differentiation and metabolic responses. Although FOXO proteins all contain an identical DNA-binding domain their cellular functions appear to be distinct, as e

  9. Reprogramming cellular events by poly(ADP-ribose)-binding proteins

    Science.gov (United States)

    Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

    2013-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

  10. Ballasted photovoltaic module and module arrays

    Science.gov (United States)

    Botkin, Jonathan; Graves, Simon; Danning, Matt

    2011-11-29

    A photovoltaic (PV) module assembly including a PV module and a ballast tray. The PV module includes a PV device and a frame. A PV laminate is assembled to the frame, and the frame includes an arm. The ballast tray is adapted for containing ballast and is removably associated with the PV module in a ballasting state where the tray is vertically under the PV laminate and vertically over the arm to impede overt displacement of the PV module. The PV module assembly can be installed to a flat commercial rooftop, with the PV module and the ballast tray both resting upon the rooftop. In some embodiments, the ballasting state includes corresponding surfaces of the arm and the tray being spaced from one another under normal (low or no wind) conditions, such that the frame is not continuously subjected to a weight of the tray.

  11. MI 4010 Thermoelectric Modules.

    Science.gov (United States)

    The report covers the design justification, physical specification and characterization of the MI 4010 module . The purpose of the contract was to...demonstrate the capability to fabricate pieceparts, process into assemblies, and test thermoelectric modules equivalent to the module used in the Hand...Held Thermal Viewer. The completed modules were also subjected to limited demonstration tests of reliability and useful life.

  12. Module utilization committee

    Science.gov (United States)

    Volkmer, K.; Praver, G.

    1984-01-01

    Photovoltaic collector modules were declared surplus to the needs of the U.S. Dept. of Energy. The Module Utilization Committee was formed to make appropriate disposition of the surplus modules on a national basis and to act as a broker for requests for these modules originating outside of the National Photovoltaics Program.

  13. Ago-allosteric modulation and other types of allostery in dimeric 7TM receptors

    DEFF Research Database (Denmark)

    Schwartz, Thue W; Holst, Birgitte

    2006-01-01

    Conventionally, an allosteric modulator is neutral in respect of efficacy and binds to a receptor site distant from the orthosteric site of the endogenous agonist. However, recently compounds being ago-allosteric modulators have been described i.e., compounds acting both as agonists on their own...... influence the potency of the endogenous agonist. It is of interest that at least some endogenous agonists can only occupy one protomer of a dimeric 7TM receptor complex at a time and thereby they leave the orthosteric binding site in the allosteric protomer free, potentially for binding of exogenous......, allosteric modulators. If the allosteric modulator is an agonist, it is an ago-allosteric modulator; if it is neutral, it is a classical enhancer. Molecular mapping in hetero-dimeric class-C receptors, where the endogenous agonist clearly binds only in one protomer, supports the notion that allosteric...

  14. Unique carbohydrate binding platforms employed by the glucan phosphatases.

    Science.gov (United States)

    Emanuelle, Shane; Brewer, M Kathryn; Meekins, David A; Gentry, Matthew S

    2016-07-01

    Glucan phosphatases are a family of enzymes that are functionally conserved at the enzymatic level in animals and plants. These enzymes bind and dephosphorylate glycogen in animals and starch in plants. While the enzymatic function is conserved, the glucan phosphatases employ distinct mechanisms to bind and dephosphorylate glycogen or starch. The founding member of the family is a bimodular human protein called laforin that is comprised of a carbohydrate binding module 20 (CBM20) followed by a dual specificity phosphatase domain. Plants contain two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). SEX4 contains a chloroplast targeting peptide, dual specificity phosphatase (DSP) domain, a CBM45, and a carboxy-terminal motif. LSF2 is comprised of simply a chloroplast targeting peptide, DSP domain, and carboxy-terminal motif. SEX4 employs an integrated DSP-CBM glucan-binding platform to engage and dephosphorylate starch. LSF2 lacks a CBM and instead utilizes two surface binding sites to bind and dephosphorylate starch. Laforin is a dimeric protein in solution and it utilizes a tetramodular architecture and cooperativity to bind and dephosphorylate glycogen. This chapter describes the biological role of glucan phosphatases in glycogen and starch metabolism and compares and contrasts their ability to bind and dephosphorylate glucans.

  15. A Unified Model of the GABA(A) Receptor Comprising Agonist and Benzodiazepine Binding Sites

    DEFF Research Database (Denmark)

    Kongsbak, Kristine Grønning; Bergmann, Rikke; Sørensen, Pernille Louise

    2013-01-01

    We present a full-length a1b2c2 GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate...

  16. Response of Fatty Acid Synthesis Genes to the Binding of Human Salivary Amylase by Streptococcus gordonii

    OpenAIRE

    Nikitkova, Anna E.; Haase, Elaine M.; Vickerman, M. Margaret; Gill, Steven R.; Scannapieco, Frank A.

    2012-01-01

    Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were di...

  17. Signal transduction of erythrocytes after specific binding of ecdysterone and cholesterol immobilized on nanodispersed magnetite

    Energy Technology Data Exchange (ETDEWEB)

    Mykhaylyk, O.M. E-mail: helek@iptelecom.net.ua; Kotzuruba, A.V.; Buchanevich, O.M.; Korduban, A.M.; Meged, E.F.; Gulaya, N.M

    2001-07-01

    Concurrent binding of cholesterol and ecdysterone immobilized on nanodispersed magnetite to intact rat erythrocytes was investigated. Several binding components on erythrocyte plasma membrane with different affinities were revealed in the range of 10{sup -15}-10{sup -8} M. The specific binding modulates signal transduction through adenylate cyclase and guanylate cyclase systems as manifested by the decrease in cAMP and increase in cGMP second messenger production.

  18. Terms of Binding

    NARCIS (Netherlands)

    Sevcenco, A.

    2006-01-01

    The present dissertation aimed at achieving two goals. First, it constitutes an attempt to widen the search for phenomena that bear relevance to the idea that binding has a syntactic residue and is not, therefore, an exclusively semantic matter. Second, it tried to provide the technical means to acc

  19. Binding and Bulgarian

    NARCIS (Netherlands)

    Schürcks-Grozeva, Lilia Lubomirova

    2003-01-01

    In haar proefschrift analyseert Lilia Schürcks de anaforische verschijnselen in de Bulgaarse taal. Het gaat dan om wederkerende aspecten, uitgedrukt bij woorden als ‘zich’ en ‘elkaar’. De situatie in het Bulgaars blijkt moeilijk in te passen in de klassieke Binding Theory van Noam Chomsky. Bron: RUG

  20. MD-2 binds cholesterol.

    Science.gov (United States)

    Choi, Soo-Ho; Kim, Jungsu; Gonen, Ayelet; Viriyakosol, Suganya; Miller, Yury I

    2016-02-19

    Cholesterol is a structural component of cellular membranes, which is transported from liver to peripheral cells in the form of cholesterol esters (CE), residing in the hydrophobic core of low-density lipoprotein. Oxidized CE (OxCE) is often found in plasma and in atherosclerotic lesions of subjects with cardiovascular disease. Our earlier studies have demonstrated that OxCE activates inflammatory responses in macrophages via toll-like receptor-4 (TLR4). Here we demonstrate that cholesterol binds to myeloid differentiation-2 (MD-2), a TLR4 ancillary molecule, which is a binding receptor for bacterial lipopolysaccharide (LPS) and is indispensable for LPS-induced TLR4 dimerization and signaling. Cholesterol binding to MD-2 was competed by LPS and by OxCE-modified BSA. Furthermore, soluble MD-2 in human plasma and MD-2 in mouse atherosclerotic lesions carried cholesterol, the finding supporting the biological significance of MD-2 cholesterol binding. These results help understand the molecular basis of TLR4 activation by OxCE and mechanisms of chronic inflammation in atherosclerosis.

  1. Sequential memory: Binding dynamics

    Science.gov (United States)

    Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

    2015-10-01

    Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories—episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

  2. Cellulose binding domain proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  3. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan;

    2005-01-01

    Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva...... to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of (125)I-labeled folate binding protein (FBP) to sections of kidney proximal tubule, known...

  4. Evidence that the modulator of the glucocorticoid-receptor complex is the endogenous molybdate factor.

    OpenAIRE

    Bodine, P V; Litwack, G

    1988-01-01

    We have recently purified the modulator of the glucocorticoid-receptor complex from rat liver. Purified modulator inhibits glucocorticoid-receptor complex activation and stabilizes the steroid-binding ability of the unoccupied glucocorticoid receptor. Since these activities are shared by exogenous sodium molybdate, modulator appears to be the endogenous factor that sodium molybdate mimics. In this report, we present additional evidence for the mechanism of action of purified modulator. (i) Mo...

  5. H-1 and N-15 resonance assignment of the second fibronectin type III module of the neural cell adhesion molecule

    DEFF Research Database (Denmark)

    Kiselyov, Vladislav V; Berezin, Vladimir; Bock, Elisabeth;

    2008-01-01

    We report here the NMR assignment of the second fibronectin type III module of the neural cell adhesion molecule (NCAM). This module has previously been shown to interact with the fibroblast growth factor receptor (FGFR), and the FGFR-binding site was mapped by NMR to the FG-loop region...... of the module. The FG-loop region also contains a putative nucleotide-binding motif, which was shown by NMR to interact with ATP. Furthermore, ATP was demonstrated to inhibit binding of the second F3 module of NCAM to FGFR....

  6. Plasmonic-Based Electrochemical Impedance Spectroscopy: Application to Molecular Binding

    Science.gov (United States)

    Lu, Jin; Wang, Wei; Wang, Shaopeng; Shan, Xiaonan; Li, Jinghong; Tao, Nongjian

    2012-01-01

    Plasmonic-based electrochemical impedance spectroscopy (P-EIS) is developed to investigate molecular binding on surfaces. Its basic principle relies on the sensitive dependence of surface plasmon resonance (SPR) signal on surface charge density, which is modulated by applying an AC potential to a SPR chip surface. The AC component of the SPR response gives the electrochemical impedance, and the DC component provides the conventional SPR detection. The plasmonic-based impedance measured over a range of frequency is in quantitative agreement with the conventional electrochemical impedance. Compared to the conventional SPR detection, P-EIS is sensitive to molecular binding taking place on the chip surface, and less sensitive to bulk refractive index changes or non-specific binding. Moreover, this new approach allows for simultaneous SPR and surface impedance analysis of molecular binding processes. PMID:22122514

  7. Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose.

    Directory of Open Access Journals (Sweden)

    Ting-Ying Jiang

    Full Text Available The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21 members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.

  8. Elements and modulation of functional dynamics.

    Science.gov (United States)

    Gibbs, Alan C

    2014-10-09

    The existing structure-function paradigm of drug discovery has been evolving toward the essential incorporation of dynamics data. This new functional dynamics paradigm emphasizes conformational entropy as a driving force of protein function and intermolecular recognition. Conformational dynamics (a proxy of conformational entropy) impacts the degree of protein (dis)order and the constitution of the conformational ensemble, the mechanisms of allostery and drug resistance, and the free energy of ligand binding. Specific protein and ligand conformations facilitate favorable, reciprocal interactions. The number of protein and ligand conformers that exhibit favorable binding interactions will vary from system to system. All binding scenarios can modulate protein dynamics by various levels of enthalpic and entropic contribution, with significant influence on the functional dynamics of the system. Analysis and consideration of resulting changes of activity, signaling, catalysis, and subsequent phenotypic outcome are powerful motivations in the drug design process.

  9. Identification and Structural Basis of Binding to Host Lung Glycogen by Streptococcal Virulence Factors

    Energy Technology Data Exchange (ETDEWEB)

    Lammerts van Bueren,A.; Higgins, M.; Wang, D.; Burke, R.; Boraston, A.

    2007-01-01

    The ability of pathogenic bacteria to recognize host glycans is often essential to their virulence. Here we report structure-function studies of previously uncharacterized glycogen-binding modules in the surface-anchored pullulanases from Streptococcus pneumoniae (SpuA) and Streptococcus pyogenes (PulA). Multivalent binding to glycogen leads to a strong interaction with alveolar type II cells in mouse lung tissue. X-ray crystal structures of the binding modules reveal a novel fusion of tandem modules into single, bivalent functional domains. In addition to indicating a structural basis for multivalent attachment, the structure of the SpuA modules in complex with carbohydrate provides insight into the molecular basis for glycogen specificity. This report provides the first evidence that intracellular lung glycogen may be a novel target of pathogenic streptococci and thus provides a rationale for the identification of the streptococcal {alpha}-glucan-metabolizing machinery as virulence factors.

  10. Thermodynamics and structural analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2

    DEFF Research Database (Denmark)

    Krintel, Christian; Frydenvang, Karla; Olsen, Lars;

    2012-01-01

    Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the ligand-binding domain and stabilize the agonist-bound conformation slow...... by the ethyl substituent of BPAM-97. These results add important information on binding affinities and thermodynamic details, and provide a new tool in development of drugs against cognitive disorders....

  11. Binding Principles A and B

    Institute of Scientific and Technical Information of China (English)

    陈源

    2014-01-01

    This paper focuses on the discussion of how Binding Principle A and Binding Principe B help with the interpretation of reference in English and Chinese. They are supposedly universal across languages.

  12. Koszul differential graded modules

    Institute of Scientific and Technical Information of China (English)

    HE JiWei; WU QuanShui

    2009-01-01

    The concept of Koszulity for differential graded (DG, for short) modules is introduced. It is shown that any bounded below DG module with bounded Ext-group to the trivial module over a Koszul DG algebra has a Koszul DG submodule (up to a shift and truncation), moreover such a DG module can be approximated by Koszul DG modules (Theorem 3.6). Let A be a Koszul DG algebra, and Dc (A) be the full triangulated subcategory of the derived category of DG A-modules generated by the object AA. If the trivial DG module kA lies in Dc(A), then the heart of the standard t-structure on Dc(A) is anti-equivalent to the category of finitely generated modules over some finite dimensional algebra. As a corollary, Dc(A) is equivalent to the bounded derived category of its heart as triangulated categories.

  13. Koszul differential graded modules

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The concept of Koszulity for differential graded (DG, for short) modules is introduced. It is shown that any bounded below DG module with bounded Ext-group to the trivial module over a Koszul DG algebra has a Koszul DG submodule (up to a shift and truncation), moreover such a DG module can be approximated by Koszul DG modules (Theorem 3.6). Let A be a Koszul DG algebra, and Dc(A) be the full triangulated subcategory of the derived category of DG A-modules generated by the object AA. If the trivial DG module kA lies in Dc(A), then the heart of the standard t-structure on Dc(A) is anti-equivalent to the category of finitely generated modules over some finite dimensional algebra. As a corollary, Dc(A) is equivalent to the bounded derived category of its heart as triangulated categories.

  14. On the influence of reward on action-effect binding

    Directory of Open Access Journals (Sweden)

    Paul Simon Muhle-Karbe

    2012-11-01

    Full Text Available Ideomotor theory states that the formation of anticipatory representations about the perceptual consequences of an action (i.e. action-effect (A-E binding provides the functional basis of voluntary action control. A host of studies has demonstrated that A-E binding occurs fast and effortlessly, yet only little is known about cognitive and affective factors that influence this learning process. In the present study, we sought to test whether the motivational value of an action modulates the acquisition of A-E associations. To this end, we associated specific actions with monetary incentives during the acquisition of novel A-E mappings. In a subsequent test phase, the degree of binding was assessed by presenting the former effect stimuli as task-irrelevant response primes in a forced-choice response task in the absence of any reward. Binding, as indexed by response priming through the former action effects, was only found for reward-related A-E mappings. Moreover, the degree to which reward associations modulated the binding strength was predicted by individuals’ trait sensitivity to reward. These observations indicate that the association of actions and their immediate outcomes depends on the motivational value of the action during learning, as well as on the motivational disposition of the individual. On a larger scale, these findings also highlight the link between ideomotor theories and reinforcement-learning theories, providing an interesting perspective for future research on anticipatory regulation of behavior.

  15. On Matlis dualizing modules

    Directory of Open Access Journals (Sweden)

    Edgar E. Enochs

    2002-01-01

    Full Text Available We consider rings admitting a Matlis dualizing module E. We argue that if R admits two such dualizing modules, then a module is reflexive with respect to one if and only if it is reflexive with respect to the other. Using this fact we argue that the number (whether finite or infinite of distinct dualizing modules equals the number of distinct invertible (R,R-bimodules. We show by example that this number can be greater than one.

  16. A peristaltic pump driven 89Zr separation module

    DEFF Research Database (Denmark)

    Siikanen, J.; Peterson, M.; Tran, T.

    2012-01-01

    To facilitate the separation of 89Zr produced in yttrium foils, an automated separation module was designed and assembled. The module separates more than 85% of produced 89Zr - activity in 3 g foils in less than 90 min. About 10 % remains in the dissolving vial. The quality of the separated 89Zr ...... activity was investigated for labeling of the HER2-binding monoclonal antibody fragment, trastuzumab-Fab....

  17. MI 6040 Thermoelectric Modules.

    Science.gov (United States)

    The report covers the design justification, physical specification and characterization of the MI 6040 module . The purpose of the thermoelectric... modules is the cooling of infrared detector arrays to temperature of 170K or colder. The completed modules were also subjected to limited demonstration tests of reliability and useful life.

  18. Gigahertz optical modulation.

    Science.gov (United States)

    Riesz, R P; Biazzo, M R

    1969-07-01

    Light pulses from a mode-locked He-Ne laser have been modulated by a LiTaO(3) electrooptic crystal mounted on a thin film substrate. The crystal was driven by pulses from a GaAs Gunn effect diode. Amplitude modulation of 20% has been achieved at 2 GHz for a single pass through the modulator.

  19. Polarization modulators for CMBPol

    Energy Technology Data Exchange (ETDEWEB)

    Ade, P A R; Savini, G [Cardiff University, School of Physics and Astronomy, Queens Buildings, The Parade, Cardiff, CF24 3AA (United Kingdom); Chuss, D T [NASA Goddard Space Flight Center, Code 665, Greenbelt, MD, 20771 (United States); Hanany, S [School of Physics and Astronomy, University of Minnesota/Twin Cities, Minneapolis, MN, 55455 (United States); Haynes, V; Pisano, G [University of Manchester, School of Physics and Astronomy - Alan Turing Building, Upper Brooke street, Manchester, M13 4PL (United Kingdom); Keating, B G [Department of Physics, University of California, San Diego, La Jolla, CA 92093-0424 (United States); Kogut, A [Code 665 Goddard Space Flight Center, Greenbelt, MD 20771 (United States); Ruhl, J E [Physics Department, Case Western Reserve University, Cleveland, OH, 44106 (United States); Wollack, E J [Observational Cosmology Laboratory, NASA/GSFC, Greenbelt, MD 20771 (United States)

    2009-03-01

    We review a number of technologies that are candidates for active polarization modulators for CMBPol. The technologies are appropriate for instruments that use bolometric detectors and include birefringent crystal-based and metal-mesh-based half-wave plates, variable phase polarization modulator, Faraday rotator, and photolithographed modulators. We also give a current account of the status of millimeter-wave orthomode transducers.

  20. Modulating lignin in plants

    Science.gov (United States)

    Apuya, Nestor; Bobzin, Steven Craig; Okamuro, Jack; Zhang, Ke

    2013-01-29

    Materials and methods for modulating (e.g., increasing or decreasing) lignin content in plants are disclosed. For example, nucleic acids encoding lignin-modulating polypeptides are disclosed as well as methods for using such nucleic acids to generate transgenic plants having a modulated lignin content.

  1. Relatively Endocoherent Modules

    Institute of Scientific and Technical Information of China (English)

    Lixin Mao

    2007-01-01

    Let MR be a right R-module over a ring R with S= End(MR)We study the coherence of the left S-module sM relative to a torsion theory for the category of right R-modules.Various results are developed, many extending known results.

  2. Carboplatin binding to histidine

    Energy Technology Data Exchange (ETDEWEB)

    Tanley, Simon W. M. [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom); Diederichs, Kay [University of Konstanz, D-78457 Konstanz (Germany); Kroon-Batenburg, Loes M. J. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Levy, Colin [University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom); Schreurs, Antoine M. M. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Helliwell, John R., E-mail: john.helliwell@manchester.ac.uk [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom)

    2014-08-29

    An X-ray crystal structure showing the binding of purely carboplatin to histidine in a model protein has finally been obtained. This required extensive crystallization trials and various novel crystal structure analyses. Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described.

  3. Bindings in working memory: The role of object-based attention.

    Science.gov (United States)

    Gao, Zaifeng; Wu, Fan; Qiu, Fangfang; He, Kaifeng; Yang, Yue; Shen, Mowei

    2017-02-01

    Over the past decade, it has been debated whether retaining bindings in working memory (WM) requires more attention than retaining constituent features, focusing on domain-general attention and space-based attention. Recently, we proposed that retaining bindings in WM needs more object-based attention than retaining constituent features (Shen, Huang, & Gao, 2015, Journal of Experimental Psychology: Human Perception and Performance, doi: 10.1037/xhp0000018 ). However, only unitized visual bindings were examined; to establish the role of object-based attention in retaining bindings in WM, more emperical evidence is required. We tested 4 new bindings that had been suggested requiring no more attention than the constituent features in the WM maintenance phase: The two constituent features of binding were stored in different WM modules (cross-module binding, Experiment 1), from auditory and visual modalities (cross-modal binding, Experiment 2), or temporally (cross-time binding, Experiments 3) or spatially (cross-space binding, Experiments 4-6) separated. In the critical condition, we added a secondary object feature-report task during the delay interval of the change-detection task, such that the secondary task competed for object-based attention with the to-be-memorized stimuli. If more object-based attention is required for retaining bindings than for retaining constituent features, the secondary task should impair the binding performance to a larger degree relative to the performance of constituent features. Indeed, Experiments 1-6 consistently revealed a significantly larger impairment for bindings than for the constituent features, suggesting that object-based attention plays a pivotal role in retaining bindings in WM.

  4. Divisible ℤ-modules

    Directory of Open Access Journals (Sweden)

    Futa Yuichi

    2016-03-01

    Full Text Available In this article, we formalize the definition of divisible ℤ-module and its properties in the Mizar system [3]. We formally prove that any non-trivial divisible ℤ-modules are not finitely-generated.We introduce a divisible ℤ-module, equivalent to a vector space of a torsion-free ℤ-module with a coefficient ring ℚ. ℤ-modules are important for lattice problems, LLL (Lenstra, Lenstra and Lovász base reduction algorithm [15], cryptographic systems with lattices [16] and coding theory [8].

  5. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Nielsen, Morten M.; Christiansen, Camilla

    2015-01-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch...... to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert...

  6. Reduced Multiplication Modules

    Indian Academy of Sciences (India)

    Karim Samei

    2011-05-01

    An -module is called a multiplication module if for each submodule of , = for some ideal of . As defined for a commutative ring , an -module is said to be reduced if the intersection of prime submodules of is zero. The prime spectrum and minimal prime submodules of the reduced module are studied. Essential submodules of are characterized via a topological property. It is shown that the Goldie dimension of is equal to the Souslin number of Spec (). Also a finitely generated module is a Baer module if and only if Spec () is an extremally disconnected space; if and only if it is a -module. It is proved that a prime submodule is minimal in if and only if for each $x\\in N,\\mathrm{Ann}(x)\

  7. Solar cell module. Taiyo denchi module

    Energy Technology Data Exchange (ETDEWEB)

    Nakano, Akihiko.

    1990-01-24

    This invention concerns a module frame of solar cell and a solar cell module using this frame. In particular, it concerns a frame and a module useful for the CdS/CdTe or CdS/CuInSe {sub 2} based cell. In the existing solar cell module, sealant is packed in between the edges of a glass substrate, a resin layer and a back protective thin film, etc. and a grooved frame of U-shaped section. For the sealant, silicon based resin and butyl rubber based resin are used many times, but either resin has defects such as their overflow from the module structure. In order to solve these defects, this invention proposes to provide stair-shaped protrusions along the four sides of the bottom of the box frame (herein after called the lower frame) of the module and at the same time, provide a groove for pooling the sealant at the portion where such protrusion meets the side wall, furthermore to provide depressions for pooling the sealant at the upper edge inside the side wall of the lower frame or to punch holes at the corners of the bottom of the lower frame. 9 figs.

  8. A unique bivalent binding and inhibition mechanism by the yatapoxvirus interleukin 18 binding protein.

    Directory of Open Access Journals (Sweden)

    Brian Krumm

    Full Text Available Interleukin 18 (IL18 is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding

  9. Analytic QCD Binding Potentials

    CERN Document Server

    Fried, H M; Grandou, T; Sheu, Y -M

    2011-01-01

    This paper applies the analytic forms of a recent non-perturbative, manifestly gauge- and Lorentz-invariant description (of the exchange of all possible virtual gluons between quarks ($Q$) and/or anti-quarks ($\\bar{Q}$) in a quenched, eikonal approximation) to extract analytic forms for the binding potentials generating a model $Q$-$\\bar{Q}$ "pion", and a model $QQQ$ "nucleon". Other, more complicated $Q$, $\\bar{Q}$ contributions to such color-singlet states may also be identified analytically. An elementary minimization technique, relevant to the ground states of such bound systems, is adopted to approximate the solutions to a more proper, but far more complicated Schroedinger/Dirac equation; the existence of possible contributions to the pion and nucleon masses due to spin, angular momentum, and "deformation" degrees of freedom is noted but not pursued. Neglecting electromagnetic and weak interactions, this analysis illustrates how the one new parameter making its appearance in this exact, realistic formali...

  10. The cyclic AMP response element modulator regulates transcription of the TCR zeta-chain

    NARCIS (Netherlands)

    Tenbrock, K; Kyttaris, VC; Ahlmann, M; Ehrchen, JA; Tolnay, M; Melkonyan, H; Mawrin, C; Roth, J; Sorg, C; Juang, YT; Tsokos, GC

    2005-01-01

    Systemic lupus erythematusus T cells display decreased amounts of TCR zeta mRNA that results in part from limited binding of the transcriptional enhancer Elf-1 to the TCR zeta promoter. We have identified a new cis-binding site for the cAMP response element (CRE) modulator (CREM) on the TCR zeta pro

  11. Rigidity of tilting modules

    DEFF Research Database (Denmark)

    Haahr Andersen, Henning; Kaneda, Masaharu

    ) modules for $U_q$ are rigid, i.e., have identical radical and socle filtrations. Moreover, we obtain the same for a large class of Weyl modules for $U_q$. On the other hand, we give examples of non-rigid indecomposable tilting modules as well as non-rigid Weyl modules. These examples are for type $B_2......$ and in this case as well as for type $A_2$ we calculate explicitly the Loewy structure for all regular Weyl modules. We also demonstrate that these results carry over to the modular case when the highest weights in question are in the so-called Jantzen region. At the same time we show by examples that as soon...... as we leave this region non-rigid tilting modules do occur....

  12. Directed network modules

    CERN Document Server

    Pálla, G; Farkas, I J; Pollner, P; Vicsek, T; Derenyi, Imre; Farkas, Illes J.; Palla, Gergely; Pollner, Peter; Vicsek, Tamas

    2007-01-01

    A search technique locating network modules, i.e., internally densely connected groups of nodes in directed networks is introduced by extending the Clique Percolation Method originally proposed for undirected networks. After giving a suitable definition for directed modules we investigate their percolation transition in the Erdos-Renyi graph both analytically and numerically. We also analyse four real-world directed networks, including Google's own webpages, an email network, a word association graph and the transcriptional regulatory network of the yeast Saccharomyces cerevisiae. The obtained directed modules are validated by additional information available for the nodes. We find that directed modules of real-world graphs inherently overlap and the investigated networks can be classified into two major groups in terms of the overlaps between the modules. Accordingly, in the word-association network and among Google's webpages the overlaps are likely to contain in-hubs, whereas the modules in the email and t...

  13. Hydrophobic kenaf nanocrystalline cellulose for the binding of curcumin.

    Science.gov (United States)

    Zainuddin, Norhidayu; Ahmad, Ishak; Kargarzadeh, Hanieh; Ramli, Suria

    2017-05-01

    Nanocrystalline cellulose (NCC) extracted from lignocellulosic materials has been actively investigated as a drug delivery excipients due to its large surface area, high aspect ratio, and biodegradability. In this study, the hydrophobically modified NCC was used as a drug delivery excipient of hydrophobic drug curcumin. The modification of NCC with a cationic surfactant, cetyl trimethylammonium bromide (CTAB) was used to modulate the loading of hydrophobic drugs that would not normally bind to NCC. The FTIR, Elemental analysis, XRD, TGA, and TEM were used to confirm the modification of NCC with CTAB. The effect of concentration of CTAB on the binding efficiency of hydrophobic drug curcumin was investigated. The amounts of curcumin bound onto the CTAB-NCC nanoparticles were analyzed by UV-vis Spectrophotometric. The result showed that the modified CTAB-NCC bound a significant amount of curcumin, in a range from 80% to 96% curcumin added. Nevertheless, at higher concentration of CTAB resulted in lower binding efficiency.

  14. A photovoltaic module

    OpenAIRE

    Krebs, Frederik C.; Sommer-Larsen, Peter

    2013-01-01

    The present invention relates to a photovoltaic module comprising a carrier substrate, said carrier substrate carrying a purely printed structure comprising printed positive and negative module terminals, a plurality of printed photovoltaic cell units each comprising one or more printed photovoltaic cells, wherein the plurality of printed photovoltaic cell units are electrically connected in series between the positive and the negative module terminals such that any two neighbouring photovolt...

  15. Investigating Quantum Modulation States

    Science.gov (United States)

    2016-03-01

    3. DATES COVERED (From - To) OCT 2012 – SEP 2015 4. TITLE AND SUBTITLE INVESTIGATING QUANTUM MODULATION STATES 5a. CONTRACT NUMBER IN-HOUSE 5b...Coherent states are the most classical of quantum states. Generation and detection of their polarization and phase modulations are well...stream cipher maps message bits onto random blocks of bits producing modulated states that are intrinsically noisy. The ciphertext so generated is

  16. Loss of selenium-binding protein 1 decreases sensitivity to clastogens and intracellular selenium content in HeLa cells

    Science.gov (United States)

    Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesized that loss of SBP1 modulates cellular selenium content and the response of ...

  17. Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Ruzanski, Christian

    2014-01-01

    Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half ...

  18. Selective Negative Allosteric Modulation Of Metabotropic Glutamate Receptors - A Structural Perspective of Ligands and Mutants

    DEFF Research Database (Denmark)

    Harpsøe, Kasper; Isberg, Vignir; Tehan, Benjamin G

    2015-01-01

    modulators. In this analysis, we make the first comprehensive structural comparison of all metabotropic glutamate receptors, placing selective negative allosteric modulators and critical mutants into the detailed context of the receptor binding sites. A better understanding of how the different m...

  19. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    Science.gov (United States)

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-06

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

  20. THERMOELECTRIC POWER MODULES.

    Science.gov (United States)

    MODULES (ELECTRONICS), GENERATORS, THERMOELECTRICITY, PERFORMANCE(ENGINEERING), TABLES(DATA), HEAT, ALUMINUM, WEIGHT, SEMICONDUCTORS, SILICON, GERMANIUM, MEASUREMENT, VOLTAGE, ELECTRICAL RESISTANCE, POWER, TEMPERATURE, TIME.

  1. Delphi Accounts Receivable Module -

    Data.gov (United States)

    Department of Transportation — Delphi accounts receivable module contains the following data elements, but are not limited to customer information, cash receipts, line of accounting details, bill...

  2. Model theory and modules

    CERN Document Server

    Prest, M

    1988-01-01

    In recent years the interplay between model theory and other branches of mathematics has led to many deep and intriguing results. In this, the first book on the topic, the theme is the interplay between model theory and the theory of modules. The book is intended to be a self-contained introduction to the subject and introduces the requisite model theory and module theory as it is needed. Dr Prest develops the basic ideas concerning what can be said about modules using the information which may be expressed in a first-order language. Later chapters discuss stability-theoretic aspects of module

  3. Prediction of tissue-specific cis-regulatory modules using Bayesian networks and regression trees

    Directory of Open Access Journals (Sweden)

    Chen Xiaoyu

    2007-12-01

    Full Text Available Abstract Background In vertebrates, a large part of gene transcriptional regulation is operated by cis-regulatory modules. These modules are believed to be regulating much of the tissue-specificity of gene expression. Results We develop a Bayesian network approach for identifying cis-regulatory modules likely to regulate tissue-specific expression. The network integrates predicted transcription factor binding site information, transcription factor expression data, and target gene expression data. At its core is a regression tree modeling the effect of combinations of transcription factors bound to a module. A new unsupervised EM-like algorithm is developed to learn the parameters of the network, including the regression tree structure. Conclusion Our approach is shown to accurately identify known human liver and erythroid-specific modules. When applied to the prediction of tissue-specific modules in 10 different tissues, the network predicts a number of important transcription factor combinations whose concerted binding is associated to specific expression.

  4. Allosteric inactivation of a trypsin-like serine protease by an antibody binding to the 37- and 70-loops

    DEFF Research Database (Denmark)

    Kromann-Hansen, Tobias; Lund, Ida K; Liu, Zhuo

    2013-01-01

    Serine protease catalytic activity is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. Inhibitory monoclonal antibodies binding to such exosites are potential therapeutics and offer opportunities...... criteria similar to the E* conformation described for other serine proteases. Hence, agents targeting serine protease conformation through binding to exosites in the 37- and 70-loops represent a new class of potential therapeutics....

  5. Direct GR Binding Sites Potentiate Clusters of TF Binding across the Human Genome.

    Science.gov (United States)

    Vockley, Christopher M; D'Ippolito, Anthony M; McDowell, Ian C; Majoros, William H; Safi, Alexias; Song, Lingyun; Crawford, Gregory E; Reddy, Timothy E

    2016-08-25

    The glucocorticoid receptor (GR) binds the human genome at >10,000 sites but only regulates the expression of hundreds of genes. To determine the functional effect of each site, we measured the glucocorticoid (GC) responsive activity of nearly all GR binding sites (GBSs) captured using chromatin immunoprecipitation (ChIP) in A549 cells. 13% of GBSs assayed had GC-induced activity. The responsive sites were defined by direct GR binding via a GC response element (GRE) and exclusively increased reporter-gene expression. Meanwhile, most GBSs lacked GC-induced reporter activity. The non-responsive sites had epigenetic features of steady-state enhancers and clustered around direct GBSs. Together, our data support a model in which clusters of GBSs observed with ChIP-seq reflect interactions between direct and tethered GBSs over tens of kilobases. We further show that those interactions can synergistically modulate the activity of direct GBSs and may therefore play a major role in driving gene activation in response to GCs.

  6. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  7. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...... for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon...... sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites...

  8. Binding Energy and Enzymatic Catalysis.

    Science.gov (United States)

    Hansen, David E.; Raines, Ronald T.

    1990-01-01

    Discussed is the fundamental role that the favorable free energy of binding of the rate-determining transition state plays in catalysis. The principle that all of the catalytic factors discussed are realized by the use of this binding energy is reviewed. (CW)

  9. Identification of collagen binding domain residues that govern catalytic activities of matrix metalloproteinase-2 (MMP-2).

    Science.gov (United States)

    Mikhailova, Margarita; Xu, Xiaoping; Robichaud, Trista K; Pal, Sanjay; Fields, Gregg B; Steffensen, Bjorn

    2012-01-01

    An innovative approach to enhance the selectivity of matrix metalloproteinase (MMP) inhibitors comprises targeting these inhibitors to catalytically required substrate binding sites (exosites) that are located outside the catalytic cleft. In MMP-2, positioning of collagen substrate molecules occurs via a unique fibronectin-like domain (CBD) that contains three distinct modular collagen binding sites. To characterize the contributions of these exosites to gelatinolysis by MMP-2, seven MMP-2 variants were generated with single, or concurrent double and triple alanine substitutions in the three fibronectin type II modules of the CBD. Circular dichroism spectroscopy verified that recombinant MMP-2 wild-type (WT) and variants had the same fold. Moreover, the MMP-2 WT and variants had the same activity on a short FRET peptide substrate that is hydrolyzed independently of CBD binding. Among single-point variants, substitution in the module 3 binding site had greatest impact on the affinity of MMP-2 for gelatin. Simultaneous substitutions in two or three CBD modules further reduced gelatin binding. The rates of gelatinolysis of MMP-2 variants were reduced by 20-40% following single-point substitutions, by 60-75% after double-point modifications, and by >90% for triple-point variants. Intriguingly, the three CBD modules contributed differentially to cleavage of dissociated α-1(I) and α-2(I) collagen chains. Importantly, kinetic analyses (k(cat)/K(m)) revealed that catalysis of a triple-helical FRET peptide substrate by MMP-2 relied primarily on the module 3 binding site. Thus, we have identified three collagen binding site residues that are essential for gelatinolysis and constitute promising targets for selective inhibition of MMP-2.

  10. Parabolic Dish Stirling Module

    Science.gov (United States)

    Washom, B.

    1984-01-01

    The design, manufacture, and assembly of a commercially designed parabolic dish Stirling 25 kWe module is examined. The cost, expected performance, design uniquenesses, and future commercial potential of this module, which is regarded as the most technically advanced in the parabolic dish industry is discussed.

  11. Cosmetology. Computerized Learning Modules.

    Science.gov (United States)

    Finnerty, Kathy, Ed.

    Intended to help reading-limited students meet course objectives, these 11 modules are based on instructional materials in cosmetology that have a higher readability equivalent. Modules cover bacteriology, chemical waving, scalp and hair massage, chemistry, hair shaping, hairstyling, chemical hair relaxing, hair coloring, skin and scalp,…

  12. Synthetic Space Vector Modulation

    Science.gov (United States)

    2013-06-01

    Modulation RF Radio Frequency SVM Space Vector Modulation VCO Voltage Controlled Oscillator VSI Voltage Source Inverter xvi THIS PAGE...examining the literature, an NE566 voltage controlled oscillator ( VCO ) chip as seen in Figure 10 was used to design a circuit that produced the

  13. The Structure of Modules

    Institute of Scientific and Technical Information of China (English)

    陈焕艮; 佟文廷

    1994-01-01

    In this paper,we introduce two concepts:projective radical and semi-reflexive radical of a module,and establish some elementary relations among subprojective,semi-reflexive and torsion-free modules Using these properties,ws give classifications of some rings.

  14. Elucidation of the mechanism of the regulatory function of the Ig1 module of the fibroblast growth factor receptor 1

    DEFF Research Database (Denmark)

    Kiselyov, Vladislav; Kochoyan, Artur; Poulsen, Flemming;

    2006-01-01

    The extracellular part of the fibroblast growth factor (FGF) receptor (FGFR) consists of up to three Ig modules (Ig1-Ig3), in which the Ig2 and Ig3 modules determine affinity and specificity for FGF and heparin. The FGFR isoforms lacking the Ig1 module have higher affinity for FGF and heparin than...... the triple Ig-module isoforms, suggesting that the Ig1 module is involved in the regulation of the FGFR-ligand interaction. We show here by surface plasmon resonance and NMR analyses that the Ig1 module binds to the Ig2 module, and identify by NMR the binding sites involved in the Ig1-Ig2 interaction....... The identified binding site in the Ig2 module was found to be in the area of the FGF-Ig2 and Ig2-heparin contact sites, thus providing direct structural evidence that the Ig1 module functions as a competitive autoinhibitor of the FGFR-ligand interaction. Furthermore, the Ig1 binding site of the Ig2 module...

  15. Ligand binding-dependent functions of the lipocalin NLaz: an in vivo study in Drosophila.

    Science.gov (United States)

    Ruiz, Mario; Ganfornina, Maria D; Correnti, Colin; Strong, Roland K; Sanchez, Diego

    2014-04-01

    Lipocalins are small extracellular proteins mostly described as lipid carriers. The Drosophila lipocalin NLaz (neural Lazarillo) modulates the IIS pathway and regulates longevity, stress resistance, and behavior. Here, we test whether a native hydrophobic pocket structure is required for NLaz to perform its functions. We use a point mutation altering the binding pocket (NLaz(L130R)) and control mutations outside NLaz binding pocket. Tryptophan fluorescence titration reveals that NLaz(L130R) loses its ability to bind ergosterol and the pheromone 7(z)-tricosene but retains retinoic acid binding. Using site-directed transgenesis in Drosophila, we test the functionality of the ligand binding-altered lipocalin at the organism level. NLaz-dependent life span reduction, oxidative stress and starvation sensitivity, aging markers accumulation, and deficient courtship are rescued by overexpression of NLaz(WT), but not of NLaz(L130R). Transcriptional responses to aging and oxidative stress show a large set of age-responsive genes dependent on the integrity of NLaz binding pocket. Inhibition of IIS activity and modulation of oxidative stress and infection-responsive genes are binding pocket-dependent processes. Control of energy metabolites on starvation appears to be, however, insensitive to the modification of the NLaz binding pocket.

  16. Characterization and DNA-binding specificities of Ralstonia TAL-like effectors

    KAUST Repository

    Li, Lixin

    2013-07-01

    Transcription activator-like effectors (TALEs) from Xanthomonas sp. have been used as customizable DNA-binding modules for genome-engineering applications. Ralstonia solanacearum TALE-like proteins (RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts specifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp. © 2013 The Author.

  17. Mn(II) binding to human serum albumin: a ¹H-NMR relaxometric study.

    Science.gov (United States)

    Fanali, Gabriella; Cao, Yu; Ascenzi, Paolo; Fasano, Mauro

    2012-12-01

    Human serum albumin (HSA) displays several metal binding sites, participating to essential and toxic metal ions disposal and transport. The major Zn(II) binding site, called Site A, is located at the I/II domain interface, with residues His67, Asn99, His247, and Asp249 contributing with five donor atoms to the metal ion coordination. Additionally, one water molecule takes part of the octahedral coordination geometry. The occurrence of the metal-coordinated water molecule allows the investigation of the metal complex geometry by water (1)H-NMR relaxation, provided that the diamagnetic Zn(II) is replaced by the paramagnetic Mn(II). Here, the (1)H-NMR relaxometric study of Mn(II) binding to HSA is reported. Mn(II) binding to HSA is modulated by Zn(II), pH, and myristate through competitive inhibition and allosteric mechanisms. The body of results indicates that the primary binding site of Zn(II) corresponds to the secondary binding site of Mn(II), i.e. the multimetal binding site A. Excess Zn(II) completely displaces Mn(II) from its primary site suggesting that the primary Mn(II) site corresponds to the secondary Zn(II) site. This uncharacterized site is functionally-linked to FA1; moreover, metal ion binding is modulated by myristate and pH. Noteworthy, water (1)H-NMR relaxometry allowed a detailed analysis of thermodynamic properties of HSA-metal ion complexes.

  18. Solar energy modulator

    Science.gov (United States)

    Hale, R. R. (Inventor); Mcdougal, A. R.

    1984-01-01

    A module is described with a receiver having a solar energy acceptance opening and supported by a mounting ring along the optic axis of a parabolic mirror in coaxial alignment for receiving solar energy from the mirror, and a solar flux modulator plate for varying the quantity of solar energy flux received by the acceptance opening of the module. The modulator plate is characterized by an annular, plate-like body, the internal diameter of which is equal to or slightly greater than the diameter of the solar energy acceptance opening of the receiver. Slave cylinders are connected to the modulator plate for supporting the plate for axial displacement along the axis of the mirror, therby shading the opening with respect to solar energy flux reflected from the surface of the mirror to the solar energy acceptance opening.

  19. Copper binding to the Alzheimer’s disease amyloid precursor protein

    OpenAIRE

    Kong, Geoffrey K.-W.; Miles, Luke A.; Crespi, Gabriela A. N.; Morton, Craig J.; Ng, Hooi Ling; Barnham, Kevin J.; McKinstry, William J.; Cappai, Roberto; Michael W. Parker

    2007-01-01

    Alzheimer’s disease is the fourth biggest killer in developed countries. Amyloid precursor protein (APP) plays a central role in the development of the disease, through the generation of a peptide called Aβ by proteolysis of the precursor protein. APP can function as a metalloprotein and modulate copper transport via its extracellular copper binding domain (CuBD). Copper binding to this domain has been shown to reduce Aβ levels and hence a molecular understanding of the interaction between me...

  20. Signal transduction by erythrocytes on specific binding of doxorubicin immobilized on nanodispersed magnetite

    Energy Technology Data Exchange (ETDEWEB)

    Mykhaylyk, Olga [Institute Applied Problems Physics and Biophysics, NAS, Sluzhbova 3, UA-03142 Kyiv (Ukraine)]. E-mail: Olga.Mykhaylyk@gmx.net; Kotzuruba, Anatoliy [Institute of Biochemistry, NAS, Leontovicha 9, UA-01030 Kyiv (Ukraine); Dudchenko, Nataliya [Institute Applied Problems Physics and Biophysics, NAS, Sluzhbova 3, UA-03142 Kyiv (Ukraine); Toerok, Gyula [Research Institute for Solid State Physics and Optics, H-1525 Budapest, P.O. Box 49 (Hungary)

    2005-05-15

    Two specific binding sites for doxorubicin were revealed at the plasma membrane of human erythrocytes on investigation of the binding of doxorubicin magnetic nanoconjugates. Free and conjugated doxorubicins modulated signal transduction in erythrocytes in a similar way. Both up-regulated nitric oxide and cyclic GMP (cGMP) and down-regulated cyclic AMP (cAMP) production and stabilize the membranes of damaged erythrocytes.

  1. Peculiarities of copper binding to alpha-synuclein.

    Science.gov (United States)

    Ahmad, Atta; Burns, Colin S; Fink, Anthony L; Uversky, Vladimir N

    2012-01-01

    Heavy metals have been implicated as the causative agents for the pathogenesis of the most prevalent neurodegenerative disease. Various mechanisms have been proposed to explain the toxic effects of metals ranging from metal-induced oxidation of protein to metal-induced changes in the protein conformation. Aggregation of a-synuclein is implicated in Parkinson's disease (PD), and various metals, including copper, constitute a prominent group of alpha-synuclein aggregation enhancers. In this study, we have systematically characterized the a-synuclein-Cu21 binding sites and analyzed the possible role of metal binding in a-synuclein fibrillation using a set of biophysical techniques, such as electron paramagnetic resonance (EPR), electron spin-echo envelope modulation (ESEEM), circular dichroism (CD), and size exclusion chromatography (SEC). Our analyses indicated that a-synuclein possesses at least two binding sites for Cu21. We have been able to locate one of the binding sites in the N-terminal region. Furthermore, based on the EPR studies of model peptides and Beta-synuclein, we concluded that the suspected His residue did not appear to participate in strong Cu21 binding.

  2. Characterization of Heparin-binding Site of Tissue Transglutaminase

    Science.gov (United States)

    Wang, Zhuo; Collighan, Russell J.; Pytel, Kamila; Rathbone, Daniel L.; Li, Xiaoling; Griffin, Martin

    2012-01-01

    Tissue transglutaminase (TG2) is a multifunctional Ca2+-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed. PMID:22298777

  3. Formyl peptide receptor chimeras define domains involved in ligand binding.

    Science.gov (United States)

    Perez, H D; Holmes, R; Vilander, L R; Adams, R R; Manzana, W; Jolley, D; Andrews, W H

    1993-02-05

    We have begun to study the structural requirements for the binding of formyl peptides to their specific receptors. As an initial approach, we constructed C5a-formyl peptide receptor chimeras. Unique (and identical) restriction sites were introduced within the transmembrane domains of these receptors that allowed for the exchange of specific areas. Four types of chimeric receptors were generated. 1) The C5a receptor was progressively substituted by the formyl peptide receptor. 2) The formyl peptide receptor was progressively substituted by the C5a receptor. 3) Specific domains of the C5a receptor were substituted by the corresponding domain of the formyl peptide receptor. 4) Specific domains of the formyl peptide receptor were replaced by the same corresponding domain of the C5a receptor. Wild type and chimeric receptors were transfected into COS 7 cells and their ability to bind formyl peptide determined, taking into account efficiency of transfection and expression of chimeric protein. Based on these results, a ligand binding model is presented in which the second, third, and fourth extracellular (and/or their transmembrane) domains together with the first transmembrane domain form a ligand binding pocket for formyl peptides. It is proposed that the amino-terminal domain plays a role by presumably providing a "lid" to the pocket. The carboxyl-terminal cytoplasmic tail appears to modulate ligand binding by regulating receptor affinity.

  4. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    Science.gov (United States)

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-01-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways. PMID:28195155

  5. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    Science.gov (United States)

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-02-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

  6. Entropic and enthalpic contributions to annexin V-membrane binding: a comprehensive quantitative model.

    Science.gov (United States)

    Jeppesen, Brian; Smith, Christina; Gibson, Donald F; Tait, Jonathan F

    2008-03-07

    Annexin V binds to membranes with very high affinity, but the factors responsible remain to be quantitatively elucidated. Analysis by isothermal microcalorimetry and calcium titration under conditions of low membrane occupancy showed that there was a strongly positive entropy change upon binding. For vesicles containing 25% phosphatidylserine at 0.15 m ionic strength, the free energy of binding was -53 kcal/mol protein, whereas the enthalpy of binding was -38 kcal/mol. Addition of 4 m urea decreased the free energy of binding by about 30% without denaturing the protein, suggesting that hydrophobic forces make a significant contribution to binding affinity. This was confirmed by mutagenesis studies that showed that binding affinity was modulated by the hydrophobicity of surface residues that are likely to enter the interfacial region upon protein-membrane binding. The change in free energy was quantitatively consistent with predictions from the Wimley-White scale of interfacial hydrophobicity. In contrast, binding affinity was not increased by making the protein surface more positively charged, nor decreased by making it more negatively charged, ruling out general ionic interactions as major contributors to binding affinity. The affinity of annexin V was the same regardless of the head group present on the anionic phospholipids tested (phosphatidylserine, phosphatidylglycerol, phosphatidylmethanol, and cardiolipin), ruling out specific interactions between the protein and non-phosphate moieties of the head group as a significant contributor to binding affinity. Analysis by fluorescence resonance energy transfer showed that multimers did not form on phosphatidylserine membranes at low occupancy, indicating that annexin-annexin interactions did not contribute to binding affinity. In summary, binding of annexin V to membranes is driven by both enthalpic and entropic forces. Dehydration of hydrophobic regions of the protein surface as they enter the interfacial region

  7. Rational targeting of subclasses of intermolecular interactions: elimination of nonspecific binding for analyte sensing.

    Science.gov (United States)

    Lane, Jordan S; Richens, Joanna L; Vere, Kelly-Ann; O'Shea, Paul

    2014-08-12

    The ability to target and control intermolecular interactions is crucial in the development of several different technologies. Here we offer a tool to rationally design liquid media systems that can modulate specific intermolecular interactions. This has broad implications in deciphering the nature of intermolecular forces in complex solutions and offers insight into the forces that govern both specific and nonspecific binding in a given system. Nonspecific binding still continues to be a problem when dealing with analyte detection across a range of different detection technologies. Here, we exemplify the problem of nonspecific binding on model membrane systems and when dealing with low-abundance protein detection on commercially available SPR technology. A range of different soluble reagents that target specific subclasses of intermolecular interactions have been tested and optimized to virtually eliminate nonspecific binding while leaving specific interactions unperturbed. Thiocyanate ions are used to target nonpolar interactions, and small reagents such as glycylglycylglycine are used to modulate the dielectric constant, which targets charge-charge and dipole interactions. We show that with rational design and careful modulation these reagents offer a step forward in dissecting the intermolecular forces that govern binding, alongside offering nonspecific binding elimination in detection systems.

  8. On Modules Whose Singular Subgenerated Modules Are Weakly Injective

    Institute of Scientific and Technical Information of China (English)

    S. Dhompongsa; J. Sanwong; S.Plubtieng; H.Tansee

    2001-01-01

    Rings over which every singular right module is injective (briefly,right SI-rings) were introduced and investigated by Goodearl. Weakly injective modules, as a generalization of injective modules, were introduced by Jain and modules are weakly injective, which we call SwI-rings. This concept is extended to SwI-modules, i.e., modules whose singular subgenerated modules are weakly injective. Several characterizations and properties of SwI-rings and SwI-modules are obtained which generalize some earlier known results on SI-rings and weakly semisimple rings.

  9. Photovoltaic module and interlocked stack of photovoltaic modules

    Science.gov (United States)

    Wares, Brian S.

    2014-09-02

    One embodiment relates to an arrangement of photovoltaic modules configured for transportation. The arrangement includes a plurality of photovoltaic modules, each photovoltaic module including a frame. A plurality of individual male alignment features and a plurality of individual female alignment features are included on each frame. Adjacent photovoltaic modules are interlocked by multiple individual male alignment features on a first module of the adjacent photovoltaic modules fitting into and being surrounded by corresponding individual female alignment features on a second module of the adjacent photovoltaic modules. Other embodiments, features and aspects are also disclosed.

  10. Interferon Lambda: Modulating Immunity in Infectious Diseases

    Science.gov (United States)

    Syedbasha, Mohammedyaseen; Egli, Adrian

    2017-01-01

    Interferon lambdas (IFN-λs; IFNL1-4) modulate immunity in the context of infections and autoimmune diseases, through a network of induced genes. IFN-λs act by binding to the heterodimeric IFN-λ receptor (IFNLR), activating a STAT phosphorylation-dependent signaling cascade. Thereby hundreds of IFN-stimulated genes are induced, which modulate various immune functions via complex forward and feedback loops. When compared to the well-characterized IFN-α signaling cascade, three important differences have been discovered. First, the IFNLR is not ubiquitously expressed: in particular, immune cells show significant variation in the expression levels of and susceptibilities to IFN-λs. Second, the binding affinities of individual IFN-λs to the IFNLR varies greatly and are generally lower compared to the binding affinities of IFN-α to its receptor. Finally, genetic variation in the form of a series of single-nucleotide polymorphisms (SNPs) linked to genes involved in the IFN-λ signaling cascade has been described and associated with the clinical course and treatment outcomes of hepatitis B and C virus infection. The clinical impact of IFN-λ signaling and the SNP variations may, however, reach far beyond viral hepatitis. Recent publications show important roles for IFN-λs in a broad range of viral infections such as human T-cell leukemia type-1 virus, rotaviruses, and influenza virus. IFN-λ also potentially modulates the course of bacterial colonization and infections as shown for Staphylococcus aureus and Mycobacterium tuberculosis. Although the immunological processes involved in controlling viral and bacterial infections are distinct, IFN-λs may interfere at various levels: as an innate immune cytokine with direct antiviral effects; or as a modulator of IFN-α-induced signaling via the suppressor of cytokine signaling 1 and the ubiquitin-specific peptidase 18 inhibitory feedback loops. In addition, the modulation of adaptive immune functions via macrophage and

  11. Cooperative binding: a multiple personality.

    Science.gov (United States)

    Martini, Johannes W R; Diambra, Luis; Habeck, Michael

    2016-06-01

    Cooperative binding has been described in many publications and has been related to or defined by several different properties of the binding behavior of the ligand to the target molecule. In addition to the commonly used Hill coefficient, other characteristics such as a sigmoidal shape of the overall titration curve in a linear plot, a change of ligand affinity of the other binding sites when a site of the target molecule becomes occupied, or complex roots of the binding polynomial have been used to define or to quantify cooperative binding. In this work, we analyze how the different properties are related in the most general model for binding curves based on the grand canonical partition function and present several examples which highlight differences between the cooperativity characterizing properties which are discussed. Our results mainly show that among the presented definitions there are not two which fully coincide. Moreover, this work poses the question whether it can make sense to distinguish between positive and negative cooperativity based on the macroscopic binding isotherm only. This article shall emphasize that scientists who investigate cooperative effects in biological systems could help avoiding misunderstandings by stating clearly which kind of cooperativity they discuss.

  12. Essentially normal Hilbert modules and Khomology Ⅲ: Homogenous quotient modules of Hardy modules on the bidisk

    Institute of Scientific and Technical Information of China (English)

    Kun-yu GUO; Peng-hui WANG

    2007-01-01

    In this paper, we study the homogenous quotient modules of the Hardy module on the bidisk. The essential normality of the homogenous quotient modules is completely characterized. We also describe the essential spectrum for a general quotient module. The paper also considers K-homology invariant defined in the case of the homogenous quotient modules on the bidisk.

  13. The Strip Module

    DEFF Research Database (Denmark)

    Pedersen, Tommy

    1996-01-01

    When the behaviour of a ship in waves is to be predicted it is convenient to have a tool which includes different approaches to the problem.The aim of this project is to develop such a tool named the strip theory module. The strip theory module will consist of submodules dependent on the I....... At last a postprocessor will be included with facilities for statistical calculations and for plots and prints of the results.The project is divided into 7 tasks where the third is to be completed.This report has two aims. To give an introduction to the project of developing a strip theory module...

  14. Analytic Hilbert modules

    CERN Document Server

    Chen, Xiaoman

    2003-01-01

    The seminal 1989 work of Douglas and Paulsen on the theory of analytic Hilbert modules precipitated a number of major research efforts. This in turn led to some intriguing and valuable results, particularly in the areas of operator theory and functional analysis. With the field now beginning to blossom, the time has come to collect those results under one cover. Written by two of the most active and often-cited researchers in the field, Analytic Hilbert Modules reports on the progress made by the authors and others, including the characteristic space theory, rigidity, the equivalence problem, the Arveson modules, extension theory, and reproducing Hilbert spaces on n-dimensional complex space.

  15. Optical modulator including grapene

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ming; Yin, Xiaobo; Zhang, Xiang

    2016-06-07

    The present invention provides for a one or more layer graphene optical modulator. In a first exemplary embodiment the optical modulator includes an optical waveguide, a nanoscale oxide spacer adjacent to a working region of the waveguide, and a monolayer graphene sheet adjacent to the spacer. In a second exemplary embodiment, the optical modulator includes at least one pair of active media, where the pair includes an oxide spacer, a first monolayer graphene sheet adjacent to a first side of the spacer, and a second monolayer graphene sheet adjacent to a second side of the spacer, and at least one optical waveguide adjacent to the pair.

  16. Microlensing modulation by binaries

    CERN Document Server

    Dubath, F; Durrer, R; Dubath, Florian; Gasparini, Maria Alice; Durrer, Ruth

    2006-01-01

    We compute the effect of the lens quadrupole on microlensing. The time dependence of the quadrupole can lead to specific modulations of the amplification signal. We study especially binary system lenses in our galaxy. The modulation is observable if the rotation period of the system is smaller than the time over which the amplification is significant and if the impact parameter of the passing light ray is sufficiently close to the Einstein radius so that the amplification is very large. Observations of this modulation can reveal important information on the quadrupole and thus on the gravitational radiation emitted by the lens.

  17. The ANTARES Optical Module

    CERN Document Server

    Amram, P; Anvar, S; Ardellier-Desages, F E; Aslanides, Elie; Aubert, Jean-Jacques; Azoulay, R; Bailey, D; Basa, S; Battaglieri, M; Bellotti, R; Benhammou, Ya; Bernard, F; Berthier, R; Bertin, V; Billault, M; Blaes, R; Bland, R W; Blondeau, F; De Botton, N R; Boulesteix, J; Brooks, B; Brunner, J; Cafagna, F; Calzas, A; Capone, A; Caponetto, L; Cârloganu, C; Carmona, E; Carr, J; Carton, P H; Cartwright, S L; Cassol, F; Cecchini, S; Ciacio, F; Circella, M; Compere, C; Cooper, S; Coyle, P; Croquette, J; Cuneo, S; Danilov, M; Van Dantzig, R; De Marzo, C; De Vita, R; Deck, P; Destelle, J J; Dispau, G; Drougou, J F; Druillole, F; Engelen, J; Feinstein, F; Festy, D; Fopma, J; Gallone, J M; Giacomelli, G; Goret, P; Gosset, L G; Gournay, J F; Heijboer, A; Hernández-Rey, J J; Herrouin, G; Hubbard, John R; Jacquet, M; De Jong, M; Karolak, M; Kooijman, P M; Kouchner, A; Kudryavtsev, V A; Lachartre, D; Lafoux, H; Lamare, P; Languillat, J C; Laubier, L; Laugier, J P; Le Guen, Y; Le Provost, H; Le Van-Suu, A; Lemoine, L; Lo Nigro, L; Lo Presti, D; Loucatos, Sotirios S; Louis, F; Lyashuk, V I; Magnier, P; Marcelin, M; Margiotta, A; Massol, A; Masullo, R; Mazéas, F; Mazeau, B; Mazure, A; McMillan, J E; Michel, J L; Migneco, E; Millot, C; Mols, P; Montanet, François; Montaruli, T; Morel, J P; Moscoso, L; Navas, S; Nezri, E; Nooren, G J L; Oberski, J; Olivetto, C; Oppelt-pohl, A; Palanque-Delabrouille, Nathalie; Payre, P; Perrin, P; Petruccetti, M; Petta, P; Piattelli, P; Poinsignon, J; Popa, V; Potheau, R; Queinec, Y; Racca, C; Raia, G; Randazzo, N; Rethore, F; Riccobene, G; Ricol, J S; Ripani, M; Roca-Blay, V; Rolin, J F; Rostovtsev, A A; Russo, G V; Sacquin, Yu; Salusti, E; Schuller, J P; Schuster, W; Soirat, J P; Suvorova, O; Spooner, N J C; Spurio, M; Stolarczyk, T; Stubert, D; Taiuti, M; Tao, Charling; Tayalati, Y; Thompson, L F; Tilav, S; Triay, R; Valente, V; Varlamov, I; Vaudaine, G; Vernin, P; De Witt-Huberts, P K A; De Wolf, E; Zakharov, V; Zavatarelli, S; De Dios-Zornoza-Gomez, Juan; Zúñiga, J

    2002-01-01

    The ANTARES collaboration is building a deep sea neutrino telescope in the Mediterranean Sea. This detector will cover a sensitive area of typically 0.1 km-squared and will be equipped with about 1000 optical modules. Each of these optical modules consists of a large area photomultiplier and its associated electronics housed in a pressure resistant glass sphere. The design of the ANTARES optical module, which is a key element of the detector, has been finalized following extensive R & D studies and is reviewed here in detail.

  18. The ANTARES optical module

    Energy Technology Data Exchange (ETDEWEB)

    Amram, P.; Anghinolfi, M.; Anvar, S.; Ardellier-Desages, F.E.; Aslanides, E.; Aubert, J.-J.; Azoulay, R.; Bailey, D.; Basa, S.; Battaglieri, M.; Bellotti, R.; Benhammou, Y.; Bernard, F.; Berthier, R.; Bertin, V.; Billault, M.; Blaes, R.; Bland, R.W.; Blondeau, F.; Botton, N. de; Boulesteix, J.; Brooks, C.B.; Brunner, J.; Cafagna, F.; Calzas, A.; Capone, A.; Caponetto, L.; Carloganu, C.; Carmona, E.; Carr, J.; Carton, P.-H.; Cartwright, S.L.; Cassol, F.; Cecchini, S.; Ciacio, F.; Circella, M.; Compere, C.; Cooper, S.; Coyle, P.; Croquette, J.; Cuneo, S.; Danilov, M.; Dantzig, R. van; De Marzo, C.; DeVita, R.; Deck, P.; Destelle, J.-J.; Dispau, G.; Drougou, J.F.; Druillole, F.; Engelen, J.; Feinstein, F.; Festy, D.; Fopma, J.; Gallone, J.-M.; Giacomelli, G.; Goret, P.; Gosset, L.; Gournay, J.-F.; Heijboer, A.; Hernandez-Rey, J.J.; Herrouin, G.; Hubbard, J.R.; Jaquet, M.; Jong, M. de; Karolak, M.; Kooijman, P.; Kouchner, A.; Kudryavtsev, V.A.; Lachartre, D.; Lafoux, H. E-mail: lafoux@cea.fr; Lamare, P.; Languillat, J.-C.; Laubier, L.; Laugier, J.-P.; Le Guen, Y.; Le Provost, H.; Le Van Suu, A.; Lemoine, L.; Lo Nigro, L.; Lo Presti, D.; Loucatos, S.; Louis, F.; Lyashuk, V.; Magnier, P.; Marcelin, M.; Margiotta, A.; Massol, A.; Masullo, R.; Mazeas, F.; Mazeau, B.; Mazure, A.; McMillan, J.E.; Michel, J.L.; Migneco, E.; Millot, C.; Mols, P.; Montanet, F.; Montaruli, T.; Morel, J.P.; Moscoso, L.; Musumeci, M.; Navas, S.; Nezri, E.; Nooren, G.J.; Oberski, J.; Olivetto, C.; Oppelt-Pohl, A.; Palanque-Delabrouille, N.; Papaleo, R.; Payre, P.; Perrin, P.; Petruccetti, M.; Petta, C.; Piattelli, P.; Poinsignon, J.; Potheau, R.; Queinec, Y.; Racca, C.; Raia, G.; Randazzo, N.; Rethore, F.; Riccobene, G.; Ricol, J.-S.; Ripani, M.; Roca-Blay, V.; Rolin, J.F.; Rostovstev, A.; Russo, G.V.; Sacquin, Y.; Salusti, E.; Schuller, J.-P.; Schuster, W.; Soirat, J.-P.; Souvorova, O.; Spooner, N.J.C.; Spurio, M.; Stolarczyk, T.; Stubert, D.; Taiuti, M.; Tao, C.; Tayalati, Y.; Thompson, L.F.

    2002-05-21

    The ANTARES collaboration is building a deep sea neutrino telescope in the Mediterranean Sea. This detector will cover a sensitive area of typically 0.1 km{sup 2} and will be equipped with about 1000 optical modules. Each of these optical modules consists of a large area photomultiplier and its associated electronics housed in a pressure resistant glass sphere. The design of the ANTARES optical module, which is a key element of the detector, has been finalized following extensive R and D studies and is reviewed here in detail.

  19. Solar cell module. Taiyo denchi module

    Energy Technology Data Exchange (ETDEWEB)

    Nakano, Akihiko; Matsumoto, Hitoshi; Komatsu, Yasumitsu; Shirai, Sadaharu.

    1989-09-29

    In the solar cell module of this invention, such junctions as CdS/CdTe or CdS/CuInSe {sub 2} are contained as a photoelectromotive force part coexists with air in a closed space which consists of glass, metal parts and a bonding resin layer; the photoelectromotive force part is coated either with a fluorine resin or a silicone resin. The fluorine resin contains a fundamental skeleton of an alternative copolymer of fluoroolefin and a hydrocarbon-based vinyl monomer; the silicone resin has three types, i.e., addition-reacted, condensated or UV-curing type, and the released oxygen is sealed in the closed space. The resin layer which adheres the glass and the metal plate is a thermoplastic resin which is polyethylene modified by copolymerization of acid anhydride. By this, the reliability of the solar cell module was enhanced. 3 figs.

  20. HIGHER FREQUENCY ULTRASONIC LIGHT MODULATORS.

    Science.gov (United States)

    LIGHT), (* MODULATORS , (*ULTRASONIC RADIATION, MODULATORS ), OPTICAL COMMUNICATIONS, BANDWIDTH, TRANSDUCERS, HIGH FREQUENCY, VERY HIGH FREQUENCY, ATTENUATION, DATA PROCESSING, OPTICAL EQUIPMENT, ANALOG COMPUTERS, THEORY.

  1. Module bay with directed flow

    Science.gov (United States)

    Torczynski, John R.

    2001-02-27

    A module bay requires less cleanroom airflow. A shaped gas inlet passage can allow cleanroom air into the module bay with flow velocity preferentially directed toward contaminant rich portions of a processing module in the module bay. Preferential gas flow direction can more efficiently purge contaminants from appropriate portions of the module bay, allowing a reduced cleanroom air flow rate for contaminant removal. A shelf extending from an air inlet slit in one wall of a module bay can direct air flowing therethrough toward contaminant-rich portions of the module bay, such as a junction between a lid and base of a processing module.

  2. CREME: Cis-Regulatory Module Explorer for the Human Genome

    Energy Technology Data Exchange (ETDEWEB)

    Loots, G G; Sharan, R; Ovcharenko, I; Ben-Hur, A

    2004-02-11

    The binding of transcription factors to specific regulatory sequence elements is a primary mechanism for controlling gene transcription. Eukaryotic genes are often regulated by several transcription factors, whose binding sites are tightly clustered and form cis-regulatory modules. In this paper we present a web-server, CREME, for identifying and visualizing cis-regulatory modules in the promoter regions of a given set of potentially co-regulated genes. CREME relies on a database of putative transcription factor binding sites that have been annotated across the human genome using a library of position weight matrices and evolutionary conservation with the mouse and rat genomes. A search algorithm is applied to this dataset to identify combinations of transcription factors whose binding sites tend to co-occur in close proximity in the promoter regions of the input gene set. The identified cis-regulatory modules are statistically scored and significant combinations are reported and graphically visualized. Our web-server is available at http://creme.dcode.org/.

  3. A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

    Science.gov (United States)

    Reddy, A. S.; Reddy, V. S.; Golovkin, M.

    2000-01-01

    Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

  4. Isothermal Containment Module

    Science.gov (United States)

    1999-01-01

    Isothermal Containment Modules are the temperature-controlling carrier that BioServe built to carry Commercial Generic Bioprocessing Apparatus (CGBA) and in the future, Space Automated Bioproduct Lab (SABL) to the International Space Station.

  5. A photovoltaic module

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to a photovoltaic module comprising a carrier substrate, said carrier substrate carrying a purely printed structure comprising printed positive and negative module terminals, a plurality of printed photovoltaic cell units each comprising one or more printed...... photovoltaic cells, wherein the plurality of printed photovoltaic cell units are electrically connected in series between the positive and the negative module terminals such that any two neighbouring photovoltaic cell units are electrically connected by a printed interconnecting electrical conductor....... The carrier substrate comprises a foil and the total thickness of the photovoltaic module is below 500 [mu]m. Moreover, the nominal voltage level between the positive and the negative terminals is at least 5 kV DC....

  6. Ultrasound Modulated Bioluminescence Tomography

    CERN Document Server

    Bal, Guillaume

    2013-01-01

    We propose a method to reconstruct the density of a luminescent source in a highly-scattering medium from ultrasound modulated optical measurements. Our approach is based on the solution to a hybrid inverse source problem for the diffusion equation.

  7. GREET Pretreatment Module

    Energy Technology Data Exchange (ETDEWEB)

    Adom, Felix K.; Dunn, Jennifer B.; Han, Jeongwoo

    2014-09-01

    A wide range of biofuels and biochemicals can be produced from biomass via different pretreatment technologies that yield sugars. This report documents the material and energy flows that occur when fermentable sugars from four lignocellulosic feedstocks (corn stover, miscanthus, switchgrass, and poplar) are produced via dilute acid pretreatment and ammonia fiber expansion. These flows are documented for inclusion in the pretreatment module of the Greenhouses Gases, Regulated Emissions, and Energy Use in Transportation (GREET) model. Process simulations of each pretreatment technology were developed in Aspen Plus. Material and energy consumption data from Aspen Plus were then compiled in the GREET pretreatment module. The module estimates the cradle-to-gate fossil energy consumption (FEC) and greenhouse gas (GHG) emissions associated with producing fermentable sugars. This report documents the data and methodology used to develop this module and the cradle-to-gate FEC and GHG emissions that result from producing fermentable sugars.

  8. Top Local Cohomology Modules

    Institute of Scientific and Technical Information of China (English)

    Mohammad T. Dibaei; Siamak Yassemi

    2007-01-01

    For a finitely generated module M over a commutative Noetherian local ring (R, m), it is shown that there exist only a finite number of non-isomorphic top localeohomology modules Hdim(M) (M) for all ideals a of RIt is also shown that for a giveninteger r ≥ 0, if Hra(R/p) is zero for all p in Supp(M), then Hia(M)=0 for all I ≥ r.

  9. Groups, rings, modules

    CERN Document Server

    Auslander, Maurice

    2014-01-01

    This classic monograph is geared toward advanced undergraduates and graduate students. The treatment presupposes some familiarity with sets, groups, rings, and vector spaces. The four-part approach begins with examinations of sets and maps, monoids and groups, categories, and rings. The second part explores unique factorization domains, general module theory, semisimple rings and modules, and Artinian rings. Part three's topics include localization and tensor products, principal ideal domains, and applications of fundamental theorem. The fourth and final part covers algebraic field extensions

  10. Asymmetric cation-binding catalysis

    DEFF Research Database (Denmark)

    Oliveira, Maria Teresa; Lee, Jiwoong

    2017-01-01

    and KCN, are selectively bound to the catalyst, providing exceptionally high enantioselectivities for kinetic resolutions, elimination reactions (fluoride base), and Strecker synthesis (cyanide nucleophile). Asymmetric cation-binding catalysis was recently expanded to silicon-based reagents, enabling...... solvents, thus increasing their applicability in synthesis. The expansion of this concept to chiral polyethers led to the emergence of asymmetric cation-binding catalysis, where chiral counter anions are generated from metal salts, particularly using BINOL-based polyethers. Alkali metal salts, namely KF...

  11. Starch-binding domains in the post-genome era.

    Science.gov (United States)

    Machovic, M; Janecek, S

    2006-12-01

    Starch belongs to the most abundant biopolymers on Earth. As a source of energy, starch is degraded by a large number of various amylolytic enzymes. However, only about 10% of them are capable of binding and degrading raw starch. These enzymes usually possess a distinct sequence-structural module, the so-called starchbinding domain (SBD). In general, all carbohydrate-binding modules (CBMs) have been classified into the CBM families. In this sequence-based classification the individual types of SBDs have been placed into seven CBM families: CBM20, CBM21, CBM25, CBM26, CBM34, CBM41 and CBM45. The family CBM20, known also as a classical C-terminal SBD of microbial amylases, is the most thoroughly studied. The three-dimensional structures have already been determined by X-ray crystallography or nuclear magnetic resonance for SBDs from five CBM families (20, 25, 26, 34 and 41), and the structure of the CBM21 has been modelled. Despite differences among the amino acid sequences, the fold of a distorted beta-barrel seems to be conserved together with a similar way of substrate binding (mainly stacking interactions between aromatic residues and glucose rings). SBDs have recently been discovered in many non-amylolytic proteins. These may, for example, have regulatory functions in starch metabolism in plants or glycogen metabolism in mammals. SBDs have also found practical uses.

  12. Absolutely Indecomposable Modules

    CERN Document Server

    Göbel, Rüdiger

    2007-01-01

    A module is called absolutely indecomposable if it is directly indecomposable in every generic extension of the universe. We want to show the existence of large abelian groups that are absolutely indecomposable. This will follow from a more general result about R-modules over a large class of commutative rings R with endomorphism ring R which remains the same when passing to a generic extension of the universe. It turns out that `large' in this context has the precise meaning, namely being smaller then the first omega-Erdos cardinal defined below. We will first apply result on large rigid trees with a similar property established by Shelah in 1982, and will prove the existence of related ` R_omega-modules' (R-modules with countably many distinguished submodules) and finally pass to R-modules. The passage through R_omega-modules has the great advantage that the proofs become very transparent essentially using a few `linear algebra' arguments accessible also for graduate students. The result gives a new constru...

  13. Photovoltaic module reliability workshop

    Energy Technology Data Exchange (ETDEWEB)

    Mrig, L. (ed.)

    1990-01-01

    The paper and presentations compiled in this volume form the Proceedings of the fourth in a series of Workshops sponsored by Solar Energy Research Institute (SERI/DOE) under the general theme of photovoltaic module reliability during the period 1986--1990. The reliability Photo Voltaic (PV) modules/systems is exceedingly important along with the initial cost and efficiency of modules if the PV technology has to make a major impact in the power generation market, and for it to compete with the conventional electricity producing technologies. The reliability of photovoltaic modules has progressed significantly in the last few years as evidenced by warranties available on commercial modules of as long as 12 years. However, there is still need for substantial research and testing required to improve module field reliability to levels of 30 years or more. Several small groups of researchers are involved in this research, development, and monitoring activity around the world. In the US, PV manufacturers, DOE laboratories, electric utilities and others are engaged in the photovoltaic reliability research and testing. This group of researchers and others interested in this field were brought together under SERI/DOE sponsorship to exchange the technical knowledge and field experience as related to current information in this important field. The papers presented here reflect this effort.

  14. Functional connectivity supporting the selective maintenance of feature-location binding in visual working memory

    Directory of Open Access Journals (Sweden)

    Sachiko eTakahama

    2014-06-01

    Full Text Available Information on an object’s features bound to its location is very important for maintaining object representations in visual working memory. Interactions with dynamic multi-dimensional objects in an external environment require complex cognitive control, including the selective maintenance of feature-location binding. Here, we used event-related functional magnetic resonance imaging to investigate brain activity and functional connectivity related to the maintenance of complex feature-location binding. Participants were required to detect task-relevant changes in feature-location binding between objects defined by color, orientation, and location. We compared a complex binding task requiring complex feature-location binding (color-orientation-location with a simple binding task in which simple feature-location binding, such as color-location, was task-relevant and the other feature was task-irrelevant. Univariate analyses showed that the dorsolateral prefrontal cortex (DLPFC, hippocampus, and frontoparietal network were activated during the maintenance of complex feature-location binding. Functional connectivity analyses indicated cooperation between the inferior precentral sulcus (infPreCS, DLPFC, and hippocampus during the maintenance of complex feature-location binding. In contrast, the connectivity for the spatial updating of simple feature-location binding determined by reanalyzing the data from Takahama et al. (2010 demonstrated that the superior parietal lobule (SPL cooperated with the DLPFC and hippocampus. These results suggest that the connectivity for complex feature-location binding does not simply reflect general memory load and that the DLPFC and hippocampus flexibly modulate the dorsal frontoparietal network, depending on the task requirements, with the infPreCS involved in the maintenance of complex feature-location binding and the SPL involved in the spatial updating of simple feature-location binding.

  15. Nanomechanical mapping of first binding steps of a virus to animal cells

    Science.gov (United States)

    Alsteens, David; Newton, Richard; Schubert, Rajib; Martinez-Martin, David; Delguste, Martin; Roska, Botond; Müller, Daniel J.

    2016-10-01

    Viral infection is initiated when a virus binds to cell surface receptors. Because the cell membrane is dynamic and heterogeneous, imaging living cells and simultaneously quantifying the first viral binding events is difficult. Here, we show an atomic force and confocal microscopy set-up that allows the surface receptor landscape of cells to be imaged and the virus binding events within the first millisecond of contact with the cell to be mapped at high resolution (virus and cell surface receptors. We find that the first bond formed between the viral glycoprotein and its cognate cell surface receptor has relatively low lifetime and free energy, but this increases as additional bonds form rapidly (≤1 ms). The formation of additional bonds occurs with positive allosteric modulation and the three binding sites of the viral glycoprotein are quickly occupied. Our quantitative approach can be readily applied to study the binding of other viruses to animal cells.

  16. Crystal structure of the single-stranded RNA binding protein HutP from Geobacillus thermodenitrificans.

    Science.gov (United States)

    Thiruselvam, Viswanathan; Sivaraman, Padavattan; Kumarevel, Thirumananseri; Ponnuswamy, Mondikalipudur Nanjappagounder

    2014-04-18

    RNA binding proteins control gene expression by the attenuation/antitermination mechanism. HutP is an RNA binding antitermination protein. It regulates the expression of hut operon when it binds with RNA by modulating the secondary structure of single-stranded hut mRNA. HutP necessitates the presence of l-histidine and divalent metal ion to bind with RNA. Herein, we report the crystal structures of ternary complex (HutP-l-histidine-Mg(2+)) and EDTA (0.5 M) treated ternary complex (HutP-l-histidine-Mg(2+)), solved at 1.9 Å and 2.5 Å resolutions, respectively, from Geobacillus thermodenitrificans. The addition of 0.5 M EDTA does not affect the overall metal-ion mediated ternary complex structure and however, the metal ions at the non-specific binding sites are chelated, as evidenced from the results of structural features.

  17. Yetter-Drinfel‘d Module and Convolution Module

    Institute of Scientific and Technical Information of China (English)

    张良云; 王栓宏

    2002-01-01

    In this paper,we first give a sufficent and necessary condition for a Hopf algebra to be a Yetter-Drinfel'd module,and prove that the finite dual of a Yetter-Drinfel'd module is still a Yetter-Drinfel'd module,Finally,we introduce a concept of convolution module.

  18. Effect of four-alpha-helix bundle cavity size on volatile anesthetic binding energetics.

    Science.gov (United States)

    Manderson, Gavin A; Michalsky, Stuart J; Johansson, Jonas S

    2003-09-30

    Currently, it is thought that inhalational anesthetics cause anesthesia by binding to ligand-gated ion channels. This is being investigated using four-alpha-helix bundles, small water-soluble analogues of the transmembrane domains of the "natural" receptor proteins. The study presented here specifically investigates how multiple alanine-to-valine substitutions (which each decrease the volume of the internal binding cavity by 38 A(3)) affect structure, stability, and anesthetic binding affinity of the four-alpha-helix bundles. Structure remains essentially unchanged when up to four alanine residues are changed to valine. However, stability increases as the number of these substitutions is increased. Anesthetic binding affinities are also affected. Halothane binds to the four-alpha-helix bundle variants with 0, 1, and 2 substitutions with equivalent affinities but binds to the variants with 3 and 4 more tightly. The same order of binding affinities was observed for chloroform, although for a particular variant, chloroform was bound less tightly. The observed differences in binding affinities may be explained in terms of a modulation of van der Waals and hydrophobic interactions between ligand and receptor. These, in turn, could result from increased four-alpha-helix bundle binding cavity hydrophobicity, a decrease in cavity size, or improved ligand/receptor shape complementarity.

  19. Ligand binding to WW tandem domains of YAP2 transcriptional regulator is under negative cooperativity.

    Science.gov (United States)

    Schuchardt, Brett J; Mikles, David C; Hoang, Lawrence M; Bhat, Vikas; McDonald, Caleb B; Sudol, Marius; Farooq, Amjad

    2014-12-01

    YES-associated protein 2 (YAP2) transcriptional regulator drives a multitude of cellular processes, including the newly discovered Hippo tumor suppressor pathway, by virtue of the ability of its WW domains to bind and recruit PPXY-containing ligands to specific subcellular compartments. Herein, we employ an array of biophysical tools to investigate allosteric communication between the WW tandem domains of YAP2. Our data show that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands. Moreover, the molecular origin of such negative cooperativity lies in an unfavorable entropic contribution to the overall free energy relative to ligand binding to isolated WW domains. Consistent with this notion, the WW tandem domains adopt a fixed spatial orientation such that the WW1 domain curves outwards and stacks onto the binding groove of the WW2 domain, thereby sterically hindering ligand binding to both itself and its tandem partner. Although ligand binding to both WW domains disrupts such interdomain stacking interaction, they reorient themselves and adopt an alternative fixed spatial orientation in the liganded state by virtue of their ability to engage laterally so as to allow their binding grooves to point outwards and away from each other. In short, while the ability of WW tandem domains to aid ligand binding is well documented, our demonstration that they may also be subject to negative binding cooperativity represents a paradigm shift in our understanding of the molecular action of this ubiquitous family of protein modules.

  20. Universal enveloping crossed module of Leibniz crossed modules and representations

    Science.gov (United States)

    Casado, Rafael F.; García-Martínez, Xabier; Ladra, Manuel

    2016-03-01

    The universal enveloping algebra functor UL: Lb → Alg, defined by Loday and Pirashvili [1], is extended to crossed modules. Then we construct an isomorphism between the category of representations of a Leibniz crossed module and the category of left modules over its universal enveloping crossed module of algebras. Note that the procedure followed in the proof for the Lie case cannot be adapted, since the actor in the category of Leibniz crossed modules does not always exist.

  1. The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

    DEFF Research Database (Denmark)

    Elmlund, Hans; Baraznenok, Vera; Lindahl, Martin

    2006-01-01

    CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8......-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II....

  2. Characterizations of Graded Distributive Modules

    Institute of Scientific and Technical Information of China (English)

    Qinghua Chen; Chang'an Li

    2002-01-01

    In this paper, we give some characterizations of graded distributive modules, prove some interesting results between graded rings (modules) and lattices under finiteness conditions, and investigate the direct sum of graded distributive modules in terms of orders of graded submodules and homomorphisms of graded factor modules.

  3. Igf binding proteins protect undifferentiated spermatogonia in the zebrafish testis against excessive differentiation

    NARCIS (Netherlands)

    Safian, Diego; Morais, Roberto D; Bogerd, Jan; Schulz, Rüdiger W

    2016-01-01

    Insulin-like growth factor (Igf) binding proteins (Igfbps) modulate the availability of Igfs for their cognate receptors. In zebrafish testes, Igf3 promotes the proliferation and differentiation of type A undifferentiated (Aund) spermatogonia, and igf3 expression is strongly elevated by Fsh, but als

  4. An Experiment Illustrating the Change in Ligand p"K"[subscript a] upon Protein Binding

    Science.gov (United States)

    Chenprakhon, Pirom; Panijpan, Bhinyo; Chaiyen, Pimchai

    2012-01-01

    The modulation of ligand p"K"[subscript a] due to its surrounding environment is a crucial feature that controls many biological phenomena. For example, the shift in the p"K"[subscript a] of substrates or catalytic residues at enzyme active sites upon substrate binding often triggers and controls enzymatic reactions. In this work, we developed an…

  5. Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

    DEFF Research Database (Denmark)

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    Carbohydrate active enzymes, particularly those that are active on polysaccharides, are often found associated with carbohydrate binding modules (CBMs), which can play several roles in supporting enzyme function, such as localizing the enzyme to the substrate. However, the presence of CBMs...

  6. Locating Temporal Functional Dynamics of Visual Short-Term Memory Binding using Graph Modular Dirichlet Energy

    Science.gov (United States)

    Smith, Keith; Ricaud, Benjamin; Shahid, Nauman; Rhodes, Stephen; Starr, John M.; Ibáñez, Augustin; Parra, Mario A.; Escudero, Javier; Vandergheynst, Pierre

    2017-02-01

    Visual short-term memory binding tasks are a promising early marker for Alzheimer’s disease (AD). To uncover functional deficits of AD in these tasks it is meaningful to first study unimpaired brain function. Electroencephalogram recordings were obtained from encoding and maintenance periods of tasks performed by healthy young volunteers. We probe the task’s transient physiological underpinnings by contrasting shape only (Shape) and shape-colour binding (Bind) conditions, displayed in the left and right sides of the screen, separately. Particularly, we introduce and implement a novel technique named Modular Dirichlet Energy (MDE) which allows robust and flexible analysis of the functional network with unprecedented temporal precision. We find that connectivity in the Bind condition is less integrated with the global network than in the Shape condition in occipital and frontal modules during the encoding period of the right screen condition. Using MDE we are able to discern driving effects in the occipital module between 100–140 ms, coinciding with the P100 visually evoked potential, followed by a driving effect in the frontal module between 140–180 ms, suggesting that the differences found constitute an information processing difference between these modules. This provides temporally precise information over a heterogeneous population in promising tasks for the detection of AD.

  7. IgG Conformer's Binding to Amyloidogenic Aggregates.

    Directory of Open Access Journals (Sweden)

    Monichan Phay

    Full Text Available Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1 pAb aggregates have greater activity than monomers (HMW species > dimers > monomers, 2 pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR interactions of F(ab regions, and 3 pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg, had up to ~200- and ~7-fold stronger binding to aggregates of Aβ and transthyretin (TTR than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated IVIg IgGs. Human pAb conformer's binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR component to pAb's activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aβ and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs.

  8. IgG Conformer's Binding to Amyloidogenic Aggregates

    Science.gov (United States)

    Phay, Monichan; Welzel, Alfred T.; Williams, Angela D.; McWilliams-Koeppen, Helen P.; Blinder, Veronika; O'Malley, Tiernan T.; Solomon, Alan; Walsh, Dominic M.; O'Nuallain, Brian

    2015-01-01

    Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs) have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1) pAb aggregates have greater activity than monomers (HMW species > dimers > monomers), 2) pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR) interactions of F(ab) regions, and 3) pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg), had up to ~200- and ~7-fold stronger binding to aggregates of Aβ and transthyretin (TTR) than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated IVIg IgGs. Human pAb conformer's binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR) component to pAb's activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aβ and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs. PMID:26367058

  9. Spatial Terahertz Modulator

    Science.gov (United States)

    Xie, Zhenwei; Wang, Xinke; Ye, Jiasheng; Feng, Shengfei; Sun, Wenfeng; Akalin, Tahsin; Zhang, Yan

    2013-11-01

    Terahertz (THz) technology is a developing and promising candidate for biological imaging, security inspection and communications, due to the low photon energy, the high transparency and the broad band properties of the THz radiation. However, a major encountered bottleneck is lack of efficient devices to manipulate the THz wave, especially to modulate the THz wave front. A wave front modulator should allow the optical or electrical control of the spatial transmission (or reflection) of an input THz wave and hence the ability to encode the information in a wave front. Here we propose a spatial THz modulator (STM) to dynamically control the THz wave front with photo-generated carriers. A computer generated THz hologram is projected onto a silicon wafer by a conventional spatial light modulator (SLM). The corresponding photo-generated carrier spatial distribution will be induced, which forms an amplitude hologram to modulate the wave front of the input THz beam. Some special intensity patterns and vortex beams are generated by using this method. This all-optical controllable STM is structure free, high resolution and broadband. It is expected to be widely used in future THz imaging and communication systems.

  10. Superconducting optical modulator

    Science.gov (United States)

    Bunt, Patricia S.; Ference, Thomas G.; Puzey, Kenneth A.; Tanner, David B.; Tache, Nacira; Varhue, Walter J.

    2000-12-01

    An optical modulator based on the physical properties of high temperature superconductors has been fabricated and tested. The modulator was constructed form a film of Yttrium Barium Copper Oxide (YBCO) grown on undoped silicon with a buffer layer of Yttria Stabilized Zirconia. Standard lithographic procedures were used to pattern the superconducting film into a micro bridge. Optical modulation was achieved by passing IR light through the composite structure normal to the micro bridge and switching the superconducting film in the bridge region between the superconducting and non-superconducting states. In the superconducting state, IR light reflects from the superconducting film surface. When a critical current is passed through the micro bridge, it causes the film in this region to switch to the non-superconducting state allowing IR light to pass through it. Superconducting materials have the potential to switch between these two states at speeds up to 1 picosecond using electrical current. Presently, fiber optic transmission capacity is limited by the rate at which optical data can be modulated. The superconducting modulator, when combined with other components, may have the potential to increase the transmission capacity of fiber optic lines.

  11. First and second generation γ-secretase modulators (GSMs) modulate amyloid-β (Aβ) peptide production through different mechanisms.

    Science.gov (United States)

    Borgegard, Tomas; Juréus, Anders; Olsson, Fredrik; Rosqvist, Susanne; Sabirsh, Alan; Rotticci, Didier; Paulsen, Kim; Klintenberg, Rebecka; Yan, Hongmei; Waldman, Magnus; Stromberg, Kia; Nord, Johan; Johansson, Jonas; Regner, Anna; Parpal, Santiago; Malinowsky, David; Radesater, Ann-Cathrin; Li, Tingsheng; Singh, Rajeshwar; Eriksson, Hakan; Lundkvist, Johan

    2012-04-01

    γ-Secretase-mediated cleavage of amyloid precursor protein (APP) results in the production of Alzheimer disease-related amyloid-β (Aβ) peptides. The Aβ42 peptide in particular plays a pivotal role in Alzheimer disease pathogenesis and represents a major drug target. Several γ-secretase modulators (GSMs), such as the nonsteroidal anti-inflammatory drugs (R)-flurbiprofen and sulindac sulfide, have been suggested to modulate the Alzheimer-related Aβ production by targeting the APP. Here, we describe novel GSMs that are selective for Aβ modulation and do not impair processing of Notch, EphB2, or EphA4. The GSMs modulate Aβ both in cell and cell-free systems as well as lower amyloidogenic Aβ42 levels in the mouse brain. Both radioligand binding and cellular cross-competition experiments reveal a competitive relationship between the AstraZeneca (AZ) GSMs and the established second generation GSM, E2012, but a noncompetitive interaction between AZ GSMs and the first generation GSMs (R)-flurbiprofen and sulindac sulfide. The binding of a (3)H-labeled AZ GSM analog does not co-localize with APP but overlaps anatomically with a γ-secretase targeting inhibitor in rodent brains. Combined, these data provide compelling evidence of a growing class of in vivo active GSMs, which are selective for Aβ modulation and have a different mechanism of action compared with the original class of GSMs described.

  12. Cholesterol binding to ion channels

    Directory of Open Access Journals (Sweden)

    Irena eLevitan

    2014-02-01

    Full Text Available Numerous studies demonstrated that membrane cholesterol is a major regulator of ion channel function. The goal of this review is to discuss significant advances that have been recently achieved in elucidating the mechanisms responsible for cholesterol regulation of ion channels. The first major insight that comes from growing number of studies that based on the sterol specificity of cholesterol effects, show that several types of ion channels (nAChR, Kir, BK, TRPV are regulated by specific sterol-protein interactions. This conclusion is supported by demonstrating direct saturable binding of cholesterol to a bacterial Kir channel. The second major advance in the field is the identification of putative cholesterol binding sites in several types of ion channels. These include sites at locations associated with the well-known cholesterol binding motif CRAC and its reversed form CARC in nAChR, BK, and TRPV, as well as novel cholesterol binding regions in Kir channels. Notably, in the majority of these channels, cholesterol is suggested to interact mainly with hydrophobic residues in non-annular regions of the channels being embedded in between transmembrane protein helices. We also discuss how identification of putative cholesterol binding sites is an essential step to understand the mechanistic basis of cholesterol-induced channel regulation. Clearly, however, these are only the first few steps in obtaining a general understanding of cholesterol-ion channels interactions and their roles in cellular and organ functions.

  13. The prion protein binds thiamine.

    Science.gov (United States)

    Perez-Pineiro, Rolando; Bjorndahl, Trent C; Berjanskii, Mark V; Hau, David; Li, Li; Huang, Alan; Lee, Rose; Gibbs, Ebrima; Ladner, Carol; Dong, Ying Wei; Abera, Ashenafi; Cashman, Neil R; Wishart, David S

    2011-11-01

    Although highly conserved throughout evolution, the exact biological function of the prion protein is still unclear. In an effort to identify the potential biological functions of the prion protein we conducted a small-molecule screening assay using the Syrian hamster prion protein [shPrP(90-232)]. The screen was performed using a library of 149 water-soluble metabolites that are known to pass through the blood-brain barrier. Using a combination of 1D NMR, fluorescence quenching and surface plasmon resonance we identified thiamine (vitamin B1) as a specific prion ligand with a binding constant of ~60 μM. Subsequent studies showed that this interaction is evolutionarily conserved, with similar binding constants being seen for mouse, hamster and human prions. Various protein construct lengths, both with and without the unstructured N-terminal region in the presence and absence of copper, were examined. This indicates that the N-terminus has no influence on the protein's ability to interact with thiamine. In addition to thiamine, the more biologically abundant forms of vitamin B1 (thiamine monophosphate and thiamine diphosphate) were also found to bind the prion protein with similar affinity. Heteronuclear NMR experiments were used to determine thiamine's interaction site, which is located between helix 1 and the preceding loop. These data, in conjunction with computer-aided docking and molecular dynamics, were used to model the thiamine-binding pharmacophore and a comparison with other thiamine binding proteins was performed to reveal the common features of interaction.

  14. The role of RNA-binding factor AUF1 in regulated gene expression and modulation of tumorigenesis%RNA 结合因子 AUF1调控基因表达及其在肿瘤发生中的双重调控作用的研究进展

    Institute of Scientific and Technical Information of China (English)

    杨颖卓; 康鹏; 高杰; 徐春林; 王诗美(综述); 吴霞(审校)

    2014-01-01

    Turn-over of messenger ribonucleic acid ( mRNA) is a major control point in gene expres-sion.In mammals,many mRNAs encode inflammatory cytokines ,oncoproteins,and G-protein-coupled receptors are destabilized by the presence of AU -rich elements ( AREs ) in their 3′-untranslated regions .Association of ARE-binding proteins(AUBPs)with these mRNAs promotes rapid mRNA degradation .ARE/poly(U)-binding factor 1(AUF1),one of the best-characterized AUBPs,binds to many ARE-mRNAs and assembles other fac-tors to recruit the mRNA degradation machinery .Most studies support an mRNA -destabilizing role for AUF1,al-though other findings suggest additional functions for this factor .However,several lines of evidence also support a role for AUF1 in the initiation and/or development of cancer .Many AUF1-targeted transcripts encode products that control pro-or anti-oncogenic processes .Numerous signaling pathways alter the composition of this AUF 1 complex of proteins to affect changes in ARE -mRNA degradation rates .This review briefly describes the roles of mRNA decay in gene expression in general and ARE -mediated decay ( AMD) in particular ,with a focus on AUF1 and the different modes of regulation that govern AUF 1 involvement in AMD.In the end,we discuss how changes in AUF1 isoform distribution,subcellular localization,and post-translational protein modifications can influence the metabolism of targeted mRNAs .%信使RNA( Messenger ribonucleic acid ,mRNA)不断更新在基因表达中具有重要作用,而且编码一些重要蛋白。本文详细阐述了RNA结合因子1(RNA-binding factor 1,AUF1)使mRNA稳定或去稳定,在转录后水平调控基因表达的可能的分子机制,归纳了AUF1各个亚型蛋白在基因表达中的独特作用的相关研究进展,并探讨了AUF1表达水平的改变在肿瘤发生中的双重调控作用。认识到AUF1发挥生物学效应的分子机制以及作用的具体信号通路,能够帮助我们揭开更多的生命谜题。

  15. NREL module energy rating methodology

    Energy Technology Data Exchange (ETDEWEB)

    Whitaker, C.; Newmiller, J.; Kroposki, B. [National Renewable Energy Laboratory, Golden, CO (United States)] [and others

    1995-11-01

    The goals of this project were to develop a tool for: evaluating one module in different climates; comparing different modules; provide a Q&D method for estimating periodic energy production; provide an achievable module rating; provide an incentive for manufacturers to optimize modules to non-STC conditions; and to have a consensus-based, NREL-sponsored activity. The approach taken was to simulate module energy for five reference days of various weather conditions. A performance model was developed.

  16. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution.

    Directory of Open Access Journals (Sweden)

    Amanda Tse

    Full Text Available Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib and promiscuous (Bosutinib, Dasatinib kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations

  17. Microfluidic screening of electrophoretic mobility shifts elucidates riboswitch binding function.

    Science.gov (United States)

    Karns, Kelly; Vogan, Jacob M; Qin, Qian; Hickey, Scott F; Wilson, Stephen C; Hammond, Ming C; Herr, Amy E

    2013-02-27

    Riboswitches are RNA sensors that change conformation upon binding small molecule metabolites, in turn modulating gene expression. Our understanding of riboswitch regulatory function would be accelerated by a high-throughput, quantitative screening tool capable of measuring riboswitch-ligand binding. We introduce a microfluidic mobility shift assay that enables precise and rapid quantitation of ligand binding and subsequent riboswitch conformational change. In 0.3% of the time required for benchtop assays (3.2 versus 1020 min), we screen and validate five candidate SAM-I riboswitches isolated from thermophilic and cryophilic bacteria. The format offers enhanced resolution of conformational change compared to slab gel formats, quantitation, and repeatability for statistical assessment of small mobility shifts, low reagent consumption, and riboswitch characterization without modification of the aptamer structure. Appreciable analytical sensitivity coupled with high-resolution separation performance allows quantitation of equilibrium dissociation constants (K(d)) for both rapidly and slowly interconverting riboswitch-ligand pairs as validated through experiments and modeling. Conformational change, triplicate mobility shift measurements, and K(d) are reported for both a known and a candidate SAM-I riboswitch with comparison to in-line probing assay results. The microfluidic mobility shift assay establishes a scalable format for the study of riboswitch-ligand binding that will advance the discovery and selection of novel riboswitches and the development of antibiotics to target bacterial riboswitches.

  18. Synthesis, pharmacological and structural characterization, and thermodynamic aspects of GluA2-positive allosteric modulators with a 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide scaffold

    DEFF Research Database (Denmark)

    Nørholm, Ann-Beth; Francotte, Pierre; Olsen, Lars

    2013-01-01

    Positive allosteric modulators of ionotropic glutamate receptors are potential compounds for treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind within the dimer interface of the ligand-binding domain (LBD) and stabilize the agonist-bound conformation, thereby slowing...

  19. Quantitative velocity modulation spectroscopy

    Science.gov (United States)

    Hodges, James N.; McCall, Benjamin J.

    2016-05-01

    Velocity Modulation Spectroscopy (VMS) is arguably the most important development in the 20th century for spectroscopic study of molecular ions. For decades, interpretation of VMS lineshapes has presented challenges due to the intrinsic covariance of fit parameters including velocity modulation amplitude, linewidth, and intensity. This limitation has stifled the growth of this technique into the quantitative realm. In this work, we show that subtle changes in the lineshape can be used to help address this complexity. This allows for determination of the linewidth, intensity relative to other transitions, velocity modulation amplitude, and electric field strength in the positive column of a glow discharge. Additionally, we explain the large homogeneous component of the linewidth that has been previously described. Using this component, the ion mobility can be determined.

  20. Models for Modules

    CERN Document Server

    Troost, Jan

    2012-01-01

    We recall the structure of the indecomposable sl(2) modules in the Bernstein-Gelfand-Gelfand category O. We show that all these modules can arise as quantized phase spaces of physical models. In particular, we demonstrate in a path integral discretization how a redefined action of the sl(2) algebra over the complex numbers can glue finite dimensional and infinite dimensional highest weight representations into indecomposable wholes. Furthermore, we discuss how projective cover representations arise in the tensor product of finite dimensional and Verma modules and give explicit tensor product decomposition rules. The tensor product spaces can be realized in terms of product path integrals. Finally, we discuss relations of our results to brane quantization and cohomological calculations in string theory.

  1. Space Experiment Module (SEM)

    Science.gov (United States)

    Brodell, Charles L.

    1999-01-01

    The Space Experiment Module (SEM) Program is an education initiative sponsored by the National Aeronautics and Space Administration (NASA) Shuttle Small Payloads Project. The program provides nationwide educational access to space for Kindergarten through University level students. The SEM program focuses on the science of zero-gravity and microgravity. Within the program, NASA provides small containers or "modules" for students to fly experiments on the Space Shuttle. The experiments are created, designed, built, and implemented by students with teacher and/or mentor guidance. Student experiment modules are flown in a "carrier" which resides in the cargo bay of the Space Shuttle. The carrier supplies power to, and the means to control and collect data from each experiment.

  2. GREET Pretreatment Module

    Energy Technology Data Exchange (ETDEWEB)

    Adom, Felix K. [Argonne National Lab. (ANL), Argonne, IL (United States). Energy Systems Division; Dunn, Jennifer B. [Argonne National Lab. (ANL), Argonne, IL (United States). Energy Systems Division; Han, Jeongwoo [Argonne National Lab. (ANL), Argonne, IL (United States). Energy Systems Division

    2014-09-01

    A wide range of biofuels and biochemicals can be produced from cellulosic biomass via different pretreatment technologies that yield sugars. Process simulations of dilute acid and ammonia fiber expansion pretreatment processes and subsequent hydrolysis were developed in Aspen Plus for four lignocellulosic feedstocks (corn stover, miscanthus, switchgrass, and poplar). This processing yields sugars that can be subsequently converted to biofuels or biochemical. Material and energy consumption data from Aspen Plus were then compiled in a new Greenhouses Gases, Regulated Emissions, and Energy Use in Transportation (GREETTM) pretreatment module. The module estimates the cradle-to-gate fossil energy consumption (FEC) and greenhouse gas (GHG) emissions associated with producing fermentable sugars. This report documents the data and methodology used to develop this module and the cradle-to-gate FEC and GHG emissions that result from producing fermentable sugars.

  3. Hollow dimension of modules

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    In this paper, we are interested in the following general question: Given a module Mwhich has finite hollow dimension and which has a finite collection of submodules Ki (1≤i≤n) such that M=K1+... +Kn, can we find an expression for the hollow dimension of Min terms of hollow dimensions of modules built up in some way from K1 Kn? We prove the following theorem:Let Mbe an amply supplemented module having finite hollow dimension and let Ki (1≤i≤n) be a finite collection of submodules of Msuch that M=K1+...+Kn. Then the hollow dimension h(M) of Mis the sum of the hollow dimensions of Ki (1≤i≤n) ifand only if Ki is a supplement of K1+...+Ki-1+Ki+1+...+Kn in Mfor each 1≤i≤n.

  4. Power module assembly

    Science.gov (United States)

    Campbell, Jeremy B [Torrance, CA; Newson, Steve [Redondo Beach, CA

    2011-11-15

    A power module assembly of the type suitable for deployment in a vehicular power inverter, wherein the power inverter has a grounded chassis, is provided. The power module assembly comprises a conductive base layer electrically coupled to the chassis, an insulating layer disposed on the conductive base layer, a first conductive node disposed on the insulating layer, a second conductive node disposed on the insulating layer, wherein the first and second conductive nodes are electrically isolated from each other. The power module assembly also comprises a first capacitor having a first electrode electrically connected to the conductive base layer, and a second electrode electrically connected to the first conductive node, and further comprises a second capacitor having a first electrode electrically connected to the conductive base layer, and a second electrode electrically connected to the second conductive node.

  5. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

    Science.gov (United States)

    Taymans, Jean-Marc; Vancraenenbroeck, Renée; Ollikainen, Petri; Beilina, Alexandra; Lobbestael, Evy; De Maeyer, Marc; Baekelandt, Veerle; Cookson, Mark R

    2011-01-01

    Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

  6. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

    Directory of Open Access Journals (Sweden)

    Jean-Marc Taymans

    Full Text Available Leucine rich repeat kinase 2 (LRRK2 is a Parkinson's disease (PD gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

  7. Galectin-3-Binding and Metastasis

    Science.gov (United States)

    Nangia-Makker, Pratima; Balan, Vitaly; Raz, Avraham

    2013-01-01

    i. Summary Galectin-3 is a member of a family of carbohydrate-binding proteins. It is present in the nucleus, the cytoplasm and also extracellular matrix of many normal and neoplastic cell types. Arrays of reports show an upregulation of this protein in transformed and metastatic cell lines (1, 2). Moreover, in many human carcinomas, an increased expression of galectin-3 correlates with progressive tumor stages (3–6). Several lines of analysis have demonstrated that the galectins participate in cell-cell and cell-matrix interactions by recognizing and binding complimentary glycoconjugates and thereby play a crucial role in normal and pathological processes. Elevated expression of the protein is associated with an increased capacity for anchorage-independent growth, homotypic aggregation, and tumor cell lung colonization (7–9). In this chapter we describe the methods of purification of galectin-3 from transformed E. coli and some of the commonly used functional assays for analyzing galectin-3 binding. PMID:22674139

  8. Stirling Module Development Overview

    Science.gov (United States)

    Livingston, F. R.

    1984-01-01

    The solar parabolic dish Stirling engine electrically generating module consists of a solar collector coupled to a Stirling engine powered electrical generator. The module is designed to convert solar power to electrical power in parallel with numerous identical units coupled to an electrical utility power grid. The power conversion assembly generates up to 25 kilowatts at 480 volts potential/3 phase/alternating current. Piston rings and seals with gas leakage have not occurred, however, operator failures resulted in two burnt out receivers, while material fatigue resulted in a broken piston rod between the piston rod seal and cap seal.

  9. Lunar Module Illustration

    Science.gov (United States)

    1969-01-01

    This concept is a cutaway illustration of the Lunar Module (LM) with detailed callouts. The LM was a two part spacecraft. Its lower or descent stage had the landing gear, engines, and fuel needed for the landing. When the LM blasted off the Moon, the descent stage served as the launching pad for its companion ascent stage, which was also home for the two astronauts on the surface of the Moon. The LM was full of gear with which to communicate, navigate, and rendezvous. It also had its own propulsion system, and an engine to lift it off the Moon and send it on a course toward the orbiting Command Module.

  10. Modulating aging and longevity

    DEFF Research Database (Denmark)

    Rattan, Suresh

    Provides information and an evaluation of a variety of approaches tried for modulating aging and longevity, including dietary supplementation with antioxidants, vitamins and hormones, genetic engineering, life-style alterations, and hormesis through mild stress. After decades of systematic....... This book provides the most up-to-date information and a critical evaluation of a variety of approaches being tried for modulating aging and longevity, including dietary supplementation with antioxidants, vitamins and hormones, genetic engineering, life-style alterations, and hormesis through mild stress...

  11. Instant node package module

    CERN Document Server

    Ali, Juzer

    2013-01-01

    Get to grips with a new technology, understand what it is and what it can do for you, and then get to work with the most important features and tasks. A practical exploration of the lifecycle of creating node modules as well as learning all of the top features that npm has to offer.Intended for readers who want to create their first node.js modules. The programming paradigm of JavaScript is not covered so a foundation in these concepts would be beneficial.

  12. Pain and modulation processes

    Directory of Open Access Journals (Sweden)

    Ghaffarpoor M

    1997-08-01

    Full Text Available Pain is one of the most important and sometimes difficult problems, that patients and physicians are encountered. It may be clinically acute or chronic, acute pain has usually definite cause and favourable response to treatment. On the other hand there are difficulties in diagnosis and management of chronic pain. Peripheral and cranial nerves convey pain impulses toward central nervous system, and modulations take place at several levels. Diagnosis of different pains, including nociceptive, nerve trunk pain and deafferentation types is essential to acceptable management. In this article we review pain pathway, neurotransmitters and modulation.

  13. Flexible programmable logic module

    Energy Technology Data Exchange (ETDEWEB)

    Robertson, Perry J. (Albuquerque, NM); Hutchinson, Robert L. (Albuquerque, NM); Pierson, Lyndon G. (Albuquerque, NM)

    2001-01-01

    The circuit module of this invention is a VME board containing a plurality of programmable logic devices (PLDs), a controlled impedance clock tree, and interconnecting buses. The PLDs are arranged to permit systolic processing of a problem by offering wide data buses and a plurality of processing nodes. The board contains a clock reference and clock distribution tree that can drive each of the PLDs with two critically timed clock references. External clock references can be used to drive additional circuit modules all operating from the same synchronous clock reference.

  14. Computational Prediction of RNA-Binding Proteins and Binding Sites.

    Science.gov (United States)

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%-8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein-RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein-RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  15. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  16. Photovoltaic module and interlocked stack of photovoltaic modules

    Science.gov (United States)

    Wares, Brian S.

    2012-09-04

    One embodiment relates to an arrangement of photovoltaic modules configured for transportation. The arrangement includes a plurality of photovoltaic modules, each photovoltaic module including a frame having at least a top member and a bottom member. A plurality of alignment features are included on the top member of each frame, and a plurality of alignment features are included on the bottom member of each frame. Adjacent photovoltaic modules are interlocked by the alignment features on the top member of a lower module fitting together with the alignment features on the bottom member of an upper module. Other embodiments, features and aspects are also disclosed.

  17. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    Science.gov (United States)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  18. Synthetic heparin-binding growth factor analogs

    Science.gov (United States)

    Pena, Louis A.; Zamora, Paul; Lin, Xinhua; Glass, John D.

    2007-01-23

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain that binds a heparin-binding growth factor receptor, covalently bound to a hydrophobic linker, which is in turn covalently bound to a non-signaling peptide that includes a heparin-binding domain. The synthetic heparin-binding growth factor analogs are useful as soluble biologics or as surface coatings for medical devices.

  19. CXC and CC Chemokines as Angiogenic Modulators in Nonhaematological Tumors

    Science.gov (United States)

    Bracarda, Sergio; Nabissi, Massimo; Massari, Francesco; Bria, Emilio; Tortora, Giampaolo; Santoni, Giorgio; Cascinu, Stefano

    2014-01-01

    Chemokines are a superfamily of structurally homologous heparin-binding proteins that includes potent inducers and inhibitors of angiogenesis. The imbalance between angiogenic and angiostatic chemokine activities can lead to abnormalities, such as chronic inflammation, dysplastic transformation, and even tumor development and spreading. In this review, we summarize the current literature regarding the role of chemokines as modulators of tumor angiogenesis and their potential role as therapeutic targets in patients with nonhaematological tumors. PMID:24971349

  20. CXC and CC Chemokines as Angiogenic Modulators in Nonhaematological Tumors

    Directory of Open Access Journals (Sweden)

    Matteo Santoni

    2014-01-01

    Full Text Available Chemokines are a superfamily of structurally homologous heparin-binding proteins that includes potent inducers and inhibitors of angiogenesis. The imbalance between angiogenic and angiostatic chemokine activities can lead to abnormalities, such as chronic inflammation, dysplastic transformation, and even tumor development and spreading. In this review, we summarize the current literature regarding the role of chemokines as modulators of tumor angiogenesis and their potential role as therapeutic targets in patients with nonhaematological tumors.

  1. Structural insights into human peroxisome proliferator activated receptor delta (PPAR-delta selective ligand binding.

    Directory of Open Access Journals (Sweden)

    Fernanda A H Batista

    Full Text Available Peroxisome proliferator activated receptors (PPARs δ, α and γ are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARδ more than 300-fold more tightly than PPARα or PPARγ but the structural basis of PPARδ:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARδ:GW0742 complex. Comparisons of the PPARδ:GW0742 complex with published structures of PPARs in complex with α and γ selective agonists and pan agonists suggests that two residues (Val312 and Ile328 in the buried hormone binding pocket play special roles in PPARδ selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARα and γ ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARδ ligand design.

  2. Scaling: An Items Module

    Science.gov (United States)

    Tong, Ye; Kolen, Michael J.

    2010-01-01

    "Scaling" is the process of constructing a score scale that associates numbers or other ordered indicators with the performance of examinees. Scaling typically is conducted to aid users in interpreting test results. This module describes different types of raw scores and scale scores, illustrates how to incorporate various sources of…

  3. Coplanar interconnection module

    Science.gov (United States)

    Steward, R. D.; Windsor, H. F.

    1970-01-01

    Module for interconnecting a semiconductor array to external leads or components incorporates a metal external heat sink for cooling the array. Heat sink, extending down from the molded block that supports the array, is immersed in a liquid nitrogen bath which is designed to maintain the desired array temperature.

  4. An Integrated Teaching Module.

    Science.gov (United States)

    Samuel, Marie R.; Seiferth, Berniece B.

    This integrated teaching module provides elementary and junior high school teachers with a "hands-on" approach to studying the Anasazi Indian. Emphasis is on creative exploration that focuses on integrating art, music, poetry, writing, geography, dance, history, anthropology, sociology, and archaeology. Replicas of artifacts,…

  5. Modelling the Photovoltaic Module

    DEFF Research Database (Denmark)

    Katsanevakis, Markos

    2011-01-01

    This paper refers into various ways in simulation the Photovoltaic (PV) module behaviour under any combination of solar irradiation and ambient temperature. There are three different approaches presented here briefly and one of them is chosen because of its good accuracy and relatively low...

  6. Multicultural Education Module.

    Science.gov (United States)

    Campbell-Whatley, Gloria D.

    The purpose of this multicultural education module is to provide faculty, teachers, and administrators with a resource that will aid them in creating an environment that is amenable to diverse social and cultural groups within U.S. school systems. The guide identifies current resources for addressing issues with respect to cultural diversity and…

  7. Paratransit: An Instructional Module.

    Science.gov (United States)

    Scalici, Anthony

    A concept-based introduction to paratransit is provided in this instructional module for undergraduate and graduate transportation-related courses for disciplines such as engineering, business, marketing, and technology. The concept of paratransit generally refers to modes of transportation other than mass transit and solo-driven automobiles. The…

  8. Computational Investigation on the Allosteric Modulation of Androgen Receptor

    Institute of Scientific and Technical Information of China (English)

    OU Min-Rui; LI Jun-Qian

    2012-01-01

    Androgens have similar structures with different biological activities. To identify molecular determinants responsible for the activity difference, we have docked six steroidal androgens to the binding site or the surface of androgen receptor by using molecular docking with computational investigation. The energy was calculated respectively based on the QM (quantum mechanics) and MM (molecular mechanics) methods. The result shows that the allosteric modulation of androgen receptor plays an important role in the binding process between androgens and receptor. The open state receptor is less stable than the close state one, but the latter is more favorable for binding with androgens. It is worthy of note that when the androgen receptors binding or without binding with androgen are in close state, they are difficult to return to their open state. This phenomenon is an exception of the well known two-state model theory in which the two states are reversible. Whether the internal of close state androgen receptor has a combination of androgen or not, the androgen receptor surface can be combined with another androgen, and their surface binding energies could be very close. The result is consistent with the experimental observations, but this phenomenon of continuous combination from open state is also an exception of the two-state model theory.

  9. Concentration-dependent effect of fibrinogen on IgG-specific antigen binding and phagocytosis.

    Science.gov (United States)

    Boehm, Tobias Konrad; Sojar, Hakimuddin; Denardin, Ernesto

    2010-01-01

    In this paper, we aim to characterize fibrinogen-IgG interactions, and explore how fibrinogen alters IgG-mediated phagocytosis. Using enzyme-linked binding assays, we found that fibrinogen binding to IgG is optimized for surfaces coated with high levels of IgG. Using a similar method, we have shown that for an antigen unable to specifically bind fibrinogen, fibrinogen enhances binding of antibodies towards that antigen. For binding of IgG antibodies to cells expressing Fc receptors, we found a bimodal binding response, where low levels of fibrinogen enhance binding of antibody to Fc receptors and high levels reduce it. This corresponds to a bimodal effect on phagocytosis of IgG-coated particles, which is inhibited in the presence of excess IgG during coating of the particles with antibodies and fibrinogen. We conclude that fibrinogen can modulate phagocytosis of IgG-coated particles in vitro by changing IgG binding behavior, and that high fibrinogen levels could negatively affect phagocytosis.

  10. A peptide derived from a trans-homophilic binding site in neural cell adhesion molecule induces neurite outgrowth and neuronal survival

    DEFF Research Database (Denmark)

    Køhler, Lene B; Soroka, Vladislav; Korshunova, Irina

    2010-01-01

    The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and synaptic plasticity. The crystal structure of a fragment of NCAM comprising the three N-terminal immunoglobulin (Ig)-like modules indicates that the first and second Ig modules bind to each other, t...

  11. Cellulose binding domain fusion proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  12. Fundamentals of spread spectrum modulation

    CERN Document Server

    Ziemer, Rodger E

    2007-01-01

    This lecture covers the fundamentals of spread spectrum modulation, which can be defined as any modulation technique that requires a transmission bandwidth much greater than the modulating signal bandwidth, independently of the bandwidth of the modulating signal. After reviewing basic digital modulation techniques, the principal forms of spread spectrum modulation are described. One of the most important components of a spread spectrum system is the spreading code, and several types and their characteristics are described. The most essential operation required at the receiver in a spread spect

  13. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

    Science.gov (United States)

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  14. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

    Directory of Open Access Journals (Sweden)

    Francisco Andrés Peralta

    2016-07-01

    Full Text Available Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators.

  15. RNA binding efficacy of theophylline, theobromine and caffeine.

    Science.gov (United States)

    Johnson, I Maria; Kumar, S G Bhuvan; Malathi, R

    2003-04-01

    The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 +/- 5%), whereas moderate and comparatively less binding activity for theobromine (45 +/- 5%) and caffeine (30 +/- 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm(-1)), theobromine (3379.8 cm(-1)) and caffeine (3343 cm(-1)) as compared to the free RNA (3341.6 cm(-1)). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (nu(C=O)) of both drug (nu(C=O)=1718, 1666 cm(-1)) as well as RNA (nu(C=O)=1699, 1658 cm(-1)) disappeared and a new vibration band appeared around 1703 cm(-1), indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theobromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.

  16. Protein binding assay for hyaluronate

    Energy Technology Data Exchange (ETDEWEB)

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  17. Coco is a dual activity modulator of TGFβ signaling

    Science.gov (United States)

    Deglincerti, Alessia; Haremaki, Tomomi; Warmflash, Aryeh; Sorre, Benoit; Brivanlou, Ali H.

    2015-01-01

    The TGFβ signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling. PMID:26116664

  18. Generic ISIS Transport Module Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The purpose of the Generic ISIS Transport Module is to provide a means to bring living specimens to and from orbit. In addition to living specimens, the module can...

  19. Bent Electro-Absorption Modulator

    DEFF Research Database (Denmark)

    2002-01-01

    The present invention relates to a method and a device for modulating optical signals based on modulating bending losses in bend, quantum well semiconductor waveguide sections. The complex refractive index of the optical active semiconducting components of the waveguide section is modulated...... components and the applied electric field in relation to the frequency of the modulated radiation, the bending losses (and possibly coupling losses) will provide extinction of light guided by the bent waveguide section. The refractive index contract may be modulated while keeping the absorption coefficient...... substantially constant and small, whereby the guided light can be modulated only by bending losses. Alternatively, the invention may be applied to enhance the extinction ratio of existing absorption modulators such as Electro-Absorption Modulators (EAMs) in which case extinction by absorption and extinction...

  20. Prime Submodules and Flat Modules

    Institute of Scientific and Technical Information of China (English)

    A.AZIZI

    2007-01-01

    In this paper, some characterizations of prime submodules in fiat modules and, particularly,in free modules are given. Furthermore, the height of prime submodules and some saturated chain of prime submodules are also given.

  1. Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin.

    Science.gov (United States)

    Bensing, Barbara A; Loukachevitch, Lioudmila V; McCulloch, Kathryn M; Yu, Hai; Vann, Kendra R; Wawrzak, Zdzislaw; Anderson, Spencer; Chen, Xi; Sullam, Paul M; Iverson, T M

    2016-04-01

    Streptococcus sanguinisis a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets.S. sanguinisexpresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site.

  2. Comparative structural analysis of lipid binding START domains.

    Directory of Open Access Journals (Sweden)

    Ann-Gerd Thorsell

    Full Text Available BACKGROUND: Steroidogenic acute regulatory (StAR protein related lipid transfer (START domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease. PRINCIPAL FINDINGS: We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains. SIGNIFICANCE: Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  3. Modulational instability of nematic phase

    Indian Academy of Sciences (India)

    T Mithun; K Porsezian

    2014-02-01

    We numerically observe the effect of homogeneous magnetic field on the modulationally stable case of polar phase in = 2 spinor Bose–Einstein condensates (BECs). Also we investigate the modulational instability of uniaxial and biaxial (BN) states of polar phase. Our observations show that the magnetic field triggers the modulational instability and demonstrate that irrespective of the magnetic field effect the uniaxial and biaxial nematic phases show modulational instability.

  4. On closed weak supplemented modules

    Institute of Scientific and Technical Information of China (English)

    ZENG Qing-yi; SHI Mei-hua

    2006-01-01

    A module M is called closed weak supplemented if for any closed submodule N of M, there is a submodule K of M such that M=K+N and K(c)N<<M. Any direct summand of closed weak supplemented module is also closed weak supplemented.Any nonsingular image of closed weak supplemented module is closed weak supplemented. Nonsingular V-rings in which all nonsingular modules are closed weak supplemented are characterized in Section 4.

  5. Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity

    DEFF Research Database (Denmark)

    Bindesbøll, Christian; Fan, Qiong; Nørgaard, Rikke C;

    2015-01-01

    Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription...... metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity....

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

    Science.gov (United States)

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

    2012-03-16

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

  7. Novel selective androgen receptor modulators: SAR studies on 6-bisalkylamino-2-quinolinones.

    Science.gov (United States)

    van Oeveren, Arjan; Motamedi, Mehrnoush; Martinborough, Esther; Zhao, Shuo; Shen, Yixing; West, Sarah; Chang, William; Kallel, Adam; Marschke, Keith B; López, Francisco J; Negro-Vilar, Andrés; Zhi, Lin

    2007-03-15

    A series of selective androgen receptor modulators (SARMs) with a wide spectrum of receptor modulating activities was developed based on optimization of the 4-substituted 6-bisalkylamino-2-quinolinones (3). Significance of the trifluoromethyl group on the side chains and its interactions with amino acid residues within the androgen receptor (AR) ligand binding domain are discussed. A representative analog (9) was tested orally in a rodent model of hypogonadism and demonstrated desirable tissue selectivity.

  8. Modulation instability: The beginning

    Science.gov (United States)

    Noskov, Roman; Belov, Pavel; Kivshar, Yuri

    2012-11-01

    The study of metal nanoparticles plays a central role in the emerging novel technologies employing optics beyond the diffraction limit. Combining strong surface plasmon resonances, high intrinsic nonlinearities and deeply subwavelength scales, arrays of metal nanoparticles offer a unique playground to develop novel concepts for light manipulation at the nanoscale. Here we suggest a novel principle to control localized optical energy in chains of nonlinear subwavelength metal nanoparticles based on the fundamental nonlinear phenomenon of modulation instability. In particular, we demonstrate that modulation instability can lead to the formation of long-lived standing and moving nonlinear localized modes of several distinct types such as bright and dark solitons, oscillons, and domain walls. We analyze the properties of these nonlinear localized modes and reveal different scenarios of their dynamics including transformation of one type of mode to another. We believe this work paves a way towards the development of nonlinear nanophotonics circuitry.

  9. Silicon optical modulators

    Directory of Open Access Journals (Sweden)

    Graham T. Reed

    2005-01-01

    Full Text Available Ever since the earliest research on optical circuits, dating back to the 1970s, there have been visions of an optical superchip (see for example1,2, containing a variety of integrated optical components to carry out light generation, modulation, manipulation, detection, and amplification (Fig. 1. The early work was associated with ferroelectric materials such as lithium niobate (LiNbO3, and III-V semiconductors such as gallium arsenide (GaAs and indium phosphide (InP based systems. LiNbO3 was interesting almost solely because of the fact that it possesses a large electro-optic coefficient3, enabling optical modulation via the Pockels effect. Alternatively, the III-V compounds were interesting because of the relative ease of laser fabrication and the prospect of optical and electronic integration.

  10. Stable local oscillator module.

    Energy Technology Data Exchange (ETDEWEB)

    Brocato, Robert Wesley

    2007-11-01

    This report gives a description of the development of a Stable Local Oscillator (StaLO) multi-chip module (MCM). It is a follow-on report to SAND2006-6414, Stable Local Oscillator Microcircuit. The StaLO accepts a 100MHz input signal and produces output signals at 1.2, 3.3, and 3.6 GHz. The circuit is built as a multi-chip module (MCM), since it makes use of integrated circuit technologies in silicon and lithium niobate as well as discrete passive components. This report describes the development of an MCM-based version of the complete StaLO, fabricated on an alumina thick film hybrid substrate.

  11. Silicon Optical Modulator Simulation

    Directory of Open Access Journals (Sweden)

    Soon Thor LIM

    2015-04-01

    Full Text Available We developed a way of predicting and analyzing high speed optical modulator. Our research adopted a bottom-up approach to consider high-speed optical links using an eye diagram. Our method leverages on modular mapping of electrical characteristics to optical characteristics, while attaining the required accuracy necessary for device footprint approaching sub-micron scales where electrical data distribution varies drastically. We calculate for the bias dependent phase shift (2pi/mm and loss (dB/mm for the optical modulator based on the real and imaginary part of complex effective indices. Subsequently, combine effectively both the electrical and optical profiles to construct the optical eye diagram which is the essential gist of signal integrity of such devices.

  12. Modulators of decision making.

    Science.gov (United States)

    Doya, Kenji

    2008-04-01

    Human and animal decisions are modulated by a variety of environmental and intrinsic contexts. Here I consider computational factors that can affect decision making and review anatomical structures and neurochemical systems that are related to contextual modulation of decision making. Expectation of a high reward can motivate a subject to go for an action despite a large cost, a decision that is influenced by dopamine in the anterior cingulate cortex. Uncertainty of action outcomes can promote risk taking and exploratory choices, in which norepinephrine and the orbitofrontal cortex appear to be involved. Predictable environments should facilitate consideration of longer-delayed rewards, which depends on serotonin in the dorsal striatum and dorsal prefrontal cortex. This article aims to sort out factors that affect the process of decision making from the viewpoint of reinforcement learning theory and to bridge between such computational needs and their neurophysiological substrates.

  13. NEUROBIOLOGICAL MODULATORS OF ANXIETY

    Directory of Open Access Journals (Sweden)

    Mohale Deepak S.

    2012-01-01

    Full Text Available Anxiety can be a core symptom of various mental/ behavioral disorders such as major depressive disorders, obsessive-compulsive disorders, panic disorder, adaptive disorder, post-traumatic stress disorder, social withdrawal disorder, and various phobias. The neuroanatomic circuits that support fear and anxiety behavior are modulated by a variety of neurochemicals, these include the peptidergic neurotransmitters, Corticotrophin releasing factor (CRF, neuropeptide Y (NPY, and substance P, the monoaminergic transmitters, Norepinephrine (NE, serotonin (5-hydroxytryptamine or 5-HT, and dopamine (DA, and the amino acid transmitters, Gamma Aminobuteric Acid (GABA and glutamate and many more. These neurochemical systems subserve important adaptive functions in preparing the organism for responding to threat or stress, by increasing vigilance, modulating memory, mobilizing energy stores, and elevating cardiovascular function. Nevertheless, these biological responses to threat and stress can become maladaptive if they are chronically or inappropriately activated.

  14. A Scalable Module System

    CERN Document Server

    Rabe, Florian

    2011-01-01

    Symbolic and logic computation systems ranging from computer algebra systems to theorem provers are finding their way into science, technology, mathematics and engineering. But such systems rely on explicitly or implicitly represented mathematical knowledge that needs to be managed to use such systems effectively. While mathematical knowledge management (MKM) "in the small" is well-studied, scaling up to large, highly interconnected corpora remains difficult. We hold that in order to realize MKM "in the large", we need representation languages and software architectures that are designed systematically with large-scale processing in mind. Therefore, we have designed and implemented the MMT language -- a module system for mathematical theories. MMT is designed as the simplest possible language that combines a module system, a foundationally uncommitted formal semantics, and web-scalable implementations. Due to a careful choice of representational primitives, MMT allows us to integrate existing representation l...

  15. Lightweight Trauma Module - LTM

    Science.gov (United States)

    Hatfield, Thomas

    2008-01-01

    Current patient movement items (PMI) supporting the military's Critical Care Air Transport Team (CCATT) mission as well as the Crew Health Care System for space (CHeCS) have significant limitations: size, weight, battery duration, and dated clinical technology. The LTM is a small, 20 lb., system integrating diagnostic and therapeutic clinical capabilities along with onboard data management, communication services and automated care algorithms to meet new Aeromedical Evacuation requirements. The Lightweight Trauma Module is an Impact Instrumentation, Inc. project with strong Industry, DoD, NASA, and Academia partnerships aimed at developing the next generation of smart and rugged critical care tools for hazardous environments ranging from the battlefield to space exploration. The LTM is a combination ventilator/critical care monitor/therapeutic system with integrated automatic control systems. Additional capabilities are provided with small external modules.

  16. -k-torsionfree modules and

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The notion of ω-k-torsionfree modules with respect to a bimoduleω is introduced, which is characterized in terms of left addR ω-approximations. The notion of ω-left approximation dimension is introduced, and the forms of k-syzygy modules being k-torsionfree modules are described.

  17. FERMI multi-chip module

    CERN Multimedia

    This FERMI multi-chip module contains five million transistors. 25 000 of these modules will handle the flood of information through parts of the ATLAS and CMS detectors at the LHC. To select interesting events for recording, crucial decisions are taken before the data leaves the detector. FERMI modules are being developed at CERN in partnership with European industry.

  18. Security Requirements for Cryptographic Modules

    Science.gov (United States)

    1999-01-01

    module interfaces; roles, services, and authentication; finite state machine model ; physical security; operating system security; cryptographic key...15 4.4 Finite State Machine Model .......................................................................................................... 17...These areas include cryptographic module specification; module interfaces; roles, services, and authentication; finite state machine model ; physical

  19. Module Utilization Committee. Final report

    Energy Technology Data Exchange (ETDEWEB)

    None

    1984-03-01

    Photovoltaic collector modules were declared surplus to the needs of the US Department of Energy. The Module Utilization Committee was formed to make appropriate disposition of the surplus modules. The final report of that committee accounts for that disposition. The membership and activities of the committee are set forth and the results of its activities are reported.

  20. Higher Level Orderings on Modules

    Institute of Scientific and Technical Information of China (English)

    Min WU; Guang Xing ZENG

    2005-01-01

    The aim of this paper is to investigate higher level orderings on modules over commutative rings. On the basis of the theory of higher level orderings on fields and commutative rings, some results involving existence of higher level orderings are generalized to the category of modules over commutative rings. Moreover, a strict intersection theorem for higher level orderings on modules is established.