Showing 3451 - 3500 of 5831 Items
Edges of the World: Photographs by Thomas Joshua Cooper
Date: 2002-01-01
Creator: Alison Ferris
Access: Open access
- "Brochure accompanies an exhibition of the same name ... from June 18 through September 1, 2002."
The cell wall-associated kinases, WAKs, as pectin receptors
Date: 2012-05-08
Creator: Bruce D. Kohorn, Susan L. Kohorn
Access: Open access
- The wall-associated kinases, WAKs, are encoded by five highly similar genes clustered in a 30-kb locus in Arabidopsis. These receptor-like proteins contain a cytoplasmic serine threonine kinase, a transmembrane domain, and a less conserved region that is bound to the cell wall and contains a series of epidermal growth factor repeats. Evidence is emerging that WAKs serve as pectin receptors, for both short oligogalacturonic acid fragments generated during pathogen exposure or wounding, and for longer pectins resident in native cell walls. This ability to bind and respond to several types of pectins correlates with a demonstrated role for WAKs in both the pathogen response and cell expansion during plant development. © 2012 Kohorn and Kohorn.
Torture under the Regime of Bashar al-Assad: Two Decades of Failed Human Rights Campaigns and Foreign Interference in Syria
Date: 2020-01-01
Creator: Olivia Giles
Access: Open access
- This honors thesis analyzes human rights campaigns to end the practice of state-sponsored torture in Syria during the presidency of Bashar al-Assad. It compares the 2000 Damascus Spring and the 2011 Arab Spring using the concept of the “contentious spiral model.” The model is based on the elements of the original “spiral model” introduced in The Power of Human Rights (1999) and the factors of contentious politics discussed in Dynamics of Contention (2001). It suggests that human rights movements that emerge from uprisings need effective mobilization by domestic and international actors. Sustained pressure from both sources should gradually force the state to make concessions until there is an absence of human rights violations. The study uses research on social movements and international politics in Syria, in addition to data on the practice of torture, to suggest that human rights campaigns to end state-sponsored torture in Syria have been unsuccessful because of the interference of Assad’s foreign alliances. These countries have helped the regime backlash against the opposition during uprisings, which has led to the fracturing of the movement. During the Damascus Spring, this interference took the form of shifting the world’s focus to other regional issues, and during the Arab Spring, Syria’s allies directly supported the Assad regime militarily, financially, and legally.
The large-scale geometry of some metabelian groups
Date: 2003-01-01
Creator: Jennifer Taback, Kevin Whyte
Access: Open access
Nonprophets: a novel This record is embargoed.
- Embargo End Date: 2025-05-14
Date: 2020-01-01
Creator: Nathan Osiason Blum
Access: Embargoed
Tame combing and almost convexity conditions
Date: 2011-12-01
Creator: Sean Cleary, Susan Hermiller, Melanie Stein, Jennifer Taback
Access: Open access
- We give the first examples of groups which admit a tame combing with linear radial tameness function with respect to any choice of finite presentation, but which are not minimally almost convex on a standard generating set. Namely, we explicitly construct such combings for Thompson's group F and the Baumslag-Solitar groups BS(1, p) with p ≥ 3. In order to make this construction for Thompson's group F, we significantly expand the understanding of the Cayley complex of this group with respect to the standard finite presentation. In particular we describe a quasigeodesic set of normal forms and combinatorially classify the arrangements of 2-cells adjacent to edges that do not lie on normal form paths. © 2010 Springer-Verlag.
Bowdoin College Catalogue (1904-1905)
Date: 1905-01-01
Access: Open access
- Bowdoin College Bulletin no. 1
Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors
Date: 1994-07-01
Creator: Tracy A. Smith, Bruce D. Kohorn
Access: Open access
- The apparatus that permits protein translocation across the internal thylakoid membranes of chloroplasts is completely unknown, even though these membranes have been the subject of extensive biochemical analysis. We have used a genetic approach to characterize the translocation of Chlamydomonas cytochrome f, a chloroplast-encoded protein that spans the thylakoid once. Mutations in the hydrophobic core of the cytochrome f signal sequence inhibit the accumulation of cytochrome f, lead to an accumulation of precursor, and impair the ability of Chlamydomonas cells to grow photosynthetically. One hydrophobic core mutant also reduces the accumulation of other thylakoid membrane proteins, but not those that translocate completely across the membrane. These results suggest that the signal sequence of cytochrome f is required and is involved in one of multiple insertion pathways. Suppressors of two signal peptide mutations describe at least two nuclear genes whose products likely describe the translocation apparatus, and selected second- site chloroplast suppressors further define regions of the cytochrome f signal peptide.
Brutal Beauty: Paintings by Walton Ford
Date: 2000-01-01
Creator: Franklin Burroughs
Access: Open access
- "Catalog accompanies the exhibition of the same name at the Bowdoin College Museum of Art, Brunswick, Maine, September 29-December 10, 2000"--P. 2
Direct selection for sequences encoding proteases of known specificity
Date: 1991-06-15
Creator: Tracy A. Smith, Bruce D. Kohorn
Access: Open access
- We have developed a simple genetic selection that could be used to isolate eukaryotic cDNAs encoding proteases that cleave within a defined amino acid sequence. The selection was developed by using the transcription factor GAL4 from Saccharomyces cerevisiae as a selectable marker, a cloned protease from tobacco etch virus (TEV), and an 18-amino acid TEV protease target sequence. In yeast, TEV protease cleaves its target even when the target is fused to internal regions of the GAL4 protein. This cleavage separates the DNA binding domain from the transcription activation domain of GAL4, rendering it transcriptionally inactive. The proteolytic cleavage can be detected phenotypically by the inability of cells to metabolize galactose. Cells expressing the TEV protease can also be selected on the suicide substrate 2-deoxygalactose. DNA binding studies show that the TEV protease decreases the activity of the GAL4/target fusion protein. Because another protease target sequence of 55 amino acids can be inserted into GAL4 without any loss of transcriptional activity, this assay offers the opportunity to use high-efficiency cDNA cloning and expression vectors to select coding sequences of other proteases from various species. The assay could also be used to help define both target specificities and functional domains of proteases.
Comparing enhancer action in cis and in trans
Date: 2012-08-01
Creator: Jack R. Bateman, Justine E. Johnson, Melissa N. Locke
Access: Open access
- Studies from diverse systems have shown that distinct interchromosomal interactions are a central component of nuclear organization. In some cases, these interactions allow an enhancer to act in trans, modulating the expression of a gene encoded on a separate chromosome held in close proximity. Despite recent advances in uncovering such phenomena, our understanding of how a regulatory element acts on another chromosome remains incomplete. Here, we describe a transgenic approach to better understand enhancer action in trans in Drosophila melanogaster. Using phiC31-based recombinase-mediated cassette exchange (RMCE), we placed transgenes carrying combinations of the simple enhancer GMR, a minimal promoter, and different fluorescent reporters at equivalent positions on homologous chromosomes so that they would pair via the endogenous somatic pairing machinery of Drosophila. Our data demonstrate that the enhancer GMR is capable of activating a promoter in trans and does so in a variegated pattern, suggesting stochastic interactions between the enhancer and the promoter when they are carried on separate chromosomes. Furthermore, we quantitatively assessed the impact of two concurrent promoter targets in cis and in trans to GMR, demonstrating that each promoter is capable of competing for the enhancer's activity, with the presence of one negatively affecting expression from the other. Finally, the single-cell resolution afforded by our approach allowed us to show that promoters in cis and in trans to GMR can both be activated in the same nucleus, implying that a single enhancer can share its activity between multiple promoter targets carried on separate chromosomes. © 2012 by the Genetics Society of America.
Adapting Greek Heroines: Penelope and Medea Access to this record is restricted to members of the Bowdoin community. Log in here to view.
Date: 2020-01-01
Creator: Ishani Agarwal
Access: Access restricted to the Bowdoin Community
Wall-associated kinase 1 (WAK1) is crosslinked in endomembranes, and transport to the cell surface requires correct cell-wall synthesis
Date: 2006-06-01
Creator: Bruce D. Kohorn, Masaru Kobayashi, Sue Johansen, Henry Perry Friedman, Andy, Fischer, Nicole Byers
Access: Open access
- The Arabidopsis thaliana wall-associated kinases (WAKs) bind to pectin with an extracellular domain and also contain a cytoplasmic protein kinase domain. WAKs are required for cell elongation and modulate sugar metabolism. This work shows that in leaf protoplasts a WAK1-GFP fusion protein accumulates in a cytoplasmic compartment that contains pectin. The WAK compartment contains markers for the Golgi, the site of pectin synthesis. The migration of WAK1-GFP to the cell surface is far slower than that of a cell surface receptor not associated with the cell wall, is influenced by the presence of fucose side chains on one or more unidentified molecules that might include pectin, and is dependent upon cellulose synthesis on the plasma membrane. WAK is crosslinked into a detergent-insoluble complex within the cytoplasmic compartment before it appears on the cell surface, and this is independent of fucose modification or cellulose synthesis. Thus, the assembly and crosslinking of WAKs may begin at an early stage within a cytoplasmic compartment rather than in the cell wall itself, and is coordinated with synthesis of surface cellulose.
Re-formando cuerpos: Las identidades femeninas en escritoras cubanas durante el Período Especial
Date: 2014-01-01
Creator: Amanda M Montenegro
Access: Open access
- Esta tesis explora cómo una variedad de autoras cubanas representan el cuerpo, las identidades femeninas y la relación entre las mujeres y la nación. Las autoras estudiadas incluyen Marilyn Bobes, Karla Suárez y Daína Chaviano. Sus narrativas ilustran y desarrollan una variedad de personajes –desde las mujeres blancas prerrevolucionarias hasta las “hijas de la revolución” afrocubanas—que representan diferentes maneras en que las mujeres construyen y reconstruyen sus identidades en la Cuba revolucionaria y hasta el comienzo del “Período especial”. Ilustran además cómo las mujeres vivieron fenómenos propios de ese período como la migración, la dolarización y el jineterismo. Así, revelan los fracasos en cuanto a la igualdad de género de la revolución, que no transformó la estructura patriarcal de la sociedad. Sin embargo, las autoras presentan una nación cubana polifacética compuesta de más que el Estado, donde las escritoras y las mujeres luchan por definirse a sí mismas. This paper explores how various female Cuban authors represent the body, female identities and the relationship between women and the nation. The authors studied include Marilyn Bobes, Karla Suárez and Daína Chaviano. Their narratives illustrate and develop a variety of characters—ranging from white prerevolutionary women to afro-Cuban “daughters of the Revolution”—which represent different ways in which women construct and reconstruct their identities during revolutionary Cuba and at the beginning of the “Special Period.” The characters also illustrate how women in particular experienced and dealt with the effects of the Special Period such as migration, dollarization and jineterismo. Thus, they reveal the failures of the Cuban Revolution regarding gender equality of the Revolution, which did not transform the patriarchal structure of society. However, the authors present a multifaceted Cuban nation comprised of more than just the State, where writers and women struggle to define themselves.
Investigating the Role of Heterochromatin Protein 1 (HP1) in the Formation of Chromosome Compartments Access to this record is restricted to members of the Bowdoin community. Log in here to view.
- Restriction End Date: 2025-06-01
Date: 2020-01-01
Creator: Diego Andres Villamarin
Access: Access restricted to the Bowdoin Community
Effects of stochasticity on the length and behaviour of ecological transients
Date: 2021-07-01
Creator: Alan Hastings, Karen C. Abbott, Kim Cuddington, Tessa B. Francis, Ying Cheng, Lai, Andrew Morozov
Access: Open access
- There is a growing recognition that ecological systems can spend extended periods of time far away from an asymptotic state, and that ecological understanding will therefore require a deeper appreciation for how long ecological transients arise. Recent work has defined classes of deterministic mechanisms that can lead to long transients. Given the ubiquity of stochasticity in ecological systems, a similar systematic treatment of transients that includes the influence of stochasticity is important. Stochasticity can of course promote the appearance of transient dynamics by preventing systems from settling permanently near their asymptotic state, but stochasticity also interacts with deterministic features to create qualitatively new dynamics. As such, stochasticity may shorten, extend or fundamentally change a system's transient dynamics. Here, we describe a general framework that is developing for understanding the range of possible outcomes when random processes impact the dynamics of ecological systems over realistic time scales. We emphasize that we can understand the ways in which stochasticity can either extend or reduce the lifetime of transients by studying the interactions between the stochastic and deterministic processes present, and we summarize both the current state of knowledge and avenues for future advances.
Species identification based on a semi-diagnostic marker: Evaluation of a simple conchological test for distinguishing blue mussels Mytilus edulis L. And M. trossulus Gould
Date: 2021-07-01
Creator: Vadim Khaitov, Julia Marchenko, Marina Katolikova, Risto Väinölä, Sarah E., Kingston, David B. Carlon, Michael Gantsevich, Petr Strelkov
Access: Open access
- Cryptic and hybridizing species may lack diagnostic taxonomic characters leaving researchers with semi-diagnostic ones. Identification based on such characters is probabilistic, the probability of correct identification depending on the species composition in a mixed population. Here we test the possibilities of applying a semi-diagnostic conchological character for distinguishing two cryptic species of blue mussels, Mytilus edulis and M. trossulus. These ecologically, stratigraphically and economically important molluscs co-occur and hybridize in many areas of the North Atlantic and the neighboring Arctic. Any cues for distinguishing them in sympatry without genotyping would save much research effort. Recently these species have been shown to statistically differ in the White Sea, where a simple character of the shell was used to distinguish two mussel morphotypes. In this paper, we analyzed the associations between morphotypes and species-specific genotypes based on an abundant material from the waters of the Kola Peninsula (White Sea, Barents Sea) and a more limited material from Norway, the Baltic Sea, Scotland and the Gulf of Maine. The performance of the “morphotype test” for species identification was formally evaluated using approaches from evidence-based medicine. Interspecific differences in the morphotype frequencies were ubiquitous and unidirectional, but their scale varied geographically (from 75% in the White Sea to 15% in the Baltic Sea). In addition, salinity-related variation of this character within M. edulis was revealed in the Arctic Barents Sea. For every studied region, we established relationships between the proportions of the morphotypes in the populations as well as between the proportions of the morphotypes in samples and the probabilities of mussels of different morphotypes being M. trossulus and M. edulis. We provide recommendations for the application of the morphotype test to mussels from unstudied contact zones and note that they may apply equally well to other taxa identified by semi-diagnostic traits.
Bowdoin College - Medical School of Maine Catalogue (1908-1909)
Date: 1909-01-01
Access: Open access
- Bowdoin College Bulletin no. 19
Bowdoin College Catalogue (1915-1916)
Date: 1916-01-01
Access: Open access
- Bowdoin College Bulletin no. 62
Convexity Properties of the Diestel-Leader Group Γ_3(2)
Date: 2014-05-01
Creator: Peter J Davids
Access: Open access
- The Diestel-Leader groups are a family of groups first introduced in 2001 by Diestel and Leader in [7]. In this paper, we demonstrate that the Diestel-Leader group Γ3(2) is not almost convex with respect to a particular generating set S. Almost convexity is a geometric property that has been shown by Cannon [3] to guarantee a solvable word problem (that is, in any almost convex group there is a finite-step algorithm to determine if two strings of generators, or “words”, represent the same group element). Our proof relies on the word length formula given by Stein and Taback in [10], and we construct a family of group elements X that contradicts the almost convexity condition. We then go on to show that Γ3(2) is minimally almost convex with respect to S.
Egg Size, Breeding Phenology, and Parental Investment in Leach’s Storm Petrels Access to this record is restricted to members of the Bowdoin community. Log in here to view.
Date: 2020-01-01
Creator: James L. O'Shea
Access: Access restricted to the Bowdoin Community
Bowdoin College Catalogue (1921-1922)
Date: 1922-01-01
Access: Open access
- Bowdoin College Bulletin no. 114
The guanine nucleotide exchange factor trio mediates axonal development in the Drosophila embryo
Date: 2000-01-01
Creator: Jack Bateman, Huidy Shu, David Van Vactor
Access: Open access
- Recent analysis of Rho subfamily GTPases in Drosophila revealed roles for Rac and Cdc42 during axonogenesis. Here, we describe the identification and characterization of the Drosophila counterpart of Trio, a guanine nucleotide exchange factor (GEF) that associates with the receptor phosphatase LAR and regulates GTPase activation in vertebrate cells. Mutants deficient in trio activity display defects in both central and peripheral axon pathways reminiscent of pheno-types observed in embryos deficient in small GTPase function. Double mutant analysis shows that trio interacts with Rac in a dose-sensitive manner but not with Rho. Moreover, reduction of trio activity potentiates the phenotype of mutations in the LAR homolog Dlar, suggesting that these proteins collaborate in orchestrating the cytoskeletal events that underlie normal axonogenesis.
Adjusting to Global change through clonal growth and epigenetic variation
Date: 2016-07-26
Creator: Richard S. Dodd, Vladimir Douhovnikoff
Access: Open access
- The earth is experiencing major changes in global and regional climates and changes are predicted to accelerate in the future. Many species will be under considerable pressure to evolve, to migrate, or be faced with extinction. Clonal plants would appear to be at a particular disadvantage due to their limited mobility and limited capacity for adaptation. However, they have outlived previous environmental shifts and clonal species have persisted for millenia. Clonal spread offers unique ecological advantages, such as resource sharing, risk sharing, and economies of scale among ramets within genotypes. We suggest that ecological attributes of clonal plants, in tandem with variation in gene regulation through epigenetic mechanisms that facilitate and optimize phenotype variation in response to environmental change may permit them to be well suited to projected conditions.
Accounting for Gender Differences in Cultural Industries: Evidence from Film and the Fine Arts Access to this record is restricted to members of the Bowdoin community. Log in here to view.
Date: 2020-01-01
Creator: Madeleine Rose Dupré
Access: Access restricted to the Bowdoin Community
Bowdoin College Catalogue (1945-1946)
Date: 1946-01-01
Access: Open access
- Bowdoin College Bulletin no. 277
Similarities and differences in circuit responses to applied Gly 1 -SIFamide and peptidergic (Gly 1 -SIFamide) neuron stimulation
Date: 2019-03-01
Creator: Dawn M. Blitz, Andrew E. Christie, Aaron P. Cook, Patsy S. Dickinson, Michael P., Nusbaum
Access: Open access
- Similarities and differences in circuit responses to applied Gly 1 -SIFamide and peptidergic (Gly 1 -SIFamide) neuron stimulation. J Neurophysiol 121: 950 –972, 2019. First published January 16, 2019; doi:10.1152/jn.00567.2018.—Microcircuit modulation by peptides is well established, but the cellular/synaptic mechanisms whereby identified neurons with identified peptide transmitters modulate microcircuits remain unknown for most systems. Here, we describe the distribution of GYRKPPFNGSIFamide (Gly 1 -SIFamide) immunoreactivity (Gly 1 -SIFamide-IR) in the stomatogastric nervous system (STNS) of the crab Cancer borealis and the Gly 1 -SIFamide actions on the two feeding-related circuits in the stomatogastric ganglion (STG). Gly 1 -SIFamide-IR localized to somata in the paired commissural ganglia (CoGs), two axons in the nerves connecting each CoG with the STG, and the CoG and STG neuropil. We identified one Gly 1 -SIFamide-IR projection neuron innervating the STG as the previously identified modulatory commissural neuron 5 (MCN5). Brief (~10 s) MCN5 stimulation excites some pyloric circuit neurons. We now find that bath applying Gly 1 -SIFamide to the isolated STG also enhanced pyloric rhythm activity and activated an imperfectly coordinated gastric mill rhythm that included unusually prolonged bursts in two circuit neurons [inferior cardiac (IC), lateral posterior gastric (LPG)]. Furthermore, longer duration (±30 s) MCN5 stimulation activated a Gly 1 -SIFamide-like gastric mill rhythm, including prolonged IC and LPG bursting. The prolonged LPG bursting decreased the coincidence of its activity with neurons to which it is electrically coupled. We also identified local circuit feedback onto the MCN5 axon terminals, which may contribute to some distinctions between the responses to MCN5 stimulation and Gly 1 -SIFamide application. Thus, MCN5 adds to the few identified projection neurons that modulate a well-defined circuit at least partly via an identified neuropeptide transmitter and provides an opportunity to study peptide regulation of electrical coupled neurons in a functional context. NEW & NOTEWORTHY Limited insight exists regarding how identified peptidergic neurons modulate microcircuits. We show that the modulatory projection neuron modulatory commissural neuron 5 (MCN5) is peptidergic, containing Gly 1 -SIFamide. MCN5 and Gly 1 -SIFamide elicit similar output from two well-defined motor circuits. Their distinct actions may result partly from circuit feedback onto the MCN5 axon terminals. Their similar actions include eliciting divergent activity patterns in normally coactive, electrically coupled neurons, providing an opportunity to examine peptide modulation of electrically coupled neurons in a functional context.
Bowdoin College Catalogue (1939-1940)
Date: 1940-01-01
Access: Open access
- Bowdoin College Bulletin no. 249
Bowdoin College Catalogue (1933-1934)
Date: 1934-01-01
Access: Open access
- Bowdoin College Bulletin no. 214
Bowdoin College Catalogue (1918-1919)
Date: 1919-01-01
Access: Open access
- Bowdoin College Bulletin no. 87
The receptor tyrosine phosphatase Dlar and integrins organize actin filaments in the Drosophila follicular epithelium
Date: 2001-09-04
Creator: Jack Bateman, R. Srekantha Reddy, Haruo Saito, David Van Vactor
Access: Open access
- Background: Regulation of actin structures is instrumental in maintaining proper cytoarchitecture in many tissues. In the follicular epithelium of Drosophila ovaries, a system of actin filaments is coordinated across the basal surface of cells encircling the oocyte. These filaments have been postulated to regulate oocyte elongation; however, the molecular components that control this cytoskeletal array are not yet understood. Results: We find that the receptor tyrosine phosphatase (RPTP) Dlar and integrins are involved in organizing basal actin filaments in follicle cells. Mutations in Dlar and the common β-integrin subunit mys cause a failure in oocyte elongation, which is correlated with a loss of proper actin filament organization. Immunolocalization shows that early in oogenesis Dlar is polarized to membranes where filaments terminate but becomes generally distributed late in development, at which time β-integrin and Enabled specifically associate with actin filament terminals. Rescue experiments point to the early period of polar Dlar localization as critical for its function. Furthermore, clonal analysis shows that loss of Dlar or mys influences actin filament polarity in wild-type cells that surround mutant tissues, suggesting that communication between neighboring cells regulates cytoskeletal organization. Finally, we find that two integrin α subunits encoded by mew and if are required for proper oocyte elongation, implying that multiple components of the ECM are instructive in coordinating actin fiber polarity. Conclusions: Dlar cooperates with integrins to coordinate actin filaments at the basal surface of the follicular epithelium. To our knowledge, this is the first direct demonstration of an RPTP's influence on the actin cytoskeleton.
Functions of the ectodomain and cytoplasmic tyrosine phosphatase domains of receptor protein tyrosine phosphatase Dlar in vivo
Date: 2003-10-01
Creator: Neil X. Krueger, R. Sreekantha Reddy, Karl Johnson, Jack Bateman, Nancy, Kaufmann, Daniella Scalice, David Van Vactor, Haruo Saito
Access: Open access
- The receptor protein tyrosine phosphatase (PTPase) Dlar has an ectodomain consisting of three immunoglobulin (Ig)-like domains and nine fibronectin type III (FnIII) repeats and a cytoplasmic domain consisting of two PTPase domains, membrane-proximal PTP-D1 and C-terminal PTP-D2. A series of mutant Dlar transgenes were introduced into the Drosophila genome via P-element transformation and were then assayed for their capacity to rescue phenotypes caused by homozygous loss-of-function genotypes. The Ig-like domains, but not the FnIII domains, are essential for survival. Conversely, the FnIII domains, but not the Ig-like domains, are required during oogenesis, suggesting that different domains of the Dlar ectodomain are involved in distinct functions during Drosophila development. All detectable PTPase activity maps to PTP-D1 in vitro. The catalytically inactive mutants of Dlar were able to rescue Dlar -/- lethality nearly as efficiently as wild-type Dlar transgenes, while this ability was impaired in the PTP-D2 deletion mutants DlarΔPTP-D2 and Dlarbypass. Dlar-C1929S, in which PTP-D2 has been inactivated, increases the frequency of bypass phenotype observed in Dlar-/- genotypes, but only if PTP-D1 is catalytically active in the transgene. These results indicate multiple roles for PTP-D2, perhaps by acting as a docking domain for downstream elements and as a regulator of PTP-D1.
Targeted identification of glycosylated proteins in the gastric pathogen helicobacter pylori (Hp)
Date: 2013-09-01
Creator: Kanokwan Champasa, Scott A. Longwell, Aimee M. Eldridge, Elizabeth A. Stemmler, Danielle H., Dube
Access: Open access
- Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen's ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host's stomach. Although Hp's glycans are linked to pathogenesis, Hp's glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp's glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp's azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.