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Sample records for bacterial adaptor membrane

  1. Analysis of Arf1 GTPase-dependent membrane binding and remodeling using the exomer secretory vesicle cargo adaptor

    Science.gov (United States)

    Paczkowski, Jon E.; Fromme, J. Christopher

    2016-01-01

    Summary Protein-protein and protein-membrane interactions play a critical role in shaping biological membranes through direct physical contact with the membrane surface. This is particularly evident in many steps of membrane trafficking, in which proteins deform the membrane and induce fission to form transport carriers. The small GTPase Arf1 and related proteins have the ability to remodel membranes by insertion of an amphipathic helix into the membrane. Arf1 and the exomer cargo adaptor coordinate cargo sorting into subset of secretory vesicle carriers in the model organism Saccharomyces cerevisiae. Here, we detail the assays we used to explore the cooperative action of Arf1 and exomer to bind and remodel membranes. We expect these methods are broadly applicable to other small GTPase/effector systems where investigation of membrane binding and remodeling is of interest. PMID:27632000

  2. Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump

    OpenAIRE

    Hinchliffe, Philip; Greene, Nicholas P.; Paterson, Neil G.; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

    2014-01-01

    Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel co...

  3. The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains

    OpenAIRE

    Fölsch, Heike; Pypaert, Marc; Maday, Sandra; Pelletier, Laurence; Mellman, Ira

    2003-01-01

    Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD μ1A or μ1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membra...

  4. Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump.

    Science.gov (United States)

    Hinchliffe, Philip; Greene, Nicholas P; Paterson, Neil G; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

    2014-08-25

    Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. PMID:24996185

  5. Filtration properties of bacterial cellulose membranes

    OpenAIRE

    Lehtonen, Janika

    2015-01-01

    Bacterial cellulose has the same molecular formula as cellulose from plant origin, but it is characterized by several unique properties including high purity, crystallinity and mechanical strength. These properties are dependent on parameters such as the bacterial strain used, the cultivation conditions and post-growth processing. The possibility to achieve bacterial cellulose membranes with different properties by varying these parameters could make bacterial cellulose an interesting materi...

  6. The clathrin adaptor complexes as a paradigm for membrane-associated allostery

    OpenAIRE

    Canagarajah, Bertram J.; Ren, Xuefeng; Bonifacino, Juan S.; Hurley, James H.

    2013-01-01

    The clathrin-associated adaptor protein (AP) complexes AP-1 and AP-2 are two members of a family of heterotetrameric assemblies that connect transmembrane protein cargo to vesicular coats. Cargo binding by AP-1 is activated by the small GTPase Arf1, while AP-2 is activated by the phosphoinositide PI(4,5)P2. The structures of both AP-1 and AP-2 have been determined in their locked and unlocked conformations. The structures show how different activators use different mechanisms to trigger simil...

  7. Bacterial cellulose membrane as separation medium

    Energy Technology Data Exchange (ETDEWEB)

    Shibazaki, Hideki; Kuga, Shigenori; Onabe, Fumihiko; Usuda, Makoto (Univ. of Toyko, (Japan). Faculty of Agriculture)

    1993-11-10

    A thin membrane of bacterial cellulose (BC) obtained from Acetobacter culture was tested for its performance as a dialysis membrane in aqueous systems. The BC membrane showed superior mechanical strength to that of a dialysis-grade regenerated cellulose membrane, allowing the use of a thinner membrane than the latter. As a result, the BC membrane gave higher permeation rates for poly(ethylene glycols) as probe solutes. The cutoff molecular weight of the original BC membrane, significantly greater than that of regenerated cellulose, could be modified by concentrated alkali treatments of the membrane. The nature of the change at the ultrastructural level caused by the alkali treatments was studied by X-ray diffraction and scanning electron microscopy.

  8. The membrane-associated adaptor protein DOK5 is upregulated in systemic sclerosis and associated with IGFBP-5-induced fibrosis.

    Directory of Open Access Journals (Sweden)

    Hidekata Yasuoka

    Full Text Available Systemic sclerosis (SSc is characterized by excessive fibrosis of the skin and internal organs due to fibroblast proliferation and excessive production of extracellular matrix (ECM. We have shown that insulin-like growth factor binding protein (IGFBP-5 plays an important role in the development of fibrosis in vitro, ex vivo, and in vivo. We identified a membrane-associated adaptor protein, downstream of tyrosine kinase/docking protein (DOK5, as an IGFBP-5-regulated target gene using gene expression profiling of primary fibroblasts expressing IGFBP-5. DOK5 is a tyrosine kinase substrate associated with intracellular signaling. Our objective was to determine the role of DOK5 in the pathogenesis of SSc and specifically in IGFBP-5-induced fibrosis. DOK5 mRNA and protein levels were increased in vitro by endogenous and exogenous IGFBP-5 in primary human fibroblasts. DOK5 upregulation required activation of the mitogen-activated protein kinase (MAPK signaling cascade. Further, IGFBP-5 triggered nuclear translocation of DOK5. DOK5 protein levels were also increased in vivo in mouse skin and lung by IGFBP-5. To determine the effect of DOK5 on fibrosis, DOK5 was expressed ex vivo in human skin in organ culture. Expression of DOK5 in human skin resulted in a significant increase in dermal thickness. Lastly, levels of DOK5 were compared in primary fibroblasts and lung tissues of patients with SSc and healthy donors. Both DOK5 mRNA and protein levels were significantly increased in fibroblasts and skin tissues of patients with SSc compared with those of healthy controls, as well as in lung tissues of SSc patients. Our findings suggest that IGFBP-5 induces its pro-fibrotic effects, at least in part, via DOK5. Furthermore, IGFBP-5 and DOK5 are both increased in SSc fibroblasts and tissues and may thus be acting in concert to promote fibrosis.

  9. Hopanoids as functional analogues of cholesterol in bacterial membranes.

    Science.gov (United States)

    Sáenz, James P; Grosser, Daniel; Bradley, Alexander S; Lagny, Thibaut J; Lavrynenko, Oksana; Broda, Martyna; Simons, Kai

    2015-09-22

    The functionality of cellular membranes relies on the molecular order imparted by lipids. In eukaryotes, sterols such as cholesterol modulate membrane order, yet they are not typically found in prokaryotes. The structurally similar bacterial hopanoids exhibit similar ordering properties as sterols in vitro, but their exact physiological role in living bacteria is relatively uncharted. We present evidence that hopanoids interact with glycolipids in bacterial outer membranes to form a highly ordered bilayer in a manner analogous to the interaction of sterols with sphingolipids in eukaryotic plasma membranes. Furthermore, multidrug transport is impaired in a hopanoid-deficient mutant of the gram-negative Methylobacterium extorquens, which introduces a link between membrane order and an energy-dependent, membrane-associated function in prokaryotes. Thus, we reveal a convergence in the architecture of bacterial and eukaryotic membranes and implicate the biosynthetic pathways of hopanoids and other order-modulating lipids as potential targets to fight pathogenic multidrug resistance.

  10. Interaction of multiple biomimetic antimicrobial polymers with model bacterial membranes

    Energy Technology Data Exchange (ETDEWEB)

    Baul, Upayan, E-mail: upayanb@imsc.res.in; Vemparala, Satyavani, E-mail: vani@imsc.res.in [The Institute of Mathematical Sciences, C.I.T. Campus, Taramani, Chennai 600113 (India); Kuroda, Kenichi, E-mail: kkuroda@umich.edu [Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109 (United States)

    2014-08-28

    Using atomistic molecular dynamics simulations, interaction of multiple synthetic random copolymers based on methacrylates on prototypical bacterial membranes is investigated. The simulations show that the cationic polymers form a micellar aggregate in water phase and the aggregate, when interacting with the bacterial membrane, induces clustering of oppositely charged anionic lipid molecules to form clusters and enhances ordering of lipid chains. The model bacterial membrane, consequently, develops lateral inhomogeneity in membrane thickness profile compared to polymer-free system. The individual polymers in the aggregate are released into the bacterial membrane in a phased manner and the simulations suggest that the most probable location of the partitioned polymers is near the 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) clusters. The partitioned polymers preferentially adopt facially amphiphilic conformations at lipid-water interface, despite lacking intrinsic secondary structures such as α-helix or β-sheet found in naturally occurring antimicrobial peptides.

  11. Differential association of the Na+/H+ exchanger regulatory factor (NHERF) family of adaptor proteins with the raft- and the non-raft brush border membrane fractions of NHE3

    NARCIS (Netherlands)

    A. Sultan (Ayesha); M. Luo (Ma); Q. Yu (Qingbao); B. Riederer (Beat Michel); W. Xia (Weiliang); M. Chen (Mingmin); S. Lissner (Simone); J.E. Gessner (Johannes); M. Donowitz (Mark); C. Chris Yun (C.); H. deJonge (Hugo); G. Lamprecht (Georg); U. Seidler (Ursula)

    2013-01-01

    textabstractBackground/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is uncl

  12. A Molecularly Complete Planar Bacterial Outer Membrane Platform.

    Science.gov (United States)

    Hsia, Chih-Yun; Chen, Linxiao; Singh, Rohit R; DeLisa, Matthew P; Daniel, Susan

    2016-01-01

    The bacterial outer membrane (OM) is a barrier containing membrane proteins and liposaccharides that fulfill crucial functions for Gram-negative bacteria. With the advent of drug-resistant bacteria, it is necessary to understand the functional role of this membrane and its constituents to enable novel drug designs. Here we report a simple method to form an OM-like supported bilayer (OM-SB), which incorporates native lipids and membrane proteins of gram-negative bacteria from outer membrane vesicles (OMVs). We characterize the formation of OM-SBs using quartz crystal microbalance with dissipation (QCM-D) and fluorescence microscopy. We show that the orientation of proteins in the OM-SB matches the native bacterial membrane, preserving the characteristic asymmetry of these membranes. As a demonstration of the utility of the OM-SB platform, we quantitatively measure antibiotic interactions between OM-SBs and polymyxin B, a cationic peptide used to treat Gram-negative infections. This data enriches understanding of the antibacterial mechanism of polymyxin B, including disruption kinetics and changes in membrane mechanical properties. Combining OM-SBs with microfluidics will enable higher throughput screening of antibiotics. With a broader view, we envision that a molecularly complete membrane-scaffold could be useful for cell-free applications employing engineered membrane proteins in bacterial membranes for myriad technological purposes. PMID:27600663

  13. Interactions between model bacterial membranes and synthetic antimicrobials.

    Science.gov (United States)

    Yang, Lihua; Mishra, Abhijit; Som, Abhigyan; Tew, Gregory N.; Wong, Gerard C. L.

    2006-03-01

    Antimicrobial peptides comprise a key component of innate immunity for a wide range of multicellular organisms. It has been shown that natural antimicrobial peptides and their analogs can permeate bacterial membranes selectively. There are a number of proposed models for this action, but the detailed molecular mechanism of the induced membrane permeation remains unclear. We investigate interactions between model bacterial membranes and a prototypical family of phenylene ethynylene-based antimicrobials with controllable hydrophilic and hydrophobic volume fractions, controllable charge placement. Preliminary results from synchrotron small angle x-ray scattering (SAXS) results will be presented.

  14. In-vitro Degradation Behaviour of Irradiated Bacterial Cellulose Membrane

    International Nuclear Information System (INIS)

    Bacterial cellulose membrane synthesized by Acetobacter xylinum in coconut water medium has potential application for Guided bone Regeneration. However, this membrane may not meet some application requirements due to its low biodegradation properties. In this paper, incorporation of gamma irradiation into the membrane is a developed strategy to increase its biodegradability properties. The in-vitro degradation study in synthetic body fluid (SBF) of the irradiated membrane has been analyzed during periods of 6 months by means of weight loss, mechanical properties and scanning electron microscopy observation compared to that the un-irradiated one. The result showed that weight loss of irradiated membrane with 25 kGy and 50 kGy and immersed in SBF solution for 6 months reached 18% and 25% respectively. While un-irradiated membrane did not give significant weight loss. Tensile strength of membranes decreases with increasing of irradiation dose and further decreases in tensile strength is observed when irradiated membrane was followed by immersion in SBF solution. Microscope electron image of cellulose membranes shows that un-irradiated bacterial cellulose membrane consists of dense ultrafine fibril network structures, while irradiation result in cleavage of fibrils network of cellulose. The fibrils network become loosely after irradiated membrane immersed in SBF solution due to released of small molecular weight carbohydrates formed during by irradiation from the structure (author)

  15. In-vitro Degradation Behaviour of Irradiated Bacterial Cellulose Membrane

    Directory of Open Access Journals (Sweden)

    D. Darwis

    2012-08-01

    Full Text Available Bacterial cellulose membrane synthesized by Acetobacter xylinum in coconut water medium has potential application for Guided bone Regeneration. However, this membrane may not meet some application requirements due to its low biodegradation properties. In this paper, incorporation of gamma irradiation into the membrane is a developed strategy to increase its biodegradability properties. The in–vitro degradation study in synthetic body fluid (SBF of the irradiated membrane has been analyzed during periods of 6 months by means of weight loss, mechanical properties and scanning electron microscopy observation compared to that the un-irradiated one. The result showed that weight loss of irradiated membrane with 25 kGy and 50 kGy and immersed in SBF solution for 6 months reached 18% and 25% respectively. While un-irradiated membrane did not give significant weight loss. Tensile strength of membranes decreases with increasing of irradiation dose and further decreases in tensile strength is observed when irradiated membrane was followed by immersion in SBF solution. Microscope electron image of cellulose membranes shows that un-irradiated bacterial cellulose membrane consists of dense ultrafine fibril network structures, while irradiation result in cleavage of fibrils network of cellulose. The fibrils network become loosely after irradiated membrane immersed in SBF solution due to released of small molecular weight carbohydrates formed during by irradiation from the structure

  16. Optically transparent membrane based on bacterial cellulose/polycaprolactone

    Directory of Open Access Journals (Sweden)

    H. S. Barud

    2013-01-01

    Full Text Available Optically transparent membranes from bacterial cellulose (BC/polycaprolactone (PCL have been prepared by impregnation of PCL acetone solution into dried BC membranes. UV-Vis measurements showed an increase on transparency in BC/PCL membrane when compared with pristine BC. The good transparency of the BC/PCL can be related to the presence of BC nanofibers associated with deposit of PCL nano-sized spherulites which are smaller than the wavelength of visible light and practically free of light scattering. XRD results show that cellulose type I structure is preserved inside the BC/PCL membrane, while the mechanical properties suggested indicated that PCL acts as a plasticizer for the BC membrane. The novel BC/PCL membrane could be used for preparation of fully biocompatible flexible display and biodegradable food packaging.

  17. Self-supported silver nanoparticles containing bacterial cellulose membranes

    Energy Technology Data Exchange (ETDEWEB)

    Barud, Hernane S.; Barrios, Celina; Regiani, Thais; Marques, Rodrigo F.C. [Institute of Chemistry-UNESP, CP 355, Zip 14801-970, Araraquara, SP, 14801-970 (Brazil); Verelst, Marc; Dexpert-Ghys, Jeannette [Centre d' Elaboration de Materiaux et d' Etudes Structurales, CEMES, UPR No. 8011 - Universite Toulouse III, B.P. 94347, 29 rue Jeanne Marvig, 31055 Toulouse Cedex (France); Messaddeq, Younes [Institute of Chemistry-UNESP, CP 355, Zip 14801-970, Araraquara, SP, 14801-970 (Brazil); Ribeiro, Sidney J.L. [Institute of Chemistry-UNESP, CP 355, Zip 14801-970, Araraquara, SP, 14801-970 (Brazil)], E-mail: sidney@iq.unesp.br

    2008-05-01

    Hydrated bacterial cellulose (BC) membranes obtained from cultures of Acetobacter xylinum were used in the preparation of silver nanoparticles containing cellulose membranes. In situ preparation of Ag nanoparticles was achieved from the hydrolytic decomposition of silver triethanolamine (TEA) complexes. Scanning electron microscopy (SEM) images and X-ray diffraction (XRD) patterns both lead to the observation of spherical metallic silver particles with mean diameter of 8 nm well adsorbed onto the BC fibriles.

  18. Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors

    OpenAIRE

    1992-01-01

    Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-...

  19. Bacterial Outer Membrane Vesicles and Vaccine Applications

    OpenAIRE

    Acevedo, Reinaldo; Fernández, Sonsire; Zayas, Caridad; Acosta, Armando; Sarmiento, Maria Elena; Valerie A. Ferro; Rosenqvist, Einar; Campa, Concepcion; Cardoso, Daniel; Garcia, Luis; Perez, Jose Luis

    2014-01-01

    Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A...

  20. BACTERIAL OUTER MEMBRANE VESICLES AND VACCINE APPLICATIONS

    OpenAIRE

    Reinaldo eAcevedo; Sonsire eFernandez; Caridad eZayas; Armando eAcosta; Maria Elena Sarmiento; Valerie A. Ferro; Einar eRosenqvist; Concepcion eCampa; Daniel eCardoso; Luis eGarcia; Jose Luis Perez

    2014-01-01

    Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of self meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cu...

  1. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    Science.gov (United States)

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane. PMID:26621472

  2. Mechanism of bacterial membrane poration by Antimicrobial Peptides

    Science.gov (United States)

    Arora, Ankita; Mishra, Abhijit

    2015-03-01

    Bacterial resistance to conventional antibiotics is a major health concern. Antimicrobial peptides (AMPs), an important component of mammalian immune system, are thought to utilize non-specific interactions to target common features on the outer membranes of pathogens; hence development of resistance to such AMPs may be less pronounced. Most AMPs are amphiphilic and cationic in nature. Most AMPs form pores in the bacterial membranes causing them to lyse, however, the exact mechanism is unknown. Here, we study the AMP CHRG01 (KSSTRGRKSSRRKK), derived from human β defensin 3 (hBD3) with all Cysteine residues substituted with Serine. Circular Dichorism studies indicate that CHRG01 shows helicity and there is change in helicity as it interacts with the lipid membrane. The AMP was effective against different species of bacteria. Leakage of cellular components from bacterial cells observed by SEM and AFM indicates AMP action by pore formation. Confocal microscopy studies on giant vesicles incubated with AMP confirm poration. The effect of this AMP on model bacterial membranes is characterized using Small Angle X-ray scattering and Fluorescence spectroscopy to elucidate the mechanism behind antimicrobial activity.

  3. Transmembrane transport of peptidoglycan precursors across model and bacterial membranes

    NARCIS (Netherlands)

    van Dam, V.; Sijbrandi, R.; Kol, M.A.; Swiezewska, E.; de Kruijff, B.; Breukink, E.J.

    2007-01-01

    Translocation of the peptidoglycan precursor Lipid II across the cytoplasmic membrane is a key step in bacterial cell wall synthesis, but hardly understood. Using NBD-labelled Lipid II, we showed by fluorescence and TLC assays that Lipid II transport does not occur spontaneously and is not induced b

  4. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    Science.gov (United States)

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients. PMID:26907692

  5. Impacts of hydrophilic colanic acid on bacterial attachment to microfiltration membranes and subsequent membrane biofouling.

    Science.gov (United States)

    Yoshida, Keitaro; Tashiro, Yosuke; May, Thithiwat; Okabe, Satoshi

    2015-06-01

    In order to examine the interactions between physicochemical properties of specific extracellular polymeric substances (EPS) and membrane biofouling, we investigated the impacts of hydrophilic colanic acid, as a model extracellular polysaccharide component, on initial bacterial attachment to different microfiltration (MF) membranes and membrane biofouling by using Escherichia coli strains producing different amounts of colanic acid. In a newly designed microtiter plate assay, the bacterial attachment by an E. coli strain RcsF(+), which produces massive amounts of colanic acid, decreased only to a hydrophobic membrane because the colanic acid made cell surfaces more hydrophilic, resulting in low cell attachment to hydrophobic membranes. The bench-scale cross-flow filtration tests followed by filtration resistance measurement revealed that RcsF(+) caused severe irreversible membrane fouling (i.e., pore-clogging), whereas less extracellular polysaccharide-producing strains caused moderate but reversible fouling to all membranes used in this study. Further cross-flow filtration tests indicated that colanic acid liberated in the bulk phase could rapidly penetrate pre-accumulated biomass layers (i.e., biofilms) and then directly clogged membrane pores. These results indicate that colanic acid, a hydrophilic extracellular polysaccharide, and possible polysaccharides with similar characteristics with colanic acid are considered as a major cause of severe irreversible membrane fouling (i.e., pore-clogging) regardless of biofilm formation (dynamic membrane).

  6. Bacterial outer membrane vesicles and vaccine applications.

    Science.gov (United States)

    Acevedo, Reinaldo; Fernández, Sonsire; Zayas, Caridad; Acosta, Armando; Sarmiento, Maria Elena; Ferro, Valerie A; Rosenqvist, Einar; Campa, Concepcion; Cardoso, Daniel; Garcia, Luis; Perez, Jose Luis

    2014-01-01

    Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A Good Manufacturing Practice (GMP) process was developed at the Finlay Institute to produce OMV from N. meningitidis serogroup B (dOMVB) using detergent extraction. Subsequently, OMV from N. meningitidis, serogroup A (dOMVA), serogroup W (dOMVW), and serogroup X (dOMVX) were obtained using this process. More recently, the extraction process has also been applied effectively for obtaining OMV on a research scale from Vibrio cholerae (dOMVC), Bordetella pertussis (dOMVBP), Mycobacterium smegmatis (dOMVSM), and BCG (dOMVBCG). The immunogenicity of the OMV has been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice has shown their protective potential. dOMVB has been evaluated with non-neisserial antigens, including with a herpes virus type 2 glycoprotein, ovalbumin, and allergens. In conclusion, OMV are proving to be more versatile than first conceived and remain an important technology for development of vaccine candidates. PMID:24715891

  7. BACTERIAL OUTER MEMBRANE VESICLES AND VACCINE APPLICATIONS

    Directory of Open Access Journals (Sweden)

    Reinaldo eAcevedo

    2014-03-01

    Full Text Available Vaccines based on outer membrane vesicles (OMV were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of self meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A Good Manufacturing Practice (GMP process was developed at the Finlay Institute to produce OMV from N. meningitidis serogroup B (dOMVB using detergent extraction. Subsequently, OMV from N. meningitidis, serogroup A (dOMVA, serogroup W (dOMVW and serogroup X (dOMVX were obtained using this process. More recently, the extraction process has also been applied effectively for obtaining OMV on a research scale from Vibrio cholerae (dOMVC, Bordetella pertussis (dOMVBP, Mycobacterium smegmatis (dOMVSM and BCG (dOMVBCG. The immunogenicity of the OMV have been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice have shown their protective potential. dOMVB has been evaluated with non-self neisserial antigens, including with a herpes virus type 2 glycoprotein, ovalbumin and allergens. In conclusion, OMV are proving to be more versatile than first conceived and remain an important technology for development of vaccine candidates.

  8. The fifth adaptor protein complex.

    OpenAIRE

    Jennifer Hirst; Barlow, Lael D.; Gabriel Casey Francisco; Sahlender, Daniela A.; Seaman, Matthew N.J.; Dacks, Joel B.; Robinson, Margaret S.

    2011-01-01

    Adaptor protein (AP) complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent manno...

  9. The Fifth Adaptor Protein Complex

    OpenAIRE

    Hirst, Jennifer; D. Barlow, Lael; Francisco, Gabriel Casey; Sahlender, Daniela A.; Seaman, Matthew N.J.; Dacks, Joel B.; Robinson, Margaret S.

    2011-01-01

    Adaptor protein (AP) complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent manno...

  10. Electrically conductive bacterial cellulose composite membranes produced by the incorporation of graphite nanoplatelets in pristine bacterial cellulose membranes

    Directory of Open Access Journals (Sweden)

    T. Zhou

    2013-09-01

    Full Text Available Graphite nanoplatelets (GNPs were utilized to improve the electrical conductivity of pristine bacterial cellulose (BC membranes. By physical and chemical methods, flake-shaped GNPs, weaving through the surface layer of web-like cellulose nanofibrils, were indeed fixed or trapped by the adjacent nanofibrils in the BC surface network, for comparison, rod-shaped multi-walled carbon nanotubes (MWCNTs were homogeneously inserted into BC membrane through the pore structures and tunnels within the BC membrane. Strong physical and chemical interaction exists between the BC nanofibrils and the particles of GNP or MWCNT even after 15 h sonication. BC membrane with 8.7 wt% incorporated GNPs reached the maximum electrical conductivity of 4.5 S/cm, while 13.9 wt% MWCNT/BC composite membrane achieved the maximum electrical conductivity of 1.2 S/cm. Compared with one dimensional (1-D MWCNTs, as long as GNPs inserted into BC membranes, the 2-D reinforcement of GNPs was proven to be more effective in improving the electrical conductivity of BC membranes thus not only break the bottleneck of further improvement of the electrical conductivity of BC-based composite membranes but also broaden the applications of BC and GNPs.

  11. Antimicrobial Bacterial Cellulose-Silver Nanoparticles Composite Membranes

    Directory of Open Access Journals (Sweden)

    Hernane S. Barud

    2011-01-01

    Full Text Available Antimicrobial bacterial cellulose-silver nanoparticles composite membranes have been obtained by “in situ” preparation of Ag nanoparticles from hydrolytic decomposition of silver nitrate solution using triethanolamine as reducing and complexing agent. The formation of silver nanoparticles was evidenced by the X-ray diffraction, scanning electron microscopy (SEM, transmission electron microscopy (TEM, and absorption in the UV-Visible (350 nm to 600 nm. Thermal and mechanical properties together with swelling behavior for water were considered. TEA concentration was observed to be important in order to obtain only Ag particles and not a mixture of silver oxides. It was also observed to control particle size and amount of silver contents in bacterial cellulose. The composite membranes exhibited strong antimicrobial activity against Gram-negative and Gram-positive bacteria.

  12. Antimicrobial Bacterial Cellulose-Silver Nanoparticles Composite Membranes

    OpenAIRE

    Barud, Hernane S.; Thaís Regiani; Rodrigo F. C. Marques; Wilton R. Lustri; Younes Messaddeq; Ribeiro, Sidney J.L.

    2011-01-01

    Antimicrobial bacterial cellulose-silver nanoparticles composite membranes have been obtained by “in situ” preparation of Ag nanoparticles from hydrolytic decomposition of silver nitrate solution using triethanolamine as reducing and complexing agent. The formation of silver nanoparticles was evidenced by the X-ray diffraction, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and absorption in the UV-Visible (350 nm to 600 nm). Thermal and mechanical properties toge...

  13. Bacterial cellulose and bacterial cellulose-vaccarin membranes for wound healing.

    Science.gov (United States)

    Qiu, Yuyu; Qiu, Liying; Cui, Jing; Wei, Qufu

    2016-02-01

    Bacterial cellulose (BC) and bacterial cellulose-vaccarin (BC-Vac) membranes were successfully produced in large scale. BC was synthesized by Gluconacetobacter xylinum. BC-Vac membranes were prepared by immersing BC in vaccarin solution. The surface morphologies of BC and BC-Vac membranes were examined by a scanning electron microscope (SEM) and an atomic force microscopy (AFM). The images showed that BC-Vac exhibited the characteristic 3D nanofibrillar network of BC matrix but there was adhesion between fibers. The mechanical properties of BC and BC-Vac membranes were evaluated and the results indicated that the adding of drug vaccarin into the BC membranes increased the malleability indicated by the increment in elongation at break compared with BC. Fourier transform infrared spectroscopy (FTIR) analysis was conducted to confirm the incorporation of vaccarin in BC-Vac and investigate the hydroxyl interactions between BC and drug vaccarin. Cell viability and cell attachment studies demonstrated that BC and BC-Vac membranes had no cytotoxicity and could be a good carrier for cell growth. The wound healing performance was examined in vivo by rat skin models. Histological observations revealed that wounds treated with BC-Vac epithelialized and regenerated faster than treated with BC. Therefore, BC-Vac was considered as a potential candidate for wound dressing materials.

  14. Bacterial cellulose and bacterial cellulose-vaccarin membranes for wound healing.

    Science.gov (United States)

    Qiu, Yuyu; Qiu, Liying; Cui, Jing; Wei, Qufu

    2016-02-01

    Bacterial cellulose (BC) and bacterial cellulose-vaccarin (BC-Vac) membranes were successfully produced in large scale. BC was synthesized by Gluconacetobacter xylinum. BC-Vac membranes were prepared by immersing BC in vaccarin solution. The surface morphologies of BC and BC-Vac membranes were examined by a scanning electron microscope (SEM) and an atomic force microscopy (AFM). The images showed that BC-Vac exhibited the characteristic 3D nanofibrillar network of BC matrix but there was adhesion between fibers. The mechanical properties of BC and BC-Vac membranes were evaluated and the results indicated that the adding of drug vaccarin into the BC membranes increased the malleability indicated by the increment in elongation at break compared with BC. Fourier transform infrared spectroscopy (FTIR) analysis was conducted to confirm the incorporation of vaccarin in BC-Vac and investigate the hydroxyl interactions between BC and drug vaccarin. Cell viability and cell attachment studies demonstrated that BC and BC-Vac membranes had no cytotoxicity and could be a good carrier for cell growth. The wound healing performance was examined in vivo by rat skin models. Histological observations revealed that wounds treated with BC-Vac epithelialized and regenerated faster than treated with BC. Therefore, BC-Vac was considered as a potential candidate for wound dressing materials. PMID:26652377

  15. Adaptor protein complexes and intracellular transport

    OpenAIRE

    2014-01-01

    The AP (adaptor protein) complexes are heterotetrameric protein complexes that mediate intracellular membrane trafficking along endocytic and secretory transport pathways. There are five different AP complexes: AP-1, AP-2 and AP-3 are clathrin-associated complexes; whereas AP-4 and AP-5 are not. These five AP complexes localize to different intracellular compartments and mediate membrane trafficking in distinct pathways. They recognize and concentrate cargo proteins into vesicular carriers th...

  16. Transmembrane transport of peptidoglycan precursors across model and bacterial membranes.

    Science.gov (United States)

    van Dam, Vincent; Sijbrandi, Robert; Kol, Matthijs; Swiezewska, Ewa; de Kruijff, Ben; Breukink, Eefjan

    2007-05-01

    Translocation of the peptidoglycan precursor Lipid II across the cytoplasmic membrane is a key step in bacterial cell wall synthesis, but hardly understood. Using NBD-labelled Lipid II, we showed by fluorescence and TLC assays that Lipid II transport does not occur spontaneously and is not induced by the presence of single spanning helical transmembrane peptides that facilitate transbilayer movement of membrane phospholipids. MurG catalysed synthesis of Lipid II from Lipid I in lipid vesicles also did not result in membrane translocation of Lipid II. These findings demonstrate that a specialized protein machinery is needed for transmembrane movement of Lipid II. In line with this, we could demonstrate Lipid II translocation in isolated Escherichia coli inner membrane vesicles and this transport could be uncoupled from the synthesis of Lipid II at low temperatures. The transport process appeared to be independent from an energy source (ATP or proton motive force). Additionally, our studies indicate that translocation of Lipid II is coupled to transglycosylation activity on the periplasmic side of the inner membrane. PMID:17501931

  17. Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane, the Periplasm and Beyond

    OpenAIRE

    Zückert, Wolfram R.

    2014-01-01

    Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., grampositive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep pro...

  18. Molecular target of synthetic antimicrobial oligomer in bacterial membranes

    Science.gov (United States)

    Yang, Lihua; Gordon, Vernita; Som, Abhigyan; Cronan, John; Tew, Gregory; Wong, Gerard

    2008-03-01

    Antimicrobial peptides comprises a key component of innate immunity for a wide range of multicellular organisms. It has been shown that natural antimicrobial peptides and their synthetic analogs have demonstrated broad-spectrum antimicrobial activity via permeating bacterial membranes selectively. Synthetic antimicrobials with tunable structure and toxicological profiles are ideal for investigations of selectivity mechanisms. We investigate interactions and self-assembly using a prototypical family of antimicrobials based on phenylene ethynylene. Results from synchrotron small angle x-ray scattering (SAXS) results and in vitro microbicidal assays on genetically modified `knock-out' bacteria will be presented.

  19. Effects of the Membrane Action of Tetralin on the Functional and Structural Properties of Artificial and Bacterial Membranes

    NARCIS (Netherlands)

    SIKKEMA, J; POOLMAN, B; KONINGS, WN; DEBONT, JAM

    1992-01-01

    Tetralin is toxic to bacterial cells at concentrations below 100-mu-mol/liter. To assess the inhibitory action of tetralin on bacterial membranes, a membrane model system, consisting of proteoliposomes in which beef heart cytochrome c oxidase was reconstituted as the proton motive force-generating m

  20. The fifth adaptor protein complex.

    Directory of Open Access Journals (Sweden)

    Jennifer Hirst

    2011-10-01

    Full Text Available Adaptor protein (AP complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolutionarily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.

  1. Biofouling control: Bacterial quorum quenching versus chlorination in membrane bioreactors.

    Science.gov (United States)

    Weerasekara, Nuwan A; Choo, Kwang-Ho; Lee, Chung-Hak

    2016-10-15

    Biofilm formation (biofouling) induced via cell-to-cell communication (quorum sensing) causes problems in membrane filtration processes. Chorine is one of the most common chemicals used to interfere with biofouling; however, biofouling control is challenging because it is a natural process. This study demonstrates biofouling control for submerged hollow fiber membranes in membrane bioreactors by means of bacterial quorum quenching (QQ) using Rhodococcus sp. BH4 with chemically enhanced backwashing. This is the first trial to bring QQ alongside chlorine injection into practice. A high chlorine dose (100 mg/L as Cl2) to the system is insufficient for preventing biofouling, but addition of the QQ bacterium is effective for disrupting biofouling that cannot be achieved by chlorination alone. QQ reduces the biologically induced metal precipitate and extracellular biopolymer levels in the biofilm, and biofouling is significantly delayed when QQ is applied in addition to chlorine dosing. QQ with chlorine injection gives synergistic effects on reducing physically and chemically reversible fouling resistances while saving substantial filtration energy. Manipulating microbial community functions with chemical treatment is an attractive tool for biofilm dispersal in membrane bioreactors.

  2. The adaptor molecule CARD9 is essential for tuberculosis control

    OpenAIRE

    Dorhoi, Anca; Desel, Christiane; Yeremeev, Vladimir; Pradl, Lydia; Brinkmann, Volker; Mollenkopf, Hans J; Hanke, Karin; Gross, Olaf; Ruland, Jürgen; Stefan H. E. Kaufmann

    2010-01-01

    The cross talk between host and pathogen starts with recognition of bacterial signatures through pattern recognition receptors (PRRs), which mobilize downstream signaling cascades. We investigated the role of the cytosolic adaptor caspase recruitment domain family, member 9 (CARD9) in tuberculosis. This adaptor was critical for full activation of innate immunity by converging signals downstream of multiple PRRs. Card9(-/-) mice succumbed early after aerosol infection, with higher mycobacteria...

  3. Dynamic bacterial communities on reverse-osmosis membranes in a full-scale desalination plant.

    Science.gov (United States)

    Manes, C-L de O; West, N; Rapenne, S; Lebaron, P

    2011-01-01

    To better understand biofouling of seawater reverse osmosis (SWRO) membranes, bacterial diversity was characterized in the intake water, in subsequently pretreated water and on SWRO membranes from a full-scale desalination plant (FSDP) during a 9 month period. 16S rRNA gene fingerprinting and sequencing revealed that bacterial communities in the water samples and on the SWRO membranes were very different. For the different sampling dates, the bacterial diversity of the active and the total bacterial fractions of the water samples remained relatively stable over the sampling period whereas the bacterial community structure on the four SWRO membrane samples was significantly different. The richness and evenness of the SWRO membrane bacterial communities increased with usage time with an increase in the Shannon diversity index of 2.2 to 3.7. In the oldest SWRO membrane (330 days), no single operational taxonomic unit (OTU) dominated and the majority of the OTUs fell into the Alphaproteobacteria or the Planctomycetes. In striking contrast, a Betaproteobacteria OTU affiliated to the genus Ideonella was dominant and exclusively found in the membrane used for the shortest time (10 days). This suggests that bacteria belonging to this genus could be one of the primary colonizers of the SWRO membrane. Knowledge of the dominant bacterial species on SWRO membranes and their dynamics should help guide culture studies for physiological characterization of biofilm forming species. PMID:21108068

  4. Dynamic bacterial communities on reverse-osmosis membranes in a full-scale desalination plant.

    Science.gov (United States)

    Manes, C-L de O; West, N; Rapenne, S; Lebaron, P

    2011-01-01

    To better understand biofouling of seawater reverse osmosis (SWRO) membranes, bacterial diversity was characterized in the intake water, in subsequently pretreated water and on SWRO membranes from a full-scale desalination plant (FSDP) during a 9 month period. 16S rRNA gene fingerprinting and sequencing revealed that bacterial communities in the water samples and on the SWRO membranes were very different. For the different sampling dates, the bacterial diversity of the active and the total bacterial fractions of the water samples remained relatively stable over the sampling period whereas the bacterial community structure on the four SWRO membrane samples was significantly different. The richness and evenness of the SWRO membrane bacterial communities increased with usage time with an increase in the Shannon diversity index of 2.2 to 3.7. In the oldest SWRO membrane (330 days), no single operational taxonomic unit (OTU) dominated and the majority of the OTUs fell into the Alphaproteobacteria or the Planctomycetes. In striking contrast, a Betaproteobacteria OTU affiliated to the genus Ideonella was dominant and exclusively found in the membrane used for the shortest time (10 days). This suggests that bacteria belonging to this genus could be one of the primary colonizers of the SWRO membrane. Knowledge of the dominant bacterial species on SWRO membranes and their dynamics should help guide culture studies for physiological characterization of biofilm forming species.

  5. Bacteriophage therapy for membrane biofouling in membrane bioreactors and antibiotic-resistant bacterial biofilms.

    Science.gov (United States)

    Bhattacharjee, Ananda Shankar; Choi, Jeongdong; Motlagh, Amir Mohaghegh; Mukherji, Sachiyo T; Goel, Ramesh

    2015-08-01

    To demonstrate elimination of bacterial biofilm on membranes to represent wastewater treatment as well as biofilm formed by antibiotic-resistant bacterial (ARB) to signify medical application, an antibiotic-resistant bacterium and its lytic bacteriophage were isolated from a full-scale wastewater treatment plant. Based on gram staining and complete 16 S rDNA sequencing, the isolated bacterium showed a more than 99% homology with Delftia tsuruhatensis, a gram-negative bacterium belonging to β-proteobacteria. The Delftia lytic phage's draft genome revealed the phage to be an N4-like phage with 59.7% G + C content. No transfer RNAs were detected for the phage suggesting that the phage is highly adapted to its host Delftia tsuruhatensis ARB-1 with regard to codon usage, and does not require additional tRNAs of its own. The gene annotation of the Delftia lytic phage found three different components of RNA polymerase (RNAP) in the genome, which is a typical characteristic of N4-like phages. The lytic phage specific to D. tsuruhatensis ARB-1 could successfully remove the biofilm formed by it on a glass slide. The water flux through the membrane of a prototype lab-scale membrane bioreactor decreased from 47 L/h m(2) to ∼15 L/h m(2) over 4 days due to a biofilm formed by D. tsuruhatensis ARB-1. However, the flux increased to 70% of the original after the lytic phage application. Overall, this research demonstrated phage therapy's great potential to solve the problem of membrane biofouling, as well as the problems posed by pathogenic biofilms in external wounds and on medical instruments.

  6. The RA domain of Ste50 adaptor protein is required for delivery of Ste11 to the plasma membrane in the filamentous growth signaling pathway of the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Truckses, Dagmar M; Bloomekatz, Joshua E; Thorner, Jeremy

    2006-02-01

    In Saccharomyces cerevisiae, pheromone response requires Ste5 scaffold protein, which ensures efficient G-protein-dependent recruitment of mitogen-activated protein kinase (MAPK) cascade components Ste11 (MAPK kinase kinase), Ste7 (MAPK kinase), and Fus3 (MAPK) to the plasma membrane for activation by Ste20 protein kinase. Ste20, which phosphorylates Ste11 to initiate signaling, is activated by binding to Cdc42 GTPase (membrane anchored via its C-terminal geranylgeranylation). Less clear is how activated and membrane-localized Ste20 contacts Ste11 to trigger invasive growth signaling, which also requires Ste7 and the MAPK Kss1, but not Ste5. Ste50 protein associates constitutively via an N-terminal sterile-alpha motif domain with Ste11, and this interaction is required for optimal invasive growth and hyperosmotic stress (high-osmolarity glycerol [HOG]) signaling but has a lesser role in pheromone response. We show that a conserved C-terminal, so-called "Ras association" (RA) domain in Ste50 is also essential for invasive growth and HOG signaling in vivo. In vitro the Ste50 RA domain is not able to associate with Ras2, but it does associate with Cdc42 and binds to a different face than does Ste20. RA domain function can be replaced by the nine C-terminal, plasma membrane-targeting residues (KKSKKCAIL) of Cdc42, and membrane-targeted Ste50 also suppresses the signaling deficiency of cdc42 alleles specifically defective in invasive growth. Thus, Ste50 serves as an adaptor to tether Ste11 to the plasma membrane and can do so via association with Cdc42, thereby permitting the encounter of Ste11 with activated Ste20. PMID:16428446

  7. Comparative pyrosequencing analysis of bacterial community change in biofilm formed on seawater reverse osmosis membrane.

    Science.gov (United States)

    Kim, In S; Lee, Jinwook; Kima, Sung-Jo; Yu, Hye-Weon; Jang, Am

    2014-01-01

    The change in bacterial community structure induced by bacterial competition and succession was investigated during seawater reverse osmosis (SWRO) in order to elucidate a possible link between the bacterial consortium on SWRO membranes and biofouling. To date, there has been no definitive characterization of the microbial diversity in SWRO in terms of distinguishing time-dependent changes in the richness or abundance of bacterial species. For bacterial succession within biofilms on the membrane surface, SWRO using a cross-flow filtration membrane test unit was operated for 5 and 100h, respectively. As results of the pyrosequencing analysis, bacterial communities differed considerably among seawater and the 5 and 100 h samples. From a total of 33,876 pyrosequences (using a 95% sequence similarity), there were less than 1% of shared species, confirming the influence of the operational time factor and lack of similarity of these communities. During SWRO operation, the abundance of Pseudomonas stutzeri BBSPN3 (GU594474) belonging to gamma-Proteobacteria suggest that biofouling of SWRO membrane might be driven by the dominant influence of a specific species. In addition, among the bacterial competition of five bacterial species (Pseudomonas aeruginosa, Bacillus sp., Rhodobacter sp., Flavobacterium sp., and Mycobacterium sp.) competing for bacterial colonization on the SWRO membrane surfaces, it was exhibited that Bacillus sp. was the most dominant. The dominant influences ofPseudomonas sp. and Bacillus sp. on biofouling during actual SWRO is decisive depending on higher removal efficiency of the seawater pretreatment. PMID:24600849

  8. Comparative pyrosequencing analysis of bacterial community change in biofilm formed on seawater reverse osmosis membrane.

    Science.gov (United States)

    Kim, In S; Lee, Jinwook; Kima, Sung-Jo; Yu, Hye-Weon; Jang, Am

    2014-01-01

    The change in bacterial community structure induced by bacterial competition and succession was investigated during seawater reverse osmosis (SWRO) in order to elucidate a possible link between the bacterial consortium on SWRO membranes and biofouling. To date, there has been no definitive characterization of the microbial diversity in SWRO in terms of distinguishing time-dependent changes in the richness or abundance of bacterial species. For bacterial succession within biofilms on the membrane surface, SWRO using a cross-flow filtration membrane test unit was operated for 5 and 100h, respectively. As results of the pyrosequencing analysis, bacterial communities differed considerably among seawater and the 5 and 100 h samples. From a total of 33,876 pyrosequences (using a 95% sequence similarity), there were less than 1% of shared species, confirming the influence of the operational time factor and lack of similarity of these communities. During SWRO operation, the abundance of Pseudomonas stutzeri BBSPN3 (GU594474) belonging to gamma-Proteobacteria suggest that biofouling of SWRO membrane might be driven by the dominant influence of a specific species. In addition, among the bacterial competition of five bacterial species (Pseudomonas aeruginosa, Bacillus sp., Rhodobacter sp., Flavobacterium sp., and Mycobacterium sp.) competing for bacterial colonization on the SWRO membrane surfaces, it was exhibited that Bacillus sp. was the most dominant. The dominant influences ofPseudomonas sp. and Bacillus sp. on biofouling during actual SWRO is decisive depending on higher removal efficiency of the seawater pretreatment.

  9. Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF Family of Adaptor Proteins with the Raft- and the Non-Raft Brush Border Membrane Fractions of NHE3

    Directory of Open Access Journals (Sweden)

    Ayesha Sultan

    2013-11-01

    Full Text Available Background/Aims: Trafficking, brush border membrane (BBM retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50, NHERF2 (E3KARP, and NHERF3 (PDZK1 with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes (“lipid rafts” were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3- mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs.

  10. Treatment of tympanic membrane perforation using bacterial cellulose: a randomized controlled trial

    OpenAIRE

    Fábio Coelho Alves Silveira; Flávia Cristina Morone Pinto; Sílvio da Silva Caldas Neto; Mariana de Carvalho Leal; Jéssica Cesário; José Lamartine de Andrade Aguiar

    2016-01-01

    ABSTRACT INTRODUCTION: Promising treatments for tympanic membrane perforation closure have been studied. Therapies derived from tissue engineering probably eliminate the need for conventional surgery. Bacterial cellulose is presented as an alternative that is safe, biocompatible, and has low toxicity. OBJECTIVES: To investigate the effect on healing of direct application of a bacterial cellulose graft on the tympanic membrane compared to the conventional approach with autologous fascia. ME...

  11. Synthesis of antimicrobial cyclodextrins bearing polyarylamino and polyalkylamino groups via click chemistry for bacterial membrane disruption.

    Science.gov (United States)

    Yamamura, Hatsuo; Sugiyama, Yuuki; Murata, Kensuke; Yokoi, Takanori; Kurata, Ryuji; Miyagawa, Atsushi; Sakamoto, Kenji; Komagoe, Keiko; Inoue, Tsuyoshi; Katsu, Takashi

    2014-05-28

    Cyclodextrin derivatives are synthesized as membrane-disrupting agents via a microwave-assisted Huisgen reaction. Their ability to permeabilize bacterial membranes depends on the amino substituents and an appropriate balance of hydrophobicity and hydrophilicity, thus enabling the preparation of derivatives with selective toxicity against bacteria.

  12. Bacterial adhesion onto nanofiltration and reverse osmosis membranes: effect of permeate flux.

    Science.gov (United States)

    Semião, Andrea J C; Habimana, Olivier; Casey, Eoin

    2014-10-15

    The influence of permeate flux on bacterial adhesion to NF and RO membranes was examined using two model Pseudomonas species, namely Pseudomonas fluorescens and Pseudomonas putida. To better understand the initial biofouling profile during NF/RO processes, deposition experiments were conducted in cross flow under permeate flux varying from 0.5 up to 120 L/(h m(2)), using six NF and RO membranes each having different surface properties. All experiments were performed at a Reynolds number of 579. Complementary adhesion experiments were performed using Pseudomonas cells grown to early-, mid- and late-exponential growth phases to evaluate the effect of bacterial cell surface properties during cell adhesion under permeate flux conditions. Results from this study show that initial bacterial adhesion is strongly dependent on the permeate flux conditions, where increased adhesion was obtained with increased permeate flux, until a maximum of 40% coverage was reached. Membrane surface properties or bacterial growth stages was further found to have little impact on bacterial adhesion to NF and RO membrane surfaces under the conditions tested. These results emphasise the importance of conducting adhesion and biofouling experiments under realistic permeate flux conditions, and raises questions about the efficacy of the methods for the evaluation of antifouling membranes in which bacterial adhesion is commonly assessed under zero-flux or low flux conditions, unrepresentative of full-scale NF/RO processes.

  13. Linearly concatenated cyclobutane (ladderane) lipids form a dense bacterial membrane

    NARCIS (Netherlands)

    Sinninghe Damsté, J.S.; Strous, M.; Rijpstra, W.I.C.; Hopmans, E.C.; Geenevasen, J.A.J.; Duin, A.C.T. van; Niftrik, L.A.; Jetten, M.S.M.

    2002-01-01

    Lipid membranes are essential to the functioning of cells, enabling the existence of concentration gradients of ions and metabolites. Microbial membrane lipids can contain three-, five-, six- and even seven-membered aliphatic rings, but four-membered aliphatic cyclobutane rings have never been obser

  14. Sorting of bacterial lipoproteins to the outer membrane by the Lol system.

    Science.gov (United States)

    Narita, Shin-ichiro; Tokuda, Hajime

    2010-01-01

    Bacterial lipoproteins comprise a subset of membrane proteins with a lipid-modified cysteine residue at their amino termini through which they are anchored to the membrane. In Gram-negative bacteria, lipoproteins are localized on either the inner or the outer membrane. The Lol system is responsible for the transport of lipoproteins to the outer membrane.The Lol system comprises an inner-membrane ABC transporter LolCDE complex, a periplasmic carrier protein, LolA, and an outer membrane receptor protein, LolB. Lipoproteins are synthesized as precursors in the cytosol and then translocated across the inner membrane by the Sec translocon to the outer leaflet of the inner membrane, where lipoprotein precursors are processed to mature lipoproteins. The LolCDE complex then mediates the release of outer membrane-specific lipoproteins from the inner membrane while the inner membrane-specific lipoproteins possessing Asp at position 2 are not released by LolCDE because it functions as a LolCDE avoidance signal, causing the retention of these lipoproteins in the inner membrane. A water-soluble lipoprotein-LolA complex is formed as a result of the release reaction mediated by LolCDE. This complex traverses the hydrophilic periplasm to reach the outer membrane, where LolB accepts a lipoprotein from LolA and then catalyzes its incorporation into the inner leaflet of the outer membrane. PMID:20419407

  15. The effect of organic loading on bacterial community composition of membrane biofilms in a submerged polyvinyl chloride membrane bioreactor.

    Science.gov (United States)

    Xia, Siqing; Li, Jixiang; He, Shuying; Xie, Kang; Wang, Xiaojia; Zhang, Yanhao; Duan, Liang; Zhang, Zhiqiang

    2010-09-01

    The effect of organic loading on bacterial community composition of membrane biofilms was investigated using a submerged polyvinyl chloride membrane bioreactor. The low and high loadings were set at 0.33 and 0.52 gCOD/(gVSSd), respectively. The results showed that membrane fouling occurred earlier and faster under the high loading conditions. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that the similarity of bacterial community in the membrane biofilms between the two loadings was 0.67, higher than that in the mixed liquors (0.52-0.55), which indicated that some specific bacteria were selected preferentially on the membranes. Clone library analysis of the membrane biofilms indicated that Betaproteobacteria and Bacteroidetes under the high loading were 54.72% and 19.81%, respectively. Microarray results further confirmed that the two bacteria were the dominant microorganisms in the high loading biofilm. The severe membrane fouling may be aroused mainly by the enrichment of the two bacteria under the high loading.

  16. Differing prevalence and diversity of bacterial species in fetal membranes from very preterm and term labor.

    Directory of Open Access Journals (Sweden)

    Hannah E Jones

    Full Text Available BACKGROUND: Intrauterine infection may play a role in preterm delivery due to spontaneous preterm labor (PTL and preterm prolonged rupture of membranes (PPROM. Because bacteria previously associated with preterm delivery are often difficult to culture, a molecular biology approach was used to identify bacterial DNA in placenta and fetal membranes. METHODOLOGY/PRINCIPAL FINDINGS: We used broad-range 16S rDNA PCR and species-specific, real-time assays to amplify bacterial DNA from fetal membranes and placenta. 74 women were recruited to the following groups: PPROM <32 weeks (n = 26; 11 caesarean; PTL with intact membranes <32 weeks (n = 19; all vaginal birth; indicated preterm delivery <32 weeks (n = 8; all caesarean; term (n = 21; 11 caesarean. 50% (5/10 of term vaginal deliveries were positive for bacterial DNA. However, little spread was observed through tissues and species diversity was restricted. Minimal bacteria were detected in term elective section or indicated preterm deliveries. Bacterial prevalence was significantly increased in samples from PTL with intact membranes [89% (17/19 versus 50% (5/10 in term vaginal delivery p = 0.03] and PPROM (CS [55% (6/11 versus 0% (0/11 in term elective CS, p = 0.01]. In addition, bacterial spread and diversity was greater in the preterm groups with 68% (13/19 PTL group having 3 or more positive samples and over 60% (12/19 showing two or more bacterial species (versus 20% (2/10 in term vaginal deliveries. Blood monocytes from women with PTL with intact membranes and PPROM who were 16S bacterial positive showed greater level of immune paresis (p = 0.03. A positive PCR result was associated with histological chorioamnionitis in preterm deliveries. CONCLUSION/SIGNIFICANCE: Bacteria are found in both preterm and term fetal membranes. A greater spread and diversity of bacterial species were found in tissues of women who had very preterm births. It is unclear to what extent the greater bacterial prevalence

  17. Bacterial Cellulose Membranes Used as Artificial Substitutes for Dural Defection in Rabbits

    OpenAIRE

    Chen Xu; Xia Ma; Shiwen Chen; Meifeng Tao; Lutao Yuan; Yao Jing

    2014-01-01

    To improve the efficacy and safety of dural repair in neurosurgical procedures, a new dural material derived from bacterial cellulose (BC) was evaluated in a rabbit model with dural defects. We prepared artificial dura mater using bacterial cellulose which was incubated and fermented from Acetobacter xylinum. The dural defects of the rabbit model were repaired with BC membranes. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering...

  18. Controlled release and antibacterial activity of tetracycline hydrochloride-loaded bacterial cellulose composite membranes.

    Science.gov (United States)

    Shao, Wei; Liu, Hui; Wang, Shuxia; Wu, Jimin; Huang, Min; Min, Huihua; Liu, Xiufeng

    2016-07-10

    Bacterial cellulose (BC) is widely used in biomedical applications. In this study, we prepared an antibiotic drug tetracycline hydrochloride (TCH)-loaded bacterial cellulose (BC) composite membranes, and evaluated the drug release, antibacterial activity and biocompatibility. The structure and morphology of the fabricated BC-TCH composite membranes were characterized using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The TCH release results show that the incorporation of BC matrix to load TCH is able to control the release. In vitro antibacterial assay demonstrate that the developed BC-TCH composites displayed excellent antibacterial activity solely associated with the loaded TCH drug. More importantly, the BC-TCH composite membranes display good biocompatibility. These characteristics of BC-TCH composite membranes indicate that they may successfully serve as wound dressings and other medical biomaterials. PMID:27106158

  19. Structural basis for alginate secretion across the bacterial outer membrane

    Energy Technology Data Exchange (ETDEWEB)

    Whitney, J.C.; Robinson, H.; Hay, I. D.; Li, C.; Eckford, P. D. W.; Amaya, M. F.; Wood, L. F.; Ohman, D. E.; Bear, C. E.; Rehm, B. H.; Howell, P. L.

    2011-08-09

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  20. Structural Basis for Alginate Secretion Across the Bacterial Outer Membrane

    Energy Technology Data Exchange (ETDEWEB)

    J Whitney; I Hay; C Li; P Eckford; H Robinson; M Amaya; L Wood; D Ohman; C Bear; et al.

    2011-12-31

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  1. Impact of ionic liquids in aqueous solution on bacterial plasma membranes studied with molecular dynamics simulations.

    Science.gov (United States)

    Lim, Geraldine S; Zidar, Jernej; Cheong, Daniel W; Jaenicke, Stephan; Klähn, Marco

    2014-09-01

    The impact of five different imidazolium-based ionic liquids (ILs) diluted in water on the properties of a bacterial plasma membrane is investigated using molecular dynamics (MD) simulations. Cations considered are 1-octyl-3-methylimidazolium (OMIM), 1-octyloxymethyl-3-methylimidazolium (OXMIM), and 1-tetradecyl-3-methylimidazolium (TDMIM), as well as the anions chloride and lactate. The atomistic model of the membrane bilayer is designed to reproduce the lipid composition of the plasma membrane of Gram-negative Escherichia coli. Spontaneous insertion of cations into the membrane is observed in all ILs. Substantially more insertions of OMIM than of OXMIM occur and the presence of chloride reduces cation insertions compared to lactate. In contrast, anions do not adsorb onto the membrane surface nor diffuse into the bilayer. Once inserted, cations are oriented in parallel to membrane lipids with cation alkyl tails embedded into the hydrophobic membrane core, while the imidazolium-ring remains mostly exposed to the solvent. Such inserted cations are strongly associated with one to two phospholipids in the membrane. The overall order of lipids decreased after OMIM and OXMIM insertions, while on the contrary the order of lipids in the vicinity of TDMIM increased. The short alkyl tails of OMIM and OXMIM generate voids in the bilayer that are filled by curling lipids. This cation induced lipid disorder also reduces the average membrane thickness. This effect is not observed after TDMIM insertions due to the similar length of cation alkyl chain and the fatty acids of the lipids. This lipid-mimicking behavior of inserted TDMIM indicates a high membrane affinity of this cation that could lead to an enhanced accumulation of cations in the membrane over time. Overall, the simulations reveal how cations are inserted into the bacterial membrane and how such insertions change its properties. Moreover, the different roles of cations and anions are highlighted and the fundamental

  2. Transport of peptidoglycan sub-units across the bacterial cytoplasmic membrane

    NARCIS (Netherlands)

    van Dam, V.

    2009-01-01

    Transport of peptidoglycan sub-units across the bacterial cytoplasmic membrane Vincent van Dam The work described in this thesis focuses on the mechanism by which the lipid-linked cell wall precursor molecule Lipid II is transported from the inner leaflet to the outer leaflet of the cytoplasmic memb

  3. Bactericidal activity of curcumin I is associated with damaging of bacterial membrane.

    Directory of Open Access Journals (Sweden)

    Poonam Tyagi

    Full Text Available Curcumin, an important constituent of turmeric, is known for various biological activities, primarily due to its antioxidant mechanism. The present study focused on the antibacterial activity of curcumin I, a significant component of commercial curcumin, against four genera of bacteria, including those that are Gram-positive (Staphylococcus aureus and Enterococcus faecalis and Gram-negative (Escherichia coli and Pseudomonas aeruginosa. These represent prominent human pathogens, particularly in hospital settings. Our study shows the strong antibacterial potential of curcumin I against all the tested bacteria from Gram-positive as well as Gram-negative groups. The integrity of the bacterial membrane was checked using two differential permeabilization indicating fluorescent probes, namely, propidium iodide and calcein. Both the membrane permeabilization assays confirmed membrane leakage in Gram-negative and Gram-positive bacteria on exposure to curcumin I. In addition, scanning electron microscopy and fluorescence microscopy were employed to confirm the membrane damages in bacterial cells on exposure to curcumin I. The present study confirms the broad-spectrum antibacterial nature of curcumin I, and its membrane damaging property. Findings from this study could provide impetus for further research on curcumin I regarding its antibiotic potential against rapidly emerging bacterial pathogens.

  4. Structure and operation of bacterial tripartite pumps.

    Science.gov (United States)

    Hinchliffe, Philip; Symmons, Martyn F; Hughes, Colin; Koronakis, Vassilis

    2013-01-01

    In bacteria such as Pseudomonas aeruginosa and Escherichia coli, tripartite membrane machineries, or pumps, determine the efflux of small noxious molecules, such as detergents, heavy metals, and antibiotics, and the export of large proteins including toxins. They are therefore influential in bacterial survival, particularly during infections caused by multidrug-resistant pathogens. In these tripartite pumps an inner membrane transporter, typically an ATPase or proton antiporter, binds and translocates export or efflux substrates. In cooperation with a periplasmic adaptor protein it recruits and opens a TolC family cell exit duct, which is anchored in the outer membrane and projects across the periplasmic space between inner and outer membranes. Assembled tripartite pumps thus span the entire bacterial cell envelope. We review the atomic structures of each of the three pump components and discuss how these have allowed high-resolution views of tripartite pump assembly, operation, and possible inhibition. PMID:23808339

  5. Loss of elongation factor P disrupts bacterial outer membrane integrity

    DEFF Research Database (Denmark)

    Zou, S Betty; Hersch, Steven J; Roy, Hervé;

    2012-01-01

    Elongation factor P (EF-P) is posttranslationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants......, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, and detergents and enhanced resistance to the compound S-nitrosoglutathione. The susceptibility phenotypes are largely explained by the enhanced membrane permeability of the efp mutant, which exhibits...... background ameliorates the detergent, antibiotic, and osmosensitivity phenotypes and restores wild-type permeability to NPN. Our data support a role for EF-P in the translational regulation of a limited number of proteins that, when perturbed, renders the cell susceptible to stress by the adventitious...

  6. The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

    Science.gov (United States)

    Shebek, Kevin; Schantz, Allen B; Sines, Ian; Lauser, Kathleen; Velegol, Stephanie; Kumar, Manish

    2015-04-21

    A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

  7. Affinities and in-plane stress forces between glycopeptide antibiotics and biomimetic bacterial membranes

    Directory of Open Access Journals (Sweden)

    Sisi Bi

    2015-03-01

    Full Text Available Understanding the molecular basis of interactions between antibiotics affecting bacterial cell wall biosynthesis and cellular membranes is important in rational drug design of new drugs to overcome resistance. However, a precise understanding of how bacteriostatic antibiotics effect action often neglects the effect of biophysical forces involved following antibiotic-receptor binding events. We have employed a combination of a label-free binding biosensor (surface plasmon resonance, SPR and a force biosensor (in-plane stress cantilever, together with model membrane systems to study the complex interplay between glycopeptide antibiotics, their cognate ligands and different model membranes. Bacterial cell wall precursor analogue N-α-Docosanoyl-ε-acetyl-Lys-d-Alanine-d-Alanine (doc-KAA was inserted into lipid layers comprised of zwitterionic or anionic lipids then exposed to either vancomycin or the membrane-anchored glycopeptide antibiotic teicoplanin. Binding affinities and kinetics of the antibiotics to these model membranes were influenced by electrostatic interactions with the different lipid backgrounds, in addition to ligand affinities. In addition, cantilever sensors coated with model membranes showed that planar surface stress changes were induced by glycopeptide antibiotics adsorption and caused compressive surface stress generation in a ligand-dependent manner.

  8. Bacterial cellulose membrane as flexible substrate for organic light emitting devices

    Energy Technology Data Exchange (ETDEWEB)

    Legnani, C.; Vilani, C. [CeDO-Organic Device Center, Dimat-Dimat, Inmetro, Duque de Caxias, RJ (Brazil); Calil, V.L. [CeDO-Organic Device Center, Dimat-Dimat, Inmetro, Duque de Caxias, RJ (Brazil); LOEM-Molecular Optoelectronic Laboratory-Physics Department-PUC-Rio, Rio de Janeiro, RJ (Brazil); Barud, H.S. [Institute of Chemistry, Sao Paulo State University-UNESP, CP 355 Araraquara, SP (Brazil); Quirino, W.G. [CeDO-Organic Device Center, Dimat-Dimat, Inmetro, Duque de Caxias, RJ (Brazil); Achete, C.A. [CeDO-Organic Device Center, Dimat-Dimat, Inmetro, Duque de Caxias, RJ (Brazil); COPPE-Programa de Engenharia Metalurgica e de Materiais, UFRJ, Rio de Janeiro, RJ (Brazil); Ribeiro, S.J.L. [Institute of Chemistry, Sao Paulo State University-UNESP, CP 355 Araraquara, SP (Brazil); Cremona, M. [CeDO-Organic Device Center, Dimat-Dimat, Inmetro, Duque de Caxias, RJ (Brazil); LOEM-Molecular Optoelectronic Laboratory-Physics Department-PUC-Rio, Rio de Janeiro, RJ (Brazil)], E-mail: cremona@fis.puc-rio.br

    2008-12-01

    Bacterial cellulose (BC) membranes produced by gram-negative, acetic acid bacteria (Gluconacetobacter xylinus), were used as flexible substrates for the fabrication of Organic Light Emitting Diodes (OLED). In order to achieve the necessary conductive properties indium tin oxide (ITO) thin films were deposited onto the membrane at room temperature using radio frequency (r.f.) magnetron sputtering with an r.f. power of 30 W, at pressure of 8 mPa in Ar atmosphere without any subsequent thermal treatment. Visible light transmittance of about 40% was observed. Resistivity, mobility and carrier concentration of deposited ITO films were 4.90 x 10{sup -4} Ohm cm, 8.08 cm{sup 2}/V-s and - 1.5 x 10{sup 21} cm{sup -3}, respectively, comparable with commercial ITO substrates. In order to demonstrate the feasibility of devices based on BC membranes three OLEDs with different substrates were produced: a reference one with commercial ITO on glass, a second one with a SiO{sub 2} thin film interlayer between the BC membrane and the ITO layer and a third one just with ITO deposited directly on the BC membrane. The observed OLED luminance ratio was: 1; 0.5; 0.25 respectively, with 2400 cd/m{sup 2} as the value for the reference OLED. These preliminary results show clearly that the functionalized biopolymer, biodegradable, biocompatible bacterial cellulose membranes can be successfully used as substrate in flexible organic optoelectronic devices.

  9. Bacterial cellulose nanofibrous membrane as thermal stable separator for lithium-ion batteries

    Science.gov (United States)

    Jiang, Fengjing; Yin, Lei; Yu, Qingchun; Zhong, Chunyan; Zhang, Junliang

    2015-04-01

    Thermal shrinkage is a severe problem for the conventional polyolefin separators. In this work, we report the excellent performance of bacterial cellulose (BC) nanofibrous membranes as separators for lithium (Li) ion batteries. Properties of BC separator including morphology, ionic conductivity, electrochemical stability, thermal stability, mechanical strength and battery charge-discharge performance are characterized and compared to a commercial separator membrane (Celgard® 2325). Because of the unique fibrous and cross-linked three-dimensional network structure, BC separator shows excellent dimensional stability up to 180 °C, good ionic conductivity and competitive battery performance.

  10. Predominant membrane localization is an essential feature of the bacterial signal recognition particle receptor

    Directory of Open Access Journals (Sweden)

    Graumann Peter

    2009-11-01

    Full Text Available Abstract Background The signal recognition particle (SRP receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR is a heterodimeric protein complex, consisting of two unrelated GTPases. The SRβ subunit is an integral membrane protein, which tethers the SRP-interacting SRα subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SRβ subunit and consists of only the SRα homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of Escherichia coli. So far, the exact function of the soluble SR in E. coli is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting. Results In the current study we have determined the contribution of soluble FtsY to co-translational targeting in vitro and have re-analysed the localization of FtsY in vivo by fluorescence microscopy. Our data show that FtsY can bind to SRP-ribosome nascent chains (RNCs in the absence of membranes. However, these soluble FtsY-SRP-RNC complexes are not efficiently targeted to the membrane. In contrast, we observed effective targeting of SRP-RNCs to membrane-bond FtsY. These data show that soluble FtsY does not contribute significantly to cotranslational targeting in E. coli. In agreement with this observation, our in vivo analyses of FtsY localization in bacterial cells by fluorescence microscopy revealed that the vast majority of FtsY was localized to the inner membrane and that soluble FtsY constituted only a negligible species in vivo. Conclusion The exact function of the SRP receptor (SR in bacteria has so far been enigmatic. Our data show that the bacterial SR is

  11. Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond.

    Science.gov (United States)

    Zückert, Wolfram R

    2014-08-01

    Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the "+2 rule". Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

  12. Pretreatment with alum or powdered activated carbon reduces bacterial predation-associated irreversible fouling of membranes.

    Science.gov (United States)

    Kim, Eun-Ho; Dwidar, Mohammed; Kwon, Young-Nam; Mitchell, Robert J

    2014-01-01

    This study evaluated the co-application of bacterial predation by Bdellovibrio bacteriovorus and either alum coagulation or powdered activated carbon adsorption to reduce fouling caused by Escherichia coli rich feed solutions in dead-end microfiltration tests. The flux increased when the samples were predated upon or treated with 100 ppm alum or PAC, but co-treatment with alum and predation gave the best flux results. The total membrane resistance caused by the predated sample was reduced six-fold when treated with 100 ppm PAC, from 11.8 to 1.98 × 10(11) m(-1), while irreversible fouling (Rp) was 2.7-fold lower. Treatment with 100 ppm alum reduced the total resistance 14.9-fold (11.8 to 0.79 × 10(11) m(-1)) while the Rp decreased 4.25-fold. SEM imaging confirmed this, with less obvious fouling of the membrane after the combined process. This study illustrates that the combination of bacterial predation and the subsequent removal of debris using coagulation or adsorption mitigates membrane biofouling and improves membrane performance. PMID:25410737

  13. Characteristics of extracellular polymeric substances and bacterial communities in an anaerobic membrane bioreactor coupled with online ultrasound equipment.

    Science.gov (United States)

    Yu, Zhiyong; Wen, Xianghua; Xu, Meilan; Huang, Xia

    2012-08-01

    Two parallel anaerobic membrane bioreactors (MBRs), integrated with or without ultrasound equipment for online membrane fouling control (US-AnMBR, or AnMBR) were established to digest waste activated sludge (WAS). The characteristics of bound extracellular polymeric substances (EPS) and bacterial communities in the systems were investigated for further understanding of the membrane fouling mechanisms. Ultrasound was an effective method for reducing cake layer resistance. A relatively high amount of bound EPS were found in the cake layer, especially for the US-AnMBR, by responding to the external forces (i.e. cross flow and ultrasound). High-throughput pyrosequencing and denaturing gradient gel electrophoresis (DGGE) were applied to analyze the bacterial diversity. Some bacterial populations contributing to membrane fouling were identified to accumulate in the cake layer, such as Peptococcaceae, Bacteroides and Syntrophobacterales. Since the ultrasounded retentate was recirculated back to the reactor, the bacterial community in the digested sludge was affected. PMID:22621809

  14. Freestanding bacterial cellulose-graphene oxide composite membranes with high mechanical strength for selective ion permeation

    Science.gov (United States)

    Fang, Qile; Zhou, Xufeng; Deng, Wei; Zheng, Zhi; Liu, Zhaoping

    2016-01-01

    Graphene oxide (GO) based membranes have been widely applied in molecular separation based on the size exclusion effect of the nanochannels formed by stacked GO sheets. However, it’s still a challenge to prepare a freestanding GO-based membrane with high mechanical strength and structural stability which is prerequisite for separation application in aqueous solution. Here, a freestanding composite membrane based on bacterial cellulose (BC) and GO is designed and prepared. BC network provides a porous skeleton to spread GO sheets and uniformly incorporates into the GO layers, which endows the BC + GO composite membrane with well water-stability, excellent tensile strength, as well as improved toughness, guaranteeing its separation applicability in water environment. The resulting BC + GO membrane exhibits obviously discrepant permeation properties for different inorganic/organic ions with different size, and in particular, it can quickly separate ions in nano-scale from angstrom-scale. Therefore, this novel composite membrane is considered to be a promising candidate in the applications of water purification, food industry, biomedicine, and pharmaceutical and fuel separation. PMID:27615451

  15. Freestanding bacterial cellulose-graphene oxide composite membranes with high mechanical strength for selective ion permeation.

    Science.gov (United States)

    Fang, Qile; Zhou, Xufeng; Deng, Wei; Zheng, Zhi; Liu, Zhaoping

    2016-01-01

    Graphene oxide (GO) based membranes have been widely applied in molecular separation based on the size exclusion effect of the nanochannels formed by stacked GO sheets. However, it's still a challenge to prepare a freestanding GO-based membrane with high mechanical strength and structural stability which is prerequisite for separation application in aqueous solution. Here, a freestanding composite membrane based on bacterial cellulose (BC) and GO is designed and prepared. BC network provides a porous skeleton to spread GO sheets and uniformly incorporates into the GO layers, which endows the BC + GO composite membrane with well water-stability, excellent tensile strength, as well as improved toughness, guaranteeing its separation applicability in water environment. The resulting BC + GO membrane exhibits obviously discrepant permeation properties for different inorganic/organic ions with different size, and in particular, it can quickly separate ions in nano-scale from angstrom-scale. Therefore, this novel composite membrane is considered to be a promising candidate in the applications of water purification, food industry, biomedicine, and pharmaceutical and fuel separation. PMID:27615451

  16. Enriched glucose and dextrin mannitol-based media modulates fibroblast behavior on bacterial cellulose membranes.

    Science.gov (United States)

    Stumpf, Taisa R; Pértile, Renata A N; Rambo, Carlos R; Porto, Luismar M

    2013-12-01

    Bacterial cellulose (BC) produced by Gluconacetobacter hansenii is a suitable biopolymer for biomedical applications. In order to modulate the properties of BC and expand its use as substrate for tissue engineering mainly in the form of biomembranes, glucose or dextrin were added into a BC fermentation mannitol-based medium (BCGl and BCDe, respectively) under static culture conditions. SEM images showed effects on fiber density and porosity on both sides of the BC membranes. Both enriched media decreased the BET surface area, water holding capacity, and rehydration rate. Fourier transform infrared (attenuated total reflectance mode) spectroscopy (FTIR-ATR) analysis revealed no change in the chemical structure of BC. L929 fibroblast cells were seeded on all BC-based membranes and evaluated in aspects of cell adhesion, proliferation and morphology. BCG1 membranes showed the highest biological performance and hold promise for the use in tissue engineering applications.

  17. Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus.

    Science.gov (United States)

    Wu, Sheng-Chi; Li, Meng-Hsun

    2015-10-01

    In this study, a novel bioreactor for producing bacterial cellulose (BC) is proposed. Traditional BC production uses static culture conditions and produces a gelatinous membrane. The potential for using various types of bioreactor, including a stirred tank, conventional airlift, and modified airlift with a rectangular wire-mesh draft tube, in large-scale production has been investigated. The BC obtained from these bioreactors is fibrous or in pellet form. Our proposed airlift bioreactor produces a membrane-type BC from Gluconacetobacter xylinus, the water-holding capacity of which is greater than that of cellulose types produced using static cultivation methods. The Young's modulus of the product can be manipulated by varying the number of net plates in the modified airlift bioreactor. The BC membrane produced using the proposed bioreactor exhibits potential for practical application.

  18. Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus.

    Science.gov (United States)

    Wu, Sheng-Chi; Li, Meng-Hsun

    2015-10-01

    In this study, a novel bioreactor for producing bacterial cellulose (BC) is proposed. Traditional BC production uses static culture conditions and produces a gelatinous membrane. The potential for using various types of bioreactor, including a stirred tank, conventional airlift, and modified airlift with a rectangular wire-mesh draft tube, in large-scale production has been investigated. The BC obtained from these bioreactors is fibrous or in pellet form. Our proposed airlift bioreactor produces a membrane-type BC from Gluconacetobacter xylinus, the water-holding capacity of which is greater than that of cellulose types produced using static cultivation methods. The Young's modulus of the product can be manipulated by varying the number of net plates in the modified airlift bioreactor. The BC membrane produced using the proposed bioreactor exhibits potential for practical application. PMID:25823854

  19. Bacterial nanocellulose/Nafion composite membranes for low temperature polymer electrolyte fuel cells

    Science.gov (United States)

    Jiang, Gao-peng; Zhang, Jing; Qiao, Jin-li; Jiang, Yong-ming; Zarrin, Hadis; Chen, Zhongwei; Hong, Feng

    2015-01-01

    Novel nanocomposite membranes aimed for both proton-exchange membrane fuel cell (PEMFC) and direct methanol fuel cell (DMFC) are presented in this work. The membranes are based on blending bacterial nanocellulose pulp and Nafion (abbreviated as BxNy, where x and y indicates the mass ratio of bacterial cellulose to Nafion). The structure and properties of BxNy membranes are characterized by FTIR, SEM, TG, DMA and EIS, along with water uptake, swelling behavior and methanol permeability tests. It is found that the BxNy composite membranes with reinforced concrete-like structure show excellent mechanical and thermal stability regardless of annealing. The water uptake plus area and volume swelling ratios are all decreased compared to Nafion membranes. The proton conductivities of pristine and annealed B1N9 are 0.071 and 0.056 S cm-1, respectively, at 30 °C and 100% humidity. Specifically, annealed B1N1 exhibited the lowest methanol permeability of 7.21 × 10-7 cm2 s-1. Through the selectivity analysis, pristine and annealed B1N7 are selected to assemble the MEAs. The performances of annealed B1N7 in PEMFC and DMFC show the maximum power densities of 106 and 3.2 mW cm-2, respectively, which are much higher than those of pristine B1N7 at 25 °C. The performances of the pristine and annealed B1N7 reach a level as high as 21.1 and 20.4 mW cm-2 at 80 °C in DMFC, respectively.

  20. Bacterial reaction centers purified with styrene maleic acid copolymer retain native membrane functional properties and display enhanced stability

    NARCIS (Netherlands)

    Swainsbury, David J K; Scheidelaar, Stefan; Van Grondelle, Rienk; Killian, J. Antoinette; Jones, Michael R.

    2014-01-01

    Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opp

  1. Structure of a bacterial type III secretion system in contact with a host membrane in situ

    Science.gov (United States)

    Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R.; Hayward, Richard D.

    2015-12-01

    Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform-ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking `pump-action' conformational changes that underpin effector injection.

  2. The participation of outer membrane proteins in the bacterial sensitivity to nanosilver.

    Science.gov (United States)

    Kędziora, Anna; Krzyżewska, Eva; Dudek, Bartłomiej; Bugla-Płoskońska, Gabriela

    2016-01-01

    The presented study is to analyze the participation of outer membrane proteins of Gram- negative bacteria in sensitivity to silver nanomaterials. The mechanism of interaction of silver with the bacterial cell is best described in this group of microorganisms. There are several theories regarding the effectiveness of antimicrobial ions and nanosilver, and at the indicated differences in the way they work. Outer membrane proteins of Gram-negative bacteria are involved in the procurement of silver from the environment and contribute to the development mechanisms of resistance to nanometals. They are measurable parameter in the field of cell phenotypic response to the presence of Gram-negative bacteria in the environment silver nanoforms: its properties, chemical composition, content or times of action. Proteomic methods (including two dimensional electrophoresis and MALDI‑TOF MS) are therefore relevant techniques for determining the susceptibility of bacteria to silver and the changes taking place in the outer membrane under the influence: uptime/exposure and physical and chemical parameters of silver nanomaterials. Many products containing nanosilver is still in the research phase in terms of physico‑chemical characteristics and biological activity, others have been already implemented in many industries. During the very fast nanotechnology developing and introduction to the market products based on the nanosilver the bacterial answer to nanosilver is needed.

  3. Bacterial Cellulose Membranes Used as Artificial Substitutes for Dural Defection in Rabbits

    Directory of Open Access Journals (Sweden)

    Chen Xu

    2014-06-01

    Full Text Available To improve the efficacy and safety of dural repair in neurosurgical procedures, a new dural material derived from bacterial cellulose (BC was evaluated in a rabbit model with dural defects. We prepared artificial dura mater using bacterial cellulose which was incubated and fermented from Acetobacter xylinum. The dural defects of the rabbit model were repaired with BC membranes. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. All animals were humanely euthanized by intravenous injection of phenobarbitone, at each time point, after the operation. Then, the histocompatibility and inflammatory effects of BC were examined by histological examination, real-time fluorescent quantitative polymerase chain reaction (PCR and Western Blot. BC membranes evenly covered the surface of brain without adhesion. There were seldom inflammatory cells surrounding the membrane during the early postoperative period. The expression of inflammatory cytokines IL-1β, IL-6 and TNF-α as well as iNOS and COX-2 were lower in the BC group compared to the control group at 7, 14 and 21 days after implantation. BC can repair dural defects in rabbit and has a decreased inflammatory response compared to traditional materials. However, the long-term effects need to be validated in larger animals.

  4. The Motion of a Single Molecule, the Lambda-Receptor, in the Bacterial Outer Membrane

    DEFF Research Database (Denmark)

    Oddershede, Lene; Dreyer, Jakob Kisbye; Grego, Sonia;

    2002-01-01

    constant of (1.5 +/- 1.0) x 10(-9) cm(2)/s and sits in a harmonic potential as if it were tethered by an elastic spring of spring constant of ~1.0 x 10(-2) pN/nm to the bacterial membrane. The purpose of the protein motion might be to facilitate transport of maltodextrins through the outer bacterial......Using optical tweezers and single particle tracking, we have revealed the motion of a single protein, the lambda-receptor, in the outer membrane of living Escherichia coli bacteria. We genetically modified the lambda-receptor placing a biotin on an extracellular site of the receptor in vivo. The...... that allows extraction of the motion of the protein from measurements of the mobility of the bead-molecule complex; these results are equally applicable to analyze bead-protein complexes in other membrane systems. Within a domain of radius approximately 25 nm, the receptor diffuses with a diffusion...

  5. Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis.

    Science.gov (United States)

    Chung, Ben C; Zhao, Jinshi; Gillespie, Robert A; Kwon, Do-Yeon; Guan, Ziqiang; Hong, Jiyong; Zhou, Pei; Lee, Seok-Yong

    2013-08-30

    MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg(2+) within the active site, and provide a structural basis of catalysis for this class of enzyme. PMID:23990562

  6. Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

    Directory of Open Access Journals (Sweden)

    Annamari Paino

    Full Text Available Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI, was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control

  7. The diversity of membrane transporters encoded in bacterial arsenic-resistance operons

    Directory of Open Access Journals (Sweden)

    Yiren Yang

    2015-05-01

    Full Text Available Transporter-facilitated arsenite extrusion is the major pathway of arsenic resistance within bacteria. So far only two types of membrane-bound transporter proteins, ArsB and ArsY (ACR3, have been well studied, although the arsenic transporters in bacteria display considerable diversity. Utilizing accumulated genome sequence data, we searched arsenic resistance (ars operons in about 2,500 bacterial strains and located over 700 membrane-bound transporters which are encoded in these operons. Sequence analysis revealed at least five distinct transporter families, with ArsY being the most dominant, followed by ArsB, ArsP (a recently reported permease family, Major Facilitator protein Superfamily (MFS and Major Intrinsic Protein (MIP. In addition, other types of transporters encoded in the ars operons were found, but in much lower frequencies. The diversity and evolutionary relationships of these transporters with regard to arsenic resistance will be discussed.

  8. Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae

    International Nuclear Information System (INIS)

    The secretion of enterotoxin by Vibrio cholerae is punctuated by the transient entry of the toxin subunits into the periplasm. In this paper, the authors show that the subunits oligomerize into an assembled holotoxin within the periplasm prior to their secretion across the outer membrane. The rate of toxin assembly was studied by pulse-labeling cells with [35S]-methionine and then monitoring the turnover of radiolabeled subunits as they assembled within the periplasm. The subunits entered the periplasm as monomers and assembled into oligomers with a half-time of ≅ 1 min. Since assembly was a rapid event compared to the rate of toxin efflux from the periplasm, which had a half-time of ≅ 13 min, they conclude that all of the subunits that pass through the periplasm assemble before they traverse the outer membrane. The average concentration of subunit monomers and assembled holotoxin within the periplasm was calculated to be ≅ 20 and ≅ 260 μg/ml, respectively. This indicates that the periplasm is a suitably concentrated milieu where spontaneous toxin assembly can occur. These findings suggest that protein movement across bacterial outer membranes, in apparent contrast to export across other biological membranes, involves translocation of polypeptides that have already folded into tertiary and even quaternary conformations

  9. Distribution and dynamics of OXPHOS complexes in the bacterial cytoplasmic membrane.

    Science.gov (United States)

    Magalon, Axel; Alberge, François

    2016-03-01

    Oxidative phosphorylation (OXPHOS) is an essential process for most living organisms mostly sustained by protein complexes embedded in the cell membrane. In order to thrive, cells need to quickly respond to changes in the metabolic demand or in their environment. An overview of the strategies that can be employed by bacterial cells to adjust the OXPHOS outcome is provided. Regulation at the level of gene expression can only provide a means to adjust the OXPHOS outcome to long-term trends in the environment. In addition, the actual view is that bioenergetic membranes are highly compartmentalized structures. This review discusses what is known about the spatial organization of OXPHOS complexes and the timescales at which they occur. As exemplified with the commensal gut bacterium Escherichia coli, three levels of spatial organization are at play: supercomplexes, membrane microdomains and polar assemblies. This review provides a particular focus on whether dynamic spatial organization can fine-tune the OXPHOS through the definition of specialized functional membrane microdomains. Putative mechanisms responsible for spatio-temporal regulation of the OXPHOS complexes are discussed. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux. PMID:26545610

  10. Reconstitution of nanomachine driving the assembly of proteins into bacterial outer membranes

    International Nuclear Information System (INIS)

    Over 9.5 million people die each year due to infectious diseases caused by pathogens. Many species of pathogenic bacteria require nanomachines acting like a molecular pump that shuttle key disease-causing molecules (proteins) from inside bacteria cells to the outside surface, priming the bacteria for infections. How such proteins are assembled remains an important question in biology. If we can inhibit the nanomachines function in transporting specific violence factors, it would disable the disease process. Therefore it is crucial to understand how the proteins are transported through the nanomachines from the periplasm to the extracellular space. Measuring the activity of the component parts of membrane-embedded nanomachines in solution is a major technological challenge. The translocation assembly module (the TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by Quartz Crystal Microbalance with Dissipation (QCM-D) and Magnetic Contrast Neutron Reflectrometry (MCNR). We show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.

  11. Plasma membrane association of three classes of bacterial toxins is mediated by a basic-hydrophobic motif.

    Science.gov (United States)

    Geissler, Brett; Ahrens, Sebastian; Satchell, Karla J F

    2012-02-01

    Plasma membrane targeting is essential for the proper function of many bacterial toxins. A conserved fourhelical bundle membrane localization domain (4HBM) was recently identified within three diverse families of toxins: clostridial glucosylating toxins, MARTX toxins and Pasteurella multocida-like toxins. When expressed in tissue culture cells or in yeast, GFP fusions to at least one 4HBM from each toxin family show significant peripheral membrane localization but with differing profiles. Both in vivo expression and in vitro binding studies reveal that the ability of these domains to localize to the plasma membrane and bind negatively charged phospholipids requires a basic-hydrophobic motif formed by the L1 and L3 loops. The different binding capacity of each 4HBM is defined by the hydrophobicity of an exposed residue within the motif. This study establishes that bacterial effectors utilize a normal host cell mechanism to locate the plasma membrane where they can then access their intracellular targets.

  12. Bacterial glyphosate resistance conferred by overexpression of an E. coli membrane efflux transporter.

    Science.gov (United States)

    Staub, Jeffrey M; Brand, Leslie; Tran, Minhtien; Kong, Yifei; Rogers, Stephen G

    2012-04-01

    Glyphosate herbicide-resistant crop plants, introduced commercially in 1994, now represent approximately 85% of the land area devoted to transgenic crops. Herbicide resistance in commercial glyphosate-resistant crops is due to expression of a variant form of a bacterial 5-enolpyruvylshikimate-3-phosphate synthase with a significantly decreased binding affinity for glyphosate at the target site of the enzyme. As a result of widespread and recurrent glyphosate use, often as the only herbicide used for weed management, increasing numbers of weedy species have evolved resistance to glyphosate. Weed resistance is most often due to changes in herbicide translocation patterns, presumed to be through the activity of an as yet unidentified membrane transporter in plants. To provide insight into glyphosate resistance mechanisms and identify a potential glyphosate transporter, we screened Escherichia coli genomic DNA for alternate sources of glyphosate resistance genes. Our search identified a single non-target gene that, when overexpressed in E. coli and Pseudomonas, confers high-level glyphosate resistance. The gene, yhhS, encodes a predicted membrane transporter of the major facilitator superfamily involved in drug efflux. We report here that an alternative mode of glyphosate resistance in E. coli is due to reduced accumulation of glyphosate in cells that overexpress this membrane transporter and discuss the implications for potential alternative resistance mechanisms in other organisms such as plants.

  13. Enriched glucose and dextrin mannitol-based media modulates fibroblast behavior on bacterial cellulose membranes

    Energy Technology Data Exchange (ETDEWEB)

    Stumpf, Taisa R.; Pértile, Renata A.N. [Integrated Technologies Laboratory, Department of Chemical and Food Engineering (Brazil); Rambo, Carlos R., E-mail: rambo@intelab.ufsc.br [Department of Electrical Engineering, Federal University of Santa Catarina, Florianópolis 88040-900 (Brazil); Porto, Luismar M. [Integrated Technologies Laboratory, Department of Chemical and Food Engineering (Brazil)

    2013-12-01

    Bacterial cellulose (BC) produced by Gluconacetobacter hansenii is a suitable biopolymer for biomedical applications. In order to modulate the properties of BC and expand its use as substrate for tissue engineering mainly in the form of biomembranes, glucose or dextrin were added into a BC fermentation mannitol-based medium (BCGl and BCDe, respectively) under static culture conditions. SEM images showed effects on fiber density and porosity on both sides of the BC membranes. Both enriched media decreased the BET surface area, water holding capacity, and rehydration rate. Fourier transform infrared (attenuated total reflectance mode) spectroscopy (FTIR-ATR) analysis revealed no change in the chemical structure of BC. L929 fibroblast cells were seeded on all BC-based membranes and evaluated in aspects of cell adhesion, proliferation and morphology. BCG1 membranes showed the highest biological performance and hold promise for the use in tissue engineering applications. - Highlights: • Glucose and dextrin were used to modify culture media for BC production. • Microarchitecture of BC was different depending on the enriching agent. • Fibroblasts adhered on the surface of BC modified microarchitectures. • Fibroblasts adhered on glucose modified BC exhibited healthy cell morphology.

  14. The Membrane Steps of Bacterial Cell Wall Synthesis as Antibiotic Targets

    Directory of Open Access Journals (Sweden)

    Yao Liu

    2016-08-01

    Full Text Available Peptidoglycan is the major component of the cell envelope of virtually all bacteria. It has structural roles and acts as a selective sieve for molecules from the outer environment. Peptidoglycan synthesis is therefore one of the most important biogenesis pathways in bacteria and has been studied extensively over the last twenty years. The pathway starts in the cytoplasm, continues in the cytoplasmic membrane and finishes in the periplasmic space, where the precursor is polymerized into the peptidoglycan layer. A number of proteins involved in this pathway, such as the Mur enzymes and the penicillin binding proteins (PBPs, have been studied and regarded as good targets for antibiotics. The present review focuses on the membrane steps of peptidoglycan synthesis that involve two enzymes, MraY and MurG, the inhibitors of these enzymes and the inhibition mechanisms. We also discuss the challenges of targeting these two cytoplasmic membrane (associated proteins in bacterial cells and the perspectives on how to overcome the issues.

  15. Flexible conductive polypyrrole nanocomposite membranes based on bacterial cellulose with amphiphobicity.

    Science.gov (United States)

    Tang, Lian; Han, Jinlu; Jiang, Zhenlin; Chen, Shiyan; Wang, Huaping

    2015-03-01

    Flexible conductive polypyrrole nanocomposite membranes based on bacterial cellulose (BC) with amphiphobicity have been successfully prepared through in situ chemical synthesis and then infiltrated with polysiloxane solution. The results suggested that polypyrrole (PPy) nanoparticles deposited on the surface of BC formed a continuous core-shell structure by taking along the BC template. After modification with polysiloxane, the surface characteristics of the conductive BC membranes changed from highly hydrophilic to hydrophobic. The AFM images revealed that the roughness of samples after polysiloxane treatment increased along with the increase of pyrrole concentration. The contact angles (CAs) data revealed that the highest water contact angle and highest oil contact angle are 160.3° and 136.7°, respectively. The conductivity of the amphiphobic membranes with excellent flexibility reached 0.32 S/cm and demonstrated a good electromagnetic shielding effectiveness with an SE of 15 dB which could be applied in electromagnetic shielding materials with self-cleaning properties. It opened a new field of potential applications of BC materials. PMID:25498630

  16. The Membrane Steps of Bacterial Cell Wall Synthesis as Antibiotic Targets.

    Science.gov (United States)

    Liu, Yao; Breukink, Eefjan

    2016-01-01

    Peptidoglycan is the major component of the cell envelope of virtually all bacteria. It has structural roles and acts as a selective sieve for molecules from the outer environment. Peptidoglycan synthesis is therefore one of the most important biogenesis pathways in bacteria and has been studied extensively over the last twenty years. The pathway starts in the cytoplasm, continues in the cytoplasmic membrane and finishes in the periplasmic space, where the precursor is polymerized into the peptidoglycan layer. A number of proteins involved in this pathway, such as the Mur enzymes and the penicillin binding proteins (PBPs), have been studied and regarded as good targets for antibiotics. The present review focuses on the membrane steps of peptidoglycan synthesis that involve two enzymes, MraY and MurG, the inhibitors of these enzymes and the inhibition mechanisms. We also discuss the challenges of targeting these two cytoplasmic membrane (associated) proteins in bacterial cells and the perspectives on how to overcome the issues. PMID:27571111

  17. Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis.

    Directory of Open Access Journals (Sweden)

    Vera Kozjak-Pavlovic

    2009-10-01

    Full Text Available The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m. Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m loss and apoptosis, demonstrating that dissipation of DeltaPsi(m is a requirement for cell death caused by neisserial infection.

  18. Study on lipid droplet dynamics in live cells and fluidity changes in model bacterial membranes using optical microscopy techniques

    OpenAIRE

    Wong, Christine Shiang Yee

    2014-01-01

    In this thesis optical microscopy techniques are used to consider aspects of viral and bacterial infections. In part 1, the physical effects of cytomegalovirus on lipid droplet dynamics in live cells are studied; in part 2, the effects of an antimicrobial peptide on the fluidity of model bacterial membranes are studied. The optical microscopy techniques used to study the effects of murine-cytomegalovirus (mCMV) on lipid droplets in live NIH/3T3 fibroblast cells in real-time are...

  19. Response Mechanisms of Bacterial Degraders to Environmental Contaminants on the Level of Cell Walls and Cytoplasmic Membrane

    Directory of Open Access Journals (Sweden)

    Slavomíra Murínová

    2014-01-01

    Full Text Available Bacterial strains living in the environment must cope with the toxic compounds originating from humans production. Surface bacterial structures, cell wall and cytoplasmic membrane, surround each bacterial cell and create selective barriers between the cell interior and the outside world. They are a first site of contact between the cell and toxic compounds. Organic pollutants are able to penetrate into cytoplasmic membrane and affect membrane physiological functions. Bacteria had to evolve adaptation mechanisms to counteract the damage originated from toxic contaminants and to prevent their accumulation in cell. This review deals with various adaptation mechanisms of bacterial cell concerning primarily the changes in cytoplasmic membrane and cell wall. Cell adaptation maintains the membrane fluidity status and ratio between bilayer/nonbilayer phospholipids as well as the efflux of toxic compounds, protein repair mechanisms, and degradation of contaminants. Low energy consumption of cell adaptation is required to provide other physiological functions. Bacteria able to survive in toxic environment could help us to clean contaminated areas when they are used in bioremediation technologies.

  20. Bacterial histo-blood group antigens contributing to genotype-dependent removal of human noroviruses with a microfiltration membrane.

    Science.gov (United States)

    Amarasiri, Mohan; Hashiba, Satoshi; Miura, Takayuki; Nakagomi, Toyoko; Nakagomi, Osamu; Ishii, Satoshi; Okabe, Satoshi; Sano, Daisuke

    2016-05-15

    We demonstrated the genotype-dependent removal of human norovirus particles with a microfiltration (MF) membrane in the presence of bacteria bearing histo-blood group antigens (HBGAs). Three genotypes (GII.3, GII.4, and GII.6) of norovirus-like particles (NoVLPs) were mixed with three bacterial strains (Enterobacter sp. SENG-6, Escherichia coli O86:K61:B7, and Staphylococcus epidermidis), respectively, and the mixture was filtered with an MF membrane having a nominal pore size of 0.45 μm. All NoVLP genotypes were rejected by the MF membrane in the presence of Enterobacter sp. SENG-6, which excreted HBGAs as extracellular polymeric substances (EPS). This MF membrane removal of NoVLPs was not significant when EPS was removed from cells of Enterobacter sp. SENG-6. GII.6 NoVLP was not rejected with the MF membrane in the presence of E. coli O86:K61:B7, but the removal of EPS of E. coli O86:K61:B7 increased the removal efficiency due to the interaction of NoVLPs with the exposed B-antigen in lipopolysaccharide (LPS) of E. coli O86:K61:B7. No MF membrane removal of all three genotypes was observed when S. epidermidis, an HBGA-negative strain, was mixed with NoVLPs. These results demonstrate that the location of HBGAs on bacterial cells is an important factor in determining the genotype-dependent removal efficiency of norovirus particles with the MF membrane. The presence of HBGAs in mixed liquor suspended solids from a membrane bioreactor (MBR) pilot plant was confirmed by immune-transmission electron microscopy, which implies that bacterial HBGAs can contribute to the genotype-dependent removal of human noroviruses with MBR using MF membrane. PMID:27095709

  1. Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification

    OpenAIRE

    1993-01-01

    The effects of methods known to perturb endocytosis from clathrin- coated pits on the localization of clathrin and HA2 adaptors in HEp-2 carcinoma cells have been studied by immunofluorescence and ultrastructural immunogold microscopy, using internalization of transferrin as a functional assay. Potassium depletion, as well as incubation in hypertonic medium, remove membrane-associated clathrin lattices: flat clathrin lattices and coated pits from the plasma membrane, and clathrin-coated vesic...

  2. Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

    Science.gov (United States)

    Jung, Anna Lena; Stoiber, Cornelia; Herkt, Christina E; Schulz, Christine; Bertrams, Wilhelm; Schmeck, Bernd

    2016-04-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host. PMID:27105429

  3. An ethanol biosensor based on a bacterial cell-immobilized eggshell membrane

    Institute of Scientific and Technical Information of China (English)

    Guang Ming Wen; Shao Min Shuang; Chuan Dong; Martin M.F. Choi

    2012-01-01

    An ethanol biosensor was fabricated based on a Methylobacterium organophilium-immobilized eggshell membrane and an oxygen (O2) electrode.A linear response for ethanol was obtained in the range of 0.050-7.5 mmol/L with a detection limit of 0.025 mmol/L (S/N =3) and a R.S.D.of 2.1%.The response time was less than 100 s at room temperature and ambient pressure.The optimal loading of bacterial cells on the biosensor membrane is 40 mg (wet weight).The optimal working conditions for the microbial biosensor are pH 7.0 phosphate buffer (50 mmol/L) at 20-25 ℃.The interference test,operational and storage stability of the biosensor are studied in detail.Finally,the biosensor is applied to determine the ethanol contents in various alcohol samples and the results are comparable to that obtained by gas chromatographic method and the results are satisfactory.Our proposed biosensor provides a convenient,simple and reliable method to determine ethanol content in alcoholic drinks.

  4. Interaction of antimicrobial peptide Plantaricin149a and four analogs with lipid bilayers and bacterial membranes

    Directory of Open Access Journals (Sweden)

    José Luiz de Souza Lopes

    2013-12-01

    Full Text Available The amidated analog of Plantaricin149, an antimicrobial peptide from Lactobacillus plantarum NRIC 149, directly interacts with negatively charged liposomes and bacterial membranes, leading to their lysis. In this study, four Pln149-analogs were synthesized with different hydrophobic groups at their N-terminus with the goal of evaluating the effect of the modifications at this region in the peptide's antimicrobial properties. The interaction of these peptides with membrane models, surface activity, their hemolytic effect on red blood cells, and antibacterial activity against microorganisms were evaluated. The analogs presented similar action of Plantaricin149a; three of them with no hemolytic effect (< 5% until 0.5 mM, in addition to the induction of a helical element when binding to negative liposomes. The N-terminus difference between the analogs and Plantaricin149a retained the antibacterial effect on S. aureus and P. aeruginosa for all peptides (MIC50 of 19 µM and 155 µM to Plantaricin149a, respectively but resulted in a different mechanism of action against the microorganisms, that was bactericidal for Plantaricin149a and bacteriostatic for the analogs. This difference was confirmed by a reduction in leakage action for the analogs. The lytic activity of Plantaricin149a is suggested to be a result of the peptide-lipid interactions from the amphipathic helix and the hydrophobic residues at the N-terminus of the antimicrobial peptide.

  5. TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88.

    Directory of Open Access Journals (Sweden)

    Hidenori Ohnishi

    Full Text Available MyD88, a Toll/interleukin-1 receptor homology (TIR domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

  6. Effect of Structure on the Interactions between Five Natural Antimicrobial Compounds and Phospholipids of Bacterial Cell Membrane on Model Monolayers

    Directory of Open Access Journals (Sweden)

    Stella W. Nowotarska

    2014-06-01

    Full Text Available Monolayers composed of bacterial phospholipids were used as model membranes to study interactions of the naturally occurring phenolic compounds 2,5-dihydroxybenzaldehyde and 2-hydroxy-5-methoxybenzaldehyde, and the plant essential oil compounds carvacrol, cinnamaldehyde, and geraniol, previously found to be active against both Gram-positive and Gram-negative pathogenic microorganisms. The lipid monolayers consist of 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE, 1,2-dihexa- decanoyl-sn-glycero-3-phospho-(1'-rac-glycerol (DPPG, and 1,1',2,2'-tetratetradecanoyl cardiolipin (cardiolipin. Surface pressure–area (π-A and surface potential–area (Δψ-A isotherms were measured to monitor changes in the thermodynamic and physical properties of the lipid monolayers. Results of the study indicated that the five compounds modified the three lipid monolayer structures by integrating into the monolayer, forming aggregates of antimicrobial –lipid complexes, reducing the packing effectiveness of the lipids, increasing the membrane fluidity, and altering the total dipole moment in the monolayer membrane model. The interactions of the five antimicrobial compounds with bacterial phospholipids depended on both the structure of the antimicrobials and the composition of the monolayers. The observed experimental results provide insight into the mechanism of the molecular interactions between naturally-occurring antimicrobial compounds and phospholipids of the bacterial cell membrane that govern activities.

  7. Graphene oxide exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal conidia by intertwining and membrane perturbation

    Science.gov (United States)

    Chen, Juanni; Peng, Hui; Wang, Xiuping; Shao, Feng; Yuan, Zhaodong; Han, Heyou

    2014-01-01

    To understand the interaction mechanism between graphene oxide (GO) and typical phytopathogens, a particular investigation was conducted about the antimicrobial activity of GO against two bacterial pathogens (P. syringae and X. campestris pv. undulosa) and two fungal pathogens (F. graminearum and F. oxysporum). The results showed that GO had a powerful effect on the reproduction of all four pathogens (killed nearly 90% of the bacteria and repressed 80% macroconidia germination along with partial cell swelling and lysis at 500 μg mL-1). A mutual mechanism is proposed in this work that GO intertwinds the bacteria and fungal spores with a wide range of aggregated graphene oxide sheets, resulting in the local perturbation of their cell membrane and inducing the decrease of the bacterial membrane potential and the leakage of electrolytes of fungal spores. It is likely that GO interacts with the pathogens by mechanically wrapping and locally damaging the cell membrane and finally causing cell lysis, which may be one of the major toxicity actions of GO against phytopathogens. The antibacterial mode proposed in this study suggests that the GO may possess antibacterial activity against more multi-resistant bacterial and fungal phytopathogens, and provides useful information about the application of GO in resisting crop diseases.To understand the interaction mechanism between graphene oxide (GO) and typical phytopathogens, a particular investigation was conducted about the antimicrobial activity of GO against two bacterial pathogens (P. syringae and X. campestris pv. undulosa) and two fungal pathogens (F. graminearum and F. oxysporum). The results showed that GO had a powerful effect on the reproduction of all four pathogens (killed nearly 90% of the bacteria and repressed 80% macroconidia germination along with partial cell swelling and lysis at 500 μg mL-1). A mutual mechanism is proposed in this work that GO intertwinds the bacteria and fungal spores with a wide range

  8. Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles.

    Science.gov (United States)

    Alves, Nathan J; Turner, Kendrick B; Medintz, Igor L; Walper, Scott A

    2016-01-01

    Bacteria possess innate machinery to transport extracellular cargo between cells as well as package virulence factors to infect host cells by secreting outer membrane vesicles (OMVs) that contain small molecules, proteins, and genetic material. These robust proteoliposomes have evolved naturally to be resistant to degradation and provide a supportive environment to extend the activity of encapsulated cargo. In this study, we sought to exploit bacterial OMV formation to package and maintain the activity of an enzyme, phosphotriesterase (PTE), under challenging storage conditions encountered for real world applications. Here we show that OMV packaged PTE maintains activity over free PTE when subjected to elevated temperatures (>100-fold more activity after 14 days at 37 °C), iterative freeze-thaw cycles (3.4-fold post four-cycles), and lyophilization (43-fold). We also demonstrate how lyophilized OMV packaged PTE can be utilized as a cell free reagent for long term environmental remediation of pesticide/chemical warfare contaminated areas. PMID:27117743

  9. Control of tick infestations in cattle vaccinated with bacterial membranes containing surface-exposed tick protective antigens.

    Science.gov (United States)

    Almazán, Consuelo; Moreno-Cantú, Orlando; Moreno-Cid, Juan A; Galindo, Ruth C; Canales, Mario; Villar, Margarita; de la Fuente, José

    2012-01-01

    Vaccines containing the Rhipicephalus (Boophilus) microplus BM86 and BM95 antigens protect cattle against tick infestations. Tick subolesin (SUB), elongation factor 1a (EF1a) and ubiquitin (UBQ) are new candidate protective antigens for the control of cattle tick infestations. Previous studies showed that R. microplus BM95 immunogenic peptides fused to the Anaplasma marginale major surface protein (MSP) 1a N-terminal region (BM95-MSP1a) for presentation on the Escherichia coli membrane were protective against R. microplus infestations in rabbits. In this study, we extended these results by expressing SUB-MSP1a, EF1a-MSP1a and UBQ-MSP1a fusion proteins on the E. coli membrane using this system and demonstrating that bacterial membranes containing the chimeric proteins BM95-MSP1a and SUB-MSP1a were protective (>60% vaccine efficacy) against experimental R. microplus and Rhipicephalus annulatus infestations in cattle. This system provides a novel, simple and cost-effective approach for the production of tick protective antigens by surface display of antigenic protein chimera on the E. coli membrane and demonstrates the possibility of using recombinant bacterial membrane fractions in vaccine preparations to protect cattle against tick infestations. PMID:22085549

  10. Protective efficacy of bacterial membranes containing surface-exposed BM95 antigenic peptides for the control of cattle tick infestations.

    Science.gov (United States)

    Canales, Mario; Labruna, Marcelo B; Soares, João F; Prudencio, Carlos R; de la Fuente, José

    2009-12-01

    The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that the recombinant chimeric protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region for presentation on the Escherichia coli membrane was protective against R. microplus infestations in rabbits. This system provides a novel and simple approach for the production of tick protective antigens by surface display of antigenic protein chimera on live E. coli and suggests the possibility of using recombinant bacterial membrane fractions for vaccination against cattle tick infestations. PMID:19835826

  11. Bacterial membrane activity of a-peptide/b-peptoid chimeras: Influence of amino acid composition and chain length on the activity against different bacterial strains

    DEFF Research Database (Denmark)

    Hein-Kristensen, Line; Knapp, Kolja M; Franzyk, Henrik;

    2011-01-01

    acid only had a minor effect on MIC values, whereas chain length had a profound influence on activity. All chimeras were less active against Serratia marcescens (MICs above 46 μM). The chimeras were bactericidal and induced leakage of ATP from Staphylococcus aureus and S. marcescens with similar time...... of onset and reduction in the number of viable cells. EDTA pre-treatment of S. marcescens and E. coli followed by treatment with chimeras resulted in pronounced killing indicating that disintegration of the Gram-negative outer membrane eliminated innate differences in susceptibility. Chimera chain length...... of the bacterial cell envelope, and the outer membrane may act as a barrier in Gram-negative bacteria. The tolerance of S. marcescens to chimeras may be due to differences in the composition of the lipopolysaccharide layer also responsible for its resistance to polymyxin B....

  12. Amphiphilic block copolymers as flexible membrane materials generating structural and functional mimics of green bacterial antenna complexes.

    Science.gov (United States)

    Collins, A M; Timlin, J A; Anthony, S M; Montaño, G A

    2016-08-11

    We describe the ability of a short-chain amphiphilic block copolymer to self-assemble to form an artificial supramolecular light-harvesting system. Specifically, we demonstrate that the 2.5 kDa, poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD), exhibits sufficient morphological flexibility as a membrane material and enables generation of mimics of three-dimensional chlorosomes as well as supported membrane bilayers containing energy acceptors. This overall architecture replicates green bacterial light-harvesting function whereby these assemblies exhibit long-range order and three-dimensional morphology similar to native chlorosomes and are capable of energy transfer internally and to external acceptors, located in a supporting biomimetic polymer membrane. Unlike native green bacterial systems that use multiple lipids as a matrix to generate the appropriate environment for chlorosome assembly and function, the described system matrix is comprised entirely of a single polymer amphiphile. This work demonstrates the potential of short-chain amphiphilic block copolymers in generating self-assembled, bio-mimetic membrane architectures, and in doing so, generates scalable, spatial-energetic landscapes for photonic applications. Finally, the results presented provide evidence of minimal requirements to induce chlorosome-like organization and function. PMID:27481550

  13. Bacterial cellulose nanofibrillar patch as a wound healing platform of tympanic membrane perforation.

    Science.gov (United States)

    Kim, Jangho; Kim, Seung Won; Park, Subeom; Lim, Ki Taek; Seonwoo, Hoon; Kim, Yeonju; Hong, Byung Hee; Choung, Yun-Hoon; Chung, Jong Hoon

    2013-11-01

    Bacterial cellulose (BC)-based biomaterials on medical device platforms have gained significant interest for tissue-engineered scaffolds or engraftment materials in regenerative medicine. In particular, BC has an ultrafine and highly pure nanofibril network structure and can be used as an efficient wound-healing platform since cell migration into a wound site is strongly meditated by the structural properties of the extracellular matrix. Here, the fabrication of a nanofibrillar patch by using BC and its application as a new wound-healing platform for traumatic tympanic membrane (TM) perforation is reported. TM perforation is a very common clinical problem worldwide and presents as conductive hearing loss and chronic perforations. The BC nanofibrillar patch can be synthesized from Gluconacetobacter xylinus; it is found that the patch contained a network of nanofibrils and is transparent. The thickness of the BC nanofibrillar patch is found to be approximately 10.33 ± 0.58 μm, and the tensile strength and Young's modulus of the BC nanofibrillar patch are 11.85 ± 2.43 and 11.90 ± 0.48 MPa, respectively, satisfying the requirements of an ideal wound-healing platform for TM regeneration. In vitro studies involving TM cells show that TM cell proliferation and migration are stimulated under the guidance of the BC nanofibrillar patch. In vivo animal studies demonstrate that the BC nanofibrillar patch promotes the rate of TM healing as well as aids in the recovery of TM function. These data demonstrate that the BC nanofibrillar patch is a useful wound-healing platform for TM perforation.

  14. Solid-state NMR Study of the YadA Membrane-Anchor Domain in the Bacterial Outer Membrane.

    Science.gov (United States)

    Shahid, Shakeel A; Nagaraj, Madhu; Chauhan, Nandini; Franks, Trent W; Bardiaux, Benjamin; Habeck, Michael; Orwick-Rydmark, Marcella; Linke, Dirk; van Rossum, Barth-J

    2015-10-19

    MAS-NMR was used to study the structure and dynamics at ambient temperatures of the membrane-anchor domain of YadA (YadA-M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA-M protein in the E. coli lipid environment by using (13) C-(13) C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA-M are unchanged relative to those in microcrystalline YadA-M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition-state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane-embedded samples indicate greater flexibility of the ASSA region in the outer-membrane preparation at physiological temperatures. This study will pave the way towards MAS-NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments. PMID:26332158

  15. Eggshell and Bacterial Cellulose Composite Membrane as Absorbent Material in Active Packaging

    OpenAIRE

    Ummartyotin, S.; Pisitsak, P.; Pechyen, C.

    2016-01-01

    Bacterial cellulose and eggshell composite was successfully developed. Eggshell was mixed with bacterial cellulose suspension and it was casted as a composite film. CaCO3 derived from eggshell was compared with its commercial availability. It can be noted that good dispersion of eggshell particle was prepared. Eggshell particle was irregular in shape with a variation in size. It existed in bacterial cellulose network. Characterization on composite was focused on thermal and mechanical propert...

  16. The influence of environmentally friendly pesticide - Eucalyptol - alone and in combination with terpinen-4-ol - on model bacterial membranes.

    Science.gov (United States)

    Hąc-Wydro, Katarzyna; Szydło, Karolina

    2016-10-01

    Eucalyptus oil is known from antimicrobial properties and due to natural origin and low toxicity it can be applied as a green pesticide. This plant extract is a mixture of compounds among which Eucalyptol prevails strongly over the number of minor components including terpinen-4-ol. However, antimicrobial effect of total extract is stronger than the effect of its major component. The mechanism of action of essential oils involves their incorporation into membrane of pathogenic cell. To investigate the effect of eucalyptus oil components on bacterial membrane in this work the influence of Eucalyptol on model lipid systems of various compositions was studied and then compared with the effect induced by the mixture of Eucalyptol and terpinen-4-ol. The analysis of the lipid monolayer properties modified by the presence of the essential oils evidenced that Eucalyptol reduces the lipids packing and decreases membrane rigidity, however, its influence is determined both by the concentration of this terpene and by the composition of lipid film. As indicated the results of calculation of the surface excess values Eucalyptol molecules tend to adsorb at the interface, however, at higher concentration in the subphase, the molecules undergo desorption at membrane-related surface pressure. By mixing of Eucalyptol and terpinen-4-ol stronger monolayer fluidization and destabilization can be achieved as well as stronger adsorption, even at higher surface pressures, occurs. However, these effects are also determined by the composition of model system. The collected data suggest that these compounds may act by penetrating bacterial membrane. Moreover, it can be proposed that although Eucalyptol is a major component of the Eucalyptus oil the antimicrobial effect of total extract is generated also by a minor component. This may explain stronger antimicrobial effect of Eucalyptus oil as compared to sole Eucalyptol confirmed in biological studies. PMID:27472454

  17. Yeast Golgi-localized, gamma-Ear-containing, ADP-ribosylation factor-binding proteins are but adaptor protein-1 is not required for cell-free transport of membrane proteins from the trans-Golgi network to the prevacuolar compartment.

    Science.gov (United States)

    Abazeed, Mohamed E; Fuller, Robert S

    2008-11-01

    Golgi-localized, gamma-Ear-containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN-PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the beta subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Delta membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Delta mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome. PMID:18784256

  18. Yeast Golgi-localized, γ-Ear–containing, ADP-Ribosylation Factor-binding Proteins Are but Adaptor Protein-1 Is Not Required for Cell-free Transport of Membrane Proteins from the Trans-Golgi Network to the Prevacuolar Compartment

    Science.gov (United States)

    Abazeed, Mohamed E.

    2008-01-01

    Golgi-localized, γ-Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN–PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the β subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Δ membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Δ mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome. PMID:18784256

  19. Guanidino groups greatly enhance the action of antimicrobial peptidomimetics against bacterial cytoplasmic membranes

    DEFF Research Database (Denmark)

    Andreev, Konstantin; Bianchi, Christopher; Laursen, Jonas Striegler;

    2014-01-01

    monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) were used to model cytoplasmic membranes of both Gram-positive and Gram-negative bacteria, while lipopolysaccharide Kdo2-lipid A monolayers were mimicking the outer membrane of Gram-negative species. We report the results...

  20. A membrane-bound matrix-metalloproteinase from Nicotiana tabacum cv. BY-2 is induced by bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Wahner Verena

    2009-06-01

    Full Text Available Abstract Background Plant matrix metalloproteinases (MMP are conserved proteolytic enzymes found in a wide range of monocotyledonous and dicotyledonous plant species. Acting on the plant extracellular matrix, they play crucial roles in many aspects of plant physiology including growth, development and the response to stresses such as pathogen attack. Results We have identified the first tobacco MMP, designated NtMMP1, and have isolated the corresponding cDNA sequence from the tobacco suspension cell line BY-2. The overall domain structure of NtMMP1 is similar to known MMP sequences, although certain features suggest it may be constitutively active rather than dependent on proteolytic processing. The protein appears to be expressed in two forms with different molecular masses, both of which are enzymatically active as determined by casein zymography. Exchanging the catalytic domain of NtMMP1 with green fluorescent protein (GFP facilitated subcellular localization by confocal laser scanning microscopy, showing the protein is normally inserted into the plasma membrane. The NtMMP1 gene is expressed constitutively at a low level but can be induced by exposure to bacterial pathogens. Conclusion Our biochemical analysis of NtMMP1 together with bioinformatic data on the primary sequence indicate that NtMMP1 is a constitutively-active protease. Given its induction in response to bacterial pathogens and its localization in the plasma membrane, we propose a role in pathogen defense at the cell periphery.

  1. Positioning of bacterial chemoreceptors.

    Science.gov (United States)

    Jones, Christopher W; Armitage, Judith P

    2015-05-01

    For optimum growth, bacteria must adapt to their environment, and one way that many species do this is by moving towards favourable conditions. To do so requires mechanisms to both physically drive movement and provide directionality to this movement. The pathways that control this directionality comprise chemoreceptors, which, along with an adaptor protein (CheW) and kinase (CheA), form large hexagonal arrays. These arrays can be formed around transmembrane receptors, resulting in arrays embedded in the inner membrane, or they can comprise soluble receptors, forming arrays in the cytoplasm. Across bacterial species, chemoreceptor arrays (both transmembrane and soluble) are localised to a variety of positions within the cell; some species with multiple arrays demonstrate this variety within individual cells. In many cases, the positioning pattern of the arrays is linked to the need for segregation of arrays between daughter cells on division, ensuring the production of chemotactically competent progeny. Multiple mechanisms have evolved to drive this segregation, including stochastic self-assembly, cellular landmarks, and the utilisation of ParA homologues. The variety of mechanisms highlights the importance of chemotaxis to motile species.

  2. Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS.

    Science.gov (United States)

    Gubbens, Jacob; Slijper, Monique; de Kruijff, Ben; de Kroon, Anton I P M

    2008-12-01

    Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al., J. Biol. Chem. 280 (2005), 28742-8]. Here, the method was applied to the evolutionarily related mitochondrial inner membrane that was isolated from the yeast Saccharomyces cerevisiae. Surprisingly, only very few proteins were found to be dissociated by trifluoroethanol of which Lpd1p, a component of multiple protein complexes localized in the mitochondrial matrix, is the most prominent. Usage of either milder or more stringent conditions did not yield any additional proteins that were released by fluorinated alcohols. This strongly suggests that membrane protein complexes in yeast are less stable in SDS solution than their E. coli counterparts, which might be due to the overall reduced hydrophobicity of mitochondrial transmembrane proteins. PMID:18817900

  3. Process Performance and Bacterial Community Structure Under Increasing Influent Disturbances in a Membrane-Aerated Biofilm Reactor.

    Science.gov (United States)

    Tian, Hailong; Yan, Yingchun; Chen, Yuewen; Wu, Xiaolei; Li, Baoan

    2016-02-01

    The membrane-aerated biofilm reactor (MABR) is a promising municipal wastewater treatment process. In this study, two cross-flow MABRs were constructed to explore the carbon and nitrogen removal performance and bacterial succession, along with changes of influent loading shock comprising flow velocity, COD, and NH4-N concentrations. Redundancy analysis revealed that the function of high flow velocity was mainly embodied in facilitating contaminants diffusion and biosorption rather than the success of overall bacterial populations (p > 0.05). In contrast, the influent NH4-N concentration contributed most to the variance of reactor efficiency and community structure (p < 0.05). Pyrosequencing results showed that Anaerolineae, and Beta- and Alphaproteobacteria were the dominant groups in biofilms for COD and NH4-N removal. Among the identified genera, Nitrosomonas and Nitrospira were the main nitrifiers, and Hyphomicrobium, Hydrogenophaga, and Rhodobacter were the key denitrifiers. Meanwhile, principal component analysis indicated that bacterial shift in MABR was probably the combination of stochastic and deterministic processes. PMID:26528534

  4. Reinforcement of epithelial cell adhesion to basement membrane by a bacterial pathogen as a new infectious stratagem.

    Science.gov (United States)

    Kim, Minsoo; Ogawa, Michinaga; Mimuro, Hitomi; Sasakawa, Chihiro

    2010-01-01

    The intestinal epithelium undergoes a rapid turnover in addition to rapid exfoliation in response to bacterial infection, thus acting as an intrinsic defense against microbial intruders. It has long been questioned how mucosal pathogens can circumvent the intestinal defense systems. Our recent discovery of a bacterial ploy used by Shigella provided us with fresh insight. Shigella delivers OspE via the type III secretion system during multiplication within epithelial cells. This effector protein reinforces epithelial adherence to the basement membrane by interacting with integrin-linked kinase (ILK), a unique intracellular Ser/Thr kinase that links the cell-adhesion receptors, integrin, and growth factors to the actin cytoskeleton. The interaction between OspE and ILK increased formation of focal adhesions (FAs) and surface levels of b1-integrin, while suppressing phosphorylation of FAK and paxillin, thus suppressing rapid turnover of FAs, reducing cell motility and promoting cell adhesion to extracellular matrix. The impact of this OspE-ILK interplay was demonstrated both in vitro and in vivo by infecting polarized epithelial cell monolayers and guinea pig colons with Shigella possessing or lacking the ospE gene. The findings thus establish a new class of virulence-associated factors, and provide new insight into the functioning of the intestinal barrier and bacterial strategies for circumventing it. PMID:21178415

  5. 'Should I stay or should I go?' Bacterial attachment vs biofilm formation on surface-modified membranes.

    Science.gov (United States)

    Bernstein, Roy; Freger, Viatcheslav; Lee, Jin-Hyung; Kim, Yong-Guy; Lee, Jintae; Herzberg, Moshe

    2014-01-01

    A number of techniques are used for testing the anti-biofouling activity of surfaces, yet the correlation between different results is often questionable. In this report, the correlation between initial bacterial deposition (fast tests, reported previously) and biofilm growth (much slower tests) was analyzed on a pristine and a surface-modified reverse osmosis membrane ESPA-1. The membrane was modified with grafted hydrophilic polymers bearing negatively charged, positively charged and zwitter-ionic moieties. Using three different bacterial strains it was found that there was no general correlation between the initial bacterial deposition rates and biofilm growth on surfaces, the reasons being different for each modified surface. For the negatively charged surface the slowest deposition due to the charge repulsion was eventually succeeded by the largest biofilm growth, probably due to secretion of extracellular polymeric substances (EPS) that mediated a strong attachment. For the positively charged surface, short-term charge attraction by quaternary amine groups led to the fastest deposition, but could be eventually overridden by their antimicrobial activity, resulting in non-consistent results where in some cases a lower biofilm formation rate was observed. The results indicate that initial deposition rates have to be used and interpreted with great care, when used for assessing the anti-biofouling activity of surfaces. However, for a weakly interacting 'low-fouling' zwitter-ionic surface, the positive correlation between initial cell deposition and biofilm growth, especially under flow, suggests that for this type of coating initial deposition tests may be fairly indicative of anti-biofouling potential.

  6. The role of lipids in membrane insertion and translocation of bacterial proteins.

    Science.gov (United States)

    van Dalen, Annemieke; de Kruijff, Ben

    2004-11-11

    Phospholipids are essential building blocks of membranes and maintain the membrane permeability barrier of cells and organelles. They provide not only the bilayer matrix in which the functional membrane proteins reside, but they also can play direct roles in many essential cellular processes. In this review, we give an overview of the lipid involvement in protein translocation across and insertion into the Escherichia coli inner membrane. We describe the key and general roles that lipids play in these processes in conjunction with the protein components involved. We focus on the Sec-mediated insertion of leader peptidase. We describe as well the more direct roles that lipids play in insertion of the small coat proteins Pf3 and M13. Finally, we focus on the role of lipids in membrane assembly of oligomeric membrane proteins, using the potassium channel KcsA as model protein. In all cases, the anionic lipids and lipids with small headgroups play important roles in either determining the efficiency of the insertion and assembly process or contributing to the directionality of the insertion process. PMID:15546660

  7. Distinct adaptor proteins assist exit of Kre2-family proteins from the yeast ER

    Directory of Open Access Journals (Sweden)

    Yoichi Noda

    2014-07-01

    Full Text Available The Svp26 protein of S. cerevisiae is an ER- and Golgi-localized integral membrane protein with 4 potential membrane-spanning domains. It functions as an adaptor protein that facilitates the ER exit of Ktr3, a mannosyltransferase required for biosynthesis of O-linked oligosaccharides, and the ER exit of Mnn2 and Mnn5, mannosyltransferases, which participate in the biosynthesis of N-linked oligosaccharides. Ktr3 belongs to the Kre2 family, which consists of 9 members of type-II membrane proteins sharing sequence similarities. In this report, we examined all Kre2 family members and found that the Golgi localizations of two others, Kre2 and Ktr1, were dependent on Svp26 by immunofluorescence microscopy and cell fractionations in sucrose density gradients. We show that Svp26 functions in facilitating the ER exit of Kre2 and Ktr1 by an in vitro COPII budding assay. Golgi localization of Ktr4 was not dependent on Svp26. Screening null mutants of the genes encoding abundant COPII membrane proteins for those showing mislocalization of Ktr4 in the ER revealed that Erv41 and Erv46 are required for the correct Golgi localization of Ktr4. We provide biochemical evidence that the Erv41-Erv46 complex functions as an adaptor protein for ER exit of Ktr4. This is the first demonstration of the molecular function of this evolutionally conserved protein complex. The domain switching experiments show that the lumenal domain of Ktr4 is responsible for recognition by the Erv41-Erv46 complex. Thus, ER exit of Kre2-family proteins is dependent on distinct adaptor proteins and our results provide new insights into the traffic of Kre2-family mannosyltransferases.

  8. Role of adaptor proteins in motor regulation and membrane transport

    NARCIS (Netherlands)

    M.A. Schlager (Max)

    2010-01-01

    markdownabstract__Abstract__ Active transport along the cytoskeleton is a process essential for proper cellular function. Although much is known about the motor proteins that generate the necessary force and the cytoskeleton that provides the cellular infrastructure, many questions still remain. Fo

  9. Negative regulation of lymphocyte activation by the adaptor protein LAX.

    Science.gov (United States)

    Zhu, Minghua; Granillo, Olivia; Wen, Renren; Yang, Kaiyong; Dai, Xuezhi; Wang, Demin; Zhang, Weiguo

    2005-05-01

    The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.

  10. Optimization of multiplexed RADseq libraries using low-cost adaptors.

    Science.gov (United States)

    Henri, Hélène; Cariou, Marie; Terraz, Gabriel; Martinez, Sonia; El Filali, Adil; Veyssiere, Marine; Duret, Laurent; Charlat, Sylvain

    2015-04-01

    Reduced representation genomics approaches, of which RADseq is currently the most popular form, offer the possibility to produce genome wide data from potentially any species, without previous genomic information. The application of RADseq to highly multiplexed libraries (including numerous specimens, and potentially numerous different species) is however limited by technical constraints. First, the cost of synthesis of Illumina adaptors including molecular identifiers (MIDs) becomes excessive when numerous specimens are to be multiplexed. Second, the necessity to empirically adjust the ratio of adaptors to genomic DNA concentration impedes the high throughput application of RADseq to heterogeneous samples, of variable DNA concentration and quality. In an attempt to solve these problems, we propose here some adjustments regarding the adaptor synthesis. First, we show that the common and unique (MID) parts of adaptors can be synthesized separately and subsequently ligated, which drastically reduces the synthesis cost, and thus allows multiplexing hundreds of specimens. Second, we show that self-ligation of adaptors, which makes the adaptor concentration so critical, can be simply prevented by using unphosphorylated adaptors, which significantly improves the ligation and sequencing yield.

  11. Selective autophagy of non-ubiquitylated targets in plants: looking for cognate receptor/adaptor proteins

    Directory of Open Access Journals (Sweden)

    Vasko eVeljanovski

    2014-06-01

    Full Text Available Cellular homeostasis is essential for the physiology of eukaryotic cells. Eukaryotic cells, including plant cells, utilize two main pathways to adjust the level of cytoplasmic components, namely the proteasomal and the lysosomal/vacuolar pathways. Macroautophagy is a lysosomal/vacuolar pathway which, until recently, was thought to be non-specific and a bulk degradation process. However, selective autophagy which can be activated in the cell under various physiological conditions, involves the specific degradation of defined macromolecules or organelles by a conserved molecular mechanism. For this process to be efficient, the mechanisms underlying the recognition and selection of the cargo to be engulfed by the double-membrane autophagosome are critical, and not yet well understood. Ubiquitin (poly-ubiquitin conjugation to the target appears to be a conserved ligand mechanism in many types of selective autophagy, and defined receptors/adaptors recognizing and regulating the autophagosomal capture of the ubiquitylated target have been characterized. However, non-proteinaceous and non-ubiquitylated cargoes are also selectively degraded by this pathway. This ubiquitin-independent selective autophagic pathway also involves receptor and/or adaptor proteins linking the cargo to the autophagic machinery. Some of these receptor/adaptor proteins including accessory autophagy-related (Atg and non-Atg proteins have been described in yeast and animal cells but not yet in plants. In this review we discuss the ubiquitin-independent cargo selection mechanisms in selective autophagy degradation of organelles and macromolecules and speculate on potential plant receptor/adaptor proteins.

  12. Radiosensitization of hypoxic bacterial cells and animal tumours by membrane active drugs and hyperthermia

    International Nuclear Information System (INIS)

    The present report deals with the results on phenothiazine derivatives such as promethazine (PMZ), trimeprazine (TMZ), trifluoperazine (TFP) and prochlorperazine (PCP) and their comparison with that of chlorpromazine (CPZ). Their efficiency in combination with hyperthermia, radiation and other anti-cancer drugs in treating murine tumors has also been presented herein. In addition, results on bacterial cells dealing with their mechanistic aspects are also included. (author). 57 refs., 27 figures, 13 tables

  13. Comparison of ammonia-oxidizing bacterial community structure in membrane-assisted bioreactors using PCR-DGGE and FISH.

    Science.gov (United States)

    Ziembińska, A; Ciesielski, S; Gnida, A; Zabczyńki, S; Surmacz-Górska, J; Miksch, K

    2012-08-01

    The ammonia-oxidizing bacterial (AOB) communities in three membrane bioreactors (MBRs) were monitored for 2 months after an acclimation period in order to investigate the influence of sludge age and medium type on AOB changeability and its connection with nitrification effectiveness. One MBR with a sludge age of 4 days was fed with a synthetic medium, whereas the other two with sludge ages of 8 and 32 days were fed with landfill leachate. The research revealed that landfill leachate can be effectively treated in an MBR with a higher sludge age for longer periods of time and that this improvement in performance was correlated with an increase in AOB biodiversity. Interestingly, the medium type has a stronger influence on AOB biocenosis formation than the sludge age. PMID:22713978

  14. Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load.

    Science.gov (United States)

    Lo, Chien-Jung; Leake, Mark C; Pilizota, Teuta; Berry, Richard M

    2007-07-01

    Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (V(m)) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure V(m) in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated V(m) using the ratio of intracellular/extracellular dye concentration. We found V(m) = -140 +/- 14 mV in Escherichia coli at external pH 7.0 (pH(ex)), decreasing to -85 +/- 10 mV at pH(ex) 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of V(m) and intracellular sodium concentration. We were able to vary the SMF between -187 +/- 15 mV and -53 +/- 15 mV by varying pH(ex) in the range 7.0-5.0 and extracellular sodium concentration in the range 1-85 mM. Rotation rates for 0.35-microm- and 1-microm-diameter beads attached to Na(+)-driven chimeric flagellar motors varied linearly with V(m). For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.

  15. Laccase immobilization on bacterial nanocellulose membranes: antimicrobial, kinetic and stability properties

    OpenAIRE

    Sampaio, Liliana M. P.; Padrão, Jorge; Faria, Jorge; Silva, João P.; Silva, Carla J.; Dourado, Fernando; Zille, Andrea

    2016-01-01

    This work studied the physical immobilization of a commercial laccase on bacterial nanocellulose (BNC) aiming to identify the laccase antibacterial properties suitable for wound dressings. Physico-chemical analysis demonstrates that the BNC structure is manly formed by pure crystalline I cellulose. The pH optimum and activation energy of free laccase depends on the substrate employed corresponding to pH 6, 7, 3 and 57, 22, 48 kJ mol1 for 2,6-dimethylphenol (DMP), catechol and 2,2 -azino-bis-(...

  16. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  17. The membrane topology of vitamin K epoxide reductase is conserved between human isoforms and the bacterial enzyme.

    Science.gov (United States)

    Cao, Zhenbo; van Lith, Marcel; Mitchell, Lorna J; Pringle, Marie Anne; Inaba, Kenji; Bulleid, Neil J

    2016-04-01

    The membrane topology of vitamin K epoxide reductase (VKOR) is controversial with data supporting both a three transmembrane and a four transmembrane model. The positioning of the transmembrane domains and the loops between these domains is critical if we are to understand the mechanism of vitamin K oxidation and its recycling by members of the thioredoxin family of proteins and the mechanism of action of warfarin, an inhibitor of VKOR. Here we show that both mammalian VKOR isoforms adopt the same topology, with the large loop between transmembrane one and two facing the lumen of the endoplasmic reticulum (ER). We used a redox sensitive green fluorescent protein (GFP) fused to the N- or C-terminus to show that these regions face the cytosol, and introduction of glycosylation sites along with mixed disulfide formation with thioredoxin-like transmembrane protein (TMX) to demonstrate ER localization of the major loop. The topology is identical with the bacterial homologue from Synechococcussp., for which the structure and mechanism of recycling has been characterized. Our results provide a resolution to the membrane topology controversy and support previous results suggesting a role for members of the ER protein disulfide isomerase (PDI) family in recycling VKOR.

  18. The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane

    Directory of Open Access Journals (Sweden)

    Cacciotto Carla

    2010-08-01

    Full Text Available Abstract Background Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of Mycoplasma agalactiae, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition. Results The selective enrichment for M. agalactiae PG2T liposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the M. agalactiae liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all M. agalactiae PG2T genes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events. Conclusions This study led to the in-depth systematic characterization of the M. agalactiae liposoluble protein component, providing useful insights into its membrane organization.

  19. A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts

    Directory of Open Access Journals (Sweden)

    Galperin Michael Y

    2005-06-01

    highest IQ, including the current leader Wolinella succinogenes, are found among the poorly studied beta-, delta- and epsilon-proteobacteria. Among all bacterial phyla, only cyanobacteria appear to be true introverts, probably due to their capacity to conduct oxygenic photosynthesis, using a complex system of intracellular membranes. The census data, available at http://www.ncbi.nlm.nih.gov/Complete_Genomes/SignalCensus.html, can be used to get an insight into metabolic and behavioral propensities of each given organism and improve prediction of the organism's properties based solely on its genome sequence.

  20. Yeast Golgi-localized, γ-Ear–containing, ADP-Ribosylation Factor-binding Proteins Are but Adaptor Protein-1 Is Not Required for Cell-free Transport of Membrane Proteins from the Trans-Golgi Network to the Prevacuolar Compartment

    OpenAIRE

    Abazeed, Mohamed E.; Fuller, Robert S.

    2008-01-01

    Golgi-localized, γ-Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. T...

  1. Is the C-terminal insertional signal in Gram-negative bacterial outer membrane proteins species-specific or not?

    Directory of Open Access Journals (Sweden)

    Paramasivam Nagarajan

    2012-09-01

    Full Text Available Abstract Background In Gram-negative bacteria, the outer membrane is composed of an asymmetric lipid bilayer of phopspholipids and lipopolysaccharides, and the transmembrane proteins that reside in this membrane are almost exclusively β-barrel proteins. These proteins are inserted into the membrane by a highly conserved and essential machinery, the BAM complex. It recognizes its substrates, unfolded outer membrane proteins (OMPs, through a C-terminal motif that has been speculated to be species-specific, based on theoretical and experimental results from only two species, Escherichia coli and Neisseria meningitidis, where it was shown on the basis of individual sequences and motifs that OMPs from the one cannot easily be over expressed in the other, unless the C-terminal motif was adapted. In order to determine whether this species specificity is a general phenomenon, we undertook a large-scale bioinformatics study on all predicted OMPs from 437 fully sequenced proteobacterial strains. Results We were able to verify the incompatibility reported between Escherichia coli and Neisseria meningitidis, using clustering techniques based on the pairwise Hellinger distance between sequence spaces for the C-terminal motifs of individual organisms. We noticed that the amino acid position reported to be responsible for this incompatibility between Escherichia coli and Neisseria meningitidis does not play a major role for determining species specificity of OMP recognition by the BAM complex. Instead, we found that the signal is more diffuse, and that for most organism pairs, the difference between the signals is hard to detect. Notable exceptions are the Neisseriales, and Helicobacter spp. For both of these organism groups, we describe the specific sequence requirements that are at the basis of the observed difference. Conclusions Based on the finding that the differences between the recognition motifs of almost all organisms are small, we assume that

  2. The late endosomal adaptor p14 is a macrophage host-defense factor against Salmonella infection.

    Science.gov (United States)

    Taub, Nicole; Nairz, Manfred; Hilber, Diana; Hess, Michael W; Weiss, Günter; Huber, Lukas A

    2012-06-01

    The outcome of an infection depends on the balance between host resistance and bacterial virulence. Here, we show that the late endosomal adaptor p14 (also known as LAMTOR2) is one of the components for cellular host defense against the intracellular pathogen Salmonella enterica serovar Typhimurium. During Salmonella infection, the complex of p14 and MP1 is required for the accurately timed transport of Salmonella through the endolysosomal system. Loss of p14 opens a time window that allows Salmonella to populate a replication niche, in which early and late antimicrobial effector systems, comprising NADPH phagocytic oxidase and inducible nitric oxide synthase, respectively, are inappropriately activated. Thus, p14 supports the accurate transport of Salmonella through the endolysosomal system, thereby limiting bacterial replication in both, professional phagocytes and in non-phagocytic cells in vitro, and helps mice to successfully battle Salmonella infection in vivo.

  3. Laccase immobilization on bacterial nanocellulose membranes: Antimicrobial, kinetic and stability properties.

    Science.gov (United States)

    Sampaio, Liliana M P; Padrão, Jorge; Faria, Jorge; Silva, João P; Silva, Carla J; Dourado, Fernando; Zille, Andrea

    2016-07-10

    This work studied the physical immobilization of a commercial laccase on bacterial nanocellulose (BNC) aiming to identify the laccase antibacterial properties suitable for wound dressings. Physico-chemical analysis demonstrates that the BNC structure is manly formed by pure crystalline Iα cellulose. The pH optimum and activation energy of free laccase depends on the substrate employed corresponding to pH 6, 7, 3 and 57, 22, 48kJmol(-1) for 2,6-dimethylphenol (DMP), catechol and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), respectively. The Michaelis-Menten constant (Km) value for the immobilized laccase (0.77mM) was found to be almost double of that of the free enzyme (0.42mM). However, the specific activities of immobilized and free laccase are similar suggesting that the cage-like structure of BNC allows entrapped laccase to maintain some flexibility and favour substrate accessibility. The results clearly show the antimicrobial effect of laccase in Gram-positive (92%) and Gram-negative (26%) bacteria and cytotoxicity acceptable for wound dressing applications. PMID:27106145

  4. Pseudomonas aeruginosa outer membrane vesicles triggered by human mucosal fluid and lysozyme can prime host tissue surfaces for bacterial adhesion

    Directory of Open Access Journals (Sweden)

    Matteo Maria Emiliano Metruccio

    2016-06-01

    Full Text Available Pseudomonas aeruginosa is a leading cause of human morbidity and mortality that often targets epithelial surfaces. Host immunocompromise, or the presence of indwelling medical devices, including contact lenses, can predispose to infection. While medical devices are known to accumulate bacterial biofilms, it is not well understood why resistant epithelial surfaces become susceptible to P. aeruginosa. Many bacteria, including P. aeruginosa, release Outer Membrane Vesicles (OMVs in response to stress that can fuse with host cells to alter their function. Here, we tested the hypothesis that mucosal fluid can trigger OMV release to compromise an epithelial barrier. This was tested using tear fluid and corneal epithelial cells in vitro and in vivo. After 1 h both human tear fluid, and the tear component lysozyme, greatly enhanced OMV release from P. aeruginosa strain PAO1 compared to PBS controls (~100 fold. TEM and SDS-PAGE showed tear fluid and lysozyme-induced OMVs were similar in size and protein composition, but differed from biofilm-harvested OMVs, the latter smaller with fewer proteins. Lysozyme-induced OMVs were cytotoxic to human corneal epithelial cells in vitro and murine corneal epithelium in vivo. OMV exposure in vivo enhanced Ly6G/C expression at the corneal surface, suggesting myeloid cell recruitment, and primed the cornea for bacterial adhesion (~4-fold, P < 0.01. Sonication disrupted OMVs retained cytotoxic activity, but did not promote adhesion, suggesting the latter required OMV-mediated events beyond cell killing. These data suggest that mucosal fluid induced P. aeruginosa OMVs could contribute to loss of epithelial barrier function during medical device-related infections.

  5. Porous rod-like MgO complex membrane with good anti-bacterial activity directed by conjugated linolenic acid polymer

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hua-Jie, E-mail: wanghuajie972001@163.com; Chen, Meng [Henan Normal University, College of Chemistry and Chemical Engineering (China); Mi, Li-Wei, E-mail: mlwzzu@163.com [Zhongyuan University of Technology, Center for Advanced Materials Research (China); Shi, Li-Hua [Anyang 101 Education Center (China); Cao, Ying, E-mail: caoying1130@sina.com [Zhongyuan University of Technology, Center for Advanced Materials Research (China)

    2016-02-15

    The problem of infection in the tissue engineering substitutes is driving us to seek new coating materials. We previously found that conjugated linolenic acid (CLnA) has well biocompatibility and excellent membrane-forming property. The objective of this study is to endow the anti-bacterial activity to CLnA membra ne by linking with MgO. The results showed that the CLnA polymer membrane can be loaded with porous rod-like MgO and such complex membrane showed anti-bacterial sensitivity against gram-positive bacteria (Staphylococcus aureus) even at the low concentration (0.15 μg/mm{sup 2}). In the present study, the best zone of inhibition got to 18.2 ± 0.8 mm when the amount of MgO reach 2.42 ± 0.58 μg/mm{sup 2}. It was deduced that the porous rod-like structure of MgO was directed by CLnA in its polymerization process. Such CLnA/MgO complex membrane can be helpful in the tissue engineering, medicine, food engineering, food preservation, etc. on the basis of its good anti-bacterial activity.

  6. Robust properties of membrane-embedded connector channel of bacterial virus phi29 DNA packaging motor.

    Science.gov (United States)

    Jing, Peng; Haque, Farzin; Vonderheide, Anne P; Montemagno, Carlo; Guo, Peixuan

    2010-10-01

    Biological systems contain highly-ordered macromolecular structures with diverse functions, inspiring their utilization in nanotechnology. A motor allows linear dsDNA viruses to package their genome into a preformed procapsid. The central component of the motor is the portal connector that acts as a pathway for the translocation of dsDNA. The elegant design of the connector and its channel motivates its application as an artificial nanopore (Nature Nanotechnology, 4, 765-772). Herein, we demonstrate the robust characteristics of the connector of the bacteriophage phi29 DNA packaging motor by single pore electrophysiological assays. The conductance of each pore is almost identical and is perfectly linear with respect to the applied voltage. Numerous transient current blockade events induced by dsDNA are consistent with the dimensions of the channel and dsDNA. Furthermore, the connector channel is stable under a wide range of experimental conditions including high salt and pH 2-12. The robust properties of the connector nanopore made it possible to develop a simple reproducible approach for connector quantification. The precise number of connectors in each sheet of the membrane was simply derived from the slopes of the plot of voltage against current. Such quantifications led to a reliable real time counting of DNA passing through the channel. The fingerprint of DNA translocation in this system has provided a new tool for future biophysical and physicochemical characterizations of DNA transportation, motion, and packaging. PMID:20523933

  7. Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB

    OpenAIRE

    Okuda, Suguru; Tokuda, Hajime

    2009-01-01

    Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detai...

  8. Membrane fluidity as a factor in production and stability of bacterial ice nuclei active at high subfreezing temperatures.

    Science.gov (United States)

    Lindow, S E

    1995-06-01

    Detailed measurements were made of the rate of appearance of bacterial ice nuclei upon cooling of suspensions of Pseudomonas syringae cells and the disappearance of ice nuclei upon warming of the cells before assay for ice nucleation activity. While no substantial change in numbers of ice nuclei active at either -5 or at -9 degrees C was observed in cells that were grown at temperatures lower than 24 degrees C and cooled to 21 degrees C before assay, large increases in -5 but not -9 degrees C ice nuclei were observed in cells grown at temperatures greater than 24 degrees C. Ice nucleation activity of cells subjected to a decrease in temperature before assay increased immediately upon temperature shift, but 8 to 12 min was required before maximum rates of increase in numbers of ice nuclei were observed. The rate of appearance of ice nuclei in cell suspensions incubated at relatively cold temperatures prior to assay was substantially less than those incubated at temperatures approaching 24 degrees C. Cells rapidly lost ice nucleation activity when warmed to above 27 degrees C before assay; the rate of loss of ice nuclei in cells grown at a given temperature increased rapidly as the temperature to which they were warmed before assay increased. Ice nuclei disappeared most rapidly when cells grown at low temperatures were warmed before assay, suggesting that ice nucleus stability was lower in highly fluid membranes. The logarithm of the half-life of ice nuclei in cells was directly related to the concentration of the membrane fluidizing agent, 2-phenethyl alcohol, in which they were suspended. PMID:7781327

  9. Bacterial cellulose membrane produced by Acetobacter sp. A10 for burn wound dressing applications.

    Science.gov (United States)

    Kwak, Moon Hwa; Kim, Ji Eun; Go, Jun; Koh, Eun Kyoung; Song, Sung Hwa; Son, Hong Joo; Kim, Hye Sung; Yun, Young Hyun; Jung, Young Jin; Hwang, Dae Youn

    2015-05-20

    Bacteria cellulose membranes (BCM) are used for wound dressings, bone grafts, tissue engineering, artificial vessels, and dental implants because of their high tensile strength, crystallinity and water holding ability. In this study, the effects of BCM application for 15 days on healing of burn wounds were investigated based on evaluation of skin regeneration and angiogenesis in burn injury skin of Sprague-Dawley (SD) rats. BCM showed a randomly organized fibrils network, 12.13 MPa tensile strength, 12.53% strain, 17.63% crystallinity, 90.2% gel fraction and 112.14 g × m(2)/h highest water vapor transmission rate (WVTR) although their swelling ratio was enhanced to 350% within 24h. In SD rats with burned skin, the skin severity score was lower in the BCM treated group than the gauze (GZ) group at all time points, while the epidermis and dermis thickness and number of blood vessels was greater in the BCM treated group. Furthermore, a significant decrease in the number of infiltrated mast cells and in vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) expression was observed in the BCM treated group at day 10 and 15. Moreover, a significant high level in collagen expression was observed in the BCM treated group at day 5 compared with GZ treated group, while low level was detected in the same group at day 10 and 15. However, the level of metabolic enzymes representing liver and kidney toxicity in the serum of BCM treated rats was maintained at levels consistent with GZ treated rats. Overall, BCM may accelerate the process of wound healing in burn injury skin of SD rats through regulation of angiogenesis and connective tissue formation as well as not induce any specific toxicity against the liver and kidney. PMID:25817683

  10. Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation

    Science.gov (United States)

    Pulido, David; Arranz-Trullén, Javier; Prats-Ejarque, Guillem; Velázquez, Diego; Torrent, Marc; Moussaoui, Mohammed; Boix, Ester

    2016-01-01

    Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1–45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance. PMID:27089320

  11. Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation.

    Science.gov (United States)

    Pulido, David; Arranz-Trullén, Javier; Prats-Ejarque, Guillem; Velázquez, Diego; Torrent, Marc; Moussaoui, Mohammed; Boix, Ester

    2016-01-01

    Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1-45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance. PMID:27089320

  12. Comprehensive comparison of bacterial communities in a membrane-free bioelectrochemical system for removing different mononitrophenols from wastewater.

    Science.gov (United States)

    Jiang, Xinbai; Shen, Jinyou; Lou, Shuai; Mu, Yang; Wang, Ning; Han, Weiqing; Sun, Xiuyun; Li, Jiansheng; Wang, Lianjun

    2016-09-01

    Membrane-free bioelectrochemical systems (MFBESs) have been developed for the degradation of nitro-aromatic contaminants, but the microbial communities that are involved have not been comprehensively investigated. In this study, the microbial communities were evaluated and compared for treating different structures of nitrophenols (NPs), i.e., o-nitrophenol (ONP), m-nitrophenol (MNP) and p-nitrophenol (PNP), in the MFBES. The results demonstrated that NPs reduction in the MFBES decreased in efficiency in the following order: ONP>MNP>PNP. Illumina MiSeq sequencing results showed that richness and diversity of bacterial species in the anodic and cathodic communities decreased when fed different NPs. Though remarkable differences in community composition were found between anodic and cathodic biofilms in the MFBES, three core genera-Treponema, Desulfovibrio and Geobacter-were dominant in the anodic or cathodic biofilm, regardless of various NPs. Other functional genera in the anodic or cathodic biofilm were selectively enriched in the MFBES treating the three NPs with different structures. PMID:27289055

  13. Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells.

    Science.gov (United States)

    Flaherty, Rebecca A; Lee, Shaun W

    2016-08-19

    Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

  14. Negative Group Velocity and Spin-Flip in Microwave Adaptors

    CERN Document Server

    Carot, Alexander; Nimtz, Guenter

    2012-01-01

    A Fabry-Perot like interferometer with two microwaveguide adaptors as reflectors creates a passive dielectric medium with a negative group delay time due to polarization shift. A rotational strain of the polarization vector by one of the adaptors is coupled with a drastic negative group velocity. The adapted rectangular and circular waveguides have the same dispersion. The input rectangular waveguide mode is linearly polarized, whereas the basic mode of the adapted circular waveguide is circularly polarized. A 667 wavelengths long circular waveguide connects the input with the output adaptor. Experiments are performed in the frequency and in the time domain. We describe, how the helical polarization change and the spin-flip of the two different circular wave modes produce the observed negative group velocity.

  15. Optimized Adaptor Polymerase Chain Reaction Method for Efficient Genomic Walking

    Institute of Scientific and Technical Information of China (English)

    Peng XU; Rui-Ying HU; Xiao-Yan DING

    2006-01-01

    Genomic walking is one of the most useful approaches in genome-related research. Three kinds of PCR-based methods are available for this purpose. However, none of them has been generally applied because they are either insensitive or inefficient. Here we present an efficient PCR protocol, an optimized adaptor PCR method for genomic walking. Using a combination of a touchdown PCR program and a special adaptor, the optimized adaptor PCR protocol achieves high sensitivity with low background noise. By applying this protocol, the insertion sites of a gene trap mouse line and two gene promoters from the incompletely sequenced Xenopus laevis genome were successfully identified with high efficiency. The general application of this protocol in genomic walking was promising.

  16. A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters.

    Science.gov (United States)

    Ma, Cheng; Hao, Zhenyu; Huysmans, Gerard; Lesiuk, Amelia; Bullough, Per; Wang, Yingying; Bartlam, Mark; Phillips, Simon E; Young, James D; Goldman, Adrian; Baldwin, Stephen A; Postis, Vincent L G

    2015-01-01

    Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a

  17. Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae.

    Science.gov (United States)

    Unterweger, Daniel; Kostiuk, Benjamin; Ötjengerdes, Rina; Wilton, Ashley; Diaz-Satizabal, Laura; Pukatzki, Stefan

    2015-08-13

    Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.

  18. The Clathrin Adaptor AP-1A Mediates Basolateral Polarity

    OpenAIRE

    Gravotta, Diego; Carvajal-Gonzalez, Jose Maria; Mattera, Rafael; Deborde, Sylvie; Banfelder, Jason R.; Bonifacino, Juan S.; Rodriguez-Boulan, Enrique

    2012-01-01

    Clathrin and the epithelial-specific clathrin adaptor AP-1B mediate basolateral trafficking in epithelia. However, several epithelia lack AP-1B and mice knocked-out for AP-1B are viable, suggesting the existence of additional mechanisms that control basolateral polarity. Here, we demonstrate a distinct role of the ubiquitous clathrin adaptor AP-1A in basolateral protein sorting. Knock-down of AP-1A causes missorting of basolateral proteins in MDCK cells but only after knock-down of AP-1B, sug...

  19. DMPD: Adaptor usage and Toll-like receptor signaling specificity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15876435 Adaptor usage and Toll-like receptor signaling specificity. Dunne A, O'Nei...sage and Toll-like receptor signaling specificity. PubmedID 15876435 Title Adaptor usage and Toll-like receptor signaling specific

  20. A novel approach to recycle bacterial culture waste for fermentation reuse via a microbial fuel cell-membrane bioreactor system.

    Science.gov (United States)

    Li, Jian; Zhu, Yuan; Zhuang, Liangpeng; Otsuka, Yuichiro; Nakamura, Masaya; Goodell, Barry; Sonoki, Tomonori; He, Zhen

    2015-09-01

    Biochemical production processes require water and nutrient resources for culture media preparation, but aqueous waste is generated after the target products are extracted. In this study, culture waste (including cells) produced from a lab-scale fermenter was fed into a microbial fuel cell-membrane bioreactor (MFC-MBR) system. Electrical energy was generated via the interaction between the microbial consortia and the solid electrode in the MFC. The treated wastewater was reclaimed in this process which was reused as a solvent and a nutrient source in subsequent fermentation. Polarization testing showed that the MFC produced a maximum current density of 37.53 A m(-3) with a maximum power density of 5.49 W m(-3). The MFC was able to generate 0.04 kWh of energy per cubic meter of culture waste treated. The lab-scale fermenters containing pure cultures of an engineered Pseudomonas spp. were used to generate 2-pyrone-4,6-dicarboxylic acid (PDC), a high value platform chemical. When the MFC-MBR-treated wastewater was used for the fermenter culture medium, a specific bacterial growth rate of 1.00 ± 0.05 h(-1) was obtained with a PDC production rate of 708.11 ± 64.70 mg PDC L(-1) h(-1). Comparable values for controls using pure water were 0.95 ± 0.06 h(-1) and 621.01 ± 22.09 mg PDC L(-1) h(-1) (P > 0.05), respectively. The results provide insight on a new approach for more sustainable bio-material production while at the same time generating energy, and suggest that the treated wastewater can be used as a solvent and a nutrient source for the fermentation production of high value platform chemicals.

  1. Src-like-adaptor protein (SLAP) differentially regulates normal and oncogenic c-Kit signaling.

    Science.gov (United States)

    Kazi, Julhash U; Agarwal, Shruti; Sun, Jianmin; Bracco, Enrico; Rönnstrand, Lars

    2014-02-01

    The Src-like-adaptor protein (SLAP) is an adaptor protein sharing considerable structural homology with Src. SLAP is expressed in a variety of cells and regulates receptor tyrosine kinase signaling by direct association. In this report, we show that SLAP associates with both wild-type and oncogenic c-Kit (c-Kit-D816V). The association involves the SLAP SH2 domain and receptor phosphotyrosine residues different from those mediating Src interaction. Association of SLAP triggers c-Kit ubiquitylation which, in turn, is followed by receptor degradation. Although SLAP depletion potentiates c-Kit downstream signaling by stabilizing the receptor, it remains non-functional in c-Kit-D816V signaling. Ligand-stimulated c-Kit or c-Kit-D816V did not alter membrane localization of SLAP. Interestingly oncogenic c-Kit-D816V, but not wild-type c-Kit, phosphorylates SLAP on residues Y120, Y258 and Y273. Physical interaction between c-Kit-D816V and SLAP is mandatory for the phosphorylation to take place. Although tyrosine-phosphorylated SLAP does not affect c-Kit-D816V signaling, mutation of these tyrosine sites to phenylalanine can restore SLAP activity. Taken together the data demonstrate that SLAP negatively regulates wild-type c-Kit signaling, but not its oncogenic counterpart, indicating a possible mechanism by which the oncogenic c-Kit bypasses the normal cellular negative feedback control.

  2. DMPD: The SAP family of adaptors in immune regulation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15541655 The SAP family of adaptors in immune regulation. Latour S, Veillette A. Se...min Immunol. 2004 Dec;16(6):409-19. (.png) (.svg) (.html) (.csml) Show The SAP family of adaptors in immune ...regulation. PubmedID 15541655 Title The SAP family of adaptors in immune regulation. Authors Latour S, Veill

  3. A dimer of the Toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signalling adaptor proteins.

    Directory of Open Access Journals (Sweden)

    Ricardo Núñez Miguel

    Full Text Available The Toll-like receptor 4 (TLR4 is a class I transmembrane receptor expressed on the surface of immune system cells. TLR4 is activated by exposure to lipopolysaccharides derived from the outer membrane of Gram negative bacteria and forms part of the innate immune response in mammals. Like other class 1 receptors, TLR4 is activated by ligand induced dimerization, and recent studies suggest that this causes concerted conformational changes in the receptor leading to self association of the cytoplasmic Toll/Interleukin 1 receptor (TIR signalling domain. This homodimerization event is proposed to provide a new scaffold that is able to bind downstream signalling adaptor proteins. TLR4 uses two different sets of adaptors; TRAM and TRIF, and Mal and MyD88. These adaptor pairs couple two distinct signalling pathways leading to the activation of interferon response factor 3 (IRF-3 and nuclear factor kappaB (NFkappaB respectively. In this paper we have generated a structural model of the TLR4 TIR dimer and used molecular docking to probe for potential sites of interaction between the receptor homodimer and the adaptor molecules. Remarkably, both the Mal and TRAM adaptors are strongly predicted to bind at two symmetry-related sites at the homodimer interface. This model of TLR4 activation is supported by extensive functional studies involving site directed mutagenesis, inhibition by cell permeable peptides and stable protein phosphorylation of receptor and adaptor TIR domains. Our results also suggest a molecular mechanism for two recent findings, the caspase 1 dependence of Mal signalling and the protective effects conferred by the Mal polymorphism Ser180Leu.

  4. An Inner Membrane Protein (Imp) of Xanthomonas oryzae pv. oryzicola Functions in Carbon Acquisition, EPS Production, Bacterial Motility and Virulence in Rice

    Institute of Scientific and Technical Information of China (English)

    CHEN Gong-you

    2014-01-01

    Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein (Imp), showed reduced virulence in rice. To explore the potential function of this gene in virulence, a deletion mutant R∆imp was constructed in the wild-type RS105. The R∆imp mutant was signiifcantly impaired for bacterial virulence and growth in planta. The mutation in imp made the pathogen insufifciently utilize glucose, fructose, mannose or pyruvate as a sole carbon source, leading to less extracellular polysaccharide (EPS) production and reduced motility. The deifciencies noted for the mutant were restored to wild-type levels when imp was introduced in trans. Transcription of imp was signiifcantly declined when hrpG and hrpX was mutated and the expression of hrpG and hrpX was also signiifcantly declined when imp was deleted. Cell sublocalization in planta showed Imp membrane-binding feature. These results suggest that Imp is a virulence factor with roles in the catabolism of sugars, EPS production, and bacterial motility.

  5. Dual layer hollow fiber PVDF ultra-filtration membranes containing Ag nano-particle loaded zeolite with longer term anti-bacterial capacity in salt water.

    Science.gov (United States)

    Shi, Huyan; Xue, Lixin; Gao, Ailin; Zhou, Qingbo

    2016-01-01

    Dual layer polyvinylidene fluoride (PVDF), antibacterial, hollow fiber, ultra-filtration composite membranes with antibacterial particles (silver (Ag) nano-particles loaded zeolite (Z-Ag)) in the outer layer were prepared with high water flux and desired pore sizes. The amounts of Ag(+) released from the composite membranes, freshly made and stored in water and salt solution, were measured. The result indicated that dual layer PVDF antibacterial hollow fiber containing Z-Ag (M-1-Ag) still possessed the ability of continuous release of Ag(+) even after exposure to water with high ionic content, showing a longer term resistance to bacterial adhesion and antibacterial activity than membrane doped with Z-Ag(+) (M-1). Results from an anti-adhesion and bacteria killing test with Escherichia coli supported that the antibacterial efficiency of dual hollow fiber PVDF membranes with Z-Ag was much higher than those with Z-Ag(+) after long time storage in water or exposure to phosphate buffered saline (PBS) solution. This novel hollow fiber membrane may find applications in constructing sea water pretreatment devices with long term antifouling capability for the desalination processes.

  6. Spiral biasing adaptor for use in Si drift detectors and Si drift detector arrays

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zheng; Chen, Wei

    2016-07-05

    A drift detector array, preferably a silicon drift detector (SDD) array, that uses a low current biasing adaptor is disclosed. The biasing adaptor is customizable for any desired geometry of the drift detector single cell with minimum drift time of carriers. The biasing adaptor has spiral shaped ion-implants that generate the desired voltage profile. The biasing adaptor can be processed on the same wafer as the drift detector array and only one biasing adaptor chip/side is needed for one drift detector array to generate the voltage profiles on the front side and back side of the detector array.

  7. Contamination of sequence databases with adaptor sequences

    Energy Technology Data Exchange (ETDEWEB)

    Yoshikawa, Takeo; Sanders, A.R.; Detera-Wadleigh, S.D. [National Institute of Mental Health, Bethesda, MD (United States)

    1997-02-01

    Because of the exponential increase in the amount of DNA sequences being added to the public databases on a daily basis, it has become imperative to identify sources of contamination rapidly. Previously, contaminations of sequence databases have been reported to alert the scientific community to the problem. These contaminations can be divided into two categories. The first category comprises host sequences that have been difficult for submitters to manage or control. Examples include anomalous sequences derived from Escherichia coli, which are inserted into the chromosomes (and plasmids) of the bacterial hosts. Insertion sequences are highly mobile and are capable of transposing themselves into plasmids during cloning manipulation. Another example of the first category is the infection with yeast genomic DNA or with bacterial DNA of some commercially available cDNA libraries from Clontech. The second category of database contamination is due to the inadvertent inclusion of nonhost sequences. This category includes incorporation of cloning-vector sequences and multicloning sites in the database submission. M13-derived artifacts have been common, since M13-based vectors have been widely used for subcloning DNA fragments. Recognizing this problem, the National Center for Biotechnology Information (NCBI) started to screen, in April 1994, all sequences directly submitted to GenBank, against a set of vector data retrieved from GenBank by use of key-word searches, such as {open_quotes}vector.{close_quotes} In this report, we present evidence for another sequence artifact that is widespread but that, to our knowledge, has not yet been reported. 11 refs., 1 tab.

  8. A role for adaptor protein-3 complex in the organization of the endocytic pathway in Dictyostelium.

    Science.gov (United States)

    Charette, Steve J; Mercanti, Valentina; Letourneur, François; Bennett, Nelly; Cosson, Pierre

    2006-11-01

    Dictyostelium discoideum cells continuously internalize extracellular material, which accumulates in well-characterized endocytic vacuoles. In this study, we describe a new endocytic compartment identified by the presence of a specific marker, the p25 protein. This compartment presents features reminiscent of mammalian recycling endosomes: it is localized in the pericentrosomal region but distinct from the Golgi apparatus. It specifically contains surface proteins that are continuously endocytosed but rapidly recycled to the cell surface and thus absent from maturing endocytic compartments. We evaluated the importance of each clathrin-associated adaptor complex in establishing a compartmentalized endocytic system by studying the phenotype of the corresponding mutants. In knockout cells for mu3, a subunit of the AP-3 clathrin-associated complex, membrane proteins normally restricted to p25-positive endosomes were mislocalized to late endocytic compartments. Our results suggest that AP-3 plays an essential role in the compartmentalization of the endocytic pathway in Dictyostelium. PMID:17010123

  9. Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rynditch A. V.

    2012-12-01

    Full Text Available Intersectin 1 (ITSN1 is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Methods. During this study we employed immunoprecipitation and confocal microscopy. Results. We have shown that endogenous ITSN1-S co-precipitates with overexpressed ITSN1-L in PC12, 293 and 293T cells. Long and short isoforms of ITSN1 also co-localize in 293T cells. Conclusions. ITSN1-L and ITSN1-S form complexes in mammalian cells.

  10. Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

    Science.gov (United States)

    Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

    2015-11-01

    The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion.

  11. Insertion mode of a novel anionic antimicrobial peptide MDpep5 (Val-Glu-Ser-Trp-Val) from Chinese traditional edible larvae of housefly and its effect on surface potential of bacterial membrane.

    Science.gov (United States)

    Tang, Ya-Li; Shi, Yong-Hui; Zhao, Wei; Hao, Gang; Le, Guo-Wei

    2008-12-01

    Antimicrobial molecules from insects may serve as a potentially significant group of antibiotics. To identify the effect of antimicrobial peptides (AMPs) on bacterial membrane and obtain further insight in the mechanism of membrane transport of AMPs, the interaction of surface potential and permeation of a novel antimicrobial peptide MDpep5 (Val-Glu-Ser-Trp-Val) from Chinese traditional edible larvae of housefly was examined using liposomes from bacterial lipids extract. Compared with the cationic AMPs, MDpep5 cannot completely disrupt membrane. The uptake of MDpep5 by bacterial liposomes was dependent on the membrane surface potential. The mutual inhibition of the transport of MDpep5 through the cell membrane was caused by the change in surface potential due to the binding of MDpep5 to the membrane. Furthermore, formation of MDpep5-enriched lipid aggregates could lead to the disorder of the bilayer structure. Based on our experimental data, we propose that MDpep5 initiated its antimicrobial activity by profoundly disordering the structure and affecting physical properties of bacterial membrane when binding to the phospholipid which accounts for its bactericidal activity.

  12. Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB.

    Science.gov (United States)

    Okuda, Suguru; Tokuda, Hajime

    2009-04-01

    Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detail. However, it remained unclear how Lol factors interact with each other to conduct very efficient lipoprotein transfer in the periplasm where ATP is not available. To address this issue, a photo-reactive phenylalanine analogue, p-benzoyl-phenylalanine, was introduced at various positions of LolA and LolB, of which the overall structures are very similar and comprise an incomplete beta-barrel with a hydrophobic cavity inside. Cells expressing LolA or LolB derivatives containing the above analogue were irradiated with UV for in vivo photo-cross-linking. These analyses revealed a hot area in the same region of LolA and LolB, through which LolA and LolB interact with each other. This area is located at the entrance of the hydrophobic cavity. Moreover, this area in LolA is involved in the interaction with a membrane subunit, LolC, whereas no cross-linking occurs between LolA and the other membrane subunit, LolE, or ATP-binding subunit LolD, despite the structural similarity between LolC and LolE. The hydrophobic cavities of LolA and LolB were both found to bind lipoproteins inside. These results indicate that the transfer of lipoproteins through Lol proteins occurs in a mouth-to-mouth manner. PMID:19307584

  13. The Analysis of the Relationship between Pregnancy Bacterial and Non-bacterial Infection and Premature Rupture of Membranes%孕期细菌性及非细菌感染与胎膜早破的关系分析

    Institute of Scientific and Technical Information of China (English)

    周敏; 瑞芬; 张碧琼

    2014-01-01

    Objective:To investigate the relationship between pregnancy bacterial and non-bacterial infection and premature rupture of membranes. Method:60 cases of maternal PROM from July 2012 to July 2013 in our hospital for treatment were selected as experimental group,60 normal maternal of inpatient expectant mothers were randomly selected at the same period in our hospital as control group.Two groups of maternal cervical and vaginal secretions cervical mycoplasma(UU),bacterial agenesis(BV),candida(VVC),chlamydia(CT) and amniotic membrane disease detection,while maternal pregnancy outcomes and postpartum infection were compared.Result:The proportion of maternal UU,BV,VVC and CT were significantly higher than those in control group(P<0.05), the rate of maternal chorioamnionitis and postpartum maternal infection in experimental group were higher than those in the control group(P<0.05),the newborns of death,premature birth,neonatal asphyxia were more than those in the control group(P<0.05).Conclusion:The PROM with UU,CT,BV,VVC,closely relates to the amniotic membrane disease,so it needs screening for early pregnancy,maternal mid-screening,to take appropriate preventive measures to reduce the incidence of premature rupture of membranes,avoid maternal and newborn children adversely affected.Pregnancy diagnosis of bacterial and non-bacterial infections is great significance for premature rupture of membranes,it is worthy of promotion for clinical.%目的:探讨孕期细菌性及非细菌感染与胎膜早破的关系。方法:通过对2012年7月-2013年7月在笔者所在医院进行治疗的60例胎膜早破的产妇为试验组,随机选取同期在笔者所在医院进行住院待产60例正常产妇为对照组。对两组产妇的宫颈和阴道分泌物进行宫颈支原体(UU)、细菌性阴道病(BV)、假丝酵母菌(VVC)、衣原体(CT)及羊膜病检测,同时比较两组产妇的妊娠结局及产后感染情况。结果:试验组产妇的UU、BV、VVC

  14. Deuterium Labeling Strategies for Creating Contrast in Structure-Function Studies of Model Bacterial Outer Membranes Using Neutron Reflectometry.

    Science.gov (United States)

    Le Brun, Anton P; Clifton, Luke A; Holt, Stephen A; Holden, Peter J; Lakey, Jeremy H

    2016-01-01

    Studying the outer membrane of Gram-negative bacteria is challenging due to the complex nature of its structure. Therefore, simplified models are required to undertake structure-function studies of processes that occur at the outer membrane/fluid interface. Model membranes can be created by immobilizing bilayers to solid supports such as gold or silicon surfaces, or as monolayers on a liquid support where the surface pressure and fluidity of the lipids can be controlled. Both model systems are amenable to having their structure probed by neutron reflectometry, a technique that provides a one-dimensional depth profile through a membrane detailing its thickness and composition. One of the strengths of neutron scattering is the ability to use contrast matching, allowing molecules containing hydrogen and those enriched with deuterium to be highlighted or matched out against the bulk isotopic composition of the solvent. Lipopolysaccharides, a major component of the outer membrane, can be isolated for incorporation into model membranes. Here, we describe the deuteration of lipopolysaccharides from rough strains of Escherichia coli for incorporation into model outer membranes, and how the use of deuterated materials enhances structural analysis of model membranes by neutron reflectometry. PMID:26791981

  15. Deuterium Labeling Strategies for Creating Contrast in Structure-Function Studies of Model Bacterial Outer Membranes Using Neutron Reflectometry.

    Science.gov (United States)

    Le Brun, Anton P; Clifton, Luke A; Holt, Stephen A; Holden, Peter J; Lakey, Jeremy H

    2016-01-01

    Studying the outer membrane of Gram-negative bacteria is challenging due to the complex nature of its structure. Therefore, simplified models are required to undertake structure-function studies of processes that occur at the outer membrane/fluid interface. Model membranes can be created by immobilizing bilayers to solid supports such as gold or silicon surfaces, or as monolayers on a liquid support where the surface pressure and fluidity of the lipids can be controlled. Both model systems are amenable to having their structure probed by neutron reflectometry, a technique that provides a one-dimensional depth profile through a membrane detailing its thickness and composition. One of the strengths of neutron scattering is the ability to use contrast matching, allowing molecules containing hydrogen and those enriched with deuterium to be highlighted or matched out against the bulk isotopic composition of the solvent. Lipopolysaccharides, a major component of the outer membrane, can be isolated for incorporation into model membranes. Here, we describe the deuteration of lipopolysaccharides from rough strains of Escherichia coli for incorporation into model outer membranes, and how the use of deuterated materials enhances structural analysis of model membranes by neutron reflectometry.

  16. Biophysical studies of the interaction of squalamine and other cationic amphiphilic molecules with bacterial and eukaryotic membranes: importance of the distribution coefficient in membrane selectivity.

    Science.gov (United States)

    Di Pasquale, Eric; Salmi-Smail, Chanaz; Brunel, Jean-Michel; Sanchez, Patrick; Fantini, Jacques; Maresca, Marc

    2010-02-01

    The interaction of squalamine (SQ) with eukaryotic and prokaryotic membranes was studied and compared with the interaction of two other cationic amphipathic antimicrobials (CAAs), i.e. the antibiotic polymyxin B (PMB) and the detergent hexadecyltrimethylammonium bromide (CTAB). Whole cell experiments showed that the three CAA have in common the ability to interact with lipopolysaccharide-containing membranes through a divalent cation sensitive process. Differences were found regarding their kinetics of membrane permeabilisation and their selectivity for bacteria, with a preferential permeabilisation of bacteria by PMB>SQ and no selectivity for CTAB. Experiments with lipid monolayers and bilayers showed that this selectivity did not correlate with a preferential interaction of the CAAs with lipids but rather relies on differences in their ability to penetrate lipid bilayers and to cause electrically active lesions. Incidentally, our results also suggest that the distribution coefficient of CAAs could be used to predict their selectivity for bacteria. PMID:19883637

  17. Action of Polymyxin B on Bacterial Membranes: Morphological Changes in the Cytoplasm and in the Outer Membrane of Salmonella typhimurium and Escherichia coli B

    Science.gov (United States)

    Schindler, Peter R. G.; Teuber, Michael

    1975-01-01

    Though the primary action of the cationic antibiotic polymyxin B is against the membrane of susceptible bacteria, severe morphological changes are detected in the cytoplasm. Using fluorescence microscopy and a mono-N-dansyl-polymyxin B derivative, we could demonstrate aggregations of the antibiotic with cellular material, possibly nucleic acids and/or ribosomes. These aggregations were only produced by minimum inhibitory or higher concentrations of the antibiotic as shown with Salmonella and Escherichia strains differing in their polymyxin susceptibility. The outer membrane of Salmonella typhimurium revealed characteristic blebs when treated with polymyxin B. This was investigated by the gentle methods of spray-freezing and freeze-etching. The obtained electron micrographs suggest that the polymyxin-induced blebs are projections of the outer monolayer of the outer membrane. A possible mechanism of penetration of polymyxin B through the cell envelope of gram-negative bacteria is presented. Images PMID:169730

  18. Molecular basis of substrate selection by the N-end rule adaptor protein ClpS

    Energy Technology Data Exchange (ETDEWEB)

    Román-Hernández, Giselle; Grant, Robert A.; Sauer, Robert T.; Baker, Tania A.; (HHMI)

    2009-06-19

    The N-end rule is a conserved degradation pathway that relates the stability of a protein to its N-terminal amino acid. Here, we present crystal structures of ClpS, the bacterial N-end rule adaptor, alone and engaged with peptides containing N-terminal phenylalanine, leucine, and tryptophan. These structures, together with a previous structure of ClpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule amino acids. In each case, the alpha-amino group and side chain of the N-terminal residue are the major determinants of recognition. The binding pocket for the N-end residue is preformed in the free adaptor, and only small adjustments are needed to accommodate N-end rule residues having substantially different sizes and shapes. M53A ClpS is known to mediate degradation of an expanded repertoire of substrates, including those with N-terminal valine or isoleucine. A structure of Met53A ClpS engaged with an N-end rule tryptophan reveals an essentially wild-type mechanism of recognition, indicating that the Met(53) side chain directly enforces specificity by clashing with and excluding beta-branched side chains. Finally, experimental and structural data suggest mechanisms that make proteins with N-terminal methionine bind very poorly to ClpS, explaining why these high-abundance proteins are not degraded via the N-end rule pathway in the cell.

  19. The influence of temperature on the behaviour of mixed bacterial contamination of the shell membrane of the hen's egg.

    OpenAIRE

    Dolman, J.; Board, R. G.

    1992-01-01

    The inner membrane of the air cell of hens' eggs was inoculated with Pseudomonas putida, Staphylococcus xylosus, Enterococcus faecalis, Escherichia coli and Salmonella enteritidis. The first mentioned eventually dominated the contamination of the albumen of eggs stored at 4, 15, and 20 degrees C. The last mentioned did so in eggs stored at 37 degrees C. The interval between inoculation of the membrane and gross contamination of the albumen was markedly influenced by site of contamination rela...

  20. The influence of temperature on the behaviour of mixed bacterial contamination of the shell membrane of the hen's egg.

    Science.gov (United States)

    Dolman, J; Board, R G

    1992-02-01

    The inner membrane of the air cell of hens' eggs was inoculated with Pseudomonas putida, Staphylococcus xylosus, Enterococcus faecalis, Escherichia coli and Salmonella enteritidis. The first mentioned eventually dominated the contamination of the albumen of eggs stored at 4, 15, and 20 degrees C. The last mentioned did so in eggs stored at 37 degrees C. The interval between inoculation of the membrane and gross contamination of the albumen was markedly influenced by site of contamination relative to yolk movement. PMID:1547832

  1. Anti-hemolytic, hemagglutination inhibition and bacterial membrane disruptive properties of selected herbal extracts attenuate virulence of Carbapenem Resistant Escherichia coli.

    Science.gov (United States)

    Thakur, Pallavi; Chawla, Raman; Narula, Alka; Goel, Rajeev; Arora, Rajesh; Sharma, Rakesh Kumar

    2016-06-01

    Expression of a multitude of virulence factors by multi-drug resistant microbial strains, e.g., Carbapenem Resistant Escherichia coli (Family: Enterobacteriaceae; Class: Gammaproteobacteria), is responsible for resistance against beta-lactam antibiotics. Hemolysin production and induction of hemagglutination by bacterial surface receptors inflicts direct cytotoxicity by destroying host phagocytic and epithelial cells. We have previously reported that Berberis aristata, Camellia sinensis, Cyperus rotundus Holarrhena antidysenterica and Andrographis paniculata are promising herbal leads for targeting Carbapenem resistant Escherichia coli. These herbal leads were analyzed for their anti-hemolytic potential by employing spectrophotometric assay of hemoglobin liberation. Anti-hemagglutination potential of the extracts was assessed by employing qualitative assay of visible RBC aggregate formation. Camellia sinensis (PTRC-31911-A) exhibited anti-hemolytic potential of 73.97 ± 0.03%, followed by Holarrhena antidysenterica (PTRC-8111-A) i.e., 68.32 ± 0.05%, Berberis aristata (PTRC-2111-A) i.e., 60.26 ± 0.05% and Cyperus rotundus (PTRC-31811-A) i.e., 53.76 ± 0.03%. Comprehensive, visual analysis of hemagglutination inhibition revealed that only Berberis aristata (PTRC-2111-A) and Camellia sinensis (PTRC-31911-A) exhibited anti-hemagglutination activity. However, Andrographis paniculata (PTRC-11611-A) exhibited none of the inhibitory activities. Furthermore, the pair wise correlation analysis of the tested activities with quantitative phytochemical descriptors revealed that an increased content of alkaloid; flavonoids; polyphenols, and decreased content of saponins supported both the activities. Additionally, flow cytometry revealed that cell membrane structures of CRE were damaged by extracts of Berberis aristata (PTRC-2111-A) and Camellia sinensis (PTRC-31911-A) at their respective Minimum Inhibitory Concentrations, thereby confirming noteworthy antibacterial

  2. Brain-derived neurotrophic factor modulation of Kv1.3 channel is disregulated by adaptor proteins Grb10 and nShc

    Directory of Open Access Journals (Sweden)

    Marks David R

    2009-01-01

    Full Text Available Abstract Background Neurotrophins are important regulators of growth and regeneration, and acutely, they can modulate the activity of voltage-gated ion channels. Previously we have shown that acute brain-derived neurotrophic factor (BDNF activation of neurotrophin receptor tyrosine kinase B (TrkB suppresses the Shaker voltage-gated potassium channel (Kv1.3 via phosphorylation of multiple tyrosine residues in the N and C terminal aspects of the channel protein. It is not known how adaptor proteins, which lack catalytic activity, but interact with members of the neurotrophic signaling pathway, might scaffold with ion channels or modulate channel activity. Results We report the co-localization of two adaptor proteins, neuronal Src homology and collagen (nShc and growth factor receptor-binding protein 10 (Grb10, with Kv1.3 channel as demonstrated through immunocytochemical approaches in the olfactory bulb (OB neural lamina. To further explore the specificity and functional ramification of adaptor/channel co-localization, we performed immunoprecipitation and Western analysis of channel, kinase, and adaptor transfected human embryonic kidney 293 cells (HEK 293. nShc formed a direct protein-protein interaction with Kv1.3 that was independent of BDNF-induced phosphorylation of Kv1.3, whereas Grb10 did not complex with Kv1.3 in HEK 293 cells. Both adaptors, however, co-immunoprecipitated with Kv1.3 in native OB. Grb10 was interestingly able to decrease the total expression of Kv1.3, particularly at the membrane surface, and subsequently eliminated the BDNF-induced phosphorylation of Kv1.3. To examine the possibility that the Src homology 2 (SH2 domains of Grb10 were directly binding to basally phosphorylated tyrosines in Kv1.3, we utilized point mutations to substitute multiple tyrosine residues with phenylalanine. Removal of the tyrosines 111–113 and 449 prevented Grb10 from decreasing Kv1.3 expression. In the absence of either adaptor protein

  3. Rhizosphere bacterial carbon turnover is higher in nucleic acids than membrane lipids: implications for understanding soil carbon cycling

    Directory of Open Access Journals (Sweden)

    Ashish A. Malik

    2015-04-01

    Full Text Available Using a pulse-chase 13CO2 plant labeling experiment we compared the flow of plant carbon into macromolecular fractions of root-associated soil microorganisms. Time dependent 13C dilution patterns in microbial cellular fractions were used to calculate their turnover time. The turnover times of microbial biomolecules were found to vary: microbial RNA (19 h and DNA (30 h turned over fastest followed by chloroform fumigation extraction-derived soluble cell lysis products (14 d, while phospholipid fatty acids (PLFAs had the slowest turnover (42 d. PLFA/NLFA 13C analyses suggest that both mutualistic arbuscular mycorrhizal and saprophytic fungi are dominant in initial plant carbon uptake. In contrast, high initial 13C enrichment in RNA hints at bacterial importance in initial C uptake due to the dominance of bacterial derived RNA in total extracts of soil RNA. To explain this discrepancy, we observed low renewal rate of bacterial lipids, which may therefore bias lipid fatty acid based interpretations of the role of bacteria in soil microbial food webs. Based on our findings, we question current assumptions regarding plant-microbe carbon flux and suggest that the rhizosphere bacterial contribution to plant assimilate uptake could be higher. This highlights the need for more detailed quantitative investigations with nucleic acid biomarkers to further validate these findings.

  4. High-resolution phylogenetic analysis of residual bacterial species of fouled membranes after NaOCl cleaning.

    Science.gov (United States)

    Navarro, Ronald R; Hori, Tomoyuki; Inaba, Tomohiro; Matsuo, Kazuyuki; Habe, Hiroshi; Ogata, Atsushi

    2016-05-01

    Biofouling is one of the major problems during wastewater treatment using membrane bioreactors (MBRs). In this regard, sodium hypochlorite (NaOCl) has been widely used to wash fouled membranes for maintenance and recovery purposes. Advanced chemical and biological characterization was conducted in this work to evaluate the performance of aqueous NaOCl solutions during washing of polyacrylonitrile membranes. Fouled membranes from MBR operations supplemented with artificial wastewater were washed with 0.1% and 0.5% aqueous NaOCl solutions for 5, 10 and 30 min. The changes in organics composition on the membrane surface were directly monitored by an attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectrometer. In addition, high-throughput Illumina sequencing of 16S rRNA genes was applied to detect any residual microorganisms. Results from ATR-FT-IR analysis indicated the complete disappearance of functional groups representing different fouling compounds after at least 30 min of treatment with 0.1% NaOCl. However, the biomolecular survey revealed the presence of residual bacteria even after 30 min of treatment with 0.5% NaOCl solution. Evaluation of microbial diversity of treated samples using Chao1, Shannon and Simpson reciprocal indices showed an increase in evenness while no significant decline in richness was observed. These implied that only the population of dominant species was mainly affected. The high-resolution phylogenetic analysis revealed the presence of numerous operational taxonomic units (OTUs) whose close relatives exhibit halotolerance. Some OTUs related to thermophilic and acid-resistant strains were also identified. Finally, the taxonomic analysis of recycled membranes that were previously washed with NaOCl also showed the presence of numerous halotolerant-related OTUs in the early stage of fouling. This further suggested the possible contribution of such chemical tolerance on their survival against NaOCl washing, which in turn

  5. In Silico understanding of the cyclodextrin–phenanthrene hybrid assemblies in both aqueous medium and bacterial membranes

    International Nuclear Information System (INIS)

    Highlights: • Two hetero-assemblies, βCD1–Phe1, and βCD2–Phe1 were observed in water solution. • Distinct membrane-binding patterns for βCD, Phe, and their complexes were found. • Minor Phe trans-membrane energy barrier confirmed its membrane penetration ability. • Huge energy barriers for βCD-involved assemblies denied their membrane penetration. • Phe separation from βCD1–Phe1 was easier than that from βCD2–Phe1. - Abstract: The explicit-solvent molecular dynamic (MD) simulation and adaptive biased forces (ABF) methods were employed to systemically study the structural and thermodynamic nature of the β-cyclodextrin (βCD) monomer, phenanthrene (Phe) monomer, and their inclusion complexes in both the aqueous and membrane environments, aiming at clarifying the atomic-level mechanisms underlying in the CD-enhanced degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria. Simulations showed that βCD and Phe monomers could associate together to construct two distinctive assemblies, i.e, βCD1–Phe1 and βCD2–Phe1, respectively. The membrane-involved equilibrium simulations and the data of potential of mean forces (PMFs) further confirmed that Phe monomer was capable of penetrating through the membranes without confronting any large energy barrier, whereas, the single βCD and βCD-involved assemblies were unable to pass across the membranes. These observations clearly suggested that βCD only served as the carrier to enhance the bioavailability of Phe rather than the co-substrate in the Phe biodegradation process. The Phe-separation PMF profiles indicated that the maximum of the Phe uptake by bacteria would be achieved by the “optimal” βCD:Phe molar ratio, which facilitated the maximal formation of βCD1–Phe1 inclusion and the minimal construction of βCD2–Phe1 complex

  6. In Silico understanding of the cyclodextrin–phenanthrene hybrid assemblies in both aqueous medium and bacterial membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Baiping [College of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024 (China); State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023 (China); Gao, Huipeng; Cao, Yafeng [College of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024 (China); Jia, Lingyun, E-mail: lyj81@dlut.edu.cn [College of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024 (China)

    2015-03-21

    Highlights: • Two hetero-assemblies, βCD{sub 1}–Phe{sub 1}, and βCD{sub 2}–Phe{sub 1} were observed in water solution. • Distinct membrane-binding patterns for βCD, Phe, and their complexes were found. • Minor Phe trans-membrane energy barrier confirmed its membrane penetration ability. • Huge energy barriers for βCD-involved assemblies denied their membrane penetration. • Phe separation from βCD{sub 1}–Phe{sub 1} was easier than that from βCD{sub 2}–Phe{sub 1}. - Abstract: The explicit-solvent molecular dynamic (MD) simulation and adaptive biased forces (ABF) methods were employed to systemically study the structural and thermodynamic nature of the β-cyclodextrin (βCD) monomer, phenanthrene (Phe) monomer, and their inclusion complexes in both the aqueous and membrane environments, aiming at clarifying the atomic-level mechanisms underlying in the CD-enhanced degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria. Simulations showed that βCD and Phe monomers could associate together to construct two distinctive assemblies, i.e, βCD{sub 1}–Phe{sub 1} and βCD{sub 2}–Phe{sub 1}, respectively. The membrane-involved equilibrium simulations and the data of potential of mean forces (PMFs) further confirmed that Phe monomer was capable of penetrating through the membranes without confronting any large energy barrier, whereas, the single βCD and βCD-involved assemblies were unable to pass across the membranes. These observations clearly suggested that βCD only served as the carrier to enhance the bioavailability of Phe rather than the co-substrate in the Phe biodegradation process. The Phe-separation PMF profiles indicated that the maximum of the Phe uptake by bacteria would be achieved by the “optimal” βCD:Phe molar ratio, which facilitated the maximal formation of βCD{sub 1}–Phe{sub 1} inclusion and the minimal construction of βCD{sub 2}–Phe{sub 1} complex.

  7. Evolutionary Genomics Suggests That CheV Is an Additional Adaptor for Accommodating Specific Chemoreceptors within the Chemotaxis Signaling Complex.

    Directory of Open Access Journals (Sweden)

    Davi R Ortega

    2016-02-01

    Full Text Available Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a "phosphate sink" possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Overall, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.

  8. Evolutionary Genomics Suggests That CheV Is an Additional Adaptor for Accommodating Specific Chemoreceptors within the Chemotaxis Signaling Complex.

    Science.gov (United States)

    Ortega, Davi R; Zhulin, Igor B

    2016-02-01

    Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a "phosphate sink" possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Overall, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.

  9. Environmental controls on the distribution of bacterial tetraether membrane lipids: Constraints on the MBT-CBT paleothermometer

    NARCIS (Netherlands)

    Peterse, F.

    2011-01-01

    Branched glycerol dialkyl glycerol tetraethers (GDGTs) are membrane lipids of soil bacteria that occur ubiquitously in soils, peats, and marine sediments. The structures of the branched GDGTs vary in the number of methyl groups (4 to 6) attached to the alkyl chains and can contain up to two cyclopen

  10. Environmental controls on the distribution of bacterial tetraether membrane lipids: constraints on the MBT-CBT paleothermometer

    NARCIS (Netherlands)

    Peterse, F.

    2011-01-01

    Over the past years, a new proxy for the reconstruction of continental paleotemperature and past soil pH has been developed based on branched glycerol dialkyl glycerol tetraethers (GDGTs). Branched GDGTs are membrane lipids of as of yet unknown bacteria that thrive in peat and soils. Their molecular

  11. Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.

    LENUS (Irish Health Repository)

    Knodler, Leigh A

    2009-11-01

    The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated. Substitution of these six lysine residues with arginine, SopB-K(6)R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB-dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro-survival kinase Akt\\/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB-K(6)R mutant. At later times, fewer SCV were decorated with SopB-K(6)R compared with SopB. Instead SopB-K(6)R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization.

  12. Environmental controls on the distribution of bacterial tetraether membrane lipids:Constraints on the MBT-CBT paleothermometer

    NARCIS (Netherlands)

    Peterse, F.

    2011-01-01

    Branched glycerol dialkyl glycerol tetraethers (GDGTs) are membrane lipids of soil bacteria that occur ubiquitously in soils, peats, and marine sediments. The structures of the branched GDGTs vary in the number of methyl groups (4 to 6) attached to the alkyl chains and can contain up to two cyclopen

  13. In silico understanding of the cyclodextrin-phenanthrene hybrid assemblies in both aqueous medium and bacterial membranes.

    Science.gov (United States)

    Ren, Baiping; Gao, Huipeng; Cao, Yafeng; Jia, Lingyun

    2015-03-21

    The explicit-solvent molecular dynamic (MD) simulation and adaptive biased forces (ABF) methods were employed to systemically study the structural and thermodynamic nature of the β-cyclodextrin (βCD) monomer, phenanthrene (Phe) monomer, and their inclusion complexes in both the aqueous and membrane environments, aiming at clarifying the atomic-level mechanisms underlying in the CD-enhanced degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria. Simulations showed that βCD and Phe monomers could associate together to construct two distinctive assemblies, i.e, βCD1-Phe1 and βCD2-Phe1, respectively. The membrane-involved equilibrium simulations and the data of potential of mean forces (PMFs) further confirmed that Phe monomer was capable of penetrating through the membranes without confronting any large energy barrier, whereas, the single βCD and βCD-involved assemblies were unable to pass across the membranes. These observations clearly suggested that βCD only served as the carrier to enhance the bioavailability of Phe rather than the co-substrate in the Phe biodegradation process. The Phe-separation PMF profiles indicated that the maximum of the Phe uptake by bacteria would be achieved by the "optimal" βCD:Phe molar ratio, which facilitated the maximal formation of βCD1-Phe1 inclusion and the minimal construction of βCD2-Phe1 complex. PMID:25497028

  14. The Role of the Clathrin Adaptor AP-1: Polarized Sorting and Beyond

    Directory of Open Access Journals (Sweden)

    Fubito Nakatsu

    2014-11-01

    Full Text Available The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis. Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells.

  15. Species-specificity of the BamA component of the bacterial outer membrane protein-assembly machinery.

    Directory of Open Access Journals (Sweden)

    Elena B Volokhina

    Full Text Available The BamA protein is the key component of the Bam complex, the assembly machinery for outer membrane proteins (OMP in gram-negative bacteria. We previously demonstrated that BamA recognizes its OMP substrates in a species-specific manner in vitro. In this work, we further studied species specificity in vivo by testing the functioning of BamA homologs of the proteobacteria Neisseria meningitidis, Neisseria gonorrhoeae, Bordetella pertussis, Burkholderia mallei, and Escherichia coli in E. coli and in N. meningitidis. We found that no BamA functioned in another species than the authentic one, except for N. gonorrhoeae BamA, which fully complemented a N. meningitidis bamA mutant. E. coli BamA was not assembled into the N. meningitidis outer membrane. In contrast, the N. meningitidis BamA protein was assembled into the outer membrane of E. coli to a significant extent and also associated with BamD, an essential accessory lipoprotein of the Bam complex.Various chimeras comprising swapped N-terminal periplasmic and C-terminal membrane-embedded domains of N. meningitidis and E. coli BamA proteins were also not functional in either host, although some of them were inserted in the OM suggesting that the two domains of BamA need to be compatible in order to function. Furthermore, conformational analysis of chimeric proteins provided evidence for a 16-stranded β-barrel conformation of the membrane-embedded domain of BamA.

  16. Environmental controls on the distribution of bacterial tetraether membrane lipids: constraints on the MBT-CBT paleothermometer

    OpenAIRE

    Peterse, F.

    2011-01-01

    Branched glycerol dialkyl glycerol tetraethers (GDGTs) are membrane lipids of soil bacteria that occur ubiquitously in soils, peats, and marine sediments. The structures of the branched GDGTs vary in the number of methyl groups (4 to 6) attached to the alkyl chains and can contain up to two cyclopentane moieties. An empirical study showed that their distribution in over 130 globally distributed soils is mainly controlled by mean annual air temperature (MAT) and soil pH, and can be expressed u...

  17. XB130: A novel adaptor protein in cancer signal transduction

    Science.gov (United States)

    ZHANG, RUIYAO; ZHANG, JINGYAO; WU, QIFEI; MENG, FANDI; LIU, CHANG

    2016-01-01

    Adaptor proteins are functional proteins that contain two or more protein-binding modules to link signaling proteins together, which affect cell growth and shape and have no enzymatic activity. The actin filament-associated protein (AFAP) family is an important member of the adaptor proteins, including AFAP1, AFAP1L1 and AFAP1L2/XB130. AFAP1 and AFAP1L1 share certain common characteristics and function as an actin-binding protein and a cSrc-activating protein. XB130 exhibits certain unique features in structure and function. The mRNA of XB130 is expressed in human spleen, thyroid, kidney, brain, lung, pancreas, liver, colon and stomach, and the most prominent disease associated with XB130 is cancer. XB130 has a controversial effect on cancer. Studies have shown that XB130 can promote cancer progression and downregulation of XB130-reduced growth of tumors derived from certain cell lines. A higher mRNA level of XB130 was shown to be associated with a better survival in non-small cell lung cancer. Previous studies have shown that XB130 can regulate cell growth, migration and invasion and possibly has the effect through the cAMP-cSrc-phosphoinositide 3-kinase/Akt pathway. Except for cancer, XB130 is also associated with other pathological or physiological procedures, such as airway repair and regeneration. PMID:26998266

  18. DMPD: Signalling adaptors used by Toll-like receptors: an update. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18706831 Signalling adaptors used by Toll-like receptors: an update. Kenny EF, O'Ne...tors used by Toll-like receptors: an update. PubmedID 18706831 Title Signalling adaptors used by Toll-like receptors: an update

  19. A Big-Five Personality Profile of the Adaptor and Innovator.

    Science.gov (United States)

    Kwang, Ng Aik; Rodrigues, Daphne

    2002-01-01

    A study explored the relationship between two creative types (adaptor and innovator) and the Big Five personality traits (extraversion, agreeableness, conscientiousness, neuroticism, and openness to experience), in 164 teachers in Singapore. Adaptors were significantly more conscientious than innovators, while innovators were significantly more…

  20. The absence of protein Y4yS affects negatively the abundance of T3SS Mesorhizobium loti secretin, RhcC2, in bacterial membranes

    Directory of Open Access Journals (Sweden)

    Virginia eMercante

    2015-01-01

    Full Text Available Mesorhizobium loti MAFF303099 has a functional type III secretion system (T3SS that is involved in the determination of nodulation competitiveness on Lotus. The M. loti T3SS cluster contains gene y4yS (mlr8765 that codes for a protein of unknown function (Y4yS. A mutation in the y4yS gene favors the M. loti symbiotic competitive ability on Lotus tenuis cv. Esmeralda and affects negatively the secretion of proteins through T3SS. Here we localize Y4yS in the bacterial membrane using a translational reporter peptide fusion. In silico analysis indicated that this protein presents a tetratricopeptide repeat (TPR domain, a signal peptide and a canonical lipobox LGCC in the N-terminal sequence. These features that are shared with proteins required for the formation of the secretin complex in type IV secretion systems and in the Tad system, together with its localization, suggest that the y4yS-encoded protein is required for the formation of the M. loti T3SS secretin (RhcC2 complex. Remarkably, analysis of RhcC2 in the wild-type and M. loti y4yS mutant strains indicated that the absence of Y4yS affects negatively the accumulation of normal levels of RhcC2 in the membrane.

  1. A potential food biopreservative, CecXJ-37N, non-covalently intercalates into the nucleotides of bacterial genomic DNA beyond membrane attack.

    Science.gov (United States)

    Liu, Dongliang; Liu, Jun; Li, Jinyao; Xia, Lijie; Yang, Jianhua; Sun, Surong; Ma, Ji; Zhang, Fuchun

    2017-02-15

    The antibacterial activities and mechanism of an amide-modified peptide CecXJ-37N were investigated in this study. CecXJ-37N showed small MICs (0.25-7.8μM) against eight harmful strains common in food industry. The α-helix proportion of CecXJ-37N increased by 11-fold in prokaryotic membrane comparable environments; cytotoxicity studies demonstrated the MHC was significantly higher than that of non-amidated isoform. Moreover, CecXJ-37N possessed stronger capacities to resist trypsin and pepsin hydrolysis within two hours. Flow cytometry and scanning electron microscopy demonstrated that CecXJ-37N induced pore-formation, morphological changes, and lysed E. coli cells. Fluorescence microscopy indicated that CecXJ-37N penetrated E. coli membrane and accumulated in cytoplasm. Further ultraviolet-visible spectroscopy suggested that CecXJ-37N changed the action mode of parental peptide interacting with bacterial genome from outside binding to a tightly non-covalent intercalation into nucleotides. Overall, this study suggested that amide-modification enhanced antimicrobial activity and reduced the cytotoxicity, thus could be potential strategies for developing novel food preservatives.

  2. A potential food biopreservative, CecXJ-37N, non-covalently intercalates into the nucleotides of bacterial genomic DNA beyond membrane attack.

    Science.gov (United States)

    Liu, Dongliang; Liu, Jun; Li, Jinyao; Xia, Lijie; Yang, Jianhua; Sun, Surong; Ma, Ji; Zhang, Fuchun

    2017-02-15

    The antibacterial activities and mechanism of an amide-modified peptide CecXJ-37N were investigated in this study. CecXJ-37N showed small MICs (0.25-7.8μM) against eight harmful strains common in food industry. The α-helix proportion of CecXJ-37N increased by 11-fold in prokaryotic membrane comparable environments; cytotoxicity studies demonstrated the MHC was significantly higher than that of non-amidated isoform. Moreover, CecXJ-37N possessed stronger capacities to resist trypsin and pepsin hydrolysis within two hours. Flow cytometry and scanning electron microscopy demonstrated that CecXJ-37N induced pore-formation, morphological changes, and lysed E. coli cells. Fluorescence microscopy indicated that CecXJ-37N penetrated E. coli membrane and accumulated in cytoplasm. Further ultraviolet-visible spectroscopy suggested that CecXJ-37N changed the action mode of parental peptide interacting with bacterial genome from outside binding to a tightly non-covalent intercalation into nucleotides. Overall, this study suggested that amide-modification enhanced antimicrobial activity and reduced the cytotoxicity, thus could be potential strategies for developing novel food preservatives. PMID:27664674

  3. Comparison of quantitative PCR and flow cytometry as cellular viability methods to study bacterial membrane permeabilization following supercritical CO2 treatment.

    Science.gov (United States)

    Tamburini, Sabrina; Ballarini, Annalisa; Ferrentino, Giovanna; Moro, Albertomaria; Foladori, Paola; Spilimbergo, Sara; Jousson, Olivier

    2013-06-01

    Foodborne illness due to bacterial pathogens is increasing worldwide as a consequence of the higher consumption of fresh and minimally processed food products, which are more easily cross-contaminated. The efficiency of food pasteurization methods is usually measured by c.f.u. plate counts, a method discriminating viable from dead cells on the basis of the ability of cells to replicate and form colonies on standard growth media, thus ignoring viable but not cultivable cells. Supercritical CO2 (SC-CO2) has recently emerged as one of the most promising fresh food pasteurization techniques, as an alternative to traditional, heat-based methods. In the present work, using three SC-CO2-treated foodborne bacteria (Listeria monocytogenes, Salmonella enterica and Escherichia coli) we tested and compared the performance of alternative viability test methods based on membrane permeability: propidium monoazide quantitative PCR (PMA-qPCR) and flow cytometry (FCM). Results were compared based on plate counts and fluorescent microscopy measurements, which showed that the former dramatically reduced the number of cultivable cells by more than 5 log units. Conversely, FCM provided a much more detailed picture of the process, as it directly quantifies the number of total cells and distinguishes among three categories, including intact, partially permeabilized and permeabilized cells. A comparison of both PMA-qPCR and FCM with plate count data indicated that only a fraction of intact cells maintained the ability to replicate in vitro. Following SC-CO2 treatment, FCM analysis revealed a markedly higher level of bacterial membrane permeabilization of L. monocytogenes with respect to E. coli and S. enterica. Furthermore, an intermediate permeabilization state in which the cellular surface was altered and biovolume increased up to 1.5-fold was observed in L. monocytogenes, but not in E. coli or S. enterica. FCM thus compared favourably with other methods and should be considered as an

  4. Heme uptake in bacterial pathogens

    OpenAIRE

    Contreras, Heidi; Chim, Nicholas; Credali, Alfredo; Goulding, Celia W.

    2014-01-01

    Iron is an essential nutrient for the survival of organisms. Bacterial pathogens possess specialized pathways to acquire heme from their human hosts. In this review, we present recent structural and biochemical data that provide mechanistic insights into several bacterial heme uptake pathways, encompassing the sequestration of heme from human hemoproteins to secreted or membrane-associated bacterial proteins, the transport of heme across bacterial membranes, and the degradation of heme within...

  5. DMPD: TIR domain-containing adaptors define the specificity of TLR signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14698224 TIR domain-containing adaptors define the specificity of TLR signaling. Ya...in-containing adaptors define the specificity of TLR signaling. PubmedID 14698224 Title TIR domain-containing adaptors define the spe...cificity of TLR signaling. Authors Yamamoto M, Takeda K,

  6. Bacterial community structure and activity of sulfate reducing bacteria in a membrane aerated biofilm analyzed by microsensor and molecular techniques.

    Science.gov (United States)

    Liu, Hong; Tan, Shuying; Sheng, Zhiya; Liu, Yang; Yu, Tong

    2014-11-01

    The activities and vertical spatial distribution of sulfate reducing bacteria (SRB) in an oxygen (O2 )-based membrane aerated biofilm (MAB) were investigated using microsensor (O2 and H2 S) measurements and molecular techniques (polymerase chain reaction-denaturing gradient gel electrophoresis [PCR-DGGE] and fluorescence in situ hybridization [FISH]). The O2 concentration profile revealed that O2 penetrated from the bottom (substratum) of the gas permeable membrane, and was gradually consumed within the biofilm until it was completely depleted near the biofilm/bulk liquid interface, indicating oxic and anoxic zone in the MAB. The H2 S concentration profile showed that H2 S production was found in the upper 285 µm of the biofilm, indicating a high activity of SRB in this region. The results from DGGE of the PCR-amplified dissimilatory sulfite reductase subunit B (dsrB) gene and FISH showed an uneven spatial distribution of SRB. The maximum SRB biomass was located in the upper biofilm. The information from the molecular analysis can be supplemented with that from microsensor measurements to better understand the microbial community and activity of SRB in the MAB.

  7. Bacterial carbonatogenesis

    International Nuclear Information System (INIS)

    Several series of experiments in the laboratory as well as in natural conditions teach that the production of carbonate particles by heterotrophic bacteria follows different ways. The 'passive' carbonatogenesis is generated by modifications of the medium that lead to the accumulation of carbonate and bicarbonate ions and to the precipitation of solid particles. The 'active' carbonatogenesis is independent of the metabolic pathways. The carbonate particles are produced by ionic exchanges through the cell membrane following still poorly known mechanisms. Carbonatogenesis appears to be the response of heterotrophic bacterial communities to an enrichment of the milieu in organic matter. The active carbonatogenesis seems to start first. It is followed by the passive one which induces the growth of initially produced particles. The yield of heterotrophic bacterial carbonatogenesis and the amounts of solid carbonates production by bacteria are potentially very high as compared to autotrophic or chemical sedimentation from marine, paralic or continental waters. Furthermore, the bacterial processes are environmentally very ubiquitous; they just require organic matter enrichment. Thus, apart from purely evaporite and autotrophic ones, all Ca and/or Mg carbonates must be considered as from heterotrophic bacterial origin. By the way, the carbon of carbonates comes from primary organic matter. Such considerations ask questions about some interpretations from isotopic data on carbonates. Finally, bacterial heterotrophic carbonatogenesis appears as a fundamental phase in the relationships between atmosphere and lithosphere and in the geo-biological evolution of Earth. (author)

  8. Adaptor protein complexes AP-1 and AP-3 are required by the HHV-7 Immunoevasin U21 for rerouting of class I MHC molecules to the lysosomal compartment.

    Directory of Open Access Journals (Sweden)

    Lisa A Kimpler

    Full Text Available The human herpesvirus-7 (HHV-7 U21 gene product binds to class I major histocompatibility complex (MHC molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes.

  9. Action of Polymyxin B on Bacterial Membranes: Phosphatidylglycerol- and Cardiolipin-Induced Susceptibility to Polymyxin B in Acholeplasma laidlawii B

    Science.gov (United States)

    Teuber, Michael; Bader, Johann

    1976-01-01

    To identify the polymyxin receptor molecules in the membranes of living microorganisms, fusion of intact Acholeplasma laidlawii B with lipid vesicles was investigated according to the procedure of Grant and McConnell (1973). The naturally polymyxin-resistant A. laidlawii B was treated with phospholipid vesicles prepared from purified phospholipids of the polymyxin-susceptible Salmonella typhimurium G30. A. laidlawii B absorbed between 15 and 45% of its own lipid content of the added tritium-labeled phospholipids without loss of viability. Association with the acidic components phosphatidylglycerol and cardiolipin produced a 10- to 30-fold increase in polymyxin susceptibility, which was not obtained with egg-phosphatidylcholine and mixed phosphatidylcholine-phosphatidylethanolamine vesicles. The polymyxin-sensitized cells bound 12 times more radioactive antibiotic than resistant cells. The phosphatidylglycerol-induced susceptibility was abolished by serum fraction V (Cohn) proteins. PMID:176930

  10. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation.

    Science.gov (United States)

    Zhong, Bo; Yang, Yan; Li, Shu; Wang, Yan-Yi; Li, Ying; Diao, Feici; Lei, Caoqi; He, Xiao; Zhang, Lu; Tien, Po; Shu, Hong-Bing

    2008-10-17

    Viral infection triggers activation of transcription factors such as NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. Here, we identified MITA as a critical mediator of virus-triggered type I IFN signaling by expression cloning. Overexpression of MITA activated IRF3, whereas knockdown of MITA inhibited virus-triggered activation of IRF3, expression of type I IFNs, and cellular antiviral response. MITA was found to localize to the outer membrane of mitochondria and to be associated with VISA, a mitochondrial protein that acts as an adaptor in virus-triggered signaling. MITA also interacted with IRF3 and recruited the kinase TBK1 to the VISA-associated complex. MITA was phosphorylated by TBK1, which is required for MITA-mediated activation of IRF3. Our results suggest that MITA is a critical mediator of virus-triggered IRF3 activation and IFN expression and further demonstrate the importance of certain mitochondrial proteins in innate antiviral immunity.

  11. Impairment of dendritic cell functions in patients with adaptor protein-3 complex deficiency.

    Science.gov (United States)

    Prandini, Alberto; Salvi, Valentina; Colombo, Francesca; Moratto, Daniele; Lorenzi, Luisa; Vermi, William; De Francesco, Maria Antonia; Notarangelo, Lucia Dora; Porta, Fulvio; Plebani, Alessandro; Facchetti, Fabio; Sozzani, Silvano; Badolato, Raffaele

    2016-06-30

    Hermansky-Pudlak syndrome type 2 (HPS2) is a primary immunodeficiency due to adaptor protein-3 (AP-3) complex deficiency. HPS2 patients present neutropenia, partial albinism, and impaired lysosomal vesicles formation in hematopoietic cells. Given the role of dendritic cells (DCs) in the immune response, we studied monocyte-derived DCs (moDCs) and plasmacytoid DCs (pDCs) in two HPS2 siblings. Mature HPS2 moDCs showed impaired expression of CD83 and DC-lysosome-associated membrane protein (LAMP), low levels of MIP1-β/CCL4, MIG/CXCL9, and severe defect of interleukin-12 (IL-12) secretion. DCs in lymph-node biopsies from the same patients showed a diffuse cytoplasm reactivity in a large fraction of DC-LAMP(+) cells, instead of the classical dot-like stain. In addition, analysis of pDC-related functions of blood-circulating mononuclear cells revealed reduced interferon-α secretion in response to herpes simplex virus-1 (HSV-1), whereas granzyme-B induction upon IL-3/IL-10 stimulation was normal. Finally, T-cell costimulatory activity, as measured by mixed lymphocyte reaction assay, was lower in patients, suggesting that function and maturation of DCs is abnormal in patients with HPS2.

  12. Molecular basis for the specific recognition of the metazoan cyclic GMP-AMP by the innate immune adaptor protein STING.

    Science.gov (United States)

    Shi, Heping; Wu, Jiaxi; Chen, Zhijian J; Chen, Chuo

    2015-07-21

    Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2'3'-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2'3'-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. Here we show that 2'3'-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Our results demonstrate that analyses of free-ligand conformations can be as important as analyses of protein conformations in understanding protein-ligand interactions.

  13. Function of FlhB, a membrane protein implicated in the bacterial flagellar type III secretion system.

    Science.gov (United States)

    Meshcheryakov, Vladimir A; Barker, Clive S; Kostyukova, Alla S; Samatey, Fadel A

    2013-01-01

    The membrane protein FlhB is a highly conserved component of the flagellar secretion system, and it plays an active role in the regulation of protein export. In this study conserved properties of FlhB that are important for its function were investigated. Replacing the flhB gene (or part of the gene) in Salmonella typhimurium with the flhB gene of the distantly related bacterium Aquifex aeolicus greatly reduces motility. However, motility can be restored to some extent by spontaneous mutations in the part of flhB gene coding for the cytoplasmic domain of Aquifex FlhB. Structural analysis suggests that these mutations destabilize the structure. The secondary structure and stability of the mutated cytoplasmic fragments of FlhB have been studied by circular dichroism spectroscopy. The results suggest that conformational flexibility could be important for FlhB function. An extragenic suppressor mutation in the fliS gene, which decreases the affinity of FliS to FliC, partially restores motility of the FlhB substitution mutants. PMID:23874605

  14. Function of FlhB, a membrane protein implicated in the bacterial flagellar type III secretion system.

    Directory of Open Access Journals (Sweden)

    Vladimir A Meshcheryakov

    Full Text Available The membrane protein FlhB is a highly conserved component of the flagellar secretion system, and it plays an active role in the regulation of protein export. In this study conserved properties of FlhB that are important for its function were investigated. Replacing the flhB gene (or part of the gene in Salmonella typhimurium with the flhB gene of the distantly related bacterium Aquifex aeolicus greatly reduces motility. However, motility can be restored to some extent by spontaneous mutations in the part of flhB gene coding for the cytoplasmic domain of Aquifex FlhB. Structural analysis suggests that these mutations destabilize the structure. The secondary structure and stability of the mutated cytoplasmic fragments of FlhB have been studied by circular dichroism spectroscopy. The results suggest that conformational flexibility could be important for FlhB function. An extragenic suppressor mutation in the fliS gene, which decreases the affinity of FliS to FliC, partially restores motility of the FlhB substitution mutants.

  15. Identification of polyvalent protective immunogens from outer membrane proteins in Vibrio parahaemolyticus to protect fish against bacterial infection.

    Science.gov (United States)

    Peng, Bo; Ye, Jin-Zhou; Han, Yi; Zeng, Li; Zhang, Jian-Ying; Li, Hui

    2016-07-01

    Vaccination is one of the most effective and economic way to prevent infectious diseases in aquaculture. The development of effective vaccines, however, is still limited, especially for polyvalent vaccines, which are against multiple species. With this regard, identification of polyvalent protective immunogens, serving as polyvalent vaccines, became a key step in vaccine development. In the current study, 17 outer membrane proteins from Vibrio parahaemolyticus were identified as immunogens. Further, four of the 17 proteins including VP2309, VP0887, VPA0548 and VP1019 were characterized as efficiently protective immunogens against V. parahaemolyticus' infection through passive and active immunizations in zebrafish. Importantly, these four proteins showed cross-protective capability against infections by Aeromonas hydrophila or/and Pseudomonas fluorescens, which shared similar epitopes with V. parahaemolyticus in homology of these proteins. Further investigation showed that the expression level of the four protective immunogens elevated in response to fish plasma in a dose-dependent manner. These results indicate that the four protective immunogens are polyvalent vaccine candidates in aquaculture. PMID:27071519

  16. Study on the isothermal forging process of MB26 magnesium alloy adaptor

    Directory of Open Access Journals (Sweden)

    Xu Wenchen

    2015-01-01

    Full Text Available The isothermal forging process is an effective method to manufacture complex-shaped components of hard-to-work materials, such as magnesium alloys. This study investigates the isothermal forging process of an MB26 magnesium alloy adaptor with three branches. The results show that two-step forging process is appropriate to form the adaptor forging, which not only improves the filling quality but also reduces the forging load compared with one-step forging process. Moreover, the flow line is distributed along the contour of the complex-shaped adaptor forging.

  17. Gene synthesis, bacterial expression, and 1H NMR spectroscopic studies of the rat outer mitochondrial membrane cytochrome b5.

    Science.gov (United States)

    Rivera, M; Barillas-Mury, C; Christensen, K A; Little, J W; Wells, M A; Walker, F A

    1992-12-01

    The gene coding for the water-soluble domain of the outer mitochondrial membrane cytochrome b5 (OM cytochrome b5) from rat liver has been synthetized and expressed in Escherichia coli. The DNA sequence was obtained by back-translating the known amino acid sequence [Lederer, F., Ghrir, R., Guiard, B., Cortial, S., & Ito, A. (1983) Eur. J. Biochem. 132, 95-102]. The recombinant OM cytochrome b5 was characterized by UV-visible, EPR, and 1H NMR spectroscopy. The UV-visible and EPR spectra of the OM cytochrome b5 are almost identical to the ones obtained from the overexpressed rat microsomal cytochrome b5 [Bodman, S. B. V., Schyler, M. A., Jollie, D. R., & Sligar, S. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9443-9447]. The one-dimensional 1H NMR spectrum of the OM cytochrome b5 indicates that the rhombic perturbation of the ferric center is essentially identical to that in the microsomal beef, rabbit, chicken, and rat cytochromes b5. Two-dimensional 1H NMR spectroscopy (NOESY) and one-dimensional NOE difference spectroscopy were used to assign the contact-shifted resonances that correspond to each of the two isomers that result from the rotation of the heme around its alpha-gamma-meso axis. The assignment of the resonances allowed the determination of the heme orientation ratio in the OM cytochrome b5, which was found to be 1.0 +/- 0.1. It is noteworthy that the two cytochromes b5 that have similar populations of the two heme isomers (large heme disorder) originate from the rat liver. PMID:1333795

  18. A Bioinformatics Analysis Reveals a Group of MocR Bacterial Transcriptional Regulators Linked to a Family of Genes Coding for Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Teresa Milano

    2016-01-01

    Full Text Available The MocR bacterial transcriptional regulators are characterized by an N-terminal domain, 60 residues long on average, possessing the winged-helix-turn-helix (wHTH architecture responsible for DNA recognition and binding, linked to a large C-terminal domain (350 residues on average that is homologous to fold type-I pyridoxal 5′-phosphate (PLP dependent enzymes like aspartate aminotransferase (AAT. These regulators are involved in the expression of genes taking part in several metabolic pathways directly or indirectly connected to PLP chemistry, many of which are still uncharacterized. A bioinformatics analysis is here reported that studied the features of a distinct group of MocR regulators predicted to be functionally linked to a family of homologous genes coding for integral membrane proteins of unknown function. This group occurs mainly in the Actinobacteria and Gammaproteobacteria phyla. An analysis of the multiple sequence alignments of their wHTH and AAT domains suggested the presence of specificity-determining positions (SDPs. Mapping of SDPs onto a homology model of the AAT domain hinted at possible structural/functional roles in effector recognition. Likewise, SDPs in wHTH domain suggested the basis of specificity of Transcription Factor Binding Site recognition. The results reported represent a framework for rational design of experiments and for bioinformatics analysis of other MocR subgroups.

  19. A Bioinformatics Analysis Reveals a Group of MocR Bacterial Transcriptional Regulators Linked to a Family of Genes Coding for Membrane Proteins

    Science.gov (United States)

    Milano, Teresa

    2016-01-01

    The MocR bacterial transcriptional regulators are characterized by an N-terminal domain, 60 residues long on average, possessing the winged-helix-turn-helix (wHTH) architecture responsible for DNA recognition and binding, linked to a large C-terminal domain (350 residues on average) that is homologous to fold type-I pyridoxal 5′-phosphate (PLP) dependent enzymes like aspartate aminotransferase (AAT). These regulators are involved in the expression of genes taking part in several metabolic pathways directly or indirectly connected to PLP chemistry, many of which are still uncharacterized. A bioinformatics analysis is here reported that studied the features of a distinct group of MocR regulators predicted to be functionally linked to a family of homologous genes coding for integral membrane proteins of unknown function. This group occurs mainly in the Actinobacteria and Gammaproteobacteria phyla. An analysis of the multiple sequence alignments of their wHTH and AAT domains suggested the presence of specificity-determining positions (SDPs). Mapping of SDPs onto a homology model of the AAT domain hinted at possible structural/functional roles in effector recognition. Likewise, SDPs in wHTH domain suggested the basis of specificity of Transcription Factor Binding Site recognition. The results reported represent a framework for rational design of experiments and for bioinformatics analysis of other MocR subgroups. PMID:27446613

  20. Increasing the efficiency of SAGE adaptor ligation by directed ligation chemistry

    Science.gov (United States)

    So, Austin P.; Turner, Robin F. B.; Haynes, Charles A.

    2004-01-01

    The ability of Serial Analysis of Gene Expression (SAGE) to provide a quantitative picture of global gene expression relies not only on the depth and accuracy of sequencing into the SAGE library, but also on the efficiency of each step required to generate the SAGE library from the starting mRNA material. The first critical step is the ligation of adaptors containing a Type IIS recognition sequence to the anchored 3′ end cDNA population that permits the release of short sequence tags (SSTs) from defined sites within the 3′ end of each transcript. Using an in vitro transcript as a template, we observed that only a small fraction of anchored 3′ end cDNA are successfully ligated with added SAGE adaptors under typical reaction conditions currently used in the SAGE protocol. Although the introduction of ∼500-fold molar excess of adaptor or the inclusion of 15% (w/v) PEG-8000 increased the yield of the adaptor-modified product, complete conversion to the desired adaptor:cDNA hetero-ligation product is not achieved. An alternative method of ligation, termed as directed ligation, is described which exploits a favourable mass-action condition created by the presence of NlaIII during ligation in combination with a novel SAGE adaptor containing a methylated base within the ligation site. Using this strategy, we were able to achieve near complete conversion of the anchored 3′ end cDNA into the desired adaptor-modified product. This new protocol therefore greatly increases the probability that a SST will be generated from every transcript, greatly enhancing the fidelity of SAGE. Directed ligation also provides a powerful means to achieve near-complete ligation of any appropriately designed adaptor to its respective target. PMID:15247329

  1. The Toll-Like receptor adaptor TRIF contributes to otitis media pathogenesis and recovery

    Directory of Open Access Journals (Sweden)

    Pak Kwang

    2009-08-01

    Full Text Available Abstract Background Toll-like receptor (TLR signalling is crucial for innate immune responses to infection. The involvement of TLRs in otitis media (OM, the most prevalent childhood disease in developed countries, has been implicated by studies in middle ear cell lines, by association studies of TLR-related gene polymorphisms, and by altered OM in mice bearing mutations in TLR genes. Activated TLRs signal via two alternative intracellular signaling molecules with differing effects; MyD88 (Myeloid differentiation primary response gene 88 inducing primarily interleukin expression and TRIF (Tir-domain-containing adaptor inducing interferon β mediating type I interferon (IFN expression. We tested the hypothesis that TRIF and type I IFN signaling play a role in OM, using a murine model of OM induced by non-typeable Haemophilus influenzae (NTHi. The ME inflammatory response to NTHi was examined in wild-type (WT and TRIF-/- mice by qPCR, gene microarray, histopathology and bacterial culture. Results Expression of TRIF mRNA was only modesty enhanced during OM, but both type I IFN signalling genes and type I IFN-inducible genes were significantly up-regulated in WT mice. TRIF-deficient mice showed reduced but more persistent mucosal hyperplasia and less leukocyte infiltration into the ME in response to NTHi infection than did WT animals. Viable bacteria could be cultured from MEs of TRIF-/- mice for much longer in the course of disease than was the case for middle ears of WT mice. Conclusion Our results demonstrate that activation of TRIF/type I IFN responses is important in both the pathogenesis and resolution of NTHi-induced OM.

  2. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex.

    Science.gov (United States)

    Bondage, Devanand D; Lin, Jer-Sheng; Ma, Lay-Sun; Kuo, Chih-Horng; Lai, Erh-Min

    2016-07-01

    Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity. PMID:27313214

  3. Surfactant Protein A Enhances Constitutive Immune Functions of Clathrin Heavy Chain and Clathrin Adaptor Protein 2.

    Science.gov (United States)

    Moulakakis, Christina; Steinhäuser, Christine; Biedziak, Dominika; Freundt, Katja; Reiling, Norbert; Stamme, Cordula

    2016-07-01

    NF-κB transcription factors are key regulators of pulmonary inflammatory disorders and repair. Constitutive lung cell type- and microenvironment-specific NF-κB/inhibitor κBα (IκB-α) regulation, however, is poorly understood. Surfactant protein (SP)-A provides both a critical homeostatic and lung defense control, in part by immune instruction of alveolar macrophages (AMs) via clathrin-mediated endocytosis. The central endocytic proteins, clathrin heavy chain (CHC) and the clathrin adaptor protein (AP) complex AP2, have pivotal alternative roles in cellular homeostasis that are endocytosis independent. Here, we dissect endocytic from alternative functions of CHC, the α-subunit of AP2, and dynamin in basal and SP-A-modified LPS signaling of macrophages. As revealed by pharmacological inhibition and RNA interference in primary AMs and RAW264.7 macrophages, respectively, CHC and α-adaptin, but not dynamin, prevent IκB-α degradation and TNF-α release, independent of their canonical role in membrane trafficking. Kinetics studies employing confocal microscopy, Western analysis, and immunomagnetic sorting revealed that SP-A transiently enhances the basal protein expression of CHC and α-adaptin, depending on early activation of protein kinase CK2 (former casein kinase II) and Akt1 in primary AMs from rats, SP-A(+/+), and SP-A(-/-) mice, as well as in vivo when intratracheally administered to SP-A(+/+) mice. Constitutive immunomodulation by SP-A, but not SP-A-mediated inhibition of LPS-induced NF-κB activity and TNF-α release, requires CHC, α-adaptin, and dynamin. Our data demonstrate that endocytic proteins constitutively restrict NF-κB activity in macrophages and provide evidence that SP-A enhances the immune regulatory capacity of these proteins, revealing a previously unknown pathway of microenvironment-specific NF-κB regulation in the lung. PMID:26771574

  4. The AP-3 adaptor complex is required for vacuolar function in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Maria Zwiewka; Elena Feraru; Barbara M(o)ller; Inhwan Hwang; Mugurel I Feraru; Jürgen Kleine-Vehn; Dolf Weijers; Ji(n) Friml

    2011-01-01

    Subcellular trafficking is required for a multitude of functions in eukaryotic cells.It involves regulation of cargo sorting,vesicle formation,trafficking and fusion processes at multiple levels.Adaptor protein (AP) complexes are key regulators of cargo sorting into vesicles in yeast and mammals but their existence and function in plants have not been demonstrated.Here we report the identification of the protein-affected trafficking 4 (pat4) mutant defective in the putative δ subunit of the AP-3 complex.pat4 and pat2,a mutant isolated from the same GFP imaging-based forward genetic screen that lacks a functional putative AP-3 β,as well as dominant negative AP-3 μ transgenic lines display undistinguishable phenotypes characterized by largely normal morphology and development,but strong intracellular accumulation of membrane proteins in aberrant vacuolar structures.All mutants are defective in morphology and function of lytic and protein storage vacuoles (PSVs) but show normal sorting of reserve proteins to PSVs.Immunoprecipitation experiments and genetic studies revealed tight functional and physical associations of putative AP-3 β and AP-3 δ subunits.Furthermore,both proteins are closely linked with putative AP-3 μ and σ subunits and several components of the clathrin and dynamin machineries.Taken together,these results demonstrate that AP complexes,similar to those in other eukaryotes,exist in plants,and that AP-3 plays a specific role in the regulation of biogenesis and function of vacuoles in plant cells.

  5. Ascent Heating Thermal Analysis on Spacecraft Adaptor Fairings

    Science.gov (United States)

    Wang, Xiao Yen; Yuko, James; Motil, Brian

    2011-01-01

    When the Crew Exploration Vehicle (CEV) is launched, the spacecraft adaptor (SA) fairings that cover the CEV service module (SM) are exposed to aero heating. Thermal analysis is performed to compute the fairing temperatures and to investigate whether the temperatures are within the material limits for nominal ascent aeroheating case. The ascent heating is analyzed by using computational fluid dynamics (CFD) and engineering codes at Marshall Space Flight Center. The aeroheating environment data used for this work is known as Thermal Environment 3 (TE3) heating data. One of the major concerns is with the SA fairings covering the CEV SM and the SM/crew launch vehicle (CLV) flange interface. The TE3 heating rate is a function of time, wall temperature, and the spatial locations. The implementation of the TE3 heating rate as boundary conditions in the thermal analysis becomes challenging. The ascent heating thermal analysis on SA fairings and SM/CLV flange interface are performed using two commercial software packages: Cullimore & Ring (C&R) Thermal Desktop (TD) 5.1 and MSC Patran 2007r1 b. TD is the pre-and post-processor for SINDA, which is a finite-difference-based solver. In TD, the geometry is built and meshed, the boundary conditions are defined, and then SINDA is used to compute temperatures. MSC Pthermal is a finite-element- based thermal solver. MSC Patran is the pre- and post-processor for Pthermal. Regarding the boundary conditions, the convection, contact resistance, and heat load can be imposed in different ways in both programs. These two software packages are used to build the thermal model for the same analysis to validate each other and show the differences in the modeling details.

  6. The bacterial lipocalins.

    Science.gov (United States)

    Bishop, R E

    2000-10-18

    The lipocalins were once regarded as a eukaryotic protein family, but new members have been recently discovered in bacteria. The first bacterial lipocalin (Blc) was identified in Escherichia coli as an outer membrane lipoprotein expressed under conditions of environmental stress. Blc is distinguished from most lipocalins by the absence of intramolecular disulfide bonds, but the presence of a membrane anchor is shared with two of its closest homologues, apolipoprotein D and lazarillo. Several common features of the membrane-anchored lipocalins suggest that each may play an important role in membrane biogenesis and repair. Additionally, Blc proteins are implicated in the dissemination of antibiotic resistance genes and in the activation of immunity. Recent genome sequencing efforts reveal the existence of at least 20 bacterial lipocalins. The lipocalins appear to have originated in Gram-negative bacteria and were probably transferred horizontally to eukaryotes from the endosymbiotic alpha-proteobacterial ancestor of the mitochondrion. The genome sequences also reveal that some bacterial lipocalins exhibit disulfide bonds and alternative modes of subcellular localization, which include targeting to the periplasmic space, the cytoplasmic membrane, and the cytosol. The relationships between bacterial lipocalin structure and function further illuminate the common biochemistry of bacterial and eukaryotic cells.

  7. Lipid raft-dependent FcepsilonRI ubiquitination regulates receptor endocytosis through the action of ubiquitin binding adaptors.

    Directory of Open Access Journals (Sweden)

    Rosa Molfetta

    Full Text Available The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcepsilonRI expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcepsilonRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcepsilonRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.

  8. Fuel combustion test in constant volume combustion chamber with built-in adaptor

    Institute of Scientific and Technical Information of China (English)

    JEONG; DongSoo; CHO; GyuBack; CHOI; SuJin; LEE; JinSoo

    2010-01-01

    Combustion tests of pre-mixture of methane and air in constant volume combustion chamber(CVCC) have been carried out by means of flame propagation photo and gas pressure measurement,the effects of CVCC body temperature,intake pressure of pre-mixture of methane and air,equivalence ratio and location of the built-in adaptor have been investigated.The whole combustion chamber can be divided into two parts,i.e.the upper combustion chamber and the lower combustion chamber,by the built-in adaptor with through hole.Owing to the built-in adaptor with through hole,jet ignition or compression ignition(auto-ignition) phenomena may occur in the lower combustion chamber,which is helpful to getting higher flame propagation velocity,higher combustion peak pressure,low cycle-to-cycle variation and more stable combustion process.

  9. Stepping motor adaptor actuator for a commercial uhv linear motion feedthrough

    International Nuclear Information System (INIS)

    An adaptor coupling has been developed that will allow the attachment of a standard stepping motor to a precision commercial (Varian) uhv linear motion feedthrough. The assembly, consisting of the motor, motor adaptor, limit switches, etc. is clamped to the feedthrough body which can be done under vacuum conditions if necessary. With a 500 step/rev. stepping motor the resolution is 1.27 μm per step. We presently use this assembly in a remote location for the precise positioning of a beam sensing monitor. 2 refs., 3 figs

  10. The Emerging and Diverse Roles of Src-Like Adaptor Proteins in Health and Disease

    Directory of Open Access Journals (Sweden)

    Nikolett Marton

    2015-01-01

    Full Text Available Although Src-like adaptor proteins (SLAP-1 and SLAP-2 were mainly studied in lymphocytes, where they act as negative regulators and provide fine control of receptor signaling, recently, several other functions of these proteins were discovered. In addition to the well-characterized immunoregulatory functions, SLAP proteins appear to have an essential role in the pathogenesis of type I hypersensitivity, osteoporosis, and numerous malignant diseases. Both adaptor proteins are expressed in a wide variety of tissues, where they have mostly inhibitory effects on multiple intracellular signaling pathways. In this review, we summarize the diverse effects of SLAP proteins.

  11. DMPD: Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17667936 Structure, function and regulation of the Toll/IL-1 receptor adaptor prote... (.svg) (.html) (.csml) Show Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. ...PubmedID 17667936 Title Structure, function and regulation of the Toll/IL-1 recep

  12. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    Science.gov (United States)

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein.

  13. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    Science.gov (United States)

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein. PMID:27231351

  14. Bacterial cellulose/boehmite composites

    International Nuclear Information System (INIS)

    Composites based on bacterial cellulose membranes and boehmite were obtained. SEM results indicate that the bacterial cellulose (BC) membranes are totally covered by boehmite and obtained XRD patterns suggest structural changes due to this boehmite addition. Thermal stability is accessed through TG curves and is dependent on boehmite content. Transparency is high comparing to pure BC as can be seen through UV-vis absorption spectroscopy. (author)

  15. Clinico-pathological Responses of Calves Associated with Infection of Pasteurella multocida Type B and the Bacterial Lipopolysaccharide and Outer Membrane Protein Immunogens

    Directory of Open Access Journals (Sweden)

    Faez Firdaus Jesse Abdullah

    2013-10-01

    Full Text Available The current study aims to investigate the Clinico-pathological responses of calves associated with the infections of Pasteurella multocida type B and the bacterial lipopolysaccharide and outer membrane protein immunogens. Alterations in the behavior of animals and pathological lesions observed following innate or experimental infections usually divulge extensive and detrimental changes in the clinical signs, organs and tissues of the animals afflicted with the disease. These alterations are imperative for Veterinary evaluation of herd health. Eight clinically healthy, non-pregnant and non-lactating Brangus cross heifers weighing 150±50 kg were used in the study. The heifers (n = 8 were divided into 4 groups of 2 calves per group. The control calves in group 1 were inoculated intramuscularly with 10 mL of sterile Phosphate Buffered Saline (PBS. Calves in group 2 were inoculated intramuscularly with 10 mL of 1012 colony forming unit (cfu of wild-type P. multocida and calves in group 3 were inoculated intravenously with 10 mL of LPS broth extract. Calves in group 4 were inoculated intramuscularly with 10 mL of OMP broth extract. All animals were observed for 48 h for clinical signs, changes in behavior and mortality pattern, including the time of death. The results divulged significant differences in the Clinico-pathological alterations. Calves inoculated with whole cell P. multocida type B: 2 showed a significant (p<0.05 increased in rectal temperature. The affected calves showed significant severe dullness (p<0.000 and significant rumen hypomotility (p<0.000 was also exhibited. The calves showed signs of hypersalivation at 14 h. There is no significant difference (p = 0.240 in pulmonary oedema in the Calves of group 2 compared to control group 1. Calves of group 4 also showed no significant difference in pulmonary oedema (p = 0.612 compared to control group 1. Calves of group 3 showed significantly moderate pulmonary oedema (p<0.000. All the

  16. The AP-3 adaptor complex is required for vacuolar function in Arabidopsis

    NARCIS (Netherlands)

    Zwiewka, M.; Feraru, E.; Moller, B.K.; Hwang, I.; Feraru, M.I.; Kleine-Vehn, J.; Weijers, D.; Friml, J.

    2011-01-01

    Subcellular trafficking is required for a multitude of functions in eukaryotic cells. It involves regulation of cargo sorting, vesicle formation, trafficking and fusion processes at multiple levels. Adaptor protein (AP) complexes are key regulators of cargo sorting into vesicles in yeast and mammals

  17. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.

    Science.gov (United States)

    Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M; Brandl, Holger; Schwich, Oliver D; Steiner, Michaela C; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M

    2016-03-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends. PMID:26944680

  18. Roles of the protruding loop of factor B essential for the localization of lipoproteins (LolB) in the anchoring of bacterial triacylated proteins to the outer membrane.

    Science.gov (United States)

    Hayashi, Yumi; Tsurumizu, Ryoji; Tsukahara, Jun; Takeda, Kazuki; Narita, Shin-ichiro; Mori, Makiko; Miki, Kunio; Tokuda, Hajime

    2014-04-11

    The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed. PMID:24569999

  19. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination.

    Directory of Open Access Journals (Sweden)

    Tonika Lam

    Full Text Available Class switch DNA recombination (CSR of the immunoglobulin heavy chain (IgH locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID expression and AID targeting to switch (S regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit and PKA-RIα (regulatory inhibitory subunit and uracil DNA glycosylase (Ung. 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198 or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR. 14-3-3 adaptors colocalized with AID and replication protein A (RPA in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr, an accessory protein of human immunodeficiency virus type-1 (HIV-1, which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

  20. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination.

    Science.gov (United States)

    Lam, Tonika; Thomas, Lisa M; White, Clayton A; Li, Guideng; Pone, Egest J; Xu, Zhenming; Casali, Paolo

    2013-01-01

    Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

  1. The adaptor molecule Nck localizes the WAVE complex to promote actin polymerization during CEACAM3-mediated phagocytosis of bacteria.

    Directory of Open Access Journals (Sweden)

    Stefan Pils

    Full Text Available BACKGROUND: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. PRINCIPAL FINDINGS: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. CONCLUSIONS: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.

  2. A transgenic Drosophila model demonstrates that the Helicobacter pylori CagA protein functions as a eukaryotic Gab adaptor.

    Directory of Open Access Journals (Sweden)

    Crystal M Botham

    2008-05-01

    Full Text Available Infection with the human gastric pathogen Helicobacter pylori is associated with a spectrum of diseases including gastritis, peptic ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. The cytotoxin-associated gene A (CagA protein of H. pylori, which is translocated into host cells via a type IV secretion system, is a major risk factor for disease development. Experiments in gastric tissue culture cells have shown that once translocated, CagA activates the phosphatase SHP-2, which is a component of receptor tyrosine kinase (RTK pathways whose over-activation is associated with cancer formation. Based on CagA's ability to activate SHP-2, it has been proposed that CagA functions as a prokaryotic mimic of the eukaryotic Grb2-associated binder (Gab adaptor protein, which normally activates SHP-2. We have developed a transgenic Drosophila model to test this hypothesis by investigating whether CagA can function in a well-characterized Gab-dependent process: the specification of photoreceptors cells in the Drosophila eye. We demonstrate that CagA expression is sufficient to rescue photoreceptor development in the absence of the Drosophila Gab homologue, Daughter of Sevenless (DOS. Furthermore, CagA's ability to promote photoreceptor development requires the SHP-2 phosphatase Corkscrew (CSW. These results provide the first demonstration that CagA functions as a Gab protein within the tissue of an organism and provide insight into CagA's oncogenic potential. Since many translocated bacterial proteins target highly conserved eukaryotic cellular processes, such as the RTK signaling pathway, the transgenic Drosophila model should be of general use for testing the in vivo function of bacterial effector proteins and for identifying the host genes through which they function.

  3. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 {mu}1A (AP-1 mu1A)

    Energy Technology Data Exchange (ETDEWEB)

    Sawasdee, Nunghathai; Junking, Mutita [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Ngaojanlar, Piengpaga [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Immunology and Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Sukomon, Nattakan; Ungsupravate, Duangporn [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Limjindaporn, Thawornchai [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Akkarapatumwong, Varaporn [Institute of Molecular Biosciences, Mahidol University at Salaya Campus, Nakorn Pathom 73170 (Thailand); Noisakran, Sansanee [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand)

    2010-10-08

    Research highlights: {yields} Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. {yields} Adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) was firstly reported to interact with kAE1. {yields} The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. {yields} AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. {yields} AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl{sup -}) and bicarbonate (HCO{sub 3}{sup -}) exchange at the basolateral membrane of kidney {alpha}-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange at the basolateral membrane and failure of proton (H{sup +}) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1

  4. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A)

    International Nuclear Information System (INIS)

    Research highlights: → Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. → Adaptor-related protein complex 1 μ1A (AP-1 mu1A) was firstly reported to interact with kAE1. → The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. → AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. → AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl-) and bicarbonate (HCO3-) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl-/HCO3- exchange at the basolateral membrane and failure of proton (H+) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells.

  5. RTK SLAP down: the emerging role of Src-like adaptor protein as a key player in receptor tyrosine kinase signaling.

    Science.gov (United States)

    Wybenga-Groot, Leanne E; McGlade, C Jane

    2015-02-01

    SLAP (Src like adaptor protein) contains adjacent Src homology 3 (SH3) and Src homology 2 (SH2) domains closely related in sequence to that of cytoplasmic Src family tyrosine kinases. Expressed most abundantly in the immune system, SLAP function has been predominantly studied in the context of lymphocyte signaling, where it functions in the Cbl dependent downregulation of antigen receptor signaling. However, accumulating evidence suggests that SLAP plays a role in the regulation of a broad range of membrane receptors including members of the receptor tyrosine kinase (RTK) family. In this review we highlight the role of SLAP in the ubiquitin dependent regulation of type III RTKs PDGFR, CSF-1R, KIT and Flt3, as well as Eph family RTKs. SLAP appears to bind activated type III and Eph RTKs via a conserved autophosphorylated juxtamembrane tyrosine motif in an SH2-dependent manner, suggesting that SLAP is important in regulating RTK signaling.

  6. Preparation and properties of polyvinyl alcohol (PVA) composites membranes based on bacterial cellulose (BC)%细菌纤维素基聚乙烯醇(BC/PVA)复合膜的制备及性能研究

    Institute of Scientific and Technical Information of China (English)

    张洪玉; 杨亮; 陆大年

    2012-01-01

    以细菌纤维素为增强体,聚乙烯醇为基体,甲醛为交联剂,在过饱和盐溶液中利用湿化学法制备BC/PVA复合膜.通过对复合膜溶胀性能、红外光谱分析、扫描电镜、热性能以及力学性能的测试分析,研究化学交联对复合膜性能的影响.结果表明:使用甲醛对复合膜进行交联处理后,在BC/PVA复合膜内形成了化学键结合,从而降低了复合膜的溶胀性能,增强了复合膜的力学性能以及热稳定性能.%Bacterial cellulose (BO/polyvinyl alcohol (PVA) composites membrane was prepared using BC as the reinforcement and PVA as the matrix materials in saturated salt solution with formaldehyde as cross-linking agent by wet chemical method. The effect of chemical cross-linking on the properties of composite membrane was investigated through the analysis of swelling property of composite membrane, infrared spectroscopic (IR), scanning electron microscope (SEM), thermal and mechanical properties tests. The results showed that the use of formaldehyde formed chemical bonds between the composite membranes, which led to the reduction of swelling property and the enhancement of the mechanical properties and thermal stability.

  7. ER Adaptor SCAP Translocates and Recruits IRF3 to Perinuclear Microsome Induced by Cytosolic Microbial DNAs

    Science.gov (United States)

    Yu, Huansha; Liu, Xing; Huang, Lulu; Wang, Qiang; Liu, Heng; Cui, Ye; Tang, Yijun; Zhang, Peng; Wang, Chen

    2016-01-01

    Stimulator of interferon genes (STING, also known as MITA, ERIS or MPYS) induces the activation of TBK1 kinase and IRF3 transcription factor, upon sensing of microbial DNAs. How IRF3 is recruited onto the STING signalosome remains unknown. We report here that silencing of the ER adaptor SCAP markedly impairs the IRF3-responsive gene expression induced by STING. Scap knockdown mice are more susceptible to HSV-1 infection. Interestingly, SCAP translocates from ER, via Golgi, to perinuclear microsome in a STING-dependent manner. Mechanistically, the N-terminal transmembrane domain of SCAP interacts with STING, and the C-terminal cytosolic domain of SCAP binds to IRF3, thus recruiting IRF3 onto STING signalosome. Mis-localization of SCAP abolishes its antiviral function. Collectively, this study characterizes SCAP as an essential adaptor in the STING signaling pathway, uncovering a critical missing link in DNAs-triggered host antiviral responses. PMID:26900919

  8. ER Adaptor SCAP Translocates and Recruits IRF3 to Perinuclear Microsome Induced by Cytosolic Microbial DNAs.

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2016-02-01

    Full Text Available Stimulator of interferon genes (STING, also known as MITA, ERIS or MPYS induces the activation of TBK1 kinase and IRF3 transcription factor, upon sensing of microbial DNAs. How IRF3 is recruited onto the STING signalosome remains unknown. We report here that silencing of the ER adaptor SCAP markedly impairs the IRF3-responsive gene expression induced by STING. Scap knockdown mice are more susceptible to HSV-1 infection. Interestingly, SCAP translocates from ER, via Golgi, to perinuclear microsome in a STING-dependent manner. Mechanistically, the N-terminal transmembrane domain of SCAP interacts with STING, and the C-terminal cytosolic domain of SCAP binds to IRF3, thus recruiting IRF3 onto STING signalosome. Mis-localization of SCAP abolishes its antiviral function. Collectively, this study characterizes SCAP as an essential adaptor in the STING signaling pathway, uncovering a critical missing link in DNAs-triggered host antiviral responses.

  9. PIP2: choreographer of actin-adaptor proteins in the HIV-1 dance

    Science.gov (United States)

    Rocha-Perugini, Vera; Gordon-Alonso, Mónica; Sánchez-Madrid, Francisco

    2014-01-01

    The actin cytoskeleton plays a key role during the replication cycle of human immunodeficiency virus-1 (HIV-1). HIV-1 infection is affected by cellular proteins that influence the clustering of viral receptors or the subcortical actin cytoskeleton. Several of these actin-adaptor proteins are controlled by the second messenger phosphatidylinositol 4,5-biphosphate (PIP2), an important regulator of actin organization. PIP2 production is induced by HIV-1 attachment and facilitates viral infection. However, the importance of PIP2 in regulating cytoskeletal proteins and thus HIV-1 infection has been overlooked. This review examines recent reports describing the roles played by actin-adaptor proteins during HIV-1 infection of CD4+ T cells, highlighting the influence of the signaling lipid PIP2 in this process. PMID:24768560

  10. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire.

    Science.gov (United States)

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-08-31

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability. PMID:20709959

  11. Role of SRC-like adaptor protein (SLAP) in immune and malignant cell signaling.

    Science.gov (United States)

    Kazi, Julhash U; Kabir, Nuzhat N; Rönnstrand, Lars

    2015-07-01

    SRC-like adaptor protein (SLAP) is an adaptor protein structurally similar to the SRC family protein kinases. Like SRC, SLAP contains an SH3 domain followed by an SH2 domain but the kinase domain has been replaced by a unique C-terminal region. SLAP is expressed in a variety of cell types. Current studies suggest that it regulates signaling of various cell surface receptors including the B cell receptor, the T cell receptor, cytokine receptors and receptor tyrosine kinases which are important regulator of immune and cancer cell signaling. SLAP targets receptors, or its associated components, by recruiting the ubiquitin machinery and thereby destabilizing signaling. SLAP directs receptors to ubiquitination-mediated degradation and controls receptors turnover as well as signaling. Thus, SLAP appears to be an important component in regulating signal transduction required for immune and malignant cells.

  12. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire.

    Science.gov (United States)

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-08-31

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability.

  13. Preparation and properties of electromagnetic functional composite membrane based on bacterial cellulose%细菌纤维素基电磁功能复合膜的制备与性能研究∗

    Institute of Scientific and Technical Information of China (English)

    韩谨潞; 李琪琪; 汤廉; 陈仕艳; 王华平

    2015-01-01

    以细菌纤维素(BC)基体材料,通过共沉淀原位复合法制备纳米钴铁氧体(CoFe2 O4)/细菌纤维素磁性复合膜;并在此基础上,通过原位化学氧化聚合法,制备了聚吡咯/钴铁氧体/细菌纤维素(PPy/CoFe2 O4/BC)复合膜,对其结构性能及应用进行研究。结果表明,PPy/CoFe2 O4/BC复合膜仍然保持了 BC的三维网状结构。当吡咯单体浓度为0.07 mol/L时,复合膜由连续的核壳结构构成,电导率稳定在0.4 S/cm 左右,其电磁屏蔽效能在25 dB左右,是一种良好的民用或商用电磁屏蔽材料。%CoFe2 O4/BC magnetic composite membranes based on bacterial cellulose (BC)were prepared through coprecipitation and in situ composition.Then,the flexible PPy/CoFe2 O4/BC functional membranes with elec-tromagnetivity were synthesized successfully through in situ chemical synthesis and the structure,properties and applications were investigated.The results revealed that the PPy/CoFe2 O4/BC membranes remained the ul-trafine network architecture of BC template.The membranes demonstrated a continue core-shell structure when the concentration of pyrrole was 0.07 mol/L.Moreover,the conductivity of composite membranes were about 0.4 S/cm and the PPy/CoFe2 O4/BC composite membranes revealed a good electromagnetic shielding efficiency with the value of SE 25 dB,which indicate a good electromagnetic interference shielding material in daily life.

  14. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire

    OpenAIRE

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D.; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-01-01

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, pe...

  15. Autoinhibition of Mint1 adaptor protein regulates amyloid precursor protein binding and processing

    OpenAIRE

    Matos, Maria F.; Xu, Yibin; Dulubova, Irina; Otwinowski, Zbyszek; Richardson, John M.; Tomchick, Diana R.; Rizo, Josep; Ho, Angela

    2012-01-01

    Mint adaptor proteins bind to the amyloid precursor protein (APP) and regulate APP processing associated with Alzheimer’s disease; however, the molecular mechanisms underlying Mint regulation in APP binding and processing remain unclear. Biochemical, biophysical, and cellular experiments now show that the Mint1 phosphotyrosine binding (PTB) domain that binds to APP is intramolecularly inhibited by the adjacent C-terminal linker region. The crystal structure of a C-terminally extended Mint1 PT...

  16. Chromatin Adaptor Brd4 Modulates E2 Transcription Activity and Protein Stability*

    OpenAIRE

    Lee, A-Young; Chiang, Cheng-Ming

    2009-01-01

    Brd4 is a chromatin adaptor containing tandem bromodomains binding to acetylated histone H3 and H4. Although Brd4 has been implicated in the transcriptional control of papillomavirus-encoded E2 protein, it is unclear how Brd4 regulates E2 function and whether the involvement of Brd4 in transactivation and transrepression is common to different types of E2 proteins. Using DNase I footprinting performed with in vitro reconstituted human papillomavirus (HPV) chromatin and...

  17. Bias in ligation-based small RNA sequencing library construction is determined by adaptor and RNA structure.

    Directory of Open Access Journals (Sweden)

    Ryan T Fuchs

    Full Text Available High-throughput sequencing (HTS has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA and other small RNAs (sRNA. Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps, specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material.

  18. The Lnk adaptor protein: a key regulator of normal and pathological hematopoiesis.

    Science.gov (United States)

    Velazquez, Laura

    2012-12-01

    The development and function of blood cells are regulated by specific growth factors/cytokines and their receptors' signaling pathways. In this way, these factors influence cell survival, proliferation and differentiation of hematopoietic cells. Central to this positive and/or negative control are the adaptor proteins. Since their identification 10 years ago, members of the Lnk adaptor protein family have proved to be important activators and/or inhibitors in the hematopoietic, immune and vascular system. In particular, the generation of animal and cellular models for the Lnk and APS proteins has helped establish the physiological role of these molecules through the identification of their specific signaling pathways and the characterization of their binding partners. Moreover, the recent identification of mutations in the LNK gene in myeloproliferative disorders, as well as the correlation of a single nucleotide polymorphism on LNK with hematological, immune and vascular diseases have suggested its involvement in the pathophysiology of these malignancies. The latter findings have thus raised the possibility of addressing Lnk signaling for the treatment of certain human diseases. This review therefore describes the pathophysiological role of this adaptor protein in hematological malignancies and the potential benefits of Lnk therapeutic targeting.

  19. The adaptor molecule Trif contributes to murine host defense during Leptospiral infection.

    Science.gov (United States)

    Jayaraman, Priya A; Devlin, Amy A; Miller, Jennifer C; Scholle, Frank

    2016-09-01

    Leptospirosis is a zoonotic disease and is caused by pathogenic species of the Leptospira genus, including Leptospira interrogans (L. interrogans). Humans, domestic and wild animals are susceptible to acute or chronic infection. The innate immune response is a critical defense mechanism against Leptospira interrogans, and has been investigated in mouse models. Murine Toll-like receptors (TLRs) have been shown to be key factors in sensing and responding to L. interrogans infection. Specifically, TLR2, TLR4 and the TLR adaptor molecule MyD88 are essential for host defense against L. interrogans; however, the role of the TLR adaptor molecule TIR-domain-containing adaptor-inducing interferon β (TRIF) in the response to L. interrogans has not been previously determined. In the present study, TRIF was found to play an important role during leptospiral infection. Following challenge with L. interrogans, Trif(-/-) mice exhibited delayed weight gain compared to wild-type mice. Moreover, Trif(-/-) mice exhibited an increase in L. interrogans burden in the kidneys, lungs, and blood at early time points (less than 7days post infection). Multiple components of the innate immune responses were dampened in response to leptospiral infection including transcription and production of cytokines, and the humoral response, which suggested that TRIF contributes to expression and production of cytokines important for the host defense against L. interrogans. PMID:27259371

  20. Science Signaling Podcast for 12 July 2016: Adaptor proteins limit signaling.

    Science.gov (United States)

    Wiley, H Steven; VanHook, Annalisa M

    2016-01-01

    This Podcast features an interview with Steven Wiley, senior author of a Research Article that appears in the 12 July 2016 issue of Science Signaling, about how the abundance of adaptor proteins and feedback regulators affect the flow of information downstream of the epidermal growth factor receptor (EGFR). Information flows through a signaling pathway by sequential interactions between core components of the pathway, many of which have enzymatic activity. Adaptor proteins do not directly participate in relaying the signal and do not have enzymatic activity, but are important for signaling because they facilitate interactions between the core components. Using quantitative methods, Shi et al demonstrated that core components of the EGFR pathway were highly abundant in both normal cells and cancer cells. However, adaptor proteins were present in much lower abundance in both cell types, indicating that it is the abundance of these proteins that limit signaling downstream of EGFR. The authors also found that differences in EGFR signaling between different cell types likely resulted from the variable abundance of feedback regulators.Listen to Podcast. PMID:27405978

  1. Versatile modes of peptide recognition by the AAA+ adaptor protein SspB

    Energy Technology Data Exchange (ETDEWEB)

    Levchenko, Igor; Grant, Robert A.; Flynn, Julia M.; Sauer, Robert T.; Baker, Tania A. (MIT)

    2010-07-19

    Energy-dependent proteases often rely on adaptor proteins to modulate substrate recognition. The SspB adaptor binds peptide sequences in the stress-response regulator RseA and in ssrA-tagged proteins and delivers these molecules to the AAA+ ClpXP protease for degradation. The structure of SspB bound to an ssrA peptide is known. Here, we report the crystal structure of a complex between SspB and its recognition peptide in RseA. Notably, the RseA sequence is positioned in the peptide-binding groove of SspB in a direction opposite to the ssrA peptide, the two peptides share only one common interaction with the adaptor, and the RseA interaction site is substantially larger than the overlapping ssrA site. This marked diversity in SspB recognition of different target proteins indicates that it is capable of highly flexible and dynamic substrate delivery.

  2. New Insights to Clathrin and Adaptor Protein 2 for the Design and Development of Therapeutic Strategies

    Directory of Open Access Journals (Sweden)

    Ebbe Toftgaard Poulsen

    2015-12-01

    Full Text Available The Amyloid Precursor Protein (APP has been extensively studied for its role as the precursor of the β-amyloid protein (Aβ in Alzheimer’s disease (AD. However, our understanding of the normal function of APP is still patchy. Emerging evidence indicates that a dysfunction in APP trafficking and degradation can be responsible for neuronal deficits and progressive degeneration in humans. We recently reported that the Y682 mutation in the 682YENPTY687 domain of APP affects its binding to specific adaptor proteins and leads to its anomalous trafficking, to defects in the autophagy machinery and to neuronal degeneration. In order to identify adaptors that influence APP function, we performed pull-down experiments followed by quantitative mass spectrometry (MS on hippocampal tissue extracts of three month-old mice incubated with either the 682YENPTY687 peptide, its mutated form, 682GENPTY687 or its phosphorylated form, 682pYENPTY687. Our experiments resulted in the identification of two proteins involved in APP internalization and trafficking: Clathrin heavy chain (hc and its Adaptor Protein 2 (AP-2. Overall our results consolidate and refine the importance of Y682 in APP normal functions from an animal model of premature aging and dementia. Additionally, they open the perspective to consider Clathrin hc and AP-2 as potential targets for the design and development of new therapeutic strategies.

  3. Recycling endosomes in apical plasma membrane domain formation and epithelial cell polarity

    NARCIS (Netherlands)

    Golachowska, Magdalena R.; Hoekstra, Dick; van IJzendoorn, Sven C. D.

    2010-01-01

    Recycling endosomes have taken central stage in the intracellular sorting and polarized trafficking of apical and basolateral plasma membrane components. Molecular players in the underlying mechanisms are now emerging, including small GTPases, class V myosins and adaptor proteins. In particular, def

  4. Membrane tension is a key determinant of bud morphology in clathrin-mediated endocytosis

    CERN Document Server

    Hassinger, Julian E; Drubin, David G; Rangamani, Padmini

    2016-01-01

    In clathrin-mediated endocytosis (CME), clathrin and various adaptor proteins coat a patch of the plasma membrane, which is reshaped to form a budded vesicle. Experimental studies have demonstrated that elevated membrane tension can inhibit bud formation by a clathrin coat. In this study, we investigate the impact of membrane tension on the mechanics of membrane budding by simulating clathrin coats that either grow in area or progressively induce greater curvature. At low membrane tension, progressively increasing the area of a curvature-generating coat causes the membrane to smoothly evolve from a flat to budded morphology, whereas the membrane remains essentially flat at high membrane tensions. Interestingly, at physiologically relevant, intermediate membrane tensions, the shape evolution of the membrane undergoes a snapthrough instability in which increasing coat area causes the membrane to "snap" from an open, U-shaped bud to a closed, $\\Omega$-shaped bud. This instability is accompanied by a large energy...

  5. Modulation of Membrane Influx and Efflux in Escherichia coli Sequence Type 131 Has an Impact on Bacterial Motility, Biofilm Formation, and Virulence in a Caenorhabditis elegans Model.

    Science.gov (United States)

    Pantel, Alix; Dunyach-Remy, Catherine; Ngba Essebe, Christelle; Mesureur, Jennifer; Sotto, Albert; Pagès, Jean-Marie; Nicolas-Chanoine, Marie-Hélène; Lavigne, Jean-Philippe

    2016-05-01

    Energy-dependent efflux overexpression and altered outer membrane permeability (influx) can promote multidrug resistance (MDR). The present study clarifies the regulatory pathways that control membrane permeability in the pandemic clone Escherichia coli sequence type 131 (ST131) and evaluates the impact of efflux and influx modulations on biofilm formation, motility, and virulence in the Caenorhabditis elegans model. Mutants of two uropathogenic E. coli (UPEC) strains, MECB5 (ST131; H30-Rx) and CFT073 (ST73), as well as a fecal strain, S250 (ST131; H22), were in vitro selected using continuous subculture in subinhibitory concentrations of ertapenem (ETP), chloramphenicol (CMP), and cefoxitin (FOX). Mutations in genes known to control permeability were shown for the two UPEC strains: MECB5-FOX (deletion of 127 bp in marR; deletion of 1 bp and insertion of an IS1 element in acrR) and CFT073-CMP (a 1-bp deletion causing a premature stop in marR). We also demonstrated that efflux phenotypes in the mutants selected with CMP and FOX were related to the AcrAB-TolC pump, but also to other efflux systems. Alteration of membrane permeability, caused by underexpression of the two major porins, OmpF and OmpC, was shown in MECB5-ETP and mutants selected with FOX. Lastly, our findings suggest that efflux pump-overproducing isolates (CMP mutants) pose a serious threat in terms of virulence (significant reduction in worm median survival) and host colonization. Lack of porins (ETP and FOX mutants) led to a high level of antibiotic resistance in an H30-Rx subclone. Nevertheless, this adaptation created a physiological disadvantage (decreased motility and ability to form biofilm) associated with a low potential for virulence. PMID:26926643

  6. Modulation of Membrane Influx and Efflux in Escherichia coli Sequence Type 131 Has an Impact on Bacterial Motility, Biofilm Formation, and Virulence in a Caenorhabditis elegans Model

    Science.gov (United States)

    Pantel, Alix; Dunyach-Remy, Catherine; Ngba Essebe, Christelle; Mesureur, Jennifer; Sotto, Albert; Nicolas-Chanoine, Marie-Hélène

    2016-01-01

    Energy-dependent efflux overexpression and altered outer membrane permeability (influx) can promote multidrug resistance (MDR). The present study clarifies the regulatory pathways that control membrane permeability in the pandemic clone Escherichia coli sequence type 131 (ST131) and evaluates the impact of efflux and influx modulations on biofilm formation, motility, and virulence in the Caenorhabditis elegans model. Mutants of two uropathogenic E. coli (UPEC) strains, MECB5 (ST131; H30-Rx) and CFT073 (ST73), as well as a fecal strain, S250 (ST131; H22), were in vitro selected using continuous subculture in subinhibitory concentrations of ertapenem (ETP), chloramphenicol (CMP), and cefoxitin (FOX). Mutations in genes known to control permeability were shown for the two UPEC strains: MECB5-FOX (deletion of 127 bp in marR; deletion of 1 bp and insertion of an IS1 element in acrR) and CFT073-CMP (a 1-bp deletion causing a premature stop in marR). We also demonstrated that efflux phenotypes in the mutants selected with CMP and FOX were related to the AcrAB-TolC pump, but also to other efflux systems. Alteration of membrane permeability, caused by underexpression of the two major porins, OmpF and OmpC, was shown in MECB5-ETP and mutants selected with FOX. Lastly, our findings suggest that efflux pump-overproducing isolates (CMP mutants) pose a serious threat in terms of virulence (significant reduction in worm median survival) and host colonization. Lack of porins (ETP and FOX mutants) led to a high level of antibiotic resistance in an H30-Rx subclone. Nevertheless, this adaptation created a physiological disadvantage (decreased motility and ability to form biofilm) associated with a low potential for virulence. PMID:26926643

  7. Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

    Institute of Scientific and Technical Information of China (English)

    Takuya Watanabe; Masumi Tsuda; Yoshinori Makino; Tassos Konstantinou; Hiroshi Nishihara; Tokifumi Majima; Akio Minami; Stephan M Feller; Shinya Tanaka

    2009-01-01

    Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extraceilular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of Crkll demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of Crkll, is critical for the induction of Gabl-Y307 phosphorylation. SH2 mutation of Crkll also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gabl, whereas Crk-SH3(N) interacted with the Gabl mutant, which lacks the clus-tered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The GabI-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxiilin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory-lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

  8. Dephosphorylation of the adaptor LAT and phospholipase C-γ by SHP-1 inhibits natural killer cell cytotoxicity.

    Science.gov (United States)

    Matalon, Omri; Fried, Sophia; Ben-Shmuel, Aviad; Pauker, Maor H; Joseph, Noah; Keizer, Danielle; Piterburg, Marina; Barda-Saad, Mira

    2016-01-01

    Natural killer (NK) cells discriminate between healthy cells and virally infected or transformed self-cells by tuning activating and inhibitory signals received through cell surface receptors. Inhibitory receptors inhibit NK cell function by recruiting and activating the tyrosine phosphatase Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-1 (SHP-1) to the plasma membrane. However, to date, the guanine nucleotide exchange factor VAV1 is the only direct SHP-1 substrate identified in NK cells. We reveal that the adaptor protein linker for activation of T cells (LAT) as well as phospholipase C-γ1 (PLC-γ1) and PLC-γ2 are SHP-1 substrates. Dephosphorylation of Tyr(132) in LAT by SHP-1 in NK cells abrogated the recruitment of PLC-γ1 and PLC-γ2 to the immunological synapse between the NK cell and a cancer cell target, which reduced NK cell degranulation and target cell killing. Furthermore, the ubiquitylation of LAT by the E3 ubiquitin ligases c-Cbl and Cbl-b, which was induced by LAT phosphorylation, led to the degradation of LAT in response to the engagement of inhibitory receptors on NK cells, which abrogated NK cell cytotoxicity. Knockdown of the Cbl proteins blocked LAT ubiquitylation, which promoted NK cell function. Expression of a ubiquitylation-resistant mutant LAT blocked inhibitory receptor signaling, enabling cells to become activated. Together, these data identify previously uncharacterized SHP-1 substrates and inhibitory mechanisms that determine the response of NK cells. PMID:27221712

  9. Arrestin-mediated endocytosis of yeast plasma membrane transporters.

    Science.gov (United States)

    Nikko, Elina; Pelham, Hugh R B

    2009-12-01

    Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters.

  10. Discs-Large and Strabismus are functionally linked to plasma membrane formation

    OpenAIRE

    Lee, Ok-Kyung; Frese, Kristopher K.; James, Jennifer S.; Chadda, Darshana; Chen, Zhi-Hong; Javier, Ronald T.; Cho, Kyung-Ok

    2003-01-01

    During early embryogenesis in Drosophila melanogaster, extensive vesicle transport occurs to build cell boundaries for 6,000 nuclei. Here we show that this important process depends on a functional complex formed between the tumour suppressor and adaptor protein Discs-Large (Dlg)1 and the integral membrane protein Strabismus (Stbm)/Van Gogh (Vang)2,3. In support of this idea, embryos with mutations in either dlg or stbm displayed severe defects in plasma membrane formation. Conversely, overex...

  11. Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

    OpenAIRE

    Kirchhausen, Tom; Owen, David; Harrison, Stephen C.

    2014-01-01

    Clathrin is a molecular scaffold for vesicular uptake of cargo at the plasma membrane, where its assembly into cage-like lattices underlies the clathrin-coated pits of classical endocytosis. This review describes the structures of clathrin, major cargo adaptors, and other proteins that participate in forming a clathrin-coated pit, loading its contents, pinching off the membrane as a lattice-enclosed vesicle, and recycling the components. It integrates as much of the structural information as ...

  12. Evidence for an evolutionary relationship between the large adaptor nucleoporin Nup192 and karyopherins.

    Science.gov (United States)

    Stuwe, Tobias; Lin, Daniel H; Collins, Leslie N; Hurt, Ed; Hoelz, André

    2014-02-18

    Nucleocytoplasmic transport is facilitated by nuclear pore complexes (NPCs), which are massive proteinaceous transport channels embedded in the nuclear envelope. Nup192 is a major component of an adaptor nucleoporin subcomplex proposed to link the NPC coat with the central transport channel. Here, we present the structure of the ∼110-kDa N-terminal domain (NTD) of Nup192 at 2.7-Å resolution. The structure reveals an open ring-shaped architecture composed of Huntingtin, EF3, PP2A, and TOR1 (HEAT) and Armadillo (ARM) repeats. A comparison of different conformations indicates that the NTD consists of two rigid halves connected by a flexible hinge. Unexpectedly, the two halves of the ring are structurally related to karyopherin-α (Kap-α) and β-karyopherin family members. Biochemically, we identify a conserved patch that binds an unstructured segment in Nup53 and show that a C-terminal tail region binds to a putative helical fragment in Nic96. The Nup53 segment that binds Nup192 is a classical nuclear localization-like sequence that interacts with Kap-α in a mutually exclusive and mechanistically distinct manner. The disruption of the Nup53 and Nic96 binding sites in vivo yields growth and mRNA export defects, revealing their critical role in proper NPC function. Surprisingly, both interactions are dispensable for NPC localization, suggesting that Nup192 possesses another nucleoporin interaction partner. These data indicate that the structured domains in the adaptor nucleoporin complex are held together by peptide interactions that resemble those found in karyopherin•cargo complexes and support the proposal that the adaptor nucleoporins arose from ancestral karyopherins. PMID:24505056

  13. Structural basis for the interaction of the adaptor protein grb14 with activated ras.

    Directory of Open Access Journals (Sweden)

    Rohini Qamra

    Full Text Available Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including a Ras-associating (RA domain, a pleckstrin-homology (PH domain, a family-specific BPS (between PH and SH2 region, and a C-terminal Src-homology-2 (SH2 domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V. The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity.

  14. Involvement of β3A Subunit of Adaptor Protein-3 in Intracellular Trafficking of Receptor-like Protein Tyrosine Phosphatase PCP-2

    Institute of Scientific and Technical Information of China (English)

    Hui DONG; Hong YUAN; Weirong JIN; Yan SHEN; Xiaojing XU; Hongyang WANG

    2007-01-01

    PCP-2 is a human receptor-like protein tyrosine phosphatase and a member of the MAM domain family cloned in human pancreatic adenocarcinoma cells. Previous studies showed that PCP-2 directly interacted with β-catenin through the juxtamembrane domain, dephosphorylated β-catenin and played an important role in the regulation of cell adhesion. Recent study showed that PCP-2 was also involved in the repression of β-catenin-induced transcriptional activity. Here we describe the interactions of PCP-2 with the β3A subunit of adaptor protein (AP)-3 and sorting nexin (SNX) 3. These protein complexes were detected using the yeast two-hybrid assay with the juxtamembrane and membrane-proximal catalytic domain of PCP-2 as "bait". Both AP-3 and SNX3 are molecules involved in intracellular trafficking of membrane receptors. The association between the β3A subunit of AP-3 and PCP-2 was further confirmed in mammalian cells. Our results suggested a possible mechanism of intracellular trafficking of PCP-2 mediated by AP-3 and SNX3 which might participate in the regulation of PCP-2 functions.

  15. Early observation of bacterial cellulose membrane for repair of dural defects in rabbits%细菌纤维素膜修复兔硬脑膜缺损的早期观察

    Institute of Scientific and Technical Information of China (English)

    徐晨; 陈世文; 田恒力; 王敢; 郭衍; 袁陆涛

    2013-01-01

    right dural defects were repaired with bacteria cellulose membrane, the left dural defects were repaired with artificial dura matter, and the tissues on the repaired sites were collected on day 7, 14 and 21 after operation for determination of expression of interleukin-1β (IL-1β) , interleukin-6 ( IL-6) and tumor necrosis factor-a (TNF-α) mRNA by RT-PCR. Results All rabbits survived without the presence of wound infection. On the patched side of group A, bacteria cellulose membrane evenly covered the surface of brain without adhesion and visible inflammatory cells, fibrous connective tissues proliferated on the outer side of bacteria cellulose membrane, fibroblasts uniformly distributed in the inner side, and new blood vessels appeared. On the unpatched side of group A, the brain was directly connected with subcutaneous tissues. In group B, RT-PCR revealed that the expression of IL-1 β and IL-6 mRNA in bacteria cellulose membrane was significantly lower than that in artificial dura matter at each time point, while the expression of TNF-α mRNA of both sides was similar. Conclusion Bacterial cellulose membrane may avoid the adhesion to the brain tissues and has slighter early inflammatory response in repair of dural defects. Bacterial cellulose membrane may be used as the ideal substitute material for dura matter.

  16. Effectiveness of Needleless Vial Adaptors and Blunt Cannulas for Drug Administration in a Microgravity Environment

    Science.gov (United States)

    Hailey, M.; Bayuse, T.

    2010-01-01

    Fluid Isolation in the medication vial: Air/ fluid isolation maneuvers were used to move the medication to the septum end of vial. This isolation may be achieved in multiple ways based on the experience of the astronaut with fluid management in microgravity. If vial adaptors/blunt cannula or syringe assembly is inserted into the to vial before fluid isolation commences, the stability of this assembly should be considered in an effort to limit the risk of "slinging off" of the vial during isolation. Alternatively, fluid isolation can be performed prior to attaching the syringe/vial adaptor assembly. Terrestrial practices for medication withdrawal from a nonvented vial require injection of an equivalent amount of air as the expected medication volume prior to withdrawing liquid. In microgravity, this action is still valid, however the injection of additional air into the vial creates a multitude of micro bubbles and increases the volume of medication mixed with air that then must be withdrawn to achieve the desired drug volume in syringe. This practice is more likely to be required when using vials >30ml in size and injection volumes >10mL. It is felt that based on the microgravity flight, the practice of air injection is more of a hindrance than help.

  17. The Adaptor Protein Rai/ShcC Promotes Astrocyte-Dependent Inflammation during Experimental Autoimmune Encephalomyelitis.

    Science.gov (United States)

    Ulivieri, Cristina; Savino, Maria Teresa; Luccarini, Ilaria; Fanigliulo, Emanuela; Aldinucci, Alessandra; Bonechi, Elena; Benagiano, Marisa; Ortensi, Barbara; Pelicci, Giuliana; D'Elios, Mario Milco; Ballerini, Clara; Baldari, Cosima Tatiana

    2016-07-15

    Th17 cells have been casually associated to the pathogenesis of autoimmune disease. We have previously demonstrated that Rai/ShcC, a member of the Shc family of adaptor proteins, negatively regulates Th17 cell differentiation and lupus autoimmunity. In this study, we have investigated the pathogenic outcome of the Th17 bias associated with Rai deficiency on multiple sclerosis development, using the experimental autoimmune encephalomyelitis (EAE) mouse model. We found that, unexpectedly, EAE was less severe in Rai(-/-) mice compared with their wild-type counterparts despite an enhanced generation of myelin-specific Th17 cells that infiltrated into the CNS. Nevertheless, when adoptively transferred into immunodeficient Rai(+/+) mice, these cells promoted a more severe disease compared with wild-type encephalitogenic Th17 cells. This paradoxical phenotype was caused by a dampened inflammatory response of astrocytes, which were found to express Rai, to IL-17. The results provide evidence that Rai plays opposite roles in Th17 cell differentiation and astrocyte activation, with the latter dominant over the former in EAE, highlighting this adaptor as a potential novel target for the therapy of multiple sclerosis. PMID:27288534

  18. The endocytic adaptor Eps15 controls marginal zone B cell numbers.

    Directory of Open Access Journals (Sweden)

    Benedetta Pozzi

    Full Text Available Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220⁺ bone marrow cells, CD19⁻ thymocytes and splenic marginal zone (MZ B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis.

  19. Synthetic protein scaffolds based on peptide motifs and cognate adaptor domains for improving metabolic productivity

    Directory of Open Access Journals (Sweden)

    Anselm H.C. Horn

    2015-11-01

    Full Text Available The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.

  20. 纳米银在细菌纤维素凝胶膜中的原位合成及性能表征%Synthesis, Structural Characteristics and Properties of Silver Nanoparticles in situ Bacterial Cellulose Gelatinous Membrane

    Institute of Scientific and Technical Information of China (English)

    吴健; 郑裕东; 高爽; 郭佳; 崔秋艳; 丁寻; 陈晓华

    2013-01-01

    Silver nanoparticles/bacterial cellulose ( n-Ag/BC) composite membranes were prepared and characterized. Silver nanoparticles were synthesized in situ through the reaction of Tollens' reagent with aldehydes , under ambient conditions in nanoporous bacterial cellulose membrances as nanoreactors. Sliver nanop-article was readily obtained and grew into the BC network by the precipitation of above reaction products. BC films were prepared and loaded with ca. 0. 14 mg/cm2 to ca. 0. 42 mg/cm2 of silver nanoparticles. XRD patterns indicated the existence of Ag0 nanoparticles in the BC, and the diameter of the silver nanoparticles is ca. 31. 8 nm. Scanning electron microscopy (SEM) images showed that the sliver nanoparticles (size range dozens nm) well dispersed in the network of BC. The sliver nanoparticle-impregnated BC membranes exhibited high hydrophilic ability and strong antimicrobial activity. Bacterial killing efficiencies of the silver loaded films were investigated against staphylococcus aureus. It was determined that as little as ca. 0. 14 mg/cm2 of silver in the BC films caused a reduction of 99% bacteria in suspensions incubated in contact with the films. Significantly, the n-Ag/BC antimicrobial membranes were good biocompatibility, and allowed the attachment and growth of the epidermal cells. The result shows an easy method to synthesis silver nanoparticles into BC membranes. The n-Ag/BC has stability, antimicrobial activity and biological properties. The preparative procedure is facile, and provides a simple route to manufacturing of useful antimicrobial membranes, which would be a good alternative for antimicrobial wound dressing.%在细菌纤维素纳米纤维网络结构中采用吐伦试剂与含醛基化合物原位反应生成纳米银颗粒,制备了纳米银/细菌纤维素(n-Ag/BC)复合凝胶膜,研究了不同反应条件对复合材料的银含量、化学结构和晶体结构的影响以及n-Ag/BC的微观结构和纳米银在纤维素网络中

  1. Regulation and function of the CD3¿ DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro

    DEFF Research Database (Denmark)

    Dietrich, J; Kastrup, J; Nielsen, B L;

    1997-01-01

    /CD3gamma chimeras; and in vitro by binding CD3gamma peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3gamma D127xxxLL131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4....../ CD3gamma molecules independently of S126. An acidic amino acid is required at position 127 and a leucine (L) is required at position 131, whereas the requirements for position 132 are more relaxed. The spacing between aspartic acid 127 (D127) and L131 is crucial for the function of the motif in vivo...... subunit clusters of differentiation (CD)3gamma. Thus, phosphorylation of CD3gamma serine 126 (S126) causes a downregulation of the TCR. In this study, we have analyzed the CD3gamma internalization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4...

  2. The Adaptor Protein Myd88 Is a Key Signaling Molecule in the Pathogenesis of Irinotecan-Induced Intestinal Mucositis.

    Directory of Open Access Journals (Sweden)

    Deysi V T Wong

    Full Text Available Intestinal mucositis is a common side effect of irinotecan-based anticancer regimens. Mucositis causes cell damage, bacterial/endotoxin translocation and production of cytokines including IL-1 and IL-18. These molecules and toll-like receptors (TLRs activate a common signaling pathway that involves the Myeloid Differentiation adaptor protein, MyD88, whose role in intestinal mucositis is unknown. Then, we evaluated the involvement of TLRs and MyD88 in the pathogenesis of irinotecan-induced intestinal mucositis. MyD88-, TLR2- or TLR9-knockout mice and C57BL/6 (WT mice were given either saline or irinotecan (75 mg/kg, i.p. for 4 days. On day 7, animal survival, diarrhea and bacteremia were assessed, and following euthanasia, samples of the ileum were obtained for morphometric analysis, myeloperoxidase (MPO assay and measurement of pro-inflammatory markers. Irinotecan reduced the animal survival (50% and induced a pronounced diarrhea, increased bacteremia, neutrophil accumulation in the intestinal tissue, intestinal damage and more than twofold increased expression of MyD88 (200%, TLR9 (400%, TRAF6 (236%, IL-1β (405%, IL-18 (365%, COX-2 (2,777% and NF-κB (245% in the WT animals when compared with saline-injected group (P<0.05. Genetic deletion of MyD88, TLR2 or TLR9 effectively controlled the signs of intestinal injury when compared with irinotecan-administered WT controls (P<0.05. In contrast to the MyD88-/- and TLR2-/- mice, the irinotecan-injected TLR9-/- mice showed a reduced survival, a marked diarrhea and an enhanced expression of IL-18 versus irinotecan-injected WT controls. Additionally, the expression of MyD88 was reduced in the TLR2-/- or TLR9-/- mice. This study shows a critical role of the MyD88-mediated TLR2 and TLR9 signaling in the pathogenesis of irinotecan-induced intestinal mucositis.

  3. Novicidin interactions with phospholipid membranes

    DEFF Research Database (Denmark)

    Balakrishnan, Vijay Shankar

    Antimicrobial peptides target bacterial cell membranes and are considered as potential antibiotics. Their interactions with cell membranes are studied using different approaches. This thesis comprises of the biophysical investigations on the antimicrobial peptide Novicidin, interacting with...... liposomes. The lipid-induced changes in the peptide due to membrane binding, and the peptide-induced changes in the membrane properties were investigated using various spectroscopic and calorimetric methods, and the structural and thermodynamic aspects of peptide-lipid interactions are discussed. This helps...... in understanding not only the antimicrobial nature of Novicidin, but also sheds light on the membrane-peptide interactions....

  4. Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

    Science.gov (United States)

    Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2014-07-01

    Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells.

  5. Bacterial Vaginosis

    Science.gov (United States)

    ... 586. Related Content STDs during Pregnancy Fact Sheet Pregnancy and HIV, Viral Hepatitis, and STD Prevention Pelvic Inflammatory Disease ( ... Bacterial Vaginosis (BV) Chlamydia Gonorrhea Genital Herpes Hepatitis HIV/AIDS & STDs Human Papillomavirus ... STDs See Also Pregnancy Reproductive ...

  6. Bacterial Meningitis

    Science.gov (United States)

    ... Schedules Preteen & Teen Vaccines Meningococcal Disease Sepsis Bacterial Meningitis Recommend on Facebook Tweet Share Compartir On this ... serious disease. Laboratory Methods for the Diagnosis of Meningitis This manual summarizes laboratory methods used to isolate, ...

  7. The Hypoxic Regulator of Sterol Synthesis Nro1 Is a Nuclear Import Adaptor

    Energy Technology Data Exchange (ETDEWEB)

    T Yeh; C Lee; L Amzel; P Espenshade; M Bianchet

    2011-12-31

    Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 {angstrom} resolution shows an all-{alpha}-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.

  8. A balance between membrane elasticity and polymerization energy sets the shape of spherical clathrin coats

    Science.gov (United States)

    Saleem, Mohammed; Morlot, Sandrine; Hohendahl, Annika; Manzi, John; Lenz, Martin; Roux, Aurélien

    2015-02-01

    In endocytosis, scaffolding is one of the mechanisms to create membrane curvature by moulding the membrane into the spherical shape of the clathrin cage. However, the impact of membrane elastic parameters on the assembly and shape of clathrin lattices has never been experimentally evaluated. Here, we show that membrane tension opposes clathrin polymerization. We reconstitute clathrin budding in vitro with giant unilamellar vesicles (GUVs), purified adaptors and clathrin. By changing the osmotic conditions, we find that clathrin coats cause extensive budding of GUVs under low membrane tension while polymerizing into shallow pits under moderate tension. High tension fully inhibits polymerization. Theoretically, we predict the tension values for which transitions between different clathrin coat shapes occur. We measure the changes in membrane tension during clathrin polymerization, and use our theoretical framework to estimate the polymerization energy from these data. Our results show that membrane tension controls clathrin-mediated budding by varying the membrane budding energy.

  9. Effectiveness of Needles Vial Adaptors and Blunt Cannulas for Drug Administration in a Microgravity Environment

    Science.gov (United States)

    Hailey, Melinda; Bayuse, Tina

    2009-01-01

    The need for a new system of injectable medications aboard the International Space Station (ISS) was identified. It is desired that this system fly medications in their original manufacturer's packaging, allowing the system to comply with United States Pharmacopeia (USP) guidelines while minimizing the resupply frequency due to medication expiration. Pre-filled syringes are desired, however, the evolving nature of the healthcare marketplace requires flexibility in the redesign. If medications must be supplied in a vial, a system is required that allows for the safe withdrawal of medication from the vial into a syringe for administration in microgravity. During two reduced gravity flights, the effectiveness of two versions of a blunt cannula and needleless vial adaptors was evaluated to facilitate the withdrawal of liquid medication from a vial into a syringe for injection. Other parameters assessed included the ability to withdraw the required amount of medication and whether this is dependent on vial size, liquid, or the total volume of fluid within the vial. Injectable medications proposed for flight on ISS were used for this evaluation. Due to differing sizes of vials and the fluid properties of the medications, the needleless vial adaptors proved to be too cumbersome to recommend for use on the ISS. The blunt cannula, specifically the plastic version, proved to be more effective at removing medication from the various sizes of vials and are the recommended hardware for ISS. Fluid isolation within the vials and syringes is an important step in preparing medication for injection regardless of the hardware used. Although isolation is a challenge in the relatively short parabolas during flight, it is not an obstacle for sustained microgravity. This presentation will provide an overview of the products tested as well as the challenges identified during the microgravity flights.

  10. The Influences of Connectors and Adaptors to Fiber-To-The-Home Network Performance

    Directory of Open Access Journals (Sweden)

    Mohammad S. Ab-Rahman

    2012-01-01

    Full Text Available Problem statement: The reliability of the entire communications network was dependent on the reliability of each single element. Connector was important devices that can affect the performance of the fiber communication. There were a large number of issues that affect the performance of fiber optic connectors in todays networks. These factors were increasingly as data rates, the number of wavelengths and transmission distances continue to escalate. Approach: Therefore this study was carried out to test on the influence of connectors and adapters to the performance of the optical network. Initially the actual attenuation of connector and adaptor were tested by using multifunction loss tester. The first two 1 m corning optical fibers with a connector at each end are measured. Then, both the 1 m corning optical fibers were joined together by an adaptor and connected to the Multifunction loss tester. Three types of wavelength are used as the source to test the attenuation of the fiber which is 1310, 1490-1550 nm. In order to measure the Bit Error Rate (BER and the power loss in optical fiber communication, a simple simulation was carried out by using software opti sys. Results: The attenuation on the connector was caused mainly by existence of impurities in the connector, less perfect connection, scattering of beam and others. These causes the parameter such as power received, Q-factor, minimum BER and also the eye-height to change. Changes in these parameters also affect the performance at the user end. It was very critical that causes of attenuation to be eliminated. Conclusion/Recommendations: From the result it can be concluded that, the greater the attenuation, the greater the decrease in power received. It also affects the Q-factor of the system where as the attenuation increase, the maximum Q-factor decreases. As for the minimum BER, minimum BER changes as the attenuation increase initially, after a maximum value it decreases as the

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

    Science.gov (United States)

    Kazi, Julhash U; Rönnstrand, Lars

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Julhash U Kazi

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

  13. Crystal structure of Toll-like receptor adaptor MAL/TIRAP reveals the molecular basis for signal transduction and disease protection

    OpenAIRE

    Valkov, Eugene; Stamp, Anna; DiMaio, Frank; Baker, David; Verstak, Brett; Roversi, Pietro; Kellie, Stuart; Sweet, Matthew J.; Mansell, Ashley; Gay, Nicholas J.; Jennifer L Martin; Kobe, Bostjan

    2011-01-01

    Initiation of the innate immune response requires agonist recognition by pathogen-recognition receptors such as the Toll-like receptors (TLRs). Toll/interleukin-1 receptor (TIR) domain-containing adaptors are critical in orchestrating the signal transduction pathways after TLR and interleukin-1 receptor activation. Myeloid differentiation primary response gene 88 (MyD88) adaptor-like (MAL)/TIR domain-containing adaptor protein (TIRAP) is involved in bridging MyD88 to TLR2 and TLR4 in response...

  14. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion...... is the first committing step in biofilm formation, and has therefore been intensely scrutinized. Much however, still remains elusive. Bacterial adhesion is a highly complex process, which is influenced by a variety of factors. In this thesis, a range of physico-chemical, molecular and environmental parameters......, which influence the transition from a planktonic lifestyle to a sessile lifestyle, have been studied. Protein conditioning film formation was found to influence bacterial adhesion and subsequent biofilm formation considerable, and an aqueous extract of fish muscle tissue was shown to significantly...

  15. Bacterial lipases

    NARCIS (Netherlands)

    Jaeger, Karl-Erich; Ransac, Stéphane; Dijkstra, Bauke W.; Colson, Charles; Heuvel, Margreet van; Misset, Onno

    1994-01-01

    Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, mea

  16. New Class of Competitive Inhibitor of Bacterial Histidine Kinases

    OpenAIRE

    Gilmour, Raymond; Foster, J. Estelle; Sheng, Qin; McClain, Jonathan R.; Riley, Anna; Sun, Pei-Ming; Ng, Wai-Leung; Yan, Dalai; Nicas, Thalia I.; Henry, Kenneth; Winkler, Malcolm E.

    2005-01-01

    Bacterial histidine kinases have been proposed as targets for the discovery of new antibiotics, yet few specific inhibitors of bacterial histidine kinases have been reported. We report here a novel thienopyridine (TEP) compound that inhibits bacterial histidine kinases competitively with respect to ATP but does not comparably inhibit mammalian serine/threonine kinases. Although it partitions into membranes and does not inhibit the growth of bacterial or mammalian cells, TEP could serve as a s...

  17. Membrane dynamics

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications for...

  18. Highly pathogenic avian influenza virus nucleoprotein interacts with TREX complex adaptor protein Aly/REF.

    Science.gov (United States)

    Balasubramaniam, Vinod R M T; Hong Wai, Tham; Ario Tejo, Bimo; Omar, Abdul Rahman; Syed Hassan, Sharifah

    2013-01-01

    We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.

  19. Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

    Science.gov (United States)

    Diezmann, F; von Kleist, L; Haucke, V; Seitz, O

    2015-08-01

    The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the β2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By

  20. Comparison Study on Finite Element Model for Structural Analysis of Lower CTS Cylinder Lifting Adaptor in ITER Assembly Tool

    International Nuclear Information System (INIS)

    The ITER tokamak assembly tools are purpose-built and specially designed to complete the ITER tokamak machine which includes; Vacuum Vessel (VV), VV Thermal Shield (VVTS) and Cryostat Thermal Shield (CTS), Toroidal Field Coil (TFC) and Poloidal Field (PF) coils, and other components contained in the cryostat. Based on the engineering design and design description documents prepared by the ITER organization (IO). In this paper, FE models to verify the structural integrity of the Lower Cryostat Thermal Shield (LCTS) Lifting Adaptor are prepared and compared with their structure analysis result. In the paper, each analysis result with difference BC applied was compared with the equivalent stress and the results of analysis were evaluated according to SM490YB's allowable stress. And the conclusions of this paper are summarized as following. - The structural stabilities of the LCTS lift adaptor have been studied using ANSYS codes for verifying structural strength for this tool

  1. Chemical-genetic disruption of clathrin function spares adaptor complex 3–dependent endosome vesicle biogenesis

    OpenAIRE

    Zlatic, Stephanie A.; Grossniklaus, Emily J.; Ryder, Pearl V.; Salazar, Gloria; Mattheyses, Alexa L.; Peden, Andrew A.; Faundez, Victor

    2013-01-01

    A role for clathrin in AP-3–dependent vesicle biogenesis has been inferred from biochemical interactions and colocalization between this adaptor and clathrin. The functionality of these molecular associations, however, is controversial. We comprehensively explore the role of clathrin in AP-3–dependent vesicle budding, using rapid chemical-genetic perturbation of clathrin function with a clathrin light chain–FKBP chimera oligomerizable by the drug AP20187. We find that AP-3 interacts and coloc...

  2. Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals.

    Science.gov (United States)

    Smith, Stephen C; Joshi, Kamal K; Zik, Justin J; Trinh, Katherine; Kamajaya, Aron; Chien, Peter; Ryan, Kathleen R

    2014-09-30

    The cell-division cycle of Caulobacter crescentus depends on periodic activation and deactivation of the essential response regulator CtrA. Although CtrA is critical for transcription during some parts of the cell cycle, its activity must be eliminated before chromosome replication because CtrA also blocks the initiation of DNA replication. CtrA activity is down-regulated both by dephosphorylation and by proteolysis, mediated by the ubiquitous ATP-dependent protease ClpXP. Here we demonstrate that proteins needed for rapid CtrA proteolysis in vivo form a phosphorylation-dependent and cyclic diguanylate (cdG)-dependent adaptor complex that accelerates CtrA degradation in vitro by ClpXP. The adaptor complex includes CpdR, a single-domain response regulator; PopA, a cdG-binding protein; and RcdA, a protein whose activity cannot be predicted. When CpdR is unphosphorylated and when PopA is bound to cdG, they work together with RcdA in an all-or-none manner to reduce the Km of CtrA proteolysis 10-fold. We further identified a set of amino acids in the receiver domain of CtrA that modulate its adaptor-mediated degradation in vitro and in vivo. Complex formation between PopA and CtrA depends on these amino acids, which reside on alpha-helix 1 of the CtrA receiver domain, and on cdG binding by PopA. These results reveal that each accessory factor plays an essential biochemical role in the regulated proteolysis of CtrA and demonstrate, to our knowledge, the first example of a multiprotein, cdG-dependent proteolytic adaptor.

  3. ATP binding to p97/VCP D1 domain regulates selective recruitment of adaptors to its proximal N-domain.

    Directory of Open Access Journals (Sweden)

    Wei Sheng Chia

    Full Text Available p97/Valosin-containing protein (VCP is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD. It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.

  4. Bacterial Ecology

    DEFF Research Database (Denmark)

    Fenchel, Tom

    2011-01-01

    Bacterial ecology is concerned with the interactions between bacteria and their biological and nonbiological environments and with the role of bacteria in biogeochemical element cycling. Many fundamental properties of bacteria are consequences of their small size. Thus, they can efficiently exploit...... biogeochemical processes are carried exclusively by bacteria. * Bacteria play an important role in all types of habitats including some that cannot support eukaryotic life....

  5. [Bacterial vaginosis].

    Science.gov (United States)

    Romero Herrero, Daniel; Andreu Domingo, Antonia

    2016-07-01

    Bacterial vaginosis (BV) is the main cause of vaginal dysbacteriosis in the women during the reproductive age. It is an entity in which many studies have focused for years and which is still open for discussion topics. This is due to the diversity of microorganisms that cause it and therefore, its difficult treatment. Bacterial vaginosis is probably the result of vaginal colonization by complex bacterial communities, many of them non-cultivable and with interdependent metabolism where anaerobic populations most likely play an important role in its pathogenesis. The main symptoms are an increase of vaginal discharge and the unpleasant smell of it. It can lead to serious consequences for women, such as an increased risk of contracting sexually transmitted infections including human immunodeficiency virus and upper genital tract and pregnancy complications. Gram stain is the gold standard for microbiological diagnosis of BV, but can also be diagnosed using the Amsel clinical criteria. It should not be considered a sexually transmitted disease but it is highly related to sex. Recurrence is the main problem of medical treatment. Apart from BV, there are other dysbacteriosis less characterized like aerobic vaginitis of which further studies are coming slowly but are achieving more attention and consensus among specialists. PMID:27474242

  6. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

    Science.gov (United States)

    Schreiber, Andreas; Huber, Matthias C.; Cölfen, Helmut; Schiller, Stefan M.

    2015-03-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based ‘adaptors/connectors’ with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties.

  7. Lymphocytes and the Dap12 adaptor are key regulators of osteoclast activation associated with gonadal failure.

    Directory of Open Access Journals (Sweden)

    Adrienne Anginot

    Full Text Available Bone resorption by osteoclasts is necessary to maintain bone homeostasis. Osteoclast differentiation from hematopoietic progenitors and their activation depend on M-CSF and RANKL, but also requires co-stimulatory signals acting through receptors associated with DAP12 and FcRgamma adaptors. Dap12 mutant mice (KDelta75 are osteopetrotic due to inactive osteoclasts but, surprisingly, these mice are more sensitive than WT mice to bone loss following an ovariectomy. Because estrogen withdrawal is known to disturb bone mass, at least in part, through lymphocyte interaction, we looked at the role of mature lymphocytes on osteoclastogenesis and bone mass in the absence of functional DAP12. Lymphocytes were found to stimulate an early osteoclast differentiation response from Dap12-deficient progenitors in vitro. In vivo, Rag1-/- mice lacking mature lymphocytes did not exhibit any bone phenotype, but lost their bone mass after ovariectomy like KDelta75 mice. KDelta75;Rag1-/- double mutant female mice exhibited a more severe osteopetrosis than Dap12-deficient animals but lost their bone mass after ovariectomy, like single mutants. These results suggest that both DAP12 and mature lymphocytes act synergistically to maintain bone mass under physiological conditions, while playing similar but not synergistic co-stimulatory roles in protecting bone loss after gonadal failure. Thus, our data support a role for lymphocytes during osteoclast differentiation and suggest that they may function as accessory cells when regular osteoclast function is compromised.

  8. Dengue virus targets the adaptor protein MITA to subvert host innate immunity.

    Science.gov (United States)

    Yu, Chia-Yi; Chang, Tsung-Hsien; Liang, Jian-Jong; Chiang, Ruei-Lin; Lee, Yi-Ling; Liao, Ching-Len; Lin, Yi-Ling

    2012-01-01

    Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN). We found that activation of interferon regulatory factor 3 (IRF3) triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA) but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓(96)G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT) stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity.

  9. Dengue virus targets the adaptor protein MITA to subvert host innate immunity.

    Directory of Open Access Journals (Sweden)

    Chia-Yi Yu

    Full Text Available Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN. We found that activation of interferon regulatory factor 3 (IRF3 triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓(96G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity.

  10. The ubiquitin ligase RNF5 regulates antiviral responses by mediating degradation of the adaptor protein MITA.

    Science.gov (United States)

    Zhong, Bo; Zhang, Lu; Lei, Caoqi; Li, Ying; Mao, Ai-Ping; Yang, Yan; Wang, Yan-Yi; Zhang, Xiao-Lian; Shu, Hong-Bing

    2009-03-20

    Viral infection activates transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. MITA (also known as STING) has recently been identified as an adaptor that links virus-sensing receptors to IRF3 activation. Here, we showed that the E3 ubiquitin ligase RNF5 interacted with MITA in a viral-infection-dependent manner. Overexpression of RNF5 inhibited virus-triggered IRF3 activation, IFNB1 expression, and cellular antiviral response, whereas knockdown of RNF5 had opposite effects. RNF5 targeted MITA at Lys150 for ubiquitination and degradation after viral infection. Both MITA and RNF5 were located at the mitochondria and endoplasmic reticulum (ER) and viral infection caused their redistribution to the ER and mitochondria, respectively. We further found that virus-induced ubiquitination and degradation of MITA by RNF5 occurred at the mitochondria. These findings suggest that RNF5 negatively regulates virus-triggered signaling by targeting MITA for ubiquitination and degradation at the mitochondria.

  11. The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation.

    Science.gov (United States)

    Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S; Nichols, Kim E

    2013-04-25

    The adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)-induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell-mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA-mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS.

  12. Transmembrane adaptor proteins in the high-affinity IgE receptor signaling

    Directory of Open Access Journals (Sweden)

    Petr eDraber

    2012-01-01

    Full Text Available Aggregation of the high-affinity IgE receptor (FcεRI initiates a cascade of signaling events leading to release of preformed inflammatory and allergy mediators and de novo synthesis and secretion of cytokines and other compounds. The first biochemically well defined step of this signaling cascade is tyrosine phosphorylation of the FcεRI subunits by Src family kinase Lyn, followed by recruitment and activation of Syk kinase. Activity of Syk is decisive for the formation of multicomponent signaling assemblies, the signalosomes, in the vicinity of the receptors. Formation of the signalosomes is dependent on the presence of transmembrane adaptor proteins (TRAPs. These proteins are characterized by a short extracellular domain, a single transmembrane domain and a cytoplasmic tail with various motifs serving as anchors for cytoplasmic signaling molecules. In mast cells five TRAPs have been identified (LAT, NTAL, LAX, PAG and GAPT; engagement of four of them (LAT, NTAL, LAX and PAG in FcεRI signaling has been documented. Here we discuss recent progress in the understanding of how TRAPs affect FcεRI-mediated mast cell signaling. The combined data indicate that individual TRAPs have irreplaceable roles in important signaling events such as calcium response, degranulation, cytokines production and chemotaxis.

  13. Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus

    Directory of Open Access Journals (Sweden)

    Skrypkina I. Ya.

    2011-06-01

    Full Text Available Aim. Previously we have identified a novel isoform of endocytic adaptor protein ITSN1 designated as ITSN122a. Western blot revealed two immunoreactive bands of 120 and 250 kDa that corresponded to ITSN1-22a. The goal of this study was to investigate the possibility of dimer formation by the novel isoform. Methods. Dimerization ability of ITSN1-22a was tested by immunoprecipitation and subsequent Western blot analysis. To specify the region responsible for dimerization, site-directed mutagenesis and truncation analysis were carried out. Inhibition of endocytosis by potassium depletion and EGF stimulation of HEK293 were performed. Results. We have found that ITSN1-22a forms dimers in HEK293 cells. The dimerization of ITSN1-22a was mediated by C-terminal domain. We showed that cysteines C1016 and C1019 were involved in homodimerization. Inhibition of clathrin-mediated endocytosis and mitogen stimulation did not affect ITSN1-22a dimer formation. Conclusions. ITSN1-22a is the only one known ITSN1 isoform, which is capable to form homodimers via disulphide bonds. This could be important for the formation of protein complexes containing ITSN1 molecules.

  14. Allostery mediates ligand binding to Grb2 adaptor in a mutually exclusive manner.

    Science.gov (United States)

    McDonald, Caleb B; El Hokayem, Jimmy; Zafar, Nawal; Balke, Jordan E; Bhat, Vikas; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Farooq, Amjad

    2013-02-01

    Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi-protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non-overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2-Sos1 and Grb2-Gab1 binary signaling complexes in concert in lieu of a composite Sos1-Grb2-Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2-Sos1 and Grb2-Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery. PMID:23334917

  15. Adaptor protein LNK is a negative regulator of brain neural stem cell proliferation after stroke.

    Science.gov (United States)

    Ahlenius, Henrik; Devaraju, Karthikeyan; Monni, Emanuela; Oki, Koichi; Wattananit, Somsak; Darsalia, Vladimer; Iosif, Robert E; Torper, Olof; Wood, James C; Braun, Sebastian; Jagemann, Lucas; Nuber, Ulrike A; Englund, Elisabet; Jacobsen, Sten-Eirik W; Lindvall, Olle; Kokaia, Zaal

    2012-04-11

    Ischemic stroke causes transient increase of neural stem and progenitor cell (NSPC) proliferation in the subventricular zone (SVZ), and migration of newly formed neuroblasts toward the damaged area where they mature to striatal neurons. The molecular mechanisms regulating this plastic response, probably involved in structural reorganization and functional recovery, are poorly understood. The adaptor protein LNK suppresses hematopoietic stem cell self-renewal, but its presence and role in the brain are poorly understood. Here we demonstrate that LNK is expressed in NSPCs in the adult mouse and human SVZ. Lnk(-/-) mice exhibited increased NSPC proliferation after stroke, but not in intact brain or following status epilepticus. Deletion of Lnk caused increased NSPC proliferation while overexpression decreased mitotic activity of these cells in vitro. We found that Lnk expression after stroke increased in SVZ through the transcription factors STAT1/3. LNK attenuated insulin-like growth factor 1 signaling by inhibition of AKT phosphorylation, resulting in reduced NSPC proliferation. Our findings identify LNK as a stroke-specific, endogenous negative regulator of NSPC proliferation, and suggest that LNK signaling is a novel mechanism influencing plastic responses in postischemic brain. PMID:22496561

  16. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    Science.gov (United States)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  17. Transmembrane adaptor molecules: a new category of lymphoid-cell markers.

    Science.gov (United States)

    Tedoldi, Sara; Paterson, Jennifer C; Hansmann, Martin-Leo; Natkunam, Yasodha; Rüdiger, Thomas; Angelisova, Pavla; Du, Ming Q; Roberton, Helen; Roncador, Giovanna; Sanchez, Lydia; Pozzobon, Michela; Masir, Noraidah; Barry, Richard; Pileri, Stefano; Mason, David Y; Marafioti, Teresa; Horejsí, Václav

    2006-01-01

    Transmembrane adaptor proteins (of which 7 have been identified so far) are involved in receptor signaling in immune cells. They have only a short extracellular region, with most of the molecule comprising a substantial intracytoplasmic region carrying multiple tyrosine residues that can be phosphorylated by Src- or Syk-family kinases. In this paper, we report an immunohistologic study of 6 of these molecules in normal and neoplastic human tissue sections and show that they are restricted to subpopulations of lymphoid cells, being present in either T cells (LAT, LIME, and TRIM), B cells (NTAL), or subsets of both cell types (PAG and SIT). Their expression in neoplastic lymphoid cells broadly reflects that of normal lymphoid tissue, including the positivity of plasma cells and myeloma/plasmacytoma for LIME, NTAL, PAG, and SIT. However, this study also revealed some reactions that may be of diagnostic/prognostic value. For example, lymphocytic lymphoma and mantle-cell lymphoma showed similar profiles but differed clearly from follicle-center lymphoma, whereas PAG tended to be selectively expressed in germinal center-derived subsets of diffuse large B-cell lymphoma. These molecules represent a potentially important addition to the panel of immunophenotypic markers detectable in routine biopsies that can be used in hematopathologic studies. PMID:16160011

  18. Interaction of Defensins with Model Cell Membranes

    Science.gov (United States)

    Sanders, Lori K.; Schmidt, Nathan W.; Yang, Lihua; Mishra, Abhijit; Gordon, Vernita D.; Selsted, Michael E.; Wong, Gerard C. L.

    2009-03-01

    Antimicrobial peptides (AMPs) comprise a key component of innate immunity for a wide range of multicellular organisms. For many AMPs, activity comes from their ability to selectively disrupt and lyse bacterial cell membranes. There are a number of proposed models for this action, but the detailed molecular mechanism of selective membrane permeation remains unclear. Theta defensins are circularized peptides with a high degree of selectivity. We investigate the interaction of model bacterial and eukaryotic cell membranes with theta defensins RTD-1, BTD-7, and compare them to protegrin PG-1, a prototypical AMP, using synchrotron small angle x-ray scattering (SAXS). The relationship between membrane composition and peptide induced changes in membrane curvature and topology is examined. By comparing the membrane phase behavior induced by these different peptides we will discuss the importance of amino acid composition and placement on membrane rearrangement.

  19. PROPERTIES OF BACTERIAL CELLULOSE AND ITS INFLUENCE ON THE PHYSICAL PROPERTIES OF PAPER

    OpenAIRE

    Wen-Hua Gao; Ke-Fu Chen; Ren-Dang Yang; Fei Yang; Wen-Jia Han

    2011-01-01

    Bacterial cellulose is a promising source of biodegradable polymers having high purity. The time required to disperse bacterial cellulose wet membranes was studied, along with evaluation by infrared spectroscopy and thermal analysis of the dispersed bacterial fiber and tests of the physical properties of the sheet. The results showed that bacterial cellulose wet membrane can be dispersed well, forming fibers when the dispersing time was 3 minutes at a suitable concentration. FT-IR results sho...

  20. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  1. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated...

  2. Hydrological Modeling Reproducibility Through Data Management and Adaptors for Model Interoperability

    Science.gov (United States)

    Turner, M. A.

    2015-12-01

    Because of a lack of centralized planning and no widely-adopted standards among hydrological modeling research groups, research communities, and the data management teams meant to support research, there is chaos when it comes to data formats, spatio-temporal resolutions, ontologies, and data availability. All this makes true scientific reproducibility and collaborative integrated modeling impossible without some glue to piece it all together. Our Virtual Watershed Integrated Modeling System provides the tools and modeling framework hydrologists need to accelerate and fortify new scientific investigations by tracking provenance and providing adaptors for integrated, collaborative hydrologic modeling and data management. Under global warming trends where water resources are under increasing stress, reproducible hydrological modeling will be increasingly important to improve transparency and understanding of the scientific facts revealed through modeling. The Virtual Watershed Data Engine is capable of ingesting a wide variety of heterogeneous model inputs, outputs, model configurations, and metadata. We will demonstrate one example, starting from real-time raw weather station data packaged with station metadata. Our integrated modeling system will then create gridded input data via geostatistical methods along with error and uncertainty estimates. These gridded data are then used as input to hydrological models, all of which are available as web services wherever feasible. Models may be integrated in a data-centric way where the outputs too are tracked and used as inputs to "downstream" models. This work is part of an ongoing collaborative Tri-state (New Mexico, Nevada, Idaho) NSF EPSCoR Project, WC-WAVE, comprised of researchers from multiple universities in each of the three states. The tools produced and presented here have been developed collaboratively alongside watershed scientists to address specific modeling problems with an eye on the bigger picture of

  3. Biophysical basis of the binding of WWOX tumor suppressor to WBP1 and WBP2 adaptors.

    Science.gov (United States)

    McDonald, Caleb B; Buffa, Laura; Bar-Mag, Tomer; Salah, Zaidoun; Bhat, Vikas; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Malhotra, Arun; Sudol, Marius; Aqeilan, Rami I; Nawaz, Zafar; Farooq, Amjad

    2012-09-01

    The WW-containing oxidoreductase (WWOX) tumor suppressor participates in a diverse array of cellular activities by virtue of its ability to recognize WW-binding protein 1 (WBP1) and WW-binding protein 2 (WBP2) signaling adaptors among a wide variety of other ligands. Herein, using a multitude of biophysical techniques, we provide evidence that while the WW1 domain of WWOX binds to PPXY motifs within WBP1 and WBP2 in a physiologically relevant manner, the WW2 domain exhibits no affinity toward any of these PPXY motifs. Importantly, our data suggest that while R25/W44 residues located within the binding pocket of a triple-stranded β-fold of WW1 domain are critical for the recognition of PPXY ligands, they are replaced by the chemically distinct E66/Y85 duo at structurally equivalent positions within the WW2 domain, thereby accounting for its failure to bind PPXY ligands. Predictably, not only does the introduction of E66R/Y85W double substitution within the WW2 domain result in gain of function but the resulting engineered domain, hereinafter referred to as WW2_RW, also appears to be a much stronger binding partner of WBP1 and WBP2 than the wild-type WW1 domain. We also show that while the WW1 domain is structurally disordered and folds upon ligand binding, the WW2 domain not only adopts a fully structured conformation but also aids stabilization and ligand binding to WW1 domain. This salient observation implies that the WW2 domain likely serves as a chaperone to augment the physiological function of WW1 domain within WWOX. Collectively, our study lays the groundwork for understanding the molecular basis of a key protein-protein interaction pertinent to human health and disease. PMID:22634283

  4. The TIR-domain containing adaptor TRAM is required for TLR7 mediated RANTES production.

    Directory of Open Access Journals (Sweden)

    Enda Shevlin

    Full Text Available Toll-like receptor 7 (TLR7 plays a vital role in the immune response to ssRNA viruses such as human rhinovirus (HRV and Influenza, against which there are currently no treatments or vaccines with long term efficacy available. Clearly, a more comprehensive understanding of the TLR7 signaling axis will contribute to its molecular targeting. TRIF related adaptor molecule (TRAM plays a vital role in TLR4 signaling by recruiting TRIF to TLR4, followed by endosomal trafficking of the complex and initiation of IRF3 dependent type I interferon production as well as NF-κB dependent pro-inflammatory cytokine production. Towards understanding the molecular mechanisms that regulate TLR7 functionality, we found that TRAM(-/- murine macrophages exhibited a transcriptional and translational impairment in TLR7 mediated RANTES, but not TNFα, production. Suppression of TRAM expression in human macrophages also resulted in an impairment in TLR7 mediated CCL5 and IFN-β, but not TNFα, gene induction. Furthermore, suppression of endogenous human TRAM expression in human macrophages significantly impaired RV16 induced CCL5 and IFNβ, but not TNFα gene induction. Additionally, TRAM-G2A dose-dependently inhibited TLR7 mediated activation of CCL5, IFNβ and IFNα reporter genes. TLR7-mediated phosphorylation and nuclear translocation of IRF3 was impaired in TRAM(-/- cells. Finally, co-immunoprecipitation studies indicated that TRAM physically interacts with MyD88 upon TLR7 stimulation, but not under basal conditions. Our results clearly demonstrate that TRAM plays a, hitherto unappreciated, role in TLR7 signaling through a novel signaling axis containing, but not limited to, MyD88, TRAM and IRF3 towards the activation of anti-viral immunity.

  5. Bacterial Hydrodynamics

    Science.gov (United States)

    Lauga, Eric

    2016-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells, yet they represent the bulk of the world's biomass and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micrometer scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically complex environments. Using hydrodynamics as an organizing framework, I review the biomechanics of bacterial motility and look ahead to future challenges.

  6. Bacterial hydrodynamics

    CERN Document Server

    Lauga, Eric

    2015-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells yet they represent the bulk of the world's biomass, and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds-number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micron scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically-complex environments. Using hydrodynamics as an organizing framework, we review the biomechanics of bacterial motility and look ahead to future challenges.

  7. 细菌纤维素膜作为生物支架构建组织工程角膜上皮的可行性研究%Feasibility of bacterial cellulose membrane as biological scaffold for construction of tissue engineering corneal epithelium

    Institute of Scientific and Technical Information of China (English)

    曹静洁; 张琛; 赵少贞; 万怡灶; 胡达

    2016-01-01

    Background Corneal transplantation is a primary method for the treatment of serious corneal diseases, but its application is limited because of the shortage of corneal donor.The study on tissue engineering corneal epithelium provides a new approach to corneal transplantation, and the biological scaffold materials for tissue engineering corneal epithelium is an issue of increasing concern.Bacterial cellulose membrane has been used in medical field,but its application in tissue engineering corneal epithelium deserves more researching.Objective This study was to evaluate the biocompatibility of bacterial cellulose membrane as a biological scaffold of tissue engineering corneal epithelium.Methods Corneal epithelium was isolated from 1 month-old New Zealand White rabbit.Corneal epithelial cells were cultured using explant method and identified by detecting the CK-3 expression using immunofluorescence technique.The second generation ceils were inoculated on bacterial cellulose membrane and culture plate, respectively, and the growth status of the cells were examined and compared under the optical microscope.The cell activity/toxicity test was performed by LIVE/DEAD cell staining kit at the third day after inoculation to evaluate the survival rate.The ultrastructure of the cell surface was examined under the scanning electron microscope.The study was performed in accordance with the ARVO Statement.Results Rabbit corneal epithelial cells grew well 1 week after primarily cultured with a cobblestone-like appearance and positive response for CK3 antibody.The cells on the bacterial cellulose membrane presented a round shape and regular arrangement and showed the green fluorescence for LIVE/DEAD test,with the survival rate 100%.Abundant leafy protrusion, microvilli and intercellular junction were seen under the scanning electron microscope.In addition, mitosis phase of cells and many filopodia between the cells and bacterial cellulose membrane were also exhibited

  8. Molecular structure, function, and dynamics of clathrin-mediated membrane traffic.

    Science.gov (United States)

    Kirchhausen, Tom; Owen, David; Harrison, Stephen C

    2014-05-01

    Clathrin is a molecular scaffold for vesicular uptake of cargo at the plasma membrane, where its assembly into cage-like lattices underlies the clathrin-coated pits of classical endocytosis. This review describes the structures of clathrin, major cargo adaptors, and other proteins that participate in forming a clathrin-coated pit, loading its contents, pinching off the membrane as a lattice-enclosed vesicle, and recycling the components. It integrates as much of the structural information as possible at the time of writing into a sketch of the principal steps in coated-pit and coated-vesicle formation.

  9. UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA.

    Science.gov (United States)

    Hautbergue, Guillaume M; Hung, Ming-Lung; Walsh, Matthew J; Snijders, Ambrosius P L; Chang, Chung-Te; Jones, Rachel; Ponting, Chris P; Dickman, Mark J; Wilson, Stuart A

    2009-12-01

    Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.

  10. Functional analyses of Src-like adaptor (SLA), a glucocorticoid-regulated gene in acute lymphoblastic leukemia.

    Science.gov (United States)

    Mansha, Muhammad; Carlet, Michela; Ploner, Christian; Gruber, Georg; Wasim, Muhammad; Wiegers, Gerrit Jan; Rainer, Johannes; Geley, Stephan; Kofler, Reinhard

    2010-04-01

    Glucocorticoids (GCs) cause apoptosis and cell cycle arrest in lymphoid cells and are used in the therapy of lymphoid malignancies. SLA (Src-like-adaptor), an inhibitor of T- and B-cell receptor signaling, is a promising candidate derived from expression profiling analyses in children with acute lymphoblastic leukemia (ALL). Over-expression and knock-down experiments in ALL in vitro model revealed that transgenic SLA alone had no effect on survival or cell cycle progression, nor did it affect sensitivity to, or kinetics of, GC-induced apoptosis. Although SLA is a prominent GC response gene, it does not seem to contribute to the anti-leukemic effects of GC.

  11. Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

    OpenAIRE

    Tunnicliffe, Richard B.; Hautbergue, Guillaume M.; Wilson, Stuart A.; Priti Kalra; Golovanov, Alexander P.

    2014-01-01

    The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range o...

  12. Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C on the regulation of Toll-like receptors, adaptor proteins and inflammatory response

    Directory of Open Access Journals (Sweden)

    Weith Andreas

    2005-11-01

    Full Text Available Abstract Background Bacterial and viral exacerbations play a crucial role in a variety of lung diseases including COPD or asthma. Since the lung epithelium is a major source of various inflammatory mediators that affect the immune response, we analyzed the inflammatory reaction of primary lung epithelial cells to different microbial molecules that are recognized by Toll-like receptors (TLR. Methods The effects of TLR ligands on primary small airway epithelial cells were analyzed in detail with respect to cytokine, chemokine and matrix metalloproteinase secretion. In addition, the regulation of the expression of TLRs and their adaptor proteins in small airway epithelial cells was investigated. Results Our data demonstrate that poly(I:C, a synthetic analog of viral dsRNA, mediated the strongest proinflammatory effects among the tested ligands, including an increased secretion of IL-6, IL-8, TNF-α, GM-CSF, GRO-α, TARC, MCP-1, MIP-3α, RANTES, IFN-β, IP-10 and ITAC as well as an increased release of MMP-1, MMP-8, MMP-9, MMP-10 and MMP-13. Furthermore, our data show that poly(I:C as well as type-1 and type-2 cytokines have a pronounced effect on the expression of TLRs and molecules involved in TLR signaling in small airway epithelial cells. Poly(I:C induced an elevated expression of TLR1, TLR2 and TLR3 and increased the gene expression of the general TLR adaptor MyD88 and IRAK-2. Simultaneously, poly(I:C decreased the expression of TLR5, TLR6 and TOLLIP. Conclusion Poly(I:C, an analog of viral dsRNA and a TLR3 ligand, triggers a strong inflammatory response in small airway epithelial cells that is likely to contribute to viral exacerbations of pulmonary diseases like asthma or COPD. The pronounced effects of poly(I:C on the expression of Toll-like receptors and molecules involved in TLR signaling is assumed to influence the immune response of the lung epithelium to viral and bacterial infections. Likewise, the regulation of TLR expression by type

  13. Risk factors for community-acquired bacterial meningitis in adults

    NARCIS (Netherlands)

    K.S. Adriani

    2015-01-01

    Bacterial meningitis is an inflammation of the meninges and occurs when bacteria invade the subarachnoid space. The meninges are the protective membranes that surround the brain and the spinal cord. Bacterial meningitis is a life-threatening disease because the proximity of the infection to the brai

  14. Downstream Toll-like receptor signaling mediates adaptor-specific cytokine expression following focal cerebral ischemia

    Directory of Open Access Journals (Sweden)

    Bolanle Famakin

    2012-07-01

    Full Text Available Abstract Background Deletion of some Toll-like receptors (TLRs affords protection against cerebral ischemia, but disruption of their known major downstream adaptors does not. To determine whether compensation in the production of downstream effectors by one pathway when the other is disrupted can explain these findings, we examined cytokine/chemokine expression and inflammatory infiltrates in wild-type (WT, MyD88−/− and TRIF-mutant mice following permanent middle cerebral artery occlusion (pMCAO. Methods Cytokine/chemokine expression was measured with a 25-plex bead array in the serum and brains of all three groups of mice at baseline (no surgery/naïve and at 3 hours and 24 hours following pMCAO. Brain inflammatory and neutrophil infiltrates were examined 24 hours following pMCAO. Results IL-6, keratinocyte chemoattractant (KC, granulocyte colony-stimulating factor (G-CSF and IL-10 were significantly decreased in MyD88−/− mice compared to WT mice following pMCAO. Significantly, decreased levels of the neutrophil chemoattractants KC and G-CSF corresponded with a trend toward fewer neutrophils in the brains of MyD88−/− mice. IP-10 was significantly decreased when either pathway was disrupted. MIP-1α was significantly decreased in TRIF-mutant mice, consistent with TRIF-dependent production. MyD88−/− mice showed elevations of a number of Th2 cytokines, such as IL-13, at baseline, which became significantly decreased following pMCAO. Conclusions Both MyD88 and TRIF mediate pathway-specific cytokine production following focal cerebral ischemia. Our results also suggest a compensatory Th2-type skew at baseline in MyD88−/− mice and a paradoxical switch to a Th1 phenotype following focal cerebral ischemia. The MyD88 pathway directs the expression of neutrophil chemoattractants following cerebral ischemia.

  15. Membrane Biophysics

    CERN Document Server

    Ashrafuzzaman, Mohammad

    2013-01-01

    Physics, mathematics and chemistry all play a vital role in understanding the true nature and functioning of biological membranes, key elements of living processes. Besides simple spectroscopic observations and electrical measurements of membranes we address in this book the phenomena of coexistence and independent existence of different membrane components using various theoretical approaches. This treatment will be helpful for readers who want to understand biological processes by applying both simple observations and fundamental scientific analysis. It provides a deep understanding of the causes and effects of processes inside membranes, and will thus eventually open new doors for high-level pharmaceutical approaches towards fighting membrane- and cell-related diseases.

  16. Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σS by ClpXP.

    Directory of Open Access Journals (Sweden)

    Dimce eMicevski

    2015-04-01

    Full Text Available In Escherichia coli, σS is the master regulator of the general stress response. The level of σS changes in response to multiple stress conditions and it is regulated at many levels including protein turnover. In the absence of stress, σS is rapidly degraded by the AAA+ protease, ClpXP in a regulated manner that depends on the adaptor protein RssB. This two-component response regulator mediates the recognition of σS and its delivery to ClpXP. The turnover of σS however, can be inhibited in a stress specific manner, by one of three anti-adaptor proteins. Each anti-adaptor binds to RssB and inhibits its activity, but how this is achieved is not fully understood at a molecular level. Here we describe details of the interaction between each anti-adaptor and RssB that leads to the stabilization of σS. By defining the domains of RssB using partial proteolysis we demonstrate that each anti-adaptor uses a distinct mode of binding to inhibit RssB activity. IraD docks specifically to the N-terminal domain of RssB, IraP interacts primarily with the C-terminal domain, while IraM interacts with both domains. Despite these differences in binding, we propose that docking of each anti-adaptor induces a conformational change in RssB, which resembles the inactive dimer of RssB. This dimer-like state of RssB not only prevents substrate binding but also triggers substrate release from a pre-bound complex.

  17. Regulation of the divalent metal ion transporter via membrane budding

    Science.gov (United States)

    Mackenzie, KimberlyD; Foot, Natalie J; Anand, Sushma; Dalton, Hazel E; Chaudhary, Natasha; Collins, Brett M; Mathivanan, Suresh; Kumar, Sharad

    2016-01-01

    The release of extracellular vesicles (EVs) is important for both normal physiology and disease. However, a basic understanding of the targeting of EV cargoes, composition and mechanism of release is lacking. Here we present evidence that the divalent metal ion transporter (DMT1) is unexpectedly regulated through release in EVs. This process involves the Nedd4-2 ubiquitin ligase, and the adaptor proteins Arrdc1 and Arrdc4 via different budding mechanisms. We show that mouse gut explants release endogenous DMT1 in EVs. Although we observed no change in the relative amount of DMT1 released in EVs from gut explants in Arrdc1 or Arrdc4 deficient mice, the extent of EVs released was significantly reduced indicating an adaptor role in biogenesis. Furthermore, using Arrdc1 or Arrdc4 knockout mouse embryonic fibroblasts, we show that both Arrdc1 and Arrdc4 are non-redundant positive regulators of EV release. Our results suggest that DMT1 release from the plasma membrane into EVs may represent a novel mechanism for the maintenance of iron homeostasis, which may also be important for the regulation of other membrane proteins. PMID:27462458

  18. Interaction between Antimicrobial Peptide LL -37 and Bacterial Membrane POPG by Molecular Dynamics Simulation%抗菌肽 LL -37与细菌膜 POPG相互作用的分子动力学模拟研究

    Institute of Scientific and Technical Information of China (English)

    毛文超; 赵立岭; 王吉华

    2016-01-01

    As a major component of the innate immune system ,human antimicrobial peptide LL -37 plays an important role in the protection of human resistance to disease .Residues 17-29 (FK -13) are consid‐ered as the core area of LL -37 antimicrobial activity .LL -37 can damage bacterial membrane with nega‐tive .LL -37 antimicrobial activity is closely related to secondary structure changes .In this work ,we studied the interaction between antimicrobial fragment FK -13 and of the bacterial membrane POPG by molecular dynamics simulation .The results show that the Coulomb interaction causes FK -13 close to the surface of the membrane rapidly and FK -13 can carpet the surface of the membrane .FK -13 has par‐tial helix structure when it interacts with membrane .In addition ,FK -13 can cause disturbance and de‐struction of the membrane and it is beneficial to exert antimicrobial activity .The residues 18 ~ 22 are key residues in the membrane function .So the work is useful for us not only understand the changes of FK -13 conformation and its damage to the membrane but also provide a theoretical basis for FK -13 as a ther‐apeutic application .%作为先天免疫系统的主要成分,人源抗菌肽L L -37在保护人类对抗疾病方面起了重要的角色。残基肽段17-29(FK -13)被认为是L L -37行使抗菌功能的核心区域,L L -37能对带负电的细菌膜产生破坏作用,其抗菌活性与二级结构的变化情况有关。用分子动力学模拟的方法研究了抗菌肽段 FK -13与带负电的细菌膜PO PG的相互作用。研究发现,库仑作用会使FK -13快速靠近膜的表面,然后像地毯一样覆盖在膜的表面;FK -13仍旧保持部分螺旋结构,并通过引起膜的扰动对膜产生破坏,以实现抗菌功能,而残基18~22是与膜作用的关键残基。研究FK -13与膜的相互作用,不仅可以从原子层面上了解L L -37对细菌膜作用的机理,也为基于L L -37

  19. The TPLATE Adaptor Complex Drives Clathrin-Mediated Endocytosis in Plants

    NARCIS (Netherlands)

    Gadeyne, A.; Sanchez-Rodriguez, C.; Rubbo, Di S.; Ketelaar, T.

    2014-01-01

    Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure.

  20. Bacterial Cellulose-Hydroxyapatite Nanocomposites for Bone Regeneration

    OpenAIRE

    Saska, S.; H.S. Barud; Gaspar, A. M. M.; Marchetto, R.; Ribeiro, S. J. L.; Y. Messaddeq

    2011-01-01

    The aim of this study was to develop and to evaluate the biological properties of bacterial cellulose-hydroxyapatite (BC-HA) nanocomposite membranes for bone regeneration. Nanocomposites were prepared from bacterial cellulose membranes sequentially incubated in solutions of CaCl2 followed by Na2HPO4. BC-HA membranes were evaluated in noncritical bone defects in rat tibiae at 1, 4, and 16 weeks. Thermogravimetric analyses showed that the amount of the mineral phase was 40%–50% of the total wei...

  1. Membrane Processes.

    Science.gov (United States)

    Pellegrin, Marie-Laure; Sadler, Mary E; Greiner, Anthony D; Aguinaldo, Jorge; Min, Kyungnan; Zhang, Kai; Arabi, Sara; Burbano, Marie S; Kent, Fraser; Shoaf, Robert

    2015-10-01

    This review, for literature published in 2014, contains information related to membrane processes for municipal and industrial applications. This review is a subsection of the Treatment Systems section of the annual Water Environment Federation literature review and covers the following topics: pretreatment, membrane bioreactor (MBR) configuration, design, nutrient removal, operation, industrial treatment, fixed film and anaerobic membrane systems, reuse, microconstituents removal, membrane technology advances, membrane fouling, and modeling. Other sub-sections of the Treatment Systems section that might relate to this literature review include: Biological Fixed-Film Systems, Activated Sludge and Other Aerobic Suspended Culture Processes, Anaerobic Processes, Water Reclamation and Reuse. The following sections might also have related information on membrane processes: Industrial Wastes, Hazardous Wastes, and Fate and Effects of Pollutants. PMID:26420079

  2. Membrane Processes.

    Science.gov (United States)

    Pellegrin, Marie-Laure; Burbano, Marie S; Sadler, Mary E; Diamond, Jason; Baker, Simon; Greiner, Anthony D; Arabi, Sara; Wong, Joseph; Doody, Alexandra; Padhye, Lokesh P; Sears, Keith; Kistenmacher, Peter; Kent, Fraser; Tootchi, Leila; Aguinaldo, Jorge; Saddredini, Sara; Schilling, Bill; Min, Kyungnan; McCandless, Robert; Danker, Bryce; Gamage, Neranga P; Wang, Sunny; Aerts, Peter

    2016-10-01

    This review, for literature published in 2015, contains information related to membrane processes for municipal and industrial applications. This review is a subsection of the Treatment Systems section of the annual Water Environment Federation literature review and covers the following topics: pretreatment, membrane bioreactor (MBR) configuration, design, nutrient removal, operation, industrial treatment, anaerobic membrane systems, reuse, microconstituents removal, membrane technology advances, membrane fouling, and modeling. Other sub-sections of the Treatment Systems section that might relate to this literature review include: Biological Fixed-Film Systems, Activated Sludge and Other Aerobic Suspended Culture Processes, Anaerobic Processes, Water Reclamation and Reuse. The following sections might also have related information on membrane processes: Industrial Wastes, Hazardous Wastes, and Fate and Effects of Pollutants. PMID:27620084

  3. Multicomponent membranes

    Science.gov (United States)

    Kulprathipanja, Santi; Kulkarni, Sudhir S.; Funk, Edward W.

    1988-01-01

    A multicomponent membrane which may be used for separating various components which are present in a fluid feed mixture comprises a mixture of a plasticizer such as a glycol and an organic polymer cast upon a porous organic polymer support. The membrane may be prepared by casting an emulsion or a solution of the plasticizer and polymer on the porous support, evaporating the solvent and recovering the membrane after curing.

  4. Membrane Tension Inhibits Deformation by Coat Proteins in Clathrin-Mediated Endocytosis

    Science.gov (United States)

    Hassinger, Julian; Drubin, David; Oster, George; Rangamani, Padmini

    2016-02-01

    In clathrin-mediated endocytosis (CME), clathrin and various adaptor proteins coat a patch of the plasma membrane, which is reshaped to form a budded vesicle. Experimental studies have demonstrated that elevated membrane tension can inhibit bud formation by a clathrin coat. In this study, we investigate the impact of membrane tension on the mechanics of membrane budding by simulating clathrin coats that either grow in area or progressively induce greater curvature. At low membrane tension, progressively increasing the area of a curvature-generating coat causes the membrane to smoothly evolve from a flat to budded morphology, whereas the membrane remains essentially flat at high membrane tensions. Interestingly, at physiologically relevant, intermediate membrane tensions, the shape evolution of the membrane undergoes a snapthrough instability in which increasing coat area causes the membrane to "snap" from an open, U-shaped bud to a closed, $\\Omega$-shaped bud. This instability is accompanied by a large energy barrier, which could cause a developing endocytic pit to stall if the binding energy of additional coat is insufficient to overcome this barrier. Similar results were found for a coat of constant area in which the spontaneous curvature progressively increases. Additionally, a pulling force on the bud, simulating a force from actin polymerization, is sufficient to drive a transition from an open to closed bud, overcoming the energy barrier opposing this transition.

  5. Association between receptor protein-tyrosine phosphatase RPTPalpha and the Grb2 adaptor. Dual Src homology (SH) 2/SH3 domain requirement and functional consequences

    DEFF Research Database (Denmark)

    Su, J; Yang, L T; Sap, J

    1996-01-01

    Receptor protein-tyrosine phosphatase RPTPalpha is found associated in vivo with the adaptor protein Grb2. Formation of this complex, which contains no detectable levels of Sos, is known to depend on a C-terminal phosphorylated tyrosine residue (Tyr798) in RPTPalpha and on the Src homology (SH) 2...

  6. Disruption of adaptor protein 2mu (AP-2mu) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing

    NARCIS (Netherlands)

    Jung, S.; Maritzen, T.; Wichmann, C.; Jing, Z.; Neef, A.; Revelo, N.H.; Al-Moyed, H.; Meese, S.; Wojcik, S.M.; Panou, I.; Bulut, H.; Schu, P.; Ficner, R.; Reisinger, E.; Rizzoli, S.O.; Neef, J.; Strenzke, N.; Haucke, V.; Moser, T.

    2015-01-01

    Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2mu (AP-2mu) is required for release site replenishment and hearing. We show that

  7. Ubc2, an Ortholog of the Yeast Ste50p Adaptor, Possesses a Basidiomycete-Specific Carboxy terminal Extension Essential for Pathogenicity Independent of Pheromone Response.

    Science.gov (United States)

    Proteins involved in the MAP kinase pathway controlling mating, morphogenesis and pathogenicity have been identified previously in the fungus Ustilago maydis. One of these, the Ubc2 adaptor protein, possesses a basidiomycete-specific structure. In addition to containing SAM and RA domains typical of...

  8. Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation

    DEFF Research Database (Denmark)

    Barres, Romain; Grémeaux, Thierry; Gual, Philippe;

    2006-01-01

    APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays...

  9. Salmonellae PhoPQ regulation of the outer membrane to resist innate immunity.

    Science.gov (United States)

    Dalebroux, Zachary D; Miller, Samuel I

    2014-02-01

    Salmonellae sense host cues to regulate properties important for bacterial survival and replication within host tissues. The PhoPQ two-component regulatory system senses phagosome acidification and cationic antimicrobial peptides (CAMP) to regulate the protein and lipid contents of the bacterial envelope that comprises an inner and outer membrane. PhoPQ-regulated lipid components of the outer membrane include lipopolysaccharides and glycerophospholipids. Envelope proteins regulated by PhoPQ, include: components of virulence associated secretion systems, the flagellar apparatus, membrane transport systems, and proteins that are likely structural components of the outer membrane. PhoPQ alteration of the bacterial surface results in increased bacterial resistance to CAMP and decreased detection by the innate immune system. This review details the molecular complexity of the bacterial cell envelope and highlights the outer membrane lipid bilayer as an environmentally regulated bacterial organelle.

  10. Bacterial and Fungal Pattern Recognition Receptors in Homologous Innate Signaling Pathways of Insects and Mammals

    Directory of Open Access Journals (Sweden)

    Bethany A Stokes

    2015-01-01

    Full Text Available In response to bacterial and fungal infections in insects and mammals, distinct families of innate immune pattern recognition receptors initiate highly complex intracellular signaling cascades. Those cascades induce a variety of immune functions that restrain the spread of microbes in the host. Insect and mammalian innate immune receptors include molecules that recognize conserved microbial molecular patterns. Innate immune recognition leads to the recruitment of adaptor molecules forming multi-protein complexes that include kinases, transcription factors and other regulatory molecules. Innate immune signaling cascades induce the expression of genes encoding antimicrobial peptides and other key factors that mount and regulate the immune response against microbial challenge. In this review, we summarize our current understanding of the bacterial and fungal pattern recognition receptors for homologous innate signaling pathways of insects and mammals in an effort to provide a framework for future studies.

  11. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J

    1994-01-01

    -phosphorylated and associated in vivo with the Grb2 protein. This association can be reproduced in stably and transiently transfected cells, as well as in vitro using recombinant Grb2 protein. Association requires the presence of an intact SH2 domain in Grb2, as well as tyrosine phosphorylation of R-PTP-alpha. This observation......Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine...... links a receptor tyrosine phosphatase with a key component of a central cellular signalling pathway and provides a basis for addressing R-PTP-alpha function....

  12. Ascent Heating Thermal Analysis on the Spacecraft Adaptor (SA) Fairings and the Interface with the Crew Launch Vehicle (CLV)

    Science.gov (United States)

    Wang, Xiao-Yen; Yuko, James; Motil, Brian

    2009-01-01

    When the crew exploration vehicle (CEV) is launched, the spacecraft adaptor (SA) fairings that cover the CEV service module (SM) are exposed to aero heating. Thermal analysis is performed to compute the fairing temperatures and to investigate whether the temperatures are within the material limits for nominal ascent aero heating case. Heating rates from Thermal Environment (TE) 3 aero heating analysis computed by engineers at Marshall Space Flight Center (MSFC) are used in the thermal analysis. Both MSC Patran 2007r1b/Pthermal and C&R Thermal Desktop 5.1/Sinda models are built to validate each other. The numerical results are also compared with those reported by Lockheed Martin (LM) and show a reasonably good agreement.

  13. A Patient-Controlled Analgesia Adaptor to Mitigate Postsurgical Pain for Combat Casualties With Multiple Limb Amputation: A Case Series.

    Science.gov (United States)

    Pasquina, Paul F; Isaacson, Brad M; Johnson, Elizabeth; Rhoades, Daniel S; Lindholm, Mark P; Grindle, Garrett G; Cooper, Rory A

    2016-08-01

    The use of explosive armaments during Operation Iraqi Freedom, Operation Enduring Freedom, and Operation New Dawn has resulted in a significant number of injured U.S. service members. These weapons often generate substantial extremity trauma requiring multiple surgical procedures to preserve life, limb, and restore function. For those individuals who require multiple surgeries, the use of patient-controlled analgesia (PCA) devices can be an effective way to achieve adequate pain management and promote successful rehabilitation and recovery during inpatient treatment. A subpopulation of patients are unable to independently control a PCA device because of severe multiple limb dysfunction and/or loss. In response to the needs of these patients, our team designed and developed a custom adaptor to assist service members who would otherwise not be able to use a PCA. Patient feedback of the device indicated a positive response, improved independence, and overall satisfaction during inpatient hospitalization. PMID:27483540

  14. Competitive and cooperative interactions mediate RNA transfer from herpesvirus saimiri ORF57 to the mammalian export adaptor ALYREF.

    Directory of Open Access Journals (Sweden)

    Richard B Tunnicliffe

    2014-02-01

    Full Text Available The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an α-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the α-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions.

  15. Bacterial sex in dental plaque

    Directory of Open Access Journals (Sweden)

    Ingar Olsen

    2013-06-01

    Full Text Available Genes are transferred between bacteria in dental plaque by transduction, conjugation, and transformation. Membrane vesicles can also provide a mechanism for horizontal gene transfer. DNA transfer is considered bacterial sex, but the transfer is not parallel to processes that we associate with sex in higher organisms. Several examples of bacterial gene transfer in the oral cavity are given in this review. How frequently this occurs in dental plaque is not clear, but evidence suggests that it affects a number of the major genera present. It has been estimated that new sequences in genomes established through horizontal gene transfer can constitute up to 30% of bacterial genomes. Gene transfer can be both inter- and intrageneric, and it can also affect transient organisms. The transferred DNA can be integrated or recombined in the recipient's chromosome or remain as an extrachromosomal inheritable element. This can make dental plaque a reservoir for antimicrobial resistance genes. The ability to transfer DNA is important for bacteria, making them better adapted to the harsh environment of the human mouth, and promoting their survival, virulence, and pathogenicity.

  16. Pembuatan Membran Selulosa Bakteri Coating Kitosan - Kolagen Untuk Aplikasi Gtr ( Guide Tissue Regeneration ) Sebagai Pembalut Luka Pada Mencit (Mus Musculus)Secara In Vivo

    OpenAIRE

    Humaira, Nadia Maulida

    2015-01-01

    Bacterial cellulose produced from the fermentation process used in the development of Acetobacter xylinum to increase efficiency of bacterial cellulose one of them in the biomedical field , is membrane . This study aimed to determine the effect concentration of chitosan-collagen, see optimum characterization of bacterial cellulose membrane coating of chitosan-collagen that can be used in the application as wound dressings in mice by In Vivo. Preparation of the bacterial cellulose membrane usi...

  17. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

    Klemm, Per; Vejborg, Rebecca Munk; Hancock, Viktoria

    2010-01-01

    Management of bacterial infections is becoming increasingly difficult due to the emergence and increasing prevalence of bacterial pathogens that are resistant to available antibiotics. Conventional antibiotics generally kill bacteria by interfering with vital cellular functions, an approach that ...

  18. Biophysical studies of membrane channel polypeptides

    CERN Document Server

    Galbraith, T P

    2001-01-01

    Membrane channels facilitate the flow of ions across biological membranes, a process which is important in numerous cellular functions. The study of large integral membrane proteins is made difficult by identification, production and purification problems, and detailed knowledge of their three-dimensional structures is relatively scarce. The study of simple 'model' membrane proteins has given valuable insight into the structures and dynamics of membrane proteins in general. The bacterial peptide gramicidin has been the subject of intense study for many years, and has provided important information into the structural basis of channel function. Peptaibols, a class of fungal membrane peptides which includes alamethicin and antiamoebin, have also been useful in relating structural details to molecular ion transport processes. Gramicidin crystals were grown in the presence of phospholipids with various headgroups and acyl chains. The diffraction patterns of the crystals obtained were processed, but found to be in...

  19. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

    Klemm, Per; Vejborg, Rebecca Munk; Hancock, Viktoria

    2010-01-01

    that imposes selection pressure for resistant bacteria. New approaches are urgently needed. Targeting bacterial virulence functions directly is an attractive alternative. An obvious target is bacterial adhesion. Bacterial adhesion to surfaces is the first step in colonization, invasion, and biofilm formation....... As such, adhesion represents the Achilles heel of crucial pathogenic functions. It follows that interference with adhesion can reduce bacterial virulence. Here, we illustrate this important topic with examples of techniques being developed that can inhibit bacterial adhesion. Some of these will become...

  20. Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Zawawi, M.S.F. [Universiti Sains Malaysia (USM) (Malaysia); Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); Dharmapatni, A.A.S.S.K.; Cantley, M.D. [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); McHugh, K.P. [University of Florida, College of Dentistry, Fl (United States); Haynes, D.R. [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); Crotti, T.N., E-mail: tania.crotti@adelaide.edu.au [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Calcineurin/NFAT inhibitors FK506 and VIVIT treated human PBMC derived osteoclasts in vitro. Black-Right-Pointing-Pointer Differential regulation of ITAM receptors and adaptor molecules by calcineurin/NFAT inhibitors. Black-Right-Pointing-Pointer FK506 and VIVIT suppress ITAM factors during late phase osteoclast differentiation. -- Abstract: Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcR{gamma}) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin ({beta}3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real

  1. Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

    International Nuclear Information System (INIS)

    Highlights: ► Calcineurin/NFAT inhibitors FK506 and VIVIT treated human PBMC derived osteoclasts in vitro. ► Differential regulation of ITAM receptors and adaptor molecules by calcineurin/NFAT inhibitors. ► FK506 and VIVIT suppress ITAM factors during late phase osteoclast differentiation. -- Abstract: Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcRγ) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin (β3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that FK506 treatment

  2. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E;

    1996-01-01

    area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane......The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  3. Hijacking of eukaryotic functions by intracellular bacterial pathogens

    OpenAIRE

    Alonso, Ana; García del Portillo, Francisco

    2004-01-01

    Intracellular bacterial pathogens have evolved as a group of microorganisms endowed with weapons to hijack many biological processes of eukaryotic cells. This review discusses how these pathogens perturb diverse host cell functions, such as cytoskeleton dynamics and organelle vesicular trafficking. Alteration of the cytoskeleton is discussed in the context of the bacterial entry process (invasion), which occurs either by activation of membrane-located host receptors ("zipper" mechani...

  4. Invertebrate and vertebrate class III myosins interact with MORN repeat-containing adaptor proteins.

    Directory of Open Access Journals (Sweden)

    Kirk L Mecklenburg

    Full Text Available In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP. Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A. In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior.

  5. Syk Kinase-Coupled C-type Lectin Receptors Engage Protein Kinase C-δ to Elicit Card9 Adaptor-Mediated Innate Immunity

    OpenAIRE

    Strasser, Dominikus; Neumann, Konstantin; Bergmann, Hanna; Marakalala, Mohlopheni J.; Guler, Reto; Rojowska, Anna; Hopfner, Karl-Peter; Brombacher, Frank; Urlaub, Henning; Baier, Gottfried; Brown, Gordon D.; Leitges, Michael; Ruland, Jürgen

    2012-01-01

    Summary C-type lectin receptors (CLRs) that couple with the kinase Syk are major pattern recognition receptors for the activation of innate immunity and host defense. CLRs recognize fungi and other forms of microbial or sterile danger, and they induce inflammatory responses through the adaptor protein Card9. The mechanisms relaying CLR proximal signals to the core Card9 module are unknown. Here we demonstrated that protein kinase C-δ (PKCδ) was activated upon Dectin-1-Syk signaling, mediated ...

  6. Nuclear IKKbeta is an adaptor protein for IkappaBalpha ubiquitination and degradation in UV-induced NF-kappaB activation.

    Science.gov (United States)

    Tsuchiya, Yoshihiro; Asano, Tomoichiro; Nakayama, Keiko; Kato, Tomohisa; Karin, Michael; Kamata, Hideaki

    2010-08-27

    Proinflammatory cytokines activate NF-kappaB using the IkappaB kinase (IKK) complex that phosphorylates inhibitory proteins (IkappaBs) at N-terminal sites resulting in their ubiquitination and degradation in the cytoplasm. Although ultraviolet (UV) irradiation does not lead to IKK activity, it activates NF-kappaB by an unknown mechanism through IkappaBalpha degradation without N-terminal phosphorylation. Here, we describe an adaptor function of nuclear IKKbeta in UV-induced IkappaBalpha degradation. UV irradiation induces the nuclear translocation of IkappaBalpha and association with IKKbeta, which constitutively interacts with beta-TrCP through heterogeneous ribonucleoprotein-U (hnRNP-U) leading to IkappaBalpha ubiquitination and degradation. Furthermore, casein kinase 2 (CK2) and p38 associate with IKKbeta and promote IkappaBalpha degradation by phosphorylation at C-terminal sites. Thus, nuclear IKKbeta acts as an adaptor protein for IkappaBalpha degradation in UV-induced NF-kappaB activation. NF-kappaB activated by the nuclear IKKbeta adaptor protein suppresses anti-apoptotic gene expression and promotes UV-induced cell death.

  7. Apolipoprotein L-I Promotes Trypanosome Lysis by Forming Pores in Lysosomal Membranes

    Science.gov (United States)

    Pérez-Morga, David; Vanhollebeke, Benoit; Paturiaux-Hanocq, Françoise; Nolan, Derek P.; Lins, Laurence; Homblé, Fabrice; Vanhamme, Luc; Tebabi, Patricia; Pays, Annette; Poelvoorde, Philippe; Jacquet, Alain; Brasseur, Robert; Pays, Etienne

    2005-07-01

    Apolipoprotein L-I is the trypanolytic factor of human serum. Here we show that this protein contains a membrane pore-forming domain functionally similar to that of bacterial colicins, flanked by a membrane-addressing domain. In lipid bilayer membranes, apolipoprotein L-I formed anion channels. In Trypanosoma brucei, apolipoprotein L-I was targeted to the lysosomal membrane and triggered depolarization of this membrane, continuous influx of chloride, and subsequent osmotic swelling of the lysosome until the trypanosome lysed.

  8. VESICULAR TRANSPORT. A structure of the COPI coat and the role of coat proteins in membrane vesicle assembly.

    Science.gov (United States)

    Dodonova, S O; Diestelkoetter-Bachert, P; von Appen, A; Hagen, W J H; Beck, R; Beck, M; Wieland, F; Briggs, J A G

    2015-07-10

    Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.

  9. Early Loss of Telomerase Action in Yeast Creates a Dependence on the DNA Damage Response Adaptor Proteins.

    Science.gov (United States)

    Jay, Kyle A; Smith, Dana L; Blackburn, Elizabeth H

    2016-07-15

    Telomeres cap the ends of chromosomes, protecting them from degradation and inappropriate DNA repair processes that can lead to genomic instability. A short telomere elicits increased telomerase action on itself that replenishes telomere length, thereby stabilizing the telomere. In the prolonged absence of telomerase activity in dividing cells, telomeres eventually become critically short, inducing a permanent cell cycle arrest (senescence). We recently showed that even early after telomerase inactivation (ETI), yeast cells have accelerated mother cell aging and mildly perturbed cell cycles. Here, we show that the complete disruption of DNA damage response (DDR) adaptor proteins in ETI cells causes severe growth defects. This synthetic-lethality phenotype was as pronounced as that caused by extensive DNA damage in wild-type cells but showed genetic dependencies distinct from such damage and was completely alleviated by SML1 deletion, which increases deoxynucleoside triphosphate (dNTP) pools. Our results indicated that these deleterious effects in ETI cells cannot be accounted for solely by the slow erosion of telomeres due to incomplete replication that leads to senescence. We propose that normally occurring telomeric DNA replication stress is resolved by telomerase activity and the DDR in two parallel pathways and that deletion of Sml1 prevents this stress. PMID:27161319

  10. Matrilin-2, an extracellular adaptor protein, is needed for the regeneration of muscle, nerve and other tissues

    Institute of Scientific and Technical Information of China (English)

    va Korpos; Ferenc Dek; Ibolya Kiss

    2015-01-01

    The extracellular matrix (ECM) performs essential functions in the differentiation, maintenance and remodeling of tissues during development and regeneration, and it undergoes dynamic chang-es during remodeling concomitant to alterations in the cell-ECM interactions. Here we discuss recent data addressing the critical role of the widely expressed ECM protein, matrilin-2 (Matn2) in the timely onset of differentiation and regeneration processes in myogenic, neural and other tissues and in tumorigenesis. As a multiadhesion adaptor protein, it interacts with other ECM proteins and integrins. Matn2 promotes neurite outgrowth, Schwann cell migration, neuromuscular junc-tion formation, skeletal muscle and liver regeneration and skin wound healing. Matn2 deposition by myoblasts is crucial for the timely induction of the global switch toward terminal myogenic differentiation during muscle regeneration by affecting transforming growth factor beta/bone morphogenetic protein 7/Smad and other signal transduction pathways. Depending on the type of tissue and the pathomechanism, Matn2 can also promote or suppress tumor growth.

  11. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects

    Science.gov (United States)

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A.

    2016-01-01

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. PMID:27257060

  12. Molecular cloning and functional analysis of the duck TIR domain-containing adaptor inducing IFN-β (TRIF) gene.

    Science.gov (United States)

    Wei, Xiaoqin; Qian, Wei; Sizhu, Suolang; Shi, Lijuan; Jin, Meilin; Zhou, Hongbo

    2016-12-01

    Toll-like receptors (TLRs) trigger the innate immune response by responding to specific components of microorganisms. The TIR domain-containing adaptor inducing IFN-β (TRIF) plays an essential role in mammalian TLR-mediated signaling. The role of TRIF in ducks (duTRIF) remains poorly understood. In this study, we cloned and characterized the full-length coding sequence of duTRIF from duck embryo fibroblasts (DEFs). In healthy ducks, duTRIF transcripts were broadly expressed in different tissues, with higher expression levels in the spleen and liver. Using quantitative real-time PCR (qRT-PCR), we demonstrated the upregulation of duTRIF in DEFs infected with AIV or DTMUV, and DEFs treated with Poly I:C or LPS. Overexpression of duTRIF was able to induce the NF-κB and IFN-β expression. Furthermore, the IFN induction function of duTRIF was impaired when Ala517 was mutated to Pro or His. Taken together, these results suggested that duTRIF regulated duck innate immune responses. PMID:27539203

  13. Positive and negative regulation of FcepsilonRI-mediated signaling by the adaptor protein LAB/NTAL.

    Science.gov (United States)

    Zhu, Minghua; Liu, Yan; Koonpaew, Surapong; Granillo, Olivia; Zhang, Weiguo

    2004-10-18

    Linker for activation of B cells (LAB, also called NTAL; a product of wbscr5 gene) is a newly identified transmembrane adaptor protein that is expressed in B cells, NK cells, and mast cells. Upon BCR activation, LAB is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT-deficient mice. To study the in vivo function of LAB, LAB-deficient mice were generated. Although disruption of the Lab gene did not affect lymphocyte development, it caused mast cells to be hyperresponsive to stimulation via the FcepsilonRI, evidenced by enhanced Erk activation, calcium mobilization, degranulation, and cytokine production. These data suggested that LAB negatively regulates mast cell function. However, mast cells that lacked both linker for activation of T cells (LAT) and LAB proteins had a more severe block in FcepsilonRI-mediated signaling than LAT(-/-) mast cells, demonstrating that LAB also shares a redundant function with LAT to play a positive role in FcepsilonRI-mediated signaling.

  14. The cytoskeleton adaptor protein ankyrin-1 is upregulated by p53 following DNA damage and alters cell migration.

    Science.gov (United States)

    Hall, A E; Lu, W-T; Godfrey, J D; Antonov, A V; Paicu, C; Moxon, S; Dalmay, T; Wilczynska, A; Muller, P A J; Bushell, M

    2016-01-01

    The integrity of the genome is maintained by a host of surveillance and repair mechanisms that are pivotal for cellular function. The tumour suppressor protein p53 is a major component of the DNA damage response pathway and plays a vital role in the maintenance of cell-cycle checkpoints. Here we show that a microRNA, miR-486, and its host gene ankyrin-1 (ANK1) are induced by p53 following DNA damage. Strikingly, the cytoskeleton adaptor protein ankyrin-1 was induced over 80-fold following DNA damage. ANK1 is upregulated in response to a variety of DNA damage agents in a range of cell types. We demonstrate that miR-486-5p is involved in controlling G1/S transition following DNA damage, whereas the induction of the ankyrin-1 protein alters the structure of the actin cytoskeleton and sustains limited cell migration during DNA damage. Importantly, we found that higher ANK1 expression correlates with decreased survival in cancer patients. Thus, these observations highlight ANK1 as an important effector downstream of the p53 pathway. PMID:27054339

  15. Identification of lysines within membrane-anchored Mga2p120 that are targets of Rsp5p ubiquitination and mediate mobilization of tethered Mga2p90

    OpenAIRE

    Bhattacharya, Sabyasachi; Shcherbik, Natalia; Vasilescu, Julian; Smith, Jeffrey C.; Figeys, Daniel; Haines, Dale S.

    2008-01-01

    Mga2p90 is an endoplasmic reticulum (ER) localized transcription factor that is released from the ER membrane by a unique ubiquitin-dependent mechanism. Mga2p90 mobilization requires poly-ubiquitination of its associating membrane-bound Mga2p120 anchor and subsequent Mga2p120-Mga2p90 complex disassembly that is mediated by ATPase Cdc48p and its heteromeric ubiquitin-binding adaptor Npl4p-Ufd1p. Although previous studies have identified the ubiquitin ligase (i.e. Rsp5p) and ligase binding site...

  16. One-way membrane trafficking of SOS in receptor-triggered Ras activation.

    Science.gov (United States)

    Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T

    2016-09-01

    SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis. PMID:27501536

  17. One-way membrane trafficking of SOS in receptor-triggered Ras activation.

    Science.gov (United States)

    Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T

    2016-09-01

    SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.

  18. Robotic membranes

    DEFF Research Database (Denmark)

    Ramsgaard Thomsen, Mette

    2008-01-01

    , Vivisection and Strange Metabolisms, were developed at the Centre for Information Technology and Architecture (CITA) at the Royal Danish Academy of Fine Arts in Copenhagen as a means of engaging intangible digital data with tactile physical material. As robotic membranes, they are a dual examination...

  19. Anisotropic Membrane Curvature Sensing by Amphipathic Peptides.

    Science.gov (United States)

    Gómez-Llobregat, Jordi; Elías-Wolff, Federico; Lindén, Martin

    2016-01-01

    Many proteins and peptides have an intrinsic capacity to sense and induce membrane curvature, and play crucial roles for organizing and remodeling cell membranes. However, the molecular driving forces behind these processes are not well understood. Here, we describe an approach to study curvature sensing by simulating the interactions of single molecules with a buckled lipid bilayer. We analyze three amphipathic antimicrobial peptides, a class of membrane-associated molecules that specifically target and destabilize bacterial membranes, and find qualitatively different sensing characteristics that would be difficult to resolve with other methods. Our findings provide evidence for direction-dependent curvature sensing mechanisms in amphipathic peptides and challenge existing theories of hydrophobic insertion. The buckling approach is generally applicable to a wide range of curvature-sensing molecules, and our results provide strong motivation to develop new experimental methods to track position and orientation of membrane proteins. PMID:26745422

  20. Anisotropic membrane curvature sensing by antibacterial peptides

    CERN Document Server

    Gómez-Llobregat, Jordi; Lindén, Martin

    2014-01-01

    Many proteins and peptides have an intrinsic capacity to sense and induce membrane curvature, and play crucial roles for organizing and remodeling cell membranes. However, the molecular driving forces behind these processes are not well understood. Here, we describe a new approach to study curvature sensing, by simulating the direction-dependent interactions of single molecules with a buckled lipid bilayer. We analyze three antimicrobial peptides, a class of membrane-associated molecules that specifically target and destabilize bacterial membranes, and find qualitatively different sensing characteristics that would be difficult to resolve with other methods. These findings provide new insights into the microscopic mechanisms of antimicrobial peptides, which might aid the development of new antibiotics. Our approach is generally applicable to a wide range of curvature sensing molecules, and our results provide strong motivation to develop new experimental methods to track position and orientation of membrane p...

  1. Disinfection of a Polyamide Nanofiltration Membrane using Ethanol

    OpenAIRE

    Heffernan, R.; Semião, A.J.C.; Desmond, P; Cao, H.; Safari, A.; Habimana, O; Casey, E.

    2013-01-01

    It is imperative that nanofiltration membranes are disinfected before they are used for laboratory-scale bacterial adhesion or biofouling experiments, yet currently no suitable disinfection protocol exists. This study aimed to determine if an ethanol treatment at a minimum inhibitory concentration (MIC) could be used to effectively disinfect nanofiltration membranes without altering membrane properties which could affect research. Two strains of bacteria, Pseudomonas fluorescens and Staphyloc...

  2. MreB-Dependent Organization of the E. coli Cytoplasmic Membrane Controls Membrane Protein Diffusion.

    Science.gov (United States)

    Oswald, Felix; Varadarajan, Aravindan; Lill, Holger; Peterman, Erwin J G; Bollen, Yves J M

    2016-03-01

    The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs. PMID:26958890

  3. Vimentin in Bacterial Infections

    Directory of Open Access Journals (Sweden)

    Tim N. Mak

    2016-04-01

    Full Text Available Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate filaments (IFs. IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria-vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial virulence factors that target vimentin to subvert its function in order to change the host cell fate in the course of a bacterial infection.

  4. The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer

    Directory of Open Access Journals (Sweden)

    Natsume Hiroko

    2012-07-01

    Full Text Available Abstract Background Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer. Methods and Results A genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2 in five highly amplified chromosome regions (with a copy number of more than 6 were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3 domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH, and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360 of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide. Conclusion These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

  5. Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells.

    Science.gov (United States)

    Reginald, Kavita; Chaoui, Karima; Roncagalli, Romain; Beau, Mathilde; Goncalves Menoita, Marisa; Monsarrat, Bernard; Burlet-Schiltz, Odile; Malissen, Marie; Gonzalez de Peredo, Anne; Malissen, Bernard

    2015-12-01

    The protein tyrosine kinase LCK plays a key role in TCR signaling, and its activity is dynamically controlled by the tyrosine kinase C-terminal Src kinase (CSK) and the tyrosine phosphatase CD45. CSK is brought in contiguity to LCK via binding to a transmembrane adaptor known as phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG). The lack of a blatant phenotype in PAG-deficient mice has impeded our understanding of the mechanisms through which PAG exerts its negative-regulatory role in TCR signaling. We used quantitative mass spectrometry and both thymocytes and CD4(+) T cells from mice in which a tag for affinity purification was knocked in the gene coding for PAG to determine the composition and dynamics of the multiprotein complexes that are found around PAG over 5 min of activation. Most of the high-confidence interactions that we observed were previously unknown. Using phosphoproteomic analysis, PAG showed low levels of tyrosine phosphorylation in resting primary mouse CD4(+) T cells; the levels of tyrosine phosphorylation increased and reached a maximum 2 min after stimulation. Analysis of the dynamics of association of the protein tyrosine phosphatase PTPN22 and lipid phosphatase SHIP-1 with PAG following T cell activation suggests that both cooperate with CSK to terminate T cell activation. Our findings provide a model of the role for PAG in mouse primary CD4(+) T cells that is consistent with recent phosphoproteomic studies of the Jurkat T cell line but difficult to reconcile with former biochemical studies indicating that PAG is constitutively phosphorylated in resting T cells and rapidly dephosphorylated once the TCR is engaged. PMID:26512138

  6. CRM1 and its ribosome export adaptor NMD3 localize to the nucleolus and affect rRNA synthesis.

    Science.gov (United States)

    Bai, Baoyan; Moore, Henna M; Laiho, Marikki

    2013-01-01

    CRM1 is an export factor that together with its adaptor NMD3 transports numerous cargo molecules from the nucleus to cytoplasm through the nuclear pore. Previous studies have suggested that CRM1 and NMD3 are detected in the nucleolus. However, their localization with subnucleolar domains or participation in the activities of the nucleolus are unclear. We demonstrate here biochemically and using imaging analyses that CRM1 and NMD3 co-localize with nucleolar marker proteins in the nucleolus. In particular, their nucleolar localization is markedly increased by inhibition of RNA polymerase I (Pol I) transcription by actinomycin D or by silencing Pol I catalytic subunit, RPA194. We show that CRM1 nucleolar localization is dependent on its activity and the expression of NMD3, whereas NMD3 nucleolar localization is independent of CRM1. This suggests that NMD3 provides nucleolar tethering of CRM1. While inhibition of CRM1 by leptomycin B inhibited processing of 28S ribosomal (r) RNA, depletion of NMD3 did not, suggesting that their effects on 28S rRNA processing are distinct. Markedly, depletion of NMD3 and inhibition of CRM1 reduced the rate of pre-47S rRNA synthesis. However, their inactivation did not lead to nucleolar disintegration, a hallmark of Pol I transcription stress, suggesting that they do not directly regulate transcription. These results indicate that CRM1 and NMD3 have complex functions in pathways that couple rRNA synthetic and processing engines and that the rRNA synthesis rate may be adjusted according to proficiency in rRNA processing and export.

  7. Molecular analysis of the prostacyclin receptor's interaction with the PDZ1 domain of its adaptor protein PDZK1.

    Directory of Open Access Journals (Sweden)

    Gabriel Birrane

    Full Text Available The prostanoid prostacyclin, or prostaglandin I2, plays an essential role in many aspects of cardiovascular disease. The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1 of PDZK1, we generated a nine residue peptide (KK(411IAACSLC(417 containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 µM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP ((411IAACSLC(417. Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 at least in vitro.

  8. The adaptor CARD9 is required for adaptive but not innate immunity to oral mucosal Candida albicans infections.

    Science.gov (United States)

    Bishu, Shrinivas; Hernández-Santos, Nydiaris; Simpson-Abelson, Michelle R; Huppler, Anna R; Conti, Heather R; Ghilardi, Nico; Mamo, Anna J; Gaffen, Sarah L

    2014-03-01

    Oropharyngeal candidiasis (OPC [thrush]) is an opportunistic infection caused by the commensal fungus Candida albicans. OPC is common in individuals with HIV/AIDS, infants, patients on chemotherapy, and individuals with congenital immune defects. Immunity to OPC is strongly dependent on the interleukin-23 (IL-23)/IL-17R axis, as mice and humans with defects in IL-17R signaling (IL17F, ACT1, IL-17RA) or in genes that direct Th17 differentiation (STAT3, STAT1, CARD9) are prone to mucocutaneous candidiasis. Conventional Th17 cells are induced in response to C. albicans infection via signals from C-type lectin receptors, which signal through the adaptor CARD9, leading to production of Th17-inducing cytokines such as IL-6, IL-1β, and IL-23. Recent data indicate that IL-17 can also be made by numerous innate cell subsets. These innate "type 17" cells resemble conventional Th17 cells, but they can be activated without need for prior antigen exposure. Because C. albicans is not a commensal organism in rodents and mice are thus naive to this fungus, we had the opportunity to assess the role of CARD9 in innate versus adaptive responses using an OPC infection model. As expected, CARD9(-/-) mice failed to mount an adaptive Th17 response following oral Candida infection. Surprisingly, however, CARD9(-/-) mice had preserved innate IL-17-dependent responses to Candida and were almost fully resistant to OPC. Thus, CARD9 is important primarily for adaptive immunity to C. albicans, whereas alternate recognition systems appear to be needed for effective innate responses. PMID:24379290

  9. Structure of bacterial respiratory complex I.

    Science.gov (United States)

    Berrisford, John M; Baradaran, Rozbeh; Sazanov, Leonid A

    2016-07-01

    Complex I (NADH:ubiquinone oxidoreductase) plays a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation. It is the largest protein assembly of respiratory chains and one of the most elaborate redox membrane proteins known. Bacterial enzyme is about half the size of mitochondrial and thus provides its important "minimal" model. Dysfunction of mitochondrial complex I is implicated in many human neurodegenerative diseases. The L-shaped complex consists of a hydrophilic arm, where electron transfer occurs, and a membrane arm, where proton translocation takes place. We have solved the crystal structures of the hydrophilic domain of complex I from Thermus thermophilus, the membrane domain from Escherichia coli and recently of the intact, entire complex I from T. thermophilus (536 kDa, 16 subunits, 9 iron-sulphur clusters, 64 transmembrane helices). The 95Å long electron transfer pathway through the enzyme proceeds from the primary electron acceptor flavin mononucleotide through seven conserved Fe-S clusters to the unusual elongated quinone-binding site at the interface with the membrane domain. Four putative proton translocation channels are found in the membrane domain, all linked by the central flexible axis containing charged residues. The redox energy of electron transfer is coupled to proton translocation by the as yet undefined mechanism proposed to involve long-range conformational changes. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt. PMID:26807915

  10. Structure of bacterial respiratory complex I.

    Science.gov (United States)

    Berrisford, John M; Baradaran, Rozbeh; Sazanov, Leonid A

    2016-07-01

    Complex I (NADH:ubiquinone oxidoreductase) plays a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation. It is the largest protein assembly of respiratory chains and one of the most elaborate redox membrane proteins known. Bacterial enzyme is about half the size of mitochondrial and thus provides its important "minimal" model. Dysfunction of mitochondrial complex I is implicated in many human neurodegenerative diseases. The L-shaped complex consists of a hydrophilic arm, where electron transfer occurs, and a membrane arm, where proton translocation takes place. We have solved the crystal structures of the hydrophilic domain of complex I from Thermus thermophilus, the membrane domain from Escherichia coli and recently of the intact, entire complex I from T. thermophilus (536 kDa, 16 subunits, 9 iron-sulphur clusters, 64 transmembrane helices). The 95Å long electron transfer pathway through the enzyme proceeds from the primary electron acceptor flavin mononucleotide through seven conserved Fe-S clusters to the unusual elongated quinone-binding site at the interface with the membrane domain. Four putative proton translocation channels are found in the membrane domain, all linked by the central flexible axis containing charged residues. The redox energy of electron transfer is coupled to proton translocation by the as yet undefined mechanism proposed to involve long-range conformational changes. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

  11. Focal Targeting of the Bacterial Envelope by Antimicrobial Peptides

    Directory of Open Access Journals (Sweden)

    Rafi eRashid

    2016-06-01

    Full Text Available Antimicrobial peptides (AMPs are utilized by both eukaryotic and prokaryotic organisms. AMPs such as the human beta defensins, human neutrophil peptides, human cathelicidin, and many bacterial bacteriocins are cationic and capable of binding to anionic regions of the bacterial surface. Cationic AMPs (CAMPs target anionic lipids (e.g. phosphatidylglycerol (PG and cardiolipins (CL in the cell membrane and anionic components (e.g. lipopolysaccharide (LPS and lipoteichoic acid (LTA of the cell envelope. Bacteria have evolved mechanisms to modify these same targets in order to resist CAMP killing, e.g. lysinylation of PG to yield cationic lysyl-PG and alanylation of LTA. Since CAMPs offer a promising therapeutic alternative to conventional antibiotics, which are becoming less effective due to rapidly emerging antibiotic resistance, there is a strong need to improve our understanding about the AMP mechanism of action. Recent literature suggests that AMPs often interact with the bacterial cell envelope at discrete foci. Here we review recent AMP literature, with an emphasis on focal interactions with bacteria, including (1 CAMP disruption mechanisms, (2 delocalization of membrane proteins and lipids by CAMPs, and (3 CAMP sensing systems and resistance mechanisms. We conclude with new approaches for studying the bacterial membrane, e.g., lipidomics, high resolution imaging and non-detergent-based membrane domain extraction.

  12. Focal Targeting of the Bacterial Envelope by Antimicrobial Peptides.

    Science.gov (United States)

    Rashid, Rafi; Veleba, Mark; Kline, Kimberly A

    2016-01-01

    Antimicrobial peptides (AMPs) are utilized by both eukaryotic and prokaryotic organisms. AMPs such as the human beta defensins, human neutrophil peptides, human cathelicidin, and many bacterial bacteriocins are cationic and capable of binding to anionic regions of the bacterial surface. Cationic AMPs (CAMPs) target anionic lipids [e.g., phosphatidylglycerol (PG) and cardiolipins (CL)] in the cell membrane and anionic components [e.g., lipopolysaccharide (LPS) and lipoteichoic acid (LTA)] of the cell envelope. Bacteria have evolved mechanisms to modify these same targets in order to resist CAMP killing, e.g., lysinylation of PG to yield cationic lysyl-PG and alanylation of LTA. Since CAMPs offer a promising therapeutic alternative to conventional antibiotics, which are becoming less effective due to rapidly emerging antibiotic resistance, there is a strong need to improve our understanding about the AMP mechanism of action. Recent literature suggests that AMPs often interact with the bacterial cell envelope at discrete foci. Here we review recent AMP literature, with an emphasis on focal interactions with bacteria, including (1) CAMP disruption mechanisms, (2) delocalization of membrane proteins and lipids by CAMPs, and (3) CAMP sensing systems and resistance mechanisms. We conclude with new approaches for studying the bacterial membrane, e.g., lipidomics, high resolution imaging, and non-detergent-based membrane domain extraction. PMID:27376064

  13. Biogenesis and Membrane Targeting of Lipoproteins.

    Science.gov (United States)

    Narita, Shin-Ichiro; Tokuda, Hajime

    2010-09-01

    Bacterial lipoproteins represent a unique class of membrane proteins, which are anchored to membranes through triacyl chains attached to the amino-terminal cysteine. They are involved in various functions localized in cell envelope. Escherichia coli possesses more than 90 species of lipoproteins, most of which are localized in the outer membrane, with others being in the inner membrane. All lipoproteins are synthesized in the cytoplasm with an N-terminal signal peptide, translocated across the inner membrane by the Sec translocon to the periplasmic surface of the inner membrane, and converted to mature lipoproteins through sequential reactions catalyzed by three lipoprotein-processing enzymes: Lgt, LspA, and Lnt. The sorting of lipoproteins to the outer membrane requires a system comprising five Lol proteins. An ATP-binding cassette transporter, LolCDE, initiates the sorting by mediating the detachment of lipoproteins from the inner membrane. Formation of the LolA-lipoprotein complex is coupled to this LolCDE-dependent release reaction. LolA accommodates the amino-terminal acyl chain of lipoproteins in its hydrophobic cavity, thereby generating a hydrophilic complex that can traverse the periplasmic space by diffusion. Lipoproteins are then transferred to LolB on the outer membrane and anchored to the inner leaflet of the outer membrane by the action of LolB. In contrast, since LolCDE does not recognize lipoproteins possessing Asp at position +2, these lipoproteins remain anchored to the inner membrane. Genes for Lol proteins are widely conserved among gram-negative bacteria, and Lol-mediated outer membrane targeting of lipoproteins is considered to be the general lipoprotein localization mechanism. PMID:26443779

  14. The Role of Lipid Domains in Bacterial Cell Processes

    Directory of Open Access Journals (Sweden)

    Katarína Muchová

    2013-02-01

    Full Text Available Membranes are vital structures for cellular life forms. As thin, hydrophobic films, they provide a physical barrier separating the aqueous cytoplasm from the outside world or from the interiors of other cellular compartments. They maintain a selective permeability for the import and export of water-soluble compounds, enabling the living cell to maintain a stable chemical environment for biological processes. Cell membranes are primarily composed of two crucial substances, lipids and proteins. Bacterial membranes can sense environmental changes or communication signals from other cells and they support different cell processes, including cell division, differentiation, protein secretion and supplementary protein functions. The original fluid mosaic model of membrane structure has been recently revised because it has become apparent that domains of different lipid composition are present in both eukaryotic and prokaryotic cell membranes. In this review, we summarize different aspects of phospholipid domain formation in bacterial membranes, mainly in Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. We describe the role of these lipid domains in membrane dynamics and the localization of specific proteins and protein complexes in relation to the regulation of cellular function.

  15. Interfering with bacterial gossip

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Tolker-Nielsen, Tim; Givskov, Michael

    2011-01-01

    defense. Antibiotics exhibit a rather limited effect on biofilms. Furthermore, antibiotics have an ‘inherent obsolescence’ because they select for development of resistance. Bacterial infections with origin in bacterial biofilms have become a serious threat in developed countries. Pseudomonas aeruginosa...... that appropriately target bacteria in their relevant habitat with the aim of mitigating their destructive impact on patients. In this review we describe molecular mechanisms involved in “bacterial gossip” (more scientifically referred to as quorum sensing (QS) and c-di-GMP signaling), virulence, biofilm formation...

  16. Peptides that influence membrane topology

    Science.gov (United States)

    Wong, Gerard C. L.

    2014-03-01

    We examine the mechanism of a range of polypeptides that influence membrane topology, including antimicrobial peptides, cell penetrating peptides, viral fusion peptides, and apoptosis proteins, and show how a combination of geometry, coordination chemistry, and soft matter physics can be used to approach a unified understanding. We will also show how such peptides can impact biomedical problems such as auto-immune diseases (psoriasis, lupus), infectious diseases (viral and bacterial infections), and mitochondrial pathologies (under-regulated apoptosis leads to neurodegenerative diseases whereas over-regulated apoptosis leads to cancer.)

  17. A Common Fold Mediates Vertebrate Defense and Bacterial Attack

    Energy Technology Data Exchange (ETDEWEB)

    Rosado, Carlos J.; Buckle, Ashley M.; Law, Ruby H.P.; Butcher, Rebecca E.; Kan, Wan-Ting; Bird, Catherina H.; Ung, Kheng; Browne, Kylie A.; Baran, Katherine; Bashtannyk-Puhalovich, Tanya A.; Faux, Noel G.; Wong, Wilson; Porter, Corrine J.; Pike, Robert N.; Ellisdon, Andrew M.; Pearce, Mary C.; Bottomley, Stephen P.; Emsley, Jonas; Smith, A. Ian; Rossjohn, Jamie; Hartland, Elizabeth L.; Voskoboinik, Ilia; Trapani, Joseph A.; Bird, Phillip I.; Dunstone, Michelle A.; Whisstock, James C. (PMCI-A); (Monash); (Nottingham)

    2008-10-02

    Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

  18. Omniphobic Membrane for Robust Membrane Distillation

    Energy Technology Data Exchange (ETDEWEB)

    Lin, SH; Nejati, S; Boo, C; Hu, YX; Osuji, CO; Ehmelech, M

    2014-11-01

    In this work, we fabricate an omniphobic microporous membrane for membrane distillation (MD) by modifying a hydrophilic glass fiber membrane with silica nanoparticles followed by surface fluorination and polymer coating. The modified glass fiber membrane exhibits an anti-wetting property not only against water but also against low surface tension organic solvents that easily wet a hydrophobic polytetrafluoroethylene (PTFE) membrane that is commonly used in MD applications. By comparing the performance of the PTFE and omniphobic membranes in direct contact MD experiments in the presence of a surfactant (sodium dodecyl sulfate, SDS), we show that SDS wets the hydrophobic PTFE membrane but not the omniphobic membrane. Our results suggest that omniphobic membranes are critical for MD applications with feed waters containing surface active species, such as oil and gas produced water, to prevent membrane pore wetting.

  19. Bacterial Sphingomyelinases and Phospholipases as Virulence Factors.

    Science.gov (United States)

    Flores-Díaz, Marietta; Monturiol-Gross, Laura; Naylor, Claire; Alape-Girón, Alberto; Flieger, Antje

    2016-09-01

    Bacterial sphingomyelinases and phospholipases are a heterogeneous group of esterases which are usually surface associated or secreted by a wide variety of Gram-positive and Gram-negative bacteria. These enzymes hydrolyze sphingomyelin and glycerophospholipids, respectively, generating products identical to the ones produced by eukaryotic enzymes which play crucial roles in distinct physiological processes, including membrane dynamics, cellular signaling, migration, growth, and death. Several bacterial sphingomyelinases and phospholipases are essential for virulence of extracellular, facultative, or obligate intracellular pathogens, as these enzymes contribute to phagosomal escape or phagosomal maturation avoidance, favoring tissue colonization, infection establishment and progression, or immune response evasion. This work presents a classification proposal for bacterial sphingomyelinases and phospholipases that considers not only their enzymatic activities but also their structural aspects. An overview of the main physiopathological activities is provided for each enzyme type, as are examples in which inactivation of a sphingomyelinase- or a phospholipase-encoding gene impairs the virulence of a pathogen. The identification of sphingomyelinases and phospholipases important for bacterial pathogenesis and the development of inhibitors for these enzymes could generate candidate vaccines and therapeutic agents, which will diminish the impacts of the associated human and animal diseases. PMID:27307578

  20. Bacterial Wound Culture

    Science.gov (United States)

    ... Home Visit Global Sites Search Help? Bacterial Wound Culture Share this page: Was this page helpful? Also known as: Aerobic Wound Culture; Anaerobic Wound Culture Formal name: Culture, wound Related ...

  1. Bacterial surface adaptation

    Science.gov (United States)

    Utada, Andrew

    2014-03-01

    Biofilms are structured multi-cellular communities that are fundamental to the biology and ecology of bacteria. Parasitic bacterial biofilms can cause lethal infections and biofouling, but commensal bacterial biofilms, such as those found in the gut, can break down otherwise indigestible plant polysaccharides and allow us to enjoy vegetables. The first step in biofilm formation, adaptation to life on a surface, requires a working knowledge of low Reynolds number fluid physics, and the coordination of biochemical signaling, polysaccharide production, and molecular motility motors. These crucial early stages of biofilm formation are at present poorly understood. By adapting methods from soft matter physics, we dissect bacterial social behavior at the single cell level for several prototypical bacterial species, including Pseudomonas aeruginosa and Vibrio cholerae.

  2. Bacterial intermediate filaments

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Cabeen, M.; Jacobs-Wagner, C.

    2009-01-01

    Crescentin, which is the founding member of a rapidly growing family of bacterial cytoskeletal proteins, was previously proposed to resemble eukaryotic intermediate filament (IF) proteins based on structural prediction and in vitro polymerization properties. Here, we demonstrate that crescentin...

  3. Bacterial Meningitis in Infants

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2004-04-01

    Full Text Available A retrospective study of 80 infantile patients (ages 30-365 days; 47 male, 33 female with culture-proven bacterial meningitis seen over a 16 year period (1986-2001 is reported from Taiwan.

  4. Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

    Science.gov (United States)

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface. PMID:23580642

  5. Bacterial Toxins as Pathogen Weapons Against Phagocytes.

    Science.gov (United States)

    do Vale, Ana; Cabanes, Didier; Sousa, Sandra

    2016-01-01

    Bacterial toxins are virulence factors that manipulate host cell functions and take over the control of vital processes of living organisms to favor microbial infection. Some toxins directly target innate immune cells, thereby annihilating a major branch of the host immune response. In this review we will focus on bacterial toxins that act from the extracellular milieu and hinder the function of macrophages and neutrophils. In particular, we will concentrate on toxins from Gram-positive and Gram-negative bacteria that manipulate cell signaling or induce cell death by either imposing direct damage to the host cells cytoplasmic membrane or enzymatically modifying key eukaryotic targets. Outcomes regarding pathogen dissemination, host damage and disease progression will be discussed.

  6. Bacterial toxins as pathogen weapons against phagocytes

    Directory of Open Access Journals (Sweden)

    Ana edo Vale

    2016-02-01

    Full Text Available Bacterial toxins are virulence factors that manipulate host cell functions and take over the control of vital processes of living organisms to favour microbial infection. Some toxins directly target innate immune cells, thereby annihilating a major branch of the host immune response. In this review we will focus on bacterial toxins that act from the extracellular milieu and hinder the function of macrophages and neutrophils. In particular, we will concentrate on toxins from Gram-positive and Gram-negative bacteria that manipulate cell signalling or induce cell death by either imposing direct damage to the host cells cytoplasmic membrane or enzymatically modifying key eukaryotic targets. Outcomes regarding pathogen dissemination, host damage and disease progression will be discussed.

  7. The protein moiety of Brucella abortus outer membrane protein 16 is a new bacterial pathogen-associated molecular pattern that activates dendritic cells in vivo, induces a Th1 immune response, and is a promising self-adjuvanting vaccine against systemic and oral acquired brucellosis.

    Science.gov (United States)

    Pasquevich, Karina A; García Samartino, Clara; Coria, Lorena M; Estein, Silvia M; Zwerdling, Astrid; Ibañez, Andrés E; Barrionuevo, Paula; Oliveira, Fernanda Souza de; Carvalho, Natalia Barbosa; Borkowski, Julia; Oliveira, Sergio Costa; Warzecha, Heribert; Giambartolomei, Guillermo H; Cassataro, Juliana

    2010-05-01

    Knowing the inherent stimulatory properties of the lipid moiety of bacterial lipoproteins, we first hypothesized that Brucella abortus outer membrane protein (Omp)16 lipoprotein would be able to elicit a protective immune response without the need of external adjuvants. In this study, we demonstrate that Omp16 administered by the i.p. route confers significant protection against B. abortus infection and that the protective response evoked is independent of the protein lipidation. To date, Omp16 is the first Brucella protein that without the requirement of external adjuvants is able to induce similar protection levels to the control live vaccine S19. Moreover, the protein portion of Omp16 (unlipidated Omp16 [U-Omp16]) elicits a protective response when administered by the oral route. Either systemic or oral immunization with U-Omp16 elicits a Th1-specific response. These abilities of U-Omp16 indicate that it is endowed with self-adjuvanting properties. The adjuvanticity of U-Omp16 could be explained, at least in part, by its capacity to activate dendritic cells in vivo. U-Omp16 is also able to stimulate dendritic cells and macrophages in vitro. The latter property and its ability to induce a protective Th1 immune response against B. abortus infection have been found to be TLR4 dependent. The facts that U-Omp16 is an oral protective Ag and possesses a mucosal self-adjuvanting property led us to develop a plant-made vaccine expressing U-Omp16. Our results indicate that plant-expressed recombinant U-Omp16 is able to confer protective immunity, when given orally, indicating that a plant-based oral vaccine expressing U-Omp16 could be a valuable approach to controlling this disease. PMID:20351187

  8. Enhancing biocompatibility of some cation selective electrodes using heparin modified bacterial cellulose.

    Science.gov (United States)

    Badr, Ibrahim H A; Abdel-Sattar, R; Keshk, Sherif M A S

    2015-12-10

    Bacterial cellulose (BC) and heparin-modified bacterial cellulose (HBC) were utilized to enhance the biocompatibility of highly thrombogenic PVC-based potassium and calcium membrane electrodes. Three types of membrane electrodes were prepared: (1) conventional PVC electrode (control), (2) PVC-based electrode sandwiched with bacterial cellulose membrane (BC-PVC), and (3) PVC-based electrode sandwiched with heparin-modified bacterial cellulose membrane (HBC-PVC). The potentiometric response characteristics of the modified potassium and calcium membrane electrodes (BC-PVC and HBC-PVC) were compared with those of the control PVC-based potassium and calcium selective electrode, respectively. Response characteristics of the modified membrane electrodes were comparable to the control PVC membrane electrode. The platelet adhesion investigations indicated that (BC) and (HBC) layers are less thrombogenic compared to PVC. Therefore, use of BC or HBC would enable the enhancement of the biocompatibility of PVC-based membrane electrodes for potassium and calcium while practically maintaining the overall electrochemical performance of the PVC sensing film. PMID:26428173

  9. Development of novel on-chip, customer-design spiral biasing adaptor on for Si drift detectors and detector arrays for X-ray and nuclear physics experiments

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zheng, E-mail: lizheng@xtu.edu.cn [School of Materials, Optoelectronics and Physics, Xiangtan University, Xiangtan, Hunan 411105 (China); Chen, Wei [Brookhaven National Laboratory, Upton, NY 11973 (United States)

    2014-11-21

    A novel on-chip, customer-design spiral biasing adaptor (SBA) has been developed. A single SBA is used for biasing a Si drift detector (SDD) and SDD array. The use of an SBA reduces the biasing current. This paper shows the calculation of the geometry of an SBA and an SDD to get the best drift field in the SDD and SDD array. Prototype SBAs have been fabricated to verify the concept. Electrical measurements on these SBAs are in agreement with the expectations. The new SDD array with an SBA can be used for X-ray detection and in nuclear physics experiments.

  10. Increasing complexity of the bacterial cytoskeleton

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Löwe, Jan

    2005-01-01

    Bacteria contain cytoskeletal elements involved in major cellular processes including DNA segregation and cell morphogenesis and division. Distant bacterial homologues of tubulin (FtsZ) and actin (MreB and ParM) not only resemble their eukaryotic counterparts structurally but also show similar...... functional characteristics, assembling into filamentous structures in a nucleotide-dependent fashion. Recent advances in fluorescence microscopic imaging have revealed that FtsZ and MreB form highly dynamic helical structures that encircle the cells along the inside of the cell membrane. With the discovery...

  11. Fusion of bacterial spheroplasts by electric fields.

    Science.gov (United States)

    Ruthe, H J; Adler, J

    1985-09-25

    Spheroplasts of Escherichia coli or Salmonella typhimurium were found to fuse in an electric field. We employed the fusion method developed by Zimmermann and Scheurich (1981): Close membrane contact between cells is established by dielectrophoresis (formation of chains of cells by an a.c. field), then membrane fusion is induced by the application of short pulses of direct current. Under optimum conditions the fusion yield was routinely 90%. Fusable spheroplasts were obtained by first growing filamentous bacteria in the presence of cephalexin, then converting these to spheroplasts by the use of lysozyme. The fusion products were viable and regenerated to the regular bacterial form. Fusion of genetically different spheroplasts resulted in strains of bacteria possessing a combination of genetic markers. Fusion could not be achieved with spheroplasts obtained by growing the cells in the presence of penicillin or by using lysozyme on bacteria of usual size. PMID:3899175

  12. The crucial role of the MyD88 adaptor protein in the inflammatory response induced by Bothrops atrox venom.

    Science.gov (United States)

    Moreira, Vanessa; Teixeira, Catarina; Borges da Silva, Henrique; D'Império Lima, Maria Regina; Dos-Santos, Maria Cristina

    2013-06-01

    Most snake accidents in North Brazil are attributed to Bothrops atrox, a snake species of the Viperidae family whose venom simultaneously induces local and systemic effects in the victims. The former are clinically more important than the latter, as they cause severe tissue lesions associated with strong inflammatory responses. Although several studies have shown that inflammatory mediators are produced in response to B. atrox venom (BaV), there is little information concerning the molecular pathways involved in innate immune system signaling. Myeloid differentiation factor 88 (MyD88) is an adaptor molecule responsible for transmitting intracellular signals from most toll-like receptors (TLRs) after they interact with pathogen-associated molecular patterns (PAMPs) or other stimuli such as endogenous damage-associated molecular patterns (DAMPs). The MyD88-dependent pathway leads to activation of transcription factors, which in turn induce synthesis of inflammatory mediators such as eicosanoids, cytokines and chemokines. The aim of this study was to investigate the involvement of MyD88 on the acute inflammatory response induced by BaV. Wild-type (WT) C57BL/6 mice and MyD88 knockout (MyD88(-/-)) mice were intraperitoneally injected with BaV. Compared to WT mice, MyD88(-/-) animals showed an impaired inflammatory response to BaV, with lower influx of polymorphonuclear and mononuclear cells to the peritoneal cavity. Furthermore, peritoneal leukocytes from BaV-injected MyD88(-/-) mice did not induce COX-2 or LTB4 protein expression and released low concentrations of PGE2. These mice also failed to produce Th1 and Th17 cytokines and CCL-2, but IL-10 levels were similar to those of BaV-injected WT mice. Our results indicate that MyD88 signaling is required for activation of the inflammatory response elicited by BaV, raising the possibility of developing new therapeutic targets to treat Bothrops sp. poisoning. PMID:23474268

  13. Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

    Science.gov (United States)

    Ross, Breyan H.; Lin, Yimo; Corales, Esteban A.; Burgos, Patricia V.; Mardones, Gonzalo A.

    2014-01-01

    Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non

  14. Morphomechanics of bacterial biofilms undergoing anisotropic differential growth

    Science.gov (United States)

    Zhang, Cheng; Li, Bo; Huang, Xiao; Ni, Yong; Feng, Xi-Qiao

    2016-10-01

    Growing bacterial biofilms exhibit a number of surface morphologies, e.g., concentric wrinkles, radial ridges, and labyrinthine networks, depending on their physiological status and nutrient access. We explore the mechanisms underlying the emergence of these greatly different morphologies. Ginzburg-Landau kinetic method and Fourier spectral method are integrated to simulate the morphological evolution of bacterial biofilms. It is shown that the morphological instability of biofilms is triggered by the stresses induced by anisotropic and heterogeneous bacterial expansion, and involves the competition between membrane energy and bending energy. Local interfacial delamination further enriches the morphologies of biofilms. Phase diagrams are established to reveal how the anisotropy and spatial heterogeneity of growth modulate the surface patterns. The mechanics of three-dimensional microbial morphogenesis may also underpin self-organization in other development systems and provide a potential strategy for engineering microscopic structures from bacterial aggregates.

  15. Electrophysiology of mycoplasma membranes.

    OpenAIRE

    Schummer, U.; Schiefer, H G

    1983-01-01

    The influence of transmembrane ion fluxes on mycoplasma membrane potentials was studied. Fluorescence intensity of potential-sensitive carbocyanine dyes was calibrated vs. electric membrane potential. Potassium and sodium ion diffusion potentials significantly contributed to mycoplasma membrane potential. Chloride ions were obviously freely permeable across mycoplasma membranes. Under growth conditions the mycoplasma membrane potential was estimated to be delta psi = - 80 mV.

  16. Formation of bacterial membrane ice-nucleating lipoglycoprotein complexes.

    OpenAIRE

    Kozloff, L. M.; Turner, M.A.; Arellano, F

    1991-01-01

    The preliminary finding that nonprotein additions to the protein product of the ice-nucleating gene of Pseudomonas syringae or Erwinia herbicola are essential for ice nucleation at the warmest temperatures has led to experiments aimed at identifying possible linkages between the ice protein and the other components. It appears that the protein is coupled to various sugars through N- and O-glycan linkages. Mannose residues are apparently bound via an N-glycan bond to the amide nitrogen of one ...

  17. The role of Na/+/ in transport processes of bacterial membranes

    Science.gov (United States)

    Lanyi, J. K.

    1979-01-01

    Until recently it was generally held that transport in bacteria was linked exclusively to proton circulation, in contrast to most eucaryotic systems, which depended on Na(+) circulation. The present review is intended to trace recent developments which have led to the discarding of this idea. The discussion covers transport of Na(+) and other cations, effects of Na(+) and Na(+) gradients on metabolite transport, properties of Na(+)-dependent transport carriers, and evolutionary considerations of Na(+) transport. It is now apparent that the transport of Na(+) is an important part of energy metabolism in bacteria, and that Na(+) gradients as well as H(+) gradients are used in these systems for the conservation and transmission of energy. Two hypotheses are proposed to explain the evolution of Na/K systems, and it is presently difficult to decide between them.

  18. From biological membranes to biomimetic model membranes

    Directory of Open Access Journals (Sweden)

    Eeman, M.

    2010-01-01

    Full Text Available Biological membranes play an essential role in the cellular protection as well as in the control and the transport of nutrients. Many mechanisms such as molecular recognition, enzymatic catalysis, cellular adhesion and membrane fusion take place into the biological membranes. In 1972, Singer et al. provided a membrane model, called fluid mosaic model, in which each leaflet of the bilayer is formed by a homogeneous environment of lipids in a fluid state including globular assembling of proteins and glycoproteins. Since its conception in 1972, many developments were brought to this model in terms of composition and molecular organization. The main development of the fluid mosaic model was made by Simons et al. (1997 and Brown et al. (1997 who suggested that membrane lipids are organized into lateral microdomains (or lipid rafts with a specific composition and a molecular dynamic that are different to the composition and the dynamic of the surrounding liquid crystalline phase. The discovery of a phase separation in the plane of the membrane has induced an explosion in the research efforts related to the biology of cell membranes but also in the development of new technologies for the study of these biological systems. Due to the high complexity of biological membranes and in order to investigate the biological processes that occur on the membrane surface or within the membrane lipid bilayer, a large number of studies are performed using biomimicking model membranes. This paper aims at revisiting the fundamental properties of biological membranes in terms of membrane composition, membrane dynamic and molecular organization, as well as at describing the most common biomimicking models that are frequently used for investigating biological processes such as membrane fusion, membrane trafficking, pore formation as well as membrane interactions at a molecular level.

  19. Inhibition of biofouling by modification of forward osmosis membrane using quaternary ammonium cation.

    Science.gov (United States)

    Park, Kang-Hee; Yu, Sang-Hyun; Kim, Han-Shin; Park, Hee-Deung

    2015-01-01

    In the operation of the forward osmosis (FO) process, biofouling of the membrane is a potentially serious problem. Development of an FO membrane with antibacterial properties could contribute to a reduction in biofouling. In this study, quaternary ammonium cation (QAC), a widely used biocidal material, was conjugated with a silane coupling agent (3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride) and used to modify an FO membrane to confer antibacterial properties. Fourier transform infrared spectroscopy (FT-IR) demonstrated that the conjugated QAC was successfully immobilized on the FO membrane via covalent bonding. Bacterial viability on the QAC-modified membrane was confirmed via colony count method and visualized via bacterial viability assay. The QAC membrane decreased the viability of Escherichia coli to 62% and Staphylococcus aureus to 77% versus the control membrane. Inhibition of biofilm formation on the QAC modified membrane was confirmed via anti-biofilm tests using the drip-flow reactor and FO unit, resulting in 64% and 68% inhibition in the QAC-modified membrane against the control membrane, respectively. The results demonstrate the effectiveness of the modified membrane in reducing bacterial viability and inhibiting biofilm formation, indicating the potential of QAC-modified membranes to decrease operation costs incurred by biofouling.

  20. EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization.

    Science.gov (United States)

    Pérez-Quintero, Luis-Alberto; Roncagalli, Romain; Guo, Huaijian; Latour, Sylvain; Davidson, Dominique; Veillette, André

    2014-04-01

    Ewing's sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase Cγ, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation.

  1. TLR adaptor MyD88 is essential for pathogen control during oral toxoplasma gondii infection but not adaptive immunity induced by a vaccine strain of the parasite.

    Science.gov (United States)

    Sukhumavasi, Woraporn; Egan, Charlotte E; Warren, Amy L; Taylor, Gregory A; Fox, Barbara A; Bzik, David J; Denkers, Eric Y

    2008-09-01

    TLR adaptor MyD88 activation is important in host resistance to Toxoplasma gondii during i.p. infection, but the function of this signaling pathway during oral infection, in which mucosal immunity assumes a predominant role, has not been examined. In this study, we show that MyD88(-/-) mice fail to control the parasite and succumb within 2 wk of oral infection. Early during infection, T cell IFN-gamma production, recruitment of neutrophils and induction of p47 GTPase IGTP (Irgm3) in the intestinal mucosa were dependent upon functional MyD88. Unexpectedly, these responses were MyD88-independent later during acute infection. In particular, CD4(+) T cell IFN-gamma reached normal levels independently of MyD88, despite continued absence of IL-12 in these animals. The i.p. vaccination of MyD88(-/-) mice with an avirulent T. gondii uracil auxotroph elicited robust IFN-gamma responses and protective immunity to challenge with a high virulence T. gondii strain. Our results demonstrate that MyD88 is required to control Toxoplasma infection, but that the parasite can trigger adaptive immunity without the need for this TLR adaptor molecule. PMID:18714019

  2. Phosphorylation of APP-CTF-AICD domains and interaction with adaptor proteins: signal transduction and/or transcriptional role--relevance for Alzheimer pathology.

    Science.gov (United States)

    Schettini, Gennaro; Govoni, Stefano; Racchi, Marco; Rodriguez, Guido

    2010-12-01

    In recent decades, the study of the amyloid precursor protein (APP) and of its proteolytic products carboxy terminal fragment (CTF), APP intracellular C-terminal domain (AICD) and amyloid beta has been mostly focussed on the role of APP as a producer of the toxic amyloid beta peptide. Here, we reconsider the role of APP suggesting, in a provocative way, the protein as a central player in a putative signalling pathway. We highlight the presence in the cytosolic tail of APP of the YENPTY motif which is typical of tyrosine kinase receptors, the phosphorylation of the tyrosine, serine and threonine residues, the kinases involved and the interaction with intracellular adaptor proteins. In particular, we examine the interaction with Shc and Grb2 regulators, which through the activation of Ras proteins elicit downstream signalling events such as the MAPK pathway. The review also addresses the interaction of APP, CTFs and AICD with other adaptor proteins and in particular with Fe65 for nuclear transcriptional activity and the importance of phosphorylation for sorting the secretases involved in the amyloidogenic or non-amyloidogenic pathways. We provide a novel perspective on Alzheimer's disease pathogenesis, focussing on the perturbation of the physiological activities of APP-CTFs and AICD as an alternative perspective from that which normally focuses on the accumulation of neurotoxic proteolytic fragments. PMID:21039524

  3. Expression of Chicken Toll-Like Receptors and Signal Adaptors in Spleen and Cecum of Young Chickens Infected with Eimeria tenella

    Institute of Scientific and Technical Information of China (English)

    ZHOU Zuo-yong; HU Shi-jun; WANG Zhi-ying; GUO Zhi-li; QIN Bo; NIE Kui

    2014-01-01

    Toll-like receptors (TLRs) are a group of highly conserved molecules which initiate the innate immune response to pathogens by recognizing structural motifs of microbes. Understanding the changes in chicken Toll-like receptors (ChTLRs) and signal adaptors expression that occur with Eimeria tenella infection will help to elucidate the molecular basis of immune control of coccidiosis caused by Eimeria. The present study detected the dynamic changes in the expression of ChTLRs and associated signal adaptors in the spleen and cecum of E. tenella-infected chickens during the early stage of infection. The results showed that the expression peak for ChTLRs, MyD88 and TRIF occurred at 12 h post-infection (hpi), ChTLR3, ChTLR15 and MyD88 mRNA expression in the spleen of E. tenella infected chickens were signiifcantly higher (P<0.05) than that of negative control chickens, and there were similar tendencies of these molecules expression in the cecum and spleen of E. tenella-infected chickens. The expression of MyD88 was upregulated at four time points in the cecum of E. tenella-infected chickens. The results of this study indicate that ChTLR3, ChTLR15 and MyD88 play a role in young chickens infected with E. tenella.

  4. Diagnosis of bacterial vaginosis

    Directory of Open Access Journals (Sweden)

    Đukić Slobodanka

    2013-01-01

    Full Text Available Bacterial vaginosis is a common, complex clinical syndrome characterized by alterations in the normal vaginal flora. When symptomatic, it is associated with a malodorous vaginal discharge and on occasion vaginal burning or itching. Under normal conditions, lactobacilli constitute 95% of the bacteria in the vagina. Bacterial vaginosis is associated with severe reduction or absence of the normal H2O2­producing lactobacilli and overgrowth of anaerobic bacteria and Gardnerella vaginalis, Atopobium vaginae, Mycoplasma hominis and Mobiluncus species. Most types of infectious disease are diagnosed by culture, by isolating an antigen or RNA/DNA from the microbe, or by serodiagnosis to determine the presence of antibodies to the microbe. Therefore, demonstration of the presence of an infectious agent is often a necessary criterion for the diagnosis of the disease. This is not the case for bacterial vaginosis, since the ultimate cause of the disease is not yet known. There are a variety of methods for the diagnosis of bacterial vaginosis but no method can at present be regarded as the best. Diagnosing bacterial vaginosis has long been based on the clinical criteria of Amsel, whereby three of four defined criteria must be satisfied. Nugent’s scoring system has been further developed and includes validation of the categories of observable bacteria structures. Up­to­date molecular tests are introduced, and better understanding of vaginal microbiome, a clear definition for bacterial vaginosis, and short­term and long­term fluctuations in vaginal microflora will help to better define molecular tests within the broader clinical context.

  5. Anaerobic membrane bioreactors: Are membranes really necessary?

    OpenAIRE

    Davila, M.; Kassab, G.; Klapwijk, A.; Van, Lier, G

    2008-01-01

    Membranes themselves represent a significant cost for the full scale application of anaerobic membrane bioreactors (AnMBR). The possibility of operating an AnMBR with a self-forming dynamic membrane generated by the substances present in the reactor liquor would translate into an important saving. A self-forming dynamic membrane only requires a support material over which a cake layer is formed, which determines the rejection properties of the system. The present research studies the applicat...

  6. Bacterial glycosyltransferase toxins.

    Science.gov (United States)

    Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-12-01

    Mono-glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide-binding proteins of the Rho family. However, toxin-induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin-catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.

  7. Distinct in vitro interaction pattern of dopamine receptor subtypes with adaptor proteins involved in post-endocytotic receptor targeting

    DEFF Research Database (Denmark)

    Heydorn, Arne; Søndergaard, Birgitte P; Hadrup, Niels;

    2004-01-01

    The mechanisms underlying targeted sorting of endocytosed receptors for recycling to the plasma membrane or degradation in lysosomes are poorly understood. In this report, the C-terminal tails of the five dopamine receptors (D1-D5) were expressed as glutathione S-transferase (GST) fusion proteins...

  8. Anaerobic membrane bioreactors: Are membranes really necessary?

    NARCIS (Netherlands)

    Davila, M.; Kassab, G.; Klapwijk, A.; Lier, van J.B.

    2008-01-01

    Membranes themselves represent a significant cost for the full scale application of anaerobic membrane bioreactors (AnMBR). The possibility of operating an AnMBR with a self-forming dynamic membrane generated by the substances present in the reactor liquor would translate into an important saving. A

  9. Thin-Membrane Sensor With Biochemical Switch

    Science.gov (United States)

    Case, George D.; Worley, Jennings F.

    1992-01-01

    Modular sensor electrochemically detects chemical or biological agent, indicating presence of agent via gate-membrane-crossing ion current triggered by chemical reaction between agent and recognition protein conjugated to channel blocker. Used in such laboratory, industrial, or field applications as detection of bacterial toxins in food, military chemical agents in air, and pesticides or other contaminants in environment. Also used in biological screening for hepatitis, acquired immune-deficiency syndrome, and like.

  10. Preparation of succinylated cellulose membranes for functionalization purposes.

    Science.gov (United States)

    Ribeiro-Viana, Renato M; Faria-Tischer, Paula C S; Tischer, Cesar A

    2016-09-01

    The anhydroglucose chains of cellulose possess hydroxyls that facilitate different chemical modification strategies to expand on, or provide new applications for membranes produced by the bacteria Gluconacetobacter xylinus. Conjugation with biomolecules such as proteins, especially by the amine groups, is of great value and interest for the production of biomaterial derivatives from bacterial cellulose. To assist in these modifications, cellulose was succinylated in order to prevent steric hindrance and to create an attachment point for conjugation. Bacterial cellulose membranes were first treated in dichloromethane and reacted with succinic anhydride through a series of conditions. The membrane structure remained intact after these first processes and the product was confirmed by Infra-Red spectroscopy and solid state nuclear magnetic resonance and characterized by X-ray diffraction, thermogravimetry and atomic force microscopy. Hydrolyzed collagen was used as a model protein of interest to be conjugated to these membranes, which furnished a biomaterial functionalized over its surface. PMID:27185111

  11. Erythrocyte membrane proteins and membrane skeleton

    Institute of Scientific and Technical Information of China (English)

    LU Yiqin; LIU Junfan

    2007-01-01

    Considerable advances in the research field of erythrocyte membrane were achieved in the recent two decades.New findings in the structure-function correlation and interactions of erythrocyte membrane proteins have attracted extensive attention.Interesting progress was also made in the molecular pathogenesis of erythrocyte membrane disorders.Advances in the composition,function and interaction of erythrocyte membrane proteins,erythrocyte membrane skeleton,and relevant diseases are briefly described and summarized here on the basis of domestic and world literatures.

  12. 革兰氏阴性细菌外膜蛋白耐药功能及其抑菌策略研究进展%An review on the antibiotics resistance function of outer membrane protein in gram-negative bacterial and related antimicrobial strategies

    Institute of Scientific and Technical Information of China (English)

    李碗芯; 孙莉娜; 林向民

    2015-01-01

    The emergence of bacterial resistance to drug use has posed serious problem worldwide. Generally, the drug resistance ca-pability of gram-negative bacteria is stronger than that of gram-positive, which is partially attributed to the antibiotics resistance of the outer membrane proteins ( OMPs) . It is well known that the specific OMPs efflux antibiotics out or inhibits uptakes of antibiot-ics, while the regulation mechanism remains uncertain. With the development of modern science and technology, especially the ap-plication of structure biology, molecular biology and proteomics, many progressive studies have unveiled the mechanisms underlying the antibiotic resistance of OMPs. This review focuses on the recent advances in the structure and antibiotic resistance mechanisms of OMP, which provides helpful insight into manufacture of new OMPs-targeting antimicrobial drug.%当前细菌耐药的形势日益严峻,已引起全世界的广泛关注.革兰氏阴性细菌的耐药性普遍比革兰氏阳性细菌强,这与镶嵌或锚定在革兰氏阴性细菌外膜上的蛋白耐药功能密切相关.目前已知外膜蛋白能够阻碍抗生素通过外膜以及将抗生素排出胞外等方式使细菌产生耐药性,但具体的耐药与调控机制还有待进一步研究.近年来,随着现代科学技术的不断发展,特别是结构生物学、分子生物学与蛋白质组学在该领域的充分运用,有关外膜蛋白功能的研究和技术取得了很大的进步.本文对近年来国内外在细菌外膜蛋白的结构与耐药机理、耐药新功能及新机制等方面的研究进展进行了概述,为揭示革兰氏阴性菌耐药形成机制奠定基础.

  13. Seizures Complicating Bacterial Meningitis

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2004-09-01

    Full Text Available The clinical data of 116 patients, 1 month to <5 years of age, admitted for bacterial meningitis, and grouped according to those with and without seizures during hospitalization, were compared in a study at Buddhist Dalin Tzu Chi General Hospital, Chang Gung Memorial Hospital and other centers in Taiwan.

  14. Bacterial extracellular lignin peroxidase

    Science.gov (United States)

    Crawford, Donald L.; Ramachandra, Muralidhara

    1993-01-01

    A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

  15. Bacterial Skin Infections

    Science.gov (United States)

    ... or scraped, the injury should be washed with soap and water and covered with a sterile bandage. Petrolatum may be applied to open areas to keep the tissue moist and to try to prevent bacterial invasion. Doctors recommend that people do not use ...

  16. Vimentin in Bacterial Infections

    DEFF Research Database (Denmark)

    Mak, Tim N; Brüggemann, Holger

    2016-01-01

    filaments (IFs). IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge...

  17. Bacterial microflora of nectarines

    Science.gov (United States)

    Microflora of fruit surfaces has been the best source of antagonists against fungi causing postharvest decays of fruit. However, there is little information on microflora colonizing surfaces of fruits other than grapes, apples, and citrus fruit. We characterized bacterial microflora on nectarine f...

  18. Modeling intraocular bacterial infections.

    Science.gov (United States)

    Astley, Roger A; Coburn, Phillip S; Parkunan, Salai Madhumathi; Callegan, Michelle C

    2016-09-01

    Bacterial endophthalmitis is an infection and inflammation of the posterior segment of the eye which can result in significant loss of visual acuity. Even with prompt antibiotic, anti-inflammatory and surgical intervention, vision and even the eye itself may be lost. For the past century, experimental animal models have been used to examine various aspects of the pathogenesis and pathophysiology of bacterial endophthalmitis, to further the development of anti-inflammatory treatment strategies, and to evaluate the pharmacokinetics and efficacies of antibiotics. Experimental models allow independent control of many parameters of infection and facilitate systematic examination of infection outcomes. While no single animal model perfectly reproduces the human pathology of bacterial endophthalmitis, investigators have successfully used these models to understand the infectious process and the host response, and have provided new information regarding therapeutic options for the treatment of bacterial endophthalmitis. This review highlights experimental animal models of endophthalmitis and correlates this information with the clinical setting. The goal is to identify knowledge gaps that may be addressed in future experimental and clinical studies focused on improvements in the therapeutic preservation of vision during and after this disease. PMID:27154427

  19. A membrane disdrometer based on membrane vibration

    International Nuclear Information System (INIS)

    Raindrop sizes were indirectly determined through the distinctive vibrations and sounds that occur when raindrops of different sizes impact on a membrane. The relationships between diameter, raindrop kinetic momentum, membrane deflection, and impact force were analyzed. Membrane deflection and vibration models were created, and the optimum membrane diameter and thickness were identified. A prototype membrane disdrometer was developed on the basis of these findings. The relationship between raindrop size and membrane vibration sound pressure was determined experimentally. The fundamental parameter of raindrop size recognition was used. The test results indicate that raindrops 0.4–2.0 mm in diameter can be detected by the proposed membrane disdrometer, and 50 raindrops per second in a zone area of 28.3 cm2 can be detected in natural rain. (paper)

  20. Does chlorination of seawater reverse osmosis membranes control biofouling?

    Science.gov (United States)

    Khan, Muhammad Tariq; Hong, Pei-Ying; Nada, Nabil; Croue, Jean Philippe

    2015-07-01

    Biofouling is the major problem of reverse osmosis (RO) membranes used for desalting seawater (SW). The use of chlorine is a conventional and common practice to control/prevent biofouling. Unlike polyamide RO membranes, cellulose triacetate (CTA) RO membranes display a high chlorine tolerance. Due to this characteristic, CTA membranes are used in most of the RO plants located in the Middle East region where the elevated seawater temperature and water quality promote the risk of membrane biofouling. However, there is no detailed study on the investigation/characterization of CTA-RO membrane fouling. In this investigation, the fouling profile of a full-scale SWRO desalination plant operating with not only continuous chlorination of raw seawater but also intermittent chlorination of CTA-RO membranes was studied. Detailed water quality and membrane fouling analyses were conducted. Profiles of microbiological, inorganic, and organic constituents of analysed fouling layers were extensively discussed. Our results clearly identified biofilm development on these membranes. The incapability of chlorination on preventing biofilm formation on SWRO membranes could be assigned to its failure in effectively reaching throughout the different regions of the permeators. This failure could have occurred due to three main factors: plugging of membrane fibers, chlorine consumption by organics accumulated on the front side fibers, or chlorine adaptation of certain bacterial populations. PMID:25917390

  1. Does Chlorination of Seawater Reverse Osmosis Membranes Control Biofouling?

    KAUST Repository

    Khan, Muhammad Tariq

    2015-04-01

    Biofouling is the major problem of reverse osmosis (RO) membranes used for desalting seawater (SW). The use of chlorine is a conventional and common practice to control/prevent biofouling. Unlike polyamide RO membranes, cellulose triacetate (CTA) RO membranes display a high chlorine tolerance. Due to this characteristic, CTA membranes are used in most of the RO plants located in the Middle East region where the elevated seawater temperature and water quality promote the risk of membrane biofouling. However, there is no detailed study on the investigation/characterization of CTA-RO membrane fouling. In this investigation, the fouling profile of a full–scale SWRO desalination plant operating with not only continuous chlorination of raw seawater but also intermittent chlorination of CTA-RO membranes was studied. Detailed water quality and membrane fouling analyses were conducted. Profiles of microbiological, inorganic, and organic constituents of analysed fouling layers were extensively discussed. Our results clearly identified biofilm development on these membranes. The incapability of chlorination on preventing biofilm formation on SWRO membranes could be assigned to its failure in effectively reaching throughout the different regions of the permeators. This failure could have occurred due to three main factors: plugging of membrane fibers, chlorine consumption by organics accumulated on the front side fibers, or chlorine adaptation of certain bacterial populations.

  2. Does chlorination of seawater reverse osmosis membranes control biofouling?

    Science.gov (United States)

    Khan, Muhammad Tariq; Hong, Pei-Ying; Nada, Nabil; Croue, Jean Philippe

    2015-07-01

    Biofouling is the major problem of reverse osmosis (RO) membranes used for desalting seawater (SW). The use of chlorine is a conventional and common practice to control/prevent biofouling. Unlike polyamide RO membranes, cellulose triacetate (CTA) RO membranes display a high chlorine tolerance. Due to this characteristic, CTA membranes are used in most of the RO plants located in the Middle East region where the elevated seawater temperature and water quality promote the risk of membrane biofouling. However, there is no detailed study on the investigation/characterization of CTA-RO membrane fouling. In this investigation, the fouling profile of a full-scale SWRO desalination plant operating with not only continuous chlorination of raw seawater but also intermittent chlorination of CTA-RO membranes was studied. Detailed water quality and membrane fouling analyses were conducted. Profiles of microbiological, inorganic, and organic constituents of analysed fouling layers were extensively discussed. Our results clearly identified biofilm development on these membranes. The incapability of chlorination on preventing biofilm formation on SWRO membranes could be assigned to its failure in effectively reaching throughout the different regions of the permeators. This failure could have occurred due to three main factors: plugging of membrane fibers, chlorine consumption by organics accumulated on the front side fibers, or chlorine adaptation of certain bacterial populations.

  3. Composite sensor membrane

    Science.gov (United States)

    Majumdar, Arun; Satyanarayana, Srinath; Yue, Min

    2008-03-18

    A sensor may include a membrane to deflect in response to a change in surface stress, where a layer on the membrane is to couple one or more probe molecules with the membrane. The membrane may deflect when a target molecule reacts with one or more probe molecules.

  4. Compositional asynchronous membrane systems

    Institute of Scientific and Technical Information of China (English)

    Cosmin Bonchis; Cornel Izbasa; Gabriel Ciobanu

    2007-01-01

    This paper presents an algorithmic way of building complex membrane systems by coupling elementary membranes. Its application seems particularly valuable in the case of asynchronous membrane systems, since the resulting membrane system remains asynchronous. The composition method is based on a handshake mechanism implemented by using antiport rules and promoters.

  5. Magnetically controlled permeability membranes

    KAUST Repository

    Kosel, Jurgen

    2013-10-31

    A bioactive material delivery system can include a thermoresponsive polymer membrane and nanowires distributed within the thermoresponsive polymer membrane. Magnetic activation of a thermoresponsive polymer membrane can take place via altering the magnetization or dimensions of nanowires dispersed or ordered within the membrane matrix.

  6. Newer systems for bacterial resistances to toxic heavy metals.

    OpenAIRE

    Silver, S; Ji, G.

    1994-01-01

    Bacterial plasmids contain specific genes for resistances to toxic heavy metal ions including Ag+, AsO2-, AsO4(3-), Cd2+, Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, and Zn2+. Recent progress with plasmid copper-resistance systems in Escherichia coli and Pseudomonas syringae show a system of four gene products, an inner membrane protein (PcoD), an outer membrane protein (PcoB), and two periplasmic Cu(2+)-binding proteins (PcoA and PcoC). Synthesis of this system is governed by two regulator...

  7. Membrane fusion proteins of type I secretion system and tripartite efflux pumps share a binding motif for TolC in gram-negative bacteria.

    Directory of Open Access Journals (Sweden)

    Minho Lee

    Full Text Available The Hly translocator complex of Escherichia coli catalyzes type I secretion of the toxin hemolysin A (HlyA. In this complex, HlyB is an inner membrane ABC (ATP Binding Cassette-type transporter, TolC is an outer membrane channel protein, and HlyD is a periplasmic adaptor anchored in the inner membrane that bridges HlyB to TolC. This tripartite organization is reminiscent of that of drug efflux systems such as AcrA-AcrB-TolC and MacA-MacB-TolC of E. coli. We have previously shown the crucial role of conserved residues located at the hairpin tip region of AcrA and MacA adaptors during assembly of their cognate systems. In this study, we investigated the role of the putative tip region of HlyD using HlyD mutants with single amino acid substitutions at the conserved positions. In vivo and in vitro data show that all mutations abolished HlyD binding to TolC and resulted in the absence of HlyA secretion. Together, our results suggest that, similarly to AcrA and MacA, HlyD interacts with TolC in a tip-to-tip manner. A general model in which these conserved interactions induce opening of TolC during drug efflux and type I secretion is discussed.

  8. Novel disk modules for membrane separation processes

    International Nuclear Information System (INIS)

    The reverse osmosis (RO) system at the Effluent Treatment Facility (ETF) at the Savannah River Site, Aiken, South Carolina has experienced fouling from trace quantities of inorganics (Al, Fe, and Si) and l.E5-l.E7/ml bacteria. The bacteria are primarily produced in an upstream Hg-removal resin bed/activated carbon bed process. The bacteria adhere to the colloidal inorganics that are in the membrane feed at their solubility limits (having been precipitated and removed upstream by a ceramic microfilter system). The resulting bacterial/inorganic foulant adheres to the membrane surface and results in high feed pressures and poor salt rejection. The feed pressure increases because the membrane system at the ETF is designed to produce a constant rate of treated water, or permeate. This is accomplished by increasing the membrane feed pressure whenever permeate flow drops. These performance losses have been attributed to bacteria present in the feed, and several potential solutions have been proposed and demonstrated here at the Savannah River Technology Center (SRTC). Advanced hybrid plate-and-frame modules have been developed that increase the applicability of membrane systems by using hydrodynamics rather than pretreatment to prevent membrane fouling

  9. Novel disk modules for membrane separation processes

    Energy Technology Data Exchange (ETDEWEB)

    Siler, J.L.

    1993-12-06

    The reverse osmosis (RO) system at the Effluent Treatment Facility (ETF) at the Savannah River Site, Aiken, South Carolina has experienced fouling from trace quantities of inorganics (Al, Fe, and Si) and l.E5-l.E7/ml bacteria. The bacteria are primarily produced in an upstream Hg-removal resin bed/activated carbon bed process. The bacteria adhere to the colloidal inorganics that are in the membrane feed at their solubility limits (having been precipitated and removed upstream by a ceramic microfilter system). The resulting bacterial/inorganic foulant adheres to the membrane surface and results in high feed pressures and poor salt rejection. The feed pressure increases because the membrane system at the ETF is designed to produce a constant rate of treated water, or permeate. This is accomplished by increasing the membrane feed pressure whenever permeate flow drops. These performance losses have been attributed to bacteria present in the feed, and several potential solutions have been proposed and demonstrated here at the Savannah River Technology Center (SRTC). Advanced hybrid plate-and-frame modules have been developed that increase the applicability of membrane systems by using hydrodynamics rather than pretreatment to prevent membrane fouling.

  10. Disposable Membrane Sensors for Biohazardous Substances

    Science.gov (United States)

    Petrov, Alexander G.

    The concept of bilayer lipid membrane sensing is elaborated. Disposable bilayer lipid membrane sensors permit both stochastic and deterministic sensing regimes. Technology for membrane sensor preparation is described in details. Stochastic membrane sensing using ion channels is described. Detection of cyano-bacterial toxins in waters by means of a stochastic sensing (ion channel induction) reveals a signature for a particular toxin type. This signature is especially well revealed by using the method of current-voltage surfaces, which establishes a bridge between stochastic and deterministic sensing by applying a time-averaging procedure. Flexoelectricity of membranes provides another way of deterministic sensing, of a stimulus-response type. The flexoelectric response is modulated by adsorption of the analyte over the membrane surface. Detection of environmental pollution of waters by heavy metal ions (Cd++ and Hg++) is thereby rendered possible. This approach is confronted with a stochastic sensing of the same metal ions by alteration of the channel open/closed probability and average channel current of two familiar channels, gramicidin and alamethicin.

  11. Transmembrane protein sorting driven by membrane curvature

    Science.gov (United States)

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-11-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  12. A Eukaryotic Sensor for Membrane Lipid Saturation.

    Science.gov (United States)

    Covino, Roberto; Ballweg, Stephanie; Stordeur, Claudius; Michaelis, Jonas B; Puth, Kristina; Wernig, Florian; Bahrami, Amir; Ernst, Andreas M; Hummer, Gerhard; Ernst, Robert

    2016-07-01

    Maintaining a fluid bilayer is essential for cell signaling and survival. Lipid saturation is a key factor determining lipid packing and membrane fluidity, and it must be tightly controlled to guarantee organelle function and identity. A dedicated eukaryotic mechanism of lipid saturation sensing, however, remains elusive. Here we show that Mga2, a transcription factor conserved among fungi, acts as a lipid-packing sensor in the ER membrane to control the production of unsaturated fatty acids. Systematic mutagenesis, molecular dynamics simulations, and electron paramagnetic resonance spectroscopy identify a pivotal role of the oligomeric transmembrane helix (TMH) of Mga2 for intra-membrane sensing, and they show that the lipid environment controls the proteolytic activation of Mga2 by stabilizing alternative rotational orientations of the TMH region. This work establishes a eukaryotic strategy of lipid saturation sensing that differs significantly from the analogous bacterial mechanism relying on hydrophobic thickness. PMID:27320200

  13. Expression of the neuronal adaptor protein X11alpha protects against memory dysfunction in a transgenic mouse model of Alzheimer's disease.

    LENUS (Irish Health Repository)

    Mitchell, Jacqueline C

    2010-01-01

    X11alpha is a neuronal-specific adaptor protein that binds to the amyloid-beta protein precursor (AbetaPP). Overexpression of X11alpha reduces Abeta production but whether X11alpha also protects against Abeta-related memory dysfunction is not known. To test this possibility, we crossed X11alpha transgenic mice with AbetaPP-Tg2576 mice. AbetaPP-Tg2576 mice produce high levels of brain Abeta and develop age-related defects in memory function that correlate with increasing Abeta load. Overexpression of X11alpha alone had no detectable adverse effect upon behavior. However, X11alpha reduced brain Abeta levels and corrected spatial reference memory defects in aged X11alpha\\/AbetaPP double transgenics. Thus, X11alpha may be a therapeutic target for Alzheimer\\'s disease.

  14. RECOMBINANT FLUORESCENT SENSOR OF HYDROGEN PEROXIDE HyPer FUSED WITH ADAPTOR PROTEIN Ruk/CIN85: DESIGNING OF EXPRESSION VECTOR AND ITS FUNCTIONAL CHARACTERIZATION

    Directory of Open Access Journals (Sweden)

    А. V. Bazalii

    2015-10-01

    Full Text Available The aim of this study was to design the expression vector encoding fluorescent sensor of hydrogen peroxide HyPer fused with adaptor protein Ruk/CIN85 as well as to check its subcellular distribution and ability to sense hydrogen peroxide. It was demonstrated that in transiently transfected HEK293 and MCF-7 cells Ruk/CIN85-HyPer is concentrated in dot-like vesicular structures of different size while HyPer is diffusely distributed throughout the cell. Using live cell fluorescence microscopy we observed gradual increase in hydrogen peroxide concentration in representative vesicular structures during the time of experiment. Thus, the developed genetic construction encoding the chimeric Ruk/CIN85-HyPer fluorescent protein represents a new tool to study localized H2O2 production in living cells.

  15. Bacterial chemoreceptors and chemoeffectors.

    Science.gov (United States)

    Bi, Shuangyu; Lai, Luhua

    2015-02-01

    Bacteria use chemotaxis signaling pathways to sense environmental changes. Escherichia coli chemotaxis system represents an ideal model that illustrates fundamental principles of biological signaling processes. Chemoreceptors are crucial signaling proteins that mediate taxis toward a wide range of chemoeffectors. Recently, in deep study of the biochemical and structural features of chemoreceptors, the organization of higher-order clusters in native cells, and the signal transduction mechanisms related to the on-off signal output provides us with general insights to understand how chemotaxis performs high sensitivity, precise adaptation, signal amplification, and wide dynamic range. Along with the increasing knowledge, bacterial chemoreceptors can be engineered to sense novel chemoeffectors, which has extensive applications in therapeutics and industry. Here we mainly review recent advances in the E. coli chemotaxis system involving structure and organization of chemoreceptors, discovery, design, and characterization of chemoeffectors, and signal recognition and transduction mechanisms. Possible strategies for changing the specificity of bacterial chemoreceptors to sense novel chemoeffectors are also discussed.

  16. Bacterial Colony Optimization

    Directory of Open Access Journals (Sweden)

    Ben Niu

    2012-01-01

    Full Text Available This paper investigates the behaviors at different developmental stages in Escherichia coli (E. coli lifecycle and developing a new biologically inspired optimization algorithm named bacterial colony optimization (BCO. BCO is based on a lifecycle model that simulates some typical behaviors of E. coli bacteria during their whole lifecycle, including chemotaxis, communication, elimination, reproduction, and migration. A newly created chemotaxis strategy combined with communication mechanism is developed to simplify the bacterial optimization, which is spread over the whole optimization process. However, the other behaviors such as elimination, reproduction, and migration are implemented only when the given conditions are satisfied. Two types of interactive communication schemas: individuals exchange schema and group exchange schema are designed to improve the optimization efficiency. In the simulation studies, a set of 12 benchmark functions belonging to three classes (unimodal, multimodal, and rotated problems are performed, and the performances of the proposed algorithms are compared with five recent evolutionary algorithms to demonstrate the superiority of BCO.

  17. [Bacterial diseases of rape].

    Science.gov (United States)

    Zakharova, O M; Mel'nychuk, M D; Dankevych, L A; Patyka, V P

    2012-01-01

    Bacterial destruction of the culture was described and its agents identified in the spring and winter rape crops. Typical symptoms are the following: browning of stem tissue and its mucilagization, chlorosis of leaves, yellowing and beginning of soft rot in the place of leaf stalks affixion to stems, loss of pigmentation (violet). Pathogenic properties of the collection strains and morphological, cultural, physiological, and biochemical properties of the agents of rape's bacterial diseases isolated by the authors have been investigated. It was found that all the isolates selected by the authors are highly or moderately aggressive towards different varieties of rape. According to the complex of phenotypic properties 44% of the total number of isolates selected by the authors are related to representatives of the genus Pseudomonas, 37% - to Xanthomonas and 19% - to Pectobacterium. PMID:23293826

  18. Bacterial transformation of terpenoids

    International Nuclear Information System (INIS)

    Data on the bacterial transformation of terpenoids published in the literature in the past decade are analyzed. Possible pathways for chemo-, regio- and stereoselective modifications of terpenoids are discussed. Considerable attention is given to new technological approaches to the synthesis of terpenoid derivatives suitable for the use in the perfume and food industry and promising as drugs and chiral intermediates for fine organic synthesis. The bibliography includes 246 references

  19. Supramolecular bacterial systems

    OpenAIRE

    Sankaran, Shrikrishnan

    2015-01-01

    For nearly over a decade, a wide variety of dynamic and responsive supramolecular architectures have been investigated and developed to address biological systems. Since the non-covalent interactions between individual molecular components in such architectures are similar to the interactions found in living systems, it was possible to integrate chemically-synthesized and naturally-occurring components to create platforms with interesting bioactive properties. Bacterial cells and recombinant ...

  20. Bacterial Colony Optimization

    OpenAIRE

    Ben Niu; Hong Wang

    2012-01-01

    This paper investigates the behaviors at different developmental stages in Escherichia coli (E. coli) lifecycle and developing a new biologically inspired optimization algorithm named bacterial colony optimization (BCO). BCO is based on a lifecycle model that simulates some typical behaviors of E. coli bacteria during their whole lifecycle, including chemotaxis, communication, elimination, reproduction, and migration. A newly created chemotaxis strategy combined with communication mechanism i...

  1. Microfluidic microbial fuel cells: from membrane to membrane free

    Science.gov (United States)

    Yang, Yang; Ye, Dingding; Li, Jun; Zhu, Xun; Liao, Qiang; Zhang, Biao

    2016-08-01

    Microfluidic microbial fuel cells (MMFCs) are small carbon-neutral devices that use self-organized bacteria to degrade organic substrates and harness energy from the waste water. Conventional MMFCs have made great strides in the past decade and have overcome some limitations, such as high capital costs and low energy output. A co-laminar flow MFC has been first proposed in 2011 with the potential to be an attractively power source to niche applications. Co-laminar MFCs typically operate without any physical membranes separating the reactants, and bacterial ecosystems can be easily manipulated by regulating the inlet conditions. This paper highlights recent accomplishments in the development of co-laminar MFCs, emphasizing basic principles, mass transport and fluid dynamics including boundary layer theory, entrance conditions and mixing zone issues. Furthermore, the development of current techniques, major challenges and the potential research directions are discussed.

  2. Membrane fluids and Dirac membrane fluids

    CERN Document Server

    Ivanov, M G

    2004-01-01

    The relation between two different methods of membrane fluid description is clarified by construction of combined method. Dirac membrane field appears naturally in new approach. It provides a possibility to consider new aspects of electrodynamics-type theories with electric and magnetic sources. The membrane fluid models automatically prohibit simulatenos existence of electric and magnetic currents. Possible applications to the dark energy problem are mentioned.

  3. Sheet Membrane Spacesuit Water Membrane Evaporator

    Science.gov (United States)

    Bue, Grant; Trevino, Luis; Zapata, Felipe; Dillion, Paul; Castillo, Juan; Vonau, Walter; Wilkes, Robert; Vogel, Matthew; Frodge, Curtis

    2013-01-01

    A document describes a sheet membrane spacesuit water membrane evaporator (SWME), which allows for the use of one common water tank that can supply cooling water to the astronaut and to the evaporator. Test data showed that heat rejection performance dropped only 6 percent after being subjected to highly contaminated water. It also exhibited robustness with respect to freezing and Martian atmospheric simulation testing. Water was allowed to freeze in the water channels during testing that simulated a water loop failure and vapor backpressure valve failure. Upon closing the backpressure valve and energizing the pump, the ice eventually thawed and water began to flow with no apparent damage to the sheet membrane. The membrane evaporator also serves to de-gas the water loop from entrained gases, thereby eliminating the need for special degassing equipment such as is needed by the current spacesuit system. As water flows through the three annular water channels, water evaporates with the vapor flowing across the hydrophobic, porous sheet membrane to the vacuum side of the membrane. The rate at which water evaporates, and therefore, the rate at which the flowing water is cooled, is a function of the difference between the water saturation pressure on the water side of the membrane, and the pressure on the vacuum side of the membrane. The primary theory is that the hydrophobic sheet membrane retains water, but permits vapor pass-through when the vapor side pressure is less than the water saturation pressure. This results in evaporative cooling of the remaining water.

  4. The structure and function of bacterial light-harvesting complexes.

    Science.gov (United States)

    Law, Christopher J; Roszak, Aleksander W; Southall, June; Gardiner, Alastair T; Isaacs, Neil W; Cogdell, Richard J

    2004-01-01

    The harvesting of solar radiation by purple photosynthetic bacteria is achieved by circular, integral membrane pigment-protein complexes. There are two main types of light-harvesting complex, termed LH2 and LH1, that function to absorb light energy and to transfer that energy rapidly and efficiently to the photochemical reaction centres where it is trapped. This mini-review describes our present understanding of the structure and function of the purple bacterial light-harvesting complexes.

  5. Characterization of biofoulants illustrates different membrane fouling mechanisms for aerobic and anaerobic membrane bioreactors

    KAUST Repository

    Xiong, Yanghui

    2015-11-17

    This study compares the membrane fouling mechanisms of aerobic (AeMBR) and anaerobic membrane bioreactors (AnMBR) of the same reactor configuration at similar operating conditions. Although both the AeMBR and AnMBR achieved more than 90% COD removal efficiency, the fouling mechanisms were different. Molecular weight (MW) fingerprint profiles showed that a majority of fragments in anaerobic soluble microbial products (SMP) were retained by the membrane and some fragments were present in both SMP and in soluble extracellular polymeric substances (EPS), suggesting that the physical retention of SMP components contributed to the AnMBR membrane fouling. One of the dominant fragments was comprised of glycoliproprotein (size 630-640 kD) and correlated in abundance in AnMBR-EPS with the extent of anaerobic membrane fouling. In contrast, all detected AeMBR-SMP fragments permeated through the membrane. Aerobic SMP and soluble EPS also showed very different fingerprinting profiles. A large amount of adenosine triphosphate was present in the AeMBR-EPS, suggesting that microbial activity arising from certain bacterial populations, such as unclassified Comamonadaceae and unclassified Chitinophagaceae, may play a role in aerobic membrane fouling. This study underlines the differences in fouling mechanisms between AeMBR and AnMBR systems and can be applied to facilitate the development of appropriate fouling control strategies.

  6. Redistribution of Cholesterol in Model Lipid Membranes in Response to the Membrane-Active Peptide Alamethicin

    Science.gov (United States)

    Heller, William; Qian, Shuo

    2013-03-01

    The cellular membrane is a heterogeneous, dynamic mixture of molecules and macromolecules that self-assemble into a tightly-regulated functional unit that provides a semipermeable barrier between the cell and its environment. Among the many compositional differences between mammalian and bacterial cell membranes that impact its physical properties, one key difference is cholesterol content, which is more prevalent in mammals. Cholesterol is an amphiphile that associates with membranes and serves to maintain its fluidity and permeability. Membrane-active peptides, such as the alpha-helical peptide alamethicin, interact with membranes in a concentration- and composition-dependent manner to form transmembrane pores that are responsible for the lytic action of the peptide. Through the use of small-angle neutron scattering and deuterium labeling, it was possible to observe a redistribution of the lipid and cholesterol in unilamellar vesicles in response to the presence of alamethicin at a peptide-to-lipid ratio of 1/200. The results demonstrate that the membrane remodeling powers of alamethicin reach beyond the membrane thinning effect to altering the localization of specific components in the bilayer, complementing the accepted two-state mechanism of pore formation. Research was supported by U. S. DOE-OBER (CSMB; FWP ERKP291) and the U. S. DOE-BES Scientific User Facilities Division (ORNL's SNS and HFIR).

  7. A bacterial toxin inhibits DNA replication elongation through a direct interaction with the β sliding clamp.

    Science.gov (United States)

    Aakre, Christopher D; Phung, Tuyen N; Huang, David; Laub, Michael T

    2013-12-12

    Toxin-antitoxin (TA) systems are ubiquitous on bacterial chromosomes, yet the mechanisms regulating their activity and the molecular targets of toxins remain incompletely defined. Here, we identify SocAB, an atypical TA system in Caulobacter crescentus. Unlike canonical TA systems, the toxin SocB is unstable and constitutively degraded by the protease ClpXP; this degradation requires the antitoxin, SocA, as a proteolytic adaptor. We find that the toxin, SocB, blocks replication elongation through an interaction with the sliding clamp, driving replication fork collapse. Mutations that suppress SocB toxicity map to either the hydrophobic cleft on the clamp that binds DNA polymerase III or a clamp-binding motif in SocB. Our findings suggest that SocB disrupts replication by outcompeting other clamp-binding proteins. Collectively, our results expand the diversity of mechanisms employed by TA systems to regulate toxin activity and inhibit bacterial growth, and they suggest that inhibiting clamp function may be a generalizable antibacterial strategy. PMID:24239291

  8. Phosphatidylglycerol is involved in protein translocation across Escherichia coli inner membranes.

    NARCIS (Netherlands)

    T. de Vrije; R.L. de Swart (Rik); W. Dowhan; J. Tommassen (Jan); B. de Kruijff

    1988-01-01

    textabstractNewly synthesized proteins to be exported out of the cytoplasm of bacterial cells have to pass across the inner membrane. In Gram-negative bacteria ATP, a membrane potential, the products of the sec genes and leader peptidases (enzymes which cleave the N-terminal signal peptides of the p

  9. Improved Fixation of Cellulose-Acetate Reverse-Osmosis Membrane for Scanning Electron Microscopy

    OpenAIRE

    Kutz, S. M.; Bentley, D L; Sinclair, N A

    1985-01-01

    Fixation of cellulose-acetate membranes with either glutaraldehyde-osmium tetroxide or glutaraldehyde-ruthenium tetroxide resulted in extensive electron beam damage. Beam damage was eliminated and the bacterial surface structure was preserved, however, when cellulose-acetate membranes were fixed with glutaraldehyderuthenium tetroxide and treated successively with thiocarbohydrazide and osmium tetroxide.

  10. Biophysical studies of membrane channel polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Galbraith, Toby Patrick

    2001-07-01

    Membrane channels facilitate the flow of ions across biological membranes, a process which is important in numerous cellular functions. The study of large integral membrane proteins is made difficult by identification, production and purification problems, and detailed knowledge of their three-dimensional structures is relatively scarce. The study of simple 'model' membrane proteins has given valuable insight into the structures and dynamics of membrane proteins in general. The bacterial peptide gramicidin has been the subject of intense study for many years, and has provided important information into the structural basis of channel function. Peptaibols, a class of fungal membrane peptides which includes alamethicin and antiamoebin, have also been useful in relating structural details to molecular ion transport processes. Gramicidin crystals were grown in the presence of phospholipids with various headgroups and acyl chains. The diffraction patterns of the crystals obtained were processed, but found to be insufficient for high-resolution structural refinement. Analysis of the crystal space group and cell dimensions suggest that gramicidin adopts the same conformation as the closed, ion-free pore form previously published. Circular dichroism studies of gramicidin in small unilamellar vesicles of phospholipids with acyl chains of varying length show that the conformation of gramicidin is affected by the width of the bilayer. The structure of antiamoebin I in deuterated methanol was determined using 2D NMR spectroscopy techniques. It was compared with previously published solid state structures of this molecule, and found to be significantly different. The solution structure may represent an intermediate pre-insertion conformation which is not feasible in the more highly ordered membrane or crystal environment. A possible mechanism for membrane insertion is proposed. (author)

  11. Alternatives to overcoming bacterial resistances: State-of-the-art.

    Science.gov (United States)

    Rios, Alessandra C; Moutinho, Carla G; Pinto, Flávio C; Del Fiol, Fernando S; Jozala, Angela; Chaud, Marco V; Vila, Marta M D C; Teixeira, José A; Balcão, Victor M

    2016-10-01

    Worldwide, bacterial resistance to chemical antibiotics has reached such a high level that endangers public health. Presently, the adoption of alternative strategies that promote the elimination of resistant microbial strains from the environment is of utmost importance. This review discusses and analyses several (potential) alternative strategies to current chemical antibiotics. Bacteriophage (or phage) therapy, although not new, makes use of strictly lytic phage particles as an alternative, or a complement, in the antimicrobial treatment of bacterial infections. It is being rediscovered as a safe method, because these biological entities devoid of any metabolic machinery do not possess any affinity whatsoever to eukaryotic cells. Lysin therapy is also recognized as an innovative antimicrobial therapeutic option, since the topical administration of preparations containing purified recombinant lysins with amounts in the order of nanograms, in infections caused by Gram-positive bacteria, demonstrated a high therapeutic potential by causing immediate lysis of the target bacterial cells. Additionally, this therapy exhibits the potential to act synergistically when combined with certain chemical antibiotics already available on the market. Another potential alternative antimicrobial therapy is based on the use of antimicrobial peptides (AMPs), amphiphilic polypeptides that cause disruption of the bacterial membrane and can be used in the treatment of bacterial, fungal and viral infections, in the prevention of biofilm formation, and as antitumoral agents. Interestingly, bacteriocins are a common strategy of bacterial defense against other bacterial agents, eliminating the potential opponents of the former and increasing the number of available nutrients in the environment for their own growth. They can be applied in the food industry as biopreservatives and as probiotics, and also in fighting multi-resistant bacterial strains. The use of antibacterial antibodies

  12. Membrane Automata with Priorities

    Institute of Scientific and Technical Information of China (English)

    Luděk Cienciala; Lucie Ciencialová

    2004-01-01

    In this paper the one-way P automata with priorities are introduced. Such automata are P systemshere the membranes are only allowed to consume objects from parent membranes, under the given conditions. The result of computation of these systems is the set of multiset sequences consumed by skin membrane intc the system. The rules associated in some order with each membrane cannot modify any objects, they can only move them through membrane. We show that P automata with priorities and two membranes can accept every recursively enumerated language.

  13. The bacterial magnetosome: a unique prokaryotic organelle.

    Science.gov (United States)

    Lower, Brian H; Bazylinski, Dennis A

    2013-01-01

    The bacterial magnetosome is a unique prokaryotic organelle comprising magnetic mineral crystals surrounded by a phospholipid bilayer. These inclusions are biomineralized by the magnetotactic bacteria which are ubiquitous, aquatic, motile microorganisms. Magnetosomes cause cells of magnetotactic bacteria to passively align and swim along the Earth's magnetic field lines, as miniature motile compass needles. These specialized compartments consist of a phospholipid bilayer membrane surrounding magnetic crystals of magnetite (Fe3O4) or greigite (Fe3S4). The morphology of these membrane-bound crystals varies by species with a nominal magnetic domain size between 35 and 120 nm. Almost all magnetotactic bacteria arrange their magnetosomes in a chain within the cell there by maximizing the magnetic dipole moment of the cell. It is presumed that magnetotactic bacteria use magnetotaxis in conjunction with chemotaxis to locate and maintain an optimum position for growth and survival based on chemistry, redox and physiology in aquatic habitats with vertical chemical concentration and redox gradients. The biosynthesis of magnetosomes is a complex process that involves several distinct steps including cytoplasmic membrane modifications, iron uptake and transport, initiation of crystallization, crystal maturation and magnetosome chain formation. While many mechanistic details remain unresolved, magnetotactic bacteria appear to contain the genetic determinants for magnetosome biomineralization within their genomes in clusters of genes that make up what is referred to as the magnetosome gene island in some species. In addition, magnetosomes contain a unique set of proteins, not present in other cellular fractions, which control the biomineralization process. Through the development of genetic systems, proteomic and genomic work, and the use of molecular and biochemical tools, the functions of a number of magnetosome membrane proteins have been demonstrated and the molecular

  14. Ankyrin-B directs membrane tethering of periaxin and is required for maintenance of lens fiber cell hexagonal shape and mechanics.

    Science.gov (United States)

    Maddala, Rupalatha; Walters, Mark; Brophy, Peter J; Bennett, Vann; Rao, Ponugoti V

    2016-01-15

    Periaxin (Prx), a PDZ domain protein expressed preferentially in myelinating Schwann cells and lens fibers, plays a key role in membrane scaffolding and cytoarchitecture. Little is known, however, about how Prx is anchored to the plasma membrane. Here we report that ankyrin-B (AnkB), a well-characterized adaptor protein involved in linking the spectrin-actin cytoskeleton to integral membrane proteins, is required for membrane association of Prx in lens fibers and colocalizes with Prx in hexagonal fiber cells. Under AnkB haploinsufficiency, Prx accumulates in the soluble fraction with a concomitant loss from the membrane-enriched fraction of mouse lenses. Moreover, AnkB haploinsufficiency induced age-dependent disruptions in fiber cell hexagonal geometry and radial alignment and decreased compressive stiffness in mouse lenses parallel to the changes observed in Prx null mouse lens. Both AnkB- and Prx-deficient mice exhibit disruptions in membrane organization of the spectrin-actin network and the dystrophin-glycoprotein complex in lens fiber cells. Taken together, these observations reveal that AnkB is required for Prx membrane anchoring and for maintenance of lens fiber cell hexagonal geometry, membrane skeleton organization, and biomechanics. PMID:26538089

  15. Biofouling ecology as a means to better understand membrane biofouling.

    Science.gov (United States)

    Vanysacker, Louise; Boerjan, Bart; Declerck, Priscilla; Vankelecom, Ivo F J

    2014-10-01

    Despite more than a decade of worldwide research on membrane fouling in membrane bioreactors, many questions remain to be answered. Biofouling, which is referred to as the unwanted deposition and growth of biofilms, remains the main problem. Due to its complexity, most of the existing anti-biofouling strategies are not completely successful. To unravel this complexity and finally to developed well-adapted control strategies, a microbial-based description of the biofouling development is needed. Therefore, in this review, the biofouling formation will be described as a typical biofilm formation in five steps including the formation of a conditioning film, the bacterial attachment, the production of extracellular polymeric substances, the biofilm maturation, and the bacterial detachment. Moreover, important processes such as hydrodynamics and bacterial communication or quorum sensing will be taken into account. It is finally discussed whether biofouling formation is an active or inactive biofilm process together with suggestion for further research.

  16. Traffic of p24 Proteins and COPII Coat Composition Mutually Influence Membrane Scaffolding.

    Science.gov (United States)

    D'Arcangelo, Jennifer G; Crissman, Jonathan; Pagant, Silvere; Čopič, Alenka; Latham, Catherine F; Snapp, Erik L; Miller, Elizabeth A

    2015-05-18

    Eukaryotic protein secretion requires efficient and accurate delivery of diverse secretory and membrane proteins. This process initiates in the ER, where vesicles are sculpted by the essential COPII coat. The Sec13p subunit of the COPII coat contributes to membrane scaffolding, which enforces curvature on the nascent vesicle. A requirement for Sec13p can be bypassed when traffic of lumenally oriented membrane proteins is abrogated. Here we sought to further explore the impact of cargo proteins on vesicle formation. We show that efficient ER export of the p24 family of proteins is a major driver of the requirement for Sec13p. The scaffolding burden presented by the p24 complex is met in part by the cargo adaptor Lst1p, which binds to a subset of cargo, including the p24 proteins. We propose that the scaffolding function of Lst1p is required to generate vesicles that can accommodate difficult cargo proteins that include large oligomeric assemblies and asymmetrically distributed membrane proteins. Vesicles that contain such cargoes are also more dependent on scaffolding by Sec13p, and may serve as a model for large carrier formation in other systems.

  17. Photometric Application of the Gram Stain Method To Characterize Natural Bacterial Populations in Aquatic Environments

    OpenAIRE

    Saida, H; Ytow, N.; Seki, H.

    1998-01-01

    The Gram stain method was applied to the photometric characterization of aquatic bacterial populations with a charge-coupled device camera and an image analyzer. Escherichia coli and Bacillus subtilis were used as standards of typical gram-negative and gram-positive bacteria, respectively. A mounting agent to obtain clear images of Gram-stained bacteria on Nuclepore membrane filters was developed. The bacterial stainability by the Gram stain was indicated by the Gram stain index (GSI), which ...

  18. Membrane fouling potentials and cellular properties of bacteria isolated from fouled membranes in a MBR treating municipal wastewater.

    Science.gov (United States)

    Ishizaki, So; Fukushima, Toshikazu; Ishii, Satoshi; Okabe, Satoshi

    2016-09-01

    Membrane fouling remains a major challenge for wider application of membrane bioreactors (MBRs) to wastewater treatment. Membrane fouling is mainly caused by microorganisms and their excreted microbial products. For development of more effective control strategies, it is important to identify and characterize the microorganisms that are responsible for membrane fouling. In this study, 41 bacterial strains were isolated from fouled microfiltration membranes in a pilot-scale MBR treating real municipal wastewater, and their membrane fouling potentials were directly measured using bench-scale cross-flow membrane filtration systems (CFMFSs) and related to their cellular properties. It was found that the fouling potential was highly strain dependent, suggesting that bacterial identification at the strain level is essential to identify key fouling-causing bacteria (FCB). The FCB showed some common cellular properties. The most prominent feature of FCB was that they formed convex colonies having swollen podgy shape and smooth lustrous surfaces with high water, hydrophilic organic matter and carbohydrate content. However, general and rigid biofilm formation potential as determined by microtiter plates and cell surface properties (i.e., hydrophobicity and surface charge) did not correlate with the fouling potential in this study. These results suggest that the fouling potential should be directly evaluated under filtration conditions, and the colony water content could be a useful indicator to identify the FCB. PMID:27232989

  19. Architecture of beta-barrel membrane proteins: analysis of trimeric porins.

    OpenAIRE

    Seshadri, K.; Garemyr, R.; Wallin, E.; von Heijne, G; Elofsson, A.

    1998-01-01

    We have analyzed the known three-dimensional structures of trimeric porins from bacterial outer membranes. The distribution of surface-exposed residues in a direction perpendicular to the membrane is similar to that in helical membrane proteins, with aliphatic residues concentrated in the central 20 A of the bilayer. Outside these residues is a layer of aromatic residues, followed by polar and charged residues. Residues in the trimer interface are more conserved than residues not in the inter...

  20. Protective role of E. coli outer membrane vesicles against antibiotics.

    Science.gov (United States)

    Kulkarni, Heramb M; Nagaraj, R; Jagannadham, Medicharla V

    2015-12-01

    The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics. PMID:26640046

  1. Protective role of E. coli outer membrane vesicles against antibiotics.

    Science.gov (United States)

    Kulkarni, Heramb M; Nagaraj, R; Jagannadham, Medicharla V

    2015-12-01

    The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics.

  2. Antibiofilm effect enhanced by modification of 1,2,3-triazole and palladium nanoparticles on polysulfone membranes

    KAUST Repository

    Cheng, Hong

    2015-08-01

    Biofouling impedes the performance of membrane bioreactors. In this study, we investigated the antifouling effects of polysulfone membranes that were modified by 1,2,3-triazole and palladium nanoparticles. The membranes to be tested were embedded within a drip flow biofilm reactor, and Pseudomonas aeruginosa PAO1 was inoculated and allowed to establish biofilm on the tested membranes. It was found that 1,2,3-triazole and palladium nanoparticles can inhibit the bacterial growth in aerobic and anaerobic conditions. The decrease in bacterial growth was observed along with a decrease in the amount of total polysaccharide and Pel polysaccharide within the biofilm matrix but not the protein content.

  3. Age and feeding system (supplemental feeding versus grazing) modulates colonic bacterial succession and host mucosal immune maturation in goats.

    Science.gov (United States)

    Jiao, J; Lu, Q; Forster, R J; Zhou, C; Wang, M; Kang, J; Tan, Z

    2016-06-01

    The gut microbiome plays important roles in the regulation of gastrointestinal tract functional development and host mucosal immune maturation. This study was conducted to test the hypothesis that age and feeding system (supplemental feeding [Sup] vs. grazing [G]) could alter colonic bacterial diversity and host mucosal immune maturation. Thirty Liuyang black goat kids ( = 4) were slaughtered on d 0, d 7 (nonrumination), d 28, d 42 (transition), and d 70 (rumination). The colonic microbiota was profiled by Miseq sequencing of the 16S rRNA gene. Host colonic mucosal immune maturation was examined using mRNA level expression of Toll-like receptors (TLR), proinflammatory cytokines, and the Toll-IL-1R (TIR) domain-containing adaptor. A correlation analysis was conducted to elucidate the relationship between bacterial diversity and fermentation parameters and host immune maturation variables. The results showed that α diversity indexes ( diversity indexes increased ( diversity and host mucosal immune maturation are age related, and concentrate supplement could alter bacterial diversity and alleviate overinflammation responses. PMID:27285927

  4. Oxygen transport membrane

    DEFF Research Database (Denmark)

    2015-01-01

    The present invention relates to a novel composite oxygen transport membrane as well as its preparation and uses thereof.......The present invention relates to a novel composite oxygen transport membrane as well as its preparation and uses thereof....

  5. Solvent resistant nanofiltration membranes

    OpenAIRE

    Dutczak, Szymon Maria

    2011-01-01

    This thesis describes preparation and characterization of membranes for organic solvent filtration (OSF). The main aim was developing membranes for solvent resistant nanofiltration (SRNF) with molecular weight cut-off below 500 g mol-1.

  6. Hybrid adsorptive membrane reactor

    Science.gov (United States)

    Tsotsis, Theodore T.; Sahimi, Muhammad; Fayyaz-Najafi, Babak; Harale, Aadesh; Park, Byoung-Gi; Liu, Paul K. T.

    2011-03-01

    A hybrid adsorbent-membrane reactor in which the chemical reaction, membrane separation, and product adsorption are coupled. Also disclosed are a dual-reactor apparatus and a process using the reactor or the apparatus.

  7. Hybrid adsorptive membrane reactor

    Science.gov (United States)

    Tsotsis, Theodore T. (Inventor); Sahimi, Muhammad (Inventor); Fayyaz-Najafi, Babak (Inventor); Harale, Aadesh (Inventor); Park, Byoung-Gi (Inventor); Liu, Paul K. T. (Inventor)

    2011-01-01

    A hybrid adsorbent-membrane reactor in which the chemical reaction, membrane separation, and product adsorption are coupled. Also disclosed are a dual-reactor apparatus and a process using the reactor or the apparatus.

  8. Bacterial Cellulose-Hydroxyapatite Nanocomposites for Bone Regeneration

    Directory of Open Access Journals (Sweden)

    S. Saska

    2011-01-01

    Full Text Available The aim of this study was to develop and to evaluate the biological properties of bacterial cellulose-hydroxyapatite (BC-HA nanocomposite membranes for bone regeneration. Nanocomposites were prepared from bacterial cellulose membranes sequentially incubated in solutions of CaCl2 followed by Na2HPO4. BC-HA membranes were evaluated in noncritical bone defects in rat tibiae at 1, 4, and 16 weeks. Thermogravimetric analyses showed that the amount of the mineral phase was 40%–50% of the total weight. Spectroscopy, electronic microscopy/energy dispersive X-ray analyses, and X-ray diffraction showed formation of HA crystals on BC nanofibres. Low crystallinity HA crystals presented Ca/P a molar ratio of 1.5 (calcium-deficient HA, similar to physiological bone. Fourier transformed infrared spectroscopy analysis showed bands assigned to phosphate and carbonate ions. In vivo tests showed no inflammatory reaction after 1 week. After 4 weeks, defects were observed to be completely filled in by new bone tissue. The BC-HA membranes were effective for bone regeneration.

  9. Membrane Curvature in Flaviviruses

    OpenAIRE

    Zhang, Wei; Kaufmann, Bärbel; Chipman, Paul R.; Kuhn, Richard J; Rossmann, Michael G.

    2013-01-01

    Coordinated interplay between membrane proteins and the lipid bilayer is required for such processes as transporter function and the entrance of enveloped viruses into host cells. In this study, three-dimensional cryo-electron microscopy density maps of mature and immature flaviviruses were analyzed to assess the curvature of the membrane leaflets and its relation to membrane-bound viral glycoproteins. The overall morphology of the viral membrane is determined by icosahedral scaffolding compo...

  10. Architecture and Characteristics of Bacterial Nanotubes.

    Science.gov (United States)

    Dubey, Gyanendra P; Malli Mohan, Ganesh Babu; Dubrovsky, Anna; Amen, Triana; Tsipshtein, Shai; Rouvinski, Alex; Rosenberg, Alex; Kaganovich, Daniel; Sherman, Eilon; Medalia, Ohad; Ben-Yehuda, Sigal

    2016-02-22

    Bacteria display an array of contact-dependent interaction systems that have evolved to facilitate direct cell-to-cell communication. We have previously identified a mode of bacterial communication mediated by nanotubes bridging neighboring cells. Here, we elucidate nanotube architecture, dynamics, and molecular components. Utilizing Bacillus subtilis as a model organism, we found that at low cell density, nanotubes exhibit remarkable complexity, existing as both intercellular tubes and extending tubes, with the latter frequently surrounding the cells in a "root-like" fashion. Observing nanotube formation in real time showed that these structures are formed in the course of minutes, displaying rapid movements. Utilizing a combination of super-resolution, light, and electron microscopy, we revealed that nanotubes are composed of chains of membranous segments harboring a continuous lumen. Furthermore, we discovered that a conserved calcineurin-like protein, YmdB, presents in nanotubes and is required for both nanotube production and intercellular molecular trade.

  11. Spontaneous bacterial peritonitis

    Institute of Scientific and Technical Information of China (English)

    Anastasios Koulaouzidis; Shivaram Bhat; Athar A Saeed

    2009-01-01

    Since its initial description in 1964, research has transformed spontaneous bacterial peritonitis (SBP) from a feared disease (with reported mortality of 90%) to a treatable complication of decompensated cirrhosis,albeit with steady prevalence and a high recurrence rate. Bacterial translocation, the key mechanism in the pathogenesis of SBP, is only possible because of the concurrent failure of defensive mechanisms in cirrhosis.Variants of SBP should be treated. Leucocyte esterase reagent strips have managed to shorten the 'tap-toshot' time, while future studies should look into their combined use with ascitic fluid pH. Third generation cephalosporins are the antibiotic of choice because they have a number of advantages. Renal dysfunction has been shown to be an independent predictor of mortality in patients with SBP. Albumin is felt to reduce the risk of renal impairment by improving effective intravascular volume, and by helping to bind proinflammatory molecules. Following a single episode of SBP, patients should have long-term antibiotic prophylaxis and be considered for liver transplantation.

  12. Model cell membranes

    DEFF Research Database (Denmark)

    Günther-Pomorski, Thomas; Nylander, Tommy; Cardenas Gomez, Marite

    2014-01-01

    The high complexity of biological membranes has motivated the development and application of a wide range of model membrane systems to study biochemical and biophysical aspects of membranes in situ under well defined conditions. The aim is to provide fundamental understanding of processes control...

  13. Membrane contactor applications

    NARCIS (Netherlands)

    Klaassen, R.; Feron, P.H.M.; Jansen, A.

    2008-01-01

    In a membrane contactor the membrane separation is completely integrated with an extraction or absorption operation in order to exploit the benefits of both technologies fully. Membrane contactor applications that have been developed can be found in both water and gas treatment. Several recently dev

  14. Antimicrobials for bacterial bioterrorism agents.

    Science.gov (United States)

    Sarkar-Tyson, Mitali; Atkins, Helen S

    2011-06-01

    The limitations of current antimicrobials for highly virulent pathogens considered as potential bioterrorism agents drives the requirement for new antimicrobials that are suitable for use in populations in the event of a deliberate release. Strategies targeting bacterial virulence offer the potential for new countermeasures to combat bacterial bioterrorism agents, including those active against a broad spectrum of pathogens. Although early in the development of antivirulence approaches, inhibitors of bacterial type III secretion systems and cell division mechanisms show promise for the future.

  15. The dynamics in the bacterial chemosensory arrays

    Science.gov (United States)

    Vaknin, Ady

    2014-08-01

    Bacterial chemoreceptors form two-dimensional sensory arrays on the cell membrane. These sensory arrays, which contain thousands of molecules, detect chemical changes in the environment of the bacterial cell and accordingly control its swimming behaviour, allowing these bacteria to track chemical gradients. It was recently demonstrated that stimulus, by ligand binding, alters the physical organization of these arrays, with dynamics that follow an apparent logarithmic time dependence. Such non-exponential dynamics is often observed in glass-like systems in which the internal dynamics slow down exponentially as the system approaches its equilibrium state. In a few of these `glassy' systems it was also demonstrated that after altering the equilibrium state of the system for a certain time tw the ensuing relaxation scales with tw. Here, we examined the relaxation of the receptor arrays in the bacterium E. coli after a perturbation by ligand binding for varying periods of times. We find that changing the time tw, during which the stimulus was present, affects mostly the deviation of the receptor arrays from equilibrium, but the dynamics of the relaxation seem to be independent of tw. A possible interpretation is discussed.

  16. Bacterial respiration of arsenic and selenium

    Science.gov (United States)

    Stolz, J.F.; Oremland, R.S.

    1999-01-01

    Oxyanions of arsenic and selenium can be used in microbial anaerobic respiration as terminal electron acceptors. The detection of arsenate and selenate respiring bacteria in numerous pristine and contaminated environments and their rapid appearance in enrichment culture suggest that they are widespread and metabolically active in nature. Although the bacterial species that have been isolated and characterized are still few in number, they are scattered throughout the bacterial domain and include Gram- positive bacteria, beta, gamma and epsilon Proteobacteria and the sole member of a deeply branching lineage of the bacteria, Chrysiogenes arsenatus. The oxidation of a number of organic substrates (i.e. acetate, lactate, pyruvate, glycerol, ethanol) or hydrogen can be coupled to the reduction of arsenate and selenate, but the actual donor used varies from species to species. Both periplasmic and membrane-associated arsenate and selenate reductases have been characterized. Although the number of subunits and molecular masses differs, they all contain molybdenum. The extent of the environmental impact on the transformation and mobilization of arsenic and selenium by microbial dissimilatory processes is only now being fully appreciated.

  17. Mechanism of activation of bacterial cellulose synthase by cyclic-di-GMP

    OpenAIRE

    Morgan, Jacob L.W.; McNamara, Joshua T.; Zimmer, Jochen

    2014-01-01

    The bacterial signaling molecule cyclic-di-GMP stimulates the synthesis of bacterial cellulose, frequently found in biofilms. Bacterial cellulose is synthesized and translocated across the inner membrane by a complex of the cellulose synthase BcsA and BcsB subunits. Here we present crystal structures of the cyclic-di-GMP-activated BcsA–B complex. The structures reveal that cyclic-di-GMP releases an auto-inhibited state of the enzyme by breaking a salt bridge which otherwise tethers a conserve...

  18. Separation membrane development

    Energy Technology Data Exchange (ETDEWEB)

    Lee, M.W. [Savannah River Technology Center, Aiken, SC (United States)

    1998-08-01

    A ceramic membrane has been developed to separate hydrogen from other gases. The method used is a sol-gel process. A thin layer of dense ceramic material is coated on a coarse ceramic filter substrate. The pore size distribution in the thin layer is controlled by a densification of the coating materials by heat treatment. The membrane has been tested by permeation measurement of the hydrogen and other gases. Selectivity of the membrane has been achieved to separate hydrogen from carbon monoxide. The permeation rate of hydrogen through the ceramic membrane was about 20 times larger than Pd-Ag membrane.

  19. Plasma membrane ATPases

    DEFF Research Database (Denmark)

    Palmgren, Michael Broberg; Bækgaard, Lone; Lopez Marques, Rosa Laura;

    2011-01-01

    The plasma membrane separates the cellular contents from the surrounding environment. Nutrients must enter through the plasma membrane in order to reach the cell interior, and toxic metabolites and several ions leave the cell by traveling across the same barrier. Biological pumps in the plasma...... membrane include ABC transporters, vacuolar (V-type) H+ pumps, and P-type pumps. These pumps all utilize ATP as a fuel for energizing pumping. This review focuses on the physiological roles of plasma membrane P-type pumps, as they represent the major ATP hydrolytic activity in this membrane....

  20. Mixed matrix membrane development.

    Science.gov (United States)

    Kulprathipanja, Santi

    2003-03-01

    Two types of mixed matrix membranes were developed by UOP in the late 1980s. The first type includes adsorbent polymers, such as silicalite-cellulose acetate (CA), NaX-CA, and AgX-CA mixed matrix membranes. The silicalite-CA has a CO(2)/H(2) selectivity of 5.15 +/- 2.2. In contrast, the CA membrane has a CO(2)/H(2) selectivity of 0.77 +/- 0.06. The second type of mixed matrix membrane is PEG-silicone rubber. The PEG-silicone rubber mixed matrix membrane has high selectivity for polar gases, such as SO(2), NH(3), and H(2)S.