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Artykuły w czasopismach na temat "Sialic Acid Binding Proteins"
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Tiralongo, Joe, Therese Wohlschlager, Evelin Tiralongo i Milton J. Kiefel. "Inhibition of Aspergillus fumigatus conidia binding to extracellular matrix proteins by sialic acids: a pH effect?" Microbiology 155, nr 9 (1.09.2009): 3100–3109. http://dx.doi.org/10.1099/mic.0.026997-0.
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Infection by Aspergillus fumigatus, which causes the life-threatening disease invasive aspergillosis, begins with the inhalation of conidia that adhere to and germinate in the lung. Previous studies have shown that A. fumigatus conidia express high levels of the negatively charged 9-carbon sugar sialic acid, and that sialic acid appears to mediate the binding of A. fumigatus conidia to basal lamina proteins. However, despite the ability of sialic acid to inhibit adherence of A. fumigatus conidia, the exact mechanism by which this binding occurs remains unresolved. Utilizing various free sialic acids and other carbohydrates, sialic acid derivatives, sialoglycoconjugates, glycoproteins, α-keto acid related compounds and amino acids we have found that the binding of A. fumigatus conidia to type IV collagen and fibrinogen was inhibited by (i) glycoproteins (in a sialic acid-independent manner), and (ii) free sialic acids, glucuronic acid and α-keto acid related compounds. However, inhibition by the latter was found to be the result of a shift in pH from neutral (pH 7.4) to acidic (less than pH 4.6) induced by the relatively high concentrations of free sialic acids, glucuronic acid and α-keto acid related compounds used in the binding assays. This suggests that previous reports describing inhibition of A. fumigatus conidia binding by free sialic acid may actually be due to a pH shift similar to that shown here. As previously reported, we found that A. fumigatus conidia express only N-acetylneuraminic acid, the most common sialic acid found in nature. However, A. fumigatus appears to do so by an alternative mechanism to that seen in other organisms. We report here that A. fumigatus (i) does not incorporate sialic acid obtained from the environment, (ii) does not synthesize and incorporate sialic acid from exogenous N-acetylmannosamine, and (iii) lacks homologues of known sialic acid biosynthesizing enzymes.
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Gangi Setty, Thanuja, Christine Cho, Sowmya Govindappa, Michael A. Apicella i S. Ramaswamy. "Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site". Acta Crystallographica Section D Biological Crystallography 70, nr 7 (24.06.2014): 1801–11. http://dx.doi.org/10.1107/s139900471400830x.
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Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteriaFusobacterium nucleatum,Pasteurella multocidaandVibrio choleraeand their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of theHaemophilus influenzaeprotein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.
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Li, Qiongyu, Yixuan Xie, Gege Xu i Carlito B. Lebrilla. "Identification of potential sialic acid binding proteins on cell membranes by proximity chemical labeling". Chemical Science 10, nr 24 (2019): 6199–209. http://dx.doi.org/10.1039/c9sc01360a.
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Hayakawa, Toshiyuki, Takashi Angata, Elliott H. Margulies, Tarjei Mikkelsen, Eric D. Green i Ajit Varki. "Gene Conversion of Sialic Acid Binding Domains in CD33-Related Siglecs by Adjacent Pseudogenes: A Novel Mechanism To Change Sialic Acid Binding Specificity." Blood 104, nr 11 (16.11.2004): 1471. http://dx.doi.org/10.1182/blood.v104.11.1471.1471.
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Abstract Siglecs (sialic acid-binding immunoglobulin superfamily lectins) are a family of cell surface receptors involved in regulating the immune response. The CD33-Related Siglecs (CD33rSiglecs, namely Siglec-3, -5 through -11 and -XII in humans) are a subgroup of these molecules found primarily on cells of the innate immune system. All are type-1 transmembrane proteins with an N-terminal sialic acid-recognizing V-set domain followed by a variable number of C-2 set domains, a transmembrane region and a cytosolic C-terminal domain that includes two tyrosine-based signaling motifs. Available data suggest an inhibitory signaling role in the innate immune response, mediated by recognition of host sialic acids as “self”. Nine of the 13 known primate Siglec genes along with 14 Siglec pseudogenes comprise the CD33-related Siglec gene cluster on human chromosome 19. Gene conversion is a mechanism for copying part of a genomic sequence into another, contributing to genetic diversity. Pseudogenes are known to play role in generating functional diversity of related genes (e.g., antibody diversity via gene conversion in chickens). We recently analyzed genomic sequences of the CD33-related Siglec gene cluster in three primates (human, chimpanzee and baboon) and found evidence for rapid evolution in this gene family (Angata et al., PNAS, in press). Additional evolutionary studies using distance-based phylogenetic trees shows evidence for three partial gene conversions between Siglec genes and adjacent Siglec pseudogenes. All three involve the coding regions for extracellular domains that mediate sialic acid recognition, and two involve a pseudogene converting a known Siglec gene. Functional analyses using recombinant proteins show marked differences in sialic acid-binding properties between the converted Siglec and its non-converted ortholog. These findings suggest that gene conversion with pseudogenes has contributed to the rapid functional evolution of the Siglecs, and provides a novel mechanism for changing sialic acid binding specificity. We hypothesize that this mechanism allows for rapid evolutionary adjustments in the recognition of endogenous sialic acids as “self”, a potential factor in controlling the innate immune response.
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Zhao, Chuankuo, i Juan Pu. "Influence of Host Sialic Acid Receptors Structure on the Host Specificity of Influenza Viruses". Viruses 14, nr 10 (28.09.2022): 2141. http://dx.doi.org/10.3390/v14102141.
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Influenza viruses need to use sialic acid receptors to invade host cells, and the α-2,3 and α-2,6 sialic acids glycosidic bonds linking the terminal sialic acids are generally considered to be the most important factors influencing the cross-species transmission of the influenza viruses. The development of methods to detect the binding of influenza virus HA proteins to sialic acid receptors, as well as the development of glycobiological techniques, has led to a richer understanding of the structure of the sialylated glycan in influenza virus hosts. It was found that, in addition to the sialic acid glycosidic bond, sialic acid variants, length of the sialylated glycan, Gal-GlcNAc-linked glycosidic bond within the sialylated glycan, and sulfation/fucosylation of the GlcNAc within the sialylated glycan all affect the binding properties of influenza viruses to the sialic acid receptors, thus indirectly affecting the host specificity of influenza viruses. This paper will review the sialic acid variants, internal structural differences of sialylated glycan molecules that affect the host specificity of influenza viruses, and distribution characteristics of sialic acid receptors in influenza virus hosts, in order to provide a more reliable theoretical basis for the in-depth investigation of cross-species transmission of influenza viruses and the development of new antiviral drugs.
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Schwegmann-Weßels, Christel, Gert Zimmer, Hubert Laude, Luis Enjuanes i Georg Herrler. "Binding of Transmissible Gastroenteritis Coronavirus to Cell Surface Sialoglycoproteins". Journal of Virology 76, nr 12 (15.06.2002): 6037–43. http://dx.doi.org/10.1128/jvi.76.12.6037-6043.2002.
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ABSTRACT The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. By comparing parental TGEV with mutant viruses deficient in the sialic acid binding activity, we determined the contributions of both binding activities to the attachment of TGEV to cultured cells. In the presence of a functional sialic acid binding activity, the amount of virus bound to two different porcine cell lines was increased sixfold compared to the binding of the mutant viruses. The attachment of parental virus was reduced to levels observed with the mutants when sialic acid containing inhibitors was present or when the cells were pretreated with neuraminidase. In virus overlay binding assays with immobilized cell surface proteins, the mutant virus only recognized pAPN. In addition, the parental virus bound to a high-molecular-mass sialoglycoprotein. The recognition of pAPN was sensitive to reducing conditions and was not dependent on sialic acid residues. On the other hand, binding to the sialic acid residues of the high-molecular-mass glycoprotein was observed regardless of whether the cellular proteins had been separated under reducing or nonreducing conditions. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells. This concept is discussed in the context of other viruses that use two different receptors to infect cells.
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Zeng, Fu-Yue, i Hans-Joachim Gabius. "Sialic Acid-Binding Proteins: Characterization, Biological Function and Application". Zeitschrift für Naturforschung C 47, nr 9-10 (1.10.1992): 641–53. http://dx.doi.org/10.1515/znc-1992-9-1001.
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The last decade has witnessed steadily growing support for the notion that the carbohydrate portion of glycoconjugates is not merely an inert structural addition to the protein or lipid backbone. Considerable attention has been given to the chemical composition of glycosidic residues in such heteropolymers. Sialic acids are frequently occurring components of oligosaccharide side chains in glycoconjugates of most higher animals and a few microorganisms. They appear to play an important role as ligands in glycobiological interactions. Mediation of a proposed protein-carbohydrate recognition will necessarily involve a binding protein with the respective specificity. Such proteins thus are able to serve as receptors for certain types of carbohydrate moieties like sialic acids in vivo. Various members of this class of proteins have already proven their value as analytical tools in studying expression and localization of defined sialoglycoconjugates. These proteins attract much attention due to both their functions in situ and their potential as laboratory tools in glycoconjugate research in areas like biochemistry or histology. We present a survey of the purification, characterization and application of this class of proteins to illustrate the status of knowledge and the current directions of research in this field.
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Gaytán, Meztlli O., Anirudh K. Singh, Shireen A. Woodiga, Surina A. Patel, Seon-Sook An, Arturo Vera-Ponce de León, Sean McGrath i in. "A novel sialic acid-binding adhesin present in multiple species contributes to the pathogenesis of Infective endocarditis". PLOS Pathogens 17, nr 1 (19.01.2021): e1009222. http://dx.doi.org/10.1371/journal.ppat.1009222.
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Bacterial binding to platelets is a key step in the development of infective endocarditis (IE). Sialic acid, a common terminal carbohydrate on host glycans, is the major receptor for streptococci on platelets. So far, all defined interactions between streptococci and sialic acid on platelets are mediated by serine-rich repeat proteins (SRRPs). However, we identified Streptococcus oralis subsp. oralis IE-isolates that bind sialic acid but lack SRRPs. In addition to binding sialic acid, some SRRP- isolates also bind the cryptic receptor β-1,4-linked galactose through a yet unknown mechanism. Using comparative genomics, we identified a novel sialic acid-binding adhesin, here named AsaA (associated with sialic acid adhesion A), present in IE-isolates lacking SRRPs. We demonstrated that S. oralis subsp. oralis AsaA is required for binding to platelets in a sialic acid-dependent manner. AsaA comprises a non-repeat region (NRR), consisting of a FIVAR/CBM and two Siglec-like and Unique domains, followed by 31 DUF1542 domains. When recombinantly expressed, Siglec-like and Unique domains competitively inhibited binding of S. oralis subsp. oralis and directly interacted with sialic acid on platelets. We further demonstrated that AsaA impacts the pathogenesis of S. oralis subsp. oralis in a rabbit model of IE. Additionally, we found AsaA orthologues in other IE-causing species and demonstrated that the NRR of AsaA from Gemella haemolysans blocked binding of S. oralis subsp. oralis, suggesting that AsaA contributes to the pathogenesis of multiple IE-causing species. Finally, our findings provide evidence that sialic acid is a key factor for bacterial-platelets interactions in a broader range of species than previously appreciated, highlighting its potential as a therapeutic target.
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Schwegmann-Wessels, Christel, Gert Zimmer, Bernd Schröder, Gerhard Breves i Georg Herrler. "Binding of Transmissible Gastroenteritis Coronavirus to Brush Border Membrane Sialoglycoproteins". Journal of Virology 77, nr 21 (1.11.2003): 11846–48. http://dx.doi.org/10.1128/jvi.77.21.11846-11848.2003.
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ABSTRACT Transmissible gastroenteritis coronavirus (TGEV) is a porcine pathogen causing enteric infections that are lethal for suckling piglets. The enterotropism of TGEV is connected with the sialic acid binding activity of the viral surface protein S. Here we show that, among porcine intestinal brush border membrane proteins, TGEV recognizes a mucin-type glycoprotein designated MGP in a sialic acid-dependent fashion. Virus binding assays with cryosections of the small intestine from a suckling piglet revealed the binding of TGEV to mucin-producing goblet cells. A nonenteropathogenic mutant virus that lacked a sialic acid binding activity was unable to bind to MGP and to attach to goblet cells. Our results suggest a role of MGP in the enteropathogenicity of TGEV.
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Marshall, P., A. Hasegawa, E. A. Davidson, V. Nussenzweig i M. Whitlow. "Interaction between complement proteins C5b-7 and erythrocyte membrane sialic acid." Journal of Experimental Medicine 184, nr 4 (1.10.1996): 1225–32. http://dx.doi.org/10.1084/jem.184.4.1225.
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The initial phase of membrane attack by complement is the interaction between C5b6, C7, and the cell membrane that leads to the insertion of C5b-7. Here we investigate the role of sialic acid residues in the assembly of C5b-7 intermediates on erythrocyte cell membranes. We find that C5b6 binds to glycophorin, whereas C5 or C6 does not bind, and desialylation of the glycophorin abolishes C5b6 binding. Complement lysis is inhibited by either masking glycophorin sialic acid with F(ab) fragments of an mAb, or by removal of the sialylated region of glycophorin by mild trypsinization. Gangliosides inhibit C5b-7 deposition when added to the aqueous phase. Asialogangliosides and synthetic gangliosides lacking the carboxylic acid residue have no inhibitory activity. We conclude that C5b6 binds to sialylated molecules on the erythrocyte surface. We propose a new model of membrane attack in which C5b6 initially binds to membranes via ionic forces. C7 then binds to C5b6, disrupting the ionic interaction and leading to the exposure of hydrophobic domains. Sialic acid is known to inhibit complement activation. Thus, these findings reveal a paradoxical role for sialic acid in complement attack; the presence of sialic acid inhibits the generation of C5b6, but once the membrane attack pathway is initiated, sialic acid enhances complement lysis.
Rozprawy doktorskie na temat "Sialic Acid Binding Proteins"
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Alias, Nadiawati. "Multivalent sialic acid binding proteins as novel therapeutics for influenza and parainfluenza infection". Thesis, University of St Andrews, 2014. http://hdl.handle.net/10023/4479.
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In nature, proteins with weak binding affinity often use a multivalency approach to enhance protein affinity via an avidity effect. Interested in this multivalency approach, we have isolated a carbohydrate binding module (CBM) that recognises sialic acid (known as a CBM40 domain) from both Vibrio cholerae (Vc) and Streptococcus pneumoniae (Sp) NanA sialidases, and generated multivalent polypeptides from them using molecular biology. Multivalent CBM40 constructs were designed either using a tandem repeat approach to produce trimeric or tetrameric forms that we call Vc3CBM and Vc4CBM, respectively, or through the addition of a trimerization domain derived from Pseudomonas aeruginosa pseudaminidase to produce three trimeric forms of proteins known as Vc-CBMTD (WT), Vc-CBMTD (Mutant) and Sp-CBMTD). Due to the position and flexibility of the linker between the trimerization domain and the CBM40 domain, site directed mutagenesis was employed to introduce a disulphide bond between the monomers at positions S164C and T83C of the CBM40 domain in order to promote a stable orientation of the binding site for easier access of sialic acids. Data from isothermal titration calorimetry (ITC) reveals that interaction of multivalent CBM40 proteins with α(2,3)-sialyllactose was mainly enthalpy driven with entropy contributing unfavorably to the interaction suggesting that these proteins establish a strong binding affinity to their ligand minimizing dissociation to produce stable multivalent molecules. However, using surface plasmon resonance (SPR), a mixed balance of entropy and enthalpy contributions was found with all constructs as determined by Van't Hoff plots. This proved that binding does not occur through a simple protein-ligand interaction but through disruption of hydrophobic and/or ionic hydration that provide the driving force to the process. Interestingly, the valency of multiple-linked polypeptides also plays an important part in the protein stabilization. However, little is known about their detailed structure when in multivalent form, as attempts to crystallize the whole protein molecule of Vc-CBMTD (WT) failed due to linker and domain flexibility. Only the trimerization domain (TD) part from Pseudomonas aeruginosa pseudaminidase was successfully crystallized and structure was determined to 3.0 Å without its CBM40 domain attached. In this thesis, we have also reported on the potential anti-influenza and anti- parainfluenza properties of these proteins, which were found to block attachment and inhibit infection of several influenza A and parainfluenza virus strains in vitro. As widely mentioned in literature, terminal sialic acids on the cell surface of mammalian host tissue provide a target for various pathogenic organisms to bind. Levels of viral inhibition were greatest against A/Udorn/72 H3N2 virus for Vc4CBM and Vc3CBM constructs with the lowest EC50 of 0.59 µM and 0.94 µM respectively, however most of the multivalent proteins tested were also effective against A/WSN/33 H1N1 and A/PR8/34 H1N1 subtypes. For parainfluenza virus, all constructs containing V. cholerae sialidase CBM40 domain showed great effect in inhibiting virus infection during cell protection assay. The best EC50 values were 0.2 µM from Vc-CBMTD (WT) followed by 1.17 µM from Vc4CBM and 1.78 µM from Vc-CBMTD (Mutant) which was against hPIV2, hPIV3 and hPIV5 infections respectively. Only a construct from S. pneumoniae sialidase known as Sp-CBMTD showed negligible effect on cell protection. All constructs were further tested for cytotoxicity in mammalian cell culture as well as undergoing an inhibition study on viral replication proteins. For the in vivo study, we also demonstrated the effectiveness of Vc4CBM to protect cotton rats and mice from hPIV3 and Streptococcus pneumoniae infections, when given intranasally in advance or on the day of infection. Therefore, these novel multivalent proteins could be promising candidates as broad-spectrum inhibitors or as a prophylactic treatment for both influenza and parainfluenza associated diseases.
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Hopkins, Adam P. "Molecular and biochemical characterisation of SiaP as a sialic acid binding protein component of a TRAP transporter of sialic acid". Thesis, University of York, 2010. http://etheses.whiterose.ac.uk/1030/.
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Sialic acid utilisation plays an important role in the growth and persistence of the obligate human mucosal pathogen Haemophilus influenzae, which causes respiratory tract infections, septicaemia and meningitis. Like many other bacteria, H. influenzae can use host-derived sialic acids as carbon, nitrogen and energy sources, but also as a terminal modification on the LPS to better evade the human immune system. H. influenzae takes up exogenous sialic acid via a tripartite ATP-independent periplasmic (TRAP) transporter, SiaPQM. This possesses an extracytoplasmic substrate binding protein (SBP), SiaP, which binds the substrate in the periplasm and delivers it to the specific membrane permease, SiaQM. SiaP contains two globular domains, which close around the substrate upon binding. Here, the mechanism of sialic acid binding by SiaP is investigated using site-directed mutagenesis of residues in the ligand binding site and analogues of sialic acid. These, and several mutations on the surface of SiaP, were investigated for their effect on transport by SiaPQM in vitro, using SiaQM reconstituted into proteoliposomes, and in vivo, using expression of siaPQM in an E. coli strain lacking its native sialic acid transporter, NanT. It is demonstrated that stabilisation of the carboxylate group of sialic acid by the totally conserved Arginine-147 is important for high-affinity ligand binding, but is not essential for transport. Mutation of Aparagine-150 to Aspartate abolishes the function of the transporter without affecting ligand binding, suggesting the existence of a critical interaction between the components of the transporter. The catabolism of the sialic acid analogues was also examined in E. coli expressing different sialic acid transporters. This indicates that a wide variety of sialic acid analogues are potential carbon sources in many pathogenic bacteria.
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Jiménez-Castells, Carmen 1982. "Capture and identification of carbohydrate-binding proteins by SPR and CREDEX-MS". Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7237.
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Carbohydrate-binding proteins of non-immunological origin -lectins- have been recognized over the last decades as decisive players in numerous biological processes, ranging from cellcell communication, fertilization, pathogen-cell adhesion to metastasis. Consequently, there is an increasing interest in finding powerful and nanosized tools to screen for these molecules and to study their carbohydrate interactions in detail. Here, two complementary approaches are described to characterize lectin-carbohydrate interactions with high sensitivity, low sample consumption, and without the need for sample labelling: SPR and CREDEX-MS. In SPR, we have developed an approach where the sugar is immobilized onto a sensor surface through a tailor-made peptide module that allows (1) to capture the lectin, (2) to characterize the interaction through kinetic and thermodynamic parameters, and (3) to identify the interacted protein by mass spectrometry. In CREDEX-MS, based on proteolytic excision of proteincarbohydrate complexes and mass spectrometric analysis, the peptides comforming the carbohydrate binding domain are identified.Las lectinas (proteínas de origen no inmune capaces de reconocer azúcares) se han revelado en las últimas décadas como participantes cruciales en multitud de procesos biológicos, tales como la comunicación célula-célula, la fertilización, la adhesión del patógeno a la célula y la metástasis, entre muchos otros. Por lo tanto, existe un gran interés en el desarrollo de técnicas analíticas potentes para el estudio de las interacciones lectina-carbohidrato. En este trabajo, se describen dos aproximaciones complementarias mediante las cuales se pueden caracterizar las interacciones lectinas-azúcar con gran sensibilidad, poca utilización de muestra y sin la necesitad de ningún marcaje. En la técnica basada en resonancia de plasmón superficial (SPR), el azúcar es inmovilizado sobre una superficie a través de un módulo peptídico, lo cual permite (1) capturar la lectina, (2) caracterizar su interacción mediante parámetros cinéticos y termodinámicos y (3) identificar posteriormente la proteína mediante espectrometría de masas. Complementariamente, la técnica CREDEX-MS, basada en la excisión proteolítica del complejo proteína-azúcar y posterior análisis por espectrometría de masas, nos permite identificar los péptidos que forman parte del dominio de unión al azúcar.
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Zhang, Mai. "Exploring the effect of sialic acid binding properties on Siglec function /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3190162.
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Freeman, Sylvie. "Studies on CD33 and sialoadhesin : sialic acid binding receptors of the haemopoietic system". Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318544.
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Ciccotosto, Silvana. "The preparation and evaluation of N-acetylneuraminic acid derivatives as probes of sialic acid-recognizing proteins". Monash University, Dept. of Medicinal Chemistry, 2004. http://arrow.monash.edu.au/hdl/1959.1/9649.
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Yao, Yujing [Verfasser]. "Influence of sialic acid modification on HIV GP120 binding and Syncytia formation / Yujing Yao". Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1176639129/34.
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Toro, Imre. "Crystal structures of two nucleic acid-binding proteins". Thesis, Open University, 2000. http://oro.open.ac.uk/58089/.
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The Crystal Structure of Sl Nuclease from Aspergillus oryzae S 1 nuclease from Aspergillus oryzae is a glycoprotein of 32 kDa molecular weight. The protein has two enzymatic activities: it is an endo-exonuclease with high specificity for single stranded nucleic acids, and it has an additional 3' -nucleotidase activity. S 1 nuclease is widely used in molecular biology as a single-strand specific nuclease due to its high stability and efficiency. It cleaves single-stranded regions of nucleic acids producing 5' -nucleotides without significant side-reactions. The crystal structure of S 1 nuclease has been determined to 1.7 A resolution by molecular replacement based on the known structure of PI nuclease from Penicillinum citrinum, which has 49 % sequence identity compared to S 1. The overall fold and the active site of S 1 nuclease is basically identical to that of PI nuclease, and also very similar to Phospholipase C from Bacillus cereus and alpha-toxin from Clostridium perfringens. The characteristic feature of this family of enzymes is a trinuclear zinc cluster in their active sites. A BLAST search in the sequence databases revealed several other protein sequences from bacteria, protozoa and plants possessing an approximately 30 % sequence identity compared to S 1 nuclease, but showing an almost complete conservation of structurally and functionally important residues. Soaking and co-crystallisation experiments with substrate analogues have been carried out in order to obtain an enzyme-substrate complex. These efforts have not resulted in the structure determination of any complexes under crystallisation conditions: no binding of substrate has been observed. Nevertheless, an enzyme mechanism has been proposed based on structural data of S 1 nuclease and nucleases with similar active sites. The Crystal Structure of an Sm-Related Protein from Archaeoglobus fulgidus In eukaryotes Sm and Sm-like proteins are the core components of the small nuclear ribonucleoprotein particles (snRNPs), which are involved in a variety of functions including rRNA processing, tRNA maturation and pre-mRNA processing. The Sm proteins are 70 to 120 amino acids long and share a common bi-partite signature sequence. The spliceosome, where the transesterification reaction of splicing occurs, is assembled by several snRNPs named after their constituting snRNA: U1, U2, U4, U5 and U6. An snRNA contains a short single stranded, uridine rich sequence motif, where the Sm proteins bind, but the three-dimensional arrangement of the Sm proteins and the mode of binding is unknown. In humans there are seven different canonical Sm proteins, which according to biochemical and electron microscopic studies seem to form a seven membered ring in vitro. Recently two crystal structures of human Sm protein dimers have been published. Interestingly Sm-related protein sequences have been found in the available genomic database of various Archaebacteria based on sequence homology. In contrast with eukaryotes only one or two Sm-related protein sequences have been identified in one organism. Their function is currently unknown, since analogous pre-mRNA splicing does not occur in Archaebacteria. Two Sm-related proteins of Archaeoglobus fulgidus have been cloned and expressed as fusion proteins. One of them called AF-Sm2 has been o crystallised utilising ammonium sulphate as precipitant and solved to 1.95 A resolution by SIRAS using a single mercury derivative. AF-Sm2 crystallises in a hexagonal space group (P6) and contains one molecule per asymmetric unit. The 77 residue long protein has a very similar fold compared to the solved human Sm protein structures: a short N-terminal a-helix followed by a five stranded, strongly bent, U-shaped ~-sheet resulting in a barrel-like overall fold. Six AF-Sm2 molecules form a ring in the crystal structure mediated by extensive hydrophobic and hydrogen-bonding interactions. Gel filtration experiments have indicated a pH dependence of oligomerisation in accordance with the crystallisation experiences. Currently the target of the Sm-related proteins of Archaeoglobus fulgidus and the stochiometry of oligomerisation in vivo is completely unknown.
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Shum, Bennett Oh Vic St Vincent's Clinical School UNSW. "Regulation of allergic asthma by fatty acid-binding proteins". Awarded by:University of New South Wales. St. Vincent's Clinical School, 2007. http://handle.unsw.edu.au/1959.4/27002.
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Fatty acid-binding proteins are small intracellular proteins with poorly defined functions in intracellular fatty acid transport. The adipocyte fatty acid-binding protein aP2 regulates systemic glucose and lipid metabolism. Using Affymetrix microarrays, we found that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by airway epithelial cells. aP2 expression was markedly increased following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13, and downregulated by the Th1 cytokine IFN-gamma. Regulation of aP2 mRNA expression by Th2 cytokines was dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2 deficient mice in a model of allergic airway inflammation, and found that infiltration of leukocytes, especially eosinophils, into the airways was highly aP2 dependent. T cell priming and peritoneal allergy was unaffected by aP2 deficiency suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-haematopoietic cells, most likely airway epithelial cells, as the site of aP2 action in allergic airway inflammation. Expression of the pro-inflammatory cytokines MCP-1 and IL-6 was impaired in cytokine activated aP2 deficient airway epithelial cells, while levels of the anti-inflammatory arachidonic acid metabolite 15-HETE was increased, providing a mechanism for the reduced airway inflammation in aP2 deficient mice. In addition to the immune functions of aP2, we found that the related fatty acid-binding protein mal1 was also upregulated by IL-4/IL-13 in airway epithelial cells, and mal1 deficient mice were protected against airway eosinophilia. Significantly, in comparison to single aP2 deficiency, mice with combined aP2-mal1 deficiency had augmented protection against airway inflammation, and bone marrow chimera experiments demonstrated that aP2-mal1 deficiency affected both non-haematopoeitic and haematopoeitic cells. In T cell priming experiments, aP2-mal1 deficiency resulted in defective cytokine profiles in antigen recall responses, suggesting compromised sensitisation to antigen as one mechanism for aP2-mal1 action in airway inflammation. Together, our data therefore demonstrates the crucial roles of fatty acid-binding proteins in airway epithelium, T cell priming and airway inflammation, and provides a new link between fatty acid signalling and allergy.
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Rowlinson, Marie-Claire. "The fatty acid and retinol binding proteins of nematodes". Thesis, University of Nottingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289452.
Pełny tekst źródłaKsiążki na temat "Sialic Acid Binding Proteins"
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Glatz, Jan F. C., i Ger J. Van Der Vusse, red. Cellular Fatty Acid-binding Proteins. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4615-3936-0.
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C, Glatz Jan F., i Vusse, G. J. van der., red. Cellular fatty-acid binding proteins. Dordrecht: Kluwer Academic Publishers, 1990.
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Glatz, Jan F. C., i Ger J. van der Vusse, red. Cellular Fatty Acid-Binding Proteins II. Boston, MA: Springer US, 1993. http://dx.doi.org/10.1007/978-1-4615-3096-1.
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Conference on High Affinity Metal-Binding Proteins in Non-Mammalian Species (1984 : Research Triangle Park) i Conference on phthalic acid esters (1984 : Guildford, England)., red. Metal-binding proteins: Phthalic acid esters. Research Triangle Park, N.C: National Institute of Environmental Health Sciences, National Institutes of Health, Dept. of Health and Human Services, 1986.
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Younis, Rafal R. Sabir. Improved assays for the reactive hexose and sialic acid contents of glycoproteins. Dublin: University College Dublin, 1996.
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C, Glatz Jan F., i Vusse, G. J. van der., red. Cellular fatty acid-binding proteins: Proceedings of the 2nd International Workshop on Fatty Acid-Binding Proteins, Maastricht, August 31 and September 1, 1992. Dordrecht: Kluwer Academic Publishers, 1993.
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Asim, Dutta-Roy, i Sener Friedrich, red. Cellular proteins and their fatty acids in health and disease. Weinheim: Wiley-VCH, 2003.
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W, Wang, i United States. National Aeronautics and Space Administration., red. A novel kinesin-like protein with a calmodulin-binding domain. [Washington, DC: National Aeronautics and Space Administration, 1996.
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Huang, Wei-chün. Structural studies of enzymes in biotin and fatty acid biosynthesis. Uppsala: Dept. of Molecular Biology, Swedish University of Agricultural Sciences, 1996.
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Khuri, Lamya Raja Tannous. The role of retinoids and retinoid binding proteins in the differentiation of the cervix. [New York]: [Columbia University], 1993.
Znajdź pełny tekst źródłaCzęści książek na temat "Sialic Acid Binding Proteins"
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Wickramasinghe, Iresha N. Ambepitiya, i M. Hélène Verheije. "Protein Histochemistry Using Coronaviral Spike Proteins: Studying Binding Profiles and Sialic Acid Requirements for Attachment to Tissues". W Coronaviruses, 155–63. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2438-7_14.
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Krempl, C., H. Laude i G. Herrler. "Is the Sialic Acid Binding Activity of the S Protein Involved in the Enteropathogenicity of Transmissible Gastroenteritis Virus?" W Advances in Experimental Medicine and Biology, 557–61. Boston, MA: Springer US, 1998. http://dx.doi.org/10.1007/978-1-4615-5331-1_72.
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Krafsur, E. S., R. D. Moon, R. Albajes, O. Alomar, Elisabetta Chiappini, John Huber, John L. Capinera i in. "Fatty Acid Binding Proteins". W Encyclopedia of Entomology, 1416–18. Dordrecht: Springer Netherlands, 2008. http://dx.doi.org/10.1007/978-1-4020-6359-6_3773.
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Kim, Cheorl-Ho. "Sialic Acid-Binding Ig-Like Lectins (Siglecs)". W Glycobiology of Innate Immunology, 311–496. Singapore: Springer Singapore, 2022. http://dx.doi.org/10.1007/978-981-16-9081-5_7.
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Medway, Christopher, i Kevin Morgan. "Sialic Acid Binding Immunoglobulin-Like Lectin-3 (CD33)". W Genetic Variants in Alzheimer's Disease, 181–90. New York, NY: Springer New York, 2013. http://dx.doi.org/10.1007/978-1-4614-7309-1_9.
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Fournier, N. C., i M. A. Richard. "Role of fatty acid-binding protein in cardiac fatty acid oxidation". W Cellular Fatty Acid-binding Proteins, 149–59. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4615-3936-0_19.
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Börchers, Torsten, Peter Højrup, Søren U. Nielsen, Peter Roepstorff, Friedrich Spener i Jens Knudsen. "Revision of the amino acid sequence of human heart fatty acid-binding protein". W Cellular Fatty Acid-binding Proteins, 127–33. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4615-3936-0_16.
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van der Vusse, Ger J., Marc van Bilsen, Jan F. C. Glatz, Danny M. Hasselbaink i Joost J. F. P. Luiken. "Critical steps in cellular fatty acid uptake and utilization". W Cellular Lipid Binding Proteins, 9–15. Boston, MA: Springer US, 2002. http://dx.doi.org/10.1007/978-1-4419-9270-3_2.
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Miller, Katherine R., i David P. Cistola. "Titration calorimetry as a binding assay for lipid-binding proteins". W Cellular Fatty Acid-Binding Proteins II, 29–37. Boston, MA: Springer US, 1993. http://dx.doi.org/10.1007/978-1-4615-3096-1_5.
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Ockner, Robert K. "Historic overview of studies on fatty acid-binding proteins". W Cellular Fatty Acid-binding Proteins, 3–9. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4615-3936-0_1.
Pełny tekst źródłaStreszczenia konferencji na temat "Sialic Acid Binding Proteins"
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Qin, Chunlin, Jan C. Brunn i William T. Butler. "MIGRATION OF SIALIC ACID-RICH PROTEINS ON SDS-PAGE IS SLOWER THAN PREDICTED FROM ACTUAL MOLECULAR WEIGHTS". W 3rd International Conference on Osteopontin and SIBLING (Small Integrin-Binding Ligand, N-linked Glycoprotein) Proteins, 2002. TheScientificWorld Ltd, 2002. http://dx.doi.org/10.1100/tsw.2002.336.
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De Marco, L., M. Mazzucato, M. G. Del Ben, U. Budde, A. B. Federici, A. Girolami i Z. M. Ruggeri. "THE PLATELET AGGREGATING PROPERTIES OF TYPE IIB VON WILLEBRAND FACTOR (vWF): THE ROLE OF PLATELET ACTIVATION, FIBRINOGEN AND TWO DISTINCT MEMBRANE RECEPTORS". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644643.
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Three preparations of purified von Willebrand factor (vWF), obtained from unrelated patients affected by type IIB von Willebrand disease, were found to have normal sialic acid content (between 129-190 nmoles/mg of vWF, as compared to 158 ± 17 nmoles/mg in four normal preparations) and to induce platelet aggregation in the presence of physiologic levels of divalent cations and without addition of ristocetin. A monoclonal antibody that blocks the vWF binding domain of the platelet glycoprotein (GP) Ib caused complete inhibition of IIB vWF-induced aggregation. On the contrary, a monoclonal antibody that blocks the receptor for adhesive proteins on the platelet GPIIb/IIIa complex failed to inhibit the initial response of platelets to high concentration of IIB vWF Moreover, IIB vWF caused agglutination of formalin-fixed platelets that was blocked only by the anti-GPIb antibody, suggesting that the binding of vWF to GPIb, even in the absence of ristocetin, results in platelet-platelet interaction that is followed by exposure of the GPIIb/IIIa receptors for adhesive proteins. Endogenous ADP, normally active platelet metabolism and fibrinogen binding to GPIIb/IIIa were necessary for maximal and irreversible platelet aggregation. In the absence of fibrinogen, however, aggregation was mediated by vWF binding to GPIIb/IIIa. A 52/48 kDa tryptic fragment containing the GPIb binding domain of normal vWF completely blocked the aggregation induced by all three IIB vWF preparations. The present study defines in detail the mechanisms involved in IIB vWF-induced platelet aggregation. Moreover, it establishes that the GPIb binding domain of normal and IIB vWF are closely related and that desialylation is not required for the direct interaction of IIB vWF with GPIb.
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Lian, E. C. Y., i F. A. Siddigui. "BINDING OF 37-DKa PLATELET AGGLUTINATING PROTEIN TO HUMAN PLATELETS". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643976.
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We have previously reported the purification of a 37-KDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura. using 125I-labeled p37, the properties of its binding to platelets were studied. The binding of p37 to washed human platelets from 4 normal subjects and two TTP patients after recovery was specific, concentration dependent and saturable. The Scatchard analysis revealed that the binding sites for p37 was about 100,000 per platelet with a dissociation constant of 48 × 10−9 M. The binding of p37 to erythrocytes was very little and non-specific. Stimulation of platelets by thrombin or ADP did not have any effect on the binding of p37 to platelets. The monoclonal antibodies to platelet GP lb (6D1) and GP Ilb-llla (10E5)(A gift of Dr. Barry coller) did not inhibit the binding of p37 to platelets. Fibrinogen (1 mg/ml) and FVIII/vWF (250 ug/ml) reduced the binding slightly. The polyclonal antibodies to p37 as well as concanavalin-A inhibited the binding of p37 to platelets through their direct interaction with p37. Other lectins such as phytohemagglutinin, potato lectin and helix pomatia lectin did not have any effect. At 40 mM, sialic acid, α-D-(+)-glucose, D-(+)-mannose and D-fructose caused 91%,44%,79%, and 63% inhibition of p37 binding respectively. D-(+)-galactose did not interfere with the binding. It is concluded that p37 binds to platelets on the sites other than GP lb and Gp IIb-IIIa and its binding to platelets is inhibited by certain sugars.
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Kitagawa, H., N. Yamamoto, G. Kosaki i H. Yamazaki. "AN IMPORTANT ROLE OF CARBOHYDRATE MOIETIES ON CANCER CELL MEMBRANE GLYCOPROTEINS IN CANCER CELL-INDUCED PLATELET AGGREGATION". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644667.
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Platelet aggregation induced by cancer cells may be an essential process in the development of hematogenous metastasis of cancers. A mechanism in HMV-I (human vaginal melanoma cell line)-induced platelet aggregation was studied by using monoclonal antibodies against membrane proteins of cancer cells or platelets. HMV-I cells or their membrana ractions induced platelet aggregation of human heparinized PRP, to which hirudin had no inhibitory effect. The platelet aggregation by HMV-I was completely lost after the pretreatment of the cells with 0.3U/ml neuraminidase for 60 min at 37°C. Preincubation of platelets with monoclonal antibodies against platelet GP lb or GP Ilb/lIIa inhibited HMV-I induced aggregation. A monoclonal antibody MB3 (igM) against another human melanoma (HMMB) which had been transplanted in nude mice was produced by hybridoma technique. Screening studies by cell binding ELISA revealed that MB3 antibody reacted with not only HMMB cells but also many other cells including HMV-I, M7609 (colon carcinoma cell line) and normal fibroblasts. Western-blot analyses showedthat MB3 antibody reacted with multiple, more than ten, proteins with molecular weights ranging from UO to 200 kDa in unreduced SDS-PAGE of HMV-I, HMMB or M7609. In contrast, when .these cells pretreated with neuraminidase were used in Western-blot, MB3 reactivity were all lost. MB3 reacted with at least three glycoproteins of human red cell membrane in Western-blot, but it did not react with human platelets. Immune adherent asgay with trypsin-treated HMV-I or HMMB cells as target cells showed negative reactivity. MB3 antibody inhibited HMV-I-induced aggregation of platelets, but did not inhibit M7609-induced aggregation which depended on thrombin generation.These results suggest that MB3 antibody may be against sialic acid-containing carbohydrate moieties of membrane glycoproteins on these cancercells and that the carbohydrate(s) may play a critical role in' cancer cell-platelet interaction.
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Kelm, Soerge, Nadine Bock, Karen Strenge i Reinhard Brossmer. "SYNTHETIC SIALIC ACID ANALOGUES BINDING TO SIGLECS WITH HIGH AFFINITY AND SELECTIVITY". W XXIst International Carbohydrate Symposium 2002. TheScientificWorld Ltd, 2002. http://dx.doi.org/10.1100/tsw.2002.484.
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McCauley, Micah, Philip R. Hardwidge, L. J. Maher III i Mark C. Williams. "DNA binding proteins that alter nucleic acid flexibility". W NanoScience + Engineering, redaktorzy Kishan Dholakia i Gabriel C. Spalding. SPIE, 2007. http://dx.doi.org/10.1117/12.736306.
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Chery, Florence, i Mark von Itzstein. "THE SYNTHESIS OF 4-MODIFIED 2-DEOXY-N-ACETYLNEURAMINIC ACIDS AS BIOLOGICAL PROBES OF SIALIC ACID RECOGNISING PROTEINS". W XXIst International Carbohydrate Symposium 2002. TheScientificWorld Ltd, 2002. http://dx.doi.org/10.1100/tsw.2002.578.
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Hamena, Soumiya, Souham Meshoul i Salima Ouadfel. "Using Chou's Pseudo-Amino Acid Composition to Predict and Classify Oxygen Binding Proteins". W 2020 6th Conference on Data Science and Machine Learning Applications (CDMA). IEEE, 2020. http://dx.doi.org/10.1109/cdma47397.2020.00017.
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Shibu, Shilpa K., i Salim A. "Prediction of RNA Binding Proteins from Amino Acid Sequences Using Machine Learning Techniques". W 2023 International Conference on Power, Instrumentation, Control and Computing (PICC). IEEE, 2023. http://dx.doi.org/10.1109/picc57976.2023.10142479.
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Farrell, Mariah, Anastasia D'Amico, Heather Fairfield Campbell, Connor Murphy i Michaela Ruth Reagan. "Abstract 2961: The novel roles of fatty acid binding proteins in multiple myeloma". W Proceedings: AACR Annual Meeting 2021; April 10-15, 2021 and May 17-21, 2021; Philadelphia, PA. American Association for Cancer Research, 2021. http://dx.doi.org/10.1158/1538-7445.am2021-2961.
Pełny tekst źródłaRaporty organizacyjne na temat "Sialic Acid Binding Proteins"
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Morrison, Mark, i Joshuah Miron. Molecular-Based Analysis of Cellulose Binding Proteins Involved with Adherence to Cellulose by Ruminococcus albus. United States Department of Agriculture, listopad 2000. http://dx.doi.org/10.32747/2000.7695844.bard.
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At the beginning of this project, it was clear that R. albus adhered tightly to cellulose and its efficient degradation of this polysaccharide was dependent on micromolar concentrations of phenylacetic acid (PAA) and phenylpropionic acid (PPA). The objectives for our research were: i) to identify how many different kinds of cellulose binding proteins are produced by Ruminococcus albus; ii) to isolate and clone the genes encoding some of these proteins from the same bacterium; iii) to determine where these various proteins were located and; iv) quantify the relative importance of these proteins in affecting the rate and extent to which the bacterium becomes attached to cellulose. BARD support has facilitated a number of breakthroughs relevant to our fundamental understanding of the adhesion process. First, R. albus possesses multiple mechanisms for adhesion to cellulose. The P.I.'s laboratory has discovered a novel cellulose-binding protein (CbpC) that belongs to the Pil-protein family, and in particular, the type 4 fimbrial proteins. We have also obtained genetic and biochemical evidence demonstrating that, in addition to CbpC-mediated adhesion, R. albus also produces a cellulosome-like complex for adhesion. These breakthroughs resulted from the isolation (in Israel and the US) of spontaneously arising mutants of R. albus strains SY3 and 8, which were completely or partially defective in adhesion to cellulose, respectively. While the SY3 mutant strain was incapable of growth with cellulose as the sole carbon source, the strain 8 mutants showed varying abilities to degrade and grow with cellulose. Biochemical and gene cloning experiments have been used in Israel and the US, respectively, to identify what are believed to be key components of a cellulosome. This combination of cellulose adhesion mechanisms has not been identified previously in any bacterium. Second, differential display, reverse transcription polymerase chain reaction (DD RT-PCR) has been developed for use with R. albus. A major limitation to cellulose research has been the intractability of cellulolytic bacteria to genetic manipulation by techniques such as transposon mutagenesis and gene displacement. The P.I.'s successfully developed DD RT- PCR, which expanded the scope of our research beyond the original objectives of the project, and a subset of the transcripts conditionally expressed in response to PAA and PPA have been identified and characterized. Third, proteins immunochemically related to the CbpC protein of R. albus 8 are present in other R. albus strains and F. intestinalis, Western immunoblots have been used to examine additional strains of R. albus, as well as other cellulolytic bacteria of ruminant origin, for production of proteins immunochemically related to the CbpC protein. The results of these experiments showed that R. albus strains SY3, 7 and B199 all possess a protein of ~25 kDa which cross-reacts with polyclonal anti-CbpC antiserum. Several strains of Butyrivibrio fibrisolvens, Ruminococcus flavefaciens strains C- 94 and FD-1, and Fibrobacter succinogenes S85 produced no proteins that cross-react with the same antiserum. Surprisingly though, F. intestinalis strain DR7 does possess a protein(s) of relatively large molecular mass (~200 kDa) that was strongly cross-reactive with the anti- CbpC antiserum. Scientifically, our studies have helped expand the scope of our fundamental understanding of adhesion mechanisms in cellulose-degrading bacteria, and validated the use of RNA-based techniques to examine physiological responses in bacteria that are nor amenable to genetic manipulations. Because efficient fiber hydrolysis by many anaerobic bacteria requires both tight adhesion to substrate and a stable cellulosome, we believe our findings are also the first step in providing the resources needed to achieve our long-term goal of increasing fiber digestibility in animals.
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Whitham, Steven A., Amit Gal-On i Victor Gaba. Post-transcriptional Regulation of Host Genes Involved with Symptom Expression in Potyviral Infections. United States Department of Agriculture, czerwiec 2012. http://dx.doi.org/10.32747/2012.7593391.bard.
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Understanding how RNA viruses cause disease symptoms in their hosts is expected to provide information that can be exploited to enhance modern agriculture. The helper component-proteinase (HC-Pro) protein of potyviruses has been implicated in symptom development. Previously, we demonstrated that symptom expression is associated with binding of duplex small-interfering-RNA (duplex-siRNA) to a highly conserved FRNK amino acid motif in the HC-Pro of Zucchini yellow mosaic virus (ZYMV). This binding activity also alters host microRNA (miRNA) profiles. In Turnip mosaic virus (TuMV), which infects the model plant Arabidopsis, mutation of the FRNK motif to FINK was lethal providing further indication of the importance of this motif to HC-Pro function. In this continuation project, our goal was to further investigate how ZYMV and TuMV cause the mis-expression of genes in cucurbits and Arabidopsis, respectively, and to correlate altered gene expression with disease symptoms. Objective 1 was to examine the roles of aromatic and positively charged residues F164RNH and K215RLF adjacent to FR180NK in small RNA binding. Objective 2 was to determine the target genes of the miRNAs which change during HC-Pro expression in infected tissues and transgenic cucumber. Objective 3 was to characterize RNA silencing mechanisms underlying differential expression of host genes. Objective 4 was to analyze the function of miRNA target genes and differentially expressed genes in potyvirus-infected tissues. We found that the charged K/R amino acid residues in the FKNH and KRLF motifs are essential for virus viability. Replacement of K to I in FKNH disrupted duplex-siRNA binding and virus infectivity, while in KRLF mutants duplex-siRNA binding was maintained and virus infectivity was limited: symptomless following a recovery phenomenon. These findings expanded the duplex-siRNA binding activity of HC-Pro to include the adjacent FRNK and FRNH sites. ZYMV causes many squash miRNAs to hyper-accumulate such as miR166, miR390, mir168, and many others. Screening of mir target genes showed that only INCURVATA-4 and PHAVOLUTA were significantly upregulated following ZYMVFRNK infection. Supporting this finding, we found similar developmental symptoms in transgenic Arabidopsis overexpressing P1-HC-Pro of a range of potyviruses to those observed in miR166 mutants. We characterized increased transcription of AGO1 in response to infection with both ZYMV strains. Differences in viral siRNA profiles and accumulation between mild and severe virus infections were characterized by Illumina sequencing, probably due to the differences in HC-Pro binding activity. We determined that the TuMV FINK mutant could accumulate and cause symptoms in dcl2 dcl4 or dcl2 dcl3 dcl4 mutants similar to TuMV FRNK in wild type Arabidopsis plants. These dcl mutant plants are defective in antiviral defenses, and the results show that factors other than HC-ProFRNK motif can induce symptoms in virus-infected plants. As a result of this work, we have a better understanding of the FRNK and FKNH amino acid motifs of HC-Pro and their contributions to the duplex-siRNA binding functions. We have identified plant genes that potentially contribute to infectivity and symptoms of virus infected plants when they are mis-expressed during potyviral infections. The results establish that there are multiple underlying molecular mechanisms that lead viral pathogenicity, some dependent on HC-Pro. The potential benefits include the development of novel strategies for controlling diseases caused by viruses, methods to ensure stable expression of transgenes in genetically improved crops, and improved potyvirus vectors for expression of proteins or peptides in plants.
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Wicker, Louise, Ilan Shomer i Uzi Merin. Membrane Processing of Citrus Extracts: Effects on Pectinesterase Activity and Cloud Stability. United States Department of Agriculture, październik 1993. http://dx.doi.org/10.32747/1993.7568754.bard.
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The U.S. team studied the role of cations and pH on thermolabile (TL-PE) and thermostable (TS-PE), permeation in ultrafiltration (UF) membranes, affinity to ion exchange membranes, mechanism of cation and pH activation, and effect on PE stability. An optimum pH and cation concentration exists for activity and UF permeation, which is specific for each cation type. Incomplete release of PE from a pectin complex resulted in low PE binding to cationic and anionic membranes. Incubation of PE at low pH increases the surface hydrophobicity, especially TL-PE, but the secondary structure of TL-PE is not greatly affected. The Israeli team showed that stable cloud colloidal constituents flocculate following the conversion of soluble to insoluble biopolymers. First, formation of pectic acid by pectinesterase activity is followed by the formation of calcium pectate gel. This process initiates a myriad of poorly defined reactions that result in juice clarification. Second, protein coagulation by heat resulted in flocculation of proteinacous bound cloud constituents, particularly after enzymatic pectin degradation. Pectinesterase activity is proposed to be an indirect cause for clarification; whereas binding of cloud constituents is the primary event in clarification by pectate gel and coagulated proteins. Understanding the mechanism of interaction of protein and pectic polymers is key to understanding cloud instability. Based on the above, it was hypothesized that the structure of pectin-protein coagulates plays a key role in cloud instability.
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Fluhr, Robert, i Maor Bar-Peled. Novel Lectin Controls Wound-responses in Arabidopsis. United States Department of Agriculture, styczeń 2012. http://dx.doi.org/10.32747/2012.7697123.bard.
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Innate immune responses in animals and plants involve receptors that recognize microbe-associated molecules. In plants, one set of this defense system is characterized by large families of TIR–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) resistance genes. The direct interaction between plant proteins harboring the TIR domain with proteins that transmit and facilitate a signaling pathway has yet to be shown. The Arabidopsis genome encodes TIR-domain containing genes that lack NBS and LRR whose functions are unknown. Here we investigated the functional role of such protein, TLW1 (TIR LECTIN WOUNDRESPONSIVE1). The TLW1 gene encodes a protein with two domains: a TIR domain linked to a lectin-containing domain. Our specific aim in this proposal was to examine the ramifications of the TL1-glycan interaction by; A) The functional characterization of TL1 activity in the context of plant wound response and B) Examine the hypothesis that wounding induced specific polysaccharides and examine them as candidates for TL-1 interactive glycan compounds. The Weizmann group showed TLW1 transcripts are rapidly induced by wounding in a JA-independent pathway and T-DNA-tagged tlw1 mutants that lack TLW1 transcripts, fail to initiate the full systemic wound response. Transcriptome methodology analysis was set up and transcriptome analyses indicates a two-fold reduced level of JA-responsive but not JA-independent transcripts. The TIR domain of TLW1 was found to interact directly with the KAT2/PED1 gene product responsible for the final b-oxidation steps in peroxisomal-basedJA biosynthesis. To identify potential binding target(s) of TL1 in plant wound response, the CCRC group first expressed recombinant TL1 in bacterial cells and optimized conditions for the protein expression. TL1 was most highly expressed in ArcticExpress cell line. Different types of extraction buffers and extraction methods were used to prepare plant extracts for TL1 binding assay. Optimized condition for glycan labeling was determined, and 2-aminobenzamide was used to label plant extracts. Sensitivity of MALDI and LC-MS using standard glycans. THAP (2,4,6- Trihydroxyacetophenone) showed minimal background peaks at positive mode of MALDI, however, it was insensitive with a minimum detection level of 100 ng. Using LC-MS, sensitivity was highly increased enough to detect 30 pmol concentration. However, patterns of total glycans displayed no significant difference between different extraction conditions when samples were separated with Dionex ICS-2000 ion chromatography system. Transgenic plants over-expressing lectin domains were generated to obtain active lectin domain in plant cells. Insertion of the overexpression construct into the plant genome was confirmed by antibiotic selection and genomic DNA PCR. However, RT-PCR analysis was not able to detect increased level of the transcripts. Binding ability of azelaic acid to recombinant TL1. Azelaic acid was detected in GST-TL1 elution fraction, however, DHB matrix has the same mass in background signals, which needs to be further tested on other matrices. The major findings showed the importance of TLW1 in regulating wound response. The findings demonstrate completely novel and unexpected TIR domain interactions and reveal a control nexus and mechanism that contributes to the propagation of wound responses in Arabidopsis. The implications are to our understanding of the function of TIR domains and to the notion that early molecular events occur systemically within minutes of a plant sustaining a wound. A WEB site (http://genome.weizmann.ac.il/hormonometer/) was set up that enables scientists to interact with a collated plant hormone database.
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Locy, Robert D., Hillel Fromm, Joe H. Cherry i Narendra K. Singh. Regulation of Arabidopsis Glutamate Decarboxylase in Response to Heat Stress: Modulation of Enzyme Activity and Gene Expression. United States Department of Agriculture, styczeń 2001. http://dx.doi.org/10.32747/2001.7575288.bard.
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Most plants accumulate the nonprotein amino acid, g-aminobutyric acid (GABA), in response to heat stress. GABA is made from glutamate in a reaction catalyzed by glutamate decarboxylase (GAD), an enzyme that has been shown by the Israeli PI to be a calmodulin (CaM) binding protein whose activity is regulated in vitro by calcium and CaM. In Arabidopsis there are at least 5 GAD genes, two isoforms of GAD, GAD1 and GAD2, are known to be expressed, both of which appear to be calmodulin-binding proteins. The role of GABA accumulation in stress tolerance remains unclear, and thus the objectives of the proposed work are intended to clarify the possible roles of GABA in stress tolerance by studying the factors which regulate the activity of GAD in vivo. Our intent was to demonstrate the factors that mediate the expression of GAD activity by analyzing the promoters of the GAD1 and GAD2 genes, to determine the role of stress induced calcium signaling in the regulation of GAD activity, to investigate the role of phosphorylation of the CaM-binding domain in the regulation of GAD activity, and to investigate whether ABA signaling could be involved in GAD regulation via the following set of original Project Objectives: 1. Construction of chimeric GAD1 and GAD2 promoter/reporter gene fusions and their utilization for determining cell-specific expression of GAD genes in Arabidopsis. 2. Utilizing transgenic plants harboring chimeric GAD1 promoter-luciferase constructs for isolating mutants in genes controlling GAD1 gene activation in response to heat shock. 3. Assess the role of Ca2+/CaM in the regulation of GAD activity in vivo in Arabidopsis. 4. Study the possible phosphorylation of GAD as a means of regulation of GAD activity. 5. Utilize ABA mutants of Arabidopsis to assess the involvement of this phytohormone in GAD activation by stress stimuli. The major conclusions of Objective 1 was that GAD1 was strongly expressed in the elongating region of the root, while GAD2 was mainly expressed along the phloem in both roots and shoots. In addition, GAD activity was found not to be transcriptionally regulated in response to heat stress. Subsequently, The Israeli side obtained a GAD1 knockout mutation, and in light of the objective 1 results it was determined that characterization of this knockout mutation would contribute more to the project than the proposed Objective 2. The major conclusion of Objective 3 is that heat-stress-induced changes in GAD activity can be explained by heat-stress-induced changes in cytosolic calcium levels. No evidence that GAD activity was transcriptionally or translationally regulated or that protein phosphorylation was involved in GAD regulation (objective 4) was obtained. Previously published data by others showing that in wheat roots ABA regulated GABA accumulation proved not to be the case in Arabidopsis (Objective 5). Consequently, we put the remaining effort in the project into the selection of mutants related to temperature adaptation and GABA utilization and attempting to characterize events resulting from GABA accumulation. A set of 3 heat sensitive mutants that appear to have GABA related mutations have been isolated and partially characterized, and a study linking GABA accumulation to growth stimulation and altered nitrate assimilation were conducted. By providing a better understanding of how GAD activity was and was not regulated in vivo, we have ruled out the use of certain genes for genetically engineering thermotolerance, and suggested other areas of endeavor related to the thrust of the project that may be more likely approaches to genetically engineering thermotolerance.
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Whitham, Steven A., Amit Gal-On i Tzahi Arazi. Functional analysis of virus and host components that mediate potyvirus-induced diseases. United States Department of Agriculture, marzec 2008. http://dx.doi.org/10.32747/2008.7591732.bard.
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The mechanisms underlying the development of symptoms in response to virus infection remain to be discovered in plants. Insight into symptoms induced by potyviruses comes from evidence implicating the potyviral HC-Pro protein in symptom development. In particular, recent studies link the development of symptoms in infected plants to HC-Pro's ability to interfere with small RNA metabolism and function in plant hosts. Moreover, mutation of the highly conserved FRNK amino acid motif to FINK in the HC-Pro of Zucchini yellow mosaic virus (ZYMV) converts a severe strain into an asymptomatic strain, but does not affect virus accumulation in cucurbit hosts. The ability of this FINK mutation to uncouple symptoms from virus accumulation creates a unique opportunity to study symptom etiology, which is usually confounded by simultaneous attenuation of both symptoms and virus accumulation. Our goal was to determine how mutations in the conserved FRNK motif affect host responses to potyvirus infection in cucurbits and Arabidopsis thaliana. Our first objective was to define those amino acids in the FRNK motif that are required for symptoms by mutating the FRNK motif in ZYMV and Turnip mosaic virus (TuMV). Symptom expression and accumulation of resulting mutant viruses in cucurbits and Arabidopsis was determined. Our second objective was to identify plant genes associated with virus disease symptoms by profiling gene expression in cucurbits and Arabidopsis in response to mutant and wild type ZYMV and TuMV, respectively. Genes from the two host species that are differentially expressed led us to focus on a subset of genes that are expected to be involved in symptom expression. Our third objective was to determine the functions of small RNA species in response to mutant and wild type HC-Pro protein expression by monitoring the accumulation of small RNAs and their targets in Arabidopsis and cucurbit plants infected with wild type and mutant TuMV and ZYMV, respectively. We have found that the maintenance of the charge of the amino acids in the FRNK motif of HC-Pro is required for symptom expression. Reduced charge (FRNA, FRNL) lessen virus symptoms, and maintain the suppression of RNA silencing. The FRNK motif is involved in binding of small RNA species including microRNAs (miRNA) and short interfering RNAs (siRNA). This binding activity mediated by the FRNK motif has a role in protecting the viral genome from degradation by the host RNA silencing system. However, it also provides a mechanism by which the FRNK motif participates in inducing the symptoms of viral infection. Small RNA species, such as miRNA and siRNA, can regulate the functions of plant genes that affect plant growth and development. Thus, this binding activity suggests a mechanism by which ZYMVHC-Pro can interfere with plant development resulting in disease symptoms. Because the host genes regulated by small RNAs are known, we have identified candidate host genes that are expected to play a role in symptoms when their regulation is disrupted during viral infections. As a result of this work, we have a better understanding of the FRNK amino acid motif of HC-Pro and its contribution to the functions of HC-Pro, and we have identified plant genes that potentially contribute to symptoms of virus infected plants when their expression becomes misregulated during potyviral infections. The results set the stage to establish the roles of specific host genes in viral pathogenicity. The potential benefits include the development of novel strategies for controlling diseases caused by viruses, methods to ensure stable expression of transgenes in genetically improved crops, and improved potyvirus vectors for expression of proteins or peptides in plants.
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Meidan, Rina, i Robert Milvae. Regulation of Bovine Corpus Luteum Function. United States Department of Agriculture, marzec 1995. http://dx.doi.org/10.32747/1995.7604935.bard.
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The main goal of this research plan was to elucidate regulatory mechanisms controlling the development, function of the bovine corpus luteum (CL). The CL contains two different sterodigenic cell types and therefore it was necessary to obtain pure cell population. A system was developed in which granulosa and theca interna cells, isolated from a preovulatory follicle, acquired characteristics typical of large (LL) and small (SL) luteal cells, respectively, as judged by several biochemical and morphological criteria. Experiments were conducted to determine the effects of granulosa cells removal on subsequent CL function, the results obtained support the concept that granulosa cells make a substaintial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. This experimental model was also used to better understand the contribution of follicular granulosa cells to subsequent luteal SCC mRNA expression. The mitochondrial cytochrome side-chain cleavage enzyme (SCC), which converts cholesterol to pregnenolone, is the first and rate-limiting enzyme of the steroidogenic pathway. Experiments were conducted to characterize the gene expression of P450scc in bovine CL. Levels of P450scc mRNA were higher during mid-luteal phase than in either the early or late luteal phases. PGF 2a injection decreased luteal P450scc mRNA in a time-dependent manner; levels were significantly reduced by 2h after treatment. CLs obtained from heifers on day 8 of the estrous cycle which had granulosa cells removed had a 45% reduction in the levels of mRNA for SCC enzymes as well as a 78% reduction in the numbers of LL cells. To characterize SCC expression in each steroidogenic cell type we utilized pure cell populations. Upon luteinization, LL expressed 2-3 fold higher amounts of both SCC enzymes mRNAs than SL. Moreover, eight days after stimulant removal, LL retained their P4 production capacity, expressed P450scc mRNA and contained this protein. In our attempts to establish the in vitro luteinization model, we had to select the prevulatory and pre-gonadotropin surge follicles. The ratio of estradiol:P4 which is often used was unreliable since P4 levels are high in atretic follicles and also in preovulatory post-gonadotropin follicles. We have therefore examined whether oxytocin (OT) levels in follicular fluids could enhance our ability to correctly and easily define follicular status. Based on E2 and OT concentrations in follicular fluids we could more accurately identify follicles that are preovulatory and post gonadotropin surge. Next we studied OT biosynthesis in granulosa cells, cells which were incubated with forskolin contained stores of the precursor indicating that forskolin (which mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release. While studying in vitro luteinization, we noticed that IGF-I induced effects were not identical to those induced by insulin despite the fact that megadoses of insulin were used. This was the first indication that the cells may secrete IGF binding protein(s) which regonize IGFs and not insulin. In a detailed study involving several techniques, we characterized the species of IGF binding proteins secreted by luteal cells. The effects of exogenous polyunsaturated fatty acids and arachidonic acid on the production of P4 and prostanoids by dispersed bovine luteal cells was examined. The addition of eicosapentaenoic acid and arachidonic acid resulted in a dose-dependent reduction in basal and LH-stimulated biosynthesis of P4 and PGI2 and an increase in production of PGF 2a and 5-HETE production. Indomethacin, an inhibitor of arachidonic acid metabolism via the production of 5-HETE was unaffected. Results of these experiments suggest that the inhibitory effect of arachidonic acid on the biosynthesis of luteal P4 is due to either a direct action of arachidonic acid, or its conversion to 5-HETE via the lipoxgenase pathway of metabolism. The detailed and important information gained by the two labs elucidated the mode of action of factors crucially important to the function of the bovine CL. The data indicate that follicular granulosa cells make a major contribution to numbers of large luteal cells, OT and basal P4 production, as well as the content of cytochrome P450 scc. Granulosa-derived large luteal cells have distinct features: when luteinized, the cell no longer possesses LH receptors, its cAMP response is diminished yet P4 synthesis is sustained. This may imply that maintenance of P4 (even in the absence of a Luteotropic signal) during critical periods such as pregnancy recognition, is dependent on the proper luteinization and function of the large luteal cell.
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Ohad, Itzhak, i Himadri Pakrasi. Role of Cytochrome B559 in Photoinhibition. United States Department of Agriculture, grudzień 1995. http://dx.doi.org/10.32747/1995.7613031.bard.
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The aim of this research project was to obtain information on the role of the cytochrome b559 in the function of Photosystem-II (PSII) with special emphasis on the light induced photo inactivation of PSII and turnover of the photochemical reaction center II protein subunit RCII-D1. The major goals of this project were: 1) Isolation and sequencing of the Chlamydomonas chloroplast psbE and psbF genes encoding the cytochrome b559 a and b subunits respectively; 2) Generation of site directed mutants and testing the effect of such mutation on the function of PSII under various light conditions; 3) To obtain further information on the mechanism of the light induced degradation and replacement of the PSII core proteins. This information shall serve as a basis for the understanding of the role of the cytochrome b559 in the process of photoinhibition and recovery of photosynthetic activity as well as during low light induced turnover of the D1 protein. Unlike in other organisms in which the psbE and psbF genes encoding the a and b subunits of cytochrome b559, are part of an operon which also includes the psbL and psbJ genes, in Chlamydomonas these genes are transcribed from different regions of the chloroplast chromosome. The charge distribution of the derived amino-acid sequences of psbE and psbF gene products differs from that of the corresponding genes in other organisms as far as the rule of "positive charge in" is concerned relative to the process of the polypeptide insertion in the thylakoid membrane. However, the sum of the charges of both subunits corresponds to the above rule possibly indicating co-insertion of both subunits in the process of cytochrome b559 assembly. A plasmid designed for the introduction of site-specific mutations into the psbF gene of C. reinhardtii. was constructed. The vector consists of a DNA fragment from the chromosome of C. reinhardtii which spans the region of the psbF gene, upstream of which the spectinomycin-resistance-conferring aadA cassette was inserted. This vector was successfully used to transform wild type C. reinhardtii cells. The spectinomycin resistant strain thus obtained can grow autotrophically and does not show significant changes as compared to the wild-type strain in PSII activity. The following mutations have been introduced in the psbF gene: H23M; H23Y; W19L and W19. The replacement of H23 involved in the heme binding to M and Y was meant to permit heme binding but eventually alter some or all of the electron transport properties of the mutated cytochrome. Tryptophane W19, a strictly conserved residue, is proximal to the heme and may interact with the tetrapyrole ring. Therefore its replacement may effect the heme properties. A change to tyrosine may have a lesser affect on the potential or electron transfer rate while a replacement of W19 by leucine is meant to introduce a more prominent disturbance in these parameters. Two of the mutants, FW19L and FH23M have segregated already and are homoplasmic. The rest are still grown under selection conditions until complete segregation will be obtained. All mutants contain assembled and functional PSII exhibiting an increased sensitivity of PSII to the light. Work is still in progress for the detailed characterization of the mutants PSII properties. A tobacco mutant, S6, obtained by Maliga and coworkers harboring the F26S mutation in the b subunit was made available to us and was characterized. Measurements of PSII charge separation and recombination, polypeptide content and electron flow indicates that this mutation indeed results in light sensitivity. Presently further work is in progress in the detailed characterization of the properties of all the above mutants. Information was obtained demonstrating that photoinactivation of PSII in vivo initiates a series of progressive changes in the properties of RCII which result in an irreversible modification of the RCII-D1 protein leading to its degradation and replacement. The cleavage process of the modified RCII-D1 protein is regulated by the occupancy of the QB site of RCII by plastoquinone. Newly synthesized D1 protein is not accumulated in a stable form unless integrated in reassembled RCII. Thus the degradation of the irreversibly modified RCII-D1 protein is essential for the recovery process. The light induced degradation of the RCII-D1 protein is rapid in mutants lacking the pD1 processing protease such as in the LF-1 mutant of the unicellular alga Scenedesmus obliquus. In this case the Mn binding site of PSII is abolished, the water oxidation process is inhibited and harmful cation radicals are formed following light induced electron flow in PSII. In such mutants photo-inactivation of PSII is rapid, it is not protected by ligands binding at the QB site and the degradation of the inactivated RCII-D1 occurs rapidly also in the dark. Furthermore the degraded D1 protein can be replaced in the dark in absence of light driven redox controlled reactions. The replacement of the RCII-D1 protein involves the de novo synthesis of the precursor protein, pD1, and its processing at the C-terminus end by an unknown processing protease. In the frame of this work, a gene previously isolated and sequenced by Dr. Pakrasi's group has been identified as encoding the RCII-pD1 C-terminus processing protease in the cyanobacterium Synechocystis sp. PCC 6803. The deduced sequence of the ctpA protein shows significant similarity to the bovine, human and insect interphotoreceptor retinoid-binding proteins. Results obtained using C. reinhardtii cells exposes to low light or series of single turnover light flashes have been also obtained indicating that the process of RCII-D1 protein turnover under non-photoinactivating conditions (low light) may be related to charge recombination in RCII due to back electron flow from the semiquinone QB- to the oxidised S2,3 states of the Mn cluster involved in the water oxidation process.
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Landau, Sergei Yan, John W. Walker, Avi Perevolotsky, Eugene D. Ungar, Butch Taylor i Daniel Waldron. Goats for maximal efficacy of brush control. United States Department of Agriculture, marzec 2008. http://dx.doi.org/10.32747/2008.7587731.bard.
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Background. Brush encroachment constitutes a serious problem in both Texas and Israel. We addressed the issue of efficacy of livestock herbivory - in the form of goat browsing - to change the ecological balance to the detriment of the shrub vegetation. Shrub consumption by goats is kept low by plant chemical defenses such as tannins and terpenes. Scientists at TAES and ARO have developed an innovative, cost-effective methodology using fecal Near Infrared Spectrometry to elucidate the dietary percentage of targeted, browse species (terpene-richredberry and blueberry juniper in the US, and tannin-rich Pistacialentiscus in Israel) for a large number of animals. The original research objectives of this project were: 1. to clarify the relative preference of goat breeds and the individual variation of goats within breeds, when consuming targeted brush species; 2. to assess the heritability of browse intake and validate the concept of breeding goat lines that exhibit high preference for chemically defended brush, using juniper as a model; 3. to clarify the relative contributions of genetics and learning on the preference for target species; 4. to identify mechanisms that are associated with greater intake of brush from the two target species; 5. to establish when the target species are the most vulnerable to grazing. (Issue no.5 was addressed only partly.) Major conclusions, solutions, achievements: Both the Israel and US scientists put significant efforts into improving and validating the technique of Fecal NIRS for predicting the botanical composition of goat diets. Israeli scientists validated the use of observational data for calibrating fecal NIRS, while US scientists established that calibrations could be used across animals differing in breed and age but that caution should be used in making comparisons between different sexes. These findings are important because the ability to select goat breeds or individuals within a breed for maximal efficiency of brush control is dependent upon accurate measurement of the botanical composition of the diet. In Israel it was found that Damascus goats consume diets more than twice richer in P. lentiscus than Mamber or Boer goats. In the US no differences were found between Angora and Boer cross goats but significant differences were found between individuals within breeds in juniper dietary percentage. In both countries, intervention strategies were found that further increased the consumption of the chemically defended plant. In Israel feeding polyethylene glycol (PEG, MW 4,000) that forms high-affinity complexes with tannins increased P. lentiscus dietary percentage an average of 7 percentage units. In the US feeding a protein supplement, which enhances rates of P450-catalyzed oxidations and therefore the rate of oxidation of monoterpenes, increased juniper consumption 5 percentage units. However, the effects of these interventions were not as large as breed or individual animal effects. Also, in a wide array of competitive tannin-binding assays in Israel with trypsin, salivary proteins did not bind more tannic acid or quebracho tannin than non-specific bovine serum albumin, parotid saliva did not bind more tannins than mixed saliva, no response of tannin-binding was found to levels of dietary tannins, and the breed effect was of minor importance, if any. These fundings strongly suggest that salivary proteins are not the first line of defense from tannin astringency in goats. In the US relatively low values for heritability and repeatability for juniper consumption were found (13% and 30%, respectively), possibly resulting from sampling error or non-genetic transfer of foraging behavior, i.e., social learning. Both alternatives seem to be true as significant variation between sequential observations were noted on the same animal and cross fostering studies conducted in Israel demonstrated that kids raised by Mamber goats showed lower propensity to consume P. lentiscus than counterparts raised by Damascus goats.
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Barash, Itamar, i Robert E. Rhoads. Translational Mechanisms that Govern Milk Protein Levels and Composition. United States Department of Agriculture, listopad 2004. http://dx.doi.org/10.32747/2004.7586474.bard.
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Original objectives: The long term objective of the project is to achieve higher content of protein in the milk of ruminants by modulating the translational machinery in the mammary gland. The first specific aim of the BARD proposal was to characterize responsiveness of various experimental systems to combination of lactogenic hormones and amino acids with particular emphasis on discrimination between the control of total protein synthesis and milk protein synthesis. Based on the results, we planned to proceed by characterizing the stage of protein synthesis in which the stimulation by lactogenic hormones and amino acid occur and finally we proposed to identify which components of the translation machinery are modified. Background to the topic: Milk protein is the most valuable component in milk, both for direct human consumption and for manufacturing cheese and other protein-based products. Attempts to augment protein content by the traditional methods of genetic selection and improved nutritional regimes have failed. The proposal was based on recent results suggesting that the limiting factor for augmenting protein synthesis in the bovine mammary gland is the efficiency of converting amino acids to milk proteins. Major conclusions, solutions, achievements: Insulin and prolactin synergistically stimulate â-casein mRNA translation by cytoplasmatic polyadenylation. The interaction between insulin and prolactin was demonstrated two decades ago as crucial for milk-protein synthesis, but the molecular mechanisms involved were not elucidated. We found in differentiated CID 9 mouse mammary epithelial cells line that insulin and prolactin synergistically increases the rate of milk protein mRNA translation. We focused on â-casein, the major milk protein, and found that the increase in â-casein mRNA translation was reflected in a shift to larger polysomes, indicating an effect on translational initiation. Inhibitors of the PI3K, mTOR, and MAPK pathways blocked insulin-stimulated total protein and â-casein synthesis but not the synergistic stimulation. Conversely, cordycepin, a polyadenylation inhibitor, abolished synergistic stimulation of protein synthesis without affecting insulin-stimulated translation. The poly(A) tract of â-casein mRNA progressively increased over 30 min of treatment with insulin plus prolactin. The 3’-untranslated region of â-casein mRNA was found to contain a cytoplasmic polyadenylation element (CPE), and in reporter constructs, this was sufficient for the translational enhancement and mRNA-specific polyadenylation. Furthermore, insulin and prolactin stimulated phosphorylation of cytoplasmic polyadenylation element binding protein (CPEB) but did not increase cytoplasmic polyadenylation.