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Sample records for membrane impairs porin

  1. Porin Loss Impacts the Host Inflammatory Response to Outer Membrane Vesicles of Klebsiella pneumoniae

    PubMed Central

    Turner, Kelli L.; Cahill, Bethaney K.; Dilello, Sarah K.; Gutel, Dedra; Brunson, Debra N.; Albertí, Sebastián

    2015-01-01

    Antibiotic-resistant strains of Klebsiella pneumoniae often exhibit porin loss. In this study, we investigated how porin loss impacted the composition of secreted outer membrane vesicles as well as their ability to trigger proinflammatory cytokine secretion by macrophages. We hypothesize that porin loss associated with antibiotic resistance will directly impact both the composition of outer membrane vesicles and their interactions with phagocytic cells. Using clonally related clinical isolates of extended-spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae with different patterns of porin expression, we demonstrated that altered expression of OmpK35 and OmpK36 results in broad alterations to the protein profile of secreted vesicles. Additionally, the level of OmpA incorporation was elevated in strains lacking a single porin. Porin loss significantly impacted macrophage inflammatory responses to purified vesicles. Outer membrane vesicles lacking both OmpK35 and OmpK36 elicited significantly lower levels of proinflammatory cytokine secretion than vesicles from strains expressing one or both porins. These data demonstrate that antibiotic resistance-associated porin loss has a broad and significant effect on both the composition of outer membrane vesicles and their interactions with phagocytic cells, which may impact bacterial survival and inflammatory reactions in the host. PMID:26666932

  2. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene.

    PubMed Central

    Duchêne, M; Schweizer, A; Lottspeich, F; Krauss, G; Marget, M; Vogel, K; von Specht, B U; Domdey, H

    1988-01-01

    Porin F is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. It forms water-filled pores of variable size. Porin F is a candidate for a vaccine against P. aeruginosa because it antigenically cross-reacts in all serotype strains of the International Antigenic Typing Scheme. We have isolated the gene for porin F from a lambda EMBL3 bacteriophage library by using oligodeoxynucleotide hybridization probes and have determined its nucleotide sequence. Different peptide sequences obtained from isolated porin F confirmed the deduced protein sequence. The mature protein consists of 326 amino acid residues and has a molecular weight of 35,250. The precursor contains an N-terminal signal peptide of 24 amino acid residues. S1 protection and primer extension experiments, together with Northern (RNA) blots, indicate that the mRNA coding for porin F is monocistronic with short untranslated regions of about 58 bases at the 5' end and about 47 bases at the 3' end. The sequences in the -10 and -35 regions upstream of the transcriptional start site are closely related to the Escherichia coli promoter consensus sequences, which explains why the porin F gene is expressed in E. coli under the control of its own promoter. The amino acid sequence of porin F is not homologous to the different E. coli porins OmpF, OmpC, LamB, and PhoE. On the other hand, a highly homologous region of 30 amino acids between the OmpA proteins of different enteric bacteria and porin F of P. aeruginosa was detected. The core region of the homology to E. coli OmpA had 11 of 12 amino acid residues in common. Images PMID:2447060

  3. Electron crystallography of PhoE porin, an outer membrane, channel- forming protein from E. coli

    SciTech Connect

    Walian, P.J.

    1989-11-01

    One approach to studying the structure of membrane proteins is the use of electron crystallography. Dr. Bing Jap has crystallized PhoE pore-forming protein (porin) from the outer membrane of escherichia coli (E. coli) into monolayer crystals. The findings of this research and those of Jap (1988, 1989) have determined these crystals to be highly ordered, yielding structural information to a resolution of better than 2.8 angstroms. The task of this thesis has been to collect and process the electron diffraction patterns necessary to generate a complete three-dimensional set of high resolution structure factor amplitudes of PhoE porin. Fourier processing of these amplitudes when combined with the corresponding phase data is expected to yield the three-dimensional structure of PhoE porin at better than 3.5 angstroms resolution. 92 refs., 33 figs., 3 tabs. (CBS)

  4. Diffusion of beta-lactam antibiotics through liposome membranes reconstituted from purified porins of the outer membrane of Pseudomonas aeruginosa.

    PubMed Central

    Satake, S; Yoshihara, E; Nakae, T

    1990-01-01

    Determination of the rates of diffusion of beta-lactam antibiotics through purified Pseudomonas aeruginosa porins C, D2, and E in liposomes yielded the following results. (i) The rates of carbapenem (imipenem and meropenem) diffusion through the protein D2 pore were roughly 2 to 70 times higher than those through other porin pores. It is not clear why the protein D2 pore allowed rapid diffusion of carbapenems. The rates of diffusion of glucosamine and triglycine through the protein D2 pore were about 14 and 4 times higher, respectively, than that of an uncharged test solute with a similar Mr, glucose. (ii) The rates of diffusion of antipseudomonal anionic beta-lactams such as piperacillin, ceftazidime, cefsulodin, and aztreonam through the protein C pore were higher than those through other porin pores. This was probably due to the slightly larger pore size and the slight anion selectivity of protein C, since the apparent exclusion limit of the protein C pore for uncharged saccharides is higher than that of other porins and the rate of diffusion of gluconic acid through the protein C pore is about double that for glucose. (iii) The rates of diffusion of cefoperazone through all three species of porin were relatively high. These results indicate that the antipseudomonal beta-lactams permeate the P. aeruginosa outer membrane via newly identified porins. Images PMID:2163239

  5. Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

    SciTech Connect

    Pszon-Bartosz, Kamila; Hansen, Jesper S.; Stibius, Karin B.; Groth, Jesper S.; Helix-Nielsen, Claus

    2011-03-04

    Research highlights: {yields} We have established a vesicle fusion efficacy assay based on the major non-specific porin of Fusobacterium nucleatum (FomA). {yields} Maximal fusion obtained was almost 150,000 porin insertions during 20 min. {yields} Incorporation can be either first order or exponential kinetics which has implications for establishing protein delivery to biomimetic membranes. -- Abstract: Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10{sup 5} FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm{sup 2} within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.

  6. Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

    PubMed Central

    Skare, J T; Champion, C I; Mirzabekov, T A; Shang, E S; Blanco, D R; Erdjument-Bromage, H; Tempst, P; Kagan, B L; Miller, J N; Lovett, M A

    1996-01-01

    The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete. PMID:8759855

  7. Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin.

    PubMed Central

    Gabay, J E; Blake, M; Niles, W D; Horwitz, M A

    1985-01-01

    We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins. Images PMID:2579942

  8. Bordetella pertussis major outer membrane porin protein forms small, anion-selective channels in lipid bilayer membranes.

    PubMed Central

    Armstrong, S K; Parr, T R; Parker, C D; Hancock, R E

    1986-01-01

    The major outer membrane protein of molecular weight 40,000 (the 40K protein) of a virulent isolate of Bordetella pertussis was purified to apparent homogeneity. The purified protein formed an oligomer band (of apparent molecular weight 90,000) on sodium dodecyl sulfate-polyacrylamide gels after solubilization at low temperatures. The porin function of this protein was characterized by the black lipid bilayer method. The 40K protein formed channels smaller than all other constitutive major outer membrane porins studied to date. The average single-channel conductance in 1 M KCl was 0.56 nS. This was less than a third of the conductance previously observed for Escherichia coli porins. Zero-current potential measurements made of the porin to determine its ion selectivity revealed the porin to be more than 100-fold selective for anions over cations. The single-channel conductance was measured as a function of salt concentration. The data could be fitted to a Lineweaver-Burk plot suggesting an anion binding site with a Kd of 1.17 M Cl- and a maximum possible conductance through the channel of 1.28 nS. Images PMID:2420780

  9. An outer membrane protein (porin) as an eliciting antigen for delayed-type hypersensitivity in murine salmonellosis.

    PubMed Central

    Udhayakumar, V; Muthukkaruppan, V R

    1987-01-01

    The porin, an outer membrane protein of Salmonella typhimurium, was found to be a suitable antigen for eliciting delayed-type hypersensitivity in mouse salmonellosis. Histological examination of the reaction site revealed that the porin was superior to other antigenic preparations in eliciting a typical delayed-type hypersensitivity reaction consisting of mononuclear cell infiltration without polymorphonuclear cell contamination. This study indicates the importance of using a suitable protein antigen from S. typhi for human application. Images PMID:3028963

  10. Outer membrane of gram-negative bacteria. XVIII. Electron microscopic studies on porin insertion sites and growth of cell surface of Salmonella typhimurium.

    PubMed Central

    Smit, J; Nikaido, H

    1978-01-01

    Salmonella typhimurium contains three "major proteins" or "porins" (34K, 35K, and 36K) in the outer membrane. A mutant strain producing only the 35K porin was first grown in media containing high concentrations of NaCl to "repress" the porin synthesis and then was shifted into a medium without NaCl. The newly made porin molecules were then labeled with the ferritin-coupled antibody at various times after the shift, and the samples were examined by whole-mount, freeze-etching, and thin-section electron microscopy. These experiments showed that newly inserted porins appeared as discrete patches uniformly distributed over the surface of the cell and, furthermore, that the sites of adhesion between the inner and outer membrane were most probably the pathway by which the newly made porin molecules appeared on cell surface. The 34K and 36K porins were also inserted in the same manner, since the appearance of new porins at discrete sites all over the cell surface was also observed when cells with wild-type porin phenotype were treated with unlabeled antibody to block existing antigenic sites, subsequently regrown, and labeled with the ferritin-coupled antibody. Since porins comprise a major portion of the densely packed, relatively immobile, "protein framework" of the outer membrane, these results lead us to conclude that the outer membrane grows predominantly by diffuse intercalation rather than by the zonal growth mechanism. Images PMID:355240

  11. Protein-translocating outer membrane porins of Gram-negative bacteria.

    PubMed

    Yen, Ming Ren; Peabody, Christopher R; Partovi, Salar M; Zhai, Yufeng; Tseng, Yi Hsiung; Saier, Milton H

    2002-05-01

    Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized. In this report, these five porin families are analyzed from structural and phylogenetic standpoints. They are the fimbrial usher protein (FUP), outer membrane factor (OMF), autotransporter (AT), two-partner secretion (TPS) and outer membrane secretin (Secretin) families. All members of these families in the current databases were identified, and all full-length homologues were multiply aligned for structural and phylogenetic analyses. The organismal distribution of homologues in each family proved to be unique with some families being restricted to proteobacteria and others being widespread in other bacterial kingdoms as well as eukaryotes. The compositions of and size differences between subfamilies provide evidence for specific orthologous relationships, which agree with available functional information and intra-subfamily phylogeny. The results reveal that horizontal transfer of genes encoding these proteins between phylogenetically distant organisms has been exceptionally rare although transfer within select bacterial kingdoms may have occurred. The resultant in silico analyses are correlated with available experimental evidence to formulate models relevant to the structures and evolutionary origins of these proteins. PMID:11988218

  12. Saccharomyces cerevisiae Porin Pore Forms Complexes with Mitochondrial Outer Membrane Proteins Om14p and Om45p

    PubMed Central

    Lauffer, Susann; Mäbert, Katrin; Czupalla, Cornelia; Pursche, Theresia; Hoflack, Bernard; Rödel, Gerhard; Krause-Buchholz, Udo

    2012-01-01

    Numerous transport processes occur between the two mitochondrial (mt) membranes due to the diverse functions and metabolic processes of the mt organelle. The metabolite and ion transport through the mt outer membrane (OM) is widely assumed to be mediated by the porin pore, whereas in the mt inner membrane (IM) specific carriers are responsible for transport processes. Here, we provide evidence by means of Blue Native (BN)-PAGE analysis, co-immunoprecipitation, and tandem affinity purification that the two mt OM proteins Om14p and Om45p associate with the porin pore. Porin molecules seem to assemble independently to build the core unit. A subpopulation of these core units interacts with Om14p and Om45p. With preparative tandem affinity purification followed by MS analysis, we could identify interaction partners of this OM complex, which are mainly localized within the mt IM and function as carriers for diverse molecules. We propose a model for the role of the two OM proteins in addressing the porin pore to bind to specific channels in the mt IM to facilitate transport of metabolites. PMID:22461620

  13. Borrelia burgdorferi BBA74, a Periplasmic Protein Associated with the Outer Membrane, Lacks Porin-Like Properties▿

    PubMed Central

    Mulay, Vishwaroop; Caimano, Melissa J.; Liveris, Dionysios; Desrosiers, Daniel C.; Radolf, Justin D.; Schwartz, Ira

    2007-01-01

    The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% α-helix with little β-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins. PMID:17189354

  14. Borrelia burgdorferi BBA74, a periplasmic protein associated with the outer membrane, lacks porin-like properties.

    PubMed

    Mulay, Vishwaroop; Caimano, Melissa J; Liveris, Dionysios; Desrosiers, Daniel C; Radolf, Justin D; Schwartz, Ira

    2007-03-01

    The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% alpha-helix with little beta-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins. PMID:17189354

  15. New Findings Concerning Vertebrate Porin

    NASA Astrophysics Data System (ADS)

    Thinnes, Friedrich P.; Reymann, Susanne

    Eukaryotic porin can be considered to be a good candidate for forming the channel component of the protein complex which, depending on the approach used, may realize its expression either as the outwardly-rectifying depolarization-induced chloride channel or as the volume-sensitive organic osmolyte-anion channel. As a basis for this proposition, we point to a series of correspondences in properties between mammalian porin and the ORDIC channel complex. Specifically, mammalian porin is expressed in the plasmalemma of different cells and chloride channels can be blocked by anti-human porin antibodies in astrocytes and endothelial cells. There is an indication of colocalisation of human porin and the cystic fibrosis (CF) gene product, CFTR, in the apical region of epithelial cells. The primary structure of porin from a CF patient was found to be normal. Cytosol and amniotic fluid fractions influence the channel characteristics of mammalian porin. Channel-active mammalian porin binds ATP and the stilbene disulphonate grouping of the chloride channel inhibitor DIDS. Human porin in black membranes is a pathway for taurine, and biogenic polyamines reduce the voltage dependence of human porin. Assuming the relationship between human porin and the ORDIC channel/VSOAC complex, studies on plasmalemma-integrated human porin have a relevance for CF research. In addition, we refer to a case study on a child with encephalomyopathy in which porin could not be detected using monoclonal anti-human porin antibodies. Our studies were based on purified and sequenced human porin from different cells and from different cell compartments. In addition, we raised antibodies against mature human porin or synthetic parts of the molecule. This provided a firm foundation for our topochemical work with which we were able to establish the multi-topological expression of eukaryotic porin channels. The data are summarized and discussed.

  16. Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins

    PubMed Central

    2009-01-01

    Background The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis. Results Isogenic m35 mutants of the type 1 strains O35E, 300 and 415 were tested for their antimicrobial susceptibility against 15 different agents. Differences in the MIC (Minimum Inhibitory Concentration) between wild-type and mutant strains were found for eight antibiotics. For ampicillin and amoxicillin, we observed a statistically significant 2.5 to 2.9-fold MIC increase (p < 0.03) in the m35 mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective m35 mutants were indistinguishable with respect to the phenotypes of autoagglutination, serum resistance, iron acquisition from human lactoferrin, adherence to and invasion of respiratory tract epithelial cells, and proinflammatory stimulation of human monocytes. DNA sequencing of m35 from the phylogenetic subpopulation type 2 strain 287 revealed 94.2% and 92.8% identity on the DNA and amino acid levels, respectively, in comparison with type 1 strains. Conclusion The increase in MIC for ampicillin and amoxicillin, respectively, in the M35-deficient mutants indicates that this porin affects the outer membrane permeability for aminopenicillins in a clinically relevant manner. The presence of IgA antibodies in healthy human donors indicates that M35 is expressed in vivo and recognized as a mucosal antigen by the human host. However, immunoblot analysis of human saliva suggests the possibility of antigenic

  17. Detection of β -Lactamases and Outer Membrane Porins among Klebsiella pneumoniae Strains Isolated in Iran.

    PubMed

    Hashemi, Ali; Fallah, Fatemeh; Erfanimanesh, Soroor; Hamedani, Parastu; Alimehr, Shadi; Goudarzi, Hossein

    2014-01-01

    This descriptive study was accomplished on 83 K. pneumoniae strains isolated from two hospitals in Tehran, Iran. Antibiotic susceptibility tests were performed by disc diffusion and broth microdilution methods. ESBLs, MBL, Amp-C, and KPC producing strains were detected by phenotypic confirmatory test, combination disk diffusion test (CDDT), Amp-C detection kit, and modified Hodge test, respectively. OXA-48, NDM-1, and CTX-M-15 genes were detected by PCR and sequencing methods. The outer membrane porins such as OmpK35 and OmpK36 were analysed by SDS-PAGE, PCR, and sequencing methods. From 83 K. pneumoniae isolates, 48 (57.5%), 3 (3.5%), 23 (28%), and 5 (6%) were ESBL, MBL, Amp-C, and KPC positive, respectively. The CTX-M-15 gene was detected in 30 (62.5%) and OXA-48 gene was found in 2 (4.1%) of the 48 ESBL-producing isolates. Two isolates harboured both OXA-48 and CTX-M-15; NDM-1 gene was not detected in this study. Outer membrane porin, OmpK35, was detected in 30 (62.5%) of 48 ESBL-producing isolates while OmpK36 was found in 35 (72.91%) of 48 ESBL-producing isolates. In this study, fosfomycin and tigecycline were more effective than other antibiotics. The high prevalence of β-lactamase-producing K. pneumoniae detected in this study is of great concern, which requires infection control measures including antibacterial management and identification of β-lactamases-producing isolates. PMID:25548718

  18. Detection of β-Lactamases and Outer Membrane Porins among Klebsiella pneumoniae Strains Isolated in Iran

    PubMed Central

    Fallah, Fatemeh; Hamedani, Parastu; Alimehr, Shadi; Goudarzi, Hossein

    2014-01-01

    This descriptive study was accomplished on 83 K. pneumoniae strains isolated from two hospitals in Tehran, Iran. Antibiotic susceptibility tests were performed by disc diffusion and broth microdilution methods. ESBLs, MBL, Amp-C, and KPC producing strains were detected by phenotypic confirmatory test, combination disk diffusion test (CDDT), Amp-C detection kit, and modified Hodge test, respectively. OXA-48, NDM-1, and CTX-M-15 genes were detected by PCR and sequencing methods. The outer membrane porins such as OmpK35 and OmpK36 were analysed by SDS-PAGE, PCR, and sequencing methods. From 83 K. pneumoniae isolates, 48 (57.5%), 3 (3.5%), 23 (28%), and 5 (6%) were ESBL, MBL, Amp-C, and KPC positive, respectively. The CTX-M-15 gene was detected in 30 (62.5%) and OXA-48 gene was found in 2 (4.1%) of the 48 ESBL-producing isolates. Two isolates harboured both OXA-48 and CTX-M-15; NDM-1 gene was not detected in this study. Outer membrane porin, OmpK35, was detected in 30 (62.5%) of 48 ESBL-producing isolates while OmpK36 was found in 35 (72.91%) of 48 ESBL-producing isolates. In this study, fosfomycin and tigecycline were more effective than other antibiotics. The high prevalence of β-lactamase-producing K. pneumoniae detected in this study is of great concern, which requires infection control measures including antibacterial management and identification of β-lactamases-producing isolates. PMID:25548718

  19. Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex.

    PubMed

    Krimmer, T; Rapaport, D; Ryan, M T; Meisinger, C; Kassenbrock, C K; Blachly-Dyson, E; Forte, M; Douglas, M G; Neupert, W; Nargang, F E; Pfanner, N

    2001-01-22

    Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore. PMID:11266446

  20. Functional Characterization of a Novel Outer Membrane Porin KpnO, Regulated by PhoBR Two-Component System in Klebsiella pneumoniae NTUH-K2044

    PubMed Central

    Srinivasan, Vijaya Bharathi; Venkataramaiah, Manjunath; Mondal, Amitabha; Vaidyanathan, Vasanth; Govil, Tanvi; Rajamohan, Govindan

    2012-01-01

    Background The diffusion of antibiotics through the outer membrane is primarily affected by the porin super family, changes contribute to antibiotic resistance. Recently we demonstrated that the CpxAR two-component signaling system alters the expression of an uncharacterized porin OmpCKP, to mediate antimicrobial resistance in K. pneumoniae. Principal Findings In this study, functional characterization of the putative porin OmpCKP (denoted kpnO) with respect to antimicrobial susceptibility and virulence was evaluated by generating an isogenic mutant, ΔkpnO in a clinical isolate of K. pneumoniae. Estimation of uronic acid content confirmed that ΔkpnO produced ∼2.0 fold lesser capsular polysaccharide than the wild-type. The ΔkpnO displayed higher sensitivity to hyper osmotic and bile conditions. Disruption of kpnO increased the susceptibility of K. pneumoniae to oxidative and nitrostative stress by ∼1.6 fold and >7 fold respectively. The loss of the Klebsiella porin led to an increase in the minimum inhibitory concentration of tetracycline (3-fold), nalidixic acid (4-fold), tobramycin (4-fold), streptomycin (10-fold), and spectinomycin (10-fold), which could be restored following complementation. The single deletion of kpnO reduced the survival of the pathogen by 50% when exposed to disinfectants. In Caenorhabditis elegans model, the kpnO mutant exhibited significantly (P<0.01) lower virulence. To dissect the role of PhoBR signaling system in regulating the expression of the kpnO, a phoBKP isogenic mutant was constructed. The phoBKP mutant exhibited impaired gastrointestinal stress response and decreased antimicrobial susceptibility. The mRNA levels of kpnO were found to be 4-fold less in phoBKP mutant compared to wild type. A regulatory role of PhoBKP for the expression of kpnO was further supported by the specific binding of PhoBKP to the putative promoter of kpnO. Conclusions and Significance Loss of PhoBR regulated porin KpnO resulted in increased

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

    PubMed

    Kozjak-Pavlovic, Vera; Dian-Lothrop, Elke A; Meinecke, Michael; Kepp, Oliver; Ross, Katharina; Rajalingam, Krishnaraj; Harsman, Anke; Hauf, Eva; Brinkmann, Volker; Günther, Dirk; Herrmann, Ines; Hurwitz, Robert; Rassow, Joachim; Wagner, Richard; Rudel, Thomas

    2009-10-01

    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. PMID:19851451

  2. Meropenem permeation through the outer membrane of Pseudomonas aeruginosa can involve pathways other than the OprD porin channel.

    PubMed

    Pérez, F J; Gimeno, C; Navarro, D; García-de-Lomas, J

    1996-01-01

    The outer membrane protein (OMP) OprD is the major channel through which carbapenems permeate the outer membrane of Pseudomonas aeruginosa. In this study, we analyzed the OMP profiles of several P. aeruginosa clinical isolates showing diminished susceptibility to imipenem while remaining susceptible to meropenem. All these isolates lacked OprD or showed a reduced expression of this porin. Susceptibility to meropenem was thus independent of the level of OprD expression, indicating that the antimicrobial could be taken up via an alternative route. The level of expression of OprC (70 kD) was also unrelated to meropenem susceptibility. Nevertheless, OMPs OprF and OprE were expressed by all isolates, suggesting that in the absence of OprD, these porins might be involved in the permeation of meropenem. PMID:8983889

  3. Selection for mutants altered in the expression or export of outer membrane porin OmpF.

    PubMed Central

    Sodergren, E J; Davidson, J; Taylor, R K; Silhavy, T J

    1985-01-01

    Strains in which the lacZ gene (which specifies beta-galactosidase) is fused to a gene encoding an envelope protein often exhibit a phenotype termed overproduction lethality. In such strains, high-level synthesis of the cognate hybrid protein interferes with the process of protein export, and this leads ultimately to cell death. A variation of this phenomenon has been discovered with lacZ fusions to the gene specifying the major outer membrane porin protein OmpF. In this case, we find that lambda transducing phage carrying an ompF-lacZ fusion will not grow on a host strain that constitutively overexpresses ompF. We have exploited this observation to develop a selection for ompF mutants. Using this protocol, we have isolated mutants altered in ompF expression and have identified mutations that block OmpF export. Our results suggest that it should be possible to adapt this selection for use with other genes specifying exported proteins. Images PMID:2987179

  4. Sialylation of Outer Membrane Porin Protein D: A Mechanistic Basis of Antibiotic Uptake in Pseudomonas aeruginosa*

    PubMed Central

    Khatua, Biswajit; Vleet, Jeremy Van; Choudhury, Biswa Pronab; Chaudhry, Rama; Mandal, Chitra

    2014-01-01

    Pseudomonas aeruginosa (PA) is an environmentally ubiquitous, extracellular, opportunistic pathogen, associated with severe infections of immune-compromised host. We demonstrated earlier the presence of both α2,3- and α2,6-linked sialic acids (Sias) on PA (PA+Sias) and normal human serum is their source of Sias. PA+Sias showed decreased complement deposition and exhibited enhanced association with immune-cells through sialic acid binding immunoglobulin like lectins (Siglecs). Such Sias-siglec-9 interaction between PA+Sias and neutrophils helped to subvert host immunity. Additionally, PA+Sias showed more resistant to β-lactam antibiotics as reflected in their minimum inhibitory concentration required to inhibit the growth of 50% than PA−Sias. Accordingly, we have affinity purified sialoglycoproteins of PA+Sias. They were electrophoresed and identified by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight mass spectrometry analysis. Sequence study indicated the presence of a few α2,6-linked, α2,3-linked, and both α2,3- and α2,6-linked sialylated proteins in PA. The outer membrane porin protein D (OprD), a specialized channel-forming protein, responsible for uptake of β-lactam antibiotics, is one such identified sialoglycoprotein. Accordingly, sialylated (OprD+Sias) and non-sialylated (OprD−Sias) porin proteins were separately purified by using anion exchange chromatography. Sialylation of purified OprD+Sias was confirmed by several analytical and biochemical procedures. Profiling of glycan structures revealed three sialylated N-glycans and two sialylated O-glycans in OprD+Sias. In contrast, OprD−Sias exhibit only one sialylated N-glycans. OprD−Sias interacts with β-lactam antibiotics more than OprD+Sias as demonstrated by surface plasmon resonance study. Lyposome-swelling assay further exhibited that antibiotics have more capability to penetrate through OprD−Sias purified from four clinical isolates of PA. Taken together, it

  5. The omp2 gene locus of Brucella abortus encodes two homologous outer membrane proteins with properties characteristic of bacterial porins.

    PubMed Central

    Marquis, H; Ficht, T A

    1993-01-01

    In Brucella abortus, a gene encoding a major cell envelope protein, omp2, is duplicated within a short segment of the large chromosomal DNA. Although both genes contain open reading frames, encoding proteins of high identity, expression from only one, omp2b, has been detected in laboratory-grown B. abortus. In the present study, we wished to determine whether omp2b encodes the previously studied Brucella porin and to characterize the omp2a gene product. Experiments were performed with Escherichia coli transformants expressing either omp2a or omp2b. Our results indicated that both gene products localized to the outer membrane of E. coli. Initial rates of transport of [14C]maltose and growth rates in the presence of maltodextrins of defined size indicated an increased hydrophilic permeability of transformants expressing omp2a. These cells were also shown to grow on maltotetraose, a molecule with a molecular mass of 667 Da. Activity consistent with the formation of pores could not be demonstrated in transformants expressing omp2b. However, Omp2b formed oligomers resistant to heat denaturation up to 70 degrees C in sodium dodecyl sulfate buffer, a property characteristic of bacterial porins. Overall, these results suggest that the omp2a gene product has pore-forming activity and that the omp2b gene encodes the previously characterized Brucella porin. Images PMID:7689540

  6. Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria.

    PubMed

    Jiang, X; Payne, M A; Cao, Z; Foster, S B; Feix, J B; Newton, S M; Klebba, P E

    1997-05-23

    Ligand-gated membrane channels selectively facilitate the entry of iron into prokaryotic cells. The essential role of iron in metabolism makes its acquisition a determinant of bacterial pathogenesis and a target for therapeutic strategies. In Gram-negative bacteria, TonB-dependent outer membrane proteins form energized, gated pores that bind iron chelates (siderophores) and internalize them. The time-resolved operation of the Escherichia coli ferric enterobactin receptor FepA was observed in vivo with electron spin resonance spectroscopy by monitoring the mobility of covalently bound nitroxide spin labels. A ligand-binding surface loop of FepA, which normally closes its transmembrane channel, exhibited energy-dependent structural changes during iron and toxin (colicin) transport. These changes were not merely associated with ligand binding, but occurred during ligand uptake through the outer membrane bilayer. The results demonstrate by a physical method that gated-porin channels open and close during membrane transport in vivo. PMID:9157886

  7. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA

    PubMed Central

    Liu, Yimo; Wang, Zheming; Liu, Juan; Levar, Caleb; Edwards, Marcus J; Babauta, Jerome T; Kennedy, David W; Shi, Zhi; Beyenal, Haluk; Bond, Daniel R; Clarke, Thomas A; Butt, Julea N; Richardson, David J; Rosso, Kevin M; Zachara, John M; Fredrickson, James K; Shi, Liang

    2014-01-01

    The multi-heme, outer membrane c-type cytochrome (c-Cyt) OmcB of Geobacter sulfurreducens was previously proposed to mediate electron transfer across the outer membrane. However, the underlying mechanism has remained uncharacterized. In G. sulfurreducens, the omcB gene is part of two tandem four-gene clusters, each is predicted to encode a transcriptional factor (OrfR/OrfS), a porin-like outer membrane protein (OmbB/OmbC), a periplasmic c-type cytochrome (OmaB/OmaC) and an outer membrane c-Cyt (OmcB/OmcC) respectively. Here, we showed that OmbB/OmbC, OmaB/OmaC and OmcB/OmcC of G. sulfurreducens PCA formed the porin-cytochrome (Pcc) protein complexes, which were involved in transferring electrons across the outer membrane. The isolated Pcc protein complexes reconstituted in proteoliposomes transferred electrons from reduced methyl viologen across the lipid bilayer of liposomes to Fe(III)-citrate and ferrihydrite. The pcc clusters were found in all eight sequenced Geobacter and 11 other bacterial genomes from six different phyla, demonstrating a widespread distribution of Pcc protein complexes in phylogenetically diverse bacteria. Deletion of ombB-omaB-omcB-orfS-ombC-omaC-omcC gene clusters had no impact on the growth of G. sulfurreducens PCA with fumarate but diminished the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite. Complementation with the ombB-omaB-omcB gene cluster restored the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite. PMID:25139405

  8. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA

    SciTech Connect

    Liu, Yimo; Wang, Zheming; Liu, Juan; Levar, Caleb; Edwards, Marcus; Babauta, Jerome T.; Kennedy, David W.; Shi, Zhi; Beyenal, Haluk; Bond, Daniel R.; Clarke, Thomas A.; Butt, Julea N.; Richardson, David J.; Rosso, Kevin M.; Zachara, John M.; Fredrickson, Jim K.; Shi, Liang

    2014-09-24

    The multiheme, outer membrane c-type cytochrome (c-Cyt) OmcB of Geobacter sulfurreducens was previously proposed to mediate electron transfer across the outer membrane. However, the underlying mechanism has remained uncharacterized. In G. sulfurreducens, the omcB gene is part of two tandem four-gene clusters, each is predicted to encode a transcriptional factor (OrfR/OrfS), a porin-like outer membrane protein (OmbB/OmbC), a periplasmic c-type cytochrome (OmaB/OmaC), and an outer membrane c-Cyt (OmcB/OmcC), respectively. Here we showed that OmbB/OmbC, OmaB/OmaC and OmcB/OmcC of G. sulfurreducens PCA formed the porin-cytochrome (Pcc) protein complexes, which were involved in transferring electrons across the outer membrane. The isolated Pcc protein complexes reconstituted in proteoliposomes transferred electrons from reduced methyl viologen across the lipid bilayer of liposomes to Fe(III)-citrate and ferrihydrite. The pcc clusters were found in all eight sequenced Geobacter and 11 other bacterial genomes from six different phyla, demonstrating a widespread distribution of Pcc protein complexes in phylogenetically diverse bacteria. Deletion of ombB-omaB-omcB-orfS-ombC-omaC-omcC gene clusters had no impact on the growth of G. sulfurreducens PCA with fumarate, but diminished the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite. Finally, complementation with the ombB-omaB-omcB gene cluster restored the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite.

  9. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA

    SciTech Connect

    Liu, Yimo; Wang, Zheming; Liu, Juan; Levar, Caleb; Edwards, Marcus; Babauta, Jerome T.; Kennedy, David W.; Shi, Zhi; Beyenal, Haluk; Bond, Daniel R.; Clarke, Thomas A.; Butt, Julea N.; Richardson, David J.; Rosso, Kevin M.; Zachara, John M.; Fredrickson, Jim K.; Shi, Liang

    2014-09-24

    The multiheme, outer membrane c-type cytochrome (c-Cyt) OmcB of Geobacter sulfurreducens was previously proposed to mediate electron transfer across the outer membrane. However, the underlying mechanism has remained uncharacterized. In G. sulfurreducens, the omcB gene is part of two tandem four-gene clusters, each is predicted to encode a transcriptional factor (OrfR/OrfS), a porin-like outer membrane protein (OmbB/OmbC), a periplasmic c-type cytochrome (OmaB/OmaC), and an outer membrane c-Cyt (OmcB/OmcC), respectively. Here we showed that OmbB/OmbC, OmaB/OmaC and OmcB/OmcC of G. sulfurreducens PCA formed the porin-cytochrome (Pcc) protein complexes, which were involved in transferring electrons across the outer membrane. The isolated Pcc protein complexes reconstituted in proteoliposomes transferred electrons from reduced methyl viologen across the lipid bilayer of liposomes to Fe(III)-citrate and ferrihydrite. The pcc clusters were found in all eight sequenced Geobacter and 11 other bacterial genomes from six different phyla, demonstrating a widespread distribution of Pcc protein complexes in phylogenetically diverse bacteria. Deletion of ombB-omaB-omcB-orfS-ombC-omaC-omcC gene clusters had no impact on the growth of G. sulfurreducens PCA with fumarate, but diminished the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite. Complementation with the ombB-omaB-omcB gene cluster restored the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite.

  10. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA

    DOE PAGESBeta

    Liu, Yimo; Wang, Zheming; Liu, Juan; Levar, Caleb; Edwards, Marcus; Babauta, Jerome T.; Kennedy, David W.; Shi, Zhi; Beyenal, Haluk; Bond, Daniel R.; et al

    2014-09-24

    The multiheme, outer membrane c-type cytochrome (c-Cyt) OmcB of Geobacter sulfurreducens was previously proposed to mediate electron transfer across the outer membrane. However, the underlying mechanism has remained uncharacterized. In G. sulfurreducens, the omcB gene is part of two tandem four-gene clusters, each is predicted to encode a transcriptional factor (OrfR/OrfS), a porin-like outer membrane protein (OmbB/OmbC), a periplasmic c-type cytochrome (OmaB/OmaC), and an outer membrane c-Cyt (OmcB/OmcC), respectively. Here we showed that OmbB/OmbC, OmaB/OmaC and OmcB/OmcC of G. sulfurreducens PCA formed the porin-cytochrome (Pcc) protein complexes, which were involved in transferring electrons across the outer membrane. The isolated Pccmore » protein complexes reconstituted in proteoliposomes transferred electrons from reduced methyl viologen across the lipid bilayer of liposomes to Fe(III)-citrate and ferrihydrite. The pcc clusters were found in all eight sequenced Geobacter and 11 other bacterial genomes from six different phyla, demonstrating a widespread distribution of Pcc protein complexes in phylogenetically diverse bacteria. Deletion of ombB-omaB-omcB-orfS-ombC-omaC-omcC gene clusters had no impact on the growth of G. sulfurreducens PCA with fumarate, but diminished the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite. Finally, complementation with the ombB-omaB-omcB gene cluster restored the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite.« less

  11. Bacterial porins stimulate bone resorption.

    PubMed Central

    Meghji, S; Henderson, B; Nair, S P; Tufano, M A

    1997-01-01

    Porins are abundant outer membrane proteins of gram-negative bacteria involved in transport of low-molecular-mass molecules. During the past decade, porins from a number of bacteria have also been shown to have proinflammatory activities including inducing the synthesis of proinflammatory mediators (cytokines, platelet-activating factor, and nitric oxide) in cultured cells and inducing inflammation in vivo. With this range of actions, it was possible that porins could also interact with bone cells to cause aberrant bone remodeling and that this could contribute to the bone destruction seen in gram-negative bone infections. By using purified preparations of Salmonella typhimurium and Pseudomonas aeruginosa porins, in the presence of polymyxin B, it was possible to induce concentration-dependent loss of calcium from cultured murine calvaria at porin concentrations in the range of 1 to 10 nM. The mechanism of action of the porins was determined by the inclusion of inhibitors of cyclooxygenase or inflammatory cytokines in the culture media. The bone-resorbing activity of both porins was not inhibited by the cyclooxygenase inhibitor indomethacin or by neutralizing the activity of tumor necrosis factor. Indeed, relatively high concentrations of these agents produced an unexpected increase in the bone resorption induced by the porins. In contrast, porin-induced bone resorption could be inhibited by relatively high concentrations of the natural inhibitor of interleukin-1 (IL-1 receptor antagonist). It appears that these porins stimulate bone resorption by a mechanism distinct from that of lipopolysaccharide, and the possibility therefore exists that porins play a role in bone destruction in gram-negative bacterial infections of bone. PMID:9119467

  12. Renaturation of Recombinant Treponema pallidum Rare Outer Membrane Protein 1 into a Trimeric, Hydrophobic, and Porin-Active Conformation

    PubMed Central

    Zhang, Hongwei H.; Blanco, David R.; Exner, Maurice M.; Shang, Ellen S.; Champion, Cheryl I.; Phillips, Martin L.; Miller, James N.; Lovett, Michael A.

    1999-01-01

    We have previously observed that while native Treponema pallidum rare outer membrane protein 1 (Tromp1) is hydrophobic and has porin activity, recombinant forms of Tromp1 do not possess these properties. In this study we show that these properties are determined by conformation and can be replicated by proper renaturation of recombinant Tromp1. Native Tromp1, but not the 47-kDa lipoprotein, extracted from whole organisms by using Triton X-114, was found to lose hydrophobicity after treatment in 8 M urea, indicating that Tromp1’s hydrophobicity is conformation dependent. Native Tromp1 was purified from 0.1% Triton X-100 extracts of whole organisms by fast-performance liquid chromatography (FPLC) and shown to have porin activity in planar lipid bilayers. Cross-linking studies of purified native Tromp1 with an 11 Å cross-linking agent showed oligomeric forms consistent with dimers and trimers. For renaturation studies of recombinant Tromp1 (rTromp1), a 31,109-Da signal-less construct was expressed in Escherichia coli and purified by FPLC. FPLC-purified rTromp1 was denatured in 8 M urea and then renatured in the presence of 0.5% Zwittergent 3,14 during dialysis to remove the urea. Renatured rTromp1 was passed through a Sephacryl S-300 gel exclusion column previously calibrated with known molecular weight standards. While all nonrenatured rTromp1 eluted from the column at approximately the position of the carbonic anhydrase protein standard (29 kDa), all renatured rTromp1 eluted at the position of the phosphorylase b protein standard (97 kDa), suggesting a trimeric conformation. Trimerization was confirmed by using an 11 Å cross-linking agent which showed both dimers and trimers similar to that of native Tromp1. Triton X-114 phase separations showed that all of renatured rTromp1, but none of nonrenatured rTromp1, phase separated exclusively into the hydrophobic detergent phase, similar to native Tromp1. Circular dichroism of nonrenatured and renatured rTromp1

  13. Outer membrane permeability in Pseudomonas cepacia: diminished porin content in a beta-lactam-resistant mutant and in resistant cystic fibrosis isolates.

    PubMed Central

    Aronoff, S C

    1988-01-01

    Since beta-lactam resistance is a feature of Pseudomonas cepacia isolates causing pulmonary infections in cystic fibrosis (CF), this study was undertaken to determine whether alterations in beta-lactam permeability mediate drug resistance in this species. A beta-lactam-susceptible non-CF isolate (strain 75-26), a resistant mutant derived from 75-26 by selection for cross-resistance to ciprofloxacin and ceftazidime, and two resistant CF isolates of P. cepacia were used. Permeability constants were calculated from the rate of nitrocefin hydrolysis in intact bacterial cells. Qualitative changes in outer membrane proteins were determined electrophoretically. The permeability constants of the mutant and the resistant CF isolates were lower than the value for the reference strain, 75-26. Whereas the lipopolysaccharide side chains were present in the test and reference strains, the resistant mutant and the CF isolates contained reduced amounts of the 36-kilodalton (kDa) outer membrane protein and failed to express the 27-kDa outer membrane protein. These observations suggest that the 27-kDa outer membrane protein may be a major porin or a major protein component of the porin complex in P. cepacia and that decreased expression of the 36-kDa outer membrane and loss of the 27-kDa porin are associated with high-level beta-lactam resistance in some CF isolates of P. cepacia. Images PMID:2855296

  14. Identification and Characterization of a Novel Porin Family Highlights a Major Difference in the Outer Membrane of Chlamydial Symbionts and Pathogens

    PubMed Central

    Aistleitner, Karin; Heinz, Christian; Hörmann, Alexandra; Heinz, Eva; Montanaro, Jacqueline; Schulz, Frederik; Maier, Elke; Pichler, Peter; Benz, Roland; Horn, Matthias

    2013-01-01

    The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P. amoebophila by in silico analysis. Two of these Protochlamydia outer membrane proteins, PomS (pc1489) and PomT (pc1077), are highly abundant in outer membrane preparations of this organism. Here we show that all four members of this putative porin family are toxic when expressed in the heterologous host Escherichia coli. Immunofluorescence analysis using antibodies against heterologously expressed PomT and PomS purified directly from elementary bodies, respectively, demonstrated the location of both proteins in the outer membrane of P. amoebophila. The location of the most abundant protein PomS was further confirmed by immuno-transmission electron microscopy. We could show that pomS is transcribed, and the corresponding protein is present in the outer membrane throughout the complete developmental cycle, suggesting an essential role for P. amoebophila. Lipid bilayer measurements demonstrated that PomS functions as a porin with anion-selectivity and a pore size similar to the Chlamydiaceae MOMP. Taken together, our results suggest that PomS, possibly in concert with PomT and other members of this porin family, is the functional equivalent of MOMP in P. amoebophila. This work contributes to our understanding of the adaptations of symbiotic and pathogenic chlamydiae to their different eukaryotic hosts. PMID:23383036

  15. Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I: OUTER MEMBRANE INSERTION, TRIMERIZATION, AND PORIN FUNCTION REQUIRE A C-TERMINAL β-BARREL DOMAIN.

    PubMed

    Anand, Arvind; LeDoyt, Morgan; Karanian, Carson; Luthra, Amit; Koszelak-Rosenblum, Mary; Malkowski, Michael G; Puthenveetil, Robbins; Vinogradova, Olga; Radolf, Justin D

    2015-05-01

    We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSP(N) and MOSP(C)) of Treponema denticola and that TprC(C) is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSP(C)-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSP(N)-like domains are tethered within the periplasm. TprF, which does not contain a MOSP(C)-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSP(N) and MOSP(C)-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSP(N)-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP. PMID:25805501

  16. Tailored Polymeric Membranes for Mycobacterium Smegmatis Porin A (MspA) Based Biosensors

    PubMed Central

    Morton, Danielle; Mortezaei, Shahab; Yemenicioglu, Sukru; Isaacman, Michael J.; Nova, Ian C.; Gundlach, Jens H.; Theogarajan, Luke

    2015-01-01

    Nanopores based on protein channels inserted into lipid membranes have paved the way towards a wide-range of inexpensive biosensors, especially for DNA sequencing. A key obstacle in using these biological ion channels as nanodevices is the poor stability of lipid bilayer membranes. Amphiphilic block copolymer membranes have emerged as a robust alternative to lipid membranes. While previous efforts have shown feasibility, we demonstrate for the first time the effect of polymer composition on MspA protein functionality. We show that membrane-protein interaction depends on the hydrophobic-hydrophilic ratio (f-ratio) of the block copolymer. These effects are particularly pronounced in asymmetric protein pores like MspA compared to the cylindrical α-Hemolysin pore. A key effect of membrane-protein interaction is the increased 1/fα noise. After first showing increases in 1/fα behaviour arise from increased substate activity, the noise power spectral density S(f) was used as a qualitative tool for understanding protein-membrane interactions in polymer membranes. Polymer compositions with f-ratios close to lipid membranes caused noise behaviour not observed in lipid membranes. However, by modifying the f-ratio using a modular synthetic approach, we were able to design a block copolymer exhibiting noise properties similar to a lipid membrane, albeit with better stability. Thus, by careful optimization, block copolymer membranes can emerge as a robust alternative for protein-pore based nano-biosensors. PMID:26413301

  17. Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

    PubMed Central

    Blanco, D R; Champion, C I; Exner, M M; Shang, E S; Skare, J T; Hancock, R E; Miller, J N; Lovett, M A

    1996-01-01

    We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure. PMID:8955283

  18. Nucleotide and derived amino acid sequences of the major porin of Comamonas acidovorans and comparison of porin primary structures.

    PubMed Central

    Gerbl-Rieger, S; Peters, J; Kellermann, J; Lottspeich, F; Baumeister, W

    1991-01-01

    The DNA sequence of the gene which codes for the major outer membrane porin (Omp32) of Comamonas acidovorans has been determined. The structural gene encodes a precursor consisting of 351 amino acid residues with a signal peptide of 19 amino acid residues. Comparisons with amino acid sequences of outer membrane proteins and porins from several other members of the class Proteobacteria and of the Chlamydia trachomatis porin and the Neurospora crassa mitochondrial porin revealed a motif of eight regions of local homology. The results of this analysis are discussed with regard to common structural features of porins. PMID:1848840

  19. A Proteomic Approach to Understand the Role of the Outer Membrane Porins in the Organic Solvent-Tolerance of Pseudomonas aeruginosa PseA

    PubMed Central

    Hemamalini, R.; Khare, Sunil

    2014-01-01

    Solvent-tolerant microbes have the unique ability to thrive in presence of organic solvents. The present study describes the effect of increasing hydrophobicity (log Pow values) of organic solvents on the outer membrane proteome of the solvent-tolerant Pseudomonas aeruginosa PseA cells. The cells were grown in a medium containing 33% (v/v) alkanes of increasing log Pow values. The outer membrane proteins were extracted by alkaline extraction from the late log phase cells and changes in the protein expression were studied by 2-D gel electrophoresis. Seven protein spots showed significant differential expression in the solvent exposed cells. The tryptic digest of the differentially regulated proteins were identified by LC-ESI MS/MS. The identity of these proteins matched with porins OprD, OprE, OprF, OprH, Opr86, LPS assembly protein and A-type flagellin. The reported pI values of these proteins were in the range of 4.94–8.67 and the molecular weights were in the range of 19.5–104.5 kDa. The results suggest significant down-regulation of the A-type flagellin, OprF and OprD and up-regulation of OprE, OprH, Opr86 and LPS assembly protein in presence of organic solvents. OprF and OprD are implicated in antibiotic uptake and outer membrane stability, whereas A-type flagellin confers motility and chemotaxis. Up-regulated OprE is an anaerobically-induced porin while Opr86 is responsible for transport of small molecules and assembly of the outer membrane proteins. Differential regulation of the above porins clearly indicates their role in adaptation to solvent exposure. PMID:25089526

  20. A proteomic approach to understand the role of the outer membrane porins in the organic solvent-tolerance of Pseudomonas aeruginosa PseA.

    PubMed

    Hemamalini, R; Khare, Sunil

    2014-01-01

    Solvent-tolerant microbes have the unique ability to thrive in presence of organic solvents. The present study describes the effect of increasing hydrophobicity (log Pow values) of organic solvents on the outer membrane proteome of the solvent-tolerant Pseudomonas aeruginosa PseA cells. The cells were grown in a medium containing 33% (v/v) alkanes of increasing log Pow values. The outer membrane proteins were extracted by alkaline extraction from the late log phase cells and changes in the protein expression were studied by 2-D gel electrophoresis. Seven protein spots showed significant differential expression in the solvent exposed cells. The tryptic digest of the differentially regulated proteins were identified by LC-ESI MS/MS. The identity of these proteins matched with porins OprD, OprE, OprF, OprH, Opr86, LPS assembly protein and A-type flagellin. The reported pI values of these proteins were in the range of 4.94-8.67 and the molecular weights were in the range of 19.5-104.5 kDa. The results suggest significant down-regulation of the A-type flagellin, OprF and OprD and up-regulation of OprE, OprH, Opr86 and LPS assembly protein in presence of organic solvents. OprF and OprD are implicated in antibiotic uptake and outer membrane stability, whereas A-type flagellin confers motility and chemotaxis. Up-regulated OprE is an anaerobically-induced porin while Opr86 is responsible for transport of small molecules and assembly of the outer membrane proteins. Differential regulation of the above porins clearly indicates their role in adaptation to solvent exposure. PMID:25089526

  1. Role of porins for uptake of antibiotics by Mycobacterium smegmatis.

    PubMed

    Danilchanka, Olga; Pavlenok, Mikhail; Niederweis, Michael

    2008-09-01

    The outer membrane of mycobacteria presents an effective permeability barrier for many antibiotics. Transport pathways across this membrane are unknown for most drugs. Here, we examined which antibiotics utilize the porin pathway across the outer membrane of the model organism Mycobacterium smegmatis. Deletion of the porins MspA and MspC drastically increased the resistance of M. smegmatis ML10 to beta-lactam antibiotics, while its beta-lactamase activity remained unchanged. These results are consistent with the ninefold-reduced outer membrane permeability of the M. smegmatis porin mutants for cephaloridine and strongly indicate that beta-lactam antibiotics rely on the porin pathway. The porin mutant ML10 accumulated less chloramphenicol and norfloxacin and was less susceptible to these antibiotics than wild-type M. smegmatis. These results demonstrated that small and hydrophilic antibiotics use the Msp porins for entering the cell. In contrast to norfloxacin, the hydrophobic moxifloxacin was 32-fold more effective in inhibiting the growth of M. smegmatis, presumably because it was able to diffuse through the lipid membrane. Structural models indicated that erythromycin, kanamycin, and vancomycin are too large to move through the MspA channel. This study presents the first experimental evidence that hydrophilic fluoroquinolones and chloramphenicol diffuse through porins in mycobacteria. Thus, mutations resulting in less efficient porins or lower porin expression levels are likely to represent a mechanism for the opportunistic pathogens M. avium, M. chelonae, and M. fortuitum, which have Msp-like porins, to acquire resistance to fluoroquinolones. PMID:18559650

  2. Reevaluation, using intact cells, of the exclusion limit and role of porin OprF in Pseudomonas aeruginosa outer membrane permeability.

    PubMed Central

    Bellido, F; Martin, N L; Siehnel, R J; Hancock, R E

    1992-01-01

    Earlier studies that used model membrane reconstitution methods have come to different conclusions regarding the exclusion limit of the outer membrane of Pseudomonas aeruginosa and whether OprF is the major channel-forming protein in the outer membrane. In this study, a 6.2-kbp SalI fragment, encoding only two cytoplasmic enzymes, alpha-galactosidase and sucrose hydrolase, and the inner membrane raffinose permease, was cloned behind the m-toluate-inducible tol promoter of vector pNM185 to create plasmid pFB71. P. aeruginosa strains harboring pFB71, when grown with inducer, produced both enzymes encoded by the insert and had acquired the ability to grow on the disaccharide melibiose and the trisaccharide raffinose. The rate of growth was dependent on the concentration and size of the saccharide and was decreased three- to fivefold by the absence of OprF, as examined by measuring the growth on melibiose and raffinose of an isogenic OprF-deficient omega insertion derivative, H636(pFB71). At high concentrations, di-, tri-, and tetrasaccharides could pass across the outer membrane to plasmolyze P. aeruginosa, as measured by light scattering and confirmed by electron microscopy. The initial rate kinetics of light-scattering changes were dependent on the size of the saccharide being used. Furthermore, the rates of change in light scattering due to raffinose and stachyose uptake across the outer membrane for strain H636 were fivefold or more lower than for its OprF-sufficient parent H103. These data are consistent with model membrane studies showing that OprF is the most predominant porin for compounds larger than disaccharides in P. aeruginosa and suggest that the exclusion limit for this porin and the outer membrane is greater than the size of a tetrasaccharide. In addition, these data confirmed the existence of other porins with a predominant function in monosaccharide uptake and a more minor function in the uptake of larger saccharides. Images PMID:1322882

  3. Hexokinase inhibits flux of fluorescently labeled ATP through mitochondrial outer membrane porin.

    PubMed

    Perevoshchikova, Irina V; Zorov, Savva D; Kotova, Elena A; Zorov, Dmitry B; Antonenko, Yuri N

    2010-06-01

    Mitochondrial function requires maintaining metabolite fluxes across the mitochondrial outer membrane, which is mediated primarily by the voltage dependent anion channel (VDAC). We applied fluorescence correlation spectroscopy (FCS) to study regulation of the VDAC functional state by monitoring distribution of fluorescently labeled ATP (BODIPY-FL-ATP) in isolated intact rat liver and heart mitochondria. Addition of mitochondria to BODIPY-FL-ATP solution resulted in accumulation of the fluorescent probe in these organelles. The addition of hexokinase II (HKII) isolated from rat heart led to a decrease in the BODIPY-FL-ATP accumulation, while a 15-residue peptide corresponding to the N-terminal domain of hexokinase did not produce this effect. Therefore, the hexokinase-induced inhibition of the ATP flow mediated by VDAC was revealed in isolated mitochondria. PMID:20412805

  4. Identification of outer membrane Porin D as a vitronectin-binding factor in cystic fibrosis clinical isolates of Pseudomonas aeruginosa

    PubMed Central

    Paulsson, Magnus; Singh, Birendra; Al-Jubair, Tamim; Su, Yu-Ching; Høiby, Niels; Riesbeck, Kristian

    2016-01-01

    Background Pseudomonas aeruginosa is a pathogen that frequently colonizes patients with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Several pathogens are known to bind vitronectin to increase their virulence. Vitronectin has been shown to enhance P. aeruginosa adhesion to host epithelial cells. Methods We screened clinical isolates from the airways of CF patients and from the bloodstream of patients with bacteremia for binding of vitronectin. Two-dimensional SDS-PAGE and a proteomic approach was used to identify vitronectin-receptors in P. aeruginosa. Results P. aeruginosa from the airways of CF patients (n=27) bound more vitronectin than bacteremic isolates (n=15, p=0.025). Porin D (OprD) was identified as a vitronectin-binding protein. A P. aeruginosa oprD transposon insertion mutant had a decreased binding to soluble and immobilized vitronectin (p ≤ 0.001). Conclusions P. aeruginosa isolates obtained from CF patients significantly bound vitronectin. Porin D was defined as a novel P. aeruginosa vitronectin-receptor, and we postulate that the Porin D-dependent interaction with vitronectin may be important for colonization. PMID:26047937

  5. Genetic definition of the substrate selectivity of outer membrane porin protein OprD of Pseudomonas aeruginosa.

    PubMed Central

    Huang, H; Hancock, R E

    1993-01-01

    Earlier studies proved that Pseudomonas aeruginosa OprD is a specific porin for basic amino acids and imipenem. It was also considered to function as a nonspecific porin that allowed the size-dependent uptake of monosaccharides and facilitation of the uptake of quinolone and other antibiotics. In the present study, we utilized P. aeruginosa strains with genetically defined levels of OprD to characterize the in vivo substrate selectivity of this porin. An oprD::omega interposon mutant was constructed by gene replacement utilizing an in vitro mutagenized cloned oprD gene. In addition, OprD was overexpressed from the lac promoter by cloning the oprD gene into the broad-host-range plasmid pUCP19. To test the substrate selectivity, strains were grown in minimal medium with limiting concentrations of the carbon sources glucose, gluconate, or pyruvate. In minimal medium with 0.5 mM gluconate, the growth rates of the parent strain H103 and its oprD::omega mutant H729 were only 60 and 20%, respectively, of that of the OprD-overexpressing strain H103(pXH2). In contrast, no significant differences were observed in the growth rates of these three strains on glucose or pyruvate, indicating that OprD selectively facilitated the transport of gluconate. To determine the role of OprD in antibiotic uptake, nine strains representing different levels of OprD and OprF were used to determine the MICs of different antibiotics. The results clearly demonstrated that OprD could be utilized by imipenem and meropenem but that, even when substantially overexpressed, it could not be significantly utilized by other beta-lactams, quinolones, or aminoglycosides. In addition, competition experiments confirmed that imipenem had common binding sites with basic amino acids in the OprD channel, but not with gluconate or glucose. Images PMID:8253668

  6. C-Terminal Region of Pseudomonas aeruginosa Outer Membrane Porin OprD Modulates Susceptibility to Meropenem

    PubMed Central

    Epp, Simone F.; Köhler, Thilo; Plésiat, Patrick; Michéa-Hamzehpour, Mehri; Frey, Joachim; Pechère, Jean-Claude

    2001-01-01

    We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of Pseudomonas aeruginosa. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 μg/ml) but susceptible to meropenem (<1 μg/ml). The oprD genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The oprD gene variants and the wild-type oprD gene were cloned and expressed in a defined oprD mutant. The meropenem MICs for strains carrying the oprD genes from clinical isolates were four times lower than that for the strain carrying the wild-type oprD gene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1 oprD gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acid l-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem. PMID:11353625

  7. Porin polypeptide contributes to surface charge of gonococci.

    PubMed Central

    Swanson, J; Dorward, D; Lubke, L; Kao, D

    1997-01-01

    Each strain of Neisseria gonorrhoeae elaborates a single porin polypeptide, with the porins expressed by different strains comprising two general classes, Por1A and Por1B. In the outer membrane, each porin molecule folds into 16 membrane-spanning beta-strands joined by top- and bottom-loop domains. Por1A and Por1B have similar membrane-spanning regions, but the eight surface-exposed top loops (I to VIII) differ in length and sequence. To determine whether porins, and especially their top loop domains, contribute to bacterial cell surface charge, strain MS11 gonococci that were identical except for expressing a recombinant Por1A, Por1B, or mosaic Por1A-1B polypeptide were compared by whole-cell electrophoresis. These porin variants displayed different electrophoretic mobilities that correlated with the net numbers of charged amino acids within surface-exposed loops of their respective porin polypeptides. The susceptibilities of porin variants to polyanionic sulfated polymers correlated roughly with gonococcal surface charge; those porin variants with diminished surface negativity showed increased sensitivity to the polyanionic sulfated compounds. These observations indicate that porin polypeptides in situ contribute to the surface charge of gonococci, and they suggest that the bacterium's interactions with large sulfated compounds are thereby affected. PMID:9171398

  8. Planar asymmetric lipid bilayers of glycosphingolipid or lipopolysaccharide on one side and phospholipids on the other: membrane potential, porin function, and complement activation.

    PubMed Central

    Wiese, A; Reiners, J O; Brandenburg, K; Kawahara, K; Zähringer, U; Seydel, U

    1996-01-01

    We have determined some physicochemical properties of the monosaccharide-type fraction (GSL-1) of glycosphingolipids, the major glycolipid components of the outer leaflet of the Gram-negative species Sphingomonas paucimobilis. These properties included the state of order of the hydrocarbon moiety, the effective molecular area, surface charge density, and intrinsic transmembrane potential profile of reconstituted planar asymmetric GSL-1/phospholipid bilayer membranes. We have, furthermore, investigated the insertion into and the function of porin channels in the reconstituted bilayers and the complement-activating capability of GSL-1 surfaces. All results were compared with respective data for deep rough mutant lipopolysaccharide of Salmonella minnesota R595. We found a remarkable agreement in most functional properties of the two glycolipids. PMID:8770208

  9. Characterization and distribution of drug resistance associated β-lactamase, membrane porin and efflux pump genes in MDR A. baumannii isolated from Zhenjiang, China

    PubMed Central

    Yang, Huijian; Huang, Lan; Barnie, Prince Amoah; Su, Zhaoliang; Mi, Zuhuang; Chen, Jianguo; Aparna, Vasudevan; Kumar, Dinesh; Xu, Huaxi

    2015-01-01

    Background: Acinetobacter baumannii (A. baumannii), especially the multidrug resistant A. baumannii (MDR-AB) is becoming a common opportunistic pathogen in hospital, and constitutes significant public health threats. This study aimed at investigating the relationship between drug resistance with expression of class A-D β-lactamase genes, mutation in membrane porin and over-expression of efflux pump genes among A. baumannii isolated from Zhengjiang, China. Methods: Antibiotic susceptibility assays were performed using Kirby-Bauer disc diffusion method. PCR was used to detect β-lactamase genes and carO, oprD, adeR, adeS. Real-time PCR was used to assess the mRNA expression level of efflux pump gene adeB. The software of DNAMAN was applied to assemble oprD and carO sequences, and the sequences were compared with those retrieved from GenBank (http://www.ncbi.nlm.nih.gov/). Results: 27 isolates (61.4%) in this study were MDR-AB, in which five β-lactamases including TEM, CTX-M-2, ADC, OXA-23 and OXA-51 were found, and the positive rate was 96.3% (26), 14.8% (4), 92.6% (25), 88.9% (24) and 92.6% (25), respectively. In addition, the expression level of adeB mRNA was significantly increased in MDR-AB, it might due to adeR mutation. Some mutations were also found in carO and oprD. Conclusion: MDR-AB showed high relationship with β-lactamase, mutation in membrane porin and overexpression of adeB, which may directly relates to the mutation in regulating gene adeR. PMID:26629028

  10. Porins increase copper susceptibility of Mycobacterium tuberculosis.

    PubMed

    Speer, Alexander; Rowland, Jennifer L; Haeili, Mehri; Niederweis, Michael; Wolschendorf, Frank

    2013-11-01

    Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 μM copper by 40% and completely suppressed growth at 15 μM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis. PMID:24013632

  11. Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis.

    PubMed

    Arunmanee, Wanatchaporn; Pathania, Monisha; Solovyova, Alexandra S; Le Brun, Anton P; Ridley, Helen; Baslé, Arnaud; van den Berg, Bert; Lakey, Jeremy H

    2016-08-23

    The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin-LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin-LPS interactions and a bridging calcium ion. PMID:27493217

  12. Bcl-2 and porin follow different pathways of TOM-dependent insertion into the mitochondrial outer membrane.

    PubMed

    Motz, Christian; Martin, Heiko; Krimmer, Thomas; Rassow, Joachim

    2002-11-01

    The bcl-2 gene encodes a 26kDa protein which functions as a central regulator of apoptosis. Here we investigated the pathway of Bcl-2alpha into the mitochondrial outer membrane using the yeast Saccharomyces cerevisiae as a model organism. We found that interactions of Bcl-2alpha with the mitochondrial import receptor Tom20 are dependent on two positively charged lysine residues in the immediate vicinity of the carboxy-terminal hydrophobic membrane anchor. The targeting function of these residues is independent of Tom22. Subsequent insertion of Bcl-2alpha into the mitochondrial outer membrane does not require Tom5 or Tom40, indicating that Bcl-2alpha bypasses the general import pore (GIP). Bcl-2alpha shows a unique pattern of interactions with the components of the mitochondrial TOM complex, demonstrating that at least two different pathways lead from the import receptor Tom20 into the mitochondrial outer membrane. PMID:12419260

  13. Neisseria meningitidis Lacking the Major Porins PorA and PorB Is Viable and Modulates Apoptosis and the Oxidative Burst of Neutrophils.

    PubMed

    Peak, Ian R; Chen, Adrienne; Jen, Freda E-C; Jennings, Courtney; Schulz, Benjamin L; Saunders, Nigel J; Khan, Arshad; Seifert, H Steven; Jennings, Michael P

    2016-08-01

    The bacterial pathogen Neisseria meningitidis expresses two major outer-membrane porins. PorA expression is subject to phase-variation (high frequency, random, on-off switching), and both PorA and PorB are antigenically variable between strains. PorA expression is variable and not correlated with meningococcal colonisation or invasive disease, whereas all naturally-occurring strains express PorB suggesting strong selection for expression. We have generated N. meningitidis strains lacking expression of both major porins, demonstrating that they are dispensable for bacterial growth in vitro. The porAB mutant strain has an exponential growth rate similar to the parental strain, as do the single porA or porB mutants, but the porAB mutant strain does not reach the same cell density in stationary phase. Proteomic analysis suggests that the double mutant strain exhibits compensatory expression changes in proteins associated with cellular redox state, energy/nutrient metabolism, and membrane stability. On solid media, there is obvious growth impairment that is rescued by addition of blood or serum from mammalian species, particularly heme. These porin mutants are not impaired in their capacity to inhibit both staurosporine-induced apoptosis and a phorbol 12-myristate 13-acetate-induced oxidative burst in human neutrophils suggesting that the porins are not the only bacterial factors that can modulate these processes in host cells. PMID:26562068

  14. Function and modulation of bacterial porins: insights from electrophysiology.

    PubMed

    Delcour, A H

    1997-06-15

    Electrophysiological techniques provide a wealth of information regarding the molecular mechanisms that underlie the function and modulation of ion channels. They have revealed that bacterial porins do not behave as static, permanently open pores but display a much more complex and dynamic behavior than anticipated from non-electrophysiological studies. The channels switch between short-lived open and closed conformations (gating activity), and can also remain in an inactivated, non-ion conducting state for prolonged periods of time. Thus the role of porins is not limited to that of a molecular filter, but is extended to the control of outer membrane permeability through the regulation of their activity. Electrophysiological studies have indeed demonstrated that both gating and inactivation are modulated by a variety of physical and chemical parameters and are highly cooperative phenomena, often involving numerous channels working in concert. Cooperativity acts as an amplification mechanism that grants a large population of porins, such as found in the outer membrane, with sensitivity to modulation by external or internal factors. By conferring permeability properties to the outer membrane, porins play a crucial role in the bacterium's antibiotic susceptibility and survival in various environmental conditions. The detailed information that electrophysiology only can provide on porin function and modulation promises to yield a more accurate description of how porin properties can be used by cells to adapt to a changing environment, and to offer mechanisms that might optimize the drug sensitivity of the microorganism. PMID:9228742

  15. Conformational analysis of the Campylobacter jejuni porin.

    PubMed Central

    Bolla, J M; Loret, E; Zalewski, M; Pagés, J M

    1995-01-01

    The major outer membrane protein (MOMP) of Campylobacter jejuni was purified to homogeneity by selective solubilization and fast protein liquid chromatography. The amino acid composition of the MOMP indicates the presence of cysteine residues. The amino-terminal sequence, determined over 31 residues, shows no significant homology with any other porin from gram-negative bacteria except in a discrete region. Immunocross-reactivity between Escherichia coli OmpC and the MOMP was analyzed, and a common antigenic site between these two porins was identified with an anti-peptide antibody. From circular dichroism and immunological investigations, the existence of a stable folded monomer, containing a high level of beta-sheet secondary structure, is evident. Conformational analyses show the presence of a native trimeric state generated by association of the three folded monomers; the stability of this trimer is reduced compared with that of E. coli porins. This study clearly reveals that the C. jejuni MOMP is related to the family of trimeric bacterial porins. PMID:7543469

  16. Structure of the monomeric outer-membrane porin OmpG in the open and closed conformation

    PubMed Central

    Yildiz, Özkan; Vinothkumar, Kutti R; Goswami, Panchali; Kühlbrandt, Werner

    2006-01-01

    OmpG, a monomeric pore-forming protein from Escherichia coli outer membranes, was refolded from inclusion bodies and crystallized in two different conformations. The OmpG channel is a 14-stranded β-barrel, with short periplasmic turns and seven extracellular loops. Crystals grown at neutral pH show the channel in the open state at 2.3 Å resolution. In the 2.7 Å structure of crystals grown at pH 5.6, the pore is blocked by loop 6, which folds across the channel. The rearrangement of loop 6 appears to be triggered by a pair of histidine residues, which repel one another at acidic pH, resulting in the breakage of neighbouring H-bonds and a lengthening of loop 6 from 10 to 17 residues. A total of 151 ordered LDAO detergent molecules were found in the 2.3 Å structure, mostly on the hydrophobic outer surface of OmpG, mimicking the outer membrane lipid bilayer, with three LDAO molecules in the open pore. In the 2.7 Å structure, OmpG binds one OG and one glucose molecule as sugar substrates in the closed pore. PMID:16888630

  17. The outer membrane porin OmpW of Acinetobacter baumannii is involved in iron uptake and colistin binding.

    PubMed

    Catel-Ferreira, Manuella; Marti, Sara; Guillon, Laurent; Jara, Luis; Coadou, Gaël; Molle, Virginie; Bouffartigues, Emeline; Bou, German; Shalk, Isabelle; Jouenne, Thierry; Vila-Farrés, Xavier; Dé, Emmanuelle

    2016-01-01

    This study was undertaken to characterize functions of the outer membrane protein OmpW, which potentially contributes to the development of colistin- and imipenem-resistance in Acinetobacter baumannii. Reconstitution of OmpW in artificial lipid bilayers showed that it forms small channels (23 pS in 1 m KCl) and markedly interacts with iron and colistin, but not with imipenem. In vivo, (55) Fe uptake assays comparing the behaviours of ΔompW mutant and wild-type strains confirmed a role for OmpW in A. baumannii iron homeostasis. However, the loss of OmpW expression did not have an impact on A. baumannii susceptibilities to colistin or imipenem. PMID:26823169

  18. Isolation, functional characterization and crystallization of Aq_1259, an outer membrane protein with porin features, from Aquifex aeolicus.

    PubMed

    Wang, Tao; Langer, Julian D; Peng, Guohong; Michel, Hartmut

    2012-12-01

    The "hypothetical protein" Aq_1259 was identified by mass spectrometry and purified from native membranes of Aquifex aeolicus. It is a 49.4kDa protein, highly homologous (>52% identity) to several conserved hypothetical proteins from other bacteria. However, none of these proteins has been characterized using biochemical or electrophysiological techniques. Based on the sequence and circular dichroism spectroscopy, the structure of Aq_1259 is predicted to be a β-barrel with 16 β-strands. The strands with loops and turns are distributed evenly through the entire sequence. The function of Aq_1259 was analyzed after incorporation into a lipid bilayer. Electrophysiological measurements revealed a pore that has a basic stationary conductance of 0.48 ± 0.038nS in a buffer with 0.5M NaH₂PO₄ at pH 6.5 and 0.2 ± 0.015nS in a buffer with 0.5M NaCl at pH 6.5. Superimposed on this is a fluctuating conductance of similar amplitude. Aq_1259 could be crystallized. The crystals diffract to a resolution of 3.4Å and belong to space group I222 with cell dimensions of a=138.3Å, b=144.6Å, c=151.8Å. PMID:22842195

  19. How Porin Heterogeneity and Trade-Offs Affect the Antibiotic Susceptibility of Gram-Negative Bacteria

    PubMed Central

    Ferenci, Thomas; Phan, Katherine

    2015-01-01

    Variations in porin proteins are common in Gram-negative pathogens. Altered or absent porins reduce access of polar antibiotics across the outer membrane and can thus contribute to antibiotic resistance. Reduced permeability has a cost however, in lowering access to nutrients. This trade-off between permeability and nutritional competence is the source of considerable natural variation in porin gate-keeping. Mutational changes in this trade-off are frequently selected, so susceptibility to detergents and antibiotics is polymorphic in environmental isolates as well as pathogens. Understanding the mechanism, costs and heterogeneity of antibiotic exclusion by porins will be crucial in combating Gram negative infections. PMID:26506392

  20. Antigenic relationship and functional properties of Yersinia porins.

    PubMed

    Vostrikova, P; Likhatskaya, G N; Novikova, D; Solovyeva, T F

    2001-01-01

    We have studied the molecular structure and functional properties of major pore-forming proteins isolated as peptidoglycan (PG)-protein complexes from four Yersinia species (Y. intermedia, Y. enterocolitica, Y. kristensenii and Y. frederiksenii) cultured as various temperatures. Despite the close antigenic relationship, Yersinia porins revealed different functional properties. When reconstituted in model membranes, the PG-protein complexes induced conductance which was different for the "cold" (grown at 6-8 degrees C) and "warm" (grown at 37 degrees C) variants of microbial cultures. We conclude that the functional state of Yersinia porins in the outer membrane depends on the cultivation temperature. PMID:11497105

  1. Porins facilitate nitric oxide-mediated killing of mycobacteria.

    PubMed

    Fabrino, Daniela Leite; Bleck, Christopher K E; Anes, Elsa; Hasilik, Andrej; Melo, Rossana C N; Niederweis, Michael; Griffiths, Gareth; Gutierrez, Maximiliano Gabriel

    2009-09-01

    Non-pathogenic mycobacteria such us Mycobacterium smegmatis reside in macrophages within phagosomes that fuse with late endocytic/lysosomal compartments. This sequential fusion process is required for the killing of non-pathogenic mycobacteria by macrophages. Porins are proteins that allow the influx of hydrophilic molecules across the mycobacterial outer membrane. Deletion of the porins MspA, MspC and MspD significantly increased survival of M. smegmatis in J774 macrophages. However, the mechanism underlying this observation is unknown. Internalization of wild-type M. smegmatis (SMR5) and the porin triple mutant (ML16) by macrophages was identical indicating that the viability of the porin mutant in vivo was enhanced. This was not due to effects on phagosome trafficking since fusion of phagosomes containing the mutant with late endocytic compartments was unaffected. Moreover, in ML16-infected macrophages, the generation of nitric oxide (NO) was similar to the wild type-infected cells. However, ML16 was significantly more resistant to the effects of NO in vitro compared to SMR5. Our data provide evidence that porins render mycobacteria vulnerable to killing by reactive nitrogen intermediates within phagosomes probably by facilitating uptake of NO across the mycobacterial outer membrane. PMID:19460455

  2. Importance of porins for biocide efficacy against Mycobacterium smegmatis.

    PubMed

    Frenzel, Elrike; Schmidt, Stefan; Niederweis, Michael; Steinhauer, Katrin

    2011-05-01

    Mycobacteria are among the microorganisms least susceptible to biocides but cause devastating diseases, such as tuberculosis, and increasingly opportunistic infections. The exceptional resistance of mycobacteria to toxic solutes is due to an unusual outer membrane, which acts as an efficient permeability barrier, in synergy with other resistance mechanisms. Porins are channel-forming proteins in the outer membrane of mycobacteria. In this study we used the alamarBlue assay to show that the deletion of Msp porins in isogenic mutants increased the resistance of Mycobacterium smegmatis to isothiazolinones (methylchloroisothiazolinone [MCI]/methylisothiazolinone [MI] and octylisothiazolinone [2-n-octyl-4-isothiazolin-3-one; OIT]), formaldehyde-releasing biocides {hexahydrotriazine [1,3,5-tris (2-hydroxyethyl)-hexahydrotriazine; HHT] and methylenbisoxazolidine [N,N'-methylene-bis-5-(methyloxazolidine); MBO]}, and the lipophilic biocides polyhexamethylene biguanide and octenidine dihydrochloride 2- to 16-fold. Furthermore, the susceptibility of the porin triple mutant against a complex disinfectant was decreased 8-fold compared to wild-type (wt) M. smegmatis. Efficacy testing in the quantitative suspension test EN 14348 revealed 100-fold improved survival of the porin mutant in the presence of this biocide. These findings underline the importance of porins for the susceptibility of M. smegmatis to biocides. PMID:21398489

  3. Importance of Porins for Biocide Efficacy against Mycobacterium smegmatis▿

    PubMed Central

    Frenzel, Elrike; Schmidt, Stefan; Niederweis, Michael; Steinhauer, Katrin

    2011-01-01

    Mycobacteria are among the microorganisms least susceptible to biocides but cause devastating diseases, such as tuberculosis, and increasingly opportunistic infections. The exceptional resistance of mycobacteria to toxic solutes is due to an unusual outer membrane, which acts as an efficient permeability barrier, in synergy with other resistance mechanisms. Porins are channel-forming proteins in the outer membrane of mycobacteria. In this study we used the alamarBlue assay to show that the deletion of Msp porins in isogenic mutants increased the resistance of Mycobacterium smegmatis to isothiazolinones (methylchloroisothiazolinone [MCI]/methylisothiazolinone [MI] and octylisothiazolinone [2-n-octyl-4-isothiazolin-3-one; OIT]), formaldehyde-releasing biocides {hexahydrotriazine [1,3,5-tris (2-hydroxyethyl)-hexahydrotriazine; HHT] and methylenbisoxazolidine [N,N′-methylene-bis-5-(methyloxazolidine); MBO]}, and the lipophilic biocides polyhexamethylene biguanide and octenidine dihydrochloride 2- to 16-fold. Furthermore, the susceptibility of the porin triple mutant against a complex disinfectant was decreased 8-fold compared to wild-type (wt) M. smegmatis. Efficacy testing in the quantitative suspension test EN 14348 revealed 100-fold improved survival of the porin mutant in the presence of this biocide. These findings underline the importance of porins for the susceptibility of M. smegmatis to biocides. PMID:21398489

  4. Cloning and in situ localization of a brain-derived porin that constitutes a large-conductance anion channel in astrocytic plasma membranes.

    PubMed Central

    Dermietzel, R; Hwang, T K; Buettner, R; Hofer, A; Dotzler, E; Kremer, M; Deutzmann, R; Thinnes, F P; Fishman, G I; Spray, D C

    1994-01-01

    We have cloned a protein from bovine brain, brain-derived voltage-dependent anion channel 1 (BR1-VDAC), that is identical to a recently sequenced plasmalemmal-bound porin from human lymphocytes. mRNA hybridization indicates that BR1-VDAC is widely distributed throughout nervous and nonnervous tissues. In situ localization substantiated that the BR1-VDAC is associated with the plasmalemma of astrocytes. A monoclonal antibody that recognizes the N terminus of the BR1-VDAC protein completely blocks an astrocytic high-conductance anion channel that has electrophysiological similarities with the mitochondrial VDAC. Since the high-conductance anion channel in astrocytes has been shown to respond to hypoosmotic solutions, its molecular identification provides the basis for a better understanding of volume regulation in brain tissue. Images Fig. 1 Fig. 3 Fig. 4 PMID:7507248

  5. Immunobiological activities of a porin fraction isolated from Fusobacterium nucleatum ATCC 10953.

    PubMed Central

    Takada, H; Ogawa, T; Yoshimura, F; Otsuka, K; Kokeguchi, S; Kato, K; Umemoto, T; Kotani, S

    1988-01-01

    From Fusobacterium nucleatum ATCC 10953 cell envelope fraction whose inner membranes had been removed by treatment with sodium N-lauroyl sarcosinate, an outer membrane protein (37,000 Mr in a native state) was prepared by extraction with lithium dodecyl sulfate. The protein thus obtained showed distinct porin activity, namely, the ability to form hydrophilic diffusion pores by incorporation into the artificial liposome membrane. The porin fraction exhibited strong immunobiological activities in the in vitro assays: B-cell mitogenicity and polyclonal B-cell activation on murine splenocytes, stimulatory effects on guinea pig peritoneal macrophages, and enhancement of the migration of human blood monocytes. The porin fraction also exhibited immunoadjuvant activity to increase the antibody production against sheep erythrocytes in the spleen of mice that were immunized by sheep erythrocytes with porin. Although chemical analyses revealed that the test porin fraction contained a considerable amount of lipopolysaccharide (LPS) (around 12% of the fraction), the studies with LPS-nonresponding C3H/HeJ mice and on the inhibitory effects of polymyxin B strongly suggest that most of the above bioactivities are due to porin protein itself, not to coexistent LPS in the porin fraction. Images PMID:2831155

  6. Role of porins in the antibiotic susceptibility of Pseudomonas aeruginosa: construction of mutants with deletions in the multiple porin genes.

    PubMed

    Yoneyama, H; Yamano, Y; Nakae, T

    1995-08-01

    We inserted deletions in the chromosomal genes of Pseudomonas aeruginosa coded for the outer membrane porins, proteins C, D2, or E1, and all possible combinations of these proteins by the gene replacement technique and selecting for imipenem-resistance. Determination of the minimum inhibitory concentrations of beta-lactams, fluoroquinolones, chloramphenicol and gentamicin in these mutants revealed that most mutants showed equal susceptibility to the porin-sufficient strain. The only exception was that imipenem and meropenem showed increased minimum inhibitory concentrations in all of the mutants lacking protein D2. These results firmly established that the P. aeruginosa porins identified so far form the pores do not accommodate the passage of most antipseudomonal antibiotics, with the exception of carbapenems. PMID:7639767

  7. High-affinity binding sites involved in the import of porin into mitochondria.

    PubMed Central

    Pfaller, R; Neupert, W

    1987-01-01

    The specific recognition by mitochondria of the precursor of porin and the insertion into the outer membrane were studied with a radiolabeled water-soluble form of porin derived from the mature protein. High-affinity binding sites had a number of 5-10 pmol/mg mitochondrial protein and a ka of 1-5 X 10(8) M-1. Binding was abolished after trypsin pretreatment of mitochondria indicating that binding sites were of protein-aceous nature. Specifically bound porin could be extracted at alkaline pH but not by high salt and was protected against low concentrations of proteinase K. It could be chased to a highly protease resistant form corresponding to mature porin. High-affinity binding sites could be extracted from mitochondria with detergent and reconstituted in asolectin-ergosterol liposomes. Water-soluble porin competed for the specific binding and import of the precursor of the ADP/ATP carrier, an inner membrane protein. We suggest that (i) binding of precursors to proteinaceous receptors serves as an initial step for recognition, (ii) the receptor for porin may also be involved in the import of precursors of inner membrane proteins, and (iii) interaction with the receptor triggers partial insertion of the precursor into the outer membrane. Images Fig. 4. PMID:2960520

  8. A model of maltodextrin transport through the sugar-specific porin, LamB, based on deletion analysis.

    PubMed

    Klebba, P E; Hofnung, M; Charbit, A

    1994-10-01

    LamB facilitates the uptake of maltose and maltodextrins across the bacterial outer membrane and acts as a general porin for small molecules. Using directed deletion mutagenesis we removed several regions of the LamB polypeptide and identified a polypeptide loop that both constricts the maltoporin channel and binds maltodextrins. In conjunction with a second sugar binding site that we identified at the rim of the channel, these data clarify, for the first time, the mechanism of transport through a substrate-specific porin. Furthermore, unlike the transverse loops of general porins, which originate from a central location in their primary structure, the loop that regulates LamB permeability originates from a C-terminal site. Thus LamB represents a second distinct class of porins in the bacterial outer membrane that is differently organized and separately evolved from OmpF-type, general porins. PMID:7925308

  9. Reproducible acquisition of Escherichia coli porin surface topographs by atomic force microscopy.

    PubMed Central

    Schabert, F A; Engel, A

    1994-01-01

    Crystalline membranes reconstituted from Escherichia coli OmpF porin and phospholipids were adsorbed to freshly cleaved mica and imaged in solution by the atomic force microscope. The extracellular as well as the periplasmic side of the porin trimers could be identified and the conditions to record topographs at 1-nm lateral and 0.1-nm vertical resolution were established. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 PMID:7696479

  10. How β-Lactam Antibiotics Enter Bacteria: A Dialogue with the Porins

    PubMed Central

    Molitor, Alexander; Bolla, Jean-Michel; Bessonov, Andrey N.; Winterhalter, Mathias; Pagès, Jean-Marie

    2009-01-01

    Background Multi-drug resistant (MDR) infections have become a major concern in hospitals worldwide. This study investigates membrane translocation, which is the first step required for drug action on internal bacterial targets. β-lactams, a major antibiotic class, use porins to pass through the outer membrane barrier of Gram-negative bacteria. Clinical reports have linked the MDR phenotype to altered membrane permeability including porin modification and efflux pump expression. Methodology/Principal Findings Here influx of β-lactams through the major Enterobacter aerogenes porin Omp36 is characterized. Conductance measurements through a single Omp36 trimer reconstituted into a planar lipid bilayer allowed us to count the passage of single β-lactam molecules. Statistical analysis of each transport event yielded the kinetic parameters of antibiotic travel through Omp36 and distinguishable translocation properties of β-lactams were quantified for ertapenem and cefepime. Expression of Omp36 in an otherwise porin-null bacterial strain is shown to confer increases in the killing rate of these antibiotics and in the corresponding bacterial susceptibility. Conclusions/Significance We propose the idea of a molecular “passport” that allows rapid transport of substrates through porins. Deciphering antibiotic translocation provides new insights for the design of novel drugs that may be highly effective at passing through the porin constriction zone. Such data may hold the key for the next generation of antibiotics capable of rapid intracellular accumulation to circumvent the further development MDR infections. PMID:19434239

  11. Porin protein of Neisseria gonorrhoeae: cloning and gene structure.

    PubMed Central

    Gotschlich, E C; Seiff, M E; Blake, M S; Koomey, M

    1987-01-01

    The outer membrane porin molecule of Neisseria gonorrhoeae is known as protein I (PI). Among different strains of gonococci there is variability of PI, and two main classes, PIA and PIB, have been recognized. A lambda gt11 bank of gonococcal DNA was screened using monoclonal antibodies directed to a PIB-type porin molecule of N. gonorrhoeae, and three immunoreactive clones were isolated. DNA sequence analysis indicated that each contained only portions of the PI structural gene, but that together they contained the complete gene, and its structure was determined. The DNA sequence predicts a protein of 348 amino acids with a typical 19 amino acid signal peptide. The PI protein resembles Escherichia coli porins in size, lack of long hydrophobic sequences, and absence of cysteine residues. Sequence homologies between PI and the E. coli porins were found, particularly in the 100 N-terminal and the 110 C-terminal amino acids. In addition to the coding sequence of PI, the complementary strand contains a large open reading frame. At the 3' end of the PI gene, immediately following an inverted repeat (probably the transcription terminator), the clone contains an unusual sequence consisting of 31 perfect repeats of the heptamer CTGTTTT. Hybridization analysis suggests that there is a single structural gene for PI and that it is homologous to the gene found in a PIA-bearing strain of gonococcus. Images PMID:2825179

  12. Crystal Structure of the Monomeric Porin OmpG

    SciTech Connect

    Subbarao,G.; van den Berg, B.

    2006-01-01

    The outer membrane (OM) of Gram-negative bacteria contains a large number of channel proteins that mediate the uptake of ions and nutrients necessary for growth and functioning of the cell. An important group of OM channel proteins are the porins, which mediate the non-specific, diffusion-based passage of small (<600 Da) polar molecules. All porins of Gram-negative bacteria that have been crystallized to date form stable trimers, with each monomer composed of a 16-stranded {beta}-barrel with a relatively narrow central pore. In contrast, the OmpG porin is unique, as it appears to function as a monomer. We have determined the X-ray crystal structure of OmpG from Escherichia coli to a resolution of 2.3 Angstroms. The structure shows a 14-stranded {beta}{beta}-barrel with a relatively simple architecture. Due to the absence of loops that fold back into the channel, OmpG has a large ({approx}13 Angstroms) central pore that is considerably wider than those of other E. coli porins, and very similar in size to that of the toxin a-hemolysin. The architecture of the channel, together with previous biochemical and other data, suggests that OmpG may form a non-specific channel for the transport of larger oligosaccharides. The structure of OmpG provides the starting point for engineering studies aiming to generate selective channels and for the development of biosensors.

  13. Porin channels in Escherichia coli: studies with beta-lactams in intact cells.

    PubMed Central

    Nikaido, H; Rosenberg, E Y; Foulds, J

    1983-01-01

    Wild-type Escherichia coli K-12 produces two porins, OmpF (protein 1a) and OmpC (protein 1b). In mutants deficient in both of these "normal" porins, secondary mutants that produce a "new" porin, protein PhoE (protein E), are selected for. We determined the properties of the channels produced by each of these porins by measuring the rates of diffusion of various cephalosporins through the outer membrane in strains producing only one porin species. We found that all porin channels retarded the diffusion of more hydrophobic cephalosporins and that with monoanionic cephalosporins a 10-fold increase in the octanol-water partition coefficient of the solute produced a 5- to 6-fold decrease in the rate of penetration. Electrical charges of the solutes had different effects on different channels. Thus, with the normal porins (i.e., OmpF and OmpC proteins) additional negative charge drastically reduced the penetration rate through the channels, whereas additional positive charge significantly accelerated the penetration. In contrast, diffusion through the PhoE channel was unaffected by the presence of an additional negative charge. We hypothesize that the relative exclusion of hydrophobic and negatively charged solutes by normal porin channels is of ecological advantage to E. coli, which must exclude hydrophobic and anionic bile salts in its natural habitat. The properties of the PhoE porin are also consistent with the recent finding (M. Argast and W. Boos, J. Bacteriol. 143:142-150, 1980; J. Tommassen and B. Lugtenberg, J. Bacteriol. 143:151-157, 1980) that its biosynthesis is derepressed by phosphate starvation; the channel may thus act as an emergency pore primarily for the uptake of phosphate and phosphorylated compounds. Images PMID:6294048

  14. Electrostatic properties of two porin channels from Escherichia coli.

    PubMed

    Karshikoff, A; Spassov, V; Cowan, S W; Ladenstein, R; Schirmer, T

    1994-07-22

    The electrostatic interactions in the channels of OmpF and PhoE porins from Escherichia coli were analysed on the basis of a macroscopic multi-dielectric model of the protein-membrane complex derived from the respective porin X-ray structures. The membrane was represented as layers of distinct dielectric constants corresponding to the aliphatic core and the polar head groups of the lipids. The pKa values of the titratable groups and the electrostatic field in the region of the channel were calculated by the finite difference technique. In spite of the differences in sequences and charge constellations, the calculated electrostatic properties of the two porins are similar in several aspects: (1) unusual titration behaviour (pKa below 7) was found for some groups of the cluster of basic residues at the constriction of the pore; (2) a number of acidic groups buried between the internal loop and the barrel wall are stabilized in their protonated forms at neutral pH; (3) there is a strong transverse electrostatic field in the channel characterized by a screw-like form. The strength of the field is greatest at the region of the constriction zone. This would facilitate the diffusion of solutes with a large dipole moment such as free amino acids. Differences between the electrostatic fields of OmpF and PhoE are mainly confined to that end of the pore that faces the cell exterior in vivo. In OmpF the electrostatic potential is close to zero in this region of the channel, whereas a positive potential was found in PhoE. It was shown that the experimentally observed difference in ion selectivity of the two porins can largely be attributed to this distinct electrostatic property. PMID:8035460

  15. In vitro trimerization of OmpF porin secreted by spheroplasts of Escherichia coli.

    PubMed Central

    Sen, K; Nikaido, H

    1990-01-01

    It is not yet clear how bacterial outer membrane proteins reach their correct destination after they are secreted across the cytoplasmic membrane. We show here that porin OmpF is secreted into the medium as a water-soluble monomeric protein by spheroplasts of Escherichia coli. Furthermore, this monomeric porin is taken up by cell envelope preparations or purified lipopolysaccharides in the presence of 0.03% Triton X-100 and is converted correctly into the mature trimeric conformation. These results appear to reproduce a part of the physiological export and targeting steps of this protein. Images PMID:1689050

  16. Paraquat Induces Cell Death Through Impairing Mitochondrial Membrane Permeability.

    PubMed

    Huang, Chuen-Lin; Chao, Chih-Chang; Lee, Yi-Chao; Lu, Mei-Kuang; Cheng, Jing-Jy; Yang, Ying-Chen; Wang, Vin-Chi; Chang, Wen-Chang; Huang, Nai-Kuei

    2016-05-01

    Paraquat (PQ) as a Parkinsonian mimetic has been demonstrated to impair dopaminergic (DAergic) neurons and is highly correlated with the etiology of Parkinson's disease (PD) where the death of DAergic neurons has been mainly attributed to impaired mitochondrial functioning. In this study, PQ-induced cytotoxicity focusing on mitochondrial membrane permeability (MMP), which has been implicated to play a part in neurodegeneration, was investigated. Primarily, PQ-induced cytotoxicity and reactive oxygen species (ROS) were inhibited by an inhibitor of NADPH oxidase (NOX), indicating the toxic effect of PQ redox cycling. Further, dibucaine and cyclosporin A which respectively inhibit mitochondrial apoptosis-induced channels (MAC) and mitochondrial permeability transition pores (mPTP) were used and found to prevent PQ-induced mitochondrial dysfunction, such as decreased mitochondrial membrane potential and increased MMP, mitochondrial ROS, and pro-apoptotic factor release. Knockdown of bax and/or bak blocked PQ-induced mitochondrial clusterization of Bax and/or Bak and cytotoxicity, demonstrating the significance of MAC which is composed of Bax and/or Bak. This clusterization coincided with the release of mitochondrial apoptotic factors before there was an increase in inner MMP, indicating that MAC may precede mPTP formation. Besides, NOX inhibitor but not dibucaine attenuated the earlier PQ-induced cytosolic ROS formation or Bax and/or Bak clusterization indicating PQ redox cycling may account for MAC formation. In this model, we have resolved for the first that PQ cytotoxicity through redox cycling may sequentially result in increased outer (MAC) and inner (mPTP) MMP and suggested MMP could be implicated as a therapeutic target in treating neurodegenerative diseases like PD. PMID:25947082

  17. Overexpression, refolding, and purification of the major immunodominant outer membrane porin OmpC from Salmonella typhi: characterization of refolded OmpC.

    PubMed

    Kumar, P D; Krishnaswamy, S

    2005-03-01

    The major immunodominant integral outer membrane protein C (OmpC) from Salmonella typhi Ty21a was overexpressed, without the signal peptide, in Escherichia coli. The protein aggregates as inclusion bodies (IBs) in the cytoplasm. OmpC from IBs was solubilized with 4 M urea and refolded. This involved rapid dilution of unfolded OmpC into a refolding buffer containing polyoxyethylene-9-lauryl ether (C(12)E(9)) and glycerol. The refolded OmpC (rfOmpC) was shown to be structurally similar to the native OmpC by SDS-PAGE, Western blotting, tryptic digestion, ultrafiltration, circular dichroism, and fluorescence spectroscopic techniques. Crystals of rfOmpC were obtained in preliminary crystallization trials. The rfOmpC also sets a stage for rational design by recombinant DNA technology for vaccine design and high resolution structure determination. PMID:15721780

  18. Transcriptional Regulation of the Outer Membrane Porin Gene ompW Reveals its Physiological Role during the Transition from the Aerobic to the Anaerobic Lifestyle of Escherichia coli.

    PubMed

    Xiao, Minfeng; Lai, Yong; Sun, Jian; Chen, Guanhua; Yan, Aixin

    2016-01-01

    Understanding bacterial physiology relies on elucidating the regulatory mechanisms and cellular functions of those differentially expressed genes in response to environmental changes. A widespread Gram-negative bacterial outer membrane protein OmpW has been implicated in the adaptation to stresses in various species. It is recently found to be present in the regulon of the global anaerobic transcription factor FNR and ArcA in Escherichia coli. However, little is known about the physiological implications of this regulatory disposition. In this study, we demonstrate that transcription of ompW is indeed mediated by a series of global regulators involved in the anaerobiosis of E. coli. We show that FNR can both activate and repress the expression of ompW through its direct binding to two distinctive sites, -81.5 and -126.5 bp respectively, on ompW promoter. ArcA also participates in repression of ompW under anaerobic condition, but in an FNR dependent manner. Additionally, ompW is also subject to the regulation by CRP and NarL which senses the availability and types of carbon sources and respiration electron acceptors in the environment respectively, implying a role of OmpW in the carbon and energy metabolism of E. coli during its anaerobic adaptation. Molecular docking reveals that OmpW can bind fumarate, an alternative electron acceptor in anaerobic respiration, with sufficient affinity. Moreover, supplement of fumarate or succinate which belongs to the C4-dicarboxylates family of metabolite, to E. coli culture rescues OmpW-mediated colicin S4 killing. Taken together, we propose that OmpW is involved in anaerobic carbon and energy metabolism to mediate the transition from aerobic to anaerobic lifestyle in E. coli. PMID:27303386

  19. Transcriptional Regulation of the Outer Membrane Porin Gene ompW Reveals its Physiological Role during the Transition from the Aerobic to the Anaerobic Lifestyle of Escherichia coli

    PubMed Central

    Xiao, Minfeng; Lai, Yong; Sun, Jian; Chen, Guanhua; Yan, Aixin

    2016-01-01

    Understanding bacterial physiology relies on elucidating the regulatory mechanisms and cellular functions of those differentially expressed genes in response to environmental changes. A widespread Gram-negative bacterial outer membrane protein OmpW has been implicated in the adaptation to stresses in various species. It is recently found to be present in the regulon of the global anaerobic transcription factor FNR and ArcA in Escherichia coli. However, little is known about the physiological implications of this regulatory disposition. In this study, we demonstrate that transcription of ompW is indeed mediated by a series of global regulators involved in the anaerobiosis of E. coli. We show that FNR can both activate and repress the expression of ompW through its direct binding to two distinctive sites, -81.5 and -126.5 bp respectively, on ompW promoter. ArcA also participates in repression of ompW under anaerobic condition, but in an FNR dependent manner. Additionally, ompW is also subject to the regulation by CRP and NarL which senses the availability and types of carbon sources and respiration electron acceptors in the environment respectively, implying a role of OmpW in the carbon and energy metabolism of E. coli during its anaerobic adaptation. Molecular docking reveals that OmpW can bind fumarate, an alternative electron acceptor in anaerobic respiration, with sufficient affinity. Moreover, supplement of fumarate or succinate which belongs to the C4-dicarboxylates family of metabolite, to E. coli culture rescues OmpW-mediated colicin S4 killing. Taken together, we propose that OmpW is involved in anaerobic carbon and energy metabolism to mediate the transition from aerobic to anaerobic lifestyle in E. coli. PMID:27303386

  20. Porin from Pseudomonas aeruginosa Induces Apoptosis in an Epithelial Cell Line Derived from Rat Seminal Vesicles

    PubMed Central

    Buommino, Elisabetta; Morelli, Francesco; Metafora, Salvatore; Rossano, Fabio; Perfetto, Brunella; Baroni, Adone; Tufano, Maria Antonietta

    1999-01-01

    Micromolar concentrations of porin, purified from the outer membranes of Pseudomonas aeruginosa, induced in vitro the classic morphological and biochemical signs of apoptosis in an epithelial cell line (SVC1) derived from the rat seminal vesicle secretory epithelium. The programmed cell death (PCD) was p53 independent and associated with significant decrease of bcl-2 expression, a marked increase of c-myc transcriptional activity, and an absence of the mRNA coding for tissue transglutaminase. The Ca2+ influx, caused by the porin treatment of SVC1 cells, appears to play an important role in the triggering of apoptosis in our biological model. The possibility that the porin property of inducing PCD plays a role in the infertility of individuals chronically infected by gram-negative bacteria is discussed. PMID:10456933

  1. The OprB porin plays a central role in carbohydrate uptake in Pseudomonas aeruginosa.

    PubMed Central

    Wylie, J L; Worobec, E A

    1995-01-01

    Using interposon mutagenesis, we have generated strains of Pseudomonas aeruginosa which lack or overexpress the substrate-selective OprB porin of this species. A marked decrease or increase in the initial uptake of glucose by these strains verified the role of OprB in facilitating the diffusion of glucose across the outer membrane of P. aeruginosa. However, we also demonstrated that the loss or overexpression of OprB had a similar effect on the uptake of three other sugars able to support the growth of this bacterium (mannitol, glycerol, and fructose). This effect was restricted to carbohydrate transport; arginine uptake was identical in mutant and wild-type strains. These results indicated that OprB cannot be considered strictly a glucose-selective porin; rather, it acts as a central component of carbohydrate transport and is more accurately described as a carbohydrate-selective porin. PMID:7768797

  2. Genomic analyses of bacterial porin-cytochrome gene clusters

    DOE PAGESBeta

    Shi, Liang; Fredrickson, James K.; Zachara, John M.

    2014-11-26

    In this study, the porin-cytochrome (Pcc) protein complex is responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III) by the dissimilatory metal-reducing bacterium Geobacter sulfurreducens PCA. The identified and characterized Pcc complex of G. sulfurreducens PCA consists of a porin-like outer-membrane protein, a periplasmic 8-heme c type cytochrome (c-Cyt) and an outer-membrane 12-heme c-Cyt, and the genes encoding the Pcc proteins are clustered in the same regions of genome (i.e., the pcc gene clusters) of G. sulfurreducens PCA. A survey of additionally microbial genomes has identified the pcc gene clusters in all sequenced Geobacter spp. and other bacteriamore » from six different phyla, including Anaeromyxobacter dehalogenans 2CP-1, A. dehalogenans 2CP-C, Anaeromyxobacter sp. K, Candidatus Kuenenia stuttgartiensis, Denitrovibrio acetiphilus DSM 12809, Desulfurispirillum indicum S5, Desulfurivibrio alkaliphilus AHT2, Desulfurobacterium thermolithotrophum DSM 11699, Desulfuromonas acetoxidans DSM 684, Ignavibacterium album JCM 16511, and Thermovibrio ammonificans HB-1. The numbers of genes in the pcc gene clusters vary, ranging from two to nine. Similar to the metal-reducing (Mtr) gene clusters of other Fe(III)-reducing bacteria, such as Shewanella spp., additional genes that encode putative c-Cyts with predicted cellular localizations at the cytoplasmic membrane, periplasm and outer membrane often associate with the pcc gene clusters. This suggests that the Pcc-associated c-Cyts may be part of the pathways for extracellular electron transfer reactions. The presence of pcc gene clusters in the microorganisms that do not reduce solid-phase Fe(III) and Mn(IV) oxides, such as D. alkaliphilus AHT2 and I. album JCM 16511, also suggests that some of the pcc gene clusters may be involved in extracellular electron transfer reactions with the substrates other than Fe(III) and Mn(IV) oxides.« less

  3. Diffusion of aminoglycoside antibiotics across the outer membrane of Escherichia coli.

    PubMed Central

    Nakae, R; Nakae, T

    1982-01-01

    The diffusion of aminoglycoside antibiotics (gentamicin, kanamycin, streptomycin, fradiomycin, lividomycin, and mannosylparomomycin) through porin pores was examined in vitro by the liposome swelling technique, using vesicle membranes reconstituted from phospholipids and purified porin trimers. Results showed that aminoglycoside antibiotics diffuse through porin-pores very efficiently, as rapidly as hexoses and disaccharides, despite the fact that the molecular weights of the aminoglycosides used were higher than or close to the exclusion limit of porin pores. The susceptibility to aminoglycoside antibiotics of mutant strains producing 3 to 4% of porin was not significantly different from that of a strain producing a wild-type quantity of porin. These results were interpreted to mean that aminoglycoside antibiotics diffuse through porin-pores very efficiently. Therefore, the diffusion of these drugs through the mutant outer membranes producing 3 to 4% of porin is not a rate-limiting step for aminoglycoside diffusion and its action. PMID:6758685

  4. Hetero-oligomeric cell wall channels (porins) of Nocardia farcinica.

    PubMed

    Kläckta, Christian; Knörzer, Philipp; Riess, Franziska; Benz, Roland

    2011-06-01

    The cell wall of Nocardia farcinica contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of N. farcinica for negatively charged antibiotics. Based on partial sequencing of the protein responsible for channel formation derived from N. farcinica ATTC 3318 we were able to identify the corresponding genes (nfa15890 and nfa15900) within the known genome of N. farcinica IFM 10152. The corresponding genes of N. farcinica ATTC 3318 were separately expressed in the Escherichia coli BL21DE3Omp8 strain and the N-terminal His10-tagged proteins were purified to homogeneity using immobilized metal affinity chromatography. The pure proteins were designated NfpANHis and NfpBNHis, for N. farcinica porin A and N. farcinica porin B. The two proteins were checked separately for channel formation in lipid bilayers. Our results clearly indicate that the proteins NfpANHis and NfpBNHis expressed in E. coli could only together form a channel in lipid bilayer membranes. This means that the cell wall channel of N. farcinica is formed by a heterooligomer. NfpA and NfpB form together a channel that may structurally be related to MspA of Mycobacterium smegmatis based on amino acid comparison and renaturation procedure. PMID:21092733

  5. Characterization of OpdH, a Pseudomonas aeruginosa Porin Involved in the Uptake of Tricarboxylates▿

    PubMed Central

    Tamber, Sandeep; Maier, Elke; Benz, Roland; Hancock, Robert E. W.

    2007-01-01

    The Pseudomonas aeruginosa outer membrane is intrinsically impermeable to many classes of antibiotics, due in part to its relative lack of general uptake pathways. Instead, this organism relies on a large number of substrate-specific uptake porins. Included in this group are the 19 members of the OprD family, which are involved in the uptake of a diverse array of metabolites. One of these porins, OpdH, has been implicated in the uptake of cis-aconitate. Here we demonstrate that this porin may also enable P. aeruginosa to take up other tricarboxylates. Isocitrate and citrate strongly and specifically induced the opdH gene via a mechanism involving derepression by the putative two-component regulatory system PA0756-PA0757. Planar bilayer analysis of purified OpdH demonstrated that it was a channel-forming protein with a large single-channel conductance (230 pS in 1 M KCl; 10-fold higher than that of OprD); however, we were unable to demonstrate the presence of a tricarboxylate binding site within the channel. Thus, these data suggest that the requirement for OpdH for efficient growth on tricarboxylates was likely due to the specific expression of this large-channel porin under particular growth conditions. PMID:17114261

  6. The mitochondrial morphology protein Mdm10 functions in assembly of the preprotein translocase of the outer membrane.

    PubMed

    Meisinger, Chris; Rissler, Michael; Chacinska, Agnieszka; Szklarz, Luiza K Sanjuán; Milenkovic, Dusanka; Kozjak, Vera; Schönfisch, Birgit; Lohaus, Christiane; Meyer, Helmut E; Yaffe, Michael P; Guiard, Bernard; Wiedemann, Nils; Pfanner, Nikolaus

    2004-07-01

    The biogenesis of mitochondrial outer membrane proteins involves the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The two known subunits of the SAM complex, Mas37 and Sam50, are required for assembly of the abundant outer membrane proteins porin and Tom40. We have identified an unexpected subunit of the SAM complex, Mdm10, which is involved in maintenance of mitochondrial morphology. Mitochondria lacking Mdm10 are selectively impaired in the final steps of the assembly pathway of Tom40, including the association of Tom40 with the receptor Tom22 and small Tom proteins, while the biogenesis of porin is not affected. Yeast mutants of TOM40, MAS37, and SAM50 also show aberrant mitochondrial morphology. We conclude that Mdm10 plays a specific role in the biogenesis of the TOM complex, indicating a connection between the mitochondrial protein assembly apparatus and the machinery for maintenance of mitochondrial morphology. PMID:15239954

  7. Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins.

    PubMed

    Rebière-Huët, J; Guérillon, J; Pimenta, A L; Di Martino, P; Orange, N; Hulen, C

    2002-09-24

    Bacterial adherence is a complex phenomenon involving specific interactions between receptors, including matricial fibronectin, and bacterial ligands. We show here that fibronectin and outer membrane proteins of Pseudomonas fluorescens were able to inhibit adherence of P. fluorescens to fibronectin-coated wells. We identified at least six fibronectin-binding proteins with molecular masses of 70, 55, 44, 37, 32 and 28 kDa. The presence of native (32 kDa) and heat-modified forms (37 kDa) of OprF was revealed by immuno-analysis and the 44-kDa band was composed of three proteins, their N-terminal sequences showing homologies with Pseudomonas aeruginosa porins (OprD, OprE1 and OprE3). PMID:12393211

  8. Role of the Novel OprD Family of Porins in Nutrient Uptake in Pseudomonas aeruginosa

    PubMed Central

    Tamber, Sandeep; Ochs, Martina M.; Hancock, Robert E. W.

    2006-01-01

    To circumvent the permeability barrier of its outer membrane, Pseudomonas aeruginosa has evolved a series of specific porins. These channels have binding sites for related classes of molecules that facilitate uptake under nutrient-limited conditions. Here, we report on the identification of a 19-member family of porins similar to the basic-amino-acid-specific porin OprD. The members of this family fell into one of two phylogenetically distinct clusters, one bearing high similarity to OprD and the other bearing most similarity to the putative phenylacetic acid uptake porin PhaK of Pseudomonas putida. Analysis of the genome context, operon arrangement, and regulation of the PhaK-like porin OpdK indicated that it might be involved in vanillate uptake. This result was confirmed by demonstrating that an opdK mutant had a deficiency in the ability to grow on vanillate as a carbon source. To extrapolate these data to other paralogues within this family, the substrate specificities of 6 of the 17 remaining OprD homologues were inferred using an approach similar to that used with opdK. The specificities determined were as follows: OpdP, glycine-glutamate; OpdC, histidine; OpdB, proline; OpdT, tyrosine; OpdH, cis-aconitate; and OpdO, pyroglutamate. Thus, members of the OprD subfamily took up amino acids and related molecules, and those characterized members most similar to PhaK were responsible for the uptake of a diverse array of organic acids. These results imply that there is a functional basis for the phylogenetic clustering of these proteins and provide a framework for studying OprD homologues in other organisms. PMID:16352820

  9. Killing of Staphylococci by θ-Defensins Involves Membrane Impairment and Activation of Autolytic Enzymes

    PubMed Central

    Wilmes, Miriam; Stockem, Marina; Bierbaum, Gabriele; Schlag, Martin; Götz, Friedrich; Tran, Dat Q.; Schaal, Justin B.; Ouellette, André J.; Selsted, Michael E.; Sahl, Hans-Georg

    2014-01-01

    θ-Defensins are cyclic antimicrobial peptides expressed in leukocytes of Old world monkeys. To get insight into their antibacterial mode of action, we studied the activity of RTDs (rhesus macaque θ-defensins) against staphylococci. We found that in contrast to other defensins, RTDs do not interfere with peptidoglycan biosynthesis, but rather induce bacterial lysis in staphylococci by interaction with the bacterial membrane and/or release of cell wall lytic enzymes. Potassium efflux experiments and membrane potential measurements revealed that the membrane impairment by RTDs strongly depends on the energization of the membrane. In addition, RTD treatment caused the release of Atl-derived cell wall lytic enzymes probably by interaction with membrane-bound lipoteichoic acid. Thus, the premature and uncontrolled activity of these enzymes contributes strongly to the overall killing by θ-defensins. Interestingly, a similar mode of action has been described for Pep5, an antimicrobial peptide of bacterial origin. PMID:25632351

  10. Molecular Basis of Filtering Carbapenems by Porins from β-Lactam-resistant Clinical Strains of Escherichia coli.

    PubMed

    Bajaj, Harsha; Scorciapino, Mariano A; Moynié, Lucile; Page, Malcolm G P; Naismith, James H; Ceccarelli, Matteo; Winterhalter, Mathias

    2016-02-01

    Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer membrane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we investigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consecutive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitterionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electrophysiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific orientation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an additional barrier by "trapping" the antibiotic in an unfavorable orientation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Identification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new antibiotics with improved permeation properties in Gram-negative bacteria. PMID:26645688

  11. Genomic analyses of bacterial porin-cytochrome gene clusters

    SciTech Connect

    Shi, Liang; Fredrickson, James K.; Zachara, John M.

    2014-11-26

    In this study, the porin-cytochrome (Pcc) protein complex is responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III) by the dissimilatory metal-reducing bacterium Geobacter sulfurreducens PCA. The identified and characterized Pcc complex of G. sulfurreducens PCA consists of a porin-like outer-membrane protein, a periplasmic 8-heme c type cytochrome (c-Cyt) and an outer-membrane 12-heme c-Cyt, and the genes encoding the Pcc proteins are clustered in the same regions of genome (i.e., the pcc gene clusters) of G. sulfurreducens PCA. A survey of additionally microbial genomes has identified the pcc gene clusters in all sequenced Geobacter spp. and other bacteria from six different phyla, including Anaeromyxobacter dehalogenans 2CP-1, A. dehalogenans 2CP-C, Anaeromyxobacter sp. K, Candidatus Kuenenia stuttgartiensis, Denitrovibrio acetiphilus DSM 12809, Desulfurispirillum indicum S5, Desulfurivibrio alkaliphilus AHT2, Desulfurobacterium thermolithotrophum DSM 11699, Desulfuromonas acetoxidans DSM 684, Ignavibacterium album JCM 16511, and Thermovibrio ammonificans HB-1. The numbers of genes in the pcc gene clusters vary, ranging from two to nine. Similar to the metal-reducing (Mtr) gene clusters of other Fe(III)-reducing bacteria, such as Shewanella spp., additional genes that encode putative c-Cyts with predicted cellular localizations at the cytoplasmic membrane, periplasm and outer membrane often associate with the pcc gene clusters. This suggests that the Pcc-associated c-Cyts may be part of the pathways for extracellular electron transfer reactions. The presence of pcc gene clusters in the microorganisms that do not reduce solid-phase Fe(III) and Mn(IV) oxides, such as D. alkaliphilus AHT2 and I. album JCM 16511, also suggests that some of the pcc gene clusters may be involved in extracellular

  12. Porin Involvement in Cephalosporin and Carbapenem Resistance of Burkholderia pseudomallei

    PubMed Central

    Aunkham, Anuwat; Schulte, Albert; Winterhalter, Mathias; Suginta, Wipa

    2014-01-01

    Background Burkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins. Principal Findings Microbiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A) enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F) inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM) suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe), increasing the ‘greasiness’ of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem). In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive. Conclusion/Significance Our results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue Tyr119 affects

  13. Proteasome Impairment Induces Recovery of Mitochondrial Membrane Potential and an Alternative Pathway of Mitochondrial Fusion

    PubMed Central

    Shirozu, Ryohei; Yashiroda, Hideki

    2015-01-01

    Mitochondria are vital and highly dynamic organelles that continuously fuse and divide to maintain mitochondrial quality. Mitochondrial dysfunction impairs cellular integrity and is known to be associated with various human diseases. However, the mechanism by which the quality of mitochondria is maintained remains largely unexplored. Here we show that impaired proteasome function recovers the growth of yeast cells lacking Fzo1, a pivotal protein for mitochondrial fusion. Decreased proteasome activity increased the mitochondrial oxidoreductase protein Mia40 and the ratio of the short isoform of mitochondrial intermembrane protein Mgm1 (s-Mgm1) to the long isoform (l-Mgm1). The increase in Mia40 restored mitochondrial membrane potential, while the increase in the s-Mgm1/l-Mgm1 ratio promoted mitochondrial fusion in an Fzo1-independent manner. Our findings demonstrate a new pathway for mitochondrial quality control that is induced by proteasome impairment. PMID:26552703

  14. Functions of the Periplasmic Loop of the Porin MspA from Mycobacterium smegmatis*

    PubMed Central

    Huff, Jason; Pavlenok, Mikhail; Sukumaran, Suja; Niederweis, Michael

    2009-01-01

    MspA is the major porin of Mycobacterium smegmatis and mediates diffusion of small and hydrophilic solutes across the outer membrane. The octameric structure of MspA, its sharply defined constriction zone, and a large periplasmic loop L6 represent novel structural features. L6 consists of 13 amino acids and is directly adjacent to the constriction zone. Deletion of 3, 5, 7, 9, and 11 amino acids of the L6 loop resulted in functional pores that restored glucose uptake and growth of a porin mutant of M. smegmatis. Lipid bilayer experiments revealed that all mutant channels were noisier than wild type (wt) MspA, indicating that L6 is required for pore stability in vitro. Voltage gating of the Escherichia coli porin OmpF was attributed to loops that collapse into the channel in response to a strong electrical field. Here, we show that deletion mutants Δ7, Δ9, and Δ11 had critical voltages similar to wt MspA. This demonstrated that the L6 loop is not the primary voltage-dependent gating mechanism of MspA. Surprisingly, large deletions in L6 resulted in 3-6-fold less extractable pores, whereas small deletions did not alter expression levels of MspA. Pores with large deletions in L6 were more permissive for glucose than smaller deletion mutants, whereas their single channel conductance was similar to that of wt MspA. These results indicate that translocation of ions through the MspA pore is governed by different mechanisms than that of neutral solutes. This is the first study identifying a molecular determinant of solute translocation in a mycobacterial porin. PMID:19208627

  15. Functions of the periplasmic loop of the porin MspA from Mycobacterium smegmatis.

    PubMed

    Huff, Jason; Pavlenok, Mikhail; Sukumaran, Suja; Niederweis, Michael

    2009-04-10

    MspA is the major porin of Mycobacterium smegmatis and mediates diffusion of small and hydrophilic solutes across the outer membrane. The octameric structure of MspA, its sharply defined constriction zone, and a large periplasmic loop L6 represent novel structural features. L6 consists of 13 amino acids and is directly adjacent to the constriction zone. Deletion of 3, 5, 7, 9, and 11 amino acids of the L6 loop resulted in functional pores that restored glucose uptake and growth of a porin mutant of M. smegmatis. Lipid bilayer experiments revealed that all mutant channels were noisier than wild type (wt) MspA, indicating that L6 is required for pore stability in vitro. Voltage gating of the Escherichia coli porin OmpF was attributed to loops that collapse into the channel in response to a strong electrical field. Here, we show that deletion mutants Delta7, Delta9, and Delta11 had critical voltages similar to wt MspA. This demonstrated that the L6 loop is not the primary voltage-dependent gating mechanism of MspA. Surprisingly, large deletions in L6 resulted in 3-6-fold less extractable pores, whereas small deletions did not alter expression levels of MspA. Pores with large deletions in L6 were more permissive for glucose than smaller deletion mutants, whereas their single channel conductance was similar to that of wt MspA. These results indicate that translocation of ions through the MspA pore is governed by different mechanisms than that of neutral solutes. This is the first study identifying a molecular determinant of solute translocation in a mycobacterial porin. PMID:19208627

  16. Structure of a putative BenF-like porin from Pseudomonas fluorescens Pf-5 at 2.6 A resolution

    SciTech Connect

    Sampathkumar, P.; Swaminathan, S.; Lu, F.; Zhao, X.; Li, Z.; Gilmore, J.; Bain, K.; Rutter, M. E.; Gheyi, T.; Schwinn, D.; Bonanno, J. B.; Pieper, U.; Fajardo, J. E.; Fiser, A.; Almo, S. C.; Chance, M. R.; Baker, D.; Atwell, S.; Thompson, D. A.; Emtage, J. S.; Wasserman, S. R.; Sali, A.; Sauder, J. M.; Burley, S. K.

    2010-11-01

    Gram-negative bacteria typically overcome poor permeability of outer membranes through general porins like OmpF and OmpC, which form water-filled transmembrane pores permitting diffusion of hydrophilic molecules with no particular selectivity. Many bacteria lacking such general porins use substrate-specific porins to overcome growth-limiting conditions and facilitate selective transport of metabolites. Exclusive reliance on substrate-specific porins yields lower membrane permeability to small molecules (<600 Da) versus that seen for Escherichia coli. In Pseudomonads, transit of most small molecules across the cell membrane is thought to be mediated by substrate-specific channels of the OprD superfamily. This property explains, at least in part, the high incidence of Pseudomonas aeruginosa antibiotic resistance. High-throughput DNA sequencing of the P. aeruginosa chromosome revealed the presence of 19 genes encoding structurally related, substrate-specific porins (with 30-45% pairwise amino acid sequence identity) that mediate transmembrane passage of small, water-soluble compounds. The OprD superfamily encompasses the eponymous OprD subfamily, which includes 9 P. aeruginosa proteins that convey basic amino acids and carbapenem antibiotics, and the OpdK subfamily, which includes 11 P. aeruginosa proteins that convey aromatic acids and other small aromatic compounds. Genome sequencing of other gram-negative bacteria has revealed additional members of the OprD and OpdK subfamilies in various organisms, including other pseudomonads. Among the many bacteria in which OprD superfamily members have been identified are P. putida, P. fluorescens Pf-5, P. syringae, and Azotobacter vinelandii, all of which share closely related genes that encode the so-called BenF-like porins. In P. putida, benF is part of an operon involved in benzoate catabolism regulated by benR. Within this operon, benK, benE, and benF genes have been suggested to contribute toward either influx or efflux

  17. Enhancement of extracellular electron transfer and bioelectricity output by synthetic porin.

    PubMed

    Yong, Yang-Chun; Yu, Yang-Yang; Yang, Yun; Liu, Jing; Wang, Jing-Yuan; Song, Hao

    2013-02-01

    The microbial fuel cell (MFC), is a promising environmental biotechnology for harvesting electricity energy from organic wastes. However, low bacterial membrane permeability of electron shuttles is a limiting factor that restricts the electron shuttle-mediated extracellular electron transfer (EET) from bacteria to electrodes, thus the electricity power output of MFCs. To this end, we heterologously expressed a porin protein OprF from Pseudomonas aeruginosa PAO1 into Escherichia coli, which dramatically increased its membrane permeability, delivering a much higher current output in MFCs than its parental strain (BL21). We found that the oprF-expression strain showed more efficient EET than its parental strain. More strikingly, the enhanced membrane permeability also rendered the oprF-expression strain an efficient usage of riboflavin as the electron shuttle, whereas its parental strain was incapable of. Our results substantiated that membrane permeability is crucial for the efficient EET, and indicated that the expression of synthetic porins could be an efficient strategy to enhance bioelectricity generation by microorganisms (including electrogenic bacteria) in MFCs. PMID:23007598

  18. Structure of Carbon Nanotube Porins in Lipid Bilayers: An in Situ Small-Angle X-ray Scattering (SAXS) Study.

    PubMed

    Tran, Ich C; Tunuguntla, Ramya H; Kim, Kyunghoon; Lee, Jonathan R I; Willey, Trevor M; Weiss, Thomas M; Noy, Aleksandr; van Buuren, Tony

    2016-07-13

    Carbon nanotube porins (CNTPs), small segments of carbon nanotubes capable of forming defined pores in lipid membranes, are important future components for bionanoelectronic devices as they could provide a robust analog of biological membrane channels. In order to control the incorporation of these CNT channels into lipid bilayers, it is important to understand the structure of the CNTPs before and after insertion into the lipid bilayer as well as the impact of such insertion on the bilayer structure. Here we employed a noninvasive in situ probe, small-angle X-ray scattering, to study the integration of CNT porins into dioleoylphosphatidylcholine bilayers. Our results show that CNTPs in solution are stabilized by a monolayer of lipid molecules wrapped around their outer surface. We also demonstrate that insertion of CNTPs into the lipid bilayer results in decreased bilayer thickness with the magnitude of this effect increasing with the concentration of CNTPs. PMID:27322135

  19. Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration

    PubMed Central

    Merkwirth, Carsten; Morbin, Michela; Brönneke, Hella S.; Jordan, Sabine D.; Rugarli, Elena I.; Langer, Thomas

    2012-01-01

    Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies

  20. Tau accumulation impairs mitophagy via increasing mitochondrial membrane potential and reducing mitochondrial Parkin

    PubMed Central

    Wang, Zhi-hao; Luo, Yu; Zhang, Xiangnan; Liu, Xiu-Ping; Feng, Qiong; Wang, Qun; Yue, Zhenyu; Chen, Zhong; Ye, Keqiang; Wang, Jian-Zhi; Liu, Gong-Ping

    2016-01-01

    Intracellular accumulation of wild type tau is a hallmark of sporadic Alzheimer's disease (AD). However, the molecular mechanisms underlying tau toxicity is not fully understood. Here, we detected mitophagy deficits evidenced by the increased levels of mitophagy markers, including COX IV, TOMM20, and the ratio of mtDNA to genomic DNA indexed as mt-Atp6/Rpl13, in the AD brains and in the human wild type full-length tau (htau) transgenic mice. More interestingly, the mitophagy deficit was only shown in the AD patients who had an increased total tau level. Further studies demonstrated that overexpression of htau induced mitophagy deficits in HEK293 cells, the primary hippocampal neurons and in the brains of C57 mice. Upon overexpression of htau, the mitochondrial membrane potential was increased and the levels of PTEN-induced kinase 1 (PINK1) and Parkin decreased in the mitochondrial fraction, while upregulation of Parkin attenuated the htau-induced mitophagy deficits. Finally, we detected a dose-dependent allocation of tau proteins into the mitochondrial outer membrane fraction along with its cytoplasmic accumulation. These data suggest that intracellular accumulation of htau induces mitophagy deficits by direct inserting into the mitochondrial membrane and thus increasing the membrane potential, which impairs the mitochondrial residence of PINK1/Parkin. Our findings reveal a novel mechanism underlying the htau-induced neuronal toxicities in AD and other tauopathies. PMID:26943044

  1. Identification of the outer membrane porin of Thermus thermophilus HB8: the channel-forming complex has an unusually high molecular mass and an extremely large single-channel conductance.

    PubMed

    Maier, E; Polleichtner, G; Boeck, B; Schinzel, R; Benz, R

    2001-01-01

    The outer membrane of the thermophilic bacterium Thermus thermophilus was isolated using sucrose step gradient centrifugation. Its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 nS in 1 M KCl. The channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis. It has a high molecular mass of 185 kDa, and its channel-forming ability resists boiling in SDS for 10 min. PMID:11133980

  2. Identification of the Outer Membrane Porin of Thermus thermophilus HB8: the Channel-Forming Complex Has an Unusually High Molecular Mass and an Extremely Large Single-Channel Conductance

    PubMed Central

    Maier, Elke; Polleichtner, Georg; Boeck, Birgit; Schinzel, Reinhard; Benz, Roland

    2001-01-01

    The outer membrane of the thermophilic bacterium Thermus thermophilus was isolated using sucrose step gradient centrifugation. Its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 nS in 1 M KCl. The channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis. It has a high molecular mass of 185 kDa, and its channel-forming ability resists boiling in SDS for 10 min. PMID:11133980

  3. Positive regulation of the Shewanella oneidensis OmpS38, a major porin facilitating anaerobic respiration, by Crp and Fur

    PubMed Central

    Gao, Tong; Ju, Lili; Yin, Jianhua; Gao, Haichun

    2015-01-01

    Major porins are among the most abundant proteins embedded in the outer membrane (OM) of Gram-negative bacteria, playing crucial roles in maintenance of membrane structural integrity and OM permeability. Although many OM proteins (especially c-type cytochromes) in Shewanella oneidensis, a research model for respiratory versatility, have been extensively studied, physiological significance of major porins remains largely unexplored. In this study, we show that OmpS38 and OmpA are two major porins, neither of which is responsive to changes in osmolarity or contributes to the intrinsic resistance to β-lactam antibiotics. However, OmpS38 but not OmpA is largely involved in respiration of non-oxygen electron acceptors. We then provide evidence that expression of ompS38 is transcribed from two promoters, the major of which is favored under anaerobic conditions while the other appears constitutive. The major promoter is under the direct control of Crp, the master regulator dictating respiration. As a result, the increase in the level of OmpS38 correlates with an elevated activity in Crp under anaerobic conditions. In addition, we show that the activity of the major promoter is also affected by Fur, presumably indirectly, the transcription factor for iron-dependent gene expression. PMID:26381456

  4. Single channel activity of OmpF-like porin from Yersinia pseudotuberculosis.

    PubMed

    Rokitskaya, Tatyana I; Kotova, Elena A; Naberezhnykh, Gennadiy A; Khomenko, Valentina A; Gorbach, Vladimir I; Firsov, Alexander M; Zelepuga, Elena A; Antonenko, Yuri N; Novikova, Olga D

    2016-04-01

    To gain a mechanistic insight in the functioning of the OmpF-like porin from Yersinia pseudotuberculosis (YOmpF), we compared the effect of pH variation on the ion channel activity of the protein in planar lipid bilayers and its binding to lipid membranes. The behavior of YOmpF channels upon acidification was similar to that previously described for Escherichia coli OmpF. In particular, a decrease in pH of the bathing solution resulted in a substantial reduction of YOmpF single channel conductance, accompanied by the emergence of subconductance states. Similar subconductance substates were elicited by the addition of lysophosphatidylcholine. This observation, made with porin channels for the first time, pointed to the relevance of lipid-protein interactions, in particular, the lipid curvature stress, to the appearance of subconductance states at acidic pH. Binding of YOmpF to membranes displayed rather modest dependence on pH, whereas the channel-forming potency of the protein tremendously decreased upon acidification. PMID:26854962

  5. Impairment of Venous Drainage on Extracorporeal Membrane Oxygenation Secondary to Air Trapping in Acute Asphyxial Asthma.

    PubMed

    Niimi, Kevin S; Lewis, Leslie S; Fanning, Jeffrey J

    2015-06-01

    The inability to adequately support a patient on extracorporeal membrane oxygenation (ECMO) due to impaired drainage is not an uncommon occurrence during support. Typically, the causes include hypovolemia, kinks in the circuit, cannula malposition, or inadequate cannula size. In this report we present an uncommon etiology of this problem. A 3-year-old female presented to our hospital in status asthmaticus and pulseless electrical activity (PEA). This was a result of dynamic hyperinflation of the lungs causing physical obstruction of venous return to the heart. Upon initiating venoarterial (VA) ECMO, we experienced inadequate drainage that did not improve despite multiple interventions. This resolved with the addition of an inhaled anesthetic gas to treat this patient's severe bronchospasm. This case illustrates the importance of considering a patient's physiology or disease state and how that may affect the mechanics of ECMO support. PMID:26405359

  6. Permeation rates of penicillins indicate that Escherichia coli porins function principally as nonspecific channels

    PubMed Central

    Kojima, Seiji; Nikaido, Hiroshi

    2013-01-01

    Small, hydrophilic compounds such as β-lactams diffuse across the outer membrane of Gram-negative bacteria through porin channels, which were originally thought to be nonspecific channels devoid of any specificity. However, since the discovery of an ampicillin-binding site within the OmpF channel in 2002, much attention has been focused on the potential specificity of the channel, where the binding site was assumed either to facilitate or to retard the penetration of β-lactams. Since the earlier studies on porin permeability were done without the knowledge of the contribution of multidrug efflux pumps in the overall flux process across the cell envelope, in this study we have carefully studied both the porin permeability and active efflux of ampicillin and benzylpenicillin. We found that the influx occurs apparently by a spontaneous passive diffusion without any indication of specific binding within the concentration range relevant to the antibiotic action of these drugs, and that the higher permeability for ampicillin is totally as expected from the gross property of this drug as a zwitterionic compound. The active efflux by AcrAB was more effective for benzylpenicillin due to the stronger affinity and high degree of positive cooperativity. Our data now give a complete quantitative picture of the influx, efflux, and periplasmic degradation (catalyzed by AmpC β-lactamase) of these two compounds, and correlate closely with the susceptibility of Escherichia coli strains used here, thus validating not only our model but also the parameters obtained in this study. PMID:23798411

  7. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells

    PubMed Central

    Shim, Su Yeon; Karri, Srinivasarao; Law, Sampson; Schatzl, Hermann M.; Gilch, Sabine

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrPSc) of the cellular prion protein (PrPc). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrPc into PrPSc. Within neurons, PrPSc accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrPSc accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases. PMID:26865414

  8. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells.

    PubMed

    Shim, Su Yeon; Karri, Srinivasarao; Law, Sampson; Schatzl, Hermann M; Gilch, Sabine

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrP(Sc)) of the cellular prion protein (PrP(c)). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrP(c) into PrP(Sc). Within neurons, PrP(Sc) accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrP(Sc) accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases. PMID:26865414

  9. Rab4GTPase modulates CFTR function by impairing channel expression at plasma membrane

    SciTech Connect

    Saxena, Sunil K. . E-mail: ssaxena@stevens.edu; Kaur, Simarna; George, Constantine

    2006-03-03

    Cystic fibrosis (CF), an autosomal recessive disorder, is caused by the disruption of biosynthesis or the function of a membrane cAMP-activated chloride channel, CFTR. CFTR regulatory mechanisms include recruitment of channel proteins to the cell surface from intracellular pools and by protein-protein interactions. Rab proteins are small GTPases involved in regulated trafficking controlling vesicle docking and fusion. Rab4 controls recycling events from endosome to the plasma membrane, fusion, and degradation. The colorectal cell line HT-29 natively expresses CFTR and responds to cAMP stimulation with an increase in CFTR-mediated currents. Rab4 over-expression in HT-29 cells inhibits both basal and cAMP-stimulated CFTR-mediated currents. GTPase-deficient Rab4Q67L and GDP locked Rab4S22N both inhibit channel activity, which appears characteristically different. Active status of Rab4 was confirmed by GTP overlay assay, while its expression was verified by Western blotting. The pull-down and immunoprecipitation experiments suggest that Rab4 physically interacts with CFTR through protein-protein interaction. Biotinylation with cell impermeant NHS-Sulfo-SS-Biotin implies that Rab4 impairs CFTR expression at cell surface. The enhanced cytosolic CFTR indicates that Rab4 expression restrains CFTR appearance at the cell membrane. The study suggests that Rab4 regulates the channel through multiple mechanisms that include protein-protein interaction, GTP/GDP exchange, and channel protein trafficking. We propose that Rab4 is a dynamic molecule with a significant role in CFTR function.

  10. Porin alterations present in non-carbapenemase-producing Enterobacteriaceae with high and intermediate levels of carbapenem resistance in Chile.

    PubMed

    Wozniak, Aniela; Villagra, Nicolás A; Undabarrena, Agustina; Gallardo, Natalia; Keller, Nicole; Moraga, Marcela; Román, Juan C; Mora, Guido C; García, Patricia

    2012-09-01

    The main goal of this work was to identify the mechanisms responsible for carbapenem resistance in 61 Chilean clinical isolates of Enterobacteriaceae (Enterobacter spp., Serratia marcescens, Morganella morganii, Escherichia coli and Klebsiella pneumoniae) with reduced susceptibility to at least one carbapenem (ertapenem, imipenem or meropenem). All of the isolates were analysed for the presence of carbapenemases, extended spectrum β-lactamases (ESBLs), AmpC enzymes and outer-membrane proteins. None of the isolates exhibited carbapenemase activity nor did they have any of the carbapenemase genes that were screened for. Most of the 61 strains produced at least one ESBL and/or one AmpC enzyme and either lost their porins or had altered porins according to sequence analysis. The distribution of ESBLs and AmpC enzymes was different among the species studied. Resistance in K. pneumoniae and E. coli isolates was associated with ESBLs; in M. morganii isolates, resistance was attributed to overexpression of an AmpC enzyme; and in Enterobacter spp. isolates, resistance was associated with both types of enzymes. In K. pneumoniae isolates, porin integrity was more a determinant of carbapenem resistance than the presence of ESBLs, whereas in isolates of Enterobacter spp., M. morganii and S. marcescens, the presence of an overexpressed AmpC enzyme was associated with higher imipenem and meropenem MIC values. Therefore, carbapenem resistance in Chilean isolates is not due to true carbapenemases but rather to a combination of porin loss/alteration and β-lactamase activity. The fact that carbapenemases were not detected in this study is unique, given that many countries in the region have already reported the presence of these enzymes. PMID:22700549

  11. Cleavage of Nidogen-1 by Cathepsin S Impairs Its Binding to Basement Membrane Partners

    PubMed Central

    Sage, Juliette; Leblanc-Noblesse, Emmanuelle; Nizard, Carine; Sasaki, Takako; Schnebert, Sylvianne; Perrier, Eric; Kurfurst, Robin; Brömme, Dieter; Lalmanach, Gilles; Lecaille, Fabien

    2012-01-01

    Cathepsin S (catS), which is expressed in normal human keratinocytes and localized close to the dermal-epidermal junction (DEJ) degrades some of major basement membrane (BM) constituents. Among them, catS readily hydrolyzed in a time and dose dependent manner human nidogen-1 (nid-1) and nidogen-2, which are key proteins in the BM structure. CatS preferentially cleaved nid-1 at both acid and neutral pH. Hydrolysis of nid-1 was hampered in murine ctss−/− spleen lysates pretreated with inhibitors of other classes of proteases. Nid-1 was cleaved within its G2 and G3 globular domains that are both involved in interactions with other BM components. Binding assays with soluble and immobilized ligands indicated that catS altered the formation of complexes between nid-1 and other BM components. Assuming that the cleavage of nid-1 impairs its ability to crosslink with BM partners and perturbs the viscoelastic properties of BM matrix, these data indicate that catS may participate in BM proteolysis, in addition to already identified proteases. PMID:22952693

  12. The Oms66 (p66) protein is a Borrelia burgdorferi porin.

    PubMed Central

    Skare, J T; Mirzabekov, T A; Shang, E S; Blanco, D R; Erdjument-Bromage, H; Bunikis, J; Bergström, S; Tempst, P; Kagan, B L; Miller, J N; Lovett, M A

    1997-01-01

    In this study we report the purification and characterization of a 66-kDa protein, designated Oms66, for outer membrane-spanning 66-kDa protein, that functions as a porin in the outer membrane (OM) of Borrelia burgdorferi. Oms66 was purified by fast-performance liquid chromatography and exhibited an average single-channel conductance of 9.62 +/- 0.37 nS in 1 M KCl, as evidenced by 581 individual insertional events in planar lipid bilayers. Electrophysiological characterization indicated that Oms66 was virtually nonselective between cations and anions and exhibited voltage-dependent closure with multiple substates. The amino acid sequence of tryptic peptides derived from purified Oms66 was identical to the deduced amino acid sequence of p66, a previously described surface-exposed protein of B. burgdorferi. Purified Oms66 was recognized by antiserum specific for p66 and serum from rabbits immune to challenge with virulent B. burgdorferi, indicating that p66 and Oms66 were identical proteins and that Oms66/p66 is an immunogenic protein in infected rabbits. In a methodology that reduces liposomal trapping and nonspecific interactions, native Oms66 was incorporated into liposomes, confirming that Oms66 is an outer membrane-spanning protein. Proteoliposomes containing Oms66 exhibited porin activity nearly identical to that of native, purified Oms66, indicating that reconstituted Oms66 retained native conformation. The use of proteoliposomes reconstituted with Oms66 and other Oms proteins provides an experimental system for determinating the relationship between conformation, protection, and biological function of these molecules. PMID:9284133

  13. Amyloid beta-peptide impairs glucose transport in hippocampal and cortical neurons: involvement of membrane lipid peroxidation.

    PubMed

    Mark, R J; Pang, Z; Geddes, J W; Uchida, K; Mattson, M P

    1997-02-01

    A deficit in glucose uptake and a deposition of amyloid beta-peptide (A beta) each occur in vulnerable brain regions in Alzheimer's disease (AD). It is not known whether mechanistic links exist between A beta deposition and impaired glucose transport. We now report that A beta impairs glucose transport in cultured rat hippocampal and cortical neurons by a mechanism involving membrane lipid peroxidation. A beta impaired 3H-deoxy-glucose transport in a concentration-dependent manner and with a time course preceding neurodegeneration. The decrease in glucose transport was followed by a decrease in cellular ATP levels. Impairment of glucose transport, ATP depletion, and cell death were each prevented in cultures pretreated with antioxidants. Exposure to FeSO4, an established inducer of lipid peroxidation, also impaired glucose transport. Immunoprecipitation and Western blot analyses showed that exposure of cultures to A beta induced conjugation of 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, to the neuronal glucose transport protein GLUT3. HNE induced a concentration-dependent impairment of glucose transport and subsequent ATP depletion. Impaired glucose transport was not caused by a decreased energy demand in the neurons, because ouabain, which inhibits Na+/K(+)-ATPase activity and thereby reduces neuronal ATP hydrolysis rate, had little or no effect on glucose transport. Collectively, the data demonstrate that lipid peroxidation mediates A beta-induced impairment of glucose transport in neurons and suggest that this action of A beta may contribute to decreased glucose uptake and neuronal degeneration in AD. PMID:8994059

  14. VDAC and the bacterial porin PorB of Neisseria gonorrhoeae share mitochondrial import pathways.

    PubMed

    Müller, Anne; Rassow, Joachim; Grimm, Jan; Machuy, Nikolaus; Meyer, Thomas F; Rudel, Thomas

    2002-04-15

    The human pathogen Neisseria gonorrhoeae induces host cell apoptosis during infection by delivering the outer membrane protein PorB to the host cell's mitochondria. PorB is a pore-forming beta-barrel protein sharing several features with the mitochondrial voltage-dependent anion channel (VDAC), which is involved in the regulation of apoptosis. Here we show that PorB of pathogenic Neisseria species produced by host cells is efficiently targeted to mitochondria. Imported PorB resides in the mitochondrial outer membrane and forms multimers with similar sizes as in the outer bacterial membrane. The mitochondria completely lose their membrane potential, a characteristic previously observed in cells infected with gonococci or treated with purified PorB. Closely related bacterial porins of non-pathogenic Neisseria mucosa or Escherichia coli remain in the cytosol. Import of PorB into mitochondria in vivo is independent of a linear signal sequence. Insertion of PorB into the mitochondrial outer membrane in vitro depends on the activity of Tom5, Tom20 and Tom40, but is independent of Tom70. Our data show that human VDAC and bacterial PorB are imported into mitochondria by a similar mechanism. PMID:11953311

  15. Triiodothyronine activates lactate oxidation without impairing fatty acid oxidation and improves weaning from extracorporeal membrane oxygenation

    SciTech Connect

    Kajimoto, Masaki; Ledee, Dolena R.; Xu, Chun; Kajimoto, Hidemi; Isern, Nancy G.; Portman, Michael A.

    2014-01-01

    Background: Extracorporeal membrane oxygenation (ECMO) provides a rescue for children with severe cardiac failure. We previously showed that triiodothyronine (T3) improves cardiac function by modulating pyruvate oxidation during weaning. This study was focused on fatty acid (FA) metabolism modulated by T3 for weaning from ECMO after cardiac injury. Methods: Nineteen immature piglets (9.1-15.3 kg) were separated into 3 groups with ECMO (6.5 hours) and wean: normal circulation (Group-C);transient coronary occlusion (10 minutes) followed by ECMO (Group-IR); and IR with T3 supplementation (Group-IR-T3). 13-Carbon labeled lactate, medium-chain and long-chain FAs were infused as oxidative substrates. Substrate fractional contribution to the citric acid cycle (FC) was analyzed by 13-Carbon nuclear magnetic resonance. Results: ECMO depressed circulating T3 levels to 40% baseline at 4 hours and were restored in Group-IR-T3. Group-IR decreased cardiac power, which was not fully restorable and 2 pigs were lost because of weaning failure. Group-IR also depressed FC-lactate, while the excellent contractile function and energy efficiency in Group-IR-T3 occurred along with a marked FC-lactate increase and [ATP]/[ADP] without either decreasing FC-FAs or elevating myocardial oxygen consumption over Group-C or -IR. Conclusions: T3 releases inhibition of lactate oxidation following ischemia-reperfusion injury without impairing FA oxidation. These findings indicate that T3 depression during ECMO is maladaptive, and that restoring levels improves metabolic flux and enhances contractile function during weaning.

  16. Genetic Analysis of Arabidopsis Mutants Impaired in Plastid Lipid Import Reveals a Role of Membrane Lipids in Chloroplast Division

    SciTech Connect

    Fan, J.; Xu, C.

    2011-03-01

    The biogenesis of photosynthetic membranes in plants relies largely on lipid import from the endoplasmic reticulum (ER) and this lipid transport process is mediated by TGD proteins in Arabidopsis. Such a dependency of chloroplast biogenesis on ER-to-plastid lipid transport was recently exemplified by analyzing double mutants between tgd1-1 or tgd4-3 and fad6 mutants. The fad6 mutants are defective in the desaturation of membrane lipids in chloroplasts and therefore dependent on import of polyunsaturated lipid precursors from the ER for constructing a competent thylakoid membrane system. In support of a critical role of TGD proteins in ER-to-plastid lipid trafficking, we showed that the introduction of the tgd mutations into fad6 mutant backgrounds led to drastic reductions in relative amounts of thylakoid lipids. Moreover, the tgd1-1 fad6 and tgd4-3 fad6 double mutants were deficient in polyunsaturated fatty acids in chloroplast membrane lipids, and severely compromised in the biogenesis of photosynthetic membrane systems. Here we report that these double mutants are severely impaired in chloroplast division. The possible role of membrane lipids in chloroplast division is discussed.

  17. Rhodococcus jostii porin A (RjpA) functions in cholate uptake.

    PubMed

    Somalinga, Vijayakumar; Mohn, William W

    2013-10-01

    RjpA in Rhodococcus jostii is the ortholog of a channel-forming porin, MspA. Deletion of rjpA delayed growth of R. jostii on cholate but not on cholesterol. Eventual growth on cholate involved increased expression of other porins, namely, RjpB, RjpC, and RjpD. Porins appear essential for the uptake of bile acids by mycolic acid bacteria. PMID:23892747

  18. 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes.

    PubMed

    Keller, J N; Mark, R J; Bruce, A J; Blanc, E; Rothstein, J D; Uchida, K; Waeg, G; Mattson, M P

    1997-10-01

    Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimer's, Parkinson's and Huntington's diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4

  19. The porin VDAC2 is the mitochondrial platform for Bax retrotranslocation.

    PubMed

    Lauterwasser, Joachim; Todt, Franziska; Zerbes, Ralf M; Nguyen, Thanh Ngoc; Craigen, William; Lazarou, Michael; van der Laan, Martin; Edlich, Frank

    2016-01-01

    The pro-apoptotic Bcl-2 protein Bax can permeabilize the outer mitochondrial membrane and therefore commit human cells to apoptosis. Bax is regulated by constant translocation to the mitochondria and retrotranslocation back into the cytosol. Bax retrotranslocation depends on pro-survival Bcl-2 proteins and stabilizes inactive Bax. Here we show that Bax retrotranslocation shuttles membrane-associated and membrane-integral Bax from isolated mitochondria. We further discover the mitochondrial porin voltage-dependent anion channel 2 (VDAC2) as essential component and platform for Bax retrotranslocation. VDAC2 ensures mitochondria-specific membrane association of Bax and in the absence of VDAC2 Bax localizes towards other cell compartments. Bax retrotranslocation is also regulated by nucleotides and calcium ions, suggesting a potential role of the transport of these ions through VDAC2 in Bax retrotranslocation. Together, our results reveal the unanticipated bifunctional role of VDAC2 to target Bax specifically to the mitochondria and ensure Bax inhibition by retrotranslocation into the cytosol. PMID:27620692

  20. Journey of poly-nucleotides through OmpF porin.

    PubMed

    Hadi-Alijanvand, Hamid; Rouhani, Maryam

    2015-05-21

    OmpF is an abundant porin in many bacteria which attracts attention as a promising biological nanopore for DNA sequencing. We study the interactions of OmpF with pentameric poly-nucleotides (poly-Ns) in silico. The poly-N molecule is forced to translocate through the lumen of OmpF. Subsequently, the structural and dynamical effects of translocation steps on protein and poly-N molecules are explored in detail. The external loops of OmpF are introduced as the main region for discrimination of poly-Ns based on their organic bases. Structural network analyses of OmpF in the presence or absence of poly-Ns characterize special residues in the structural network of porin. These residues pave the way for engineering OmpF protein. The poly-N-specific pattern of OmpF's local conductance is detected in the current study. Computing the potential of mean force for translocation steps, we define the energetic barrier ahead of poly-N to move through OmpF's lumen. We suggest that fast translocation of the examined poly-N molecules through OmpF seems unattainable by small external driving forces. Our computational results suggest some abilities for OmpF porin like OmpF's potential for being used in poly-N sequencing. PMID:25965338

  1. Purification and characterization of protein H, the major porin of Pasteurella multocida.

    PubMed Central

    Chevalier, G; Duclohier, H; Thomas, D; Shechter, E; Wróblewski, H

    1993-01-01

    Protein H (B. Lugtenberg, R. van Boxtel, D. Evenberg, M. de Jong, P. Storm, and J. Frik, Infect. Immun. 52:175-182, 1986) is the major polypeptide of the outer membrane of Pasteurella multocida, a bacterium pathogenic for humans and animals. We have purified this protein to homogeneity by size exclusion chromatography after selective extraction with surfactants and demonstrated its pore-forming ability after reincorporation into planar lipid bilayers. In these experiments, the current through the pores was a linear function of the applied voltage in the range of -50 to +50 mV. Voltages beyond +/- 50 mV tended to partially close the channels, giving rise to apparent negative resistances. These observations suggest that protein H channels are probably not voltage regulated in vivo. With the patch clamp technique, single-channel conductance fluctuations of 0.33 nS were recorded in 1 M KCl. Electrophoretic and circular dichroism analyses showed that protein H forms homotrimers stable in sodium dodecyl sulfate at room temperature, with a high content of beta-sheet secondary structure. Upon boiling, the trimers were fully dissociated into monomers with an increase of alpha helix and irregular structure, at the expense of beta sheets. The apparent molecular mass of fully denatured monomers ranged between 37 and 41.8 kDa, depending on the electrophoretic system used for analysis. The trimeric arrangement of protein H was confirmed by image analysis of negatively stained, two-dimensional crystal arrays. This morphological study revealed, in agreement with electrophoretical data, a trimeric structure with an overall diameter of 7.7 nm. Each monomer appeared to contain a pore with an average diameter of 1 nm. Quantitative comparisons revealed that the amino acid composition (hydropathy index of -0.40) and the N-terminal sequence (determined over 36 residues) of protein H are similar to those of bacterial general porins, notably porin P2 of Haemophilus influenzae. We conclude

  2. Structure-kinetic relationship of carbapenem antibacterials permeating through E. coli OmpC porin.

    PubMed

    Tran, Que-Tien; Pearlstein, Robert A; Williams, Sarah; Reilly, John; Krucker, Thomas; Erdemli, Gül

    2014-11-01

    The emergence of Gram-negative "superbugs" exhibiting resistance to known antibacterials poses a major public health concern. Low molecular weight Gram-negative antibacterials are believed to penetrate the outer bacterial membrane (OM) through porin channels. Therefore, intracellular exposure needed to drive antibacterial target occupancy should depend critically on the translocation rates through these proteins and avoidance of efflux pumps. We used electrophysiology to study the structure-translocation kinetics relationships of a set of carbapenem antibacterials through purified porin OmpC reconstituted in phospholipid bilayers. We also studied the relative susceptibility of OmpC+ and OmpC- E. coli to these compounds as an orthogonal test of translocation. Carbapenems exhibit good efficacy in OmpC-expressing E. coli cells compared with other known antibacterials. Ertapenem, which contains an additional acidic group compared to other analogs, exhibits the fastest entry into OmpC (k(on) ≈ 2 × 10(4) M(-1) s(-1)). Zwitterionic compounds with highly polar groups attached to the penem-2 ring, including panipenem, imipenem and doripenem exhibit faster k(on) (>10(4) M(-1) s(-1)), while meropenem and biapenem with fewer exposed polar groups exhibit slower k(on) (∼5 × 10(3) M(-1) s(-1)). Tebipenem pivoxil and razupenem exhibit ∼13-fold slower k(on) (∼1.5 × 10(3) M(-1) s(-1)) than ertapenem. Overall, our results suggest that (a) OmpC serves as an important route of entry of these antibacterials into E. coli cells; and (b) that the structure-kinetic relationships of carbapenem translocation are governed by H-bond acceptor/donor composition (in accordance with our previous findings that the enthalpic cost of transferring water from the constriction zone to bulk solvent increases in the presence of exposed nonpolar groups). PMID:25082756

  3. Outer membrane protein purification.

    PubMed

    Arigita, C; Jiskoot, W; Graaf, M R; Kersten, G F

    2001-01-01

    The major outer membrane proteins (OMPs) from Neisseria meningitidis, which are expressed at high levels, are subdivided in five classes based on molecular weight (1,2) (see Table 1). Table 1 Major Meningococcal Outer-Membrane Proteins Outer-membrane proteins Name Molecular maass Function/characteristics Class 1 PorA 44-47 kDa Porin Class 2/3 PorB 37-42 kDa Porin Class 4 Rmp Reductionmodifiableprotein, unknown Class 5 Opa 26-30 kDa Adhesion,opacity protein Opc 25 kDa Invasion, opacity protein Iron-regulated proteins Mirp 37 kDa Iron acquisition (?);majoriron-regulatedprotein FrpB 70 kDa Ferric enterobactin receptor (also FetA) Adapted from ref. (1). PMID:21336748

  4. Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor.

    PubMed

    Clinton, Ryan W; Francy, Christopher A; Ramachandran, Rajesh; Qi, Xin; Mears, Jason A

    2016-01-01

    Mitochondrial fission is a crucial cellular process mediated by the mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1). During mitochondrial division, Drp1 is recruited from the cytosol to the outer mitochondrial membrane by one, or several, integral membrane proteins. One such Drp1 partner protein, mitochondrial fission factor (Mff), is essential for mitochondrial division, but its mechanism of action remains unexplored. Previous studies have been limited by a weak interaction between Drp1 and Mff in vitro. Through refined in vitro reconstitution approaches and multiple independent assays, we show that removal of the regulatory variable domain (VD) in Drp1 enhances formation of a functional Drp1-Mff copolymer. This protein assembly exhibits greatly stimulated cooperative GTPase activity in solution. Moreover, when Mff was anchored to a lipid template, to mimic a more physiologic environment, significant stimulation of GTPase activity was observed with both WT and ΔVD Drp1. Contrary to recent findings, we show that premature Drp1 self-assembly in solution impairs functional interactions with membrane-anchored Mff. Instead, dimeric Drp1 species are selectively recruited by Mff to initiate assembly of a functional fission complex. Correspondingly, we also found that the coiled-coil motif in Mff is not essential for Drp1 interactions, but rather serves to augment cooperative self-assembly of Drp1 proximal to the membrane. Taken together, our findings provide a mechanism wherein the multimeric states of both Mff and Drp1 regulate their collaborative interaction. PMID:26578514

  5. Interleukin-1 and interleukin-6 gene expression in human monocytes stimulated with Salmonella typhimurium porins.

    PubMed Central

    Galdiero, M; Cipollaro de L'ero, G; Donnarumma, G; Marcatili, A; Galdiero, F

    1995-01-01

    The aim of this study was to verify whether Salmonella typhimurium porins can affect the expression of interleukin-1 (IL-1) and interleukin-6 (IL-6) genes. Human monocytes were treated with porins, and total RNAs were analysed by Northern blotting to evaluate the expression of IL-1 alpha, IL-1 beta and IL-6 in both treated and untreated cell cultures. Porins induced a significant increase in IL-1 and IL-6 transcripts. This increase was related to the dose of porins, and it peaked 5 hr after treatment. The same results were obtained when polymyxin B was added to the porin preparation to eliminate eventual traces of lipopolysaccharide (LPS) associated with porins. The porins-mediated increase in interleukin transcripts did not require de novo protein synthesis, and it was because of the enhanced half-life of IL-1 and IL-6 mRNAs, rather an increased rate of gene transcription. These data suggest that porins may affect inflammatory and immunological responses by enhancing the expression of cytokine genes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8567029

  6. Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC

    PubMed Central

    Hacker, Christian; Howell, Matthew; Bhella, David; Lucocq, John

    2013-01-01

    Summary Microsporidia are obligate intracellular parasites with extremely reduced genomes and a dependence on host-derived ATP. The microsporidium Encephalitozoon cuniculi proliferates within a membranous vacuole and we investigated how the ATP supply is optimized at the vacuole–host interface. Using spatial EM quantification (stereology), we found a single layer of mitochondria coating substantial proportions of the parasitophorous vacuole. Mitochondrial binding occurred preferentially over the vegetative ‘meront’ stages of the parasite, which bulged into the cytoplasm, thereby increasing the membrane surface available for mitochondrial interaction. In a broken cell system mitochondrial binding was maintained and was typified by electron dense structures (< 10 nm long) bridging between outer mitochondrial and vacuole membranes. In broken cells mitochondrial binding was sensitive to a range of protease treatments. The function of directly bound mitochondria, as measured by the membrane potential sensitive dye JC-1, was indistinguishable from other mitochondria in the cell although there was a generalized depression of the membrane potential in infected cells. Finally, quantitative immuno-EM revealed that the ATP-delivering mitochondrial porin, VDAC, was concentrated atthe mitochondria-vacuole interaction site. Thus E. cuniculi appears to maximize ATP supply by direct binding of mitochondria to the parasitophorous vacuole bringing this organelle within 0.020 microns of the growing vegetative form of the parasite. ATP-delivery is further enhanced by clustering of ATP transporting porins in those regions of the outer mitochondrial membrane lying closest to the parasite. PMID:24245785

  7. Treatment of membrane surfaces with oriented monolayers for improved renovation of impaired water. Final report

    SciTech Connect

    Speaker, L.M.; Russo, G.M.; Quinn, K.B.; Wolf, B.L.; Whitt, T.P.

    1991-04-21

    AFT (antifouling technology) for separatory membranes was initially demonstrated in 1981. The effort summarized here was designed to test AFT against humates and alternative foulants, enhancing the fundamental understanding of fouling and fouling propensity. Investigator surface-modified membranes with Langmuir-Blodgett (LB) films (oriented monomolecular films) and monitored responses of selectivities and throughputs to challenges. The materials bank included 18 surfactants, five different types of separatory membranes, and 15 likely foulants. At least 300 membranes were surface-modified and their permeation characteristics followed in more than 150 computer-controlled 23-h challenges, in most cases with aqueous sodium humate. LB-system vibration problems may have compromised the quality and fouling resistance of some coatings. Both selectivities and cumulative permeate volumes of membranes LB-coated with 11-perfluorohexyl undecanoic acid, 11-perfluoroheptyl undecanyl pyridinium bromide, and 11-perfluorooctyl undecanyl pyridinium bromide were superior to those of controls, indicating that LB treatment had simultaneously increased both selectivity and fouling-resistance. More data is being analyzed; as the database expands, investigators expect to contribute to the mechanisitic understanding of fouling and fouling prevention, as well as developing selection criteria for the best (least fouling) combinations of AFT treatment, membrane, and feed stream.

  8. Cloning, Sequencing, and Role in Serum Susceptibility of Porin II from Mesophilic Aeromonas hydrophila

    PubMed Central

    Nogueras, Maria Mercé; Merino, Susana; Aguilar, Alicia; Benedi, Vicente Javier; Tomás, Juan M.

    2000-01-01

    We cloned and sequenced the structural gene for Aeromonas hydrophila porin II from strain AH-3 (serogroup O:34). The genetic position of this gene, like that of ompF in Escherichia coli, is adjacent to aspC and transcribed in the same direction. However, upstream of the porin II gene no similarities with E. coli were found. We obtained defined insertion mutants in porin II gene either in A. hydrophila (O:34) or A. veronii sobria (serogroup O:11) serum-resistant or -sensitive strains. Furthermore, we complemented these mutants with a plasmid harboring only the porin II gene, which allowed us to define the role of porin II as an important surface molecule involved in serum susceptibility and C1q binding in these strains. PMID:10722573

  9. Monogalactosyldiacylglycerol deficiency in Arabidopsis affects pigment composition in the prolamellar body and impairs thylakoid membrane energization and photoprotection in leaves.

    PubMed

    Aronsson, Henrik; Schöttler, Mark A; Kelly, Amélie A; Sundqvist, Christer; Dörmann, Peter; Karim, Sazzad; Jarvis, Paul

    2008-09-01

    Monogalactosyldiacylglycerol (MGDG) is the major lipid constituent of chloroplast membranes and has been proposed to act directly in several important plastidic processes, particularly during photosynthesis. In this study, the effect of MGDG deficiency, as observed in the monogalactosyldiacylglycerol synthase1-1 (mgd1-1) mutant, on chloroplast protein targeting, phototransformation of pigments, and photosynthetic light reactions was analyzed. The targeting of plastid proteins into or across the envelope, or into the thylakoid membrane, was not different from wild-type in the mgd1 mutant, suggesting that the residual amount of MGDG in mgd1 was sufficient to maintain functional targeting mechanisms. In dark-grown plants, the ratio of bound protochlorophyllide (Pchlide, F656) to free Pchlide (F631) was increased in mgd1 compared to the wild type. Increased levels of the photoconvertible pigment-protein complex (F656), which is photoprotective and suppresses photooxidative damage caused by an excess of free Pchlide, may be an adaptive response to the mgd1 mutation. Leaves of mgd1 suffered from a massively impaired capacity for thermal dissipation of excess light due to an inefficient operation of the xanthophyll cycle; the mutant contained less zeaxanthin and more violaxanthin than wild type after 60 min of high-light exposure and suffered from increased photosystem II photoinhibition. This is attributable to an increased conductivity of the thylakoid membrane at high light intensities, so that the proton motive force is reduced and the thylakoid lumen is less acidic than in wild type. Thus, the pH-dependent activation of the violaxanthin de-epoxidase and of the PsbS protein is impaired. PMID:18641085

  10. Acute cholesterol depletion impairs functional expression of tissue factor in fibroblasts: modulation of tissue factor activity by membrane cholesterol

    PubMed Central

    Mandal, Samir K.; Iakhiaev, Alexei; Pendurthi, Usha R.; Mohan Rao, L. Vijaya

    2010-01-01

    Cholesterol, in addition to providing rigidity to the fluid membrane, plays a critical role in receptor function, endocytosis, recycling, and signal transduction. In the present study, we examined the effect of membrane cholesterol on functional expression of tissue factor (TF), a cellular receptor for clotting factor VIIa. Depletion of cholesterol in human fibroblasts (WI-38) with methyl-β-cyclodextrin–reduced TF activity at the cell surface. Binding studies with radiolabeled VIIa and TF monoclonal antibody (mAB) revealed that reduced TF activity in cholesterol-depleted cells stems from the impairment of VIIa interaction with TF rather than the loss of TF receptors at the cell surface. Repletion of cholesterol-depleted cells with cholesterol restored TF function. Loss of caveolar structure on cholesterol removal is not responsible for reduced TF activity. Solubilization of cellular TF in different detergents indicated that a substantial portion of TF in fibroblasts is associated with noncaveolar lipid rafts. Cholesterol depletion studies showed that the TF association with these rafts is cholesterol dependent. Overall, the data presented herein suggest that membrane cholesterol functions as a positive regulator of TF function by maintaining TF receptors, probably in noncaveolar lipid rafts, in a high-affinity state for VIIa binding. PMID:15328160

  11. Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

    PubMed

    Raffaele, Sylvain; Bayer, Emmanuelle; Lafarge, David; Cluzet, Stéphanie; German Retana, Sylvie; Boubekeur, Tamy; Leborgne-Castel, Nathalie; Carde, Jean-Pierre; Lherminier, Jeannine; Noirot, Elodie; Satiat-Jeunemaître, Béatrice; Laroche-Traineau, Jeanny; Moreau, Patrick; Ott, Thomas; Maule, Andrew J; Reymond, Philippe; Simon-Plas, Françoise; Farmer, Edward E; Bessoule, Jean-Jacques; Mongrand, Sébastien

    2009-05-01

    Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane. PMID:19470590

  12. Effects of impaired membrane interactions on α-synuclein aggregation and neurotoxicity

    PubMed Central

    Ysselstein, Daniel; Joshi, Mehul; Mishra, Vartika; Griggs, Amy M.; Asiago, Josephat M.; McCabe, George P.; Stanciu, Lia A.; Post, Carol Beth; Rochet, Jean-Christophe

    2015-01-01

    The post-mortem brains of individuals with Parkinson’s disease (PD) and other synucleinopathy disorders are characterized by the presence of aggregated forms of the presynaptic protein α-synuclein (aSyn). Understanding the molecular mechanism of aSyn aggregation is essential for the development of neuroprotective strategies to treat these diseases. In this study, we examined how interactions between aSyn and phospholipid vesicles influence the protein’s aggregation and toxicity to dopaminergic neurons. Two-dimensional NMR data revealed that two familial aSyn mutants, A30P and G51D, populated an exposed, membrane-bound conformer in which the central hydrophobic region was dissociated from the bilayer to a greater extent than in the case of wild-type aSyn. A30P and G51D had a greater propensity to undergo membrane-induced aggregation and elicited greater toxicity to primary dopaminergic neurons compared to the wild-type protein. In contrast, the non-familial aSyn mutant A29E exhibited a weak propensity to aggregate in the presence of phospholipid vesicles or to elicit neurotoxicity, despite adopting a relatively exposed membrane-bound conformation. Our findings suggest that the aggregation of exposed, membrane-bound aSyn conformers plays a key role in the protein’s neurotoxicity in PD and other synucleinopathy disorders. PMID:25931201

  13. Relationship of structural to functional impairment during alveolar-capillary membrane development.

    PubMed

    Ahlfeld, Shawn K; Gao, Yong; Conway, Simon J; Tepper, Robert S

    2015-04-01

    Bronchopulmonary dysplasia is a chronic lung disease of extreme preterm infants and results in impaired gas exchange. Although bronchopulmonary dysplasia is characterized histologically by alveolar-capillary simplification in animal models, it is clinically defined by impaired gas diffusion. With the use of a developmentally relevant model, we correlated alveolar-capillary structural simplification with reduced functional gas exchange as measured by the diffusing factor for carbon monoxide (DFCO). Neonatal mouse pups were exposed to >90% hyperoxia or room air during postnatal days 0 to 7, and then all pups were returned to room air from days 7 to 56. At day 56, DFCO was measured as the ratio of carbon monoxide uptake to neon dilution, and lungs were fixed for histologic assessment of alveolar-capillary development. Neonatal hyperoxia exposure inhibited alveolar-capillary septal development as evidenced by significantly increased mean linear intercept, increased airspace-to-septal ratio, decreased nodal density, and decreased pulmonary microvasculature. Importantly, alveolar-capillary structural deficits in hyperoxia-exposed pups were accompanied by a significant 28% decrease in DFCO (0.555 versus 0.400; P < 0.0001). In addition, DFCO was highly and significantly correlated with structural measures of reduced alveolar-capillary growth. Simplification of alveolar-capillary structure is highly correlated with impaired gas exchange function. Current mechanistic and therapeutic animal models of inhibited alveolar development may benefit from application of DFCO as an alternative physiologic indicator of alveolar-capillary development. PMID:25661110

  14. Hypercapnic acidosis impairs plasma membrane wound resealing in ventilator-injured lungs.

    PubMed

    Doerr, Clinton H; Gajic, Ognjen; Berrios, Jorge C; Caples, Sean; Abdel, Matthew; Lymp, James F; Hubmayr, Rolf D

    2005-06-15

    The objective of this study was to assess the effects of hypercapnic acidosis on lung cell injury and repair by confocal microscopy in a model of ventilator-induced lung injury. Three groups of normocapnic, hypocapnic, and hypercapnic rat lungs were perfused ex vivo, either during or after injurious ventilation, with a solution containing the membrane-impermeant label propidium iodide. In lungs labeled during injurious ventilation, propidium iodide fluorescence identifies all cells with plasma membrane wounds, both permanent and transient, whereas in lungs labeled after injurious ventilation propidium iodide fluorescence identifies only cells with permanent plasma membrane wounds. Hypercapnia minimized the adverse effects of high-volume ventilation on vascular barrier function, whereas hypocapnia had the opposite effect. Despite CO2-dependent differences in lung mechanics and edema the number of injured subpleural cells per alveolus was similar in the three groups (0.48 +/- 0.34 versus 0.51 +/- 0.19 versus 0.43 +/- 0.20 for hypocapnia, normocapnia, and hypercapnia, respectively). However, compared with normocapnia the probability of wound repair was significantly reduced in hypercapnic lungs (63 versus 38%; p < 0.02). This finding was subsequently confirmed in alveolar epithelial cell scratch models. The potential relevance of these observations for lung inflammation and remodeling after mechanical injury is discussed. PMID:15695495

  15. Deficiency in the inner mitochondrial membrane peptidase 2-like (Immp21) gene increases ischemic brain damage and impairs mitochondrial function

    PubMed Central

    Ma, Yi; Mehta, Suresh L.; Lu, Baisong; Andy Li, P.

    2011-01-01

    Mitochondrial dysfunction plays an important role in mediating ischemic brain damage. Immp2l is an inner mitochondrial membrane peptidase that processes mitochondrial proteins cytochrome c1 (Cyc1). Homozygous mutation of Immp2l (Immp2lTg(Tyr)979Ove or Immp2l−/−) elevates mitochondrial membrane potential, increases superoxide (•O2−) production in the brain and impairs fertility. The objectives of this study are to explore the effects of heterozygous mutation of lmmp2l (Immp2l+/−) on ischemic outcome and to determine the influence of Immp2l deficiency on brain mitochondria after stroke. Male Immp2l+/− and wild-type (WT) mice were subjected to 1-h focal cerebral ischemia. Their brains were harvested after 5 and 24-h of reperfusion. The results showed that infarct volume and DNA oxidative damage significantly increased in the Immp2l+/− mice. There were no obvious cerebral vasculature abnormalities between the two types of mice viewed by Indian ink perfusion. The increased damage in Immp2l+/− mice was associated with early increase in •O2− production. Mitochondrial respiratory rate, total mitochondrial respiratory capacity and mitochondrial respiratory complex activities were decreased at 5-h of recirculation in Immp2l+/− mice compared to WT mice. Our results suggest that lmmp2l deficiency increases ischemic brain damage by enhancing •O2− production and damaging mitochondrial functional performance. PMID:21824519

  16. Cell killing by simian virus 40: impairment of membrane formation and function.

    PubMed Central

    Norkin, L C

    1977-01-01

    Simian virus 40 infection of the CV-1 line of green monkey kidney cells results in the release of mitochondrial malic dehydrogenase as early as 24 h. Released malic dehydrogenase is detected in the cytoplasm prior to its appearance in the overlay medium. Infected cells lose the ability to consume oxygen between 48 and 56 h, and damage to the elctron transport system is indicated. Nevertheless, cellular ATP levels remain high as late as 72 h. Infection leads to a stimulation of membrane phospholipid synthesis, which reaches a peak at about 32 h. This is followed by a severe decline in new membrane synthesis, which correlates in time with the release of cytoplasmic lactic dehydrogenase into the overlay media. Lactic dehydrogenase release precedes the accumulation of trypan blue-stainable cells by about 6 h. Infection had no effect on the turnover of prelabeled membrane phospholipids. An early simian virus 40 mutant, tsA58, and a late mutant, tsB11, are both less effective than wild-type virus at causing reduced levels of phospholipid synthesis, enzyme release, and the accumulation of trypan blue-stainable cells. Another late mutant, tsB8, is similar to wild-type virus in these respects. At 64 h, there is no detectable cell-associated lactic dehydrogenase and nearly all the cells are trypan blue stainable. Nevertheless, at concentrations of deoxyglucose in the medium below the transport Km, deoxyglucose uptake was similar in infected and control cultures. With higher concentrations of deoxyglucose in the medium, uptake by the infected cultures exceeded that by the control cultures. PMID:191651

  17. The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions*

    PubMed Central

    Münch, Daniela; Müller, Anna; Schneider, Tanja; Kohl, Bastian; Wenzel, Michaela; Bandow, Julia Elisabeth; Maffioli, Sonia; Sosio, Margherita; Donadio, Stefano; Wimmer, Reinhard; Sahl, Hans-Georg

    2014-01-01

    The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell. PMID:24627484

  18. The lantibiotic NAI-107 binds to bactoprenol-bound cell wall precursors and impairs membrane functions.

    PubMed

    Münch, Daniela; Müller, Anna; Schneider, Tanja; Kohl, Bastian; Wenzel, Michaela; Bandow, Julia Elisabeth; Maffioli, Sonia; Sosio, Margherita; Donadio, Stefano; Wimmer, Reinhard; Sahl, Hans-Georg

    2014-04-25

    The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell. PMID:24627484

  19. Cloning and nucleotide sequence of anaerobically induced porin protein E1 (OprE) of Pseudomonas aeruginosa PAO1.

    PubMed

    Yamano, Y; Nishikawa, T; Komatsu, Y

    1993-05-01

    The porin oprE gene of Pseudomonas aeruginosa PAO1 was isolated. Its nucleotide sequence indicated that the structural gene of 1383 nucleotide residues encodes a precursor consisting of 460 amino acid residues with a signal peptide of 29 amino acid residues, which was confirmed by the N-terminal 23-amino-acid sequence and the reaction with anti-OprE polyclonal antiserum. Anaerobiosis induced OprE production at the transcription level. The transcription start site was determined to be 40 nucleotides upstream from the ATG initiation codon. The control region contained an appropriately situated E sigma 54 recognition site and the putative second half of an ANR box. The amino acid sequence of OprE had some clusters of sequence homologous with that of OprD of P. aeruginosa, which might be responsible for the outer membrane permeability of imipenem and basic amino acids. PMID:8394980

  20. Partitioning of differently sized poly(ethylene glycol)s into OmpF porin.

    PubMed Central

    Rostovtseva, Tatiana K; Nestorovich, Ekaterina M; Bezrukov, Sergey M

    2002-01-01

    To understand the physics of polymer equilibrium and dynamics in the confines of ion channel pores, we study partitioning of poly(ethylene glycol)s (PEGs) of different molecular weights into the bacterial porin, OmpF. Thermodynamic and kinetic parameters of partitioning are deduced from the effects of polymer addition on ion currents through single OmpF channels reconstituted into planar lipid bilayer membranes. The equilibrium partition coefficient is inferred from the average reduction of channel conductance in the presence of PEG; rates of polymer exchange between the pore and the bulk are estimated from PEG-induced conductance noise. Partition coefficient as a function of polymer weight is best fitted by a "compressed exponential" with the compression factor of 1.65. This finding demonstrates that PEG partitioning into the OmpF channel pore has sharper dependence on polymer molecular weight than predictions of hard-sphere, random-flight, or scaling models. A 1360-Da polymer separates regimes of partitioning and exclusion. Comparison of its characteristic size with the size of a 2200-Da polymer previously found to separate these regimes for the alpha-toxin shows good agreement with the x-ray structural data for these channels. The PEG-induced conductance noise is compatible with the polymer mobility reduced inside the OmpF pore by an order of magnitude relatively to its value in bulk solution. PMID:11751305

  1. Metalloido-porins: Essentiality of Nodulin 26-like intrinsic proteins in metalloid transport.

    PubMed

    Pommerrenig, Benjamin; Diehn, Till Arvid; Bienert, Gerd Patrick

    2015-09-01

    Metalloids are a group of physiologically important elements ranging from the essential to the highly toxic. Arsenic, antimony, germanium, and tellurium are highly toxic to plants themselves and to consumers of metalloid-contaminated plants. Boron, silicon, and selenium fulfill essential or beneficial functions in plants. However, when present at high concentrations, boron and selenium cause toxicity symptoms that are detrimental to plant fitness and yield. Consequently, all plants require efficient membrane transport systems to control the uptake and extrusion of metalloids into or out of the plant and their distribution within the plant body. Several Nodulin 26-like intrinsic proteins (NIPs) that belong to the aquaporin plant water channel protein family facilitate the diffusion of uncharged metalloid species. Genetic, physiological, and molecular evidence is that NIPs from primitive to higher plants not only transport all environmentally important metalloids, but that these proteins have a major role in the uptake, translocation, and extrusion of metalloids in plants. As most of the metalloid-permeable NIP aquaporins are impermeable or are poorly permeable to water, these NIP channel proteins should be considered as physiologically essential metalloido-porins. PMID:26259189

  2. Crystal structures of the OmpF porin: function in a colicin translocon

    SciTech Connect

    Yamashita, Eiki; Zhalnina, Mariya V.; Zakharov, Stanislav D.; Sharma, Onkar; Cramer, William A.

    2008-11-18

    The OmpF porin in the Escherichia coli outer membrane (OM) is required for the cytotoxic action of group A colicins, which are proposed to insert their translocation and active domains through OmpF pores. A crystal structure was sought of OmpF with an inserted colicin segment. A 1.6 {angstrom} OmpF structure, obtained from crystals formed in 1 M Mg{sup 2+}, has one Mg{sup 2+} bound in the selectivity filter between Asp113 and Glu117 of loop 3. Co-crystallization of OmpF with the unfolded 83 residue glycine-rich N-terminal segment of colicin E3 (T83) that occludes OmpF ion channels yielded a 3.0 {angstrom} structure with inserted T83, which was obtained without Mg{sup 2+} as was T83 binding to OmpF. The incremental electron density could be modelled as an extended poly-glycine peptide of at least seven residues. It overlapped the Mg{sup 2+} binding site obtained without T83, explaining the absence of peptide binding in the presence of Mg{sup 2+}. Involvement of OmpF in colicin passage through the OM was further documented by immuno-extraction of an OM complex, the colicin translocon, consisting of colicin E3, BtuB and OmpF.

  3. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I

    PubMed Central

    Zhou, Zhongjun; Apte, Suneel S.; Soininen, Raija; Cao, Renhai; Baaklini, George Y.; Rauser, Richard W.; Wang, Jianming; Cao, Yihai; Tryggvason, Karl

    2000-01-01

    Membrane-type matrix metalloproteinase I (MT1-MMP)-deficient mice were found to have severe defects in skeletal development and angiogenesis. The craniofacial, axial, and appendicular skeletons were severely affected, leading to a short and domed skull, marked deceleration of postnatal growth, and death by 3 wk of age. Shortening of bones is a consequence of decreased chondrocyte proliferation in the proliferative zone of the growth plates. Defective vascular invasion of cartilage leads to enlargement of hypertrophic zones of growth plates and delayed formation of secondary ossification centers in long bones. In an in vivo corneal angiogenesis assay, null mice did not have angiogenic response to implanted FGF-2, suggesting that the defect in angiogenesis is not restricted to cartilage alone. In tissues from null mice, activation of latent matrix metalloproteinase 2 was deficient, suggesting that MT1-MMP is essential for its activation in vivo. PMID:10737763

  4. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I.

    PubMed

    Zhou, Z; Apte, S S; Soininen, R; Cao, R; Baaklini, G Y; Rauser, R W; Wang, J; Cao, Y; Tryggvason, K

    2000-04-11

    Membrane-type matrix metalloproteinase I (MT1-MMP)-deficient mice were found to have severe defects in skeletal development and angiogenesis. The craniofacial, axial, and appendicular skeletons were severely affected, leading to a short and domed skull, marked deceleration of postnatal growth, and death by 3 wk of age. Shortening of bones is a consequence of decreased chondrocyte proliferation in the proliferative zone of the growth plates. Defective vascular invasion of cartilage leads to enlargement of hypertrophic zones of growth plates and delayed formation of secondary ossification centers in long bones. In an in vivo corneal angiogenesis assay, null mice did not have angiogenic response to implanted FGF-2, suggesting that the defect in angiogenesis is not restricted to cartilage alone. In tissues from null mice, activation of latent matrix metalloproteinase 2 was deficient, suggesting that MT1-MMP is essential for its activation in vivo. PMID:10737763

  5. Interaction between complement subcomponent C1q and the Klebsiella pneumoniae porin OmpK36.

    PubMed Central

    Albertí, S; Marqués, G; Hernández-Allés, S; Rubires, X; Tomás, J M; Vivanco, F; Benedí, V J

    1996-01-01

    The interaction between C1q, a subcomponent of the complement classical pathway component C1, and OmpK36, a porin protein from Klebsiella pneumoniae, was studied in a solid-phase direct-binding assay, inhibition assays with the purified globular and collagen-like regions of C1q, and cross-linking experiments. We have shown that the binding of C1q to the OmpK36 porin of the serum-sensitive strain K. pneumoniae KT707 occurs in an in vivo situation and that this binding leads to activation of the complement classical pathway and the subsequent deposition of complement components C3b and C5b-9 on the OmpK36 porin. Scatchard analysis of the binding of [125I]C1q to the OmpK36 porin showed two binding sites with dissociation constants of 1.5 and 75 nM. The decrease of [125I]C1q binding to the OmpK36 porin in buffer with increasing salt concentrations and the pIs of the C1q subcomponent (10.3) and OmpK36 porin (4.5) suggest that charged amino acids are involved in the binding phenomenon. In inhibition assays, only the globular regions of C1q inhibited the interaction between C1q and OmpK36 porin, demonstrating that C1q binds to porin through its globular region and not through the collagen-like stalks. PMID:8890231

  6. Pore formation in lipid bilayer membranes made of phosphatidylinositol and oxidized cholesterol followed by means of alternating current.

    PubMed Central

    Gallucci, E; Micelli, S; Monticelli, G

    1996-01-01

    The kinetics of porin incorporation into black lipid membranes (BLM) made of phosphatidylinositol (PI) or oxidized cholesterol (Ox Ch) were studied by means of alternating current; the set-up was able to acquire resistance and capacitance simultaneously by means of a mixed double-frequency approach at 1 Hz and 1 KHz, respectively. Conductance was dependent on the interaction between protein-forming pores and lipids. For PI membranes below a porin concentration of 12.54 ng/ml, there was no membrane conductivity, whereas at 200 ng/ml a steady-state value was reached. Different behavior was displayed by Ox Ch membranes, in which a concentration of 12.54 ng/ml was sufficient to reach a steady state. The incorporation kinetics when porin was added after membrane formation were sigmoidal. When porin was present in the medium before membrane formation, the kinetics were sigmoidal for PI membranes but became exponential for Ox Ch membranes. Furthermore, for BLM made of PI, the conductance-versus-porin concentration relationship is sigmoidal, with a Hill coefficient of 5.6 +/- 0.07, which is functional evidence corroborating the six-channel repeating units seen previously. For BLM made of Ox Ch, this relationship followed a binding isotherm curve with a Hill coefficient of 0.934 +/- 0.129. PMID:8842220

  7. Gonococcal porin IB activates NF-kappaB in human urethral epithelium and increases the expression of host antiapoptotic factors.

    PubMed

    Binnicker, Matthew J; Williams, Richard D; Apicella, Michael A

    2004-11-01

    Infection of human urethral epithelial cells (UECs) with Neisseria gonorrhoeae increases the transcription of several host antiapoptotic genes, including bfl-1, cox-2, and c-IAP-2. In order to identify the bacterial factor(s) responsible for eliciting these changes, the transcriptional status of apoptotic machinery was monitored in UECs challenged with certain gonococcal membrane components. Initially, we observed that infection of UECs with gentamicin-killed gonococci increased the expression of the antiapoptotic Bcl-2 family member, bfl-1. This observation indicated that viable, replicating bacteria are not required for induction of antiapoptotic gene expression. Confirming this observation, treatment of UECs with purified gonococcal membrane increased the expression of bfl-1, cox-2, and c-IAP-2. This finding suggested that a factor or multiple factors present in the outer membrane (OM) are responsible for altering UEC antiapoptotic gene expression. Interestingly, treatment of UECs with gonococcal porin IB (PorB IB), a major constituent of the OM, significantly increased the transcription of bfl-1, cox-2, and c-IAP-2. The upregulation of these genes by PorB IB was determined to be dependent on NF-kappaB activation, as inhibiting NF-kappaB blocked induced expression of these genes. This work demonstrates the altered expression of host apoptotic factors in response to gonococcal PorB IB and supports a model whereby UEC cell death may be modulated as a potential mechanism of bacterial survival and proliferation. PMID:15501771

  8. Trends of the Major Porin Gene (ompF) Evolution: Insight from the Genus Yersinia

    PubMed Central

    Stenkova, Anna M.; Isaeva, Marina P.; Shubin, Felix N.; Rasskazov, Valeri A.; Rakin, Alexander V.

    2011-01-01

    OmpF is one of the major general porins of Enterobacteriaceae that belongs to the first line of bacterial defense and interactions with the biotic as well as abiotic environments. Porins are surface exposed and their structures strongly reflect the history of multiple interactions with the environmental challenges. Unfortunately, little is known on diversity of porin genes of Enterobacteriaceae and the genus Yersinia especially. We analyzed the sequences of the ompF gene from 73 Yersinia strains covering 14 known species. The phylogenetic analysis placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree. Very high congruence in the tree topologies was observed for Y. enterocolitica, Y. kristensenii, Y. ruckeri, indicating that intragenic recombination in these species had no effect on the ompF gene. A significant level of intra- and interspecies recombination was found for Y. aleksiciae, Y. intermedia and Y. mollaretii. Our analysis shows that the ompF gene of Yersinia has evolved with nonrandom mutational rate under purifying selection. However, several surface loops in the OmpF porin contain positively selected sites, which very likely reflect adaptive diversification Yersinia to their ecological niches. To our knowledge, this is a first investigation of diversity of the porin gene covering the whole genus of the family Enterobacteriaceae. This study demonstrates that recombination and positive selection both contribute to evolution of ompF, but the relative contribution of these evolutionary forces are different among Yersinia species. PMID:21655186

  9. Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth

    PubMed Central

    2009-01-01

    Background Highly pathogenic mycobacteria like Mycobacterium tuberculosis are characterised by their slow growth and their ability to reside and multiply in the very hostile phagosomal environment and a correlation between the growth rate of mycobacteria and their pathogenicity has been hypothesised. Here, porin genes from M. fortuitum were cloned and characterised to address their impact on the growth rate of fast-growing and pathogenic mycobacteria. Results Two genes encoding porins orthologous to MspA from M. smegmatis, porM1 and porM2, were cloned from M. fortuitum strains, which were originally isolated from human patients. Both porin genes were at least partially able to complement the mutations of a M. smegmatis mutant strain lacking the genes mspA and mspC with respect to the growth rate. PorM1 and porM2 were present in different strains of M. fortuitum including the type strain. Comparative expression analysis of porM genes revealed divergent porin expression among analysed M. fortuitum strains. Repression of the expression of porins by antisense technique decreased the growth rates of different M. fortuitum. The effects of over-expression of porM1 as well as porM2 varied depending on the strain and the concentration of antibiotic added to the medium and indicated that PorM1 and PorM2 enhance the growth of M. fortuitum strains, but also the diffusion of the antibiotic kanamycin into the cells. Conclusion This study demonstrates the important role of porin expression in growth as well as antibiotic susceptibility of the opportunistic bacterium M. fortuitum. PMID:19203364

  10. Development of an enhanced chromosomal expression system based on porin synthesis operon for halophile Halomonas sp.

    PubMed

    Yin, Jin; Fu, Xiao-Zhi; Wu, Qiong; Chen, Jin-Chun; Chen, Guo-Qiang

    2014-11-01

    Since halophile Halomonas spp. can grow contamination free in seawater under unsterile and continuous conditions, it holds great promise for industrial biotechnology to produce low-cost chemicals in an economic way. Yet, metabolic engineering methods are urgently needed for Halomonas spp. It is commonly known that chromosomal expression is more stable yet weaker than plasmid one is. To overcome this challenge, a novel chromosomal expression method was developed for halophile Halomonas TD01 and its derivatives based on a strongly expressed porin gene as a site for external gene integration. The gene of interest was inserted downstream the porin gene, forming an artificial operon porin-inserted gene. This chromosome expression system was proven functional by some examples: First, chromosomal expression of heterologous polyhydroxybutyrate (PHB) synthase gene phaC Re from Ralstonia eutropha completely restored the PHB accumulation level in endogenous phaC knockout mutant of Halomonas TD01. The integrated phaC Re was expressed at the highest level when inserted at the locus of porin compared with insertions in other chromosome locations. Second, an inducible expression system was constructed in phaC-deleted Halomonas TD01 by integrating the lac repressor gene (lacI) into the porin site in the host chromosome. The native porin promoter was inserted with the key 21 bp DNA of lac operator (lacO) sequence to become an inducible promoter encoded in a plasmid. This inducible system allowed on-off switch of gene expression in Halomonas TD strains. Thus, the stable and strong chromosomal expression method in Halomonas TD spp. was established. PMID:25070598

  11. Proinflammatory signal transduction pathway induced by Shigella flexneri porins in caco-2 cells

    PubMed Central

    Elena, Grimaldi; Giovanna, Donnarumma; Brunella, Perfetto; De Anna, Filippis; Alessandro, Melito; Antonietta, Tufano Maria

    2009-01-01

    The recognition of bacterial components on the intestinal epithelial cells occurs through the toll-like receptors and is followed by the induction of an effective innate immune response. We analyzed receptor expression and signaling pathways involved in activation of human colon adenocarcinoma cells after stimulation with porins and LPS of Shigella flexneri. We also analyzed the expression and production of some cytokines, of intercellular adhesion molecule-1, of antimicrobial peptides human β-defensins, and of the inducible form of nitric oxide synthase. Our data demonstrate that TLR2 is involved in porin recognition, whereas TLR4 with MD2, is required for LPS recognition. PMID:24031417

  12. Native Escherichia coli OmpF Porin Surfaces Probed by Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Schabert, Frank A.; Henn, Christian; Engel, Andreas

    1995-04-01

    Topographs of two dimensional porin OmpF crystals reconstituted in the presence of lipids were recorded in solution by atomic force microscopy (AFM) to a lateral resolution of 10 angstroms and a vertical resolution of 1 angstrom. Protein-protein interactions were demonstrated on the basis of the AFM results and earlier crystallographic findings. To assess protein-lipid interactions, the bilayer was modeled with kinked lipids by fitting the head groups to contours determined with AFM. Finally, two conformations of the extracellular porin surface were detected at forces of 0.1 nanonewton, demonstrating the potential of AFM to monitor conformational changes with high resolution.

  13. Impaired Intestinal Calcium Absorption in Protein 4.1R-deficient Mice Due to Altered Expression of Plasma Membrane Calcium ATPase 1b (PMCA1b)*

    PubMed Central

    Liu, Congrong; Weng, Haibao; Chen, Lixiang; Yang, Shaomin; Wang, Hua; Debnath, Gargi; Guo, Xinhua; Wu, Liancheng; Mohandas, Narla; An, Xiuli

    2013-01-01

    Protein 4.1R was first identified in the erythrocyte membrane skeleton. It is now known that the protein is expressed in a variety of epithelial cell lines and in the epithelia of many tissues, including the small intestine. However, the physiological function of 4.1R in the epithelial cells of the small intestine has not so far been explored. Here, we show that 4.1R knock-out mice exhibited a significantly impaired small intestinal calcium absorption that resulted in secondary hyperparathyroidism as evidenced by increased serum 1,25-(OH)2-vitamin D3 and parathyroid hormone levels, decreased serum calcium levels, hyperplasia of the parathyroid, and demineralization of the bones. 4.1R is located on the basolateral membrane of enterocytes, where it co-localizes with PMCA1b (plasma membrane calcium ATPase 1b). Expression of PMCA1b in enterocytes was decreased in 4.1−/− mice. 4.1R directly associated with PMCA1b, and the association involved the membrane-binding domain of 4.1R and the second intracellular loop and C terminus of PMCA1b. Our findings have enabled us to define a functional role for 4.1R in small intestinal calcium absorption through regulation of membrane expression of PMCA1b. PMID:23460639

  14. Combination of IMP-4 metallo-beta-lactamase production and porin deficiency causes carbapenem resistance in a Klebsiella oxytoca clinical isolate.

    PubMed

    Chen, Li-Rong; Zhou, Hong-Wei; Cai, Jia-Chang; Zhang, Rong; Chen, Gong-Xiang

    2009-10-01

    This study shows for the first time the mechanism of carbapenem resistance of a Klebsiella oxytoca clinical isolate ZC101 recovered from a Zhejiang University Hospital in Hangzhou, China. MIC values of imipenem, meropenem, and ertapenem for K. oxytoca ZC101 were 16, 16, and 128 microg/mL, respectively. Conjugation experiments demonstrated the transferability of a resistance determinant from K. oxytoca ZC101 to Escherichia coli EC600. Results from isoelectric focusing, polymerase chain reactions, and DNA sequencing confirmed that K. oxytoca ZC101 produced IMP-4 metallo-beta-lactamase (MBL) and CTX-M-14 extended-spectrum beta-lactamase, whereas E. coli transconjugant only produced the IMP-4. Amplification of integron revealed that bla(IMP-4) gene is located within a class I integron that was carried in a plasmid approximately 55 kb in size. Sodium dodecyl sulfate polyacrylamide gel electrophoresis profiling of outer membrane proteins of K. oxytoca ZC101 indicated lack of expression of the OmpK36 porin. DNA sequence analysis of ompK36 gene of K. oxytoca ZC101 showed the gene was disrupted by an insertion sequence IS5. In all, the results show that plasmid-mediated IMP-4 MBL production combined with the loss of OmpK36 porin caused the resistance in K. oxytoca ZC101 to carbapenems. PMID:19748427

  15. The Acinetobacter baumannii Omp33-36 Porin Is a Virulence Factor That Induces Apoptosis and Modulates Autophagy in Human Cells

    PubMed Central

    Rumbo, Carlos; Fernández Moreira, Esteban; Soares, Nelson Cruz; Carvajal, Micaela; Santillana, Elena; Beceiro, Alejandro; Romero, Antonio

    2014-01-01

    Acinetobacter baumannii is an extracellular opportunistic human pathogen that is becoming increasingly problematic in hospitals. In the present study, we demonstrate that the A. baumannii Omp 33- to 36-kDa protein (Omp33-36) is a porin that acts as a channel for the passage of water. The protein is found on the cell surface and is released along with other porins in the outer membrane vesicles (OMVs). In immune and connective cell tissue, this protein induced apoptosis by activation of caspases and modulation of autophagy, with the consequent accumulation of p62/SQSTM1 (sequestosome 1) and LC3B-II (confirmed by use of autophagy inhibitors). Blockage of autophagy enables the bacterium to persist intracellularly (inside autophagosomes), with the subsequent development of cytotoxicity. Finally, we used macrophages and a mouse model of systemic infection to confirm that Omp33-36 is a virulence factor in A. baumannii. Overall, the study findings show that Omp33-36 plays an important role in the pathogenesis of A. baumannii infections. PMID:25156738

  16. Decreased outer membrane permeability protects mycobacteria from killing by ubiquitin-derived peptides.

    PubMed

    Purdy, Georgiana E; Niederweis, Michael; Russell, David G

    2009-09-01

    Ubiquitin-derived peptides are bactericidal in vitro and contribute to the mycobactericidal activity of the lysosome. To further define interactions of ubiquitin-derived peptides with mycobacteria, we screened for mutants with increased resistance to the bactericidal activity of the synthetic ubiquitin-derived peptide Ub2. The four Ub2-resistant Mycobacterium smegmatis mutants were also resistant to the bactericidal action of other antimicrobial peptides and macrophages. Two mutants were in the mspA gene encoding the main M. smegmatis porin. Using a translocation-deficient MspA point mutant, we showed that susceptibility of M. smegmatis to Ub2 was independent of MspA channel activity. Instead, the M. smegmatis Ub2-resistant mutants shared a common phenotype of decreased cell wall permeability compared with wild-type bacteria. Expression of mspA rendered Mycobacterium tuberculosis CDC1551 more susceptible both to ubiquitin-derived peptides in vitro and to lysosomal killing in macrophages. Finally, biochemical assays designed to assess membrane integrity indicated that Ub2 treatment impairs membrane function of M. smegmatis and M. tuberculosis cells. The M. smegmatis Ub2-resistant mutants were more resistant than wild-type M. smegmatis to this damage. We conclude that Ub2 targets mycobacterial membranes and that reduced membrane permeability provides mycobacteria intrinsic resistance against antimicrobial compounds including bactericidal ubiquitin-derived peptides. PMID:19682257

  17. Development of novel cell surface display in Corynebacterium glutamicum using porin.

    PubMed

    Tateno, Toshihiro; Hatada, Kazuki; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-09-01

    We have developed a novel cell surface display in Corynebacterium glutamicum using porin proteins as anchor proteins. Porins are localized at C. glutamicum mycolic acid layer and exist as a hexamer. We used alpha-amylase from Streptococcus bovis 148 (AmyA) as a model protein to be displayed on the C. glutamicum cell surface. AmyA was fused to the C terminus of the porins PorB, PorC, or PorH. Expression vectors using fused proteins under the control of the cspB promoter were constructed and introduced into the C. glutamicum Cm strain. Immunostaining microscopy and flow cytometric analysis revealed that PorB-AmyA, PorC-AmyA, and PorH-AmyA were displayed on the C. glutamicum cell surface. AmyA activity was only detected in the cell fraction of C. glutamicum cells that displayed AmyA fused to PorB, PorC or PorH and AmyA activity was not detected in the supernatants of C. glutamicum culture broths after 72 h cultivation. Thus, we have demonstrated that C. glutamicum porins are very efficient anchor proteins for protein display in C. glutamicum. PMID:19430772

  18. Antibodies to porin antigens of Salmonella typhi induced during typhoid infection in humans.

    PubMed Central

    Calderón, I; Lobos, S R; Rojas, H A; Palomino, C; Rodríguez, L H; Mora, G C

    1986-01-01

    Immunoglobulin G (IgG)- and IgM-specific antibody titers against Salmonella typhi Ty2 porins have been measured in 30 paired typhoid sera by enzyme-linked immunosorbent assay. These studies have found that IgG serum titers of acute and convalescent sera were 625 and 5,000 times higher, respectively than the control serum titers. The same typhoid sera were titrated with S. typhi Ty2 flagellin and S. typhi lipopolysaccharide. The titers against these antigens were considerably lower than those against the porins. The highest IgM-specific titer has also been found against porins in convalescent-phase sera. However, the largest increase in IgM-specific titer compared with the control group titer was obtained against flagellin during the acute phase of typhoid. The lowest increases in antibody titer were obtained with the IgM-specific anti-lipopolysaccharide in both types of sera. This may be because many normal individuals in endemic areas already have IgM titers against lipopolysaccharide. This study has provided good evidence that porins are excellent antigens and that IgG-specific antiporin titers may be of diagnostic value in typhoid infections in endemic areas. PMID:3007360

  19. Loss of Mammal-specific Tectorial Membrane Component Carcinoembryonic Antigen Cell Adhesion Molecule 16 (CEACAM16) Leads to Hearing Impairment at Low and High Frequencies*

    PubMed Central

    Kammerer, Robert; Rüttiger, Lukas; Riesenberg, Rainer; Schäuble, Constanze; Krupar, Rosemarie; Kamp, Annegret; Sunami, Kishiko; Eisenried, Andreas; Hennenberg, Martin; Grunert, Fritz; Bress, Andreas; Battaglia, Sebastiano; Schrewe, Heinrich; Knipper, Marlies; Schneider, Marlon R.; Zimmermann, Wolfgang

    2012-01-01

    The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function, we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16−/− mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal days 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16−/− mice tectorial membranes were significantly more often stretched out as compared with wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxyl-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 can probably form higher order structures with other tectorial membrane proteins such as α-tectorin and β-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea, allowing hearing over an extended frequency range. PMID:22544735

  20. Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins.

    PubMed Central

    Nikaido, H; Rosenberg, E Y

    1983-01-01

    Rates of diffusion of uncharged and charged solute molecules through porin channels were determined by using liposomes reconstituted from egg phosphatidylcholine and purified Escherichia coli porins OmpF (protein 1a), OmpC (protein 1b), and PhoE (protein E). All three porin proteins appeared to produce channels of similar size, although the OmpF channel appeared to be 7 to 9% larger than the OmpC and PhoE channels in an equivalent radius. Hydrophobicity of the solute retarded the penetration through all three channels in a similar manner. The presence of one negative charge on the solute resulted in about a threefold reduction in penetration rates through OmpF and OmpC channels, whereas it produced two- to tenfold acceleration of diffusion through the PhoE channel. The addition of the second negatively charged group to the solutes decreased the diffusion rates through OmpF and OmpC channels further, whereas diffusion through the PhoE channel was not affected much. These results suggest that PhoE specializes in the uptake of negatively charged solutes. At the present level of resolution, no sign of true solute specificity was found in OmpF and OmpC channels; peptides, for example, diffused through both of these channels at rates expected from their molecular size, hydrophobicity, and charge. However, the OmpF porin channel allowed influx of more solute molecules per unit time than did the equivalent weight of the OmpC porin when the flux was driven by a concentration gradient of the same size. This apparent difference in "efficiency" became more pronounced with larger solutes, and it is likely to be the consequence of the difference in the sizes of OmpF and OmpC channels. PMID:6294049

  1. Loss of the F-BAR protein CIP4 reduces platelet production by impairing membrane-cytoskeleton remodeling

    PubMed Central

    Chen, Yolande; Aardema, Jorie; Kale, Sayali; Whichard, Zakary L.; Awomolo, Arinola; Blanchard, Elisabeth; Chang, Brian; Myers, David R.; Ju, Lining; Tran, Reginald; Reece, David; Christensen, Hilary; Boukour, Siham; Debili, Najet; Strom, Ted S.; Rawlings, David; Vázquez, Francisco X.; Voth, Gregory A.; Zhu, Cheng; Kahr, Walter H. A.; Lam, Wilbur A.

    2013-01-01

    Megakaryocytes generate platelets through extensive reorganization of the cytoskeleton and plasma membrane. Cdc42 interacting protein 4 (CIP4) is an F-BAR protein that localizes to membrane phospholipids through its BAR domain and interacts with Wiskott-Aldrich Syndrome Protein (WASP) via its SRC homology 3 domain. F-BAR proteins promote actin polymerization and membrane tubulation. To study its function, we generated CIP4-null mice that displayed thrombocytopenia similar to that of WAS− mice. The number of megakaryocytes and their progenitors was not affected. However, the number of proplatelet protrusions was reduced in CIP4-null, but not WAS−, megakaryocytes. Electron micrographs of CIP4-null megakaryocytes showed an altered demarcation membrane system. Silencing of CIP4, not WASP, expression resulted in fewer proplatelet-like extensions. Fluorescence anisotropy studies showed that loss of CIP4 resulted in a more rigid membrane. Micropipette aspiration demonstrated decreased cortical actin tension in megakaryocytic cells with reduced CIP4 or WASP protein. These studies support a new biophysical mechanism for platelet biogenesis whereby CIP4 enhances the complex, dynamic reorganization of the plasma membrane (WASP independent) and actin cortex network (as known for WASP and cortical actin) to reduce the work required for generating proplatelets. CIP4 is a new component in the highly coordinated system of megakaryocytic membrane and cytoskeletal remodeling affecting platelet production. PMID:23881916

  2. Biogenesis of the preprotein translocase of the outer mitochondrial membrane: protein kinase A phosphorylates the precursor of Tom40 and impairs its import.

    PubMed

    Rao, Sanjana; Schmidt, Oliver; Harbauer, Angelika B; Schönfisch, Birgit; Guiard, Bernard; Pfanner, Nikolaus; Meisinger, Chris

    2012-05-01

    The preprotein translocase of the outer mitochondrial membrane (TOM) functions as the main entry gate for the import of nuclear-encoded proteins into mitochondria. The major subunits of the TOM complex are the three receptors Tom20, Tom22, and Tom70 and the central channel-forming protein Tom40. Cytosolic kinases have been shown to regulate the biogenesis and activity of the Tom receptors. Casein kinase 2 stimulates the biogenesis of Tom22 and Tom20, whereas protein kinase A (PKA) impairs the receptor function of Tom70. Here we report that PKA exerts an inhibitory effect on the biogenesis of the β-barrel protein Tom40. Tom40 is synthesized as precursor on cytosolic ribosomes and subsequently imported into mitochondria. We show that PKA phosphorylates the precursor of Tom40. The phosphorylated Tom40 precursor is impaired in import into mitochondria, whereas the nonphosphorylated precursor is efficiently imported. We conclude that PKA plays a dual role in the regulation of the TOM complex. Phosphorylation by PKA not only impairs the receptor activity of Tom70, but it also inhibits the biogenesis of the channel protein Tom40. PMID:22419819

  3. Remorin, a Solanaceae Protein Resident in Membrane Rafts and Plasmodesmata, Impairs Potato virus X Movement[W

    PubMed Central

    Raffaele, Sylvain; Bayer, Emmanuelle; Lafarge, David; Cluzet, Stéphanie; German Retana, Sylvie; Boubekeur, Tamy; Leborgne-Castel, Nathalie; Carde, Jean-Pierre; Lherminier, Jeannine; Noirot, Elodie; Satiat-Jeunemaître, Béatrice; Laroche-Traineau, Jeanny; Moreau, Patrick; Ott, Thomas; Maule, Andrew J.; Reymond, Philippe; Simon-Plas, Françoise; Farmer, Edward E.; Bessoule, Jean-Jacques; Mongrand, Sébastien

    2009-01-01

    Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in ∼70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane. PMID:19470590

  4. Single Point Mutation in Bin/Amphiphysin/Rvs (BAR) Sequence of Endophilin Impairs Dimerization, Membrane Shaping, and Src Homology 3 Domain-mediated Partnership*

    PubMed Central

    Gortat, Anna; San-Roman, Mabel Jouve; Vannier, Christian; Schmidt, Anne A.

    2012-01-01

    Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are essential players in the dynamics of intracellular compartments. The BAR domain is an evolutionarily conserved dimeric module characterized by a crescent-shaped structure whose intrinsic curvature, flexibility, and ability to assemble into highly ordered oligomers contribute to inducing the curvature of target membranes. Endophilins, diverging into A and B subgroups, are BAR and SH3 domain-containing proteins. They exert activities in membrane dynamic processes such as endocytosis, autophagy, mitochondrial dynamics, and permeabilization during apoptosis. Here, we report on the involvement of the third α-helix of the endophilin A BAR sequence in dimerization and identify leucine 215 as a key residue within a network of hydrophobic interactions stabilizing the entire BAR dimer interface. With the combination of N-terminal truncation retaining the high dimerization capacity of the third α-helices of endophilin A and leucine 215 substitution by aspartate (L215D), we demonstrate the essential role of BAR sequence-mediated dimerization on SH3 domain partnership. In comparison with wild type, full-length endophilin A2 heterodimers with one protomer bearing the L215D substitution exhibit very significant changes in membrane binding and shaping activities as well as a dramatic decrease of SH3 domain partnership. This suggests that subtle changes in the conformation and/or rigidity of the BAR domain impact both the control of membrane curvature and downstream binding to effectors. Finally, we show that expression, in mammalian cells, of endophilin A2 bearing the L215D substitution impairs the endocytic recycling of transferrin receptors. PMID:22167186

  5. Direct observation of gold nanoparticle assemblies with the porin MspA on mica.

    PubMed

    Basel, Matthew T; Dani, Raj Kumar; Kang, Myungshim; Pavlenok, Mikhail; Chikan, Viktor; Smith, Paul E; Niederweis, Michael; Bossmann, Stefan H

    2009-02-24

    The octameric porin MspA from Mycobacterium smegmatis is sufficiently stable to form a nonmembrane-supported stand-alone porin on mica surfaces. About 98% of all MspA octamers were found to stand upright on mica, with their periplasmic loop regions bound to the hydrophilic mica surface. Both, small (d = 3.7 nm) and large (d = 17 nm) gold nanoparticles bind to MspA, however, in different positions: small gold nanoparticles bind within the MspA pore, whereas the large gold nanoparticles bind to the upper region of MspA. These experiments demonstrate that gold nanoparticles can be positioned at different, well-defined distances from the underlying surface using the MspA pore as a template. These findings represent a significant step toward the use of electrically insulating stable proteins in combination with metal nanoparticles in nanodevices. PMID:19236086

  6. Antibiotic translocation through porins studied in planar lipid bilayers using parallel platforms.

    PubMed

    Weichbrodt, Conrad; Bajaj, Harsha; Baaken, Gerhard; Wang, Jiajun; Guinot, Serap; Kreir, Mohamed; Behrends, Jan C; Winterhalter, Mathias; Fertig, Niels

    2015-07-21

    In general, the method of choice to characterize the conductance properties of channel-forming bacterial porins is electrophysiology. Here, the classical method is to reconstitute single porins into planar lipid bilayers to derive functional information from the observed channel conductance. In addition to an estimated pore size, ion selectivity or transport properties in general are of importance. For the latter, measuring the ion current fluctuation can provide some information about the mode of transport of charged molecules penetrating the proteins. For instance, increasing the external voltage modifies the residence time in the channel: charged molecules with the ability to permeate through channels will travel faster whereas non-permeating molecules get pushed to the constriction zone with enhanced residence time. Here, we are interested in the ability of antibiotics to permeate channels and compare different techniques to reveal fast events. PMID:25834843

  7. Adenine nucleotide translocator isoforms 1 and 2 are differently distributed in the mitochondrial inner membrane and have distinct affinities to cyclophilin D.

    PubMed Central

    Vyssokikh, M Y; Katz, A; Rueck, A; Wuensch, C; Dörner, A; Zorov, D B; Brdiczka, D

    2001-01-01

    Different isoforms of the adenine nucleotide translocase (ANT) are expressed in a tissue-specific manner. It was assumed that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be differently distributed within the membrane structures that constitute the peripheral inner membrane or the crista membrane. To discriminate between ANT originating from peripheral or from cristal inner membranes we made use of the fact that complexes between porin, the outer-membrane pore protein, and the ANT can be generated. Such complexes between porin and the ANT in the peripheral inner membrane were induced in rat heart mitochondria and isolated from rat brain and kidney. Using ANT-isotype-specific antibodies and sequence analysis of the N-terminal end, it was discovered that the peripheral inner membrane contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively ANT-2. Cyclophilin was co-purified with the porin-ANT complexes, whereas it was absent in the crista-derived ANT. This suggested that ANT-1 might have a higher affinity for cyclophilin. This specific intra-mitochondrial distribution of the two ANT isotypes and cyclophilin D suggests specific functions of the peripheral and crista-forming parts of the inner membrane and the two ANT isotypes therein. PMID:11513733

  8. Ultrafast proton transport in sub-1-nm diameter carbon nanotube porins.

    PubMed

    Tunuguntla, Ramya H; Allen, Frances I; Kim, Kyunghoon; Belliveau, Allison; Noy, Aleksandr

    2016-07-01

    Proton transport plays an important role in many biological processes due to the ability of protons to rapidly translocate along chains of hydrogen-bonded water molecules. Molecular dynamics simulations have predicted that confinement in hydrophobic nanochannels should enhance the rate of proton transport. Here, we show that 0.8-nm-diameter carbon nanotube porins, which promote the formation of one-dimensional water wires, can support proton transport rates exceeding those of bulk water by an order of magnitude. The transport rates in these narrow nanotube pores also exceed those of biological channels and Nafion. With larger 1.5-nm-diameter nanotube porins, proton transport rates comparable to bulk water are observed. We also show that the proton conductance of these channels can be modulated by the presence of Ca(2+) ions. Our results illustrate the potential of small-diameter carbon nanotube porins as a proton conductor material and suggest that strong spatial confinement is a key factor in enabling efficient proton transport. PMID:27043198

  9. Ultrafast proton transport in sub-1-nm diameter carbon nanotube porins

    NASA Astrophysics Data System (ADS)

    Tunuguntla, Ramya H.; Allen, Frances I.; Kim, Kyunghoon; Belliveau, Allison; Noy, Aleksandr

    2016-07-01

    Proton transport plays an important role in many biological processes due to the ability of protons to rapidly translocate along chains of hydrogen-bonded water molecules. Molecular dynamics simulations have predicted that confinement in hydrophobic nanochannels should enhance the rate of proton transport. Here, we show that 0.8-nm-diameter carbon nanotube porins, which promote the formation of one-dimensional water wires, can support proton transport rates exceeding those of bulk water by an order of magnitude. The transport rates in these narrow nanotube pores also exceed those of biological channels and Nafion. With larger 1.5-nm-diameter nanotube porins, proton transport rates comparable to bulk water are observed. We also show that the proton conductance of these channels can be modulated by the presence of Ca2+ ions. Our results illustrate the potential of small-diameter carbon nanotube porins as a proton conductor material and suggest that strong spatial confinement is a key factor in enabling efficient proton transport.

  10. Asymmetric pore occupancy in crystal structure of OmpF porin from Salmonella typhi.

    PubMed

    Balasubramaniam, D; Arockiasamy, Arulandu; Kumar, P D; Sharma, Amit; Krishnaswamy, S

    2012-06-01

    OmpF is a major general diffusion porin of Salmonella typhi, a Gram-negative bacterium, which is an obligatory human pathogen causing typhoid. The structure of S. typhi Ty21a OmpF (PDB Id: 3NSG) determined at 2.8 Å resolution by X-ray crystallography shows a 16-stranded β-barrel with three β-barrel monomers associated to form a trimer. The packing observed in S. typhi Ty21a rfOmpF crystals has not been observed earlier in other porin structures. The variations seen in the loop regions provide a starting point for using the S. typhi OmpF for structure-based multi-valent vaccine design. Along one side of the S. typhi Ty21a OmpF pore there exists a staircase arrangement of basic residues (20R, 60R, 62K, 65R, 77R, 130R and 16K), which also contribute, to the electrostatic potential in the pore. This structure suggests the presence of asymmetric electrostatics in the porin oligomer. Moreover, antibiotic translocation, permeability and reduced uptake in the case of mutants can be understood based on the structure paving the way for designing new antibiotics. PMID:22525817

  11. MspA Porin-Gold Nanoparticle Assemblies: Enhanced Binding through a Controlled Cysteine Mutation.

    PubMed

    Dani, Raj Kumar; Kang, Myungshim; Kalita, Mausam; Smith, Paul E; Bossmann, Stefan H; Chikan, Viktor

    2008-04-01

    In this study, the interactions of two gold nanoparticles of different sizes (average diameters of 3.7 +/- 2.6 and 17 +/- 3 nm) with octameric mycobacterial porin A from Mycobacterium smegmatis (MspA) and a mutant of MspA featuring a cysteine mutation in position 126 (Q126C) are investigated. From the observation of enhanced photoluminescence quenching, it is inferred that the presence of eight cysteines in the MspA Q126C mutant significantly enhances the binding of selected small gold nanoparticles within the inner pore of MspA. The large gold nanoparticle/porin complex shows photoluminescence enhancement, which is expected since the larger nanoparticles cannot dock within the homopore of MspA due to size exclusion. In addition to the fluorescence experiments, observation of energy transfer from the small gold nanoparticles to the MspA shows the close proximity of the small gold nanoparticles with the porin. Interestingly, the energy transfer of the large nanoparticle/MspA complex is completely missing. From high-performance liquid chromatography data, the estimated binding constants for small Au@MspA, large Au@MspA, small Au@MspAcys, and large Au@MspAcys are 1.3 x 10 (9), 2.22 x 10 (10), > 10 (12) (irreversible), and 1.7 x 10 (10), respectively. PMID:18318505

  12. Branched Chain Amino Acids Induce Apoptosis in Neural Cells without Mitochondrial Membrane Depolarization or Cytochrome c Release: Implications for Neurological Impairment Associated with Maple Syrup Urine Disease

    PubMed Central

    Jouvet, Philippe; Rustin, Pierre; Taylor, Deanna L.; Pocock, Jennifer M.; Felderhoff-Mueser, Ursula; Mazarakis, Nicholas D.; Sarraf, Catherine; Joashi, Umesh; Kozma, Mary; Greenwood, Kirsty; Edwards, A. David; Mehmet, Huseyin

    2000-01-01

    Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a deficiency in branched chain α-keto acid dehydrogenase that can result in neurodegenerative sequelae in human infants. In the present study, increased concentrations of MSUD metabolites, in particular α-keto isocaproic acid, specifically induced apoptosis in glial and neuronal cells in culture. Apoptosis was associated with a reduction in cell respiration but without impairment of respiratory chain function, without early changes in mitochondrial membrane potential and without cytochrome c release into the cytosol. Significantly, α-keto isocaproic acid also triggered neuronal apoptosis in vivo after intracerebral injection into the developing rat brain. These findings suggest that MSUD neurodegeneration may result, at least in part, from an accumulation of branched chain amino acids and their α-keto acid derivatives that trigger apoptosis through a cytochrome c-independent pathway. PMID:10793161

  13. Cosuppression of a Plasma Membrane H+-ATPase Isoform Impairs Sucrose Translocation, Stomatal Opening, Plant Growth, and Male Fertility

    PubMed Central

    Zhao, Rongmin; Dielen, Vincent; Kinet, Jean-Marie; Boutry, Marc

    2000-01-01

    The plasma membrane H+-ATPase builds up a pH and potential gradient across the plasma membrane, thus activating a series of secondary ion and metabolite transporters. pma4 (for plasma membrane H+-ATPase 4), the most widely expressed H+-ATPase isogene in Nicotiana plumbaginifolia, was overexpressed in tobacco. Plants that overexpressed PMA4 showed no major changes in plant growth under normal conditions. However, two transformants were identified by their stunted growth, slow leaf initiation, delayed stem bolting and flowering, and male sterility. Protein gel blot analysis showed that expression of the endogenous and transgenic pma4 was cosuppressed. Cosuppression was developmentally regulated because PMA4 was still present in developing leaves but was not detected in mature leaves. The glucose and fructose content increased threefold, whereas the sucrose content remained unchanged. The rate of sucrose exudation from mature leaves was reduced threefold and the sugar content of apical buds was reduced twofold, suggesting failure of sucrose loading and translocation to the sink tissues. Cosuppression of PMA4 also affected the guard cells, stomatal opening, and photosynthesis in mature leaves. These results show that a single H+-ATPase isoform plays a major role in several transport-dependent physiological processes. PMID:10760242

  14. Impaired surface membrane insertion of homo- and heterodimeric human muscle chloride channels carrying amino-terminal myotonia-causing mutations

    PubMed Central

    Ronstedt, Katharina; Sternberg, Damien; Detro-Dassen, Silvia; Gramkow, Thomas; Begemann, Birgit; Becher, Toni; Kilian, Petra; Grieschat, Matthias; Machtens, Jan-Philipp; Schmalzing, Günther; Fischer, Martin; Fahlke, Christoph

    2015-01-01

    Mutations in the muscle chloride channel gene (CLCN1) cause myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement. We here studied the functional consequences of four disease-causing mutations that predict amino acid substitutions Q43R, S70L, Y137D and Q160H. Wild-type (WT) and mutant hClC-1 channels were heterologously expressed as YFP or CFP fusion protein in HEK293T cells and analyzed by whole-cell patch clamp and fluorescence recordings on individual cells. Q43R, Y137D and Q160H, but not S70L reduced macroscopic current amplitudes, but left channel gating and unitary current amplitudes unaffected. We developed a novel assay combining electrophysiological and fluorescence measurements at the single-cell level in order to measure the probability of ion channel surface membrane insertion. With the exception of S70L, all tested mutations significantly reduced the relative number of homodimeric hClC-1 channels in the surface membrane. The strongest effect was seen for Q43R that reduced the surface insertion probability by more than 99% in Q43R homodimeric channels and by 92 ± 3% in heterodimeric WT/Q43R channels compared to homodimeric WT channels. The new method offers a sensitive approach to investigate mutations that were reported to cause channelopathies, but display only minor changes in ion channel function. PMID:26502825

  15. Role of outer membrane proteins in imipenem diffusion in Pseudomonas aeruginosa.

    PubMed

    Lian, Z; Tianjue, Y

    1999-03-01

    The present study identified the properties of porins in the outer membrane in Pseudomonas aeruginosa, and showed the role of outer membrane in determining imipenem diffusion in Pseudomonas aeruginosa. The molecular weight of the major outer membrane protein was analyzed by SDS-PAGE. The purification of the porins in Pseudomonas aeruginosa was achieved by DEAE ion-exchange HPLC. The purified outer membrane proteins were reconstituted with phosphatidylcholine and dicetylphosphate into membrane vesicles, and were tested by the liposomes swelling method for the diffusion of imipenem. The permeability assay showed that OprC (70 kD), OprD2 (46 kD), and OprE (43 kD) were the channel-forming proteins. But only OprD2 was thought to be the likely route of imipenem diffusion. PMID:12899386

  16. Increased susceptibility to beta-lactam antibiotics and decreased porin content caused by envB mutations of Salmonella typhimurium.

    PubMed Central

    Oppezzo, O J; Avanzati, B; Antón, D N

    1991-01-01

    Isogenic derivatives carrying envB6, envB9, or envB+ alleles were obtained from a strain of Salmonella typhimurium that was partially resistant to mecillinam, a beta-lactam antibiotic specific for penicillin-binding protein 2 (PBP 2). Testing of the isogenic strains with several antibacterial agents demonstrated that envB mutations either increased resistance (mecillinam) or did not affect the response (imipemen) to beta-lactams that act primarily on PBP 2, while susceptibilities to beta-lactams that act on PBP 1B, PBP 3, or both were increased. Furthermore, the susceptibilities of envB strains to hydrophobic compounds such as rifampin, novobiocin, or chloramphenicol were not modified, even though their susceptibilities to deoxycholate and crystal violet were enhanced. Outer cell membranes of envB mutants presented a 50% reduction in protein content compared with that of the isogenic envB+ strains, and OmpF and OmpD porins were particularly affected by the reduction. No alteration in the amount or pattern of periplasmic proteins was noticed, and lipopolysaccharides from envB mutants appeared to be normal by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. By using derivatives that produced a plasmid-encoded beta-lactamase, it was demonstrated that envB cells are slightly less permeable to cephalothin than envB+ bacteria are. It is concluded that the high susceptibility of envB mutants to beta-lactams is due to the increased effectiveness of the antibiotics on PBP 1B, PBP 3, or both. Images PMID:1656857

  17. Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation.

    PubMed

    Khan, Junaid; Sharma, Praveen K; Mukhopadhaya, Arunika

    2015-11-01

    Polarization of the monocytes and macrophages toward the M1 and M2 states is important for hosts' defense against the pathogens. Moreover, it plays a crucial role to resolve the overwhelming inflammatory responses that can be harmful to the host. Polarization of macrophages/monocytes can be induced by pathogen-associated molecular patterns (PAMPs). PAMP-mediated monocyte/macrophage polarization is important during the infection, as pathogen can suppress host immune system by altering the polarization status of the macrophages/monocytes. OmpU, an outer membrane porin protein of Vibrio cholerae, possesses the ability to induce pro-inflammatory responses in monocytes/macrophages. It is also able to down-regulate the LPS-mediated activation of the monocytes/macrophages. Such observation leads us to believe that OmpU may induce a state that can be called as M1/M2-intermediate state. In the present study, we evaluated a set of M1 and M2 markers in RAW 264.7 murine macrophage cell line, and THP-1 human monocytic cell line, in response to the purified OmpU protein. We observed that OmpU, as a PAMP, induced M1-polarization by activating the Toll-like receptor (TLR) signaling pathway. OmpU induced formation of TLR1/TLR2-heterodimers. OmpU-mediated TLR-activation led to the MyD88 recruitment to the TLR1/TLR2 complex. MyD88, in turn, recruited IRAK1. Ultimately, OmpU-mediated signaling led to the activation and subsequent nuclear translocation of the NFκB p65 subunit. We also observed that blocking of the TLR1, TLR2, IRAK1, and NFκB affected OmpU-mediated production of M1-associated pro-inflammatory cytokines such as TNFα and IL-6. PMID:26093918

  18. Effect of phenol-induced changes in lipid composition on conformation of OmpF-like porin of Yersinia pseudotuberculosis.

    PubMed

    Sanina, Nina; Nina, Sanina; Davydova, Ludmila; Ludmila, Davydova; Bakholdina, Svetlana; Svetlana, Bakholdina; Novikova, Olga; Olga, Novikova; Pornyagina, Olga; Olga, Pornyagina; Solov'eva, Tamara; Tamara, Solov'eva; Shnyrov, Valery; Valery, Shnyrov; Bogdanov, Mikhail; Mikhail, Bogdanov

    2013-07-11

    The present work aimed to compare the effects of different lysophosphatidylethanolamine (LPE) content in lipids derived from Yersinia pseudotuberculosis cells exposed and not exposed to phenol on the conformation of OmpF-like porin of these bacteria. Differential scanning calorimetry and intrinsic protein fluorescence showed that the 2.5-fold increase of LPE content and the corresponding increase in the phase transition temperature of bacterial lipids were accompanied by enhanced protein thermostability. Integral conformational rearrangement of protein was supported by drastic changes in the microenvironment of the tryptophan residues, likely resulting in a convergence of monomers in trimeric porin and exposure of outer tryptophan residues to the water environment. These conformational changes may impede the porin channel permeability under stress conditions in bacteria. PMID:23742936

  19. Plasma membrane coenzyme Q: evidence for a role in autism

    PubMed Central

    Crane, Frederick L; Löw, Hans; Sun, Iris; Navas, Placido; Gvozdjáková, Anna

    2014-01-01

    Background The Voltage Dependent Anion Channel (VDAC) is involved in control of autism. Treatments, including coenzyme Q, have had some success on autism control. Data sources Correlation of porin redox activity and expression of autism is based on extensive literature, especially studies of antibodies, identification of cytosolic nicotinamide adenine dinucleotide reduced (NADH) dehydrogenase activity in the VDAC, and evidence for extreme sensitivity of the dehydrogenase to a mercurial. Evidence for a coenzyme Q requirement came from extraction and analog inhibition of NADH ferricyanide reductase in the erythrocyte plasma membrane, done in 1994, and reinterpreted when it was identified in VDAC in 2004. The effects of ubiquinol (the QH2 – reduced form of coenzyme Q) in children with autism were studied. Results A new role for coenzyme Q in the porin channels has implications on autism. Ubiquinol, the more active form of coenzyme Q, produces favorable response in children with autism. Agents which affected electron transport in porin show parallel effects in autism. Conclusion We propose a hypothesis that autism is controlled by a coenzyme Q-dependent redox system in the porin channels; this conclusion is based on the effects of agents that positively or negatively affect electron transport and the symptoms of autism. The full understanding of the mechanism of their control needs to be established. PMID:24920882

  20. Cigarette smoke decreases mitochondrial porin expression and steroidogenesis

    SciTech Connect

    Bose, Mahuya; Whittal, Randy M.; Gairola, C. Gary; Bose, Himangshu S.

    2008-03-01

    Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer to inner mitochondrial membrane for steroidogenesis. Here, we investigated the effect of cigarette smoke (CS) on steroidogenesis using adrenal mitochondria isolated from mice chronically exposed to CS. Steroidogenesis was decreased approximately 78% in CS-exposed mitochondria, as measured by synthesis of the steroid hormone precursor pregnenolone. This effect was accompanied by decreased mitochondrial import of {sup 35}S-StAR. Further characterization of the imported {sup 35}S-StAR by native gradient PAGE revealed the presence of a high molecular weight complex in both control and CS-exposed groups. Following density gradient fractionation of {sup 35}S-StAR that had been extracted from control mitochondria, precursor StAR could be found in fractions 2-6 and smaller-sized StAR complexes in fractions 6-13. In the CS-exposed group, the appearance of precursor shifted from fraction 1-6 and the smaller complexes in fractions 6-9 disappeared. Mass spectrometric analysis revealed that the {sup 35}S-StAR-associated protein complex was composed of several resident matrix proteins as well as the OMM resident, VDAC. VDAC expression was greatly reduced by CS, and blockage of VDAC with Koenig's polyanion decreased pregnenolone synthesis in isolated mitochondria. Taken together, these results suggest that VDAC may participate in steroidogenesis by promoting StAR interaction with the OMM and that CS may inhibit steroidogenesis by reducing VDAC-StAR interactions.

  1. Chronic dietary exposure to chlorpyrifos causes behavioral impairments, low activity of brain membrane-bound acetylcholinesterase, and increased brain acetylcholinesterase-R mRNA.

    PubMed

    López-Granero, Caridad; Cardona, Diana; Giménez, Estela; Lozano, Rafael; Barril, José; Sánchez-Santed, Fernando; Cañadas, Fernando

    2013-06-01

    Chlorpyrifos (CPF) is an organophosphate (OP) insecticide that is metabolically activated to the highly toxic chlorpyrifos oxon. Dietary exposure is the main route of intoxication for non-occupational exposures. However, only limited behavioral effects of chronic dietary exposure have been investigated. Therefore, male Wistar rats were fed a dose of 5mg/kg/day of CPF for thirty-one weeks. Animals were evaluated in spatial learning and impulsivity tasks after 21 weeks of CPF dietary exposure and one week after exposure ended, respectively. In addition, the degree of inhibition of brain acetylcholinesterase (AChE) was evaluated for both the soluble and particulate forms of the enzyme, as well as AChE gene expression. Also, brain acylpeptide hydrolase (APH) was investigated as an alternative target for OP-mediated effects. All variables were evaluated at various time points in response to CPF diet and after exposure ended. Results from behavioral procedures suggest cognitive and emotional disorders. Moreover, low levels of activity representing membrane-bound oligomeric forms (tetramers) were also observed. In addition, increased brain AChE-R mRNA levels were detected after four weeks of CPF dietary exposure. However, no changes in levels of brain APH were observed among groups. In conclusion, our data point to a relationship between cognitive impairments and changes in AChE forms, specifically to a high inhibition of the particulate form and a modification of alternative splicing of mRNA during CPF dietary exposure. PMID:23545134

  2. Deficient guanine nucleotide regulatory unit activity in cultured fibroblast membranes from patients with pseudohypoparathyroidism type I. A cause of impaired synthesis of 3',5'-cyclic AMP by intact and broken cells

    PubMed Central

    Levine, Michael A.; Eil, Charles; Downs, Robert W.; Spiegel, Allen M.

    1983-01-01

    Deficient activity of the guanine nucleotide regulatory protein (G unit), an integral component of the membrane-bound adenylate cyclase complex, has been implicated as the biochemical lesion in many patients with pseudohypoparathyroidism (PHP) type I. In addition to renal resistance to parathyroid hormone in this disorder, there is decreased responsiveness of diverse tissues to hormones that act via 3',5'-cyclic AMP (cAMP). To assess whether a deficiency of G units could account for impaired adenylate cyclase activity, we studied cAMP production in intact cultured fibroblasts and fibroblast plasma membranes from five patients with PHP in response to several activators of adenylate cyclase. The number of G units in PHP fibroblast membranes, measured by cholera toxin-dependent [32P]ADP ribosylation of G-unit peptides, as well as the G-unit activity, determined by the ability of detergent extracts to reconstitute adenylate cyclase activity in G-unit-deficient S49 CYC- membranes, were found to be markedly reduced compared with control membranes (43 and 40%, respectively), The activation of fibroblast membrane adenylate cyclase by effectors that act directly through the G unit (guanosine triphosphate, guanosine 5'-0-[3-thiotriphosphate] [GTP-γ-S], NaF) was significantly greater in control membranes than in membranes from patients with PHP. Moreover, we found that hormone (prostaglandin E1) stimulated adenylate cyclase activity was also greater in control membranes than in PHP membranes. Neither the apparent affinity of membrane adenylate cyclase for GTP-γ-S (apparent Km =5 X 10-8 M) nor the rate of enzyme activation by GTP-γ-S was significantly different in fibroblast membranes from control subjects and patients with PHP. In contrast to the notable differences in hormone and G-unit-activated adenylate cyclase shown in fibroblast membranes from PHP patients and control subjects, the intrinsic catalytic activity of membranes, as determined by forskolin

  3. Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria

    PubMed Central

    Commandeur, Susanna; van der Weerd, Robert; Sparrius, Marion; Weerdenburg, Eveline; Alber, Marina; Kalscheuer, Rainer; Piersma, Sander R.; Abdallah, Abdallah M.; Abd El Ghany, Moataz; Abdel-Haleem, Alyaa M.; Pain, Arnab; Jiménez, Connie R.; Bitter, Wilbert; Houben, Edith N.G.

    2015-01-01

    Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow

  4. Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria.

    PubMed

    Ates, Louis S; Ummels, Roy; Commandeur, Susanna; van de Weerd, Robert; van der Weerd, Robert; Sparrius, Marion; Weerdenburg, Eveline; Alber, Marina; Kalscheuer, Rainer; Piersma, Sander R; Abdallah, Abdallah M; Abd El Ghany, Moataz; Abdel-Haleem, Alyaa M; Pain, Arnab; Jiménez, Connie R; Bitter, Wilbert; Houben, Edith N G

    2015-05-01

    Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow

  5. Multistep Mechanism of Chloride Translocation in a Strongly Anion-Selective Porin Channel

    PubMed Central

    Zachariae, Ulrich; Helms, Volkhard; Engelhardt, Harald

    2003-01-01

    The strongly anion-selective porin channel Omp32 from the bacterium Delftia acidovorans differs from other unspecific porins by its pronounced selectivity for anions and its particularly small channel cross-section. Multinanosecond molecular dynamics simulations of chloride ion movement in this pore protein suggest that translocated anions interact intimately with the charges of a “basic ladder”, whose dynamics lead the anions in a stepwise manner through the constriction zone of the channel. The ladder-steps comprise the central clustered arginine groups and flanking basic residues at its exoplasmic and periplasmic sides. The computed free energy profile of ion movement in and around the constriction zone shows a corresponding succession of free energy minima and barriers. A number of polar atoms from other amino acids contribute to the coordination of Cl− at certain sites and to its temporary immobilization in the channel. A special binding site occurs at the transition of the constriction zone to the periplasmic funnel, binding the chloride ion over significant lengths of time. The results from our MD study offer a possible explanation for the nonlinear conductance properties and unusual salt-dependent characteristics of Omp32 observed earlier in experimental measurements. PMID:12885642

  6. Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

    PubMed Central

    Fernández, Lucía

    2012-01-01

    Summary: The substantial use of antibiotics in the clinic, combined with a dearth of new antibiotic classes, has led to a gradual increase in the resistance of bacterial pathogens to these compounds. Among the various mechanisms by which bacteria endure the action of antibiotics, those affecting influx and efflux are of particular importance, as they limit the interaction of the drug with its intracellular targets and, consequently, its deleterious effects on the cell. This review evaluates the impact of porins and efflux pumps on two major types of resistance, namely, mutational and adaptive types of resistance, both of which are regarded as key phenomena in the global rise of antibiotic resistance among pathogenic microorganisms. In particular, we explain how adaptive and mutational events can dramatically influence the outcome of antibiotic therapy by altering the mechanisms of influx and efflux of antibiotics. The identification of porins and pumps as major resistance markers has opened new possibilities for the development of novel therapeutic strategies directed specifically against these mechanisms. PMID:23034325

  7. ExbBD-Dependent Transport of Maltodextrins through the Novel MalA Protein across the Outer Membrane of Caulobacter crescentus

    PubMed Central

    Neugebauer, Heidi; Herrmann, Christina; Kammer, Winfried; Schwarz, Gerold; Nordheim, Alfred; Braun, Volkmar

    2005-01-01

    Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe3+ and vitamin B12—the substrates hitherto known to be transported by TonB-dependent transporters—the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [14C]maltodextrins from [14C]maltose to [14C]maltopentaose. [14C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a Kd of 0.2 μM, while the second transport had a Kd of 5 μM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 μM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [14C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe3+-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe3+-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of

  8. Structural and dynamical properties of the porins OmpF and OmpC: insights from molecular simulations

    NASA Astrophysics Data System (ADS)

    Kumar, Amit; Hajjar, Eric; Ruggerone, Paolo; Ceccarelli, Matteo

    2010-11-01

    In this paper we investigate the structural and dynamical properties of the two major porins (OmpF and OmpC) in Escherichia coli, using molecular dynamics (MD) simulations. In particular we characterized the atomic fluctuations, correlated motions, temperature dependence, solvent-accessible cross-sectional area and water dynamics in the key regions of the two channels. Our in-depth analysis allows us to highlight the importance of both the key conserved and substituted residues between OmpF and OmpC. The latter is characterized by a narrower and longer constriction region with respect to OmpF. OmpC also showed a higher stability upon increasing temperature. We then present the results of transport properties by using accelerated MD simulations to probe the diffusion of norfloxacin (a fluoroquinolone antibiotic) through the two porins OmpF/OmpC. Our study constitutes a step forward towards understanding the structure-function relationship of the two porins' channels. This will benefit the research of antibacterials with improved permeation properties and nanopores that aim to use these porins as sensing systems.

  9. 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.

    PubMed

    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

  10. Origami in outer membrane mimetics: correlating the first detailed images of refolded VDAC with over 20 years of biochemical data.

    PubMed

    Summers, William A T; Court, Deborah A

    2010-06-01

    Mitochondrial porin forms an aqueous pore in the outer membrane, through which selective passage of small metabolites and ions occurs, thereby regulating both mitochondrial function and cellular respiration. Investigations of the structure and function of porin have been performed with whole mitochondria, membrane vesicles, artificial membranes, and in detergent solutions, resulting in numerous models of porin structure. The mechanisms by which this protein functions are undoubtedly linked to its structure, which remained elusive until 2008, with reports of 3 high-resolution structures of this voltage-dependent, anion-selective channel (VDAC). The barrel structure is relatively simple yet unique: it is arranged as 19 anti-parallel beta-strands, with beta-strands 1 and 19 aligned parallel to each other to close the barrel. The N-terminal helical component is located within the lumen of the channel, although its precise structure and location in the lumen varies. With the basic barrel structure in hand, the data obtained in attempts to model the structure and understand porin over the past 20 years can be re-evaluated. Herein, using the mammalian VDAC structures as templates, the amassed electrophysiological and biochemical information has been reassessed with respect to the functional mechanisms of VDAC activity, with a focus on voltage-dependent gating. PMID:20555384

  11. Why do the outer membrane proteins OmpF from E. coli and OprP from P. aeruginosa prefer trimers? Simulation studies.

    PubMed

    Niramitranon, Jitti; Sansom, Mark Sp; Pongprayoon, Prapasiri

    2016-04-01

    Porins are water-filled protein channels across the outer membrane of gram-negative bacteria. They facilitate the uptake of nutrients and essential ions. Solutes are filtered by a constriction loop L3 at the mid of a pore. Porins are heat-stable and resistant to toxic agents and detergents. Most porins are trimer, but no clear explanation why trimeric form is preferable. In this work, we thus studied effects of oligomerization on porin structure and function in microscopic detail. A well-studied OmpF (general porin from Escherichia coli) and well-characterised OprP (phosphate-specific pore from Pseudomonas aeruginosa) are used as samples from 2 types of porins found in gram-negative bacteria. MD simulations of trimeric and monomeric pores in pure water and 1M NaCl solution were performed. With a salt solution, the external electric field was applied to mimic a transmembrane potential. Expectedly, OprP is more stable than OmpF. Interestingly, being a monomer turns OmpF into an anion-selective pore. The dislocation of D113's side chain on L3 in OmpF causes the disruption of cation pathway resulting in the reduction of cation influx. In contrast, OprP's structure and function are less dependent on oligomeric states. Both monomeric and trimeric OprP can maintain their anion selectivity. Our findings suggest that trimerization is crucial for both structure and function of general porin OmpF, whereas being trimer in substrate-specific channel OprP supports a pore function. PMID:26895142

  12. The structural biology of β-barrel membrane proteins: a summary of recent reports

    PubMed Central

    Fairman, James W.; Noinaj, Nicholas; Buchanan, Susan K.

    2011-01-01

    The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts all contain transmembrane β-barrel proteins. These β-barrel proteins serve essential functions in cargo transport and signaling and are also vital for membrane biogenesis. They have also been adapted to perform a diverse set of important cellular functions including acting as porins, transporters, enzymes, virulence factors and receptors. Recent structures of transmembrane β-barrels include that of a full length autotransporter (EstA), a bacterial heme transporter complex (HasR), a bacterial porin in complex with several ligands (PorB), and the mitochondrial voltage-dependent anion channel (VDAC) from both mouse and human. These represent only a few of the interesting structures of β-barrel membrane proteins recently elucidated. However, they demonstrate many of the advancements made within the field of transmembrane protein structure in the past few years. PMID:21719274

  13. Nanoscopic surfactant behavior of the porin MspA in aqueous media.

    PubMed

    Perera, Ayomi S; Wang, Hongwang; Shrestha, Tej B; Troyer, Deryl L; Bossmann, Stefan H

    2013-01-01

    The mycobacterial porin MspA is one of the most stable channel proteins known to date. MspA forms vesicles at low concentrations in aqueous buffers. Evidence from dynamic light scattering, transmission electron microscopy and zeta-potential measurements by electrophoretic light scattering indicate that MspA behaves like a nanoscale surfactant. The extreme thermostability of MspA allows these investigations to be carried out at temperatures as high as 343 K, at which most other proteins would quickly denature. The principles of vesicle formation of MspA as a function of temperature and the underlying thermodynamic factors are discussed here. The results obtained provide crucial evidence in support of the hypothesis that, during vesicle formation, nanoscopic surfactant molecules, such as MspA, deviate from the principles underlined in classical surface chemistry. PMID:23766950

  14. Rapid detection of porins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry

    PubMed Central

    Hu, Yan-Yan; Cai, Jia-Chang; Zhou, Hong-Wei; Zhang, Rong; Chen, Gong-Xiang

    2015-01-01

    The rapid and cost-efficient determination of carbapenem resistance is an important prerequisite for the choice of an adequate antibiotic therapy. A MALDI-TOF MS-based assay was set up to detect porins in the current study. A loss of the components of porin alone such as OmpK35/OmpK36 or together with the production of carbapenemases will augment the carbapenem resistance. Ten strains of Escherichia coli and eight strains of Klebsiella pneumoniae were conducted for both sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF MS analysis. MALDI-TOF/TOF MS analysis was then performed to verify the correspondence of proteins between SDS-PAGE and MALDI-TOF MS. The results indicated that the mass spectrum of ca. 35,000, 37,000, and 38,000-m/z peaks of E. coli ATCC 25922 corresponded to OmpA, OmpC, and OmpF with molecular weight of approximately ca. 38, 40, and 41 kDa in SDS-PAGE gel, respectively. The band of OmpC and OmpF porins were unable to be distinguished by SDS-PAGE, whereas it was easy to be differentiated by MALDI-TOF MS. As for K. pneumoniae isolates, the mass spectrum of ca. 36,000 and 38,600-m/z peaks was observed corresponding to OmpA and OmpK36 with molecular weight of approximately ca. 40 and 42 kDa in SDS-PAGE gel, respectively. Porin OmpK35 was not observed in the current SDS-PAGE, while a 37,000-m/z peak was found in K. pneumoniae ATCC 13883 and carbapenem-susceptible strains by MALDI-TOF MS which was presumed to be the characteristic peak of the OmpK35 porin. Compared with SDS-PAGE, MALDI-TOF MS is able to rapidly identify the porin-deficient strains within half an hour with better sensitivity, less cost, and is easier to operate and has less interference. PMID:26300858

  15. Outer Membrane Permeability and Antibiotic Resistance

    PubMed Central

    Delcour, Anne H.

    2009-01-01

    Summary To date most antibiotics are targeted at intracellular processes, and must be able to penetrate the bacterial cell envelope. In particular, the outer membrane of Gram-negative bacteria provides a formidable barrier that must be overcome. There are essentially two pathways that antibiotics can take through the outer membrane: a lipid-mediated pathway for hydrophobic antibiotics, and general diffusion porins for hydrophilic antibiotics. The lipid and protein compositions of the outer membrane have a strong impact on the sensitivity of bacteria to many types of antibiotics, and drug resistance involving modifications of these macromolecules is common. This review will describe the molecular mechanisms for permeation of antibiotics through the outer membrane, and the strategies that bacteria have deployed to resist antibiotics by modifications of these pathways. PMID:19100346

  16. Ultrastructure of the outer membrane of Salmonella typhimurium bacteriocin-resistant mutants deficient in the 33K protein.

    PubMed Central

    Lounatmaa, K

    1979-01-01

    Outer membrane mutants of Salmonella typhimurium deficient in one, two, or three of the 33,000-dalton (33K), 34K, and 36K outer membrane proteins (7) were studied by using thin sectioning and freeze-fracturing electron microscopy techniques. The outer concave fracture face of all mutants deficient in the 33K protein had numerous particleless patches. In contrast to all previously examined 34K to 36K-deficient mutants, the 33K-deficient mutants showed marked heterogeneity in the size and distribution of such "empty" patches between cells of a culture. One mutant was deficient in both the 33K and the 34K to 36K "porin" protein complex; its outer membrane had very large particleless smooth areas. It is concluded that the 33K protein on one hand and the porin on the other are both able to form intramembraneous particles. Images PMID:378982

  17. Construtcion of Neisseria gonorrhoeae porin B plasmid recombinant and its expression in E. coli.

    PubMed

    Song, Qifa; Liao, Fang; Ye, Siying; Cui, Bing; Xiong, Ping

    2005-01-01

    A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E. coli DE3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E. coli DE3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28% of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect EISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed. PMID:16201262

  18. IL-4 and IL-13 induce protection from complement and melittin in endothelial cells despite initial loss of cytoplasmic proteins: membrane resealing impairs quantifying cytotoxicity with the lactate dehydrogenase permeability assay.

    PubMed

    Benson, Barbara A; Vercellotti, Gregory M; Dalmasso, Agustin P

    2015-01-01

    Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL-4 and IL-13 through metabolic activation. However, despite this resistance, the complement-treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore, to define the apparent discrepancy of permeability changes vis-à-vis the protection from killing, we now investigated whether IL-4 and IL-13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore-forming protein melittin. Primary cultures of ECs were pre-treated with IL-4 or IL-13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. We found that IL-4-/IL-13-induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine-treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. We conclude that the protection induced by IL-4/IL-13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin. PMID:26031609

  19. IL-4 and IL-13 induce protection from complement and melittin in endothelial cells despite initial loss of cytoplasmic proteins: Membrane resealing impairs quantifying cytotoxicity with the lactate dehydrogenase permeability assay

    PubMed Central

    Benson, Barbara A.; Vercellotti, Gregory M.; Dalmasso, Agustin P.

    2015-01-01

    Background Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL-4 and IL-13 through metabolic activation. However, despite this resistance, the complement-treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore to define the apparent discrepancy of permeability changes vis-à-vis the protection from killing we now investigated whether IL-4 and IL-13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore-forming protein melittin. Methods Primary cultures of ECs were pretreated with IL-4 or IL-13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. Results We found that IL-4/IL-13 induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine-treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. Conclusion We conclude that the protection induced by IL-4/IL-13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin. PMID:26031609

  20. Stochastic transport through carbon nanotubes in lipid bilayers and live cell membranes

    NASA Astrophysics Data System (ADS)

    Geng, Jia; Kim, Kyunghoon; Zhang, Jianfei; Escalada, Artur; Tunuguntla, Ramya; Comolli, Luis R.; Allen, Frances I.; Shnyrova, Anna V.; Cho, Kang Rae; Munoz, Dayannara; Wang, Y. Morris; Grigoropoulos, Costas P.; Ajo-Franklin, Caroline M.; Frolov, Vadim A.; Noy, Aleksandr

    2014-10-01

    There is much interest in developing synthetic analogues of biological membrane channels with high efficiency and exquisite selectivity for transporting ions and molecules. Bottom-up and top-down methods can produce nanopores of a size comparable to that of endogenous protein channels, but replicating their affinity and transport properties remains challenging. In principle, carbon nanotubes (CNTs) should be an ideal membrane channel platform: they exhibit excellent transport properties and their narrow hydrophobic inner pores mimic structural motifs typical of biological channels. Moreover, simulations predict that CNTs with a length comparable to the thickness of a lipid bilayer membrane can self-insert into the membrane. Functionalized CNTs have indeed been found to penetrate lipid membranes and cell walls, and short tubes have been forced into membranes to create sensors, yet membrane transport applications of short CNTs remain underexplored. Here we show that short CNTs spontaneously insert into lipid bilayers and live cell membranes to form channels that exhibit a unitary conductance of 70-100 picosiemens under physiological conditions. Despite their structural simplicity, these `CNT porins' transport water, protons, small ions and DNA, stochastically switch between metastable conductance substates, and display characteristic macromolecule-induced ionic current blockades. We also show that local channel and membrane charges can control the conductance and ion selectivity of the CNT porins, thereby establishing these nanopores as a promising biomimetic platform for developing cell interfaces, studying transport in biological channels, and creating stochastic sensors.

  1. Impaired processing of human pro-islet amyloid polypeptide is not a causative factor for fibril formation or membrane damage in vitro.

    PubMed

    Khemtémourian, Lucie; Lahoz Casarramona, Gemma; Suylen, Dennis P L; Hackeng, Tilman M; Meeldijk, Johannes D; de Kruijff, Ben; Höppener, Jo W M; Killian, J Antoinette

    2009-11-24

    Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus (DM2). hIAPP is synthesized by islet beta-cells initially as a preprohormone, processing of which occurs in several steps. It has been suggested that in DM2 this processing is defective and that aggregation of the processing intermediates prohIAPP and prohIAPP(1-48) may represent the initial step in formation of islet amyloid. Here we investigate this possibility by analyzing the aggregation, the structure, and the membrane interaction of mature hIAPP and its precursors, prohIAPP and prohIAPP(1-48), in vitro. Our data reveal that both precursors form amyloid fibrils in solution but not in the presence of membranes. This inhibition is in contrast to the catalyzing effect of membranes on fibril formation of mature hIAPP. Importantly, in the presence of membranes, both precursors are able to inhibit fibrillogenesis of mature hIAPP. These differences in behavior between mature hIAPP and its precursors are most likely related to differences in their mode of membrane insertion. Both precursors insert efficiently and adopt an alpha-helical structure even with a high lipid/peptide ratio, while mature hIAPP rapidly adopts a beta-sheet conformation. Furthermore, while mature hIAPP affects the barrier properties of lipid vesicles, neither of the precursors is able to induce membrane leakage. Our study suggests that the hIAPP precursors prohIAPP and prohIAPP(1-48) do not serve as amyloid initiators but rather prevent aggregation and membrane damage of mature hIAPP in early stages of its biosynthesis and intracellular transport. PMID:19817482

  2. Effect of porin loss on the activity of tigecycline against Klebsiella pneumoniae producing extended-spectrum beta-lactamases or plasmid-mediated AmpC-type beta-lactamases.

    PubMed

    Conejo, M Carmen; Hernández, J Ramón; Pascual, Alvaro

    2008-07-01

    Tigecycline showed excellent in vitro activity against 50 clinical isolates of Klebsiella pneumoniae producing extended-spectrum beta-lactamases, plasmid-mediated AmpC-type beta-lactamases, or both. This activity was not affected by porin loss. Porin loss, however, did affect the activity of imipenem against strains that expressed both types of enzymes. PMID:18339509

  3. Carbapenem-resistant Pseudomonas aeruginosa strains from a Spanish hospital: characterization of metallo-beta-lactamases, porin OprD and integrons.

    PubMed

    Rojo-Bezares, Beatriz; Estepa, Vanesa; Cebollada, Rocío; de Toro, María; Somalo, Sergio; Seral, Cristina; Castillo, Francisco Javier; Torres, Carmen; Sáenz, Yolanda

    2014-05-01

    Molecular typing and mechanisms of carbapenem resistance such as alterations in porin OprD and presence of metallo-beta-lactamases (MBLs), as well as integrons have been studied in a collection of carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from a Spanish hospital. One hundred and twenty-three CRPA isolates were recovered from different samples of 80 patients. Clonal relationship among CRPA was analyzed by SpeI-PFGE. Susceptibility testing to 11 antibiotics and MBL phenotype was determined by microdilution, IP/IPI E-test and double disc method. The oprD gene was studied by PCR and sequencing, and mutations were determined comparing with P. aeruginosa PAO1 sequence. Characterization of MBLs, and class 1 and 2 integrons were studied by PCR and sequencing. SDS-PAGE analysis of outer membrane proteins of selected strains was performed. Seventy-four-per-cent of patients with CRPA were hospitalised in the ICU setting and 50% had long hospitalization stays. Sixty-four different PFGE patterns were detected, and 87 CRPA strains were further analyzed. MBL phenotype was detected in 43 of 87 strains (49.4%), which contained blaVIM-2 gene inside class 1 integrons. VIM-2-producing strains belonged to lineages ST175, ST235, and ST973. A great diversity of nucleotide insertions, deletions, and mutations in oprD gene, and the presence of a new insertion sequence (ISPa45) truncating oprD were identified among CRPA strains. Class 1 integrons were detected in 75% of CRPA strains, blaVIM-2 and the new arrangement aac(3)-Ia+ISPa34+aadA1 (named as In661) being the most frequent gene-cassette arrays detected. Other gene cassettes detected in integrons were: aadB, aadA6, aadA7, aac(6')-Ib', and blaOXA-46. PMID:24594145

  4. Salmonella Typhi OmpS1 and OmpS2 porins are potent protective immunogens with adjuvant properties

    PubMed Central

    Moreno-Eutimio, Mario A; Tenorio-Calvo, Alejandra; Pastelin-Palacios, Rodolfo; Perez-Shibayama, Christian; Gil-Cruz, Cristina; López-Santiago, Rubén; Baeza, Isabel; Fernández-Mora, Marcos; Bonifaz, Laura; Isibasi, Armando; Calva, Edmundo; López-Macías, Constantino

    2013-01-01

    Salmonella enterica serovar Typhi (S. Typhi) is the causal agent of typhoid fever, a disease that primarily affects developing countries. Various antigens from this bacterium have been reported to be targets of the immune response. Recently, the S. Typhi genome has been shown to encode two porins – OmpS1 and OmpS2 – which are expressed at low levels under in vitro culture conditions. In this study, we demonstrate that immunizing mice with either OmpS1 or OmpS2 induced production of specific, long-term antibody titres and conferred protection against S. Typhi challenge; in particular, OmpS1 was more immunogenic and conferred greater protective effects than OmpS2. We also found that OmpS1 is a Toll-like receptor 4 (TLR4) agonist, whereas OmpS2 is a TLR2 and TLR4 agonist. Both porins induced the production of tumour necrosis factor and interleukin-6, and OmpS2 was also able to induce interleukin-10 production. Furthermore, OmpS1 induced the over-expression of MHC II molecules in dendritic cells and OmpS2 induced the over-expression of CD40 molecules in macrophages and dendritic cells. Co-immunization of OmpS1 or OmpS2 with ovalbumin (OVA) increased anti-OVA antibody titres, the duration and isotype diversity of the OVA-specific antibody response, and the proliferation of T lymphocytes. These porins also had adjuvant effects on the antibody response when co-immunized with either the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus [A(H1N1)pdm09]. Taken together, the data indicate that OmpS1 and OmpS2, despite being expressed at low levels under in vitro culture conditions, are potent protective immunogens with intrinsic adjuvant properties. PMID:23432484

  5. Eicosapentaenoic acid membrane incorporation impairs ABCA1-dependent cholesterol efflux via a protein kinase A signaling pathway in primary human macrophages.

    PubMed

    Fournier, Natalie; Tardivel, Sylviane; Benoist, Jean-François; Vedie, Benoît; Rousseau-Ralliard, Delphine; Nowak, Maxime; Allaoui, Fatima; Paul, Jean-Louis

    2016-04-01

    A diet rich in n-3/n-6 polyunsaturated fatty acids (PUFAs) is cardioprotective. Dietary PUFAs affect the cellular phospholipids composition, which may influence the function of membrane proteins. We investigated the impact of the membrane incorporation of several PUFAs on ABCA1-mediated cholesterol efflux, a key antiatherogenic pathway. Arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (-28% for 70μM of EPA in HMDM), without any alterations in ABCA1 expression. These results show that PUFA membrane incorporation does not have the same consequences on cholesterol efflux from mouse and human macrophages. The EPA-treated HMDM exhibited strong phospholipid composition changes, with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which is associated with a decreased level of AA. In HMDM, EPA reduced the ATPase activity of the membrane transporter. Moreover, the activation of adenylate cyclase by forskolin and the inhibition of cAMP phosphodiesterase by isobutylmethylxanthine restored ABCA1 cholesterol efflux in EPA-treated human macrophages. In conclusion, EPA membrane incorporation reduces ABCA1 functionality in mouse macrophages as well as in primary human macrophages and this effect seems to be PKA-dependent in human macrophages. PMID:26776055

  6. Pseudomonas aeruginosa Porin OprF Exists in Two Different Conformations*

    PubMed Central

    Sugawara, Etsuko; Nestorovich, Ekaterina M.; Bezrukov, Sergey M.; Nikaido, Hiroshi

    2010-01-01

    The major nonspecific porin of Pseudomonas aeruginosa, OprF, produces a large channel yet allows only a slow diffusion of various solutes. Here we provide an explanation of this apparent paradox. We first show, by introduction of tobacco etch virus protease cleavage site in the middle of OprF protein, that most of OprF population folds as a two-domain protein with an N-terminal β-barrel domain and a C-terminal periplasmic domain rich in α-helices. However, sedimentation of unilamellar proteoliposomes through an iso-osmotic gradient showed that only about 5% of the OprF population produced open channels. Gel filtration showed that the open channel conformers tended to occur in oligomeric associations. Because the open channel conformer is likely to fold as a single domain protein with a large β-barrel, we reasoned that residues near the C terminus may be exposed on cell surface in this conformer. Introduction of a cysteine residue at position 312 produced a functional mutant protein. By using bulky biotinylation reagents on intact cells, we showed that this cysteine residue was not exposed on cell surface in most of the OprF population. However, the minority OprF population that was biotinylated in such experiments was enriched for the conformer with pore-forming activity and had a 10-fold higher pore-forming specific activity than the bulk OprF population. Finally trypsin treatment, which preferentially cleaves the C-terminal domain of the two-domain conformer, did not affect the pore-forming activity of OprF nor did it digest the minority conformer whose residue 312 is exposed on cell surface. PMID:16595653

  7. Vibrio cholerae porin OmpU induces LPS tolerance by attenuating TLR-mediated signaling.

    PubMed

    Sakharwade, Sanica C; Mukhopadhaya, Arunika

    2015-12-01

    Porins can act as pathogen-associated molecular patterns, can be recognized by the host immune system and modulate immune responses. Vibrio choleraeporin OmpU aids in bacterial survival in the human gut by increasing resistance against bile acids and anti-microbial peptides. V. choleraeOmpU is pro-inflammatory in nature. However, interestingly, it can also down-regulate LPS-mediated pro-inflammatory responses. In this study, we have explored how OmpU-pretreatment affects LPS-mediated responses. Our study indicates that OmpU-pretreatment followed by LPS-activation does not induce M2-polarization of macrophages/monocytes. Further, OmpU attenuates LPS-mediated TLR2/TLR6 signaling by decreasing the association of TLRs along with recruitment of MyD88 and IRAKs to the receptor complex. This results in decreased translocation of NFκB in the nucleus. Additionally, OmpU-pretreatment up-regulates expression of IRAK-M, a negative regulator of TLR signaling, in RAW 264.7 mouse macrophage cells upon LPS-stimulation. Suppressor cytokine IL-10 is partially involved in OmpU-induced down-regulation of LPS-mediated TNFα production in human PBMCs. Furthermore, OmpU-pretreatment also affects macrophage function, by enhancing phagocytosis in LPS-treated RAW 264.7 cells, and down-regulates LPS-induced cell surface expression of co-stimulatory molecules. Altogether, OmpU causes suppression of LPS-mediated responses by attenuating the LPS-mediated TLR signaling pathway. PMID:26454478

  8. Pseudomonas aeruginosa porin OprF exists in two different conformations.

    PubMed

    Sugawara, Etsuko; Nestorovich, Ekaterina M; Bezrukov, Sergey M; Nikaido, Hiroshi

    2006-06-16

    The major nonspecific porin of Pseudomonas aeruginosa, OprF, produces a large channel yet allows only a slow diffusion of various solutes. Here we provide an explanation of this apparent paradox. We first show, by introduction of tobacco etch virus protease cleavage site in the middle of OprF protein, that most of OprF population folds as a two-domain protein with an N-terminal beta-barrel domain and a C-terminal periplasmic domain rich in alpha-helices. However, sedimentation of unilamellar proteoliposomes through an iso-osmotic gradient showed that only about 5% of the OprF population produced open channels. Gel filtration showed that the open channel conformers tended to occur in oligomeric associations. Because the open channel conformer is likely to fold as a single domain protein with a large beta-barrel, we reasoned that residues near the C terminus may be exposed on cell surface in this conformer. Introduction of a cysteine residue at position 312 produced a functional mutant protein. By using bulky biotinylation reagents on intact cells, we showed that this cysteine residue was not exposed on cell surface in most of the OprF population. However, the minority OprF population that was biotinylated in such experiments was enriched for the conformer with pore-forming activity and had a 10-fold higher pore-forming specific activity than the bulk OprF population. Finally trypsin treatment, which preferentially cleaves the C-terminal domain of the two-domain conformer, did not affect the pore-forming activity of OprF nor did it digest the minority conformer whose residue 312 is exposed on cell surface. PMID:16595653

  9. Proteomic characterization of the outer membrane vesicle of Pseudomonas putida KT2440.

    PubMed

    Choi, Chi-Won; Park, Edmond Changkyun; Yun, Sung Ho; Lee, Sang-Yeop; Lee, Yeol Gyun; Hong, Yeonhee; Park, Kyeong Ryang; Kim, Sang-Hyun; Kim, Gun-Hwa; Kim, Seung Il

    2014-10-01

    Outer membrane vesicles (OMVs) are produced by various pathogenic Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In this study, we isolated OMVs from a representative soil bacterium, Pseudomonas putida KT2440, which has a biodegradative activity toward various aromatic compounds. Proteomic analysis identified the outer membrane proteins (OMPs) OprC, OprD, OprE, OprF, OprH, OprG, and OprW as major components of the OMV of P. putida KT2440. The production of OMVs was dependent on the nutrient availability in the culture media, and the up- or down-regulation of specific OMPs was observed according to the culture conditions. In particular, porins (e.g., benzoate-specific porin, BenF-like porin) and enzymes (e.g., catechol 1,2-dioxygenase, benzoate dioxygenase) for benzoate degradation were uniquely found in OMVs prepared from P. putida KT2440 that were cultured in media containing benzoate as the energy source. OMVs of P. putida KT2440 showed low pathological activity toward cultured cells that originated from human lung cells, which suggests their potential as adjuvants or OMV vaccine carriers. Our results suggest that the protein composition of the OMVs of P. putida KT2440 reflects the characteristics of the total proteome of P. putida KT2440. PMID:25198519

  10. Cell envelope of Bordetella pertussis: immunological and biochemical analyses and characterization of a major outer membrane porin protein

    SciTech Connect

    Armstrong, S.K.

    1986-01-01

    Surface molecules of Bordetella pertussis which may be important in metabolism, pathogenesis, and immunity to whooping cough were examined using cell fractionation and /sup 125/I cell surface labeling. Antigenic envelope proteins were examined by immunofluorescence microscopy and Western blotting procedures using monoclonal antibodies and convalescent sera. A surface protein with a high M/sub r/, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis but was absent in virulent B. pertussis strains. At least three envelope proteins were found only in virulent B. pertussis strains and were absent or diminished in avirulent and most phenotypically modulated strains. Transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Two dimensional gel electrophoresis revealed at least five heat modifiable proteins which migrated as higher or lower M/sub r/ moieties if solubilized at 25/sup 0/C instead of 100/sup 0/C.

  11. Substrate Specificity within a Family of Outer Membrane Carboxylate Channels

    SciTech Connect

    Eren, Elif; Vijayaraghavan, Jagamya; Liu, Jiaming; Cheneke, Belete R.; Touw, Debra S.; Lepore, Bryan W.; Indic, Mridhu; Movileanu, Liviu; van den Berg, Bert; Dutzler, Raimund

    2012-01-17

    Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM) that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.

  12. Functional characterization of Pseudomonas fluorescens OprE and OprQ membrane proteins.

    PubMed

    Jaouen, Thomas; Coquet, Laurent; Marvin-Guy, Laure; Orange, Nicole; Chevalier, Sylvie; Dé, Emmanuelle

    2006-08-01

    Outer membrane (OM) proteins of the OprD family may enable bacteria of the genus Pseudomonas to adapt to various environments by modulating OM permeability. The OprE and OprQ porins from P. fluorescens strain MF0 were purified and identified by MALDI-TOF mass spectrometry and N-terminal and internal microsequencing. These proteins, when reconstituted in an artificial planar lipid bilayer, induced similar ion channels with low single-conductance values. Secondary structure prediction of both proteins showed similar folding patterns into a 16 transmembrane beta-strands barrel but a highly variable amino-acid composition and length for their putative external loops implicated in porin function. Both proteins were overexpressed under poor oxygenation conditions, but not by using several amino acids as sole carbon source, indicating a different specificity for these proteins compared to the paradigm of this protein family, OprD. PMID:16777062

  13. Characteristics of Sucrose Transport through the Sucrose-Specific Porin ScrY Studied by Molecular Dynamics Simulations.

    PubMed

    Sun, Liping; Bertelshofer, Franziska; Greiner, Günther; Böckmann, Rainer A

    2016-01-01

    Sucrose-specific porin (ScrY) is a transmembrane protein that allows for the uptake of sucrose under growth-limiting conditions. The crystal structure of ScrY was resolved before by X-ray crystallography, both in its uncomplexed form and with bound sucrose. However, little is known about the molecular characteristics of the transport mechanism of ScrY. To date, there has not yet been any clear demonstration for sucrose transport through the ScrY. Here, the dynamics of the ScrY trimer embedded in a phospholipid bilayer as well as the characteristics of sucrose translocation were investigated by means of atomistic molecular dynamics (MD) simulations. The potential of mean force (PMF) for sucrose translocation through the pore showed two main energy barriers within the constriction region of ScrY. Energy decomposition allowed to pinpoint three aspartic acids as key residues opposing the passage of sucrose, all located within the L3 loop. Mutation of two aspartic acids to uncharged residues resulted in an accordingly modified electrostatics and decreased PMF barrier. The chosen methodology and results will aid in the design of porins with modified transport specificities. PMID:26913282

  14. Characteristics of Sucrose Transport through the Sucrose-Specific Porin ScrY Studied by Molecular Dynamics Simulations

    PubMed Central

    Sun, Liping; Bertelshofer, Franziska; Greiner, Günther; Böckmann, Rainer A.

    2016-01-01

    Sucrose-specific porin (ScrY) is a transmembrane protein that allows for the uptake of sucrose under growth-limiting conditions. The crystal structure of ScrY was resolved before by X-ray crystallography, both in its uncomplexed form and with bound sucrose. However, little is known about the molecular characteristics of the transport mechanism of ScrY. To date, there has not yet been any clear demonstration for sucrose transport through the ScrY. Here, the dynamics of the ScrY trimer embedded in a phospholipid bilayer as well as the characteristics of sucrose translocation were investigated by means of atomistic molecular dynamics (MD) simulations. The potential of mean force (PMF) for sucrose translocation through the pore showed two main energy barriers within the constriction region of ScrY. Energy decomposition allowed to pinpoint three aspartic acids as key residues opposing the passage of sucrose, all located within the L3 loop. Mutation of two aspartic acids to uncharged residues resulted in an accordingly modified electrostatics and decreased PMF barrier. The chosen methodology and results will aid in the design of porins with modified transport specificities. PMID:26913282

  15. Purification of integral outer-membrane protein OmpC, a surface antigen from Salmonella typhi for structure-function studies: a method applicable to enterobacterial major outer-membrane protein.

    PubMed

    Arockiasamy, A; Krishnaswamy, S

    2000-07-15

    Extraction of the outer-membrane porin, OmpC, from Salmonella typhi Ty21a was done by using a modified salt-extraction procedure. It was possible to extract only the major outer-membrane protein (OMP) from the crude membrane using this method. Aberrant lipopolysaccharide (LPS) production in the galE mutant Ty21a has resulted in more isoforms of OmpC and subsequently led to anomalous mobility in SDS-PAGE. The purity of the preparation was confirmed by denaturing urea SDS-PAGE and N-terminal sequencing. The major OMP extracts had LPS of both bound and free forms. The free form of LPS could be removed by gel filtration and the bound form, largely, was removed using ion-exchange chromatography and by passing through ultrafiltration devices. This method has been used to extract the native trimer of OmpC, the major OMP, in a large scale, for structure-function studies. S. typhi Ty21a OmpC preparation yielded reproducible diffraction-quality crystals. Extracts of porin from wild-type Escherichia coli HB101, grown under high osmolarity conditions, showed a single species of OMP on SDS-PAGE. This suggests the possible application of the method to other gram-negative bacterial porins. PMID:10929809

  16. Loss of the Arabidopsis thaliana P4-ATPases ALA6 and ALA7 impairs pollen fitness and alters the pollen tube plasma membrane.

    PubMed

    McDowell, Stephen C; López-Marqués, Rosa L; Cohen, Taylor; Brown, Elizabeth; Rosenberg, Alexa; Palmgren, Michael G; Harper, Jeffrey F

    2015-01-01

    Members of the P4 subfamily of P-type ATPases are thought to create and maintain lipid asymmetry in biological membranes by flipping specific lipids between membrane leaflets. In Arabidopsis, 7 of the 12 Aminophospholipid ATPase (ALA) family members are expressed in pollen. Here we show that double knockout of ALA6 and ALA7 (ala6/7) results in siliques with a ~2-fold reduction in seed set with a high frequency of empty seed positions near the bottom. Seed set was reduced to near zero when plants were grown under a hot/cold temperature stress. Reciprocal crosses indicate that the ala6/7 reproductive deficiencies are due to a defect related to pollen transmission. In-vitro growth assays provide evidence that ala6/7 pollen tubes are short and slow, with ~2-fold reductions in both maximal growth rate and overall length relative to wild-type. Outcrosses show that when ala6/7 pollen are in competition with wild-type pollen, they have a near 0% success rate in fertilizing ovules near the bottom of the pistil, consistent with ala6/7 pollen having short and slow growth defects. The ala6/7 phenotypes were rescued by the expression of either an ALA6-YFP or GFP-ALA6 fusion protein, which showed localization to both the plasma membrane and highly-mobile endomembrane structures. A mass spectrometry analysis of mature pollen grains revealed significant differences between ala6/7 and wild-type, both in the relative abundance of lipid classes and in the average number of double bonds present in acyl side chains. A change in the properties of the ala6/7 plasma membrane was also indicated by a ~10-fold reduction of labeling by lipophilic FM-dyes relative to wild-type. Together, these results indicate that ALA6 and ALA7 provide redundant activities that function to directly or indirectly change the distribution and abundance of lipids in pollen, and support a model in which ALA6 and ALA7 are critical for pollen fitness under normal and temperature-stress conditions. PMID:25954280

  17. Loss of the Arabidopsis thaliana P4-ATPases ALA6 and ALA7 impairs pollen fitness and alters the pollen tube plasma membrane

    PubMed Central

    McDowell, Stephen C.; López-Marqués, Rosa L.; Cohen, Taylor; Brown, Elizabeth; Rosenberg, Alexa; Palmgren, Michael G.; Harper, Jeffrey F.

    2015-01-01

    Members of the P4 subfamily of P-type ATPases are thought to create and maintain lipid asymmetry in biological membranes by flipping specific lipids between membrane leaflets. In Arabidopsis, 7 of the 12 Aminophospholipid ATPase (ALA) family members are expressed in pollen. Here we show that double knockout of ALA6 and ALA7 (ala6/7) results in siliques with a ~2-fold reduction in seed set with a high frequency of empty seed positions near the bottom. Seed set was reduced to near zero when plants were grown under a hot/cold temperature stress. Reciprocal crosses indicate that the ala6/7 reproductive deficiencies are due to a defect related to pollen transmission. In-vitro growth assays provide evidence that ala6/7 pollen tubes are short and slow, with ~2-fold reductions in both maximal growth rate and overall length relative to wild-type. Outcrosses show that when ala6/7 pollen are in competition with wild-type pollen, they have a near 0% success rate in fertilizing ovules near the bottom of the pistil, consistent with ala6/7 pollen having short and slow growth defects. The ala6/7 phenotypes were rescued by the expression of either an ALA6-YFP or GFP-ALA6 fusion protein, which showed localization to both the plasma membrane and highly-mobile endomembrane structures. A mass spectrometry analysis of mature pollen grains revealed significant differences between ala6/7 and wild-type, both in the relative abundance of lipid classes and in the average number of double bonds present in acyl side chains. A change in the properties of the ala6/7 plasma membrane was also indicated by a ~10-fold reduction of labeling by lipophilic FM-dyes relative to wild-type. Together, these results indicate that ALA6 and ALA7 provide redundant activities that function to directly or indirectly change the distribution and abundance of lipids in pollen, and support a model in which ALA6 and ALA7 are critical for pollen fitness under normal and temperature-stress conditions. PMID:25954280

  18. Identification of membrane proteins in the hyperthermophilic archaeon Pyrococcus furiosus using proteomics and prediction programs.

    SciTech Connect

    Holden, J. F.; Poole, F. L.; Tollaksen, S. L.; Giometti, C. S.; Lim, H.; Yates, J. R.; Adams, M. W. W.; Biosciences Division; Univ. of Georgia; The Scnpps Research Inst.

    2001-01-01

    Cell-free extracts from the hyperthermophilic archaeon Pyrococcus furiosus were separated into membrane and cytoplasmic fractions and each was analyzed by 2D-gel electrophoresis. A total of 66 proteins were identified, 32 in the membrane fraction and 34 in the cytoplasmic fraction. Six prediction programs were used to predict the subcellular locations of these proteins. Three were based on signal-peptides (SignalP, TargetP, and SOSUISignal) and three on transmembrane-spanning a-helices (TSEG, SOSUI, and PRED-TMR2). A consensus of the six programs predicted that 23 of the 32 proteins (72%) from the membrane fraction should be in the membrane and that all of the proteins from the cytoplasmic fraction should be in the cytoplasm. Two membrane-associated proteins predicted to be cytoplasmic by the programs are also predicted to consist primarily of transmembrane-spanning {beta}-sheets using porin protein models, suggesting that they are, in fact, membrane components. An ATPase subunit homolog found in the membrane fraction, although predicted to be cytoplasmic, is most likely complexed with other ATPase subunits in the membrane fraction. An additional three proteins predicted to be cytoplasmic but found in the membrane fraction, may be cytoplasmic contaminants. These include a chaperone homolog that may have attached to denatured membrane proteins during cell fractionation. Omitting these three proteins would boost the membrane-protein predictability of the models to near 80%. A consensus prediction using all six programs for all 2242 ORFs in the P. furiosus genome estimates that 24% of the ORF products are found in the membrane. However, this is likely to be a minimum value due to the programs' inability to recognize certain membrane-related proteins, such as subunits associated with membrane complexes and porin-type proteins.

  19. Psychological stress impairs Na+-dependent glucose absorption and increases GLUT2 expression in the rat jejunal brush-border membrane.

    PubMed

    Boudry, Gaëlle; Cheeseman, Christopher I; Perdue, Mary H

    2007-02-01

    Chronic psychological stress impacts many functions of the gastrointestinal tract. However, the effect of stress on nutrient absorption is poorly documented. This study was designed to investigate glucose transporters in rats submitted to different periods of water-avoidance stress (WAS). Rats were subjected to WAS (1 h/day) for 1, 5, or 10 consecutive days. Four hours after the last WAS session, rats were killed and segments of jejunum were mounted in Ussing chambers to study electrophysiological properties of the jejunum and Na+-dependent glucose absorption kinetics. Mucosa was obtained to prepare brush-border membrane vesicles (BBMV) used to measure [14C]fructose uptake as well as sodium-glucose transporter 1 (SGLT-1) and GLUT2 expression by Western blot analysis. Exposure of animals to WAS induced a decrease in Na+-dependent glucose absorption Vmax after 1, 5, and 10 days without any change in SGLT-1 expression. Potential difference across the jejunum was decreased for all stressed groups. Furthermore, we observed an increase in phloretin-sensitive uptake of [14C]fructose by BBMV after 1, 5, or 10 days of WAS, which was not present in control animals. This suggested the abnormal appearance of GLUT2 in the brush border, which was confirmed by Western blot analysis. We concluded that psychological stress induces major changes in glucose transport with a decrease in Na+-dependent glucose absorption and an increase in GLUT2 expression at the brush-border membrane level. PMID:17053095

  20. Susceptibility of Pseudomonas aeruginosa isolates to ceftazidime is unrelated to the expression of the outer membrane protein OprC.

    PubMed

    Pérez, F J; Navarro, D; Gimeno, C; García-de-Lomas, J

    1997-01-01

    Previously, it has been postulated that the porin OprC facilitates the diffusion of ceftazidime through the outer membrane of Pseudomonas aeruginosa. To further investigate this claim, the outer membrane protein (OMP) profiles of 22 ceftazidime-susceptible clinical isolates were analyzed. No correlation was found between MIC values and the level of expression of OprC. Further, OprC was either undetectable or expressed in reduced amounts in 12 isolates. In contrast, OprF and OprE were present in all isolates studied. This study suggests that OprC is dispensable for the permeation of ceftazidime through the outer membrane of P. aeruginosa. PMID:8996738

  1. Salicylate-inducible antibiotic resistance in Pseudomonas cepacia associated with absence of a pore-forming outer membrane protein.

    PubMed Central

    Burns, J L; Clark, D K

    1992-01-01

    The most common mechanism of antibiotic resistance in multiply resistant Pseudomonas cepacia is decreased porin-mediated outer membrane permeability. In some gram-negative organisms this form of antibiotic resistance can be induced by growth in the presence of weak acids, such as salicylates, which suppress porin synthesis. To determine the effects of salicylates on outer membrane permeability of P. cepacia, a susceptible laboratory strain, 249-2, was grown in 10 mM sodium salicylate. Antibiotic susceptibility and uptake, as well as outer membrane protein patterns, were compared between strain 249-2 grown with and without salicylates. The MICs of chloramphenicol, trimethoprim, ciprofloxacin, and ceftazidime were compared between organisms grown in standard and salicylate-containing medium and are as follows: chloramphenicol, 12.5 versus 100 micrograms/ml; trimethoprim, 0.78 versus 3.125 micrograms/ml; ciprofloxacin, 0.4 versus 1.56 micrograms/ml; ceftazidime, 3.125 versus 3.125 micrograms/ml. The permeability of beta-lactam antibiotics was calculated from the rate of hydrolysis of the chromogenic cephalosporin, PADAC. There was no significant difference between strains grown in the presence and absence of salicylate. By using high-pressure liquid chromatography quantitation of loss from culture medium, the effect of 10 mM salicylate on the cellular permeability of chloramphenicol was measured in strain 249-2 by introduction of a plasmid which encodes production of chloramphenicol acetyltransferase. After 1 h of incubation, 18.5% +/- 1.54% versus 70.1% +/- 3.52%, and after 2 h, 4.20% +/- 1.65% versus 41.90% +/- 2.16% remained in supernatants from organisms grown in the absence and presence of 10 mM salicylate, respectively. Outer membrane protein pattern analysis demonstrated the absence of a protein of apparent molecular weight of 40,000 when strain 249-2 was grown in the presence of 10 mM salicylate. To determine whether this protein functioned as a porin

  2. Porin-mediated antibiotic resistance in Neisseria gonorrhoeae: ion, solute, and antibiotic permeation through PIB proteins with penB mutations.

    PubMed

    Olesky, Melanie; Zhao, Shuqing; Rosenberg, Robert L; Nicholas, Robert A

    2006-04-01

    Neisseria gonorrhoeae has two porins, PIA and PIB, whose genes (porA and porB, respectively) are alleles of a single por locus. We recently demonstrated that penB mutations at positions 120 and 121 in PIB, which are presumed to reside in loop 3 that forms the pore constriction zone, confer intermediate-level resistance to penicillin and tetracycline (M. Olesky, M. Hobbs, and R. A. Nicholas, Antimicrob. Agents Chemother. 46:2811-2820, 2002). In the present study, we investigated the electrophysiological properties as well as solute and antibiotic permeation rates of recombinant PIB proteins containing penB mutations (G120K, G120D/A121D, G120P/A121P, and G120R/A121H). In planar lipid bilayers, the predominant conducting state of each porin variant was 30 to 40% of the wild type, even though the anion selectivity and maximum channel conductance of each PIB variant was similar to that of the wild type. Liposome-swelling experiments revealed no significant differences in the permeation of sugars or beta-lactam antibiotics through the wild type or PIB variants. Although these results are seemingly contradictory with the ability of these variants to increase antibiotic resistance, they are consistent with MIC data showing that these porin mutations confer resistance only in strains containing an mtrR mutation, which increases expression of the MtrC-MtrD-MtrE efflux pump. Moreover, both the mtrR and penB mutations were required to decrease in vivo permeation rates below those observed in the parental strain containing either mtrR or porin mutations alone. Thus, these data demonstrate a novel mechanism of porin-mediated resistance in which mutations in PIB have no affect on antibiotic permeation alone but instead act synergistically with the MtrC-MtrD-MtrE efflux pump in the development of antibiotic resistance in gonococci. PMID:16547016

  3. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    SciTech Connect

    Timmins, P.A.; Pebay-Peyroula, E.

    1994-12-31

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H{sub 2}O/D{sub 2}O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

  4. Spilanthol from Acmella Oleracea Lowers the Intracellular Levels of cAMP Impairing NKCC2 Phosphorylation and Water Channel AQP2 Membrane Expression in Mouse Kidney

    PubMed Central

    Gerbino, Andrea; Schena, Giorgia; Milano, Serena; Milella, Luigi; Barbosa, Alan Franco; Armentano, Francesca; Procino, Giuseppe; Svelto, Maria; Carmosino, Monica

    2016-01-01

    Acmella oleracea is well recognized in Brazilian traditional medicine as diuretic, although few scientific data have been published to support this effect. Aim of this study was to determine the molecular effect of Acmella oleracea extract and its main alkylamide spilanthol on two major processes involved in the urine concentrating mechanism: Na-K-2Cl symporter (NKCC2) activity in the thick ascending limb and water channel aquaporin 2 accumulation at the apical plasma membrane of collecting duct cells. Phosphorylation of NKCC2 was evaluated as index of its activation by Western blotting. Rate of aquaporin 2 apical expression was analyzed by confocal laser microscopy. Spilanthol-induced intracellular signalling events were dissected by video-imaging experiments. Exposure to spilanthol reduced the basal phosphorylation level of NKCC2 both in freshly isolated mouse kidney slices and in NKCC2-expresing HEK293 cells. In addition, exposure to spilanthol strongly reduced both desmopressin and low Cl−-dependent increase in NKCC2 phosphorylation in mouse kidney slices and NKCC2-expressing HEK293 cells, respectively. Similarly, spilanthol reduced both desmopressin- and forskolin-stimulated aquaporin 2 accumulation at the apical plasma membrane of collecting duct in mouse kidney slice and MCD4 cells, respectively. Of note, when orally administered, spilanthol induced a significant increase in both urine output and salt urinary excretion associated with a markedly reduced urine osmolality compared with control mice. Finally, at cellular level, spilanthol rapidly reduced or reversed basal and agonist-increased cAMP levels through a mechanism involving increases in intracellular [Ca2+]. In conclusion, spilanthol-induced inhibition of cAMP production negatively modulates urine-concentrating mechanisms thus holding great promise for its use as diuretic. PMID:27213818

  5. Spilanthol from Acmella Oleracea Lowers the Intracellular Levels of cAMP Impairing NKCC2 Phosphorylation and Water Channel AQP2 Membrane Expression in Mouse Kidney.

    PubMed

    Gerbino, Andrea; Schena, Giorgia; Milano, Serena; Milella, Luigi; Barbosa, Alan Franco; Armentano, Francesca; Procino, Giuseppe; Svelto, Maria; Carmosino, Monica

    2016-01-01

    Acmella oleracea is well recognized in Brazilian traditional medicine as diuretic, although few scientific data have been published to support this effect. Aim of this study was to determine the molecular effect of Acmella oleracea extract and its main alkylamide spilanthol on two major processes involved in the urine concentrating mechanism: Na-K-2Cl symporter (NKCC2) activity in the thick ascending limb and water channel aquaporin 2 accumulation at the apical plasma membrane of collecting duct cells. Phosphorylation of NKCC2 was evaluated as index of its activation by Western blotting. Rate of aquaporin 2 apical expression was analyzed by confocal laser microscopy. Spilanthol-induced intracellular signalling events were dissected by video-imaging experiments. Exposure to spilanthol reduced the basal phosphorylation level of NKCC2 both in freshly isolated mouse kidney slices and in NKCC2-expresing HEK293 cells. In addition, exposure to spilanthol strongly reduced both desmopressin and low Cl--dependent increase in NKCC2 phosphorylation in mouse kidney slices and NKCC2-expressing HEK293 cells, respectively. Similarly, spilanthol reduced both desmopressin- and forskolin-stimulated aquaporin 2 accumulation at the apical plasma membrane of collecting duct in mouse kidney slice and MCD4 cells, respectively. Of note, when orally administered, spilanthol induced a significant increase in both urine output and salt urinary excretion associated with a markedly reduced urine osmolality compared with control mice. Finally, at cellular level, spilanthol rapidly reduced or reversed basal and agonist-increased cAMP levels through a mechanism involving increases in intracellular [Ca2+]. In conclusion, spilanthol-induced inhibition of cAMP production negatively modulates urine-concentrating mechanisms thus holding great promise for its use as diuretic. PMID:27213818

  6. Rv1698 of Mycobacterium tuberculosis represents a new class of channel-forming outer membrane proteins.

    PubMed

    Siroy, Axel; Mailaender, Claudia; Harder, Daniel; Koerber, Stephanie; Wolschendorf, Frank; Danilchanka, Olga; Wang, Ying; Heinz, Christian; Niederweis, Michael

    2008-06-27

    Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 +/- 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes. PMID:18434314

  7. Experimental type II diabetes and related models of impaired glucose metabolism differentially regulate glucose transporters at the proximal tubule brush border membrane.

    PubMed

    Chichger, Havovi; Cleasby, Mark E; Srai, Surjit K; Unwin, Robert J; Debnam, Edward S; Marks, Joanne

    2016-06-01

    What is the central question of this study? Although SGLT2 inhibitors represent a promising treatment for patients suffering from diabetic nephropathy, the influence of metabolic disruption on the expression and function of glucose transporters is largely unknown. What is the main finding and its importance? In vivo models of metabolic disruption (Goto-Kakizaki type II diabetic rat and junk-food diet) demonstrate increased expression of SGLT1, SGLT2 and GLUT2 in the proximal tubule brush border. In the type II diabetic model, this is accompanied by increased SGLT- and GLUT-mediated glucose uptake. A fasted model of metabolic disruption (high-fat diet) demonstrated increased GLUT2 expression only. The differential alterations of glucose transporters in response to varying metabolic stress offer insight into the therapeutic value of inhibitors. SGLT2 inhibitors are now in clinical use to reduce hyperglycaemia in type II diabetes. However, renal glucose reabsorption across the brush border membrane (BBM) is not completely understood in diabetes. Increased consumption of a Western diet is strongly linked to type II diabetes. This study aimed to investigate the adaptations that occur in renal glucose transporters in response to experimental models of diet-induced insulin resistance. The study used Goto-Kakizaki type II diabetic rats and normal rats rendered insulin resistant using junk-food or high-fat diets. Levels of protein kinase C-βI (PKC-βI), GLUT2, SGLT1 and SGLT2 were determined by Western blotting of purified renal BBM. GLUT- and SGLT-mediated d-[(3) H]glucose uptake by BBM vesicles was measured in the presence and absence of the SGLT inhibitor phlorizin. GLUT- and SGLT-mediated glucose transport was elevated in type II diabetic rats, accompanied by increased expression of GLUT2, its upstream regulator PKC-βI and SGLT1 protein. Junk-food and high-fat diet feeding also caused higher membrane expression of GLUT2 and its upstream regulator PKC

  8. Molecular cloning and characterization of the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa.

    PubMed

    Okamoto, K; Gotoh, N; Tsujimoto, H; Yamada, H; Yoshihara, E; Nakae, T; Nishino, T

    1999-01-01

    We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1. The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602. Computer-aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P. aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin. Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild-type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other beta-lactam antibiotics. PMID:10338201

  9. Role of outer membrane barrier in efflux-mediated tetracycline resistance of Escherichia coli.

    PubMed Central

    Thanassi, D G; Suh, G S; Nikaido, H

    1995-01-01

    Accumulation of tetracycline in Escherichia coli was studied to determine its permeation pathway and to provide a basis for understanding efflux-mediated resistance. Passage of tetracycline across the outer membrane appeared to occur preferentially via the porin OmpF, with tetracycline in its magnesium-bound form. Rapid efflux of magnesium-chelated tetracycline from the periplasm was observed. In E. coli cells that do not contain exogenous tetracycline resistance genes, the steady-state level of tetracycline accumulation was decreased when porins were absent or when the fraction of Mg(2+)-chelated tetracycline was small. This is best explained by assuming the presence of a low-level endogenous active efflux system that bypasses the outer membrane barrier. When influx of tetracycline is slowed, this efflux is able to reduce the accumulation of tetracycline in the cytoplasm. In contrast, we found no evidence of a special outer membrane bypass mechanism for high-level efflux via the Tet protein, which is an inner membrane efflux pump coded for by exogenous tetA genes. Fractionation and equilibrium density gradient centrifugation experiments showed that the Tet protein is not localized to regions of inner and outer membrane adhesion. Furthermore, a high concentration of tetracycline was found in the compartment that rapidly equilibrated with the medium, most probably the periplasm, of Tet-containing E. coli cells, and the level of tetracycline accumulation in Tet-containing cells was not diminished by the mutational loss of the OmpF porin. These results suggest that the Tet protein, in contrast to the endogenous efflux system(s), pumps magnesium-chelated tetracycline into the periplasm. A quantitative model of tetracycline fluxes in E. coli cells of various types is presented. PMID:7860612

  10. A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations.

    PubMed

    Calì, Tito; Lopreiato, Raffaele; Shimony, Joshua; Vineyard, Marisa; Frizzarin, Martina; Zanni, Ginevra; Zanotti, Giuseppe; Brini, Marisa; Shinawi, Marwan; Carafoli, Ernesto

    2015-06-26

    The particular importance of Ca(2+) signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca(2+) ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca(2+). A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca(2+) ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca(2+) transients generated by cell stimulation and impairs its Ca(2+) extrusion function under conditions of low resting cytosolic Ca(2+) as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca(2+)-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca(2+) homeostasis and the previous finding that PMCAs act as digenic modulators in Ca(2+)-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype. PMID:25953895

  11. OmpW of Caulobacter crescentus Functions as an Outer Membrane Channel for Cations

    PubMed Central

    Benz, Roland; Jones, Michael D.; Younas, Farhan; Maier, Elke; Modi, Niraj; Mentele, Reinhard; Lottspeich, Friedrich; Kleinekathöfer, Ulrich; Smit, John

    2015-01-01

    Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures

  12. Effects of a siderophore receptor and porin proteins-based vaccination on fecal shedding of Escherichia coli O157:H7 in experimentally inoculated cattle.

    PubMed

    Thornton, A B; Thomson, D U; Loneragan, G H; Fox, J T; Burkhardt, D T; Emery, D A; Nagaraja, T G

    2009-04-01

    The efficacy of a vaccine containing outer membrane siderophore receptor and porin (SRP) proteins for reducing fecal prevalence and shedding of Escherichia coli O157:H7 was evaluated in cattle inoculated with E. coli O157:H7. Thirty calves were randomly assigned to one of two groups, and on days 1 and 21 these calves were given subcutaneous injections of either a placebo (control) or the vaccine. Blood was collected weekly to monitor the serum anti-SRP antibody titers. Two weeks after the second vaccination, calves were orally inoculated with a mixture of five strains of nalidixic acid-resistant (NalR) E. coli O157:H7. Fecal samples and rectoanal mucosal swabs were collected daily for the first 5 days and then three times each week for the following 4 weeks to determine the presence and enumerate the fecal concentration of NalR E. coli O157:H7. At necropsy on day 35, gut contents and tissue swabs were collected to determine the presence and concentration of NalR E. coli O157:H7. Vaccinated cattle had significantly higher anti-SRP antibody titers than did control cattle, with a significant treatment x week interaction (P < 0.01). Vaccination of cattle with the SRP protein tended to decrease fecal concentration (1.9 versus 1.6 log CFU/g) of NalR E. coli O157:H7 (P = 0.10). The number of calves that were fecal culture positive for E. coli O157:H7 was lower (P = 0.05) in the vaccinated group than in the control group. The E. coli O157:H7 SRP vaccine tended to reduce fecal prevalence and concentration of E. coli O157:H7 in cattle orally inoculated with NalR E. coli 0157:H7 and may be a useful prehavest intervention strategy. Future research must be conducted on natural prevalence in feedlot operations to further evaluate the efficacy of this novel vaccine. PMID:19435240

  13. Characterization of the Outer Membrane Protein OprF of Pseudomonas aeruginosa in a Lipopolysaccharide Membrane by Computer Simulation

    SciTech Connect

    Straatsma, TP; Soares, Thereza A.

    2009-02-01

    The N-terminal domain of outer membrane protein OprF of Pseudomonas aeruginosa forms a membrane spanning eight-stranded anti-parallel β-barrel domain that folds into a membrane channel with low conductance. The structure of this protein has been modeled after the crystal structure of the homologous protein OmpA of Escherichia coli. A number of molecular dynamics simulations have been carried out for the homology modeled structure of OprF in an explicit molecular model for the rough lipopolysaccharide (LPS) outer membrane of P. aeruginosa. The structural stability of the outer membrane model as a result of the strong electrostatic interactions compared to simple lipid bilayers is restricting both the conformational flexibility and the lateral diffusion of the porin in the membrane. Constricting side-chain interactions within the pore are similar to those found in reported simulations of the protein in a solvated lipid bilayer membrane. Because of the strong interactions between the loop regions of OprF and functional groups in the saccharide core of the LPS, the entrance to the channel from the extracellular space is widened compared to the lipid bilayer simulations in which the loops are extruding in the solvent. The specific electrostatic signature of the LPS membrane, which results in a net intrinsic dipole across the membrane, is found to be altered by the presence of OprF, resulting in a small electrically positive patch at the position of the channel.

  14. Hearing Impairments

    NASA Astrophysics Data System (ADS)

    Cavender, Anna; Ladner, Richard E.

    For many people with hearing impairments, the degree of hearing loss is only a small aspect of their disability and does not necessarily determine the types of accessibility solutions or accommodations that may be required. For some people, the ability to adjust the audio volume may be sufficient. For others, translation to a signed language may be more appropriate. For still others, access to text alternatives may be the best solution. Because of these differences, it is important for researchers in Web accessibility to understand that people with hearing impairments may have very different cultural-linguistic traditions and personal backgrounds.

  15. Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

    PubMed Central

    Skare, J T; Shang, E S; Foley, D M; Blanco, D R; Champion, C I; Mirzabekov, T; Sokolov, Y; Kagan, B L; Miller, J N; Lovett, M A

    1995-01-01

    We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease. Images PMID:7593626

  16. Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli.

    PubMed

    Cama, Jehangir; Bajaj, Harsha; Pagliara, Stefano; Maier, Theresa; Braun, Yvonne; Winterhalter, Mathias; Keyser, Ulrich F

    2015-11-01

    Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. We present a semiquantitative optofluidic assay for studying the uptake of autofluorescent drug molecules in single liposomes. We studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on average. We estimate a flux of about 10 norfloxacin molecules per second per OmpF trimer in the presence of a 1 mM concentration gradient of norfloxacin. We also performed single channel electrophysiology measurements and found that the application of transmembrane voltages causes an electric field driven uptake in addition to concentration driven diffusion. We use our results to propose a physical mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics. PMID:26478537

  17. Outer membrane proteins of pathogenic spirochetes

    PubMed Central

    Cullen, Paul A.; Haake, David A.; Adler, Ben

    2009-01-01

    Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed. We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance. A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given. A final section is included which covers experimental considerations in spirochetal outer membrane biology. This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure. A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis. PMID:15449605

  18. Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia.

    PubMed Central

    Kropinski, A M; Parr, T R; Angus, B L; Hancock, R E; Ghiorse, W C; Greenberg, E P

    1987-01-01

    The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl. Images PMID:3025168

  19. Characterization of Porin Expression in Klebsiella pneumoniae Carbapenemase (KPC)-Producing K. pneumoniae Identifies Isolates Most Susceptible to the Combination of Colistin and Carbapenems

    PubMed Central

    Hong, Jae H.; Cheng, Shaoji; Shields, Ryan K.; Chen, Liang; Doi, Yohei; Zhao, Yanan; Perlin, David S.; Kreiswirth, Barry N.; Nguyen, M. Hong

    2013-01-01

    We characterized carbapenem resistance mechanisms among 12 Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (referred to here as KPC K. pneumoniae) clinical isolates and evaluated their effects on the activity of 2- and 3-drug combinations of colistin, doripenem, and ertapenem. All isolates were resistant to ertapenem and doripenem; 75% (9/12) were resistant to colistin. Isolates belonged to the ST258 clonal group and harbored blaKPC-2, blaSHV-12, and blaTEM-1. As determined by time-kill assays, doripenem (8 μg/ml) and ertapenem (2 μg/ml) were inactive against 92% (11/12) and 100% (12/12) of isolates, respectively. Colistin (2.5 μg/ml) exerted some activity (range, 0.39 to 2.5 log10) against 78% (7/9) of colistin-resistant isolates. Colistin-ertapenem, colistin-doripenem, and colistin-doripenem-ertapenem exhibited synergy against 42% (5/12), 50% (6/12), and 67% (8/12) of isolates, respectively. Expression of ompK35 and ompK36 porins correlated with each other (R2 = 0.80). Levels of porin expression did not correlate with colistin-doripenem or colistin-ertapenem synergy. However, synergy with colistin-doripenem-ertapenem was more likely against isolates with high porin expression than those with low expression (100% [8/8] versus 0% [0/4]; P = 0.002). Moreover, bactericidal activity (area under the bacterial killing curve) against isolates with high porin expression was greater for colistin-doripenem-ertapenem than colistin-doripenem or colistin-ertapenem (P ≤ 0.049). In conclusion, colistin-carbapenem combinations may provide optimal activity against KPC K. pneumoniae, including colistin-resistant isolates. Screening for porin expression may identify isolates that are most likely to respond to a triple combination of colistin-doripenem-ertapenem. In the future, molecular characterization of KPC K. pneumoniae isolates may be a practical tool for identifying effective combination regimens. PMID:23459476

  20. Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants

    NASA Astrophysics Data System (ADS)

    Schulz, G. E.; Dreyer, M.; Klein, C.; Kreusch, A.; Mittl, P.; Mu¨ller, C. W.; Mu¨ller-Dieckmann, J.; Muller, Y. A.; Proba, K.; Schlauderer, G.; Spu¨rgin, P.; Stehle, T.; Weiss, M. S.

    1992-08-01

    Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8A˚resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0A˚resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.

  1. Effects of conjugated and unconjugated bile acids on the activity of the Vibrio cholerae porin OmpT.

    PubMed

    Pagel, Melissa; Delcour, Anne H

    2011-01-01

    During infection, the enteric pathogen Vibrio cholerae encounters a bile-containing environment. Previous studies have shown that bile and/or bile acids exert several effects on the virulence and physiology of the bacterial cells. These observations have led to the suggestion that bile acids may play a signaling role in infection. We have previously reported that the bile component deoxycholic acid blocks the general diffusion porin OmpT in a dose-dependent manner, presumably as it transits through the pore. V. cholerae colonizes the distal jejunum and ileum, where a mixture of various conjugated and unconjugated bile acids are found. In this work, we have used patch clamp electrophysiology to investigate the effects of six bile acids on OmpT. Two bile acids (deoxycholic and chenodeoxycholic acids) were found to block OmpT at physiological concentrations below 1 mM, while glycodeoxycholic acid was mildly effective and cholic, lithocholic and taurodeoxycholic acids were ineffective in this range. The block was also voltage-dependent. These observations suggest the presence of a specific binding site inside the OmpT pore. Since deconjugation is due to the activity of the endogenous flora, the preferential uptake of some unconjugated bile acids by OmpT may signal the presence of a hospitable environment. The results are also discussed in terms of the possible molecular interactions between the penetrating bile acid molecule and the channel wall. PMID:21067451

  2. Relative importances of outer membrane permeability and group 1 beta-lactamase as determinants of meropenem and imipenem activities against Enterobacter cloacae.

    PubMed Central

    Cornaglia, G; Russell, K; Satta, G; Fontana, R

    1995-01-01

    The roles of outer membrane permeability and Bush group 1 beta-lactamase activity in determining Enterobacter cloacae susceptibility to either meropenem or imipenem were investigated. A beta-lactamase-deficient strain was obtained by mutagenesis from a clinical isolate of E. cloacae, and a porin-deficient strain was selected from this mutant with cefoxitin. Both strains were transformed with the plasmid pAA20R, which contained the gene coding for the carbapenem-hydrolyzing CphA beta-lactamase, and the carbapenem permeability coefficients were measured by the Zimmermann and Rosselet technique (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368-372, 1977). The permeability coefficient of meropenem was roughly half that of imipenem in the normally permeable strain and almost seven times lower than that of imipenem in the porin-deficient strain. In the porin-deficient strain, the virtual absence of porins caused the MICs of meropenem to increase from 8 to 16 times, while it did not affect the MICs of imipenem. Conversely, the beta-lactamase affected imipenem but not meropenem activity: meropenem showed a similar activity in the parent strain and in the beta-lactamase-deficient mutant with both a low- and high-density inoculum, whereas imipenem was 16 times less active against the parent strain when the high-density inoculum was used. It is concluded that outer membrane permeability and stability to group 1 beta-lactamase have different impacts on the activities of meropenem and imipenem against E. cloacae. PMID:7726496

  3. Molecular cloning, sequencing, and expression of omp-40, the gene coding for the major outer membrane protein from the acidophilic bacterium Thiobacillus ferrooxidans.

    PubMed

    Guiliani, N; Jerez, C A

    2000-06-01

    Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane. PMID:10831405

  4. All Vision Impairment

    MedlinePlus

    ... Jobs Home > Statistics and Data > All Vision Impairment All Vision Impairment Vision Impairment Defined Vision impairment is ... being blind by the U.S. definition.) The category “All Vision Impairment” includes both low vision and blindness. ...

  5. Molecular Basis of Bacterial Outer Membrane Permeability Revisited

    PubMed Central

    Nikaido, Hiroshi

    2003-01-01

    Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions. PMID:14665678

  6. Proteome analysis of mitochondrial outer membrane from Neurospora crassa

    SciTech Connect

    Schmitt, Simone; Prokisch, Holger; Schlunk, Tilman; Camp, David G.; Ahting, Uwe; Waizenegger, Thomas; Scharfe, Curt M.; Meitinger, Thomas; Imhof, Axel; Neupert, Walter; Oefner, Peter J.; Rapaport, Doron

    2006-01-01

    The mitochondrial outer membrane mediates numerous interactions between the metabolic and genetic systems of mitochondria and the rest of the eukaryotic cell. We performed a proteomic study to discover novel functions of components of the mitochondrial outer membrane. Proteins of highly pure outer membrane vesicles (OMV) from Neurospora crassa were identified by a combination of liquid chromatography tandem mass spectrometry of tryptic peptide digests and gel electrophoresis of solubilized OMV proteins, followed by their identification using MALDI-MS peptide fingerprinting. Among the 30 proteins found in at least three of four separate analyses were 23 proteins with known functions in the outer membrane. These included components of the import machinery (the TOM and TOB complexes), a pore-forming component (Porin), and proteins that control fusion and fission of the organelle. In addition, proteins playing a role in various biosynthetic pathways, whose intracellular location had not been established previously, could be localized to the mitochondrial outer membrane. Thus, the proteome of the outer membrane can help in identifying new mitochondria-related functions.

  7. Outbreak Caused by an Ertapenem-Resistant, CTX-M-15-Producing Klebsiella pneumoniae Sequence Type 101 Clone Carrying an OmpK36 Porin Variant

    PubMed Central

    Poulou, Aggeliki; Voulgari, Evangelia; Vrioni, Georgia; Koumaki, Vasiliki; Xidopoulos, Grigorios; Chatzipantazi, Vasiliki; Markou, Fani

    2013-01-01

    Although numerous studies have documented outbreaks of carbapenem-resistant Klebsiella pneumoniae (CRKP) possessing various carbapenemases, reports on outbreaks due to CRKP possessing extended-spectrum β-lactamases (ESBLs) and/or AmpCs with porin lesions have been limited. Here, we describe an outbreak caused by an ertapenem-resistant, CTX-M-15-producing clonal K. pneumoniae strain expressing an OmpK36 porin variant. From May 2012 to November 2012, 37 ertapenem-resistant K. pneumoniae isolates phenotypically negative for carbapenemase production were recovered from 19 patients hospitalized in the intensive care unit of a Greek hospital. The isolates were either susceptible or intermediate to other carbapenems and resistant to all remaining β-lactams but cefotetan. Phenotypic and molecular analysis revealed the presence in all isolates of the blaCTX-M-15 gene on a conjugative 100-kb plasmid, disruption in the expression of the ompK35 gene, and the production of an Ompk36 porin variant. The index case was a patient admitted from another hospital. Active surveillance upon admission and on a weekly basis was immediately initiated; environmental samples were also periodically tested. Molecular typing showed that all clinical isolates as well as two ertapenem-resistant environmental K. pneumoniae isolates belonged to the same clonal type and were assigned to multilocus sequence typing (MLST) sequence type 101 (ST101). As all colonized/infected patients were hospitalized during overlapping periods, cross-infection was considered the main route for the dissemination of the outbreak strain. Despite reinforcement of infection control measures and active surveillance, the outbreak lasted approximately 7 months. Identification of hidden carriers upon admission and by screening on a weekly basis was found valuable for early recognition and subsequent successful management of the outbreak. PMID:23850951

  8. Structure, Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa.

    PubMed

    Modi, Niraj; Ganguly, Sonalli; Bárcena-Uribarri, Iván; Benz, Roland; van den Berg, Bert; Kleinekathöfer, Ulrich

    2015-10-01

    The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier between cell and environment. For nutrient acquisition, the OM contains a number of channels that mediate uptake of small molecules by diffusion. Many of these channels are specific, i.e., they prefer certain substrates over others. In electrophysiological experiments, the OM channels OprP and OprO from Pseudomonas aeruginosa show a specificity for phosphate and diphosphate, respectively. In this study we use x-ray crystallography, free-energy molecular dynamics (MD) simulations, and electrophysiology to uncover the atomic basis for the different substrate specificity of these highly similar channels. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. In OprP there are two tyrosine residues, Y62 and Y114, whereas the corresponding residues in OprO are phenylalanine F62 and aspartate D114. To probe the importance of these two residues in generating the different substrate specificities, the double mutants were generated in silico and in vitro. Applied-field MD simulations and electrophysiological experiments demonstrated that the double mutations interchange the phosphate and diphosphate specificities of OprP and OprO. Our findings outline a possible strategy to rationally design channel specificity by modification of a small number of residues that may be applicable to other pores as well. PMID:26445443

  9. Contribution of Efflux Pumps, Porins, and β-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

    PubMed Central

    Rumbo, C.; Gato, E.; López, M.; Ruiz de Alegría, C.; Fernández-Cuenca, F.; Martínez-Martínez, L.; Vila, J.; Pachón, J.; Cisneros, J. M.; Rodríguez-Baño, J.; Pascual, A.

    2013-01-01

    We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system. PMID:23939894

  10. Contribution of efflux pumps, porins, and β-lactamases to multidrug resistance in clinical isolates of Acinetobacter baumannii.

    PubMed

    Rumbo, C; Gato, E; López, M; Ruiz de Alegría, C; Fernández-Cuenca, F; Martínez-Martínez, L; Vila, J; Pachón, J; Cisneros, J M; Rodríguez-Baño, J; Pascual, A; Bou, G; Tomás, M

    2013-11-01

    We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system. PMID:23939894

  11. Functional microdomains in bacterial membranes.

    PubMed

    López, Daniel; Kolter, Roberto

    2010-09-01

    The membranes of eukaryotic cells harbor microdomains known as lipid rafts that contain a variety of signaling and transport proteins. Here we show that bacterial membranes contain microdomains functionally similar to those of eukaryotic cells. These membrane microdomains from diverse bacteria harbor homologs of Flotillin-1, a eukaryotic protein found exclusively in lipid rafts, along with proteins involved in signaling and transport. Inhibition of lipid raft formation through the action of zaragozic acid--a known inhibitor of squalene synthases--impaired biofilm formation and protein secretion but not cell viability. The orchestration of physiological processes in microdomains may be a more widespread feature of membranes than previously appreciated. PMID:20713508

  12. Functional microdomains in bacterial membranes

    PubMed Central

    López, Daniel; Kolter, Roberto

    2010-01-01

    The membranes of eukaryotic cells harbor microdomains known as lipid rafts that contain a variety of signaling and transport proteins. Here we show that bacterial membranes contain microdomains functionally similar to those of eukaryotic cells. These membrane microdomains from diverse bacteria harbor homologs of Flotillin-1, a eukaryotic protein found exclusively in lipid rafts, along with proteins involved in signaling and transport. Inhibition of lipid raft formation through the action of zaragozic acid—a known inhibitor of squalene synthases—impaired biofilm formation and protein secretion but not cell viability. The orchestration of physiological processes in microdomains may be a more widespread feature of membranes than previously appreciated. PMID:20713508

  13. Membrane stabilizer

    DOEpatents

    Mingenbach, William A.

    1988-01-01

    A device is provided for stabilizing a flexible membrane secured within a frame, wherein a plurality of elongated arms are disposed radially from a central hub which penetrates the membrane, said arms imposing alternately against opposite sides of the membrane, thus warping and tensioning the membrane into a condition of improved stability. The membrane may be an opaque or translucent sheet or other material.

  14. The role of the outer membrane of Gram-negative bacteria in antibiotic resistance: Ajax' shield or Achilles' heel?

    PubMed

    Page, Malcolm G P

    2012-01-01

    There has been an enormous increase in our knowledge of the fundamental steps in the biosynthesis and assembly of the outer membrane of Gram-negative bacteria. Lipopolysaccharide is a major component of the outer membrane of Gram-negative bacteria as is peptidoglycan. Porins, efflux pumps and other transport proteins of the outer membrane are also present. It is clear that there are numerous essential proteins that have the potential to be targets for novel antimicrobial agents. Progress, however, has been slow. Much of the emphasis has been on cytoplasmic processes that were better understood earlier on, but have the drawback that two penetration barriers, with different permeability properties, have to be crossed. With the increased understanding of the late-stage events occurring in the periplasm, it may be possible to shift focus to these more accessible targets. Nevertheless, getting drugs across the outer membrane will remain a challenge to the ingenuity of the medicinal chemist. PMID:23090596

  15. Immunoblot detection of class-specific humoral immune response to outer membrane proteins isolated from Salmonella typhi in humans with typhoid fever.

    PubMed Central

    Ortiz, V; Isibasi, A; García-Ortigoza, E; Kumate, J

    1989-01-01

    The studies reported here were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce a humoral immune response in humans with typhoid fever. OMPs were isolated with the nonionic detergent Triton X-100 and were found to be contaminated with approximately 4% lipopolysaccharide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns showed protein bands with molecular size ranges from 17 to 70 kilodaltons; the major groups of proteins were those that correspond to the porins and OmpA of gram-negative bacteria. Rabbit antiserum to OMPs or to S. typhi recognized OMPs after absorption with lipopolysaccharide. Sera from patients with typhoid fever contained immunoglobulin M antibodies which reacted with a protein of 28 kilodaltons and immunoglobulin G antibodies which reacted mainly with the porins, as determined by immunoblotting. These results indicate that the porins are the major immunogenic OMPs from S. typhi and that the immune response induced in the infection could be related to the protective status. Images PMID:2768450

  16. Mycobacterium tuberculosis Rv0899 defines a family of membrane proteins widespread in nitrogen-fixing bacteria

    PubMed Central

    Marassi, Francesca M.

    2011-01-01

    The Mycobacterium tuberculosis membrane protein Rv0899 confers adaptation of the bacterium to acidic environments. Due to strong sequence homology of its C-terminus to bacterial OmpA-like domains, Rv0899 has been proposed to constitute an outer membrane porin of M. tuberculosis. However, OmpA-like domains are widespread in a wide variety of bacterial proteins with different functions. Furthermore, the three-dimensional structure of Rv0899 does not contain a transmembrane β-barrel, and recent evidence demonstrates that it does not have porin activity. Instead, the rv0899 gene is part of an operon (rv0899-rv0901) that is required for fast ammonia secretion, pH neutralization and growth of M. tuberculosis in acidic environments. The mechanism whereby these functions are accomplished is not known. To gain further functional insights, a targeted search of the genomic databases was performed for proteins with sequence similarity beyond the OmpA-like C-terminus. The results presented here, show that Rv0899-like proteins are widespread in bacteria with functions in nitrogen metabolism, adaptation to nutrient poor environments, and/or establishing symbiosis with the host organism, and appear to form a protein family. These findings suggest that M. tuberculosis Rv0899 may also assist similar processes and lend further support to its role in ammonia secretion and M. tuberculosis adaptation to the host environment. PMID:21905117

  17. Participation of the Salmonella OmpD Porin in the Infection of RAW264.7 Macrophages and BALB/c Mice

    PubMed Central

    Monsalva, Debbie; Bustamante, Victor H.; Luraschi, Roberto; Alegría-Arcos, Melissa; Almonacid, Daniel E.; Aguayo, Daniel; Calderón, Iván L.; Gil, Fernando; Santiviago, Carlos A.; Morales, Eduardo H.; Calva, Edmundo; Saavedra, Claudia P.

    2014-01-01

    Salmonella Typhimurium is the etiological agent of gastroenteritis in humans and enteric fever in mice. Inside these hosts, Salmonella must overcome hostile conditions to develop a successful infection, a process in which the levels of porins may be critical. Herein, the role of the Salmonella Typhimurium porin OmpD in the infection process was assessed for adherence, invasion and proliferation in RAW264.7 mouse macrophages and in BALB/c mice. In cultured macrophages, a ΔompD strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain. In contrast, overexpression of ompD caused a reduction in bacterial proliferation but did not affect adherence or invasion. In the murine model, the ΔompD strain showed increased ability to survive and replicate in target organs of infection. The ompD transcript levels showed a down-regulation when Salmonella resided within cultured macrophages and when it colonized target organs in infected mice. Additionally, cultured macrophages infected with the ΔompD strain produced lower levels of reactive oxygen species, suggesting that down-regulation of ompD could favor replication of Salmonella inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host. PMID:25360745

  18. A structural study of ion permeation in OmpF porin from anomalous X-ray diffraction and molecular dynamics simulations

    PubMed Central

    Blachowicz, Lydia; Roux, Benoît

    2013-01-01

    OmpF, a multiionic porin from Escherichia coli, is a useful protypical model system for addressing general questions about electrostatic interactions in the confinement of an aqueous molecular pore. Here, favorable anion locations in the OmpF pore were mapped by anomalous X-ray scattering of Br− ions from four different crystal structures and compared with Mg2+ sites and Rb+ sites from a previous anomalous diffraction study to provide a complete picture of cation and anion transfer paths along the OmpF channel. By comparing structures with various crystallization conditions, we find that anions bind in discrete clusters along the entire length of the OmpF pore, whereas cations find conserved binding sites with the extracellular, surface-exposed loops. Results from molecular dynamics simulations are consistent with the experimental data and help highlight the critical residues that preferentially contact either cations or anions during permeation. Analysis of these results provides new insights into the molecular mechanisms that determine ion selectivity in OmpF porin. PMID:24106986

  19. Evidence that the outer membrane protein gene nmpC of Escherichia coli K-12 lies within the defective qsr' prophage.

    PubMed Central

    Highton, P J; Chang, Y; Marcotte, W R; Schnaitman, C A

    1985-01-01

    Recombinants between phage lambda and the defective qsr' prophage of Escherichia coli K-12 were made in an nmpC (p+) mutant strain and in the nmpC+ parent. The outer membrane of strains lysogenic for recombinant qsr' phage derived from the nmpC (p+) strain contained a new protein identical in electrophoretic mobility to the NmpC porin and to the Lc porin encoded by phage PA-2. Lysogens of qsr' recombinants from the nmpC+ strain and lysogens of lambda p4, which carries the qsr' region, did not produce this protein. When observed by electron microscopy, the DNA acquired from the qsr' prophage showed homology with the region of the DNA molecule of phage PA-2 which contains the lc gene. Relative to that of the recombinant from the nmpC (p+) mutant, the DNA molecule of the recombinant from the nmpC+ parent contained an insertion near the lc gene. These results were supported by blot hybridization analysis of the E. coli chromosome with probes derived from the lc gene of phage PA-2. A sequence homologous to the lc gene was found at the nmpC locus, and the parental strains contained an insertion, tentatively identified as IS5B, located near the 3' end of the porin coding sequence. We conclude that the structural gene for the NmpC porin protein is located within the defective qsr' prophage at 12.5 min on the E. coli K-12 map and that this gene can be activated by loss of an insertion element. Images PMID:2984173

  20. Membrane stabilizer

    DOEpatents

    Mingenbach, W.A.

    1988-02-09

    A device is provided for stabilizing a flexible membrane secured within a frame, wherein a plurality of elongated arms are disposed radially from a central hub which penetrates the membrane, said arms imposing alternately against opposite sides of the membrane, thus warping and tensioning the membrane into a condition of improved stability. The membrane may be an opaque or translucent sheet or other material. 10 figs.

  1. Detergent-associated Solution Conformations of Helical and Beta-barrel Membrane Proteins

    SciTech Connect

    Mo, Yiming; Lee, Byung-Kwon; Ankner, John Francis; Becker, Jeffrey Marvin; Heller, William T

    2008-01-01

    Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the ?-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.

  2. Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

    PubMed Central

    Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

    1995-01-01

    The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

  3. Membrane tension and membrane fusion.

    PubMed

    Kozlov, Michael M; Chernomordik, Leonid V

    2015-08-01

    Diverse cell biological processes that involve shaping and remodeling of cell membranes are regulated by membrane lateral tension. Here we focus on the role of tension in driving membrane fusion. We discuss the physics of membrane tension, forces that can generate the tension in plasma membrane of a cell, and the hypothesis that tension powers expansion of membrane fusion pores in late stages of cell-to-cell and exocytotic fusion. We propose that fusion pore expansion can require unusually large membrane tensions or, alternatively, low line tensions of the pore resulting from accumulation in the pore rim of membrane-bending proteins. Increase of the inter-membrane distance facilitates the reaction. PMID:26282924

  4. Membrane tethering

    PubMed Central

    Chia, Pei Zhi Cheryl

    2014-01-01

    Membrane trafficking depends on transport vesicles and carriers docking and fusing with the target organelle for the delivery of cargo. Membrane tethers and small guanosine triphosphatases (GTPases) mediate the docking of transport vesicles/carriers to enhance the efficiency of the subsequent SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion event with the target membrane bilayer. Different classes of membrane tethers and their specific intracellular location throughout the endomembrane system are now well defined. Recent biochemical and structural studies have led to a deeper understanding of the mechanism by which membrane tethers mediate docking of membrane carriers as well as an appreciation of the role of tethers in coordinating the correct SNARE complex and in regulating the organization of membrane compartments. This review will summarize the properties and roles of membrane tethers of both secretory and endocytic systems. PMID:25343031

  5. Assessment of the convergence of molecular dynamics simulations of lipopolysaccharide membranes

    SciTech Connect

    Soares, Thereza A.; Straatsma, TP

    2008-03-01

    The outer membrane of Gram-negative bacteria is composed of a phospholipid inner leaflet and a lipopolysaccharide outer leaflet. The chemical structure of lipopolysaccharide confers an asymmetric character to outer membranes that has been shown to play an important role in the in the electrical properties of porins, low permeability and intrinsic antibiotic resistance of Gram-negative bacteria. In the present work, atomistic molecular dynamics simulations of two different configurations of the outer membrane of Pseudomonas aeruginosa under periodic boundary conditions were carried out in order to i) validate model-derived properties against the available experimental data, ii) identify the properties whose dynamics can be sampled on nanosecond timescales, and iii) evaluate the dependence of the convergence of structural and dynamical properties on the initial configuration of the system, within the chosen force field and simulation conditions. Because the relaxation times associated with the motions of individual LPS monomers in outer membranes is very slow, the two initial configurations do not converge to a common ensemble of configuration on the nanosecond time scale. However, a number of properties of the outer membrane that will significantly impact the structural and internal dynamics of transmembrane proteins, most notably the electrostatic potential and molecular density, do converge within the simulated time scale. For these properties, a good agreement with the available experimental data was found. Such molecular model, capable of accounting for the high asymmetry and low fluidity characteristics of outer membranes, will certainly benefit future atomistic simulations of outer membrane proteins.

  6. Molecular Evolution of the Yersinia Major Outer Membrane Protein C (OmpC).

    PubMed

    Stenkova, Anna M; Bystritskaya, Evgeniya P; Guzev, Konstantin V; Rakin, Alexander V; Isaeva, Marina P

    2016-01-01

    The genus Yersinia includes species with a wide range of eukaryotic hosts (from fish, insects, and plants to mammals and humans). One of the major outer membrane proteins, the porin OmpC, is preferentially expressed in the host gut, where osmotic pressure, temperature, and the concentrations of nutrients and toxic products are relatively high. We consider here the molecular evolution and phylogeny of Yersinia ompC. The maximum likelihood gene tree reflects the macroevolution processes occurring within the genus Yersinia. Positive selection and horizontal gene transfer are the key factors of ompC diversification, and intraspecies recombination was revealed in two Yersinia species. The impact of recombination on ompC evolution was different from that of another major porin gene, ompF, possibly due to the emergence of additional functions and conservation of the basic transport function. The predicted antigenic determinants of OmpC were located in rapidly evolving regions, which may indicate the evolutionary mechanisms of Yersinia adaptation to the host immune system. PMID:27578962

  7. Molecular Evolution of the Yersinia Major Outer Membrane Protein C (OmpC)

    PubMed Central

    Stenkova, Anna M.; Bystritskaya, Evgeniya P.; Guzev, Konstantin V.; Rakin, Alexander V.; Isaeva, Marina P.

    2016-01-01

    The genus Yersinia includes species with a wide range of eukaryotic hosts (from fish, insects, and plants to mammals and humans). One of the major outer membrane proteins, the porin OmpC, is preferentially expressed in the host gut, where osmotic pressure, temperature, and the concentrations of nutrients and toxic products are relatively high. We consider here the molecular evolution and phylogeny of Yersinia ompC. The maximum likelihood gene tree reflects the macroevolution processes occurring within the genus Yersinia. Positive selection and horizontal gene transfer are the key factors of ompC diversification, and intraspecies recombination was revealed in two Yersinia species. The impact of recombination on ompC evolution was different from that of another major porin gene, ompF, possibly due to the emergence of additional functions and conservation of the basic transport function. The predicted antigenic determinants of OmpC were located in rapidly evolving regions, which may indicate the evolutionary mechanisms of Yersinia adaptation to the host immune system. PMID:27578962

  8. Key diffusion mechanisms involved in regulating bidirectional water permeation across E. coli outer membrane lectin

    PubMed Central

    Sachdeva, Shivangi; Kolimi, Narendar; Nair, Sanjana Anilkumar; Rathinavelan, Thenmalarchelvi

    2016-01-01

    Capsular polysaccharides (CPSs) are major bacterial virulent determinants that facilitate host immune evasion. E. coli group1 K30CPS is noncovalently attached to bacterial surface by Wzi, a lectin. Intriguingly, structure based phylogenetic analysis indicates that Wzi falls into porin superfamily. Molecular dynamics (MD) simulations further shed light on dual role of Wzi as it also functions as a bidirectional passive water specific porin. Such a functional role of Wzi was not realized earlier, due to the occluded pore. While five water specific entry points distributed across extracellular & periplasmic faces regulate the water diffusion involving different mechanisms, a luminal hydrophobic plug governs water permeation across the channel. Coincidently, MD observed open state structure of “YQF” triad is seen in sugar-binding site of sodium-galactose cotransporters, implicating its involvement in K30CPS surface anchorage. Importance of Loop 5 (L5) in membrane insertion is yet another highlight. Change in water diffusion pattern of periplasmic substitution mutants suggests Wzi’s role in osmoregulation by aiding in K30CPS hydration, corroborating earlier functional studies. Water molecules located inside β-barrel of Wzi crystal structure further strengthens the role of Wzi in osmoregulation. Thus, interrupting water diffusion or L5 insertion may reduce bacterial virulence. PMID:27320406

  9. Comparative Proteome Analysis of Spontaneous Outer Membrane Vesicles and Purified Outer Membranes of Neisseria meningitidis

    PubMed Central

    Otto, Andreas; Becher, Dörte; Vogel, Ulrich

    2013-01-01

    Outer membrane vesicles (OMVs) of Gram-negative bacteria receive increasing attention because of various biological functions and their use as vaccines. However, the mechanisms of OMV release and selective sorting of proteins into OMVs remain unclear. Comprehensive quantitative proteome comparisons between spontaneous OMVs (SOMVs) and the outer membrane (OM) have not been conducted so far. Here, we established a protocol for metabolic labeling of neisserial proteins with 15N. SOMV and OM proteins labeled with 15N were used as an internal standard for proteomic comparison of the SOMVs and OMs of two different strains. This labeling approach, coupled with high-sensitivity mass spectrometry, allowed us to comprehensively unravel the proteome of the SOMVs and OMs. We quantified the relative distribution of 155 proteins between SOMVs and the OM. Complement regulatory proteins, autotransporters, proteins involved in iron and zinc acquisition, and a two-partner secretion system were enriched in SOMVs. The highly abundant porins PorA and PorB and proteins connecting the OM with peptidoglycan or the inner membrane, such as RmpM, MtrE, and PilQ, were depleted in SOMVs. Furthermore, the three lytic transglycosylases MltA, MltB, and Slt were less abundant in SOMVs. In conclusion, SOMVs are likely to be released from surface areas with a low local abundance of membrane-anchoring proteins and lytic transglycosylases. The enrichment of complement regulatory proteins, autotransporters, and trace metal binding and transport proteins needs to be explored in the context of the pathogenesis of meningococcal disease. PMID:23893116

  10. Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains.

    PubMed Central

    Verstreate, D R; Winter, A J

    1984-01-01

    Outer membrane proteins were solubilized from 49 strains of Brucella abortus by sequential extraction of physically disrupted cells with N-lauroylsarcosinate and a dipolar ionic detergent (Verstreate et al., Infect. Immun. 35:979-989, 1982). The strains tested included standard agglutination test strain 1119, virulent strain 2308, and eight reference strains representing each of the biotypes; the remainder were isolates from cattle in North America with natural infections and included biotypes 1, 2, and 4. Three principal protein groups with apparent molecular weights of 88,000 to 94,000 (group 1), 35,000 to 40,000 (group 2, now established as porins [Douglas et al., Infect. Immun. 44:16-21, 1984]), and 25,000 to 30,000 (group 3) were observed in every strain. Some variability in banding patterns occurred among strains, but intrastrain variation was sufficient to preclude the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of outer membrane proteins for differentiating among strains of B. abortus. One antigen ([b]) was shared among the porin proteins, and three others ([c], ([d], and ([ e]) were shared among the group 3 proteins of all of the strains tested, indicating that these relationships are probably species wide. These results suggest that it may be possible to use outer membrane proteins from a representative strain of B. abortus in a vaccine for species-wide immunization. Images PMID:6434426

  11. Speech impairment (adult)

    MedlinePlus

    Language impairment; Impairment of speech; Inability to speak; Aphasia; Dysarthria; Slurred speech; Dysphonia voice disorders ... disorders develop gradually, but anyone can develop a speech and ... suddenly, usually in a trauma. APHASIA Alzheimer disease ...

  12. Mild Cognitive Impairment

    MedlinePlus

    ... Research Portfolio (IADRP) AMP-AD Detecting Cognitive Impairment Database ... Mild cognitive impairment (MCI) is a condition in which people have more memory or other thinking problems than normal for their ...

  13. Speech impairment (adult)

    MedlinePlus

    Language impairment; Impairment of speech; Inability to speak; Aphasia; Dysarthria; Slurred speech; Dysphonia voice disorders ... Common speech and language disorders include: APHASIA Aphasia is ... understand or express spoken or written language. It commonly ...

  14. The Origin and Early Evolution of Membrane Proteins

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

    2006-01-01

    The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

  15. Molecular dynamics simulations of membrane proteins under asymmetric ionic concentrations

    PubMed Central

    Khalili-Araghi, Fatemeh; Ziervogel, Brigitte; Gumbart, James C.

    2013-01-01

    A computational method is developed to allow molecular dynamics simulations of biomembrane systems under realistic ionic gradients and asymmetric salt concentrations while maintaining the conventional periodic boundary conditions required to minimize finite-size effects in an all-atom explicit solvent representation. The method, which consists of introducing a nonperiodic energy step acting on the ionic species at the edge of the simulation cell, is first tested with illustrative applications to a simple membrane slab model and a phospholipid membrane bilayer. The nonperiodic energy-step method is then used to calculate the reversal potential of the bacterial porin OmpF, a large cation-specific β-barrel channel, by simulating the I-V curve under an asymmetric 10:1 KCl concentration gradient. The calculated reversal potential of 28.6 mV is found to be in excellent agreement with the values of 26–27 mV measured from lipid bilayer experiments, thereby demonstrating that the method allows realistic simulations of nonequilibrium membrane transport with quantitative accuracy. As a final example, the pore domain of Kv1.2, a highly selective voltage-activated K+ channel, is simulated in a lipid bilayer under conditions that recreate, for the first time, the physiological K+ and Na+ concentration gradients and the electrostatic potential difference of living cells. PMID:24081985

  16. The Impaired Social Worker.

    ERIC Educational Resources Information Center

    Reamer, Frederic G.

    1992-01-01

    Discusses concept of the impaired professional; reviews research on various types of impairment (personality disorders, depression and other emotional problems, marital problems, and physical illness), prevalence and causes of impairment, and responses to it; and outlines model assessment and action plan for social workers who encounter an…

  17. ABE. The Hearing Impaired.

    ERIC Educational Resources Information Center

    Carver, L. Sue

    This handbook was written to help teachers of adult basic education (ABE) adapt their teaching methods for hearing impaired persons. Written in a narrative format, the guide covers the following topics: ABE for the hearing impaired, hints for working with the hearing impaired without an interpreter, peer pairing, interpreters in the classroom…

  18. Adapting for Impaired Patrons.

    ERIC Educational Resources Information Center

    Schuyler, Michael

    1999-01-01

    Describes how a library, with an MCI Corporation grant, approached the process of setting up computers for the visually impaired. Discusses preparations, which included hiring a visually-impaired user as a consultant and contacting the VIP (Visually Impaired Persons) group; equipment; problems with the graphical user interface; and training.…

  19. Antibiotic Trapping by Plasmid-Encoded CMY-2 β-Lactamase Combined with Reduced Outer Membrane Permeability as a Mechanism of Carbapenem Resistance in Escherichia coli

    PubMed Central

    van der Bij, Akke K.; van Boxtel, Ria; Pitout, Johann D. D.; van Ulsen, Peter; Melles, Damian C.; Tommassen, Jan

    2013-01-01

    A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CR E. coli lacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CR E. coli possessed blaCTX-M-15 and blaOXA-1. The CR E. coli strain additionally harbored blaCMY-2 and demonstrated a >15-fold increase in β-lactamase activity against nitrocefin, but no hydrolysis of meropenem was detected. However, nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation either in vitro or in vivo with meropenem, indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into various E. coli strains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance. In conclusion, carbapenem resistance emerged by a combination of elevated β-lactamase production and lack of porin expression. Due to the reduced outer membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the large amount of the β-lactamase. This study, therefore, provides evidence that the mechanism of “trapping” by CMY-2 β-lactamase plays a role in carbapenem resistance. PMID:23733461

  20. Identification of outer membrane proteins of Mycobacterium tuberculosis.

    PubMed

    Song, Houhui; Sandie, Reatha; Wang, Ying; Andrade-Navarro, Miguel A; Niederweis, Michael

    2008-11-01

    The cell wall of mycobacteria includes an unusual outer membrane of extremely low permeability. While Escherichia coli uses more than 60 proteins to functionalize its outer membrane, only two mycobacterial outer membrane proteins (OMPs) are known. The porin MspA of Mycobacterium smegmatis provided the proof of principle that integral mycobacterial OMPs share the beta-barrel structure, the absence of hydrophobic alpha-helices and the presence of a signal peptide with OMPs of gram-negative bacteria. These properties were exploited in a multi-step bioinformatic approach to predict OMPs of M. tuberculosis. A secondary structure analysis was performed for 587 proteins of M. tuberculosis predicted to be exported. Scores were calculated for the beta-strand content and the amphiphilicity of the beta-strands. Reference OMPs of gram-negative bacteria defined threshold values for these parameters that were met by 144 proteins of unknown function of M. tuberculosis. Two of them were verified as OMPs by a novel two-step experimental approach. Rv1698 and Rv1973 were detected only in the total membrane fraction of M. bovis BCG in Western blot experiments, while proteinase K digestion of whole cells showed the surface accessibility of these proteins. These findings established that Rv1698 and Rv1973 are indeed localized in the outer membrane and tripled the number of known OMPs of M. tuberculosis. Significantly, these results provide evidence for the usefulness of the bioinformatic approach to predict mycobacterial OMPs and indicate that M. tuberculosis likely has many OMPs with beta-barrel structure. Our findings pave the way to identify the set of proteins which functionalize the outer membrane of M. tuberculosis. PMID:18439872

  1. Solute Transport Proteins and the Outer Membrane Protein NmpC Contribute to Heat Resistance of Escherichia coli AW1.7▿

    PubMed Central

    Ruan, Lifang; Pleitner, Aaron; Gänzle, Michael G.; McMullen, Lynn M.

    2011-01-01

    This study aimed to elucidate determinants of heat resistance in Escherichia coli by comparing the composition of membrane lipids, as well as gene expression, in heat-resistant E. coli AW1.7 and heat-sensitive E. coli GGG10 with or without heat shock. The survival of E. coli AW1.7 at late exponential phase was 100-fold higher than that of E. coli GGG10 after incubation at 60°C for 15 min. The cytoplasmic membrane of E. coli AW1.7 contained a higher proportion of saturated and cyclopropane fatty acids than that of E. coli GGG10. Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked cultures was performed to compare gene expression in these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR. DnaK and 30S and 50S ribosomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50°C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane properties confirmed that NmpC is present in the outer membrane of E. coli AW1.7 but not in that of E. coli GGG10. Expression of NmpC in E. coli GGG10 increased survival at 60°C 50- to 1,000-fold. In conclusion, the outer membrane porin NmpC contributes to heat resistance in E. coli AW1.7, but the heat resistance of this strain is dependent on additional factors, which likely include the composition of membrane lipids, as well as solute transport proteins. PMID:21398480

  2. Memory Impairment in Children with Language Impairment

    ERIC Educational Resources Information Center

    Baird, Gillian; Dworzynski, Katharina; Slonims, Vicky; Simonoff, Emily

    2010-01-01

    Aim: The aim of this study was to assess whether any memory impairment co-occurring with language impairment is global, affecting both verbal and visual domains, or domain specific. Method: Visual and verbal memory, learning, and processing speed were assessed in children aged 6 years to 16 years 11 months (mean 9y 9m, SD 2y 6mo) with current,…

  3. Membrane Processes.

    PubMed

    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

  4. Membrane Processes.

    PubMed

    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

  5. Multicomponent membranes

    DOEpatents

    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.

  6. Global Analysis of the Membrane Subproteome of Pseudomonas aeruginosa using Liquid Chromatography-Tandem Mass Spectrometry

    SciTech Connect

    Blonder, Josip; Goshe, Michael B.; Xiao, Wenzhong; Camp, David G.; Wingerd, Mark A.; Davis, Ronald W.; Smith, Richard D.

    2004-05-30

    Pseudomonas aeruginosa is one of the most significant opportunistic bacterial pathogens in humans causing infections and premature death in patients with cystic fibrosis, AIDS, severe burns, organ transplants or cancer. Liquid chromatography coupled online with tandem mass spectrometry (LC-MS/MS) was used for the large-scale proteomic analysis of the P. aeruginosa membrane subproteome. Concomitantly, an affinity labeling technique, using iodoacetyl-PEO biotin to tag cysteinyl-containing proteins, permitted the enrichment and detection of lower abundance membrane proteins. The application of these approaches resulted in the identification of 786 proteins. A total of 333 proteins (42%) had a minimum of one transmembrane domain (TMD; ranging from 1 to 14) and 195 proteins were classified as hydrophobic based on their positive GRAVY values (ranging from 0.01 to 1.32). Key integral inner and outer membrane proteins involved in adaptation and antibiotic resistance were conclusively identified, including the detection of 53% of all predicted opr-type porins (outer integral membrane proteins) and all the components of the mexA-mexB-oprM transmembrane protein complex. This work represents the most comprehensive qualitative proteomic analysis of the membrane subproteome of P. aeruginosa and for prokaryotes in general to date.

  7. PLGA-microencapsulation protects Salmonella typhi outer membrane proteins from acidic degradation and increases their mucosal immunogenicity.

    PubMed

    Carreño, Juan Manuel; Perez-Shibayama, Christian; Gil-Cruz, Cristina; Printz, Andrea; Pastelin, Rodolfo; Isibasi, Armando; Chariatte, Dominic; Tanoue, Yutaka; Lopez-Macias, Constantino; Gander, Bruno; Ludewig, Burkhard

    2016-07-29

    Salmonella (S.) enterica infections are an important global health problem with more than 20 million individuals suffering from enteric fever annually and more than 200,000 lethal cases per year. Although enteric fever can be treated appropriately with antibiotics, an increasing number of antibiotic resistant Salmonella strains is detected. While two vaccines against typhoid fever are currently on the market, their availability in subtropical endemic areas is limited because these products need to be kept in uninterrupted cold chains. Hence, the development of a thermally stable vaccine that induces mucosal immune responses would greatly improve human health in endemic areas. Here, we have combined the high structural stability of Salmonella typhi outer membrane proteins (porins) with their microencapsulation into poly(lactic-co-glycolic acid) (PLGA) to generate an orally applicable vaccine. Encapsulated porins were protected from acidic degradation and exhibited enhanced immunogenicity following oral administration. In particular, the vaccine elicited strong S. typhi-specific B cell responses in Peyer's patches and mesenteric lymph nodes. In sum, PLGA microencapsulation substantially improved the efficacy of oral vaccination against S. typhi. PMID:27372155

  8. Channel Formation by CarO, the Carbapenem Resistance-Associated Outer Membrane Protein of Acinetobacter baumannii

    PubMed Central

    Siroy, Axel; Molle, Virginie; Lemaître-Guillier, Christelle; Vallenet, David; Pestel-Caron, Martine; Cozzone, Alain J.; Jouenne, Thierry; Dé, Emmanuelle

    2005-01-01

    It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (Mw and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a β-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels. PMID:16304148

  9. Molecular cloning and characterization of the structural gene for protein I, the major outer membrane protein of Neisseria gonorrhoeae.

    PubMed Central

    Carbonetti, N H; Sparling, P F

    1987-01-01

    Protein I (P.I) is the major outer membrane protein of Neisseria gonorrhoeae and serves as a porin. By using oligonucleotide probes derived from the known amino-terminal sequence of the mature protein, we have cloned the gene encoding the P.I of gonococcal strain FA19 in three overlapping fragments and determined the DNA sequence. The gene sequence predicts a protein with characteristics typical of the porins of other Gram-negative bacteria. A clone expressing P.I in Escherichia coli was obtained by removing a portion of the P.I gene promoter and reconstructing the entire P.I gene in a position just downstream from a phage T7 promoter. Expression of P.I was then achieved by introducing this recombinant plasmid into an E. coli strain containing an inducible T7 polymerase gene. The clone produced a protein that was identical in size to native P.I and reacted with anti-P.I monoclonal antibodies. Prolonged expression of the protein apparently was lethal for E. coli, possibly explaining failures to clone an intact P.I gene with its own promoter. Images PMID:3122212

  10. Transport Across Chloroplast Membranes: Optimizing Photosynthesis for Adverse Environmental Conditions.

    PubMed

    Pottosin, Igor; Shabala, Sergey

    2016-03-01

    Chloroplasts are central to solar light harvesting and photosynthesis. Optimal chloroplast functioning is vitally dependent on a very intensive traffic of metabolites and ions between the cytosol and stroma, and should be attuned for adverse environmental conditions. This is achieved by an orchestrated regulation of a variety of transport systems located at chloroplast membranes such as porines, solute channels, ion-specific cation and anion channels, and various primary and secondary active transport systems. In this review we describe the molecular nature and functional properties of the inner and outer envelope and thylakoid membrane channels and transporters. We then discuss how their orchestrated regulation affects thylakoid structure, electron transport and excitation energy transfer, proton-motive force partition, ion homeostasis, stromal pH regulation, and volume regulation. We link the activity of key cation and anion transport systems with stress-specific signaling processes in chloroplasts, and discuss how these signals interact with the signals generated in other organelles to optimize the cell performance, with a special emphasis on Ca(2+) and reactive oxygen species signaling. PMID:26597501

  11. The Origin and Early Evolution of Membrane Channels

    NASA Astrophysics Data System (ADS)

    Pohorille, Andrew; Schweighofer, Karl; Wilson, Michael A.

    2005-02-01

    The origin and early evolution of ion channels are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly greater complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Bacteria, and Archaea). We discuss the potassiumsodium- calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

  12. Protein folding in the periplasm in the absence of primary oxidant DsbA: modulation of redox potential in periplasmic space via OmpL porin

    PubMed Central

    Dartigalongue, Claire; Nikaido, Hiroshi; Raina, Satish

    2000-01-01

    Disulfide bond formation in Escherichia coli is a catalyzed reaction accomplished by DsbA. We found that null mutations in a new porin gene, ompL, allowed a total bypass of the DsbA requirement for protein oxidation. These mutations acted as extragenic null suppressors for dsbA, and restored normal folding of alkaline phosphatase and relieved sensitivity to dithiothreitol. ompL dsbA double mutants were completely like wild-type mutants in terms of motility and lack of mucoidy. This suppression was not dependent on DsbC and DsbG, since the oxidation status of these proteins was unaltered in ompL dsbA strains. Purified OmpL allowed diffusion of small solutes, including sugars, but the suppression was not dependent on the carbon sources used. Suppression by ompL null mutations required DsbB, leading us to propose a hypothesis that DsbB oxidizes yet unidentified, low-molecular-weight redox agents in the periplasm. These oxidized agents accumulate and substitute for DsbA if their leakage into the medium is prevented by the absence of OmpL, presumed to form a specific channel for their diffusion. PMID:11080145

  13. Hydrogen peroxide and hypochlorous acid influx through the major S. Typhimurium porin OmpD is affected by substitution of key residues of the channel.

    PubMed

    Aguayo, Daniel; Pacheco, Nicolás; Morales, Eduardo H; Collao, Bernardo; Luraschi, Roberto; Cabezas, Carolina; Calderón, Paulina; González-Nilo, Fernando; Gil, Fernando; Calderón, Iván L; Saavedra, Claudia P

    2015-02-15

    OmpD is the major Salmonella enterica serovar Typhimurium (S. Typhimurium) porin and mediates hydrogen peroxide (H2O2) influx. The results described herein extend this finding to hypochlorous acid (HOCl), another reactive oxygen species that is also part of the oxidative burst generated by the phagosome. S. Typhimurium cells lacking OmpD show decreased HOCl influx, and OmpD-reconstituted proteoliposomes show an increase in the uptake of the toxic compound. To understand this physiologically relevant process, we investigated the role of key OmpD residues in H2O2 and NaOCl transport. Using a theoretical approach, residue K16 was defined as a major contributor to the channel electrostatic properties, and E111 was shown to directly participate in the size-exclusion limit of the channel. Together, we provide theoretical, genetic, and biochemical evidence that OmpD mediates H2O2 and NaOCl uptake, and that key residues of the channel are implicated in this process. PMID:25600570

  14. Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus

    PubMed Central

    Duperthuy, Marylise; Schmitt, Paulina; Garzón, Edwin; Caro, Audrey; Rosa, Rafael D.; Le Roux, Frédérique; Lautrédou-Audouy, Nicole; Got, Patrice; Romestand, Bernard; de Lorgeril, Julien; Kieffer-Jaquinod, Sylvie; Bachère, Evelyne; Destoumieux-Garzón, Delphine

    2011-01-01

    OmpU porins are increasingly recognized as key determinants of pathogenic host Vibrio interactions. Although mechanisms remain incompletely understood, various species, including the human pathogen Vibrio cholera, require OmpU for host colonization and virulence. We have shown previously that OmpU is essential for virulence in the oyster pathogen Vibrio splendidus LGP32. Here, we showed that V. splendidus LGP32 invades the oyster immune cells, the hemocytes, through subversion of host-cell actin cytoskeleton. In this process, OmpU serves as an adhesin/invasin required for β-integrin recognition and host cell invasion. Furthermore, the major protein of oyster plasma, the extracellular superoxide dismutase Cg-EcSOD, is used as an opsonin mediating the OmpU-promoted phagocytosis through its RGD sequence. Finally, the endocytosed bacteria were found to survive intracellularly, evading the host defense by preventing acidic vacuole formation and limiting reactive oxygen species production. We conclude that (i) V. splendidus is a facultative intracellular pathogen that manipulates host defense mechanisms to enter and survive in host immune cells, and (ii) that OmpU is a major determinant of host cell invasion in Vibrio species, used by V. splendidus LGP32 to attach and invade oyster hemocytes through opsonisation by the oyster plasma Cg-EcSOD. PMID:21282662

  15. Crystalline Membranes

    NASA Technical Reports Server (NTRS)

    Tsapatsis, Michael (Inventor); Lai, Zhiping (Inventor)

    2008-01-01

    In certain aspects, the invention features methods for forming crystalline membranes (e.g., a membrane of a framework material, such as a zeolite) by inducing secondary growth in a layer of oriented seed crystals. The rate of growth of the seed crystals in the plane of the substrate is controlled to be comparable to the rate of growth out of the plane. As a result, a crystalline membrane can form a substantially continuous layer including grains of uniform crystallographic orientation that extend through the depth of the layer.

  16. The Transition from Closed to Open Conformation of Treponema pallidum Outer Membrane-associated Lipoprotein TP0453 Involves Membrane Sensing and Integration by Two Amphipathic Helices*

    PubMed Central

    Luthra, Amit; Zhu, Guangyu; Desrosiers, Daniel C.; Eggers, Christian H.; Mulay, Vishwaroop; Anand, Arvind; McArthur, Fiona A.; Romano, Fabian B.; Caimano, Melissa J.; Heuck, Alejandro P.; Malkowski, Michael G.; Radolf, Justin D.

    2011-01-01

    The molecular architecture and composition of the outer membrane (OM) of Treponema pallidum (Tp), the noncultivable agent of venereal syphilis, differ considerably from those of typical Gram-negative bacteria. Several years ago we described TP0453, the only lipoprotein associated with the inner leaflet of the Tp OM. Whereas polypeptides of other treponemal lipoproteins are hydrophilic, non-lipidated TP0453 can integrate into membranes, a property attributed to its multiple amphipathic helices (AHs). Furthermore, membrane integration of the TP0453 polypeptide was found to increase membrane permeability, suggesting the molecule functions in a porin-like manner. To better understand the mechanism of membrane integration of TP0453 and its physiological role in Tp OM biogenesis, we solved its crystal structure and used mutagenesis to identify membrane insertion elements. The crystal structure of TP0453 consists of an α/β/α-fold and includes five stably folded AHs. In high concentrations of detergent, TP0453 transitions from a closed to open conformation by lateral movement of two groups of AHs, exposing a large hydrophobic cavity. Triton X-114 phase partitioning, liposome floatation assay, and bis-1-anilino-8-naphthalenesulfonate binding revealed that two adjacent AHs are critical for membrane sensing/integration. Using terbium-dipicolinic acid complex-loaded large unilamellar vesicles, we found that TP0453 increased efflux of fluorophore only at acidic pH. Gel filtration and cross-linking experiments demonstrated that one AH critical for membrane sensing/insertion also forms a dimeric interface. Based on structural dynamics and comparison with Mycobacterium tuberculosis lipoproteins LprG and LppX, we propose that TP0453 functions as a carrier of lipids, glycolipids, and/or derivatives during OM biogenesis. PMID:21965687

  17. A conformational landscape for alginate secretion across the outer membrane of Pseudomonas aeruginosa

    SciTech Connect

    Tan, Jingquan; Rouse, Sarah L.; Li, Dianfan; Pye, Valerie E.; Vogeley, Lutz; Brinth, Alette R.; El Arnaout, Toufic; Whitney, John C.; Howell, P. Lynne; Sansom, Mark S. P.; Caffrey, Martin

    2014-08-01

    Crystal structures of the β-barrel porin AlgE reveal a mechanism whereby alginate is exported from P. aeruginosa for biofilm formation. The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common β-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE.

  18. Education for the Hearing Impaired (Auditorily Impaired).

    ERIC Educational Resources Information Center

    World Federation of the Deaf, Rome (Italy).

    Education for the hearing impaired is discussed in nine conference papers. J. N. Howarth describes "The Education of Deaf Children in Schools for Hearing Pupils in the United Kingdom" and A.I.Dyachkov of the U.S.S.R. outlines Didactical Principles of Educating the Deaf in the Light of their Rehabilitation Goal." Seven papers from Poland are also…

  19. Development or Impairment?

    ERIC Educational Resources Information Center

    Hakansson, Gisela

    2010-01-01

    Joanne Paradis' Keynote Article on bilingualism and specific language impairment (SLI) is an impressive overview of research in language acquisition and language impairment. Studying different populations is crucial both for theorizing about language acquisition mechanisms, and for practical purposes of diagnosing and supporting children with…

  20. Biological membranes

    PubMed Central

    Watson, Helen

    2015-01-01

    Biological membranes allow life as we know it to exist. They form cells and enable separation between the inside and outside of an organism, controlling by means of their selective permeability which substances enter and leave. By allowing gradients of ions to be created across them, membranes also enable living organisms to generate energy. In addition, they control the flow of messages between cells by sending, receiving and processing information in the form of chemical and electrical signals. This essay summarizes the structure and function of membranes and the proteins within them, and describes their role in trafficking and transport, and their involvement in health and disease. Techniques for studying membranes are also discussed. PMID:26504250

  1. Membranous nephropathy

    MedlinePlus

    ... to reduce cholesterol and triglyceride levels (most often statins) may be recommended. A low-salt diet may ... of membranous nephropathy Your symptoms get worse or don't go away You develop new symptoms You have ...

  2. Impairment in Non-Word Repetition: A Marker for Language Impairment or Reading Impairment?

    ERIC Educational Resources Information Center

    Baird, Gillian; Slonims, Vicky; Simonoff, Emily; Dworzynski, Katharina

    2011-01-01

    Aim: A deficit in non-word repetition (NWR), a measure of short-term phonological memory proposed as a marker for language impairment, is found not only in language impairment but also in reading impairment. We evaluated the strength of association between language impairment and reading impairment in children with current, past, and no language…

  3. Hearing or speech impairment - resources

    MedlinePlus

    Resources - hearing or speech impairment ... The following organizations are good resources for information on hearing impairment or speech impairment: Alexander Graham Bell Association for the Deaf and Hard of Hearing -- www.agbell. ...

  4. In Silico Structure and Sequence Analysis of Bacterial Porins and Specific Diffusion Channels for Hydrophilic Molecules: Conservation, Multimericity and Multifunctionality.

    PubMed

    Vollan, Hilde S; Tannæs, Tone; Vriend, Gert; Bukholm, Geir

    2016-01-01

    Diffusion channels are involved in the selective uptake of nutrients and form the largest outer membrane protein (OMP) family in Gram-negative bacteria. Differences in pore size and amino acid composition contribute to the specificity. Structure-based multiple sequence alignments shed light on the structure-function relations for all eight subclasses. Entropy-variability analysis results are correlated to known structural and functional aspects, such as structural integrity, multimericity, specificity and biological niche adaptation. The high mutation rate in their surface-exposed loops is likely an important mechanism for host immune system evasion. Multiple sequence alignments for each subclass revealed conserved residue positions that are involved in substrate recognition and specificity. An analysis of monomeric protein channels revealed particular sequence patterns of amino acids that were observed in other classes at multimeric interfaces. This adds to the emerging evidence that all members of the family exist in a multimeric state. Our findings are important for understanding the role of members of this family in a wide range of bacterial processes, including bacterial food uptake, survival and adaptation mechanisms. PMID:27110766

  5. In Silico Structure and Sequence Analysis of Bacterial Porins and Specific Diffusion Channels for Hydrophilic Molecules: Conservation, Multimericity and Multifunctionality

    PubMed Central

    Vollan, Hilde S.; Tannæs, Tone; Vriend, Gert; Bukholm, Geir

    2016-01-01

    Diffusion channels are involved in the selective uptake of nutrients and form the largest outer membrane protein (OMP) family in Gram-negative bacteria. Differences in pore size and amino acid composition contribute to the specificity. Structure-based multiple sequence alignments shed light on the structure-function relations for all eight subclasses. Entropy-variability analysis results are correlated to known structural and functional aspects, such as structural integrity, multimericity, specificity and biological niche adaptation. The high mutation rate in their surface-exposed loops is likely an important mechanism for host immune system evasion. Multiple sequence alignments for each subclass revealed conserved residue positions that are involved in substrate recognition and specificity. An analysis of monomeric protein channels revealed particular sequence patterns of amino acids that were observed in other classes at multimeric interfaces. This adds to the emerging evidence that all members of the family exist in a multimeric state. Our findings are important for understanding the role of members of this family in a wide range of bacterial processes, including bacterial food uptake, survival and adaptation mechanisms. PMID:27110766

  6. Impairments to Vision

    MedlinePlus

    ... an external Non-Government web site. Impairments to Vision Normal Vision Diabetic Retinopathy Age-related Macular Degeneration In this ... pictures, fixate on the nose to simulate the vision loss. In diabetic retinopathy, the blood vessels in ...

  7. Kids' Quest: Vision Impairment

    MedlinePlus

    ... important job. Â Return to Steps World-Wide Web Search Kids Health: What is Vision Impairment What ... for the Blind (AFB) created the Braille Bug web site to teach sighted children about braille, and ...

  8. Mild Cognitive Impairment

    MedlinePlus

    ... other people their age. This condition is called mild cognitive impairment, or MCI. People with MCI can take care of themselves and do their normal activities. MCI memory problems may include Losing things often Forgetting ...

  9. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    SciTech Connect

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S. E-mail: mkukrishna@rediffmail.com

    2005-08-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V{sub M}) of 3.3 Å{sup 3} Da{sup −1}, corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

  10. Hearing Impairment and Retirement

    PubMed Central

    Fischer, Mary E; Cruickshanks, Karen J; Pinto, Alex; Klein, Barbara E K; Klein, Ronald; Dalton, Dayna S

    2013-01-01

    BACKGROUND Many factors influence the decision to retire including age, insurance and pension availability along with physical and mental health. Hearing impairment may be one such factor. PURPOSE The purpose of this study was to compare the 15 year retirement rate among subjects with and without hearing impairment. RESEARCH DESIGN Prospective, population-based study STUDY SAMPLE Subjects were participants in the Epidemiology of Hearing Loss Study (EHLS), a longitudinal investigation of age-related hearing loss. Participants who were working full- or part-time in 1993–1995 were included (n=1410, mean age=57.8 years). DATA COLLECTION AND ANALYSIS Data from four EHLS phases (1993–1995, 1998–2000, 2003–2005, and 2009–2010) were analyzed in 2010–2012. Hearing impairment was defined as a pure tone threshold average (at 0.5,1,2 and 4 kHz) greater than 25 dB HL in the worse ear. Employment status was determined at each of the four phases. Kaplan-Meier estimates of the cumulative incidence of retirement were calculated and Cox discrete-time modeling was used to determine the effect of hearing impairment on the rate of retirement. RESULTS The cumulative incidence of retirement was significantly (p < 0.02) higher in those with a hearing impairment (77%) compared to those without a hearing impairment (74%). After adjustment for age, gender, self-reported health, and history of chronic disease, there was no significant difference in the rate of retirement between those with and without a hearing impairment (Hazard Ratio (HR) = 0.9, 95% Confidence Interval (CI) = 0.7, 1.1). Similar results were observed when hearing aid users were excluded, when hearing impairment was based on the better ear thresholds, and when analyses were restricted to those less than 65 years of age and working full-time at baseline. Participants with a hearing impairment were less likely to state that the main reason for retirement was that the time seemed right. CONCLUSIONS Hearing impairment

  11. Membrane magic

    SciTech Connect

    Buecker, B.

    2005-09-01

    The Kansas Power and Light Co.'s La Cyne generating station has found success with membrane filtration water pretreatment technology. The article recounts the process followed in late 2004 to install a Pall Aria 4 microfilter in Unit 1 makeup water system at the plant to produce cleaner water for reverse osmosis feed. 2 figs., 2 photos.

  12. Utility of biological membranes as indicators for radiation exposure: alterations in membrane structure and function over time.

    PubMed

    Dainiak, N; Tan, B J

    1995-05-01

    In addition to interacting with genomic DNA, ionizing radiation may directly and indirectly alter the structure and function of components of the plasma membrane of eukaryotic cells. Water radiolysis generates reactive species, including superoxide, hypochlorous acid and chloride radicals that may in turn react with biological membranes, as well as with cellular DNA. Reaction of plasma membrane lipids with molecular oxygen results in lipid peroxidation of both reconstituted membranes and biological membranes, an effect that increases with decreasing dose rate. Both ionizing radiation and ultraviolet light alter functions of membrane-anchored molecules, including adhesion molecules, histocompatibility complex antigens and membrane-bound growth factors. The latter growth factors represent a repertoire of growth and differentiation signals that are expressed in a nondiffusible fashion at the cell surface, and in soluble forms appearing after cleavage of their extracellular domain. The importance of cell-cell signaling via the membrane-anchored form of growth factors is becoming increasingly recognized. Expression of membrane-bound hematopoietic cytokines by eukaryotic cells is impaired after exposure to ultraviolet light, a defect in cell-cell signaling that may lead to impaired hematopoiesis. While studies suggest that permanent changes in membrane structure and function may result from radiation-induced injury to the plasma membrane and reconstituted "pure" membranes, reversibility of these defects over time requires additional study. PMID:7488940

  13. Outer membrane protein OmpQ of Bordetella bronchiseptica is required for mature biofilm formation.

    PubMed

    Cattelan, Natalia; Villalba, María Inés; Parisi, Gustavo; Arnal, Laura; Serra, Diego Omar; Aguilar, Mario; Yantorno, Osvaldo

    2016-02-01

    Bordetella bronchiseptica, an aerobic Gram-negative bacterium, is capable of colonizing the respiratory tract of diverse animals and chronically persists inside the hosts by forming biofilm. Most known virulence factors in Bordetella species are regulated by the BvgAS two-component transduction system. The Bvg-activated proteins play a critical role during host infection. OmpQ is an outer membrane porin protein which is expressed under BvgAS control. Here, we studied the contribution of OmpQ to the biofilm formation process by B. bronchiseptica. We found that the lack of expression of OmpQ did not affect the growth kinetics and final biomass of B. bronchiseptica under planktonic growth conditions. The ΔompQ mutant strain displayed no differences in attachment level and in early steps of biofilm formation. However, deletion of the ompQ gene attenuated the ability of B. bronchiseptica to form a mature biofilm. Analysis of ompQ gene expression during the biofilm formation process by B. bronchiseptica showed a dynamic expression pattern, with an increase of biofilm culture at 48 h. Moreover, we demonstrated that the addition of serum anti-OmpQ had the potential to reduce the biofilm biomass formation in a dose-dependent manner. In conclusion, we showed for the first time, to the best of our knowledge, evidence of the contribution of OmpQ to a process of importance for B. bronchiseptica pathobiology. Our results indicate that OmpQ plays a role during the biofilm development process, particularly at later stages of development, and that this porin could be a potential target for strategies of biofilm formation inhibition. PMID:26673448

  14. Deciphering the Function of the Outer Membrane Protein OprD Homologue of Acinetobacter baumannii

    PubMed Central

    Catel-Ferreira, Manuella; Nehmé, Rony; Molle, Virginie; Aranda, Jesús; Bouffartigues, Emeline; Chevalier, Sylvie; Bou, Germán; Jouenne, Thierry

    2012-01-01

    The increasing number of carbapenem-resistant Acinetobacter baumannii isolates is a major cause for concern which restricts therapeutic options to treat severe infections caused by this emerging pathogen. To identify the molecular mechanisms involved in carbapenem resistance, we studied the contribution of an outer membrane protein homologue of the Pseudomonas aeruginosa OprD porin. Suspected to be the preferred pathway of carbapenems in A. baumannii, the oprD homologue gene was inactivated in strain ATCC 17978. Comparison of wild-type and mutant strains did not confirm the expected increased resistance to any antibiotic tested. OprD homologue sequence analysis revealed that this protein actually belongs to an OprD subgroup but is closer to the P. aeruginosa OprQ protein, with which it could share some functions, e.g., allowing bacterial survival under low-iron or -magnesium growth conditions or under poor oxygenation. We thus overexpressed and purified a recombinant OprD homologue protein to further examine its functional properties. As a specific channel, this porin presented rather low single-channel conductance, i.e., 28 pS in 1 M KCl, and was partially closed by micro- and millimolar concentrations of Fe3+ and Mg2+, respectively, but not by imipenem and meropenem or basic amino acids. The A. baumannii OprD homologue is likely not involved in the carbapenem resistance mechanism, but as an OprQ-like protein, it could contribute to the adaptation of this bacterium to magnesium- and/or iron-depleted environments. PMID:22564848

  15. Structure and supramolecular architecture of membrane channel-forming peptides.

    PubMed

    Spach, G; Duclohier, H; Molle, G; Valleton, J M

    1989-01-01

    Peptides gathering together to induce channels in lipid bilayers may be classified in several categories according to the spatial structures involved. For example, gramicidin A forms intramolecular tubes, alamethicin, bundles of helical rods with intermolecular pores, porins (being proteins, properly speaking) are rich in beta-sheets that may form barrels, whereas cyclic peptides might stack together resulting in the formation of pores. The chemical structure of these compounds is now well characterized. The transmembrane electrical signals that they transmit are also typical of the particular supramolecular configurations (or architecture). Investigations in this field are thus relevant to structure-function relationship studies due to the availability of natural or synthetic analogues allowing the measurement of the influence of physico-chemical parameters upon the energy profiles of the pores. Consequently, questions such as the existence and probabilities of conductance substrates, their voltage-dependence and their ion or molecular selectivity can be tackled. Today, the loosest aspect of these studies lies in the actual molecular conformations and architecture in the membranes of the peptide aggregates, the knowledge of which remains imprecise, even 'at rest' in the best-studied cases. This review attempts to point out still unresolved questions and to propose some plausible approaches concerning, for example: 1) the configurations of the molecular aggregates responsible for ion transfer; 2) the mechanisms for channel-opening and closing (gating); 3) the eventual cooperative phenomena between channels, via the bilayer or interfacial components. Possible applications of these structures will be tentatively outlined. PMID:2470416

  16. High-level carbapenem-resistant OXA-48-producing Klebsiella pneumoniae with a novel OmpK36 variant and low-level, carbapenem-resistant, non-porin-deficient, OXA-181-producing Escherichia coli from Thailand.

    PubMed

    Lunha, Kamonwan; Chanawong, Aroonwadee; Lulitanond, Aroonlug; Wilailuckana, Chotechana; Charoensri, Nicha; Wonglakorn, Lumyai; Saenjamla, Pimjai; Chaimanee, Prajuab; Angkititrakul, Sunpetch; Chetchotisakd, Ploenchan

    2016-06-01

    Five blaOXA-48-like-carrying Enterobacteriaceae isolates collected from two Thai patients in December 2012 were characterized. Three Klebsiella pneumoniae isolates giving two different pulsed-field gel electrophoresis patterns and sequence types (ST11 and ST37) from patient 1 harbored blaOXA-48 locating on Tn1999.2, whereas two Escherichia coli isolates with the same pulsotype and ST5 from Patient 2 carried ISEcp1-associated blaOXA-181. One K. pneumoniae strain had blaSHV-12, blaDHA-1, qnrB, and qnrS, while another strain harbored blaCTX-M-15, qnrS and aac(6')-Ib-cr. The E. coli strain contained blaCTX-M-15, blaCMY-2, qnrS, and aac(6')-Ib-cr. Interestingly, the OXA-48 producers with a novel OmpK36 variant by a substitution of Gly to Asp in the L3 loop-borne PEFXG motif exhibited high-level resistance to ertapenem, imipenem, and meropenem. In contrast, the OXA-181 producer with non-porin-deficient background showed low-level resistance to ertapenem only. Both patients died because of either septic shock or pneumonia. This study showed the impact of OXA-48-like carbapenemases in porin-defective clinical isolate background, which may lead to serious therapeutic problems in the near future. PMID:27041106

  17. HEARING IMPAIRMENT IN CHILDREN

    PubMed Central

    Kohlmoos, H. W.

    1953-01-01

    Abnormal behavior in children may frequently be caused by impairment of hearing. Early detection of the impairment and of the cause are of utmost importance, not only to prevent irreversible changes where that is possible, but to permit early beginning of special training for children who are permanently deaf. Recent studies have shown that deafness of infants may follow rubella in the mother in early pregnancy, or kernicterus caused by Rh incompatibilities. Measures to control these disorders are being investigated. Adequate and careful treatment of diseases of the nose, as well as surgical drainage of infected ears when necessary, are important factors in the prevention of hearing loss in children. PMID:13009516

  18. [Membranous nephropathy].

    PubMed

    Mercadal, Lucile

    2013-12-01

    Membranous nephropathy is characterized by immune complex deposits on the outer side of the glomerular basement membrane. Activation of complement and of oxidation lead to basement membrane lesions. The most frequent form is idiopathic. At 5 and 10 years, renal survival is around 90 and 65% respectively. A prognostic model based on proteinuria, level and duration, progression of renal failure in a few months can refine prognosis. The urinary excretion of C5b-9, β2 and α1 microglobuline and IgG are strong predictors of outcome. Symptomatic treatment is based on anticoagulation in case of nephrotic syndrome, angiotensin conversion enzyme inhibitors, angiotensin II receptor blockers and statins. Immunosuppressive therapy should be discussed for patients having a high risk of progression. Corticoids alone has no indication. Treatment should include a simultaneous association or more often alternating corticoids and alkylant agent for a minimum of 6 months. Adrenocorticoid stimulating hormone and steroids plus mycophenolate mofetil may be equally effective. Steroids plus alkylant decrease the risk of end stage renal failure. Cyclosporine and tacrolimus decrease proteinuria but are associated with a high risk of recurrence at time of withdrawal and are nephrotoxic. Rituximab evaluated on open studies needs further evaluations to define its use. PMID:24315535

  19. Ion Channels in Native Chloroplast Membranes: Challenges and Potential for Direct Patch-Clamp Studies

    PubMed Central

    Pottosin, Igor; Dobrovinskaya, Oxana

    2015-01-01

    Photosynthesis without any doubt depends on the activity of the chloroplast ion channels. The thylakoid ion channels participate in the fine partitioning of the light-generated proton-motive force (p.m.f.). By regulating, therefore, luminal pH, they affect the linear electron flow and non-photochemical quenching. Stromal ion homeostasis and signaling, on the other hand, depend on the activity of both thylakoid and envelope ion channels. Experimentally, intact chloroplasts and swollen thylakoids were proven to be suitable for direct measurements of the ion channels activity via conventional patch-clamp technique; yet, such studies became infrequent, although their potential is far from being exhausted. In this paper we wish to summarize existing challenges for direct patch-clamping of native chloroplast membranes as well as present available results on the activity of thylakoid Cl− (ClC?) and divalent cation-permeable channels, along with their tentative roles in the p.m.f. partitioning, volume regulation, and stromal Ca2+ and Mg2+ dynamics. Patch-clamping of the intact envelope revealed both large-conductance porin-like channels, likely located in the outer envelope membrane and smaller conductance channels, more compatible with the inner envelope location. Possible equivalent model for the sandwich-like arrangement of the two envelope membranes within the patch electrode will be discussed, along with peculiar properties of the fast-activated cation channel in the context of the stromal pH control. PMID:26733887

  20. Specific Language Impairment

    MedlinePlus

    ... to distinguish between children who are struggling to learn a new language and children with true language impairments. After studying a large group of Hispanic children who speak English as a second language, NIDCD-funded researchers have developed a dual ...

  1. Cognitive Impairment After Stroke

    PubMed Central

    Gauba, Charu; Chaudhari, Dinesh

    2015-01-01

    Background: Vascular dementia is extremely common and contributes to stroke-associated morbidity and mortality. The study of vascular dementia may help to plan preventive interventions. Aims: To study the frequency of cognitive impairment after stroke in a series of consecutive patients with acute stroke, along with factors which influence it. Methods: Fifty adults with acute infarct or hemorrhage (as seen on computed tomography of the brain) were included in the study. The National Institute of Health Stroke Scale (NIHSS) and Barthel’s Index scores were done. Cognitive testing was done by PGI Battery of Brain Dysfunction (PGI-BBD) and Short Form of the Informant Questionnaire on Cognitive Decline in the Elderly (SIQCODE). Statistical analysis was by Student’s t-test, Chi-square test, Fisher’s exact test, and Mann-Whitney U test. Results: Mean age of patients was 61.82 years; males and ischemic strokes predominated. Dementia was seen in 30%, cognitive impairment no dementia (CIND) in 42%, and normal cognition in 28% patients. Factors associated with vascular cognitive impairment included old age, male sex, low education, hemorrhages, recurrent or severe stroke, silent infarcts, severe cortical atrophy, and left hemispheric or subcortical involvement. Conclusions: Up to 72% of patients have some form of cognitive impairment after a stroke. Secondary stroke prevention could reduce the incidence of vascular dementia. PMID:26543693

  2. Lipid membranes for membrane proteins.

    PubMed

    Kukol, Andreas

    2015-01-01

    The molecular dynamics (MD) simulation of membrane proteins requires the setup of an accurate representation of lipid bilayers. This chapter describes the setup of a lipid bilayer system from scratch using generally available tools, starting with a definition of the lipid molecule POPE, generation of a lipid bilayer, energy minimization, MD simulation, and data analysis. The data analysis includes the calculation of area and volume per lipid, deuterium order parameters, self-diffusion constant, and the electron density profile. PMID:25330959

  3. Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane

    PubMed Central

    Bohnert, Maria; Wenz, Lena-Sophie; Zerbes, Ralf M.; Horvath, Susanne E.; Stroud, David A.; von der Malsburg, Karina; Müller, Judith M.; Oeljeklaus, Silke; Perschil, Inge; Warscheid, Bettina; Chacinska, Agnieszka; Veenhuis, Marten; van der Klei, Ida J.; Daum, Günther; Wiedemann, Nils; Becker, Thomas; Pfanner, Nikolaus; van der Laan, Martin

    2012-01-01

    Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins. PMID:22918945

  4. Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane.

    PubMed Central

    Akins, D R; Robinson, E; Shevchenko, D; Elkins, C; Cox, D L; Radolf, J D

    1997-01-01

    Treponema pallidum rare outer membrane protein 1 (Tromp1) has extensive sequence homology with substrate-binding proteins of ATP-binding cassette transporters. Because such proteins typically are periplasmic or cytoplasmic membrane associated, experiments were conducted to clarify Tromp1's physicochemical properties and cellular location in T. pallidum. Comparison of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities of (i) native Tromp1 and Tromp1 synthesized by coupled in vitro transcription-translation and (ii) native Tromp1 and recombinant Tromp1 lacking the N-terminal signal sequence revealed that the native protein is not processed. Other studies demonstrated that recombinant Tromp1 lacks three basic porin-like properties: (i) the ability to form aqueous channels in liposomes which permit the influx of small hydrophilic solutes, (ii) an extensive beta-sheet secondary structure, and (iii) amphiphilicity. Subsurface localization of native Tromp1 was demonstrated by immunofluorescence analysis of treponemes encapsulated in gel microdroplets, while opsonization assays failed to detect surface-exposed Tromp1. Incubation of motile treponemes with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarine, a photoactivatable, lipophilic probe, also did not result in the detection of Tromp1 within the outer membranes of intact treponemes but, instead, resulted in the labeling of a basic 30.5-kDa presumptive outer membrane protein. Finally, analysis of fractionated treponemes revealed that native Tromp1 is associated predominantly with cell cylinders. These findings comprise a body of evidence that Tromp1 actually is anchored by an uncleaved signal sequence to the periplasmic face of the T. pallidum cytoplasmic membrane, where it likely subserves a transport-related function. PMID:9260949

  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.

    PubMed

    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. Omniphobic Membrane for Robust Membrane Distillation

    SciTech Connect

    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.

  7. MicL, a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein

    PubMed Central

    Guo, Monica S.; Updegrove, Taylor B.; Gogol, Emily B.; Shabalina, Svetlana A.; Gross, Carol A.; Storz, Gisela

    2014-01-01

    In enteric bacteria, the transcription factor σE maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σE-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σE activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σE regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σE to repress the synthesis of all abundant outer membrane proteins in response to stress. PMID:25030700

  8. Cholesterol reduction impairs exocytosis of synaptic vesicles.

    PubMed

    Linetti, Anna; Fratangeli, Alessandra; Taverna, Elena; Valnegri, Pamela; Francolini, Maura; Cappello, Valentina; Matteoli, Michela; Passafaro, Maria; Rosa, Patrizia

    2010-02-15

    Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Ca(v)2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons. PMID:20103534

  9. Grammatical Impairments in PPA

    PubMed Central

    Thompson, Cynthia K.; Mack, Jennifer E.

    2015-01-01

    Background Grammatical impairments are commonly observed in the agrammatic subtype of primary progressive aphasia (PPA-G), whereas grammatical processing is relatively preserved in logopenic (PPA-L) and semantic (PPA-S) subtypes. Aims We review research on grammatical deficits in PPA and associated neural mechanisms, with discussion focused on production and comprehension of four aspects of morphosyntactic structure: grammatical morphology, functional categories, verbs and verb argument structure, and complex syntactic structures. We also address assessment of grammatical deficits in PPA, with emphasis on behavioral tests of grammatical processing. Finally, we address research examining the effects of treatment for progressive grammatical impairments. Main Contribution PPA-G is associated with grammatical deficits that are evident across linguistic domains in both production and comprehension. PPA-G is associated with damage to regions including the left inferior frontal gyrus (IFG) and dorsal white matter tracts, which have been linked to impaired comprehension and production of complex sentences. Detailing grammatical deficits in PPA is important for estimating the trajectory of language decline and associated neuropathology. We, therefore, highlight several new assessment tools for examining different aspects of morphosyntactic processing in PPA. Conclusions Individuals with PPA-G present with agrammatic deficit patterns distinct from those associated with PPA-L and PPA-S, but similar to those seen in agrammatism resulting from stroke, and patterns of cortical atrophy and white matter changes associated with PPA-G have been identified. Methods for clinical evaluation of agrammatism, focusing on comprehension and production of grammatical morphology, functional categories, verbs and verb argument structure, and complex syntactic structures are recommended and tools for this are emerging in the literature. Further research is needed to investigate the real

  10. Phosphorus removal using nanofiltration membranes.

    PubMed

    Leo, C P; Chai, W K; Mohammad, A W; Qi, Y; Hoedley, A F A; Chai, S P

    2011-01-01

    A high concentration of phosphorus in wastewater may lead to excessive algae growth and deoxygenation of the water. In this work, nanofiltration (NF) of phosphorus-rich solutions is studied in order to investigate its potential in removing and recycling phosphorus. Wastewater samples from a pulp and paper plant were first analyzed. Commercial membranes (DK5, MPF34, NF90, NF270, NF200) were characterized and tested in permeability and phosphorus removal experiments. NF90 membranes offer the highest rejection of phosphorus; a rejection of more than 70% phosphorus was achieved for a feed containing 2.5 g/L of phosphorus at a pH <2. Additionally, NF90, NF200 and NF270 membranes show higher permeability than DK5 and MPF34 membranes. The separation performance of NF90 is slightly affected by phosphorus concentration and pressure, which may be due to concentration polarization and fouling. By adjusting the pH to 2 or adding sulfuric acid, the separation performance of NF90 was improved in removing phosphorus. However, the presence of acetic acid significantly impairs the rejection of phosphorus. PMID:22053475

  11. C1q binding and activation of the complement classical pathway by Klebsiella pneumoniae outer membrane proteins.

    PubMed Central

    Albertí, S; Marqués, G; Camprubí, S; Merino, S; Tomás, J M; Vivanco, F; Benedí, V J

    1993-01-01

    The mechanisms of killing of Klebsiella pneumoniae serum-sensitive strains in nonimmune serum by the complement classical pathway have been studied. The bacterial cell surface components that bind C1q more efficiently were identified as two major outer membrane proteins, presumably the porins of this bacterial species. These two outer membrane proteins were isolated from a representative serum-sensitive strain. We have demonstrated that in their purified form, they bind C1q and activate the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted in factor D). Binding of C1q to other components of the bacterial outer membrane, in particular the rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the two outer membrane proteins. The antibody-independent binding of C1q to serum-sensitive strains was independent of the presence of capsular polysaccharide, while strains possessing lipopolysaccharide O antigen bind less C1q and are resistant to complement-mediated killing. Images PMID:8432605

  12. Surface hydrolysis of sphingomyelin by the outer membrane protein Rv0888 supports replication of Mycobacterium tuberculosis in macrophages.

    PubMed

    Speer, Alexander; Sun, Jim; Danilchanka, Olga; Meikle, Virginia; Rowland, Jennifer L; Walter, Kerstin; Buck, Bradford R; Pavlenok, Mikhail; Hölscher, Christoph; Ehrt, Sabine; Niederweis, Michael

    2015-09-01

    Sphingomyelinases secreted by pathogenic bacteria play important roles in host-pathogen interactions ranging from interfering with phagocytosis and oxidative burst to iron acquisition. This study shows that the Mtb protein Rv0888 possesses potent sphingomyelinase activity cleaving sphingomyelin, a major lipid in eukaryotic cells, into ceramide and phosphocholine, which are then utilized by Mtb as carbon, nitrogen and phosphorus sources, respectively. An Mtb rv0888 deletion mutant did not grow on sphingomyelin as a sole carbon source anymore and replicated poorly in macrophages indicating that Mtb utilizes sphingomyelin during infection. Rv0888 is an unusual membrane protein with a surface-exposed C-terminal sphingomyelinase domain and a putative N-terminal channel domain that mediated glucose and phosphocholine uptake across the outer membrane in an M. smegmatis porin mutant. Hence, we propose to name Rv0888 as SpmT (sphingomyelinase of Mycobacterium tuberculosis). Erythrocyte membranes contain up to 27% sphingomyelin. The finding that Rv0888 accounts for half of Mtb's hemolytic activity is consistent with its sphingomyelinase activity and the observation that Rv0888 levels are increased in the presence of erythrocytes and sphingomyelin by 5- and 100-fold, respectively. Thus, Rv0888 is a novel outer membrane protein that enables Mtb to utilize sphingomyelin as a source of several essential nutrients during intracellular growth. PMID:26036301

  13. Hearing or speech impairment - resources

    MedlinePlus

    ... resources for information on hearing impairment or speech impairment: Alexander Graham Bell Association for the Deaf and Hard of Hearing -- www.agbell.org American Speech-Language-Hearing Association -- www.asha.org/public Center for ...

  14. Chemistry for the Visually Impaired.

    ERIC Educational Resources Information Center

    Ratliff, Judy L.

    1997-01-01

    Discusses modifications to general education or introductory chemistry courses that allow visually impaired students to participate productively. Describes a strategy for teaching about elements and density, and the construction of a conductivity tester for visually impaired students. (JRH)

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

    PubMed

    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. PMID:26351677

  16. Hopanoids as functional analogues of cholesterol in bacterial membranes

    PubMed Central

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

    2015-01-01

    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. PMID:26351677

  17. Mechanisms of membrane toxicity of hydrocarbons.

    PubMed Central

    Sikkema, J; de Bont, J A; Poolman, B

    1995-01-01

    Microbial transformations of cyclic hydrocarbons have received much attention during the past three decades. Interest in the degradation of environmental pollutants as well as in applications of microorganisms in the catalysis of chemical reactions has stimulated research in this area. The metabolic pathways of various aromatics, cycloalkanes, and terpenes in different microorganisms have been elucidated, and the genetics of several of these routes have been clarified. The toxicity of these compounds to microorganisms is very important in the microbial degradation of hydrocarbons, but not many researchers have studied the mechanism of this toxic action. In this review, we present general ideas derived from the various reports mentioning toxic effects. Most importantly, lipophilic hydrocarbons accumulate in the membrane lipid bilayer, affecting the structural and functional properties of these membranes. As a result of accumulated hydrocarbon molecules, the membrane loses its integrity, and an increase in permeability to protons and ions has been observed in several instances. Consequently, dissipation of the proton motive force and impairment of intracellular pH homeostasis occur. In addition to the effects of lipophilic compounds on the lipid part of the membrane, proteins embedded in the membrane are affected. The effects on the membrane-embedded proteins probably result to a large extent from changes in the lipid environment; however, direct effects of lipophilic compounds on membrane proteins have also been observed. Finally, the effectiveness of changes in membrane lipid composition, modification of outer membrane lipopolysaccharide, altered cell wall constituents, and active excretion systems in reducing the membrane concentrations of lipophilic compounds is discussed. Also, the adaptations (e.g., increase in lipid ordering, change in lipid/protein ratio) that compensate for the changes in membrane structure are treated. PMID:7603409

  18. Neurological Impairment: Nomenclature and Consequences.

    ERIC Educational Resources Information Center

    Spears, Catherine E.; Weber, Robert E.

    Neurological impairment as discussed includes a range of disabilities referred to as neurological impairment: minimal brain dysfunction/damage, developmental disability, perceptual handicap, learning disability, hyperkinetic behavioral syndrome, and others. Defined are causes of neurological impairment and methods of diagnosis. Symptoms…

  19. Membrane channel gene expression in human costal and articular chondrocytes

    PubMed Central

    Asmar, A.; Barrett-Jolley, R.; Werner, A.; Kelly, R.; Stacey, M.

    2016-01-01

    ABSTRACT Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca2+ activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought. PMID:27116676

  20. The impaired radiologist.

    PubMed

    Magnavita, N; Magnavita, G; Bergamaschi, A

    2010-08-01

    The concept of the "impaired physician" is an oxymoron. Physicians are by definition bearers of health, which can lead to overlooking the possibility of them contracting an illness that reduces their diagnostic and therapeutic abilities, with a consequent danger to their patients' health. The clinical reasons for which a radiologist may constitute a danger to patients can be divided into two categories: infectious blood-borne diseases, which can be transmitted to the patient during interventional radiology procedures; and neurodegenerative and psychiatric disorders, including alcohol and drug abuse, which temporarily or permanently impair the faculty of judgement. All radiologists have a duty to periodically verify their own state of health and seek help as soon as possible when they fear it may be a danger. This individual responsibility towards one's own patients is flanked by the health and safety requirements provided by European regulations for radiologists who are employers, directors or department heads. The occupational health physician plays a key role in identifying and managing the impaired radiologist. PMID:20221712

  1. Diabetes and Cognitive Impairment.

    PubMed

    Zilliox, Lindsay A; Chadrasekaran, Krish; Kwan, Justin Y; Russell, James W

    2016-09-01

    Both type 1 (T1DM) and type 2 diabetes mellitus (T2DM) have been associated with reduced performance on multiple domains of cognitive function and with evidence of abnormal structural and functional brain magnetic resonance imaging (MRI). Cognitive deficits may occur at the very earliest stages of diabetes and are further exacerbated by the metabolic syndrome. The duration of diabetes and glycemic control may have an impact on the type and severity of cognitive impairment, but as yet we cannot predict who is at greatest risk of developing cognitive impairment. The pathophysiology of cognitive impairment is multifactorial, although dysfunction in each interconnecting pathway ultimately leads to discordance in metabolic signaling. The pathophysiology includes defects in insulin signaling, autonomic function, neuroinflammatory pathways, mitochondrial (Mt) metabolism, the sirtuin-peroxisome proliferator-activated receptor-gamma co-activator 1α (SIRT-PGC-1α) axis, and Tau signaling. Several promising therapies have been identified in pre-clinical studies, but remain to be validated in clinical trials. PMID:27491830

  2. Anion permselective membrane

    NASA Technical Reports Server (NTRS)

    Hodgdon, R. B.; Waite, W. A.

    1980-01-01

    The efforts on the synthesis of polymer anion redox membranes were mainly concentrated in two areas, membrane development and membrane fabrication. Membrane development covered the preparation and evaluation of experimental membranes systems with improved resistance stability and/or lower permeability. Membrane fabrication covered the laboratory scale production of prime candidate membranes in quantities of up to two hundred and sizes up to 18 inches x 18 inches (46 cm x 46 cm). These small (10 in x 11 in) and medium sized membranes were mainly for assembly into multicell units. Improvements in processing procedures and techniques for preparing such membrane sets lifted yields to over 90 percent.

  3. Selecting a Roof Membrane.

    ERIC Educational Resources Information Center

    Waldron, Larry W.

    1990-01-01

    Offers a brief synopsis of the unique characteristics of the following roof membranes: (1) built-up roofing; (2) elastoplastic membranes; (3) modified bitumen membranes; (4) liquid applied membranes; and (5) metal roofing. A chart compares the characteristics of the raw membranes only. (MLF)

  4. Magnetic Membrane System

    DOEpatents

    McElfresh, Michael W.; ; Lucas, Matthew S.

    2004-12-30

    The present invention provides a membrane with magnetic particles. In one embodiment the membrane is created by mixing particles in a non-magnetic base. The membrane may act as an actuator, a sensor, a pump, a valve, or other device. A magnet is operatively connected to the membrane. The magnet acts on and changes the shape of the membrane.

  5. Doripenem MICs and ompK36 Porin Genotypes of Sequence Type 258, KPC-Producing Klebsiella pneumoniae May Predict Responses to Carbapenem-Colistin Combination Therapy among Patients with Bacteremia

    PubMed Central

    Shields, Ryan K.; Potoski, Brian A.; Press, Ellen G.; Chen, Liang; Kreiswirth, Barry N.; Clarke, Lloyd G.; Eschenauer, Gregory A.; Clancy, Cornelius J.

    2014-01-01

    Treatment failures of a carbapenem-colistin regimen among patients with bacteremia due to sequence type 258 (ST258), KPC-2-producing Klebsiella pneumoniae were significantly more likely if both agents were inactive in vitro, as defined by a colistin MIC of >2 μg/ml and the presence of either a major ompK36 porin mutation (guanine and alanine insertions at amino acids 134 and 135 [ins aa 134–135 GD], IS5 promoter insertion [P = 0.007]) or a doripenem MIC of >8 μg/ml (P = 0.01). Major ompK36 mutations among KPC-K. pneumoniae strains are important determinants of carbapenem-colistin responses in vitro and in vivo. PMID:25534733

  6. Membrane Systems in Cyanobacteria

    SciTech Connect

    Liberton, Michelle L.; Pakrasi, Himadri B.

    2008-01-01

    Cyanobacteria are photosynthetic prokaryotes with highly differentiated membrane systems. In addition to a Gram-negative-type cell envelope with plasma membrane and outer membrane separated by a periplasmic space, cyanobacteria have an internal system of thylakoid membranes where the fully functional electron transfer chains of photosynthesis and respiration reside. The presence of different membrane systems lends these cells a unique complexity among bacteria. Cyanobacteria must be able to reorganize the membranes, synthesize new membrane lipids, and properly target proteins to the correct membrane system. The outer membrane, plasma membrane, and thylakoid membranes each have specialized roles in the cyanobacterial cell. Understanding the organization, functionality, protein composition and dynamics of the membrane systems remains a great challenge in cyanobacterial cell biology.

  7. The outer membrane protein Omp35 affects the reduction of Fe(III), nitrate, and fumarate by Shewanella oneidensis MR-1

    PubMed Central

    Maier, Tamara M; Myers, Charles R

    2004-01-01

    Background Shewanella oneidensis MR-1 uses several electron acceptors to support anaerobic respiration including insoluble species such as iron(III) and manganese(IV) oxides, and soluble species such as nitrate, fumarate, dimethylsulfoxide and many others. MR-1 has complex branched electron transport chains that include components in the cytoplasmic membrane, periplasm, and outer membrane (OM). Previous studies have implicated a role for anaerobically upregulated OM electron transport components in the use of insoluble electron acceptors, and have suggested that other OM components may also contribute to insoluble electron acceptor use. In this study, the role for an anaerobically upregulated 35-kDa OM protein (Omp35) in the use of anaerobic electron acceptors was explored. Results Omp35 was purified from the OM of anaerobically grown cells, the gene encoding Omp35 was identified, and an omp35 null mutant (OMP35-1) was isolated and characterized. Although OMP35-1 grew on all electron acceptors tested, a significant lag was seen when grown on fumarate, nitrate, and Fe(III). Complementation studies confirmed that the phenotype of OMP35-1 was due to the loss of Omp35. Despite its requirement for wild-type rates of electron acceptor use, analysis of Omp35 protein and predicted sequence did not identify any electron transport moieties or predicted motifs. OMP35-1 had normal levels and distribution of known electron transport components including quinones, cytochromes, and fumarate reductase. Omp35 is related to putative porins from MR-1 and S. frigidimarina as well as to the PorA porin from Neisseria meningitidis. Subcellular fraction analysis confirmed that Omp35 is an OM protein. The seven-fold anaerobic upregulation of Omp35 is mediated post-transcriptionally. Conclusion Omp35 is a putative porin in the OM of MR-1 that is markedly upregulated anaerobically by a post-transcriptional mechanism. Omp35 is required for normal rates of growth on Fe(III), fumarate, and

  8. Outer Membrane Proteome of Burkholderia pseudomallei and Burkholderia mallei From Diverse Growth Conditions

    PubMed Central

    Schell, Mark A.; Zhao, Peng; Wells, Lance

    2016-01-01

    Burkholderia mallei and Burkholderia pseudomallei are closely related, aerosol-infective human pathogens that cause life-threatening diseases. Biochemical analyses requiring large-scale growth and manipulation at biosafety level 3 under select agent regulations are cumbersome and hazardous. We developed a simple, safe, and rapid method to prepare highly purified outer membrane (OM) fragments from these pathogens. Shotgun proteomic analyses of OMs by trypsin shaving and mass spectrometry identified >155 proteins, the majority of which are clearly outer membrane proteins (OMPs). These included: 13 porins, 4 secretins for virulence factor export, 11 efflux pumps, multiple components of a Type VI secreton, metal transport receptors, polysaccharide exporters, and hypothetical OMPs of unknown function. We also identified 20 OMPs in each pathogen that are abundant under a wide variety of conditions, including in serum and with macrophages, suggesting these are fundamental for growth and survival and may represent prime drug or vaccine targets. Comparison of the OM proteomes of B. mallei and B. pseudomallei showed many similarities but also revealed a few differences, perhaps reflecting evolution of B. mallei away from environmental survival toward host-adaptation. PMID:21391724

  9. Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

    PubMed Central

    Clémençon, Benjamin

    2012-01-01

    The existence of a mitochondrial interactosome (MI) has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK) in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp) and inorganic phosphate (PiC) carriers as well as the VDAC (or mitochondrial porin) catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1) under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003. PMID:22408429

  10. Increased outer membrane resistance to ethylenediaminetetraacetate and cations in novel lipid A mutants.

    PubMed Central

    Vaara, M

    1981-01-01

    Polymyxin-resistant pmrA mutants of Salmonella typhimurium differed from their parents in that they were resistant to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-lysozyme, tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-deoxycholate, and tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-bacitracin. Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate released about 50% less lipopolysaccharide from the pmrA strains than from the parental strains when the bacteria were grown in L-broth containing 2 mM Ca2+. Protamine, polylysine, octapeptin, benzalkonium chloride, cold NaCl, cold MgCl2, or cold tris(hydroxymethyl)aminomethane hydrochloride (pH 7.2) caused no leakage or markedly less leakage of periplasmic beta-lactamase from a pmrA mutant than from its parent strain. pmrA mutants were more resistant than their parent strains to protamine and polylysine but not to octapeptin or benzalkonium chloride, as measured by the ability of these agents to kill the bacteria or to sensitize them to deoxycholate-induced lysis. The pmrA strains did not differ from their parent strains in sensitivity to several antibiotics, in porin function (as measured by cephaloridine diffusion across the outer membrane), or in outer membrane-associated phospholipase A activity. PMID:6795177

  11. The visually impaired child.

    PubMed

    Thompson, Lisa; Kaufman, Lawrence M

    2003-02-01

    This article discusses the causes of childhood blindness and how the primary care provider may begin the appropriate steps toward diagnosing and managing the visually impaired child. Community resources (see Box 3) and low-vision programs in schools should be used so that parents do not need to reinvent strategies to raise a blind child. Worldwide, childhood blindness, which places is a tremendous burden on families and communities of the third world, is mostly preventable with improved hygiene, diet, and immunization. PMID:12713115

  12. Impaired Muscle Mitochondrial Biogenesis and Myogenesis in Spinal Muscular Atrophy

    PubMed Central

    Ripolone, Michela; Ronchi, Dario; Violano, Raffaella; Vallejo, Dionis; Fagiolari, Gigliola; Barca, Emanuele; Lucchini, Valeria; Colombo, Irene; Villa, Luisa; Berardinelli, Angela; Balottin, Umberto; Morandi, Lucia; Mora, Marina; Bordoni, Andreina; Fortunato, Francesco; Corti, Stefania; Parisi, Daniela; Toscano, Antonio; Sciacco, Monica; DiMauro, Salvatore; Comi, Giacomo P.; Moggio, Maurizio

    2016-01-01

    , implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P < .001), COX4 (26.6%; P < .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P < .001). Conversely, the levels of expression of 3 myogenic regulatory factors—muscle-specificmyogenic factor 5, myoblast determination 1, and myogenin—were higher in muscles from patients with SMA compared with muscles from age-matched controls (P < .05). CONCLUSIONS AND RELEVANCE Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes. PMID:25844556

  13. Specific language impairment.

    PubMed

    Kamhi, Alan G; Clark, Mary Kristen

    2013-01-01

    The acquisition of language is one of the most important achievements in young children, in part because most children appear to acquire language with little effort. Some children are not so fortunate, however. There is a large group of children who also have difficulty learning language, but do not have obvious neurological, cognitive, sensory, emotional, or environmental deficits. Clinicians often refer to these children as language disordered or language impaired. Researchers tend to refer to these children as specific language impaired (SLI). Children with SLI have intrigued researchers for many years because there is no obvious reason for their language learning difficulties. SLI has been found to be an enduring condition that begins in early childhood and often persists into adolescence and adulthood. The language problems of children with SLI are not limited to spoken language; they also affect reading and writing and thus much of academic learning. Knowledge of the characteristics of SLI should aid physicians, pediatricians, and early childhood specialists to identify these children during the preschool years and ensure that they receive appropriate services. With high-quality language intervention and literacy instruction, most children with SLI should be able to perform and function adequately in school and beyond. PMID:23622167

  14. Dimethylfumarate Impairs Neutrophil Functions.

    PubMed

    Müller, Susen; Behnen, Martina; Bieber, Katja; Möller, Sonja; Hellberg, Lars; Witte, Mareike; Hänsel, Martin; Zillikens, Detlef; Solbach, Werner; Laskay, Tamás; Ludwig, Ralf J

    2016-01-01

    Host defense against pathogens relies on neutrophil activation. Inadequate neutrophil activation is often associated with chronic inflammatory diseases. Neutrophils also constitute a significant portion of infiltrating cells in chronic inflammatory diseases, for example, psoriasis and multiple sclerosis. Fumarates improve the latter diseases, which so far has been attributed to the effects on lymphocytes and dendritic cells. Here, we focused on the effects of dimethylfumarate (DMF) on neutrophils. In vitro, DMF inhibited neutrophil activation, including changes in surface marker expression, reactive oxygen species production, formation of neutrophil extracellular traps, and migration. Phagocytic ability and autoantibody-induced, neutrophil-dependent tissue injury ex vivo was also impaired by DMF. Regarding the mode of action, DMF modulates-in a stimulus-dependent manner-neutrophil activation using the phosphoinositide 3-kinase/Akt-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways. For in vivo validation, mouse models of epidermolysis bullosa acquisita, an organ-specific autoimmune disease caused by autoantibodies to type VII collagen, were employed. In the presence of DMF, blistering induced by injection of anti-type VII collagen antibodies into mice was significantly impaired. DMF treatment of mice with clinically already-manifested epidermolysis bullosa acquisita led to disease improvement. Collectively, we demonstrate a profound inhibitory activity of DMF on neutrophil functions. These findings encourage wider use of DMF in patients with neutrophil-mediated diseases. PMID:26763431

  15. Vascular cognitive impairment and dementia.

    PubMed

    Gorelick, Philip B; Counts, Scott E; Nyenhuis, David

    2016-05-01

    Vascular contributions to cognitive impairment are receiving heightened attention as potentially modifiable factors for dementias of later life. These factors have now been linked not only to vascular cognitive disorders but also Alzheimer's disease. In this chapter we review 3 related topics that address vascular contributions to cognitive impairment: 1. vascular pathogenesis and mechanisms; 2. neuropsychological and neuroimaging phenotypic manifestations of cerebrovascular disease; and 3. prospects for prevention of cognitive impairment of later life based on cardiovascular and stroke risk modification. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock. PMID:26704177

  16. Composite sensor membrane

    DOEpatents

    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.

  17. Impaired Verb Fluency: A Sign of Mild Cognitive Impairment

    ERIC Educational Resources Information Center

    Ostberg, Per; Fernaeus, Sven-Erik; Hellstrom, Ake; Bogdanovic, Nenad; Wahlund, Lars Olof

    2005-01-01

    We assessed verb fluency vs. noun and letter-based fluency in 199 subjects referred for cognitive complaints including Subjective Cognitive Impairment, Mild Cognitive Impairment, and Alzheimer's disease. ANCOVAs and factor analyses identified verb, noun, and letter-based fluency as distinct tasks. Verb fluency performance in Mild Cognitive…

  18. Astrocytes Underlie Neuroinflammatory Memory Impairment.

    PubMed

    Osso, Lindsay A; Chan, Jonah R

    2015-12-17

    Neuroinflammation is being increasingly recognized as a potential mediator of cognitive impairments in various neurological conditions. Habbas et al. demonstrate that the pro-inflammatory cytokine tumor necrosis factor alpha signals through astrocytes to alter synaptic transmission and impair cognition in a mouse model of multiple sclerosis. PMID:26687350

  19. Statins impair glucose uptake in human cells

    PubMed Central

    Nowis, Dominika; Malenda, Agata; Furs, Karolina; Oleszczak, Bozenna; Sadowski, Radoslaw; Chlebowska, Justyna; Firczuk, Malgorzata; Bujnicki, Janusz M; Staruch, Adam D; Zagozdzon, Radoslaw; Glodkowska-Mrowka, Eliza; Szablewski, Leszek; Golab, Jakub

    2014-01-01

    Objective Considering the increasing number of clinical observations indicating hyperglycemic effects of statins, this study was designed to measure the influence of statins on the uptake of glucose analogs by human cells derived from liver, adipose tissue, and skeletal muscle. Design Flow cytometry and scintillation counting were used to measure the uptake of fluorescently labeled or tritiated glucose analogs by differentiated visceral preadipocytes, skeletal muscle cells, skeletal muscle myoblasts, and contact-inhibited human hepatocellular carcinoma cells. A bioinformatics approach was used to predict the structure of human glucose transporter 1 (GLUT1) and to identify the presence of putative cholesterol-binding (cholesterol recognition/interaction amino acid consensus (CRAC)) motifs within this transporter. Mutagenesis of CRAC motifs in SLC2A1 gene and limited proteolysis of membrane GLUT1 were used to determine the molecular effects of statins. Results Statins significantly inhibit the uptake of glucose analogs in all cell types. Similar effects are induced by methyl-β-cyclodextrin, which removes membrane cholesterol. Statin effects can be rescued by addition of mevalonic acid, or supplementation with exogenous cholesterol. Limited proteolysis of GLUT1 and mutagenesis of CRAC motifs revealed that statins induce conformational changes in GLUTs. Conclusions Statins impair glucose uptake by cells involved in regulation of glucose homeostasis by inducing cholesterol-dependent conformational changes in GLUTs. This molecular mechanism might explain hyperglycemic effects of statins observed in clinical trials. PMID:25452863

  20. Sheet Membrane Spacesuit Water Membrane Evaporator

    NASA Technical Reports Server (NTRS)

    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.

  1. Methanotroph outer membrane preparation.

    PubMed

    Karlsen, Odd A; Berven, Frode S; Jensen, Harald B; Fjellbirkeland, Anne

    2011-01-01

    All presently known methanotrophs are gram-negative bacteria suggesting that they are surrounded by a two-layered membrane: an inner or cytoplasmic membrane and an outer membrane. In the methanotroph Methylococcus capsulatus (Bath), separation of the two membranes has allowed studies on protein and lipid composition of the outer membrane. Its outer membrane can be isolated from purified cell envelopes by selective solubilization of the inner membranes with the detergent Triton X-100. The proteins associated with the outer membrane can further be fractionated into integral and tightly associated proteins and peripheral loosely associated proteins. We present here protocols for this fractionation and show how the proteins associated with the outer leaflet of the outer membrane can be isolated and identified by whole-cell biotin surface labeling. PMID:21419921

  2. A novel mutation, cog, which results in production of a new porin protein (OmpG) of Escherichia coli K-12.

    PubMed Central

    Misra, R; Benson, S A

    1989-01-01

    A mutant of Escherichia coli K-12 which produces a new outer membrane protein, OmpG, was isolated and genetically and biochemically characterized. The presence of OmpG allows growth on maltodextrins in the absence of the LamB maltoporin. The data obtained from in vivo growth and uptake experiments suggested that the presence of the OmpG protein results in an increase in outer membrane permeability for small hydrophilic compounds. In light of these findings, we suggest that OmpG is a porinlike protein. The mutation which results in the expression of OmpG has been termed cog (for control of OmpG) and mapped to 29 min on the E. coli chromosome. Diploid analysis shows that the mutant cog-192 allele is recessive for both the Dex+ and OmpG+ phenotypes. We propose that the cog mutation destroys a negative regulatory function and therefore derepresses ompG expression. Images PMID:2473977

  3. Membrane selectivity in pervaporation

    SciTech Connect

    Kujawski, W.

    1996-06-01

    A qualitative description is presented of pervaporation which discusses the initial preferential sorption into the membrane, diffusion of liquid, phase transition from liquid to vapor phase, followed by diffusion of vapors and fast desorption from the other side of the membrane. The overall separation of each pervaporation step was calculated in terms of separation factor {alpha}. The results show that in the case of hydrophilic membranes (i.e., dense polyamide-6 membrane and ion-exchange membrane PESS-1) and water-ethanol mixtures, the phase transition step decreases the overall separation. Also, diffusion through the membrane is unfavorable to water at a low concentration range.

  4. Transport of AMPA receptors during long-term potentiation is impaired in rats with hepatic encephalopathy.

    PubMed

    Monfort, Pilar; Piedrafita, Blanca; Felipo, Vicente

    2009-12-01

    Cognitive function is impaired in patients with hepatic encephalopathy. Learning ability is also impaired in rats with hepatic encephalopathy due to portacaval shunts. Long-term potentiation (LTP) in hippocampus, considered the basis of some forms of learning and memory, is impaired in rats with portacaval shunt. We analyzed the mechanisms by which LTP is impaired in these rats. In control rats, application of the tetanus to induce LTP increases phosphorylation of Thr286 of calcium-calmodulin dependent protein kinase II. This activates the kinase which phosphorylates the GluR1 subunit of AMPA receptors in Ser831 and induces its translocation to the post-synaptic densities. All these steps are completely prevented in rats with hepatic encephalopathy in which the tetanus does not induce phosphorylation of CaMKII or GluR1 nor translocation of this subunit to the post-synaptic membrane. This would explain the impairment in LTP in these rats. PMID:19450629

  5. Specific Language Impairment in Families: Evidence for Co-Occurrence with Reading Impairments.

    ERIC Educational Resources Information Center

    Flax, Judy F.; Realpe-Bonilla, Teresa; Hirsch, Linda S.; Brzustowicz, Linda M.; Bartlett, Christopher W.; Tallal, Paula

    2003-01-01

    Two family aggregation studies involving 25 children (ages 5-10) with specific language impairment (SLI) report the occurrence and co-occurrence of oral language impairments and reading impairments. Results indicate that when language impairments occur within families of SLI probands, these impairments generally co-occur with reading impairments.…

  6. Synthesis of outer membrane proteins in cpxA cpxB mutants of Escherichia coli K-12.

    PubMed Central

    McEwen, J; Sambucetti, L; Silverman, P M

    1983-01-01

    Two major proteins, the murein lipoprotein and the OmpF matrix porin, are deficient in the outer membrane of cpxA cpxB mutants of Escherichia coli K-12. We present evidence that the cpx mutations prevent or retard the translocation of these proteins to the outer membrane. The mutations had no effect on the rate of lipoprotein synthesis. Mutant cells labeled for 5 min with radioactive arginine accumulated as much lipoprotein as otherwise isogenic cpxA+ cpxB+ cells. This lipoprotein accumulated as such; no material synthesized in mutant cells and reactive with antilipoprotein antibodies had the electrophoretic mobility of prolipoprotein. Hence, the initial stages of prolipoprotein insertion into the inner membrane leading to its cleavage to lipoprotein appeared normal. However, after a long labeling interval, mutant cells were deficient in free lipoprotein and lacked lipoprotein covalently bound to peptidoglycan, suggesting that little if any of the lipoprotein synthesized in mutant cells reaches the outer membrane. Immunoreactive OmpF protein could also be detected in extracts of mutant cells labeled for 5 min, but the amount that accumulated was severalfold less in mutant cells than in cpxA+ cpxB+ cells. Analysis of beta-galactosidase synthesis from ompF-lacZ fusion genes showed this difference to be the result of a reduced rate of ompF transcription in mutant cells. Even so, little or none of the ompF protein synthesized in mutant cells was incorporated into the outer membrane. Images PMID:6339479

  7. Ionene membrane battery separator

    NASA Technical Reports Server (NTRS)

    Moacanin, J.; Tom, H. Y.

    1969-01-01

    Ionic transport characteristics of ionenes, insoluble membranes from soluble polyelectrolyte compositions, are studied for possible application in a battery separator. Effectiveness of the thin film of separator membrane essentially determines battery lifetime.

  8. Composite zeolite membranes

    DOEpatents

    Nenoff, Tina M.; Thoma, Steven G.; Ashley, Carol S.; Reed, Scott T.

    2002-01-01

    A new class of composite zeolite membranes and synthesis techniques therefor has been invented. These membranes are essentially defect-free, and exhibit large levels of transmembrane flux and of chemical and isotopic selectivity.

  9. Hybrid adsorptive membrane reactor

    DOEpatents

    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.

  10. Supertubes and Superconducting Membranes

    SciTech Connect

    Cordero, Ruben; Miguel-Pilar, Zelin

    2007-02-09

    We show the equivalence between configurations that arise from string theory of type IIA, called supertubes, and superconducting membranes at the bosonic level. We find equilibrium and oscillating configurations for a tubular membrane carrying a current along its axis.

  11. Premature rupture of membranes

    MedlinePlus

    ... When the water breaks early, it is called premature rupture of membranes (PROM). Most women will go ... th week of pregnancy, it is called preterm premature rupture of membranes (PPROM). The earlier your water ...

  12. Hybrid adsorptive membrane reactor

    NASA Technical Reports Server (NTRS)

    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.

  13. Supported inorganic membranes

    DOEpatents

    Sehgal, Rakesh; Brinker, Charles Jeffrey

    1998-01-01

    Supported inorganic membranes capable of molecular sieving, and methods for their production, are provided. The subject membranes exhibit high flux and high selectivity. The subject membranes are substantially defect free and less than about 100 nm thick. The pores of the subject membranes have an average critical pore radius of less than about 5 .ANG., and have a narrow pore size distribution. The subject membranes are prepared by coating a porous substrate with a polymeric sol, preferably under conditions of low relative pressure of the liquid constituents of the sol. The coated substrate is dried and calcined to produce the subject supported membrane. Also provided are methods of derivatizing the surface of supported inorganic membranes with metal alkoxides. The subject membranes find use in a variety of applications, such as the separation of constituents of gaseous streams, as catalysts and catalyst supports, and the like.

  14. Loss of SNAP29 Impairs Endocytic Recycling and Cell Motility

    PubMed Central

    Rapaport, Debora; Lugassy, Yevgenia; Sprecher, Eli; Horowitz, Mia

    2010-01-01

    Intracellular membrane trafficking depends on the ordered formation and consumption of transport intermediates and requires that membranes fuse with each other in a tightly regulated and highly specific manner. Membrane anchored SNAREs assemble into SNARE complexes that bring membranes together to promote fusion. SNAP29 is a ubiquitous synaptosomal-associated SNARE protein. It interacts with several syntaxins and with the EH domain containing protein EHD1. Loss of functional SNAP29 results in CEDNIK syndrome (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma). Using fibroblast cell lines derived from CEDNIK patients, we show that SNAP29 mediates endocytic recycling of transferrin and β1-integrin. Impaired β1-integrin recycling affected cell motility, as reflected by changes in cell spreading and wound healing. No major changes were detected in exocytosis of VSVG protein from the Golgi apparatus, although the Golgi system acquired a dispersed morphology in SNAP29 deficient cells. Our results emphasize the importance of SNAP29 mediated membrane fusion in endocytic recycling and consequently, in cell motility. PMID:20305790

  15. Composite fuel cell membranes

    DOEpatents

    Plowman, Keith R.; Rehg, Timothy J.; Davis, Larry W.; Carl, William P.; Cisar, Alan J.; Eastland, Charles S.

    1997-01-01

    A bilayer or trilayer composite ion exchange membrane suitable for use in a fuel cell. The composite membrane has a high equivalent weight thick layer in order to provide sufficient strength and low equivalent weight surface layers for improved electrical performance in a fuel cell. In use, the composite membrane is provided with electrode surface layers. The composite membrane can be composed of a sulfonic fluoropolymer in both core and surface layers.

  16. Composite fuel cell membranes

    DOEpatents

    Plowman, K.R.; Rehg, T.J.; Davis, L.W.; Carl, W.P.; Cisar, A.J.; Eastland, C.S.

    1997-08-05

    A bilayer or trilayer composite ion exchange membrane is described suitable for use in a fuel cell. The composite membrane has a high equivalent weight thick layer in order to provide sufficient strength and low equivalent weight surface layers for improved electrical performance in a fuel cell. In use, the composite membrane is provided with electrode surface layers. The composite membrane can be composed of a sulfonic fluoropolymer in both core and surface layers.

  17. Impaired cortical mitochondrial function following TBI precedes behavioral changes

    PubMed Central

    Watson, William D.; Buonora, John E.; Yarnell, Angela M.; Lucky, Jessica J.; D’Acchille, Michaela I.; McMullen, David C.; Boston, Andrew G.; Kuczmarski, Andrew V.; Kean, William S.; Verma, Ajay; Grunberg, Neil E.; Cole, Jeffrey T.

    2014-01-01

    Traumatic brain injury (TBI) pathophysiology can be attributed to either the immediate, primary physical injury, or the delayed, secondary injury which begins minutes to hours after the initial injury and can persist for several months or longer. Because these secondary cascades are delayed and last for a significant time period post-TBI, they are primary research targets for new therapeutics. To investigate changes in mitochondrial function after a brain injury, both the cortical impact site and ipsilateral hippocampus of adult male rats 7 and 17 days after a controlled cortical impact (CCI) injury were examined. State 3, state 4, and uncoupler-stimulated rates of oxygen consumption, respiratory control ratios (RCRs) were measured and membrane potential quantified, and all were significantly decreased in 7 day post-TBI cortical mitochondria. By contrast, hippocampal mitochondria at 7 days showed only non-significant decreases in rates of oxygen consumption and membrane potential. NADH oxidase activities measured in disrupted mitochondria were normal in both injured cortex and hippocampus at 7 days post-CCI. Respiratory and phosphorylation capacities at 17 days post-CCI were comparable to naïve animals for both cortical and hippocampus mitochondria. However, unlike oxidative phosphorylation, membrane potential of mitochondria in the cortical lining of the impact site did not recover at 17 days, suggesting that while diminished cortical membrane potential at 17 days does not adversely affect mitochondrial capacity to synthesize ATP, it may negatively impact other membrane potential-sensitive mitochondrial functions. Memory status, as assessed by a passive avoidance paradigm, was not significantly impaired until 17 days after injury. These results indicate pronounced disturbances in cortical mitochondrial function 7 days after CCI which precede the behavioral impairment observed at 17 days. PMID:24550822

  18. ANTIBODIES TO INTESTINAL MICROVILLOUS MEMBRANES

    PubMed Central

    Mackenzie, Iain L.; Donaldson, Robert M.; Kopp, William L.; Trier, Jerry S.

    1968-01-01

    Microvillous membranes isolated from the distal, but not proximal, half of hamster small bowel induced in rabbits the formation of antisera which inhibited intrinsic factor-mediated uptake of vitamin B12 by hamster brush borders. The extent of inhibition was directly proportional to the concentration of antiserum, and an excess of IF-bound vitamin B12 could overcome the inhibitory effect. The inhibitory factor was absorbed from antisera by brush borders isolated from the distal, but not proximal, half of the hamster intestine. Fractionation of antisera by gel filtration and DEAE-cellulose chromatography established that immunoglobulin G contained the inhibitory factor. Antisera capable of completely blocking uptake of IF-bound vitamin B12 did not react with hamster IF or with the IF-vitamin B12 complex, did not inhibit brush border disaccharidase activity and did not impair glucose transport by everted sacs of hamster intestine. These results demonstrate that an antibody to distal microvillous membranes competes with the IF-vitamin B12 complex for a specific binding site or receptor located on the surface of distal hamster intestine. PMID:19867301

  19. Brucella outer membrane lipoprotein shares antigenic determinants with Escherichia coli Braun lipoprotein and is exposed on the cell surface.

    PubMed Central

    Gómez-Miguel, M J; Moriyón, I; López, J

    1987-01-01

    In an enzyme-linked immunosorbent assay (ELISA), purified Brucella abortus and Escherichia coli peptidoglycan-linked lipoproteins gave a strong cross-reaction with sera from rabbits hyperimmunized with the heterologous lipoprotein. When smooth E. coli cells were used as ELISA antigens, the immunological cross-reaction was not observed unless the cells were treated to remove lipopolysaccharide and other outer membrane components. In contrast, intact cells from smooth strains of B. abortus and Brucella melitensis bound anti-lipoprotein immunoglobulin G, and the controls performed by ELISA showed that this reaction was not due to antibodies to the lipopolysaccharide, group 3 outer membrane proteins, or porins. Electron microscopy of cells labeled with antilipoprotein serum and protein A-colloidal gold showed specific labeling of smooth cells from both B. abortus and B. melitensis, even though unspecific labeling by nonimmune serum was observed with rough B. abortus. These results confirm the close similarity between E. coli and Brucella peptidoglycan-linked lipoproteins and show that, in contrast to E. coli, the lipoprotein of B. abortus and B. melitensis is partially exposed on the surface of smooth cells. Images PMID:2432014

  20. Genetic demonstration that the plasma membrane maxianion channel and voltage-dependent anion channels are unrelated proteins.

    PubMed

    Sabirov, Ravshan Z; Sheiko, Tatiana; Liu, Hongtao; Deng, Defeng; Okada, Yasunobu; Craigen, William J

    2006-01-27

    The maxianion channel is widely expressed in many cell types, where it fulfills a general physiological function as an ATP-conductive gate for cell-to-cell purinergic signaling. Establishing the molecular identity of this channel is crucial to understanding the mechanisms of regulated ATP release. A mitochondrial porin (voltage-dependent anion channel (VDAC)) located in the plasma membrane has long been considered as the molecule underlying the maxianion channel activity, based upon similarities in the biophysical properties of these two channels and the purported presence of VDAC protein in the plasma membrane. We have deleted each of the three genes encoding the VDAC isoforms individually and collectively and demonstrate that maxianion channel (approximately 400 picosiemens) activity in VDAC-deficient mouse fibroblasts is unaltered. The channel activity is similar in VDAC1/VDAC3-double-deficient cells and in double-deficient cells with the VDAC2 protein depleted by RNA interference. VDAC deletion slightly down-regulated, but never abolished, the swelling-induced ATP release. The lack of correlation between VDAC protein expression and maxianion channel activity strongly argues against the long held hypothesis of plasmalemmal VDAC being the maxianion channel. PMID:16291750

  1. Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa.

    PubMed

    Adewoye, L O; Worobec, E A

    2000-08-01

    The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins. PMID:10940570

  2. Meniscus membranes for separations

    DOEpatents

    Dye, Robert C.; Jorgensen, Betty; Pesiri, David R.

    2004-01-27

    Gas separation membranes, especially meniscus-shaped membranes for gas separations are disclosed together with the use of such meniscus-shaped membranes for applications such as thermal gas valves, pre-concentration of a gas stream, and selective pre-screening of a gas stream. In addition, a rapid screening system for simultaneously screening polymer materials for effectiveness in gas separation is provided.

  3. Meniscus Membranes For Separation

    DOEpatents

    Dye, Robert C.; Jorgensen, Betty; Pesiri, David R.

    2005-09-20

    Gas separation membranes, especially meniscus-shaped membranes for gas separations are disclosed together with the use of such meniscus-shaped membranes for applications such as thermal gas valves, pre-concentration of a gas stream, and selective pre-screening of a gas stream. In addition, a rapid screening system for simultaneously screening polymer materials for effectiveness in gas separation is provided.

  4. Cadmium sulfide membranes

    DOEpatents

    Spanhel, Lubomir; Anderson, Marc A.

    1992-07-07

    A method is described for the creation of novel q-effect cadmium sulfide membranes. The membranes are made by first creating a dilute cadmium sulfide colloid in aqueous suspension and then removing the water and excess salts therefrom. The cadmium sulfide membrane thus produced is luminescent at room temperature and may have application in laser fabrication.

  5. Cadmium sulfide membranes

    DOEpatents

    Spanhel, Lubomir; Anderson, Marc A.

    1991-10-22

    A method is described for the creation of novel q-effect cadmium sulfide membranes. The membranes are made by first creating a dilute cadmium sulfide colloid in aqueous suspension and then removing the water and excess salts therefrom. The cadmium sulfide membrane thus produced is luminescent at room temperature and may have application in laser fabrication.

  6. Polyphosphazene semipermeable membranes

    DOEpatents

    Allen, Charles A.; McCaffrey, Robert R.; Cummings, Daniel G.; Grey, Alan E.; Jessup, Janine S.; McAtee, Richard E.

    1988-01-01

    A semipermeable, inorganic membrane is disclosed; the membrane is prepared from a phosphazene polymer and, by the selective substitution of the constituent groups bound to the phosphorous in the polymer structure, the selective passage of fluid from a feedstream can be controlled. Resistance to high temperatures and harsh chemical environments is observed in the use of the phosphazene polymers as semipermeable membranes.

  7. Water vapor diffusion membranes

    NASA Technical Reports Server (NTRS)

    Holland, F. F., Jr.; Smith, J. K.

    1974-01-01

    The program is reported, which was designed to define the membrane technology of the vapor diffusion water recovery process and to test this technology using commercially available or experimental membranes. One membrane was selected, on the basis of the defined technology, and was subjected to a 30-day demonstration trial.

  8. Psychiatric Issues in Cognitive Impairment

    PubMed Central

    Aarsland, Dag; Taylor, John-Paul; Weintraub, Daniel

    2015-01-01

    Neuropsychiatric symptoms (NPS) such as depression, hallucinations and apathy commonly occur in Parkinson’s disease (PD) and have major clinical consequences including a negative impact on quality of life. This review discusses the epidemiology, clinical features, diagnostic procedures and treatment issues of NPS in PD and related disorders in the perspective of cognitive impairment, focusing on depression, anxiety, visual hallucinations, apathy, sleep disturbances, impulse control disorder and non-motor fluctuations. The majority of NPS are more common in PD patients with dementia, possibly related to shared underlying pathologies. Recent studies also suggest that NPS are associated with mild cognitive impairment in PD, in particular with the amnestic type. Accurate diagnosis of NPS is important but can be difficult, due to overlapping symptoms and similar appearance of symptoms of motor symptoms of parkinsonism, cognitive impairment, mood disorders and apathy. There are few systematic studies focusing on the management of NPS in PD with cognitive impairment. PMID:24757113

  9. [Cognitive Impairment in Multiple Sclerosis].

    PubMed

    Niino, Masaaki; Miyazaki, Yusei

    2016-04-01

    While cognitive impairment is a major symptom of multiple sclerosis (MS), it is commonly overlooked. This may be explained by the fact that it is difficult to evaluate cognitive function in patients with MS using screening batteries for the detection of dementia such as the mini-mental state examination. Further more, cognitive impairment in MS typically involves domain-specific deficits such as imparement of sustained attention and information processing speed rather than global cognitive decline. Cognitive impairment may influence the daily living and social lines of affected patients. This review discusses the characteristics of cognitive impairment, appropreate tests to evaluate its symptoms, and the current status of clinical trials for the treatment of MS. PMID:27056855

  10. [Drug-induced Cognitive Impairment].

    PubMed

    Shinohara, Moeko; Yamada, Masahito

    2016-04-01

    Elderly people are more likely than young people to develop cognitive impairments associated with medication use. One of the reasons for this is that renal and liver functions are often impaired in elderly people. Dementia and delirium (an acute confused state) are known to be associated with drug toxicity. Anticholinergic medications are common causes of both acute and chronic cognitive impairment. Psychoactive drugs, antidepressants and anticonvulsants can cause dementia and delirium. In addition, non-psychoactive drugs such as histamine H2 receptor antagonists, corticosteroids, NSAIDs (nonsteroidal anti-inflammatory agent), and cardiac medications, may cause acute or chronic cognitive impairment. Early diagnosis and withdrawal of the offending agent are essential for the prevention of drug-induced dementia and delirium. PMID:27056860

  11. A new method for modeling rough membrane surface and calculation of interfacial interactions.

    PubMed

    Zhao, Leihong; Zhang, Meijia; He, Yiming; Chen, Jianrong; Hong, Huachang; Liao, Bao-Qiang; Lin, Hongjun

    2016-01-01

    Membrane fouling control necessitates the establishment of an effective method to assess interfacial interactions between foulants and rough surface membrane. This study proposed a new method which includes a rigorous mathematical equation for modeling membrane surface morphology, and combination of surface element integration (SEI) method and the composite Simpson's approach for assessment of interfacial interactions. The new method provides a complete solution to quantitatively calculate interfacial interactions between foulants and rough surface membrane. Application of this method in a membrane bioreactor (MBR) showed that, high calculation accuracy could be achieved by setting high segment number, and moreover, the strength of three energy components and energy barrier was remarkably impaired by the existence of roughness on the membrane surface, indicating that membrane surface morphology exerted profound effects on membrane fouling in the MBR. Good agreement between calculation prediction and fouling phenomena was found, suggesting the feasibility of this method. PMID:26519696

  12. Two outer membrane proteins are bovine lactoferrin-binding proteins in Mannheimia haemolytica A1.

    PubMed

    Samaniego-Barrón, Luisa; Luna-Castro, Sarahí; Piña-Vázquez, Carolina; Suárez-Güemes, Francisco; de la Garza, Mireya

    2016-01-01

    Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 μM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine. PMID:27599994

  13. Crystal Structure of the Outer Membrane Protein OpdK from Pseudomonas aeruginosa

    SciTech Connect

    Biswas,S.; Mohammad, M.; Movileanu, L.; van den Berg, B.

    2008-01-01

    In Gram-negative bacteria that do not have porins, most water-soluble and small molecules are taken up by substrate-specific channels belonging to the OprD family. We report here the X-ray crystal structure of OpdK, an OprD family member implicated in the uptake of vanillate and related small aromatic acids. The OpdK structure reveals a monomeric, 18-stranded {beta} barrel with a kidney-shaped central pore. The OpdK pore constriction is relatively wide for a substrate-specific channel ({approx}8 Angstroms diameter), and it is lined by a positively charged patch of arginine residues on one side and an electronegative pocket on the opposite side--features likely to be important for substrate selection. Single-channel electrical recordings of OpdK show binding of vanillate to the channel, and they suggest that OpdK forms labile trimers in the outer membrane. Comparison of the OpdK structure with that of Pseudomonas aeruginosa OprD provides the first qualitative insights into the different substrate specificities of these closely related channels.

  14. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

    SciTech Connect

    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-10-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for (/sup 3/H)diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with (/sup 3/H)flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines.

  15. Permissive linker insertion sites in the outer membrane protein of 987P fimbriae of Escherichia coli.

    PubMed Central

    Schifferli, D M; Alrutz, M A

    1994-01-01

    The FasD protein is essential for the biogenesis of 987P fimbriae of Escherichia coli. In this study, subcellular fractionation was used to demonstrate that FasD is an outer membrane protein. In addition, the accessibility of FasD to proteases established the presence of surface-exposed FasD domains on both sides of the outer membrane. The fasD gene was sequenced, and the deduced amino acid sequence was shown to share homologous domains with a family of outer membrane proteins from various fimbrial systems. Similar to porins, fimbrial outer membrane proteins are relatively polar, lack typical hydrophobic membrane-spanning domains, and posses secondary structures predicted to be rich in turns and amphipathic beta-sheets. On the basis of the experimental data and structural predictions, FasD is postulated to consist essentially of surface-exposed turns and loops and membrane-spanning interacting amphipathic beta-strands. In an attempt to test this prediction, the fasD gene was submitted to random in-frame linker insertion mutagenesis. Preliminary experiments demonstrated that it was possible to produce fasD mutants, whose products remain functional for fimbrial export and assembly. Subsequently, 11 fasD alleles, containing linker inserts encoding beta-turn-inducing residues, were shown to express functional proteins. The insertion sites were designated permissive sites. The inserts used are expected to be least detrimental to the function of FasD when they are inserted into surface-exposed domains not directly involved in fimbrial export. In contrast, FasD is not expected to accommodate such residues in its amphipathic beta-strands without being destabilized in the membrane and losing function. All permissive sites were sequenced and shown to be located in or one residue away from predicted turns. In contrast, 5 of 10 sequenced nonpermissive sites were mapped to predicted amphipathic beta-strands. These results are consistent with the structural predictions for Fas

  16. Premenstrual changes. Impaired hormonal homeostasis.

    PubMed

    Halbreich, U; Alt, I H; Paul, L

    1988-03-01

    Premenstrual changes (PMCs) in mood and behavior are very prevalent. Nonetheless, their pathophysiology is still obscure and no proven treatment is yet available. Evaluation of the plethora of available data leads to the suggestion that PMCs may result from a temporary impairment of homeostasis among a multitude of systems. This impairment is triggered by a differential pace and magnitude of change-over-time in levels of several hormones and other substances during the luteal phase. PMID:3288473

  17. Chemistry for the Visually Impaired

    NASA Astrophysics Data System (ADS)

    Ratliff, Judy L.

    1997-06-01

    Methods used to try to provide a valuable experience for visually impaired students in a general education or an introductory chemistry class are discussed. Modifications that can be made cheaply and with little time commitment which will allow visually impaired students to participate productively in the laboratory are examined. A conductivity tester that cost less than $4.00 to construct, is easy to assemble, very rugged, and provides a great deal of entertainment for sighted and non-sighted students is described.

  18. Regulation of membrane trafficking by signalling on endosomal and lysosomal membranes

    PubMed Central

    Li, Xinran; Garrity, Abigail G; Xu, Haoxing

    2013-01-01

    Endosomal and lysosomal membrane trafficking requires the coordination of multiple signalling events to control cargo sorting and processing, and endosome maturation. The initiation and termination of signalling events in endosomes and lysosomes is not well understood, but several key regulators have been identified, which include small GTPases, phosphoinositides, and Ca2+. Small GTPases act as master regulators and molecular switches in a GTP-dependent manner, initiating signalling cascades to regulate the direction and specificity of endosomal trafficking. Phosphoinositides are membrane-bound lipids that indicate vesicular identities for recruiting specific cytoplasmic proteins to endosomal membranes, thus allowing specificity of membrane fusion, fission, and cargo sorting to occur within and between specific vesicle compartments. In addition, phosphoinositides regulate the function of membrane proteins such as ion channels and transporters in a compartment-specific manner to mediate transport and signalling. Finally, Ca2+, a locally acting second messenger released from intracellular ion channels, may provide precise spatiotemporal regulation of endosomal signalling and trafficking events. Small GTPase signalling can regulate phosphoinositide conversion during endosome maturation, and electrophysiological studies on isolated endosomes have shown that endosomal and lysosomal Ca2+ channels are directly modulated by endosomal lipids. Thus trafficking and maturation of endosomes and lysosomes can be precisely regulated by dynamic changes in GTPases and membrane lipids, as well as Ca2+ signalling. Importantly, impaired phosphoinositide and Ca2+ signalling can cause endosomal and lysosomal trafficking defects at the cellular level, and a spectrum of lysosome storage diseases. PMID:23878375

  19. Tracking Membrane Protein Association in Model Membranes

    PubMed Central

    Reffay, Myriam; Gambin, Yann; Benabdelhak, Houssain; Phan, Gilles; Taulier, Nicolas; Ducruix, Arnaud; Hodges, Robert S.; Urbach, Wladimir

    2009-01-01

    Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue. We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well. After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 Å, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We

  20. Elastic membranes in confinement

    NASA Astrophysics Data System (ADS)

    Bostwick, Joshua; Miksis, Michael; Davis, Stephen

    2014-11-01

    An elastic membrane stretched between two walls takes a shape defined by its length and the volume of fluid it encloses. Many biological structures, such as cells, mitochondria and DNA, have finer internal structure in which a membrane (or elastic member) is geometrically ``confined'' by another object. We study the shape stability of elastic membranes in a ``confining'' box and introduce repulsive van der Waals forces to prevent the membrane from intersecting the wall. We aim to define the parameter space associated with mitochondria-like deformations. We compare the confined to `unconfined' solutions and show how the structure and stability of the membrane shapes changes with the system parameters.

  1. Separation membrane development

    SciTech Connect

    Lee, M.W.

    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.

  2. Membrane Flotation Assay

    PubMed Central

    Vogt, Dorothee A; Ott, Melanie

    2016-01-01

    Many postitive-stranded RNA viruses, such as Hepatitis C virus (HCV), highjack cellular membranes, including the Golgi, ER, mitchondria, lipid droplets, and utilize them for replication of their RNA genome or assembly of new virions. By investigating how viral proteins associate with cellular membranes we will better understand the roles of cellular membranes in the viral life cycle. Our lab has focused specifically on the role of lipid droplets and lipid-rich membranes in the life cycle of HCV. To analyze the role of lipid-rich membranes in HCV RNA replication, we utilized a membrane flotation assay based on an 10–20–30% iodixanol density gradient developed by Yeaman et al. (2001). This gradient results in a linear increase in density over almost the entire length of the gradient, and membrane particles are separated in the gradient based on their buoyant characteristics. To preserve membranes in the lysate, cells are broken mechanically in a buffer lacking detergent. The cell lysate is loaded on the bottom of the gradient, overlaid with the gradient, and membranes float up as the iodixanol gradient self-generates. The lipid content of membranes and the concentration of associated proteins will determine the separation of different membranes within the gradient. After centrifugation, fractions can be sampled from the top of the gradient and analyzed using standard SDS-PAGE and western blot analysis for proteins of interest.

  3. Water Membrane Evaporator

    NASA Technical Reports Server (NTRS)

    Ungar, Eugene K.; Almlie, Jay C.

    2010-01-01

    A water membrane evaporator (WME) has been conceived and tested as an alternative to the contamination-sensitive and corrosion-prone evaporators currently used for dissipating heat from space vehicles. The WME consists mainly of the following components: An outer stainless-steel screen that provides structural support for the components mentioned next; Inside and in contact with the stainless-steel screen, a hydrophobic membrane that is permeable to water vapor; Inside and in contact with the hydrophobic membrane, a hydrophilic membrane that transports the liquid feedwater to the inner surface of the hydrophobic membrane; Inside and in contact with the hydrophilic membrane, an annular array of tubes through which flows the spacecraft coolant carrying the heat to be dissipated; and An inner exclusion tube that limits the volume of feedwater in the WME. In operation, a pressurized feedwater reservoir is connected to the volume between the exclusion tube and the coolant tubes. Feedwater fills the volume, saturates the hydrophilic membrane, and is retained by the hydrophobic membrane. The outside of the WME is exposed to space vacuum. Heat from the spacecraft coolant is conducted through the tube walls and the water-saturated hydrophilic membrane to the liquid/vapor interface at the hydrophobic membrane, causing water to evaporate to space. Makeup water flows into the hydrophilic membrane through gaps between the coolant tubes.

  4. Catalytic membranes beckon

    SciTech Connect

    Caruana, C.M.

    1994-11-01

    Chemical engineers here and abroad are finding that the marriage of catalysts and membranes holds promise for faster and more specific reactions, although commercialization of this technology is several years away. Catalytic membrane reactors (CMRs) combine a heterogeneous catalyst and a permselective membrane. Reactions performed by CMRs provide higher yields--sometimes as much as 50% higher--because of better reaction selectivity--as opposed to separation selectivity. CMRs also can work at very high temperatures, using ceramic materials that would not be possible with organic membranes. Although the use of CMRs is not widespread presently, the development of new membranes--particularly porous ceramic and zeolite membranes--will increase the potential to improve yields of many catalytic processes. The paper discusses ongoing studies, metal and advanced materials for membranes, the need for continued research, hydrogen recovery from coal-derived gases, catalytic oxidation of sulfides, CMRs for water purification, and oxidative coupling of methane.

  5. Dystypia: isolated typing impairment without aphasia, apraxia or visuospatial impairment.

    PubMed

    Otsuki, Mika; Soma, Yoshiaki; Arihiro, Shoji; Watanabe, Yoshimasa; Moriwaki, Hiroshi; Naritomi, Hiroaki

    2002-01-01

    We report a 60-year-old right-handed Japanese man who showed an isolated persistent typing impairment without aphasia, agraphia, apraxia or any other neuropsychological deficit. We coined the term 'dystypia' for this peculiar neuropsychological manifestation. The symptom was caused by an infarction in the left frontal lobe involving the foot of the second frontal convolution and the frontal operculum. The patient's typing impairment was not attributable to a disturbance of the linguistic process, since he had no aphasia or agraphia. The impairment was not attributable to the impairment of the motor execution process either, since he had no apraxia. Thus, his typing impairment was deduced to be based on a disturbance of the intermediate process where the linguistic phonological information is converted into the corresponding performance. We hypothesized that there is a specific process for typing which branches from the motor programming process presented in neurolinguistic models. The foot of the left second frontal convolution and the operculum may play an important role in the manifestation of 'dystypia'. PMID:11914550

  6. Epigenetic modification of PKMζ rescues aging-related cognitive impairment

    PubMed Central

    Chen, Chen; Meng, Shi-Qiu; Xue, Yan-Xue; Han, Ying; Sun, Cheng-Yu; Deng, Jia-Hui; Chen, Na; Bao, Yan-Ping; Zhang, Fei-Long; Cao, Lin-Lin; Zhu, Wei-Guo; Shi, Jie; Song, Wei-Hong; Lu, Lin

    2016-01-01

    Cognition is impacted by aging. However, the mechanisms that underlie aging-associated cognitive impairment are unclear. Here we showed that cognitive decline in aged rats was associated with changes in DNA methylation of protein kinase Mζ (PKMζ) in the prelimbic cortex (PrL). PKMζ is a crucial molecule involved in the maintenance of long-term memory. Using different behavioral models, we confirmed that aged rats exhibited cognitive impairment in memory retention test 24 h after training, and overexpression of PKMζ in the PrL rescued cognitive impairment in aged rats. After fear conditioning, the protein levels of PKMζ and the membrane expression of GluR2 increased in the PrL in young and adult rats but not in aged rats, and the levels of methylated PKMζ DNA in the PrL decreased in all age groups, whereas the levels of unmethylated PKMζ DNA increased only in young and adult rats. We also found that environmentally enriched housing reversed the hypermethylation of PKMζ and restored cognitive performance in aged rats. Inactivation of PKMζ prevented the potentiating effects of environmental enrichment on memory retention in aged rats. These results indicated that PKMζ might be a potential target for the treatment of aging-related cognitive impairment, suggesting a potential therapeutic avenue. PMID:26926225

  7. Anti-Outer membrane protein C antibodies in colorectal neoplasia.

    PubMed

    Kohoutova, D; Drahosova, M; Cihak, M; Moravkova, P; Bures, J

    2016-07-01

    Sporadic colorectal cancer (CRC) represents an enormous problem worldwide. Large intestinal microbiota play an important role in the colorectal carcinogenesis. The aim of the study was to investigate anti-Outer membrane protein C (anti-OmpC) antibodies, aimed at porin C, which is embedded in the outer membrane of gram-negative bacteria, in patients with colorectal adenoma (CRA), CRC and controls. The study included 22 patients with CRA (11 men, 11 women, aged 26-79, mean 65 ± 12), 11 patients with CRC (9 men, 2 women, aged 50-83, mean 66 ± 11) and 45 controls, blood donors (24 men, 21 women, aged 20-58, mean 38 ± 10). Serum anti-OmpC antibodies were investigated by means of ELISA. Values of 0-20 U/mL were considered to be negative; values >25 U/mL were assessed as positive. A total of 9/11 (82 %) patients with CRC had positive anti-OmpC antibodies. Anti-OmpC antibodies were negative or grey-zone in 37/45 (82 %) controls. Serum anti-OmpC were found to be significantly higher in patients with CRC (median 42.4, interquartile range (IQR) 22.2) compared to controls (median 18.3, IQR 12.4), p < 0.001. No statistically significant difference in anti-OmpC was found between controls (median 18.3, IQR 12.4) and CRA patients (median 17.7, IQR 16.5), p = 0.326. Anti-OmpC were significantly higher in patients with CRC (median 42.4, IQR 22.2) compared to patients with CRA (median 17.7, IQR 16.5), p = 0.011. Positivity of anti-OmpC antibodies was found in patients with CRC, which supports the contribution of gram-negative large intestinal microbiota to the pathogenesis of CRC. PMID:26612659

  8. Polymers at membranes

    NASA Astrophysics Data System (ADS)

    Breidenich, Markus

    2000-11-01

    The surface of biological cells consists of a lipid membrane and a large amount of various proteins and polymers, which are embedded in the membrane or attached to it. We investigate how membranes are influenced by polymers, which are anchored to the membrane by one end. The entropic pressure exerted by the polymer induces a curvature, which bends the membrane away from the polymer. The resulting membrane shape profile is a cone in the vicinity of the anchor segment and a catenoid far away from it. The perturbative calculations are confirmed by Monte-Carlo simulations. An additional attractive interaction between polymer and membrane reduces the entropically induced curvature. In the limit of strong adsorption, the polymer is localized directly on the membrane surface and does not induce any pressure, i.e. the membrane curvature vanishes. If the polymer is not anchored directly on the membrane surface, but in a non-vanishing anchoring distance, the membrane bends towards the polymer for strong adsorption. In the last part of the thesis, we study membranes under the influence of non-anchored polymers in solution. In the limit of pure steric interactions between the membrane and free polymers, the membrane curves towards the polymers (in contrast to the case of anchored polymers). In the limit of strong adsorption the membrane bends away from the polymers. Die Oberfläche biologischer Zellen besteht aus einer Lipidmembran und einer Vielzahl von Proteinen und Polymeren, die in die Membran eingebaut sind. Die Beeinflussung der Membran durch Polymere, die mit einem Ende an der Membran verankert sind, wird im Rahmen dieser Arbeit anhand eines vereinfachten biomimetischen Systems studiert. Der entropische Druck, den das Polymer durch Stöße auf die Membran ausübt, führt dazu, dass sich die Membran vom Polymer weg krümmt. Die resultierende Membranform ist ein Kegel in der Nähe des Ankers und ein Katenoid in grossem Abstand vom Ankerpunkt. Monte Carlo-Simulationen best

  9. Effects of the membrane action of tetralin on the functional and structural properties of artificial and bacterial membranes.

    PubMed Central

    Sikkema, J; Poolman, B; Konings, W N; de Bont, J A

    1992-01-01

    Tetralin is toxic to bacterial cells at concentrations below 100 mumol/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 mechanism, and several gram-positive and gram-negative bacteria were studied. Because of its hydrophobicity, tetralin partitioned into lipid membranes preferentially (lipid/buffer partition coefficient of tetralin is approximately 1,100). The excessive accumulation of tetralin caused expansion of the membrane and impairment of different membrane functions. Studies with proteoliposomes and intact cells indicated that tetralin makes the membrane permeable for ions (protons) and inhibits the respiratory enzymes, which leads to a partial dissipation of the pH gradient and electrical potential. The effect of tetralin on the components of the proton motive force as well as disruption of protein-lipid interaction(s) could lead to impairment of various metabolic functions and to low growth rates. The data offer an explanation for the difficulty in isolating and cultivating microorganisms in media containing tetralin or other lipophilic compounds. PMID:1314806

  10. Topology of the membrane protein LamB by epitope tagging and a comparison with the X-ray model.

    PubMed

    Newton, S M; Klebba, P E; Michel, V; Hofnung, M; Charbit, A

    1996-06-01

    We previously developed a genetic approach to study, with a single antibody, the topology of the outer membrane protein LamB, an Escherichia coli porin with specificity towards maltodextrins and a receptor for bacteriophage lambda. Our initial procedure consisted of inserting at random the same reporter epitope (the C3 neutralization epitope from poliovirus) into permissive sites of LamB (i.e., sites which tolerate insertions without deleterious effects on the protein activities or the cell). A specific monoclonal antibody was then used to examine the position of the inserted epitope with respect to the protein and the membrane. In the present work, we set up a site-directed procedure to insert the C3 epitope at new sites in order to distinguish between two-dimensional folding models. This allowed us to identify two new surface loops of LamB and to predict another periplasmic exposed region. The results obtained by random and directed epitope tagging are analyzed in light of the recently published X-ray structure of the LamB protein. Study of 23 hybrid LamB-C3 proteins led to the direct identification of five of the nine external loops (L4, L5, L6, L7, and L9) and led to the prediction of four periplasmic loops (I1, I4, I5, and I8) of LamB. Nine of the hybrid proteins did not lead to topological conclusions, and none led to the wrong predictions or conclusions. The comparison indicates that parts of models based on secondary structure predictions alone are not reliable and points to the importance of experimental data in the establishment of outer membrane protein topological models. The advantages and limitations of genetic foreign epitope insertion for the study of integral membrane proteins are discussed. PMID:8655540

  11. Membrane with supported internal passages

    NASA Technical Reports Server (NTRS)

    Gonzalez-Martin, Anuncia (Inventor); Salinas, Carlos E. (Inventor); Cisar, Alan J. (Inventor); Hitchens, G. Duncan (Inventor); Murphy, Oliver J. (Inventor)

    2000-01-01

    The invention provides an improved proton exchange membrane for use in electrochemical cells having internal passages parallel to the membrane surface comprising permanent tubes preferably placed at the ends of the fluid passages. The invention also provides an apparatus and process for making the membrane, membrane and electrode assemblies fabricated using the membrane, and the application of the membrane and electrode assemblies to a variety of devices, both electrochemical and otherwise. The passages in the membrane extend from one edge of the membrane to another and allow fluid flow through the membrane and give access directly to the membrane.

  12. 20 CFR 404.1598 - If you become disabled by another impairment(s).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false If you become disabled by another impairment... Disability § 404.1598 If you become disabled by another impairment(s). If a new severe impairment(s) begins... are still disabled under § 404.1594....

  13. 20 CFR 404.1598 - If you become disabled by another impairment(s).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false If you become disabled by another impairment... Disability § 404.1598 If you become disabled by another impairment(s). If a new severe impairment(s) begins... are still disabled under § 404.1594....

  14. 20 CFR 404.1598 - If you become disabled by another impairment(s).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 20 Employees' Benefits 2 2013-04-01 2013-04-01 false If you become disabled by another impairment... Disability § 404.1598 If you become disabled by another impairment(s). If a new severe impairment(s) begins... are still disabled under § 404.1594....

  15. 20 CFR 404.1598 - If you become disabled by another impairment(s).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 20 Employees' Benefits 2 2012-04-01 2012-04-01 false If you become disabled by another impairment... Disability § 404.1598 If you become disabled by another impairment(s). If a new severe impairment(s) begins... are still disabled under § 404.1594....

  16. 20 CFR 404.1598 - If you become disabled by another impairment(s).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false If you become disabled by another impairment... Disability § 404.1598 If you become disabled by another impairment(s). If a new severe impairment(s) begins... are still disabled under § 404.1594....

  17. Lacking power impairs executive functions.

    PubMed

    Smith, Pamela K; Jostmann, Nils B; Galinsky, Adam D; van Dijk, Wilco W

    2008-05-01

    Four experiments explored whether lacking power impairs executive functioning, testing the hypothesis that the cognitive presses of powerlessness increase vulnerability to performance decrements during complex executive tasks. In the first three experiments, low power impaired performance on executive-function tasks: The powerless were less effective than the powerful at updating (Experiment 1), inhibiting (Experiment 2), and planning (Experiment 3). Existing research suggests that the powerless have difficulty distinguishing between what is goal relevant and what is goal irrelevant in the environment. A fourth experiment established that the executive-function impairment associated with low power is driven by goal neglect. The current research implies that the cognitive alterations arising from powerlessness may help foster stable social hierarchies and that empowering employees may reduce costly organizational errors. PMID:18466404

  18. Poxvirus Membrane Biogenesis

    PubMed Central

    2015-01-01

    Poxviruses differ from most DNA viruses by replicating entirely within the cytoplasm. The first discernible viral structures are crescents and spherical immature virions containing a single lipoprotein membrane bilayer with an external honeycomb lattice. Because this viral membrane displays no obvious continuity with a cellular organelle, a de novo origin was suggested. Nevertheless, transient connections between viral and cellular membranes could be difficult to resolve. Despite the absence of direct evidence, the intermediate compartment (ERGIC) between the endoplasmic reticulum (ER) and Golgi apparatus and the ER itself were considered possible sources of crescent membranes. A break-through in understanding poxvirus membrane biogenesis has come from recent studies of the abortive replication of several vaccinia virus null mutants. Novel images showing continuity between viral crescents and the ER and the accumulation of immature virions in the expanded ER lumen provide the first direct evidence for a cellular origin of this poxvirus membrane. PMID:25728299

  19. Poxvirus membrane biogenesis.

    PubMed

    Moss, Bernard

    2015-05-01

    Poxviruses differ from most DNA viruses by replicating entirely within the cytoplasm. The first discernible viral structures are crescents and spherical immature virions containing a single lipoprotein membrane bilayer with an external honeycomb lattice. Because this viral membrane displays no obvious continuity with a cellular organelle, a de novo origin was suggested. Nevertheless, transient connections between viral and cellular membranes could be difficult to resolve. Despite the absence of direct evidence, the intermediate compartment (ERGIC) between the endoplasmic reticulum (ER) and Golgi apparatus and the ER itself were considered possible sources of crescent membranes. A break-through in understanding poxvirus membrane biogenesis has come from recent studies of the abortive replication of several vaccinia virus null mutants. Novel images showing continuity between viral crescents and the ER and the accumulation of immature virions in the expanded ER lumen provide the first direct evidence for a cellular origin of this poxvirus membrane. PMID:25728299

  20. Substituted polyacetylene separation membrane

    DOEpatents

    Pinnau, Ingo; Morisato, Atsushi

    1998-01-13

    A separation membrane useful for gas separation, particularly separation of C.sub.2+ hydrocarbons from natural gas. The invention encompasses the membrane itself, methods of making it and processes for using it. The membrane comprises a polymer having repeating units of a hydrocarbon-based, disubstituted polyacetylene, having the general formula: ##STR1## wherein R.sub.1 is chosen from the group consisting of C.sub.1 -C.sub.4 alkyl and phenyl, and wherein R.sub.2 is chosen from the group consisting of hydrogen and phenyl. In the most preferred embodiment, the membrane comprises poly(4-methyl-2-pentyne) ›PMP!. The membrane exhibits good chemical resistance and has super-glassy properties with regard to separating certain large, condensable permeant species from smaller, less-condensable permeant species. The membranes may also be useful in other fluid separations.

  1. Substituted polyacetylene separation membrane

    DOEpatents

    Pinnau, I.; Morisato, Atsushi

    1998-01-13

    A separation membrane is described which is useful for gas separation, particularly separation of C{sub 2+} hydrocarbons from natural gas. The invention encompasses the membrane itself, methods of making it and processes for using it. The membrane comprises a polymer having repeating units of a hydrocarbon-based, disubstituted polyacetylene, having the general formula shown in the accompanying diagram, wherein R{sub 1} is chosen from the group consisting of C{sub 1}-C{sub 4} alkyl and phenyl, and wherein R{sub 2} is chosen from the group consisting of hydrogen and phenyl. In the most preferred embodiment, the membrane comprises poly(4-methyl-2-pentyne) [PMP]. The membrane exhibits good chemical resistance and has super-glassy properties with regard to separating certain large, condensable permeant species from smaller, less-condensable permeant species. The membranes may also be useful in other fluid separations. 4 figs.

  2. Anion permselective membrane

    NASA Technical Reports Server (NTRS)

    Alexander, S.; Hodgdon, R. B.

    1977-01-01

    The objective of NAS 3-20108 was the development and evaluation of improved anion selective membranes useful as efficient separators in a redox power storage cell system being constructed. The program was divided into three parts, (a) optimization of the selected candidate membrane systems, (b) investigation of alternative membrane/polymer systems, and (c) characterization of candidate membranes. The major synthesis effort was aimed at improving and optimizing as far as possible each candidate system with respect to three critical membrane properties essential for good redox cell performance. Substantial improvements were made in 5 candidate membrane systems. The critical synthesis variables of cross-link density, monomer ratio, and solvent composition were examined over a wide range. In addition, eight alternative polymer systems were investigated, two of which attained candidate status. Three other alternatives showed potential but required further research and development. Each candidate system was optimized for selectivity.

  3. Vascular contributions to cognitive impairment

    PubMed Central

    Flores, Alan

    2015-01-01

    Summary Unlike many neurodegenerative causes of cognitive impairment and dementia, vascular damage is preventable. Despite the heterogeneity of vascular cognitive impairment (VCI) and the complexity of its clinical presentations, the potential for limiting progression and changing the trajectory of damage makes it all the more important for physicians to be educated about the syndrome and to remain vigilant when taking care of patients. In this review, we outline an approach to patients with possible VCI, summarize current treatment and prevention guidelines, and provide an overview with case examples. PMID:26124978

  4. Visitation arrangements for impaired parents.

    PubMed

    Montgomery, Stephen A; Street, David F

    2011-07-01

    Forensic mental health professionals are frequently asked to evaluate the parenting skills of divorcing parents because the court seeks help in determining the custody, visitation, and parenting time arrangements for the children. When one of the parents is impaired, the court wants to know the way to help the children have a good relationship with that parent and keep the children safe. There is little empirical research to answer such questions. In this article, the authors describe their methodology for providing useful clinical information to the court to help guide their decisions regarding visitation with impaired parents. PMID:21683915

  5. Driver Compensation: Impairment or Improvement?

    PubMed

    Young, Richard A

    2015-12-01

    Strayer et al.'s conclusion that their "cognitive distraction scale" for auditory-vocal tasks indicates "significant impairments to driving" is not supported by their data. Additional analysis demonstrates that slower brake reaction times during auditory-vocal tasks were fully compensated for by longer following distances to the lead car. Naturalistic driving data demonstrate that cellular conversation decreases crash risk, the opposite of the article's assumption. Hence, the scale's internal and external validities for indicating driving impairment are highly questionable. PMID:26534851

  6. BDE-154 induces mitochondrial permeability transition and impairs mitochondrial bioenergetics.

    PubMed

    Pereira, Lílian Cristina; Miranda, Luiz Felippe Cabral; de Souza, Alecsandra Oliveira; Dorta, Daniel Junqueira

    2014-01-01

    Brominated flame retardants are used in various consumer goods to make these materials difficult to burn. Polybrominated diphenyl ethers (PBDE), which are representative of this class of retardants, consist of two benzene rings linked by an oxygen atom, and contain between 1 and 10 bromine atoms in their chemical structure, with the possibility of up to 209 different congeners. Among these congeners, BDE-154 (hexa-BDE) is persistent in the environment and easy to detect in the biota, but no apparent information regarding the mechanism underlying action and toxicity is available. Mitochondria, as the main energy-producing organelles, play an important role in the maintenance of various cellular functions. Therefore, mitochondria were used in the present study as an experimental model to determine the effects of BDE-154 congener at concentrations ranging from 0.1 μM to 50 μM. Our results demonstrated that BDE-154 interacts with the mitochondrial membrane, preferably by inserting into the hydrophobic core of the mitochondrial membrane, which partially inhibits respiration, dissipates Δψ, and permeabilizes the inner mitochondrial membrane to deplete ATP. These effects are more pronounced at concentrations equal to or higher than 10 μM. Results also showed that BDE-154 did not induce reactive oxygen species (ROS) accumulation within the mitochondria, indicating the absence of oxidative stress. Therefore, BDE-154 impairs mitochondrial bioenergetics and permeabilizes the mitochondrial membrane, potentially leading to cell death but not via mechanisms involving oxidative stress. PMID:24555644

  7. Anion permselective membrane

    NASA Technical Reports Server (NTRS)

    Hodgdon, R. B.; Waite, W. A.; Alexander, S. S.

    1984-01-01

    Two polymer ion exchange membranes were synthesized to fulfill the needs of both electrical resistivity and anolyte/catholyte separation for utility load leveling utilizing the DOE/NASA mixed electrolyte REDOX battery. Both membranes were shown to meet mixed electrolyte utility load leveling criteria. Several modifications of an anion exchange membrane failed to meet utility load leveling REDOX battery criteria using the unmixed electrolyte REDOX cell.

  8. Anion permselective membrane

    NASA Astrophysics Data System (ADS)

    Hodgdon, R. B.; Waite, W. A.; Alexander, S. S.

    1984-07-01

    Two polymer ion exchange membranes were synthesized to fulfill the needs of both electrical resistivity and anolyte/catholyte separation for utility load leveling utilizing the DOE/NASA mixed electrolyte REDOX battery. Both membranes were shown to meet mixed electrolyte utility load leveling criteria. Several modifications of an anion exchange membrane failed to meet utility load leveling REDOX battery criteria using the unmixed electrolyte REDOX cell.

  9. Siloxane-grafted membranes

    DOEpatents

    Friesen, Dwayne T.; Obligin, Alan S.

    1989-01-01

    Composite cellulosic semipermeable membranes are disclosed which are the covalently bonded reaction product of an asymmetric cellulosic semipermeable membrane and a polysiloxane containing reactive functional groups. The two reactants chemically bond by ether, ester, amide or acrylate linkages to form a siloxane-grafted cellulosic membrane having superior selectivity and flux stability. Selectivity may be enhanced by wetting the surface with a swelling agent such as water.

  10. Polyarylether composition and membrane

    DOEpatents

    Hung, Joyce; Brunelle, Daniel Joseph; Harmon, Marianne Elisabeth; Moore, David Roger; Stone, Joshua James; Zhou, Hongyi; Suriano, Joseph Anthony

    2010-11-09

    A composition including a polyarylether copolymer is provided. The copolymer includes a polyarylether backbone; and a sulfonated oligomeric group bonded to the polyarylether suitable for use as a cation conducting membrane. Method of bonding a sulfonated oligomeric group to the polyarylether backbone to form a polyarylether copolymer. The membrane may be formed from the polyarylether copolymer composition. The chain length of the sulfonated oligomeric group may be controlled to affect or control the ion conductivity of the membrane.

  11. Siloxane-grafted membranes

    DOEpatents

    Friesen, D.T.; Obligin, A.S.

    1989-10-31

    Composite cellulosic semipermeable membranes are disclosed which are the covalently bonded reaction product of an asymmetric cellulosic semipermeable membrane and a polysiloxane containing reactive functional group. The two reactants chemically bond by ether, ester, amide or acrylate linkages to form a siloxane-grafted cellulosic membrane having superior selectivity and flux stability. Selectivity may be enhanced by wetting the surface with a swelling agent such as water.

  12. Proteins causing membrane fouling in membrane bioreactors.

    PubMed

    Miyoshi, Taro; Nagai, Yuhei; Aizawa, Tomoyasu; Kimura, Katsuki; Watanabe, Yoshimasa

    2015-01-01

    In this study, the details of proteins causing membrane fouling in membrane bioreactors (MBRs) treating real municipal wastewater were investigated. Two separate pilot-scale MBRs were continuously operated under significantly different operating conditions; one MBR was a submerged type whereas the other was a side-stream type. The submerged and side-stream MBRs were operated for 20 and 10 days, respectively. At the end of continuous operation, the foulants were extracted from the fouled membranes. The proteins contained in the extracted foulants were enriched by using the combination of crude concentration with an ultrafiltration membrane and trichloroacetic acid precipitation, and then separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The N-terminal amino acid sequencing analysis of the proteins which formed intensive spots on the 2D-PAGE gels allowed us to partially identify one protein (OmpA family protein originated from genus Brevundimonas or Riemerella anatipestifer) from the foulant obtained from the submerged MBR, and two proteins (OprD and OprF originated from genus Pseudomonas) from that obtained from the side-stream MBR. Despite the significant difference in operating conditions of the two MBRs, all proteins identified in this study belong to β-barrel protein. These findings strongly suggest the importance of β-barrel proteins in developing membrane fouling in MBRs. PMID:26360742

  13. Biomolecular membrane protein crystallization

    NASA Astrophysics Data System (ADS)

    Reddy Bolla, Jani; Su, Chih-Chia; Yu, Edward W.

    2012-07-01

    Integral membrane proteins comprise approximately 30% of the sequenced genomes, and there is an immediate need for their high-resolution structural information. Currently, the most reliable approach to obtain these structures is X-ray crystallography. However, obtaining crystals of membrane proteins that diffract to high resolution appears to be quite challenging, and remains a major obstacle in structural determination. This brief review summarizes a variety of methodologies for use in crystallizing these membrane proteins. Hopefully, by introducing the available methods, techniques, and providing a general understanding of membrane proteins, a rational decision can be made about now to crystallize these complex materials.

  14. Plant cell membranes

    SciTech Connect

    Packer, L.; Douce, R.

    1987-01-01

    The contents of this book are: Cells, Protoplasts, Vacuoles and Liposomes; Tonoplasts; Nuclei, Endolplasmic Reticulum, and Plasma Membrane; Peroxisomes; Plastids; Teneral Physical and Biochemical Methods; and Mitochondira.

  15. Drugging Membrane Protein Interactions.

    PubMed

    Yin, Hang; Flynn, Aaron D

    2016-07-11

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind cells to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally "undruggable" regions of membrane proteins, enabling modulation of protein-protein, protein-lipid, and protein-nucleic acid interactions. In this review, we survey the state of the art of high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  16. Membrane Tension Control

    NASA Technical Reports Server (NTRS)

    Su, Ji (Inventor); Harrison, Joycelyn S. (Inventor)

    2005-01-01

    An electrostrictive polymer actuator comprises an electrostrictive polymer with a tailorable Poisson's ratio. The electrostrictive polymer is electroded on its upper and lower surfaces and bonded to an upper material layer. The assembly is rolled tightly and capped at its ends. In a membrane structure having a membrane, a supporting frame and a plurality of threads connecting the membrane to the frame, an actuator can be integrated into one or more of the plurality of threads. The electrostrictive polymer actuator displaces along its longitudinal axis, thereby affecting movement of the membrane surface.

  17. Gas separation membranes

    DOEpatents

    Schell, William J.

    1979-01-01

    A dry, fabric supported, polymeric gas separation membrane, such as cellulose acetate, is prepared by casting a solution of the polymer onto a shrinkable fabric preferably formed of synthetic polymers such as polyester or polyamide filaments before washing, stretching or calendering (so called griege goods). The supported membrane is then subjected to gelling, annealing, and drying by solvent exchange. During the processing steps, both the fabric support and the membrane shrink a preselected, controlled amount which prevents curling, wrinkling or cracking of the membrane in flat form or when spirally wound into a gas separation element.

  18. Anion exchange membrane

    DOEpatents

    Verkade, John G; Wadhwa, Kuldeep; Kong, Xueqian; Schmidt-Rohr, Klaus

    2013-05-07

    An anion exchange membrane and fuel cell incorporating the anion exchange membrane are detailed in which proazaphosphatrane and azaphosphatrane cations are covalently bonded to a sulfonated fluoropolymer support along with anionic counterions. A positive charge is dispersed in the aforementioned cations which are buried in the support to reduce the cation-anion interactions and increase the mobility of hydroxide ions, for example, across the membrane. The anion exchange membrane has the ability to operate at high temperatures and in highly alkaline environments with high conductivity and low resistance.

  19. A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles

    PubMed Central

    Song, Tianyan; Mika, Franziska; Lindmark, Barbro; Liu, Zhi; Schild, Stefan; Bishop, Anne; Zhu, Jun; Camilli, Andrew; Johansson, Jörgen; Vogel, Jörg; Wai, Sun Nyunt

    2008-01-01

    We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5′ region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, σE, suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract. PMID:18681937

  20. Recombinant outer membrane protein C of Aeromonas hydrophila elicits mixed immune response and generates agglutinating antibodies.

    PubMed

    Yadav, Sunita Kumari; Meena, Jitendra Kumar; Sharma, Mahima; Dixit, Aparna

    2016-08-01

    Aeromonas hydrophila is a gram-negative fish pathogenic bacterium, also responsible for causing opportunistic pathological conditions in humans. It causes a number of diseases in fish due to which the fish industry incurs huge economic losses annually. Due to problems of antibiotic resistance, and the rapidity with which the infection spreads among fishes, vaccination remains the most effective strategy to combat this infection in fish populations. Among various virulence factors associated with bacterial virulence, outer membrane proteins have been widely evaluated for their vaccine potential owing to their surface exposure and related role in pathogenicity. In the present study, we have investigated the immunogenic potential of a non-specific porin, outer membrane protein C (OmpC) whose expression is regulated by the two-component regulatory system and plays a major role in the survival of A. hydrophila under different osmolaric conditions. The full-length gene (~1 kb) encoding OmpC of A. hydrophila was cloned, characterized and expressed in E. coli. High yield (~112 mg/L at shake flask level) of the recombinant OmpC (rOmpC) (~40 kDa) of A. hydrophila was obtained upon purification from inclusion bodies using Ni(2+)-NTA affinity chromatography. Immunization with purified rOmpC in murine model generated high endpoint (>1:40,000) titers. IgG isotyping, ELISA and ELISPOT assay indicated mixed immune response with a TH2 bias. Also, the anti-rOmpC antibodies were able to agglutinate A. hydrophila in vitro and exhibited specific cross-reactivity with different Aeromonas strains, which will facilitate easy detection of different Aeromonas isolates in infected samples. Taken together, these data clearly indicate that rOmpC could serve as an effective vaccine against different strains of Aeromonas, a highly heterogenous group of bacteria. PMID:27328672

  1. A conformational landscape for alginate secretion across the outer membrane of Pseudomonas aeruginosa.

    PubMed

    Tan, Jingquan; Rouse, Sarah L; Li, Dianfan; Pye, Valerie E; Vogeley, Lutz; Brinth, Alette R; El Arnaout, Toufic; Whitney, John C; Howell, P Lynne; Sansom, Mark S P; Caffrey, Martin

    2014-08-01

    The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common β-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE. PMID:25084326

  2. Language Impairment in Autistic Children.

    ERIC Educational Resources Information Center

    Deaton, Ann Virginia

    Discussed is the language impairment of children with infantile autism. The speech patterns of autistic children, including echolalia, pronomial reversal, silent language, and voice imitation, are described. The clinical picture of the autistic child is compared to that of children with such other disorders as deafness, retardation, and…

  3. Oceanography for the Visually Impaired

    ERIC Educational Resources Information Center

    Fraser, Kate

    2008-01-01

    Amy Bower is a physical oceanographer and senior scientist at the Woods Hole Oceanographic Institution (WHOI) in Woods Hole, Massachusetts--she has also been legally blind for 14 years. Through her partnership with the Perkins School for the Blind in Watertown, Massachusetts, the oldest K-12 school for the visually impaired in the United States,…

  4. [Cognitive impairment in multiple sclerosis].

    PubMed

    Ochi, Hirofumi

    2014-10-01

    Cognitive impairment may occur in up to 70% of all patients with multiple sclerosis (MS). Although MS can affect various sites within the central nervous system, a specific pattern of cognitive deficits tends to be seen, especially in the early stages of the disease. These deficits include problems with attention, information processing speed, and working memory. This constellation of deficits can occur with any disease course, and a minimal correlation has been found between physical disability assessed by EDSS and cognitive impairment. Many studies have shown that cognitive impairment is correlated with brain lesion volume, as well as brain atrophy. There are promising neuroimaging indicators that may be useful for identifying patients at risk for cognitive impairment, such as diffusion tensor imaging, the magnetization transfer ratio, and N-acetyl aspartate levels. Cognitive dysfunction is associated with adverse effects on quality of life, employment status, and social activities. Today, there are three avenues for treatment: disease modifying therapies, symptomatic treatments, and cognitive rehabilitation. Unfortunately, data linking therapeutic interventions are limited. A better understanding of cognitive function and its correlation with disease mechanisms will assist in providing a new comprehensive treatment strategy that begins immediately with the diagnosis of MS. PMID:25296874

  5. Cognitive impairments may mimic delusions.

    PubMed

    Eterović, Marija; Kozarić-Kovačić, Dragica

    2015-12-01

    Delusions are often recognized as key to the concept of psychosis. What is delusion is one of the basic questions of psychopathology. The common denominator of definitions of delusions is the divergence between the strong conviction in the delusional belief and superior evidences to the contrary which are continually ignored. An implicit, sustainably unspoken assumption is that the person with delusional belief has cognitive capacities to process the (counter-)arguments relevant to their delusion. However, individual's cognitive capacities are not being emphasized when delusions are evaluated. Moreover, the impact of cognitive decline on formation of delusions is neglected, both in theory and practice. We elaborate that cognitive deficits may facilitate, oppose, or mimic delusions. We focus on the last, which can lead to diagnosing as delusion what could be explained by cognitive decline and better called pseudo-delusion. The risk is significant when cognition is impaired, as in demented people; an issue which has not yet been debated. True delusions are incompatible with person's cognitive capacities, i.e., if we take into account person's cognitive status, we still cannot understand how the person holds the strange belief with an extraordinary conviction. Pseudo-delusions would be beliefs, thoughts or judgments that at first seem delusional (they are false, subculturally atypical beliefs that are strongly maintained in the face of counterargument), but lose the essence of delusions after we take cognitive impairment into account. Pseudo-delusions could actually be explained or understood by person's cognitive impairments, they "fit into" them. The reported reality-based contents of delusions in the elderly, poor response to antipsychotics and lack of association with early or family history of psychiatric disorders could in part be accounted for by the bias of misdiagnosing the cognitive impairment as the delusion. Not recognizing that the cognitive impairment

  6. Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells

    PubMed Central

    2013-01-01

    Background Protein PIII is one of the major outer membrane proteins of Neisseria gonorrhoeae, 95% identical to RmpM (reduction modifiable protein M) or class 4 protein of Neisseria meningitidis. RmpM is known to be a membrane protein associated by non-covalent bonds to the peptidoglycan layer and interacting with PorA/PorB porin complexes resulting in the stabilization of the bacterial membrane. The C-terminal domain of PIII (and RmpM) is highly homologous to members of the OmpA family, known to have a role in adhesion/invasion in many bacterial species. The contribution of PIII in the membrane architecture and its role in the interaction with epithelial cells has never been investigated. Results We generated a ΔpIII knock-out mutant strain and evaluated the effects of the loss of PIII expression on bacterial morphology and on outer membrane composition. Deletion of the pIII gene does not cause any alteration in bacterial morphology or sensitivity to detergents. Moreover, the expression profile of the main membrane proteins remains the same for the wild-type and knock-out strains, with the exception of the NG1873 which is not exported to the outer membrane and accumulates in the inner membrane in the ΔpIII knock-out mutant strain. We also show that purified PIII protein is able to bind human cervical and urethral cells and that the ΔpIII knock-out mutant strain has a lower ability to adhere to human cervical and urethral cells. Conclusion Here we demonstrated that the PIII protein does not play a key structural role in the membrane organization of gonococcus and does not induce major effects on the expression of the main outer membrane proteins. However, in the PIII knock-out strain, the NG1873 protein is not localized in the outer membrane as it is in the wild-type strain suggesting a possible interaction of PIII with NG1873. The evidence that PIII binds to human epithelial cells derived from the female and male genital tract highlights a possible role of PIII

  7. Anion permselective membrane

    NASA Technical Reports Server (NTRS)

    Hodgdon, R. B.; Waite, W. A.

    1982-01-01

    The synthesis and fabrication of polymeric anion permselective membranes for redox systems are discussed. Variations of the prime candidate anion membrane formulation to achieve better resistance and/or lower permeability were explored. Processing parameters were evaluated to lower cost and fabricate larger sizes. The processing techniques to produce more membranes per batch were successfully integrated with the fabrication of larger membranes. Membranes of about 107 cm x 51 cm were made in excellent yield. Several measurements were made on the larger sample membranes. Among the data developed were water transport and transference numbers for these prime candidate membranes at 20 C. Other work done on this system included characterization of a number of specimens of candidate membranes which had been returned after service lives of up to sixteen months. Work with new polymer constituents, with new N.P.'s, catalysts and backing fabrics is discussed. Some work was also done to evaluate other proportions of the ingredients of the prime candidate system. The adoption of a flow selectivity test at elevated temperature was explored.

  8. Membrane in cancer cells

    SciTech Connect

    Galeotti, T.; Cittadini, A.; Neri, G.; Scarpa, A.

    1988-01-01

    This book contains papers presented at a conference on membranes in cancer cells. Topics covered include Oncogenies, hormones, and free-radical processes in malignant transformation in vitro and Superoxide onion may trigger DNA strand breaks in human granulorytes by acting as a membrane target.

  9. Microporous alumina ceramic membranes

    DOEpatents

    Anderson, M.A.; Guangyao Sheng.

    1993-05-04

    Several methods are disclosed for the preparation microporous alumina ceramic membranes. For the first time, porous alumina membranes are made which have mean pore sizes less than 100 Angstroms and substantially no pores larger than that size. The methods are based on improved sol-gel techniques.

  10. Microporous alumina ceramic membranes

    DOEpatents

    Anderson, Marc A.; Sheng, Guangyao

    1993-01-01

    Several methods are disclosed for the preparation microporous alumina ceramic membranes. For the first time, porous alumina membranes are made which have mean pore sizes less than 100 Angstroms and substantially no pores larger than that size. The methods are based on improved sol-gel techniques.

  11. Polymide gas separation membranes

    DOEpatents

    Ding, Yong; Bikson, Benjamin; Nelson, Joyce Katz

    2004-09-14

    Soluble polyamic acid salt (PAAS) precursors comprised of tertiary and quaternary amines, ammonium cations, sulfonium cations, or phosphonium cations, are prepared and fabricated into membranes that are subsequently imidized and converted into rigid-rod polyimide articles, such as membranes with desirable gas separation properties. A method of enhancing solubility of PAAS polymers in alcohols is also disclosed.

  12. Membrane module assembly

    DOEpatents

    Kaschemekat, Jurgen

    1994-01-01

    A membrane module assembly adapted to provide a flow path for the incoming feed stream that forces it into prolonged heat-exchanging contact with a heating or cooling mechanism. Membrane separation processes employing the module assembly are also disclosed. The assembly is particularly useful for gas separation or pervaporation.

  13. Membrane module assembly

    DOEpatents

    Kaschemekat, J.

    1994-03-15

    A membrane module assembly is described which is adapted to provide a flow path for the incoming feed stream that forces it into prolonged heat-exchanging contact with a heating or cooling mechanism. Membrane separation processes employing the module assembly are also disclosed. The assembly is particularly useful for gas separation or pervaporation. 2 figures.

  14. Membrane Transport Phenomena (MTP)

    NASA Technical Reports Server (NTRS)

    Mason, Larry W.

    1996-01-01

    The development of the seal between the membrane and the Fluid Optical Cells (FOC) has been a high priority activity. This seal occurs at an interface in the instrument where three key functions must be realized: (1) physical membrane support, (2) fluid sealing, and (3) unobscured optical transmission.

  15. Permeable membrane experiment

    NASA Technical Reports Server (NTRS)

    Slavin, Thomas J.; Cao, Tuan Q.; Kliss, Mark H.

    1993-01-01

    The purpose of the Permeable Membrane Experiment is to gather flight data on three areas of membrane performance that are influenced by the presence of gravity. These areas are: (1) Liquid/gas phase separation, (2) gas bubble interference with diffusion through porous membranes and (3) wetting characteristics of hydrophilic membrane surfaces. These data are important in understaning the behavior of membrane/liquid/gas interfaces where surface tension forces predominate. The data will be compared with 1-g data already obtained and with predicted micrograviity behavior. The data will be used to develop designs for phase separation and plant nutrient delivery systems and will be available to the life support community for use in developing technologies which employ membranes. A conceptual design has been developed to conduct three membrane experiments, in sequence, aboard a single Complex Autonomous Payload (CAP) carrier to be carried in the Shuttle Orbiter payload bay. One experiment is conducted for each of the three membrane performance areas under study. These experiments are discussed in this paper.

  16. Viral Membrane Scission

    PubMed Central

    Rossman, Jeremy S.; Lamb, Robert A.

    2014-01-01

    Virus budding is a complex, multistep process in which viral proteins make specific alterations in membrane curvature. Many different viral proteins can deform the membrane and form a budding virion, but very few can mediate membrane scission to complete the budding process. As a result, enveloped viruses have developed numerous ways of facilitating membrane scission, including hijacking host cellular scission machinery and expressing their own scission proteins. These proteins mediate scission in very different ways, though the biophysical mechanics underlying their actions may be similar. In this review, we explore the mechanisms of membrane scission and the ways in which enveloped viruses use these systems to mediate the release of budding virions. PMID:24099087

  17. Ion-conducting membranes

    DOEpatents

    Masel, Richard L.; Chen, Qingmei; Liu, Zengcai; Kutz, Robert

    2016-06-21

    An ion conducting polymeric composition mixture comprises a copolymer of styrene and vinylbenzyl-R.sub.s. R.sub.s is selected from the group consisting of imidazoliums and pyridiniums. The composition contains 10%-90% by weight of vinylbenzyl-R.sub.s. The composition can further comprise a polyolefin comprising substituted polyolefins, a polymer comprising cyclic amine groups, a polymer comprising at least one of a phenylene group and a phenyl group, a polyamide, and/or the reaction product of a constituent having two carbon-carbon double bonds. The composition can be in the form of a membrane. In a preferred embodiment, the membrane is a Helper Membrane that increases the faradaic efficiency of an electrochemical cell into which the membrane is incorporated, and also allows product formation at lower voltages than in cells without the Helper Membrane.

  18. Anton permselective membrane

    NASA Technical Reports Server (NTRS)

    Alexander, S. S.; Hodgdon, R. B.; Waite, W. A.

    1979-01-01

    Experimental composite membranes were synthesized on a lab scale consisting of a thin layer of anion permselective resin supported by and bonded to a porous physically strong and conductive substrate film. These showed good selectivity and also substantially lower electrical resistivities than the homogenous candidate membranes optimized in the previous contract. A wide range of resin porosities were examined for three candidate membrane systems, CDIL, CP4L, and A3L to identify the formulation giving the best overall redox cell performance. Candidate anion membranes showed large increases in resistivity after a short time of immersion in concentrated FeCl/HCl solution. Largely on the basis of resistance stability the CDIL formulation was selected as prime candidate and about thirty-five membranes (one foot square) were produced for experimental static and dynamic evaluation.

  19. Elastic membranes in confinement.

    PubMed

    Bostwick, J B; Miksis, M J; Davis, S H

    2016-07-01

    An elastic membrane stretched between two walls takes a shape defined by its length and the volume of fluid it encloses. Many biological structures, such as cells, mitochondria and coiled DNA, have fine internal structure in which a membrane (or elastic member) is geometrically 'confined' by another object. Here, the two-dimensional shape of an elastic membrane in a 'confining' box is studied by introducing a repulsive confinement pressure that prevents the membrane from intersecting the wall. The stage is set by contrasting confined and unconfined solutions. Continuation methods are then used to compute response diagrams, from which we identify the particular membrane mechanics that generate mitochondria-like shapes. Large confinement pressures yield complex response diagrams with secondary bifurcations and multiple turning points where modal identities may change. Regions in parameter space where such behaviour occurs are then mapped. PMID:27440257

  20. OXYGEN TRANSPORT CERAMIC MEMBRANES

    SciTech Connect

    Dr. Sukumar Bandopadhyay; Dr. Nagendra Nagabhushana

    2002-04-01

    This report covers the following tasks: Task 1--Design, fabricate and evaluate ceramic to metal seals based on graded ceramic powder/metal braze joints; Task 2--Evaluate the effect of defect configuration on ceramic membrane conductivity and long term chemical and structural stability; Task 3--Determine materials mechanical properties under conditions of high temperatures and reactive atmospheres; Task 4--Evaluate phase stability and thermal expansion of candidate perovskite membranes and develop techniques to support these materials on porous metal structures; Task 5--Assess the microstructure of membrane materials to evaluate the effects of vacancy-impurity association, defect clusters, and vacancy-dopant association on the membrane performance and stability; and Task 6--Measure kinetics of oxygen uptake and transport in ceramic membrane materials under commercially relevant conditions using isotope labeling techniques.

  1. Membrane projection lithography

    DOEpatents

    Burckel, David Bruce; Davids, Paul S; Resnick, Paul J; Draper, Bruce L

    2015-03-17

    The various technologies presented herein relate to a three dimensional manufacturing technique for application with semiconductor technologies. A membrane layer can be formed over a cavity. An opening can be formed in the membrane such that the membrane can act as a mask layer to the underlying wall surfaces and bottom surface of the cavity. A beam to facilitate an operation comprising any of implantation, etching or deposition can be directed through the opening onto the underlying surface, with the opening acting as a mask to control the area of the underlying surfaces on which any of implantation occurs, material is removed, and/or material is deposited. The membrane can be removed, a new membrane placed over the cavity and a new opening formed to facilitate another implantation, etching, or deposition operation. By changing the direction of the beam different wall/bottom surfaces can be utilized to form a plurality of structures.

  2. Functional impairment in social anxiety disorder.

    PubMed

    Aderka, Idan M; Hofmann, Stefan G; Nickerson, Angela; Hermesh, Haggai; Gilboa-Schechtman, Eva; Marom, Sofi

    2012-04-01

    The present study examined functional impairment among treatment seekers with social anxiety disorder (SAD). We investigated the effects of diagnostic subtypes of SAD and comorbidity with mood and anxiety disorders on impairment. In addition, we used cluster analysis procedures to empirically identify subgroups of individuals with distinct patterns of impairment. Participants were 216 treatment-seeking individuals with SAD. Clinical interviews were undertaken to determine diagnoses of anxiety disorders and major depressive disorder, and a battery of self-report measures was administered to index symptoms of social anxiety, depression and extent of impairment. Results indicated that individuals with the generalized subtype of SAD had greater impairment in all three life domains compared to individuals with the nongeneralized subtype. Comorbidity with mood disorders was associated with greater impairment than SAD alone, but comorbidity with anxiety disorders was not. Four distinct impairment profiles emerged from the cluster analysis: primary work/studies impairment, primary social life impairment, both work/studies and social impairment, and impairment in all domains. Findings from this study suggest that SAD is associated with substantial impairment across multiple domains, and that individuals with SAD present diverse impairment profiles. These profiles may inform subtyping of the disorder as well as therapeutic interventions. PMID:22306132

  3. Functional Implications of Plasma Membrane Condensation for T Cell Activation

    PubMed Central

    Quinn, Carmel M.; Engelhardt, Karin; Williamson, David; Grewal, Thomas; Jessup, Wendy; Harder, Thomas; Gaus, Katharina

    2008-01-01

    The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process. PMID:18509459

  4. [Cognitive impairment in Parkinson's disease].

    PubMed

    Tachibana, Hisao

    2013-01-01

    Cognitive impairment is a common finding in Parkinson's disease (PD), even in the early stages. The concept of mild cognitive impairment (MCI) in PD was recently formalized with diagnosis being reached after impairments in neuropsychological tasks become significant in at least one domain. The brain profile of cognitive deficits involves executive functions (e. g., planning, set shifting, set maintenance, problem solving), attention and memory function. Memory deficits are characterized by impairments in delayed recall, temporal ordering and conditional associate learning. PD patients demonstrate relatively preserved recognition. Visuospatial dysfunctions have also been reported, while language is largely preserved. The existence of two distinct mild cognitive syndromes has also been suggested. One of these affects mainly the frontostriatal executive deficits that are modulated by dopaminergic medications and by a genetically determined level of prefrontal cortex dopamine release. The other affects the more-posterior cortical abilities, such as visuospatial and memory functions, and is suggested to be associated with an increased risk for conversion to dementia. Cross-sectional studies have commonly reported dementia in 20-30% of PD patients, although the 8-year cumulative incidence of dementia may be as high as 78%. Factors associated with dementia in PD are age at onset, age at the time of examination, akinetic-rigid form PD, depression, hallucination, rapid eye movement sleep behavioral disorder and severe olfactory deficits. Clinical features generally involve the same type of deficits as those found in MCI patients, which are more severe and more extensive. The phenomenology of the dementia syndrome is similar to that seen in dementia with Lewy bodies, and clinicopathological correlation studies have revealed varying results with regard to neurochemical deficits and the pathological substrate underlying cognitive impairment and dementia. Early cognitive

  5. Methamphetamine Alters Brain Structures, Impairs Mental Flexibility

    MedlinePlus

    ... Alters Brain Structures, Impairs Mental Flexibility Methamphetamine Alters Brain Structures, Impairs Mental Flexibility Email Facebook Twitter March ... methamphetamine use, such as tobacco smoking. Can the Brain Recover? The UCLA study’s findings underscore the importance ...

  6. The clinical features of thin basement membrane nephropathy.

    PubMed

    Gregory, Martin C

    2005-05-01

    Thin basement membrane nephropathy (TBMN) is a common, lifelong condition affecting the kidneys that is characterized by microscopic glomerular hematuria, minimal or no proteinuria, and normal renal function. It often is discovered incidentally, and usually has an excellent prognosis. Many cases are familial and show autosomal-dominant inheritance. The defining characteristic is a glomerular basement membrane (GBM) that is thinned to about half its normal thickness on ultrastructural examination of the renal biopsy specimen. However, occasionally patients with TBMN develop marked proteinuria or renal impairment. It is unclear whether individuals with TBMN and impaired renal function represent part of the spectrum of TBMN associated with heterozygous COL4A3 or COL4A4 mutations, or if their disease is caused by mutations of other genes, or whether it is caused by a second coexistent renal lesion or is misdiagnosed Alport syndrome. PMID:15880323

  7. Auditory processing efficiency deficits in children with developmental language impairments

    NASA Astrophysics Data System (ADS)

    Hartley, Douglas E. H.; Moore, David R.

    2002-12-01

    The ``temporal processing hypothesis'' suggests that individuals with specific language impairments (SLIs) and dyslexia have severe deficits in processing rapidly presented or brief sensory information, both within the auditory and visual domains. This hypothesis has been supported through evidence that language-impaired individuals have excess auditory backward masking. This paper presents an analysis of masking results from several studies in terms of a model of temporal resolution. Results from this modeling suggest that the masking results can be better explained by an ``auditory efficiency'' hypothesis. If impaired or immature listeners have a normal temporal window, but require a higher signal-to-noise level (poor processing efficiency), this hypothesis predicts the observed small deficits in the simultaneous masking task, and the much larger deficits in backward and forward masking tasks amongst those listeners. The difference in performance on these masking tasks is predictable from the compressive nonlinearity of the basilar membrane. The model also correctly predicts that backward masking (i) is more prone to training effects, (ii) has greater inter- and intrasubject variability, and (iii) increases less with masker level than do other masking tasks. These findings provide a new perspective on the mechanisms underlying communication disorders and auditory masking.

  8. Beta-adrenergic blockade and atrio--ventricular conduction impairment.

    PubMed

    Giudicelli, J F; Lhoste, F; Boissier, J R

    1975-04-01

    Atrio--ventricular conduction and its modifications induced by six Beta-adrenergic blocking agents have been investigated in the dog. Premature atrial stimuli (St2) were applied at variable intervals following regular stimuli (St1) ensuring atrial pacing; atrial (AERP), nodoventricular (NERP) and global (GERP) effective refractory periods as well as global functional refractory period (GFRP) were determined before and after administration of each of the six drugs. When Beta-blockade was produced with d,1-propranolol which hwas membrane stabilizing effects (MSE) but no intrinsic sympathomimetic activity (ISA) or with sotalol, which has neither MSE nor ISA, all parameters were significantly increased. When Beta-blockade was achieved with pindolol or practolol, which have only a poor Beta-adrenolytic potency and no ISA. Alprenolol showed intermediate effects. Thus, it appears that Beta-blockade and not MSE, is responsible for the onset of A-V conduction impairment but that ISA, probably through a metabolic mechanism, affords protection against this impairment. On the other hand, measurement of ventricular effective refractory period (VERP) has shown that at the Purkinje-free junction, it is MSE which is mainly involved in conduction impairment. PMID:238853

  9. Ordered ceramic membranes

    SciTech Connect

    Anderson, M.A.; Hill, C.G. Jr.; Zeltner, W.A.

    1991-10-01

    Ceramic membranes have been formed from colloidal sols coated on porous clay supports. These supported membranes have been characterized in terms of their permeabilities and permselectivities to various aqueous test solutions. The thermal stabilities and pore structures of these membranes have been characterized by preparing unsupported membranes of the correpsonding material and performing N{sub 2} adsorption-desorption and X-ray diffraction studies on these membranes. To date, membranes have been prepared from a variety of oxides, including TiO{sub 2}, SiO{sub 2}, ZrO{sub 2}, and Al{sub 2}O{sub 3}, as well as Zr-, Fe-, and Nb-doped TiO{sub 2}. In many of these membranes pore diameters are less than 2 nm, while in others the pore diameters are between 3 and 5 nm. Procedures for fabricating porous clay supports with reproducible permeabilities for pure water are also discussed. 30 refs., 59 figs., 22 tabs.

  10. Hollow fiber catalytic membranes

    SciTech Connect

    Ma, Yi Hua; Moser, W.; Shelekhin, A.; Pien, Shyhing

    1993-09-01

    The objective of the present research is to investigate the possibility of the enhancement of the H{sub 2}S thermal decomposition in the IGCC system by employing the hollow fiber catalytic membrane reactor. To accomplish the objective, the following major components in the analysis of the high temperature membrane reactor must be investigated: high-temperature stability of the porous glass membrane; catalytic properties of MoS{sub 2} and of the porous glass membrane; catalytic decomposition of H{sub 2}S in a packed bed reactor; catalytic decomposition of 100%, 8.6%, and 1.1% H{sub 2}S gas mixtures in the membrane reactor. The study has been shown that the conversion of the H{sub 2}S can be increased in the packed bed membrane reactor compared to the equilibrium conversion on the shell side. The development of a mathematical model for the proposed process is in progress. The model will enable optimization of the H{sub 2}S decomposition. These conditions include selectivity factors and pressure drop across the membrane.

  11. Supported double membranes

    PubMed Central

    Murray, David H.; Tamm, Lukas K.; Kiessling, Volker

    2009-01-01

    Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been proven to be useful tools for the investigation of complex biological functions in a significantly less complex membrane environment. In this study, we introduce a supported double membrane system that should be useful for studies that target biological processes in the proximity of two lipid bilayers such as the periplasm of bacteria and mitochondria or the small cleft between pre-and postsynaptic neuronal membranes. Large unilamellar vesicles (LUV) were tethered to a preformed supported bilayer by a biotin-streptavidin tether. We show from single particle tracking (SPT) experiments that these vesicle are mobile above the plane of the supported membrane. At higher concentrations, the tethered vesicles fuse to form a second continuous bilayer on top of the supported bilayer. The distance between the two bilayers was determined by fluorescence interference contrast (FLIC) microscopy to be between 16 and 24 nm. The lateral diffusion of labeled lipids in the second bilayer was very similar to that in supported membranes. SPT experiments with reconstituted syntaxin-1A show that the mobility of transmembrane proteins was not improved when compared with solid supported membranes. PMID:19236921

  12. Electroporation of cell membranes.

    PubMed

    Tsong, T Y

    1991-08-01

    Electric pulses of intensity in kilovolts per centimeter and of duration in microseconds to milliseconds cause a temporary loss of the semipermeability of cell membranes, thus leading to ion leakage, escape of metabolites, and increased uptake by cells of drugs, molecular probes, and DNA. A generally accepted term describing this phenomenon is "electroporation." Other effects of a high-intensity electric field on cell membranes include membrane fusions, bleb formation, cell lysis... etc. Electroporation and its related phenomena reflect the basic bioelectrochemistry of cell membranes and are thus important for the study of membrane structure and function. These phenomena also occur in such events as electric injury, electrocution, and cardiac procedures involving electric shocks. Electroporation has found applications in: (a) introduction of plasmids or foreign DNA into living cells for gene transfections, (b) fusion of cells to prepare heterokaryons, hybridoma, hybrid embryos... etc., (c) insertion of proteins into cell membranes, (d) improving drug delivery and hence effectiveness in chemotherapy of cancerous cells, (e) constructing animal model by fusing human cells with animal tissues, (f) activation of membrane transporters and enzymes, and (g) alteration of genetic expression in living cells. A brief review of mechanistic studies of electroporation is given. PMID:1912274

  13. Electroporation of cell membranes.

    PubMed Central

    Tsong, T Y

    1991-01-01

    Electric pulses of intensity in kilovolts per centimeter and of duration in microseconds to milliseconds cause a temporary loss of the semipermeability of cell membranes, thus leading to ion leakage, escape of metabolites, and increased uptake by cells of drugs, molecular probes, and DNA. A generally accepted term describing this phenomenon is "electroporation." Other effects of a high-intensity electric field on cell membranes include membrane fusions, bleb formation, cell lysis... etc. Electroporation and its related phenomena reflect the basic bioelectrochemistry of cell membranes and are thus important for the study of membrane structure and function. These phenomena also occur in such events as electric injury, electrocution, and cardiac procedures involving electric shocks. Electroporation has found applications in: (a) introduction of plasmids or foreign DNA into living cells for gene transfections, (b) fusion of cells to prepare heterokaryons, hybridoma, hybrid embryos... etc., (c) insertion of proteins into cell membranes, (d) improving drug delivery and hence effectiveness in chemotherapy of cancerous cells, (e) constructing animal model by fusing human cells with animal tissues, (f) activation of membrane transporters and enzymes, and (g) alteration of genetic expression in living cells. A brief review of mechanistic studies of electroporation is given. PMID:1912274

  14. Catalytic nanoporous membranes

    DOEpatents

    Pellin, Michael J; Hryn, John N; Elam, Jeffrey W

    2013-08-27

    A nanoporous catalytic membrane which displays several unique features Including pores which can go through the entire thickness of the membrane. The membrane has a higher catalytic and product selectivity than conventional catalysts. Anodic aluminum oxide (AAO) membranes serve as the catalyst substrate. This substrate is then subjected to Atomic Layer Deposition (ALD), which allows the controlled narrowing of the pores from 40 nm to 10 nm in the substrate by deposition of a preparatory material. Subsequent deposition of a catalytic layer on the inner surfaces of the pores reduces pore sizes to less than 10 nm and allows for a higher degree of reaction selectivity. The small pore sizes allow control over which molecules enter the pores, and the flow-through feature can allow for partial oxidation of reactant species as opposed to complete oxidation. A nanoporous separation membrane, produced by ALD is also provided for use in gaseous and liquid separations. The membrane has a high flow rate of material with 100% selectivity. Also provided is a method for producing a catalytic membrane having flow-through pores and discreet catalytic clusters adhering to the inside surfaces of the pores.

  15. Molecular cloning, expression, and primary sequence of outer membrane protein P2 of Haemophilus influenzae type b.

    PubMed Central

    Munson, R; Tolan, R W

    1989-01-01

    The structural gene for the porin of Haemophilus influenzae type b, designated outer membrane protein P2, was cloned, and the DNA sequence was determined. An oligonucleotide probe generated by reverse translation of N-terminal amino acid sequence data from the purified protein was used to screen genomic DNA. The probe detected a single EcoRI fragment of approximately 1,700 base pairs which was cloned to lambda gt11 and then into M13 and partially sequenced. The derived amino acid sequence indicated that we had cloned the N-terminal portion of the P2 gene. An overlapping approximately 1,600-base-pair PvuII genomic fragment was cloned into M13, and the sequence of the remainder of the P2 gene was determined. The gene for P2 was then reconstructed under the control of the T7 promoter and expressed in Escherichia coli. The N-terminal sequence of the purified protein corresponds to residues 21 through 34 of the derived amino acid sequence. Thus, the protein is synthesized with a 20-amino-acid leader peptide. The Mr of the processed protein is 37,782, in good agreement with the estimate of 37,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:2535836

  16. Factors That Impair Wound Healing

    PubMed Central

    Anderson, Kristin; Hamm, Rose L.

    2014-01-01

    The body's response to tissue injury in a healthy individual is an intricate, sequential physiologic process that results in timely healing with full re-epithelialization, resolution of drainage, and return of function to the affected tissue. Chronic wounds, however, do not follow this sequence of events and can challenge the most experienced clinician if the underlying factors that are impairing wound healing are not identified. The purpose of this article is to present recent information about factors that impair wound healing with the underlying pathophysiological mechanism that interferes with the response to tissue injury. These factors include co-morbidities (diabetes, obesity, protein energy malnutrition), medications (steroids, non-steroidal anti-inflammatory drugs or NSAIDs, anti-rejection medications), oncology interventions (radiation, chemotherapy), and life style habits (smoking, alcohol abuse). Successful treatment of any chronic wound depends upon identification and management of the factors for each individual. PMID:26199879

  17. Nanoengineered membrane electrode assembly interface

    DOEpatents

    Song, Yujiang; Shelnutt, John A

    2013-08-06

    A membrane electrode structure suitable for use in a membrane electrode assembly (MEA) that comprises membrane-affixed metal nanoparticles whose formation is controlled by a photochemical process that controls deposition of the metal nanoparticles using a photocatalyst integrated with a polymer electrolyte membrane, such as an ionomer membrane. Impregnation of the polymer membrane with the photocatalyst prior to metal deposition greatly reduces the required amount of metal precursor in the deposition reaction solution by restricting metal reduction substantially to the formation of metal nanoparticles affixed on or near the surface of the polymer membrane with minimal formation of metallic particles not directly associated with the membrane.

  18. CENTRIFUGAL MEMBRANE FILTRATION

    SciTech Connect

    Daniel J. Stepan; Bradley G. Stevens; Melanie D. Hetland

    1999-10-01

    The overall project consists of several integrated research phases related to the applicability, continued development, demonstration, and commercialization of the SpinTek centrifugal membrane filtration process. Work performed during this reporting period consisted of Phase 2 evaluation of the SpinTek centrifugal membrane filtration technology and Phase 3, Technology Partnering. During Phase 1 testing conducted at the EERC using the SpinTek ST-IIL unit operating on a surrogate tank waste, a solids cake developed on the membrane surface. The solids cake was observed where linear membrane velocities were less than 17.5 ft/s and reduced the unobstructed membrane surface area up to 25%, reducing overall filtration performance. The primary goal of the Phase 2 research effort was to enhance filtration performance through the development and testing of alternative turbulence promoter designs. The turbulence promoters were designed to generate a shear force across the entire membrane surface sufficient to maintain a self-cleaning membrane capability and improve filtration efficiency and long-term performance. Specific Phase 2 research activities included the following: System modifications to accommodate an 11-in.-diameter, two-disk rotating membrane assembly; Development and fabrication of alternative turbulence promoter designs; Testing and evaluation of the existing and alternative turbulence promoters under selected operating conditions using a statistically designed test matrix; and Data reduction and analysis; The objective of Phase 3 research was to demonstrate the effectiveness of SpinTek's centrifugal membrane filtration as a pretreatment to remove suspended solids from a liquid waste upstream of 3M's WWL cartridge technology for the selective removal of technetium (Tc).

  19. Membrane photobiophysics and photochemistry

    NASA Astrophysics Data System (ADS)

    Ti Tien, H.

    Life, as we know it, depends on solar energy, in particular in the visible range of the solar spectrum. However, visible light alone is useless to the living organism unless a means is available for its capture, transformation, and utilization. Nature, through its long evolution, has perfected a process known as photosynthesis by which visible light is transduced into electrical/ chemical energy. However, the heart of Nature's energy transducer is the thylakoid membrane whose molecular organization was not known until early in the 1960s. Then it was established that the bilayer lipid membrane was central to the design of the thylakoid membrane. To explain the light-driven reactions from water oxidation to carbon dioxide reduction, the so-called Z-scheme was proposed. Concurrent with the establishment of Mitchell's Chemiosmotic Hypothesis for electron transfer and phosphorylation, the experimental bilayer lipid membrane (BLM) system was developed in 1960. But what are the fundamental biophysical mechanisms involved in the phototransduction via pigmented bilayer lipid membrane-based transducers? One of the main purposes of this review is to consider these questions. A second main purpose is to introduce to the reader the experimental aspects of lipid bilayers in the investigation of photoactive biomembranes. The areas covered in this review include a brief summary of the laws of photochemistry relevant to membrane photobiophysics and photobiology. The current exploitation of the BLM system in relation to the thylakoid membrane and to the visual receptor membrane will be considered in turn. The purple membrane of H. Halobium is then discussed. Consideration will also be given to dye-sensitized BLMs, semiconducting BLMs, and BLMs formed from liquid crystals. A common characteristic in the topics covered in this review is the desire to stimulate further studies in the use of the BLM system, not only for the fundamental understanding of biomembranes, but also towards

  20. The Relationship between Visual Impairment and Gestures.

    ERIC Educational Resources Information Center

    Frame, Melissa J.

    2000-01-01

    A study found the gestural activity of 15 adolescents with visual impairments differed from that of 15 adolescents with sight. Subjects with visual impairments used more adapters (especially finger-to-hand gestures) and fewer conversational gestures. Differences in gestural activity by degree of visual impairment and grade in school were also…

  1. Evaluating the Visually Impaired: Neuropsychological Techniques.

    ERIC Educational Resources Information Center

    Price, J. R.; And Others

    1987-01-01

    Assessment of nonvisual neuropsychological impairments in visually impaired persons can be achieved through modification of existing intelligence, memory, sensory-motor, personality, language, and achievement tests so that they do not require vision or penalize visually impaired persons. The Halstead-Reitan and Luria-Nebraska neuropsychological…

  2. Familial Aggregation in Specific Language Impairment.

    ERIC Educational Resources Information Center

    Tallal, Paula; Hirsch, Linda S.; Realpe-Bonilla, Teresa; Miller, Steve; Brzustowicz, Linda M.; Bartlett, Christopher; Flax, Judy F.

    2001-01-01

    A case-control family study design examined the current language-related abilities of all biological, primary relatives of probands (N=22) with specific language impairment (SLI) and of matched controls. Impairment rates for family members of SLI probands was significantly higher than for controls. Also, impairment rates estimated from a family…

  3. Alcohol and the Physically Impaired: Special Focus.

    ERIC Educational Resources Information Center

    Boros, Alexander, Ed.

    1989-01-01

    The articles in this special issue explore the connections between the dual disabilities of alcohol abuse and physical impairment, and reflect progress made in exploring the causes and treatments of alcohol abuse among the physically impaired. Selected articles include: "Results of a Model Intervention Program for Physically Impaired Persons"…

  4. Nature Trails for the Visually Impaired.

    ERIC Educational Resources Information Center

    Schwartz, Jonathan R.

    Many interpretive nature trails have been established for the visually impaired in recent years. The objectives of the investigation were to (a) identify what has been done in the past in the way of nature trail design for the visually impaired, (b) compare this with what professional workers for the visually impaired consider important in the…

  5. Mobile Device Impairment ... Similar Problems, Similar Solutions?

    ERIC Educational Resources Information Center

    Harper, Simon; Yesilada, Yeliz; Chen, Tianyi

    2011-01-01

    Previous studies have defined a new type of impairment in which an able-bodied user's behaviour is impaired by both the characteristics of a device and the environment in which it is used. This behavioural change is defined as a situationally-induced impairment and is often associated with small devices used in a mobile setting or constrained…

  6. 20 CFR 416.923 - Multiple impairments.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Determining Disability and Blindness Evaluation of Disability § 416.923 Multiple impairments. In... impairments, the combined impact of the impairments will be considered throughout the disability...

  7. 20 CFR 416.923 - Multiple impairments.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Determining Disability and Blindness Evaluation of Disability § 416.923 Multiple impairments. In... impairments, the combined impact of the impairments will be considered throughout the disability...

  8. 20 CFR 416.923 - Multiple impairments.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Determining Disability and Blindness Evaluation of Disability § 416.923 Multiple impairments. In... impairments, the combined impact of the impairments will be considered throughout the disability...

  9. 20 CFR 416.923 - Multiple impairments.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Determining Disability and Blindness Evaluation of Disability § 416.923 Multiple impairments. In... impairments, the combined impact of the impairments will be considered throughout the disability...

  10. 20 CFR 416.923 - Multiple impairments.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Determining Disability and Blindness Evaluation of Disability § 416.923 Multiple impairments. In... impairments, the combined impact of the impairments will be considered throughout the disability...

  11. Cortical Visual Impairment: New Directions

    PubMed Central

    Good, William V.

    2009-01-01

    Cortical visual impairment is the leading cause of bilateral low vision in children in the U.S., yet very little research is being done to find new diagnostic measures and treatments. Dr. Velma Dobson's pioneering work on visual assessments of developmentally delayed children stands out as highly significant in this field. Future research will assess new diagnostic measures, including advanced imaging techniques. In addition, research will evaluate methods to prevent, treat, and rehabilitate infants and children afflicted with this condition. PMID:19417710

  12. Cyclic membrane separation process

    DOEpatents

    Bowser, John

    2004-04-13

    A cyclic process for controlling environmental emissions of volatile organic compounds (VOC) from vapor recovery in storage and dispensing operations of liquids maintains a vacuum in the storage tank ullage. In one of a two-part cyclic process ullage vapor is discharged through a vapor recovery system in which VOC are stripped from vented gas with a selectively gas permeable membrane. In the other part, the membrane is inoperative while gas pressure rises in the ullage. Ambient air is charged to the membrane separation unit during the latter part of the cycle.

  13. Rotating bubble membrane radiator

    DOEpatents

    Webb, Brent J.; Coomes, Edmund P.

    1988-12-06

    A heat radiator useful for expelling waste heat from a power generating system aboard a space vehicle is disclosed. Liquid to be cooled is passed to the interior of a rotating bubble membrane radiator, where it is sprayed into the interior of the bubble. Liquid impacting upon the interior surface of the bubble is cooled and the heat radiated from the outer surface of the membrane. Cooled liquid is collected by the action of centrifical force about the equator of the rotating membrane and returned to the power system. Details regarding a complete space power system employing the radiator are given.

  14. Cholesterol dynamics in membranes.

    PubMed Central

    Yeagle, P L; Albert, A D; Boesze-Battaglia, K; Young, J; Frye, J

    1990-01-01

    Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased

  15. Neural Membrane Signaling Platforms

    PubMed Central

    Wallace, Ron

    2010-01-01

    Throughout much of the history of biology, the cell membrane was functionally defined as a semi-permeable barrier separating aqueous compartments, and an anchoring site for proteins. Little attention was devoted to its possible regulatory role in intracellular molecular processes and neuron electrical signaling. This article reviews the history of membrane studies and the current state of the art. Emphasis is placed on natural and artificial membrane studies of electric field effects on molecular organization, especially as these may relate to impulse propagation in neurons. Implications of these studies for new designs in artificial intelligence are briefly examined. PMID:20640161

  16. Composite metal membrane

    DOEpatents

    Peachey, Nathaniel M.; Dye, Robert C.; Snow, Ronny C.; Birdsell, Stephan A.

    1998-01-01

    A composite metal membrane including a first metal layer of Group IVB met or Group VB metals, the first metal layer sandwiched between two layers of an oriented metal of palladium, platinum or alloys thereof is provided together with a process for the recovery of hydrogen from a gaseous mixture including contacting a hydrogen-containing gaseous mixture with a first side of a nonporous composite metal membrane including a first metal of Group IVB metals or Group VB metals, the first metal layer sandwiched between two layers of an oriented metal of palladium, platinum or alloys thereof, and, separating hydrogen from a second side of the nonporous composite metal membrane.

  17. Composite metal membrane

    DOEpatents

    Peachey, N.M.; Dye, R.C.; Snow, R.C.; Birdsell, S.A.

    1998-04-14

    A composite metal membrane including a first metal layer of Group IVB met or Group VB metals, the first metal layer sandwiched between two layers of an oriented metal of palladium, platinum or alloys thereof is provided together with a process for the recovery of hydrogen from a gaseous mixture including contacting a hydrogen-containing gaseous mixture with a first side of a nonporous composite metal membrane including a first metal of Group IVB metals or Group VB metals, the first metal layer sandwiched between two layers of an oriented metal of palladium, platinum or alloys thereof, and, separating hydrogen from a second side of the nonporous composite metal membrane.

  18. Cyclic membrane separation process

    DOEpatents

    Nemser, Stuart M.

    2005-05-03

    A cyclic process for controlling environmental emissions of volatile organic compounds (VOC) from vapor recovery in storage and dispensing operations of liquids maintains a vacuum in the storage tank ullage. In the first part of a two-part cyclic process ullage vapor is discharged through a vapor recovery system in which VOC are stripped from vented gas with a selectively gas permeable membrane. In the second part, the membrane is inoperative while gas pressure rises in the ullage. In one aspect of this invention, a vacuum is drawn in the membrane separation unit thus reducing overall VOC emissions.

  19. Cognitive impairment in Parkinson's disease.

    PubMed

    Cosgrove, Jeremy; Alty, Jane Elizabeth; Jamieson, Stuart

    2015-04-01

    Cognitive impairment is a significant non-motor symptom of Parkinson's disease (PD). Longitudinal cohort studies have demonstrated that approximately 50% of those with PD develop dementia after 10 years, increasing to over 80% after 20 years. Deficits in cognition can be identified at the time of PD diagnosis in some patients and this mild cognitive impairment (PD-MCI) has been studied extensively over the last decade. Although PD-MCI is a risk factor for developing Parkinson's disease dementia there is evidence to suggest that PD-MCI might consist of distinct subtypes with different pathophysiologies and prognoses. The major pathological correlate of Parkinson's disease dementia is Lewy body deposition in the limbic system and neocortex although Alzheimer's related pathology is also an important contributor. Pathological damage causes alteration to neurotransmitter systems within the brain, producing behavioural change. Management of cognitive impairment in PD requires a multidisciplinary approach and accurate communication with patients and relatives is essential. PMID:25814509

  20. Mandatory notification of impaired doctors.

    PubMed

    Beran, R G

    2014-12-01

    Mandatory reporting of impaired doctors is compulsory in Australasia. Australian Health Practitioner Regulation Agency guidelines for notification claim high benchmark though the Royal Australasian College of Surgeons and the Royal Australasian College of Physicians suggest they still obstruct doctors seeking help. Western Australia excludes mandatory reporting of practitioner-patients. This study examines reporting, consequences and international experiences with notification. Depressed doctors avoid diagnosis and treatment, fearing consequences, yet are more prone to marital problems, substance dependence and needing psychotherapy. South African research confirms isolation of impaired doctors and delayed seeking help with definable characteristics of those at risk. New Zealand data acknowledge: errors occur; questionable contribution from mandatory reporting; issues concerning competence assessment; favouring reporting to senior colleagues or self-intervention to compliance with mandatory reporting. UK found an anaesthetist guilty of professional misconduct for not reporting and sanctioned doctors regarding Harold Shipman. Australians are reluctant to report, fearing legalistic intrusion into care. Australian research confirmed definable characteristics for doctors with psychiatric illness or alcohol abuse. Exposure to legal medicine evokes personal disenchantment for doctors involved. Medicine poses barriers for impaired doctors. Spanish and UK doctors do not use general practitioners and may have suboptimal care. US and European doctors self-medicate using samples. US drug-dependent doctors also prescribe for spouses. Junior doctors are losing empathy with the profession. UK doctors favour private care, avoiding public scrutiny. NZ and Brazil created specific services for doctors, which appear effective. Mandatory reporting may be counterproductive requiring reappraisal. PMID:25442756