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991.
Deng H Bannister TD Jin L Babine RE Quinn J Nagafuji P Celatka CA Lin J Lazarova TI Rynkiewicz MJ Bibbins F Pandey P Gorga J Meyers HV Abdel-Meguid SS Strickler JE 《Bioorganic & medicinal chemistry letters》2006,16(11):3049-3054
Using an alpha-ketothiazole arginine moiety as a key recognition element, a series of small peptidomimetic molecules was designed and synthesized, and their co-crystal structures with factor XIa were studied in an effort to develop smaller, less peptidic inhibitors as antithrombotic agents. 相似文献
992.
David Schubert Federico Herrera Robert Cumming Jessica Read† William Low† Pamela Maher Wolfgang H. Fischer† 《Journal of neurochemistry》2009,109(2):427-435
Proteins that are released from cells consist of those in the extracellular matrix, as well as extracellular signaling and adhesion molecules. The majority of these extracellular proteins are, however, unknown. To determine their identity, we have used a proteomics approach to define proteins released from neurons, astrocytes and neural precursor cells. Using two-dimensional gels and liquid chromatography/mass spectrometry technology, it is shown that while astrocytes release a relatively small number of proteins, neurons and neuronal precursor cells release a larger number of proteins with more functional diversity. Although there is overlap between the different cell types, the exact composition of the extracellular protein pool is unique for each cell population. The various subsets of extracellular neural proteins include those involved in cellular Redox regulation and chaperones. In addition, many proteolytic enzymes are found outside of the cell. These data show that the extracellular space within the nervous system has a more diverse protein composition than previously thought. 相似文献
993.
994.
Design and expression of a dimeric form of human immunodeficiency virus type 1 antibody 2G12 with increased neutralization potency 下载免费PDF全文
West AP Galimidi RP Foglesong CP Gnanapragasam PN Huey-Tubman KE Klein JS Suzuki MD Tiangco NE Vielmetter J Bjorkman PJ 《Journal of virology》2009,83(1):98-104
The antigen-binding fragment of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) antibody 2G12 has an unusual three-dimensional (3D) domain-swapped structure with two aligned combining sites that facilitates recognition of its carbohydrate epitope on gp120. When expressed as an intact immunoglobulin G (IgG), 2G12 formed typical IgG monomers containing two combining sites and a small fraction of a higher-molecular-weight species, which showed a significant increase in neutralization potency (50- to 80-fold compared to 2G12 monomer) across a range of clade A and B strains of HIV-1. Here we show that the higher-molecular-weight species corresponds to a 2G12 dimer containing four combining sites and present a model for how intermolecular 3D domain swapping could create a 2G12 dimer. Based on the structural model for a 3D domain-swapped 2G12 dimer, we designed and tested a series of 2G12 mutants predicted to increase the ratio of 2G12 dimer to monomer. We report a mutation that effectively increases the 2G12 dimer/monomer ratio without decreasing the expression yield. Increasing the proportion of 2G12 dimer compared to monomer could lead to a more potent reagent for gene therapy or passive immunization. 相似文献
995.
Denise Daley Mathieu Lemire Loubna Akhabir Moira Chan-Yeung Jian Qing He Treena McDonald Andrew Sandford Dorota Stefanowicz Ben Tripp David Zamar Yohan Bosse Vincent Ferretti Alexandre Montpetit Marie-Catherine Tessier Allan Becker Anita L. Kozyrskyj John Beilby Pamela A. McCaskie Bill Musk Nicole Warrington Alan James Catherine Laprise Lyle J. Palmer Peter D. Paré Thomas J. Hudson 《Human genetics》2009,125(4):445-459
996.
997.
The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis‐acting replication determinant—the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non‐rDNA fragment containing two closely associated replicators, ARS1‐A (0.8 kb) and ARS1‐B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA‐independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non‐rDNA ARS1 chromosome changed across the cell cycle. In G2 phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1‐A/B. Remarkably, ORC and Mcm6 associated with just the ARS1‐A replicator in G1 phase when pre‐replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non‐rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase. 相似文献
998.
Gabriela Vanina Villanova Pamela Cribb Ariel Mariano Silber Sergio Schenkman 《International journal for parasitology》2009,39(6):665-673
Histone tail post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) regulate many cellular processes. Among these modifications, phosphorylation, methylation and acetylation have already been described in trypanosomatid histones. Bromodomains, together with chromodomains and histone-binding SANT domains, were proposed to be responsible for “histone code” reading. The Trypanosoma cruzi genome encodes four coding sequences (CDSs) that contain a bromodomain, named TcBDF1-4. Here we show that one of those, TcBDF2, is expressed in discrete regions inside the nucleus of all the parasite life cycle stages and binds H4 and H2A purified histones from T. cruzi. Immunolocalization experiments using both anti-histone H4 acetylated peptides and anti-TcBDF2 antibodies determined that TcBDF2 co-localizes with histone H4 acetylated at lysines K10 and K14. TcDBF2 and K10 acetylated H4 interaction was confirmed by co-immunoprecipitation. It is also shown that TcBDF2 was accumulated after UV irradiation of T. cruzi epimastigotes. These results suggest that TcBDF2 could be taking part in a chromatin remodelling complex in T. cruzi. 相似文献
999.
Juliya Kalinina Sara A. Byron Helen P. Makarenkova Shaun K. Olsen Anna V. Eliseenkova William J. Larochelle Mohanraj Dhanabal Steven Blais David M. Ornitz Loren A. Day Thomas A. Neubert Pamela M. Pollock Moosa Mohammadi 《Molecular and cellular biology》2009,29(17):4663-4678
Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20''s receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.Fibroblast growth factor (FGF) signaling plays pleiotropic roles throughout the life spans of mammalian organisms, ranging from germ cell maturation, mesoderm induction, body plan formation, and organogenesis during embryonic development to serum phosphate homeostasis and glucose, bile acid, lipid, and cholesterol metabolism in the adult (3, 23, 27, 28, 57, 60, 62). The diversity of FGF signaling is underscored by virtue of the fact that aberrant FGF signaling leads to a wide array of human diseases, including skeletal and olfactory/reproductive syndromes, phosphate wasting disorders, and cancer (16, 60, 67). Recent data also implicate dysregulated FGF signaling in the etiology of neurodegenerative disorders, such as major depressive disorder and Parkinson''s disease (10, 63, 64).Based on pairwise sequence homology and phylogeny, the 18 bona fide mammalian FGFs (FGF1 to FGF10 and FGF16 to FGF23) are divided into six subfamilies (45). Five FGF subfamilies have high-to-moderate affinity for pericellular heparan sulfate (HS) glycosaminoglycans and thus diffuse locally within tissues to act in a paracrine fashion, whereas the poor affinity of the FGF19 subfamily for HS enables this subfamily to act in an endocrine manner (28, 38). All FGFs share a core homology region of about 120 amino acids, which fold into 12 antiparallel β strands (β1 to β12) that are arranged into three sets of four-stranded β sheets (β-trefoil fold) (39). The globular FGF core domain is flanked by highly divergent N- and C-terminal extensions, which are the principal regions responsible for the different biology of FGFs.FGFs exert their diverse actions by binding and activating FGF receptors (FGFRs) in an HS-dependent fashion (51, 53, 69). There are four distinct mammalian FGFR genes (FGFR1 to FGFR4), each coding for a single-pass transmembrane tyrosine kinase receptor whose ectodomain consists of three immunoglobulin-like domains (D1 to D3) connected by flexible linkers and whose intracellular domain contains the conserved tyrosine kinase domain flanked by the juxtamembrane (JM) and C-terminal regions (38). The 210-amino-acid-long D2-D3 segment of the ectodomain is both necessary and sufficient for ligand binding (20, 51, 52, 58, 70).FGF signaling is tightly regulated by spatial and temporal expression of ligands, receptors, HS cofactors, and most critically by means of FGF-FGFR binding specificity. The tissue-specific alternative splicing in the D3 domain of FGFR1 to FGFR3 is the main mechanism by which FGF-FGFR binding specificity is regulated. This splicing event gives rise to epithelial “b” isoforms (FGFR1b to FGFR3b) and mesenchymal “c” isoforms (FGFR1c to FGFR3c) (24, 25, 47, 68), which differ from one another at the primary sequences of their key ligand binding regions and thus in their FGF binding specificity/promiscuity profiles. Most FGFs are also expressed in either epithelial or mesenchymal tissues and exhibit specificity for FGFR isoforms expressed in the opposite tissues. This results in the establishment of a bidirectional signaling loop between the epithelium and mesenchyme that is essential for organogenesis and tissue homeostasis. It is well established that FGF7 and FGF10, which are expressed exclusively in the mesenchyme, activate specifically FGFR2b to mediate mesenchymal-to-epithelial signaling in the lung, prostate, and lacrimal, mammary, and salivary glands (19, 29, 35, 36, 59). Several lines of genetic and biochemical evidence suggest that the members of the FGF9 subfamily, which includes FGF9, FGF16, and FGF20, convey the reciprocal signaling from the epithelium to the mesenchyme. In the prostate, the epithelial-specific FGF9 has been shown to activate mesenchymal FGFR3c isoforms (25). In the heart, FGF9, FGF16, and FGF20 in the epicardium and endocardium stimulate myocardial proliferation and differentiation in vivo, acting redundantly through FGFR1c and FGFR2c (32). Analysis of FGF9-deficient mice has identified FGF9 as a reciprocal epithelial-to-mesenchymal signal required for morphogenesis of the lung, cecum, small intestine, and inner ear (14, 49, 65, 71). In addition, studies in zebra fish show that FGF16 and FGF20 are apical ectodermal ridge factors that are required for pectoral fin bud outgrowth and, in general, for cell proliferation and differentiation of the mesenchyme (41, 66).In light of the key role of the FGF9 subfamily in tissue homeostasis, it is essential to investigate the molecular mechanisms by which the activity of this subfamily is regulated. Our previous structural and in vitro studies of FGF9 showed that homodimerization masks FGF9''s key receptor binding sites, suggesting that ligand dimerization may autoinhibit FGF9''s biologic activity (50). In this report, we show that, like FGF9, FGF20 also homodimerizes in the crystal and in solution. Characterization of the dimer interface mutations in vitro and in living cells demonstrates that ligand homodimerization autoinhibits FGF9 and FGF20 signaling by suppressing both receptor binding and HS-dependent diffusion in the extracellular matrix (ECM). Our study is the first to implicate ligand dimerization as an autoregulatory mechanism in growth factor bioactivity. 相似文献
1000.
Rhea N. Coler Ajay Bhatia Jean-Francois Maisonneuve Peter Probst Brenda Barth Pamela Ovendale Hang Fang Mark Alderson Yves Lobet Joe Cohen Pascal Mettens & Steven G. Reed 《FEMS immunology and medical microbiology》2009,55(2):258-270
Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide, with over 91 million cases estimated annually. An effective subunit vaccine against Chlamydia may require a multivalent subunit cocktail of antigens in a single formulation for broad coverage of a heterogeneous major histocompatibility complex population. Herein, we describe the identification of novel C. trachomatis antigens by CD4+ and CD8+ T-cell expression cloning, serological expression cloning, and an in silico analysis of the C. trachomatis genome. These antigens elicited human CD4+ T-cell responses, and a subset proved to be immunogenic and protective when administered as immunoprophylactic vaccines against C. trachomatis challenge. Candidate vaccines consisting of the prioritized C. trachomatis antigens adjuvanted in a GlaxoSmithKline proprietary AS01B adjuvant were prioritized based on induction of solid protection against challenge in C57BL/6 and BALB/c mice with C. trachomatis . Some of the vaccines prevented bacterial shedding and colonization of the upper genital tract to varying degrees by mechanisms that may include CD4+ T cells. 相似文献