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Sample records for membrane impairs porin

  1. Salmonella typhimurium contains an anion-selective outer membrane porin induced by phosphate starvation.

    PubMed Central

    Bauer, K; Benz, R; Brass, J; Boos, W

    1985-01-01

    A mutant of Salmonella typhimurium was selected that is constitutive for the pho regulon. It exhibited constitutive glycerol-3-phosphate transport activity and synthesized a new outer membrane porin. Upon measurement of porin activity in black lipid films, it exhibited anion selectivity. It therefore appears analogous to the Escherichia coli PhoE porin. Images PMID:2981826

  2. 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

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

    PubMed

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

    2015-12-14

    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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. Subunit constituent of the porin trimers that form the permeability channels in the outer membrane of Salmonella typhimurium.

    PubMed Central

    Ishii, J; Nakae, T

    1980-01-01

    The polypeptide composition of the functional porin trimers that produced the permeability channels in the outer membrane of Salmonella typhimurium was examined on two-dimensional slab gels. The results suggested that the majority of porin trimers from strains producing mixed species of porin polypeptides consisted of homologous subunit polypeptides. The present results do not exclude the possibility that a small fraction of porin trimer is constructed from heterologous subunit polypeptides. Images PMID:6246065

  5. Characterisation of channels induced in planar bilayer membranes by detergent solubilised Escherichia coli porins.

    PubMed

    Lakey, J H; Watts, J P; Lea, E J

    1985-07-25

    Purified OmpF, OmpC, NmpC, PhoE and Lc (Protein 2) porins from the Escherichia coli outer membrane were incorporated into planar phospholipid bilayer membranes and the permeability properties of the pores studied. Triton X-100 solubilised porin samples showed large and reproducible increases in membrane conductivity composed of discreet single-channel events. The magnitude of the cation selectivity found for the porins was in the order OmpC greater than OmpF greater than NmpC = Lc; PhoE was anion selective. For the cation selective porins the cation/anion permeability ratios in a variety of solutes ranged from 6 to 35. Further information on the internal structure of the porins was obtained by examination of the single-channel conductance and this was used to interpret macroscopic observations and to estimate single-channel diameters. The same porins solubilised in SDS exhibited slight conductance increase with no observable single-channel activity. Use of on-line microcomputer techniques confirmed the ohmic current vs. voltage behaviour for all the single porin channels examined.

  6. Amelioration of the Fitness Costs of Antibiotic Resistance Due To Reduced Outer Membrane Permeability by Upregulation of Alternative Porins.

    PubMed

    Knopp, Michael; Andersson, Dan I

    2015-12-01

    The fitness cost of antibiotic resistance is a key parameter in determining the evolutionary success of resistant bacteria. Studies of the effect of antibiotic resistance on bacterial fitness are heavily biased toward target alterations. Here we investigated how the costs in the form of a severely impaired growth rate associated with resistance due to absence of two major outer membrane porins can be genetically compensated. We performed an evolution experiment with 16 lineages of a double mutant of Escherichia coli with the ompCF genes deleted, and reduced fitness and increased resistance to different classes of antibiotics, including the carbapenems ertapenem and meropenem. After serial passage for only 250 generations, the relative growth rate increased from 0.85 to 0.99 (susceptible wild type set to 1.0). Compensation of the costs followed two different adaptive pathways where upregulation of expression of alternative porins bypassed the need for functional OmpCF porins. The first compensatory mechanism involved mutations in the phoR and pstS genes, causing constitutive high-level expression of the PhoE porin. The second mechanism involved mutations in the hfq and chiX genes that disrupted Hfq-dependent small RNA regulation, causing overexpression of the ChiP porin. Although susceptibility was restored in compensated mutants with PhoE overexpression, evolved mutants with high ChiP expression maintained the resistance phenotype. Our findings may explain why porin composition is often altered in resistant clinical isolates and provide new insights into how bypass mechanisms may allow genetic adaptation to a common multidrug resistance mechanism. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. 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

  8. Nonlinear and asymmetric open channel characteristics of an ion-selective porin in planar membranes.

    PubMed Central

    Mathes, A; Engelhardt, H

    1998-01-01

    The open channel characteristics of the bacterial porin Omp32 from Comamonas acidovorans were investigated by means of conductance measurements in planar lipid bilayers of the Montal-Mueller type. Particularly at low salt conditions (< or = 30 mM KCl) Omp32 exhibited some unusual asymmetric and nonlinear functional properties. Current-voltage relationship measurements showed that conductance depends on the orientation of porin molecules and is a nonlinear function of the applied membrane potential. Conductance also depends on the salt concentration in a manner not common to porins and the salt concentration modulates the nonlinearity of conductance-voltage relationships. Omp32 is strongly anion-selective. The nonlinear and asymmetric conductance of the open channel is a new observation in porins. PMID:9726928

  9. Enhancement of macrophage-mediated tumor cell killing by bacterial outer membrane proteins (porins).

    PubMed Central

    Weinberg, J B; Ribi, E; Wheat, R W

    1983-01-01

    Various microbial products are known to influence the function of mouse peritoneal macrophages. Lipopolysaccharide (LPS) and certain lipid A-associated proteins are known to enhance the tumoricidal effects of macrophages. The purpose of this study was to determine whether porins (outer membrane proteins) of Salmonella typhimurium G30/C21 would influence the activity of macrophages from lipid A-responsive and -unresponsive mice. Porins, extracted by a combined sodium dodecyl sulfate-EDTA method from cell walls, were free of LPS as determined by Limulus amebocyte lysate assay and appeared as a band at approximately 36,000 molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In tumor cell killing assays done under LPS-free conditions, the porins in doses of 1 to 10 ng/ml enhanced the tumoricidal effect of macrophages from bacillus Calmette-Guérin-infected C3H/HeN or C3H/HeJ mice. Protein-free LPS enhanced the tumoricidal activity of macrophages from bacillus Calmette-Guérin-infected C3H/HeN but not C3H/HeJ mice. The tumoricidal-enhancing activity of protein-free LPS was blocked by the lipid A-binding antibiotic polymyxin B sulfate, but the effects of porins were not altered by the polymyxin B sulfate. These results suggest that porins, proteins known to alter membrane function, may alter macrophage function by interaction with macrophage membranes. Images PMID:6311745

  10. 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)

  11. 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.

  12. Approaches to the characterization of membrane channel proteins (porins) by UV MALDI-MS

    NASA Astrophysics Data System (ADS)

    Schnaible, Volker; Michels, Jenny; Zeth, Kornelius; Freigang, Jorg; Welte, Wolfram; Buhler, Stefan; Glocker, Michael O.; Przybylski, Michael

    1997-12-01

    Suitable detergents and sample preparation conditions have been characterized for the analysis of membrane channel proteins (porins) by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). Using the matrix [alpha]-cyano-4-hydroxycinnamic acid (HCCA), the best results were obtained with non-ionic detergents such as n-octylglucopyranoside. Ionic detergents such as sodium dodecylsulphate (SDS) had the most adverse effects on MALDI-MS analyses both for water-soluble and for membrane proteins. For the analysis of membrane proteins, a sample preparation procedure by ultrasonic treatment has been developed which yielded high signal intensities, particularly for proteins for which MALDI spectra, were difficult to obtain by the standard dried-droplet technique. For some high molecular weight porins, successful MALDI-MS analyses were only possible with the ultrasonication procedure.

  13. Outer membrane and porin characteristics of Serratia marcescens grown in vitro and in rat intraperitoneal diffusion chambers.

    PubMed Central

    Malouin, F; Campbell, G D; Halpenny, M; Becker, G W; Parr, T R

    1990-01-01

    The composition and antibiotic permeability barrier of the outer membrane of Serratia marcescens were assessed in cells grown in vivo and in vitro. Intraperitoneal diffusion chambers implanted in rats were used for the in vivo cultivation of bacteria. Outer membranes isolated from log-phase bacterial cells recovered from these chambers were compared with membranes isolated from cells grown in vitro. Analysis revealed that the suspected 41-kilodalton porin and the OmpA protein were recovered on sodium dodecyl sulfate-polyacrylamide gels in equal quantities. Several high-molecular-weight proteins, thought to be iron starvation induced, appeared in the diffusion chamber-grown cells. The outer membrane permeability barriers to cephaloridine were similar in in vivo- and in vitro-grown cells based on permeability coefficient calculations. The permeability coefficient of cephaloridine in S. marcescens cells (30.3 x 10(-5) to 38.9 x 10(-5) cm s-1) was greater than that obtained for an Escherichia coli strain expressing only porin OmpC but smaller than those obtained for the E. coli wild type and a strain expressing only porin OmpF. Functional characterization of the suspected porin was performed by using the planar lipid bilayer technology. The sodium dodecyl sulfate-0.4 M NaCl-soluble porin from both in vitro- and in vivo-grown cells showed an average single-channel conductance in 1 M KCl of 1.6. A partial amino acid sequence (19 residues) was obtained for the S. marcescens porin. The sequence showed a very high homology to the E. coli OmpC porin. These data identified the S. marcescens outer membrane 41-kilodalton protein as a porin by both functional and amino acid analyses. Also, the methodology used allowed for efficient growth and recovery of diffusion chamber-grown bacterial cells and permitted identification of specific in vivo-induced changes in bacterial cell membrane composition. Images PMID:2157667

  14. Role of the mar-sox-rob Regulon in Regulating Outer Membrane Porin Expression▿†

    PubMed Central

    Chubiz, Lon M.; Rao, Christopher V.

    2011-01-01

    Multiple factors control the expression of the outer membrane porins OmpF and OmpC in Escherichia coli. In this work, we investigated the role of the mar-sox-rob regulon in regulating outer membrane porin expression in response to salicylate. We provide both genetic and physiological evidence that MarA and Rob can independently activate micF transcription in response to salicylate, leading to reduced OmpF expression. MarA was also found to repress OmpF expression through a MicF-independent pathway. In the case of OmpC, we found that its transcription was moderately increased in response to salicylate. However, this increase was independent of MarA and Rob. Finally, we found that the reduction in OmpF expression in a tolC mutant is due primarily to Rob. Collectively, this work further clarifies the coordinated role of MarA and Rob in regulating the expression of the outer membrane porins. PMID:21398557

  15. The major outer membrane protein of Acidovorax delafieldii is an anion-selective porin.

    PubMed

    Brunen, M; Engelhardt, H; Schmid, A; Benz, R

    1991-07-01

    The major outer membrane protein (Omp34) of Acidovorax delafieldii (formerly Pseudomonas delafieldii) was purified to homogeneity and was characterized biochemically and functionally. The polypeptide has an apparent molecular weight (Mr) of 34,000, and it forms stable oligomers at pH 9.0 in the presence of 10% octylpolyoxyethylene or 2% lithium dodecyl sulfate below 70 degrees C. The intact protein has a characteristic secondary structure composition, as revealed by Fourier transforming infrared spectroscopy (about 60% beta sheet). These features and the amino acid composition are typical for porins. The purified Omp34 is associated with 1 to 2 mol of lipopolysaccharide per mol of the monomer. Pore-forming activity was demonstrated with lipid bilayer experiments. Single-channel and selectivity measurements showed that the protein forms highly anion-selective channels. The unusual dependence of the single-channel conductance on salt concentration suggests that the porin complexes bear positive surface charges, accumulating negatively charged counterions at the pore mouth.

  16. 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.

  17. 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

  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. Purification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis.

    PubMed

    Roussel, G; Matagne, A; De Bolle, X; Perpète, E A; Michaux, C

    2012-06-01

    Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella β-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-β-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination.

  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. 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

  2. Effect of various mild surfactants on the reassembly of an oligomeric integral membrane protein OmpF porin.

    PubMed

    Watanabe, Yasushi

    2002-03-01

    Reassembly of OmpF porin from its denatured monomer into the sodium dodecyl sulfate-resistant species was investigated by using 27 kinds of mild surfactants. Polyethyleneoxide-type surfactants with a hydrophilic-lipophilic balance value of 10.8-14.6 induced the trimerization of denatured OmpF porin. Dimerization and trimerization were induced by non-polyethyleneoxide-type mild surfactants that are generally used for membrane protein solubilization. The dependence of surfactant concentrations on reassembly was estimated to obtain a minimal concentration required for the reassembly of the protein. Extensive reassembly (to approximately 85% yield) into dimer (a putative assembly intermediate) was observed at a protein concentration of 0.05 mg/ml in 7 mg/ml n-octyl-beta-D-glucopyranoside and 1 mg/ml sodium dodecyl sulfate. This condition will be useful for the studies of the dimer and dimerization of OmpF porin. The role of mixed micelle system on the protein renaturation was discussed.

  3. Comparison of outer membrane porin proteins produced by Escherichia coli and Salmonella typhimurium.

    PubMed Central

    Lee, D R; Schnaitman, C A

    1980-01-01

    The OmpC, OmpF, and Lc (NmpC) porin proteins of Escherichia coli K-12 have been shown to be similar to the OmpC (36K), OmpF (35K) and OmpD (34K) porin proteins of Salmnella typhimurium LT2 in terms of function, regulation of expression, and, in the case of OmpC and OmpF proteins, equivalence of the genetic loci determining their production. However, the corresponding pairs of proteins from these two species showed only limited similarity in peptide maps and no similarity in terms of migration on polyacrylamide gels. PMID:6247332

  4. Extramitochondrial porin: facts and hypotheses.

    PubMed

    Báthori, G; Parolini, I; Szabó, I; Tombola, F; Messina, A; Oliva, M; Sargiacomo, M; De Pinto, V; Zoratti, M

    2000-02-01

    Mitochondrial porin, or VDAC, is a pore-forming protein abundant in the outer mitochondrial membrane. Several publications have reported extramitochondrial localizations as well, but the evidence was considered insufficient by many, and the presence of porin in nonmitochondrial cellular compartments has remained in doubt for a long time. We have now obtained new data indicating that the plasma membrane of hematopoietic cells contains porin, probably located mostly in caveolae or caveolae-like domains. Porin was purified from the plasma membrane of intact cells by a procedure utilizing the membrane-impermeable labeling reagent NH-SS-biotin and streptavidin affinity chromatography, and shown to have the same properties as mitochondrial porin. A channel with properties similar to that of isolated VDAC was observed by patch-clamping intact cells. This review discusses the evidence supporting extramitochondrial localization, the putative identification of the plasma membrane porin with the "maxi" chloride channel, the hypothetical mechanisms of sorting porin to various cellular membrane structures, and its possible functions.

  5. OpnS, an outer membrane porin of Xenorhabdus nematophila, confers a competitive advantage for growth in the insect host.

    PubMed

    van der Hoeven, Ransome; Forst, Steven

    2009-09-01

    The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded beta-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the DeltaopnS strain. Coinjection of the wild-type and DeltaopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or DeltaopnS strain were colonized by the wild-type strain. In addition, the DeltaopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The DeltaopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment.

  6. 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.

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

    DOE PAGES

    Liu, Yimo; Wang, Zheming; Liu, Juan; ...

    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

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

    PubMed

    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

    2014-12-01

    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.

  9. 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

  10. Voltage-dependent anion channels (VDACs, porin) expressed in the plasma membrane regulate the differentiation and function of human osteoclasts.

    PubMed

    Kotake, Shigeru; Yago, Toru; Kawamoto, Manabu; Nanke, Yuki

    2013-01-01

    Fewer molecules have been identified on human than murine osteoclasts, the former differing from murine osteoclasts in many ways. We show that voltage-dependent anion channels (VDACs, porin) are expressed in the plasma membrane of human osteoclasts. A search for novel proteins expressed in the plasma membrane of human osteoclasts identified VDAC. Anti-VDAC antibodies inhibited human osteoclastogenesis in vitro. VDAC expression was detected in membranes by immunoelectron microscopy and immunocytochemical double staining. The VDAC protein functions as a Cl(-) channel. VDACs regulate bone resorption, which show using Osteologic™ plates. The epitope of the antibody lay within a 10-amino acid sequence in the VDAC. The findings suggest that the VDAC is, at least partly, a novel Cl(-) channel regulating the differentiation and function of human osteoclasts. VDACs may play a crucial role in acidifying the resorption lacunae between osteoclasts and bone. Inhibitors of VDACs could be used to treat diseases involving increased resorption, such as osteoporosis, rheumatoid arthritis, and Paget's disease. © 2012 International Federation for Cell Biology.

  11. Incubation temperature, osmolarity, and salicylate affect the expression of resistance-nodulation-division efflux pumps and outer membrane porins in Acinetobacter baumannii ATCC19606T.

    PubMed

    Bazyleu, Andrei; Kumar, Ayush

    2014-08-01

    In this study, we examined the impact of various environmental conditions on the expression of resistance-nodulation-division (RND) efflux pumps and outer membrane (OM) porins, two key determinants of Acinetobacter baumannii's intrinsic resistance, an organism known to cause various multidrug resistant infections in immunocompromised individuals. Quantitative RT-PCR was used to analyze the expression of adeB, adeG, and adeJ (genes encoding RND pumps) and 33 kDa, carO, and oprD (genes encoding OM porins) of A. baumannii ATCC19606(T) under different incubation temperatures (30, 37, and 42 °C) and in the presence of high osmolarity and salicylate. Downregulation of all three RND pumps was observed at 30 °C, while downregulation of all three porins tested was observed at increased osmolarity. Downregulation of RND efflux pumps, particularly AdeABC, was consistent with increased susceptibility to antibiotics that are substrates of this pump. Expression of the adeR response regulator gene of the AdeRS system, the activator of the AdeABC pump, was also analyzed. Our work shows that various environmental stress conditions can influence the expression of RND pumps and porins in A. baumannii ATCC19606(T) and thus may play a role in the modulation of its antibiotic resistance.

  12. 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

    2015-05-08

    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.

  13. Functional Expression in Escherichia coli and Membrane Topology of Porin HopE, a Member of a Large Family of Conserved Proteins in Helicobacter pylori

    PubMed Central

    Bina, Jim; Bains, Manjeet; Hancock, Robert E. W.

    2000-01-01

    HopE is one of the smallest members of a family of 31 outer membrane proteins in Helicobacter pylori and has been shown to function as a porin. In this study it was cloned into Escherichia coli where it was expressed in the outer membrane, as confirmed by indirect immunofluorescence using HopE-specific antibodies. HopE purified from E. coli reconstituted channels in planar bilayer membranes that were the same size as those formed by HopE purified from H. pylori. A model of the membrane topology of HopE was constructed and indicated that this protein formed a β-barrel with 16 transmembrane amphipathic β-strands. The accuracy of this model was tested by linker insertion mutagenesis, assuming that, like other porins, amino acid insertions were not tolerated in the transmembrane β-strands but were tolerated in the adjoining loop regions. Generally, the results obtained with a series of 12 insertions of the sequence RSKDV and two substitutions were consistent with the topological model. The preponderance of amino acids that were conserved in the extended family of HopE paralogs were predicted to be within the membrane and comprised 45% of all residues in the membrane. PMID:10762234

  14. One-step purification and porin transport activity of the major outer membrane proteins P2 from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis.

    PubMed

    Kattner, Christof; Pfennig, Sabrina; Massari, Paola; Tanabe, Mikio

    2015-03-01

    Bacterial porins are major outer membrane proteins that function as essential solute transporters between the bacteria and the extracellular environment. Structural features of porins are also recognized by eukaryotic cell receptors involved in innate and adaptive immunity. To better investigate the function of porins, proper refolding is necessary following purification from inclusion bodies [1, 2]. Using a single-step size exclusion chromatographic method, we have purified three major porins from pathogenic bacteria, the OmpP2 (P2) from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis, at high yield and report their unique solute transport activity with size exclusion limit. Furthermore, we have optimized their purification method and achieved improvement of their thermostability for facilitating functional and structural analyses.

  15. 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.

  16. 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

  17. Outer Membrane Permeability of Cyanobacterium Synechocystis sp. Strain PCC 6803: Studies of Passive Diffusion of Small Organic Nutrients Reveal the Absence of Classical Porins and Intrinsically Low Permeability.

    PubMed

    Kowata, Hikaru; Tochigi, Saeko; Takahashi, Hideyuki; Kojima, Seiji

    2017-10-01

    The outer membrane of heterotrophic Gram-negative bacteria plays the role of a selective permeability barrier that prevents the influx of toxic compounds while allowing the nonspecific passage of small hydrophilic nutrients through porin channels. Compared with heterotrophic Gram-negative bacteria, the outer membrane properties of cyanobacteria, which are Gram-negative photoautotrophs, are not clearly understood. In this study, using small carbohydrates, amino acids, and inorganic ions as permeation probes, we determined the outer membrane permeability of Synechocystis sp. strain PCC 6803 in intact cells and in proteoliposomes reconstituted with outer membrane proteins. The permeability of this cyanobacterium was >20-fold lower than that of Escherichia coli The predominant outer membrane proteins Slr1841, Slr1908, and Slr0042 were not permeable to organic nutrients and allowed only the passage of inorganic ions. Only the less abundant outer membrane protein Slr1270, a homolog of the E. coli export channel TolC, was permeable to organic solutes. The activity of Slr1270 as a channel was verified in a recombinant Slr1270-producing E. coli outer membrane. The lack of putative porins and the low outer membrane permeability appear to suit the cyanobacterial autotrophic lifestyle; the highly impermeable outer membrane would be advantageous to cellular survival by protecting the cell from toxic compounds, especially when the cellular physiology is not dependent on the uptake of organic nutrients.IMPORTANCE Because the outer membrane of Gram-negative bacteria affects the flux rates for various substances into and out of the cell, its permeability is closely associated with cellular physiology. The outer membrane properties of cyanobacteria, which are photoautotrophic Gram-negative bacteria, are not clearly understood. Here, we examined the outer membrane of Synechocystis sp. strain PCC 6803. We revealed that it is relatively permeable to inorganic ions but is markedly less

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

    PubMed

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

    2015-09-01

    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. 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 were used to identify vitronectin-receptors in P. aeruginosa. 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). 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. Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  19. Effect of natural polymorphism on structure and function of the Yersinia pestis outer membrane porin F (OmpF protein): a computational study.

    PubMed

    Shaban, Hiba; Na, Insing; Kislichkina, Angelina A; Dentovskaya, Svetlana V; Anisimov, Andrey P; Uversky, Vladimir N

    2016-09-03

    The Yersinia pestis outer membrane porin F (OmpF) is a transmembrane protein located in the outer membrane of this Gram-negative bacterium which is the causative agent of plague, where it plays a significant role in controlling the selective permeability of the membrane. The amino acid sequences of OmpF proteins from 48 Y. pestis strains representing all currently available phylogenetic groups of this Gram-negative bacterium were recently deduced. Comparison of these amino acid sequences revealed that the OmpF can be present in four isoforms, the pestis-pestis type, and the pestis-microtus types I, II, and III. OmpF of the most recent pestis-pestis type has an alanine residue at the position 148, where all the pestis-microtus types have threonine there (T148A polymorphism). The variability of different pestis-microtus types is caused by an additional polymorphism at the 193rd position, where the OmpFs of the pestis-microtus type II and type III have isoleucine-glycine (IG(+)193) or isoleucine-glycine-isoleucine-glycine (IGIG(+)193) insertions, respectively (IG(+)193 and IGIG(+)193 polymorphism). To investigate potential effects of these sequence polymorphisms on the structural properties of the OmpF protein, we conducted multi-level computational analysis of its isoforms. Analysis of the I-TASSER-generated 3D-models revealed that the Yersinia OmpF is very similar to other non-specific enterobacterial porins. The T148A polymorphism affected a loop located in the external vestibule of the OmpF channel, whereas IG(+)193 and IGIG(+)193 polymorphisms affected one of its β-strands. Our analysis also suggested that polymorphism has moderate effect on the predicted local intrinsic disorder predisposition of OmpF, but might have some functional implementations.

  20. 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

  1. Porins Increase Copper Susceptibility of Mycobacterium tuberculosis

    PubMed Central

    Speer, Alexander; Rowland, Jennifer L.; Haeili, Mehri; Niederweis, Michael

    2013-01-01

    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

  2. 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.

  3. Antibiotic uptake through membrane channels: role of Providencia stuartii OmpPst1 porin in carbapenem resistance.

    PubMed

    Bajaj, Harsha; Tran, Que-Tien; Mahendran, Kozhinjampara R; Nasrallah, Chady; Colletier, Jacques-Phillippe; Davin-Regli, Anne; Bolla, Jean-Michel; Pagès, Jean-Marie; Winterhalter, Mathias

    2012-12-21

    The role of major porin OmpPst1 of Providencia stuartii in antibiotic susceptibility for two carbapenems is investigated by combining high-resolution conductance measurements, liposome swelling, and microbiological assays. Reconstitution of a single OmpPst1 into a planar lipid bilayer and measuring the ion current, in the presence of imipenem, revealed a concentration-dependent decrease in conductance, whereas meropenem produced well-resolved short ion current blockages. Liposome swelling assays suggested a small flux of imipenem in contrast to a rapid permeation of meropenem. The lower antibiotic susceptibility of P. stuartii to imipenem compared to meropenem correlated well with the decreased level of permeation of the former through the OmpPst1 channel.

  4. Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation.

    PubMed

    Ficarra, Florencia A; Grandellis, Carolina; Galván, Estela M; Ielpi, Luis; Feil, Regina; Lunn, John E; Gottig, Natalia; Ottado, Jorgelina

    2016-05-25

    Xanthomonas citri ssp. citri (Xcc) causes canker disease in citrus, and biofilm formation is critical for the disease cycle. OprB (Outer membrane protein B) has been shown previously to be more abundant in Xcc biofilms compared with the planktonic state. In this work, we showed that the loss of OprB in an oprB mutant abolishes bacterial biofilm formation and adherence to the host, and also compromises virulence and efficient epiphytic survival of the bacteria. Moreover, the oprB mutant is impaired in bacterial stress resistance. OprB belongs to a family of carbohydrate transport proteins, and the uptake of glucose is decreased in the mutant strain, indicating that OprB transports glucose. Loss of OprB leads to increased production of xanthan exopolysaccharide, and the carbohydrate intermediates of xanthan biosynthesis are also elevated in the mutant. The xanthan produced by the mutant has a higher viscosity and, unlike wild-type xanthan, completely lacks pyruvylation. Overall, these results suggest that Xcc reprogrammes its carbon metabolism when it senses a shortage of glucose input. The participation of OprB in the process of biofilm formation and virulence, as well as in metabolic changes to redirect the carbon flux, is discussed. Our results demonstrate the importance of environmental nutrient supply and glucose uptake via OprB for Xcc virulence.

  5. Induction of immune responses by two recombinant proteins of brucella abortus, outer membrane proteins 2b porin and Cu/Zn superoxide dismutase, in mouse model.

    PubMed

    Sung, Kyung Yong; Jung, Myunghwan; Shin, Min-Kyoung; Park, Hyun-Eui; Lee, Jin Ju; Kim, Suk; Yoo, Han Sang

    2014-06-28

    The diagnosis of Brucella abortus is mainly based on serological methods using antibody against LPS, which has diagnostic problems. Therefore, to solve this problem, we evaluated two proteins of B. abortus, Cu/Zn superoxide dismutase (SodC) and outer membrane proteins 2b porin (Omp2b). The genes were cloned and expressed in a pMAL system, and the recombinant proteins, rOmp2b and rSodC, were purified as fusion forms with maltosebinding protein. The identity of the proteins was confirmed by SDS-PAGE and Western blot analysis with sera of mice infected with B. abortus. Production of cytokines and nitric oxide (NO) was investigated in RAW 264.7 cells and mouse splenocytes after stimulation with the proteins. Moreover, cellular and humoral immune responses were investigated in BALB/c mice after immunization with the proteins. TNF-α, IL-6, and NO were significantly inducible in RAW 264.7 cells. Splenocytes of naive mice produced IFN-γ and IL-4 significantly by stimulation. Moreover, number of IgG, IFN-γ, and IL-4 producing cells were increased in immunized mice with the two proteins. Production of IgG and IgM with rOmp2b was higher than those with rSodC in immunized mice. These results suggest that the two recombinant proteins of B. abortus may be potential LPS-free proteins for diagnosis.

  6. Role of Porins for Uptake of Antibiotics by Mycobacterium smegmatis▿ †

    PubMed Central

    Danilchanka, Olga; Pavlenok, Mikhail; Niederweis, Michael

    2008-01-01

    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 β-lactam antibiotics, while its β-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 β-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

  7. Human cell mediated immunity to porins from Salmonella typhi.

    PubMed

    Blanco, F; Isibasi, A; Raúl González, C; Ortiz, V; Paniagua, J; Arreguín, C; Kumate, J

    1993-01-01

    The current studies were undertaken to assess the role of the porins and outer membrane proteins (OMP) in the human immune response to Salmonella typhi 9, 12 Vi:d. Experiments were performed to determinate the lymphocyte activation response to porins in individuals who had been vaccinated against typhoid fever. 10 healthy volunteers were studied before and 10 days after oral or subcutaneous immunisation. Five patients with typhoid fever were also studied. Lymphocyte activation was measured by the 3H thymidine incorporation assay. Individuals with typhoid fever as well as those immunised with oral vaccine responded well to porins and outer membrane proteins, as opposed to those immunised with the subcutaneous vaccine. These results suggest that the porins and OMP play a role in the cellular immune response against Salmonella typhi.

  8. Two-Step Folding of Recombinant Mitochondrial Porin in Detergent

    PubMed Central

    Bay, Denice C.; O'Neil, Joe D.; Court, Deborah A.

    2008-01-01

    Precise information regarding the transmembrane topology of mitochondrial porin is essential for understanding the mechanisms by which this protein functions. Porin acts as a channel in the outer membrane and interacts with small solutes and proteins to regulate mitochondrial function. The acquisition of high-resolution structural data requires a method of maintaining high concentrations of unaggregated, properly folded porin. In the current studies, several mixed detergent systems were analyzed for their ability to fold Neurospora mitochondrial porin expressed in and isolated from Escherichia coli. A mixture of sodium dodecyl sulfate and dodecyl-β-d-maltopyranoside in a 1:6 molar ratio supports a β-strand-rich conformation. In this state, the two tryptophan residues in the protein reside in hydrophobic environments, and about half of the nine tyrosines are solvent exposed. Most importantly, heat-labile tertiary contacts, as detected by near-UV circular dichroism spectropolarimetry, in the sodium dodecyl sulfate/dodecyl-β-d-maltopyranoside-solubilized porin are very similar to those of the protein following functional reconstitution into liposomes. Similarly, both forms are protease resistant. Thus, a method has been identified with the potential to solubilize high concentrations of mitochondrial porin in a state virtually indistinguishable from the membrane-embedded form. PMID:17872960

  9. 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

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

    PubMed

    Ferenci, Thomas; Phan, Katherine

    2015-10-21

    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.

  11. Large-Conductance Transmembrane Porin Made from DNA Origami.

    PubMed

    Göpfrich, Kerstin; Li, Chen-Yu; Ricci, Maria; Bhamidimarri, Satya Prathyusha; Yoo, Jejoong; Gyenes, Bertalan; Ohmann, Alexander; Winterhalter, Mathias; Aksimentiev, Aleksei; Keyser, Ulrich F

    2016-09-27

    DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance 10-fold compared to previous man-made channels. In our design, 19 cholesterol tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins' large ionic conductance.

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

    PubMed Central

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

    2016-01-01

    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

  13. TolA central domain interacts with Escherichia coli porins.

    PubMed Central

    Derouiche, R; Gavioli, M; Bénédetti, H; Prilipov, A; Lazdunski, C; Lloubès, R

    1996-01-01

    TolA is an inner membrane protein with three domains: a transmembrane N-terminus and periplasmic central and C-terminal domains. The interaction of TolA with outer membrane porins of Escherichia coli was investigated. Western blot analyses of cell extracts with anti-TolA antibodies indicated that TolA forms high molecular weight complexes specifically with trimeric OmpF, OmpC, PhoE and LamB, but not with OmpA. The interaction of purified TolA domains with purified porins was also studied. TolA interacted with OmpF, PhoE and LamB porins via its central domain, but not with either their denatured monomeric forms or OmpA. Moreover, the presence or absence of lipopolysaccharides associated with trimeric porins did not modify the interactions. These results suggest that the specific interaction of TolA with outer membrane porins might be relevant to the function of Tol proteins. Images PMID:8978668

  14. Characterization of the refolding and reassembly of an integral membrane protein OmpF porin by low-angle laser light scattering photometry coupled with high-performance gel chromatography.

    PubMed

    Watanabe, Yasushi

    2002-06-28

    The refolding and reassembly of an integral membrane protein OmpF porin denatured in sodium dodecylsulfate (SDS) into its stable species by the addition of n-octyl-beta-D-glucopyranoside (OG) have been studied by means of circular dichroism (CD) spectroscopy and low-angle laser light scattering photometry coupled with high-performance gel chromatography. The minimal concentration where change in the secondary structure was induced by the addition of OG was found to be 6.0 mg/ml in CD experiments. A species unfolded further than the SDS-denatured form of this protein was observed at an early stage (5-15 min) of refolding just above the minimal OG concentration. In addition, the CD spectrum of protein species obtained above the minimal OG concentration showed that the protein is composed of a beta-structure which is different from the native structure of this protein. In light scattering experiments, no changes in molecular assemblies were observed when the OG concentration was below its minimal refolding concentration determined by CD measurements. Above the minimal concentration, a compact monomeric species was observed when denatured OmpF porin was incubated for 5 min at 25 degrees C in a refolding medium containing 1 mg/ml SDS and 7 mg/ml OG, and then injected into columns equilibrated with the refolding medium. After an incubation of 24 h before injection into the columns, predominant dimerization of this protein was observed in addition to incorrect aggregation.

  15. 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.

  16. 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

  17. Role of porins in intrinsic antibiotic resistance of Pseudomonas cepacia.

    PubMed Central

    Parr, T R; Moore, R A; Moore, L V; Hancock, R E

    1987-01-01

    The measured outer membrane permeability of Pseudomonas cepacia to the beta-lactam nitrocefin was low: approximately 10 times less than that of Escherichia coli and comparable to that of Pseudomonas aeruginosa. The purified P. cepacia porin demonstrated an average single channel conductance in 1 M KCl of 0.23 nS. Images PMID:3032087

  18. Diagnosis of typhoid fever: detection of Salmonella typhi porins-specific antibodies by inhibition ELISA.

    PubMed Central

    Nandakumar, K S; Palanivel, V; Muthukkaruppan, V

    1993-01-01

    Porins are highly immunogenic outer membrane proteins of Salmonella. Sera from typhoid patients contained a high level of IgG antibodies directed to porins of Salm. typhi. Since porins are highly conserved proteins, anti-porins antibodies both from typhoid patients and healthy normals reacted with porins from several Gram-negative bacteria. Therefore, in order to improve the specificity of detecting Salm. typhi porins-specific antibodies, an inhibition ELISA was developed using enzyme-conjugated MoAbs (MP1 and MPN4) specific to Salm. typhi porins. Sera from typhoid patients with positive haemoculture (16 out of 17) inhibited the binding of MP1 to porins, thus showing a positive test for typhoid, whereas sera from patients with other Gram-negative bacterial infections (n = 7) and from healthy volunteers (66 out of 67) were found to be negative. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of this assay were 94.1, 98.7, 97.8, 94.1 and 98.7% respectively. The validity of our inhibition ELISA for typhoid was higher than that of the Widal test. The diagnosis of typhoid fever as early as 3 days after the onset of fever, using a single specimen is possible. PMID:8222322

  19. 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

  20. Functional characterization of an N-terminally-truncated mitochondrial porin expressed in Neurospora crassa.

    PubMed

    Shuvo, Sabbir R; Kovaltchouk, Uliana; Zubaer, Abdullah; Kumar, Ayush; Summers, William A T; Donald, Lynda J; Hausner, Georg; Court, Deborah A

    2017-08-01

    Mitochondrial porin, which forms voltage-dependent anion-selective channels (VDAC) in the outer membrane, can be folded into a 19-β-stranded barrel. The N terminus of the protein is external to the barrel and contains α-helical structure. Targeted modifications of the N-terminal region have been assessed in artificial membranes, leading to different models for gating in vitro. However, the in vivo requirements for gating and the N-terminal segment of porin are less well-understood. Using Neurospora crassa porin as a model, the effects of a partial deletion of the N-terminal segment were investigated. The protein, ΔN2-12porin, is assembled into the outer membrane, albeit at lower levels than the wild-type protein. The resulting strain displays electron transport chain deficiencies, concomitant expression of alternative oxidase, and decreased growth rates. Nonetheless, its mitochondrial genome does not contain any significant mutations. Most of the genes that are expressed in high levels in porin-less N. crassa are expressed at levels similar to that of wild type or are slightly increased in ΔN2-12porin strains. Thus, although the N-terminal segment of VDAC is required for complete function in vivo, low levels of a protein lacking part of the N terminus are able to rescue some of the defects associated with the absence of porin.

  1. [Elaboration of an immunosorbent for the purification of porins from Salmonella typhi 9, 12, Vi:d].

    PubMed

    Pelayo, R; Isibasi, A; Paniagua, J; Ortíz, V; Muy, M; González, C; Blanco, F; Islas, S; Kumate, J

    1989-01-01

    The current work was undertaken to purify porins of Salmonella typhi, which are outer membrane proteins (OMPs) that induce protection in mice against challenge with the bacteria in mucin. OMPs, isolated with a non-ionic detergent, had a 4% contamination with LPS (endotoxin) and molecular sizes ranging from 17 to 70 KDa. Porins (Mw 38-41 KDa) were isolated from OMPs preparative SDS-PAGE. Anti-porins antisera were raised in rabbits and specific IgG was purified, which was coupled to Sepharose-CNBr. This immunosorbent was used to purify LPS-free porins.

  2. Porins Are Required for Uptake of Phosphates by Mycobacterium smegmatis▿

    PubMed Central

    Wolschendorf, Frank; Mahfoud, Maysa; Niederweis, Michael

    2007-01-01

    Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membrane (OM) porin MspA also reduced the phosphatase activity in whole cells compared to that in lysed cells. A similar result was obtained for M. smegmatis that overexpressed endogenous alkaline phosphatase, indicating that PhoA is not a surface protein, contrary to a previous report. The uptake of phosphate by a mutant lacking the porins MspA and MspC was twofold lower than that by wild-type M. smegmatis. Strikingly, the loss of these porins resulted in a severe growth defect of M. smegmatis on low-phosphate plates. We concluded that the OM of M. smegmatis represents a permeability barrier for phosphates and that Msp porins are the only OM channels for the diffusion of phosphate in M. smegmatis. However, phosphate diffusion through Msp pores is rather inefficient as shown by the 10-fold lower permeability of M. smegmatis for phosphate compared to that for glucose. This is likely due to the negative charges in the constriction zone of Msp porins. The phosphatase activity in whole cells of Mycobacterium bovis BCG was significantly less than that in lysed cells, indicating a similar uptake pathway for phosphates in slow-growing mycobacteria. However, porins that could mediate the diffusion of phosphates across the OM of M. bovis BCG and Mycobacterium tuberculosis are unknown. PMID:17209034

  3. Understanding Voltage Gating of Providencia stuartii Porins at Atomic Level.

    PubMed

    Song, Wanling; Bajaj, Harsha; Nasrallah, Chady; Jiang, Hualiang; Winterhalter, Mathias; Colletier, Jacques-Philippe; Xu, Yechun

    2015-05-01

    Bacterial porins are water-filled β-barrel channels that allow translocation of solutes across the outer membrane. They feature a constriction zone, contributed by the plunging of extracellular loop 3 (L3) into the channel lumen. Porins are generally in the open state, but undergo gating in response to external voltages. To date the underlying mechanism is unclear. Here we report results from molecular dynamics simulations on the two porins of Providenica stuartii, Omp-Pst1 and Omp-Pst2, which display distinct voltage sensitivities. Voltage gating was observed in Omp-Pst2, where the binding of cations in-between L3 and the barrel wall results in exposing a conserved aromatic residue in the channel lumen, thereby halting ion permeation. Comparison of Omp-Pst1 and Omp-Pst2 structures and trajectories suggests that their sensitivity to voltage is encoded in the hydrogen-bonding network anchoring L3 onto the barrel wall, as we observed that it is the strength of this network that governs the probability of cations binding behind L3. That Omp-Pst2 gating is observed only when ions flow against the electrostatic potential gradient of the channel furthermore suggests a possible role for this porin in the regulation of charge distribution across the outer membrane and bacterial homeostasis.

  4. Large-Conductance Transmembrane Porin Made from DNA Origami

    PubMed Central

    2016-01-01

    DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance 10-fold compared to previous man-made channels. In our design, 19 cholesterol tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins’ large ionic conductance. PMID:27504755

  5. 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

  6. Immunobiological activities of Helicobacter pylori porins.

    PubMed Central

    Tufano, M A; Rossano, F; Catalanotti, P; Liguori, G; Capasso, C; Ceccarelli, M T; Marinelli, P

    1994-01-01

    Studies were carried out on some biological activities of Helicobacter pylori porins in vitro. We extracted and purified a porin with an apparent molecular mass of 30 kDa. Human polymorphonuclear leukocytes preincubated with H. pylori porins showed a decrease of chemotaxis, of adherence to nylon wool, and of chemiluminescence. Used as chemotaxins in place of zymosan-activated serum or as chemotaxinogens in place of zymosan, the porins induced polymorphonuclear leukocyte migration. Human monocytes and lymphocytes cultivated in the presence of H. pylori porins released cytokines. Release of the various cytokines studied was obtained with differentiated kinetics and at various porin concentrations. Starting only 3 h after culture, tumor necrosis factor alpha is released quickly, reaching a peak at 18 h, at a porin concentration of 1 microgram/ml/10(6) cells. Interleukin-6 (IL-6) appears later, with a peak at 10 micrograms/ml/10(6) cells, while IL-8 is released after 6 h of culture, with a peak at 24 h, at a porin concentration of 10 micrograms/ml/10(6) cells, while IL-8 is released after 6 h of culture, with a peak at 24 h, at a porin concentration of 10 micrograms/ml/10(6) cells. Lymphocytes stimulated by H. pylori porins release gamma interferon after 18 h of culture at higher concentrations of porins (20 micrograms/ml/10(6) cells). Granulocyte macrophage colony-stimulating factor is released from 6 to 48 h at a concentration of 1 microgram/ml/10(6) cells, while both IL-3 and IL-4 are released after 18 h of culture at different porin concentrations (0.1 and 1 microgram/ml/10(6) cells, respectively). Our results lead us to think that during H. pylori infection, surface components, porins in particular, are able to induce a series of chain reactions ranging from the inflammatory to the immunological responses. Images PMID:8132346

  7. 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

  8. Salmonella porins induce a sustained, lifelong specific bactericidal antibody memory response

    PubMed Central

    Secundino, Ismael; López-Macías, Constantino; Cervantes-Barragán, Luisa; Gil-Cruz, Cristina; Ríos-Sarabia, Nora; Pastelin-Palacios, Rodolfo; Angel Villasis-Keever, Miguel; Becker, Ingeborg; Luis Puente, José; Calva, Edmundo; Isibasi, Armando

    2006-01-01

    We examined the ability of porins from Salmonella enterica serovar typhi to induce a long-term antibody response in BALB/c mice. These porins triggered a strong lifelong production of immunoglobulin G (IgG) antibody in the absence of exogenous adjuvant. Analysis of the IgG subclasses produced during this antibody response revealed the presence of the subclasses IgG2b, IgG1, IgG2a and weak IgG3. Despite the high homology of porins, the long-lasting anti-S. typhi porin sera did not cross-react with S. typhimurium. Notably, the antiporin sera showed a sustained lifelong bactericidal-binding activity to the wild-type S. typhi strain, whereas porin-specific antibody titres measured by enzyme-linked immunosorbent assay (ELISA) decreased with time. Because our porin preparations contained the outer membrane proteins C and F (OmpC and OmpF), we evaluated the individual contribution of each porin to the long-lasting antibody response. OmpC and OmpF induced long-lasting antibody titres, measured by ELISA, which were sustained for 300 days. In contrast, although OmpC induced sustained high bactericidal antibody titres for 300 days, postimmunization, the bactericidal antibody titre induced by OmpF was not detected at day 180. These results indicate that OmpC is the main protein responsible for the antibody-mediated memory bactericidal response induced by porins. Taken together, our results show that porins are strong immunogens that confer lifelong specific bactericidal antibody responses in the absence of added adjuvant. PMID:16423041

  9. Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC.

    PubMed Central

    Singh, S P; Upshaw, Y; Abdullah, T; Singh, S R; Klebba, P E

    1992-01-01

    The immunochemistry and structure of enteric bacterial porins are critical to the understanding of the immune response to bacterial infection. We raised 41 monoclonal antibodies (MAbs) to Salmonella typhimurium OmpD and OmpC porin trimers and monomers. Enzyme-linked immunosorbent assays, immunoprecipitations, and/or Western immunoblot techniques indicated that 39 MAbs (11 anti-trimer and 28 anti-monomer) in the panel are porin specific and one binds to the lipopolysaccharide; the specificity of the remaining MAb probably lies in the porin-lipopolysaccharide complex. Among the porin-specific MAbs, 10 bound cell-surface-exposed epitopes, one reacted with a periplasmic epitope, and the remaining 28 recognized determinants that are buried within the outer membrane bilayer. Many of the MAbs reacting with surface-exposed epitopes were highly specific, recognizing only the homologous porin trimers; this suggests that the cell-surface-exposed regions of porins tends to be quite different among S. typhimurium OmpF, OmpC, and OmpD porins. Immunological cross-reaction showed that S. typhimurium OmpD was very closely related to Escherichia coli NmpC and to the Lc porin of bacteriophage PA-2. Immunologically, E. coli OmpG and protein K also appear to belong to the family of closely related porins including E. coli OmpF, OmpC, PhoE, and NmpC and S. typhimurium OmpF, OmpC, and OmpD. It appears, however, that S. typhimurium "PhoE" is not closely related to this group. Finally, about one-third of the MAbs that presumably recognize buried epitopes reacted with porin domains that are widely conserved in 13 species of the family Enterobacteriaceae, but apparently not in the seven nonenterobacterial species tested. These data are evaluated in relation to host immune response to infection by gram-negative bacteria. Images PMID:1312535

  10. OmpK26, a Novel Porin Associated with Carbapenem Resistance in Klebsiella pneumoniae▿

    PubMed Central

    García-Sureda, Laura; Doménech-Sánchez, Antonio; Barbier, Mariette; Juan, Carlos; Gascó, Joan; Albertí, Sebastián

    2011-01-01

    Clinical isolates of Klebsiella pneumoniae resistant to carbapenems are being isolated with increasing frequency. Loss of the expression of the major nonspecific porins OmpK35/36 is a frequent feature in these isolates. In this study, we looked for porins that could compensate for the loss of the major porins in carbapenem-resistant organisms. Comparison of the outer membrane proteins from two K. pneumoniae clinical isogenic isolates that are susceptible (KpCS-1) and resistant (KpCR-1) to carbapenems revealed the absence of OmpK35/36 and the presence of a new 26-kDa protein in the resistant isolate. An identical result was obtained when another pair of isogenic isolates that are homoresistant (Kpn-3) and heteroresistant (Kpn-17) to carbapenems were compared. Mass spectrometry and DNA sequencing analysis demonstrated that this new protein, designated OmpK26, is a small monomeric oligogalacturonate-specific porin that belongs to the KdgM family of porins. Insertion-duplication mutagenesis of the OmpK26 coding gene, yjhA, in the carbapenem-resistant, porin-deficient isolate KpCR-1 caused the expression of OmpK36 and the reversion to the carbapenem-susceptible phenotype, suggesting that OmpK26 is indispensable for KpCR-1 to lose OmpK36 and become resistant to these antibiotics. Moreover, loss of the major porin and expression of OmpK26 reduced in vitro fitness and attenuated virulence in a murine model of acute systemic infection. Altogether, these results indicate that expression of the oligogalacturonate-specific porin OmpK26 compensates for the absence of OmpK35/36 and allows carbapenem resistance in K. pneumoniae but cannot restore the fitness of the microorganism. PMID:21807980

  11. Humoral immune response in breeding hens and protective immunity provided by administration of purified Salmonella Gallinarum porins.

    PubMed

    Gómez-Verduzco, G; Téllez, G; Quintana, A L; Isibasi, A; Ortiz-Navarrete, V

    2010-03-01

    The current studies were undertaken to assess the ability of humoral immune response in breeding hens to provide protective maternal antibody in the progeny. A highly purified outer membrane protein, 34 kDa, was isolated from a virulent strain of Salmonella Gallinarum. Cross-reactivity was observed between this protein and Salmonella Typhi porins; thus we consider this outer membrane protein as a Salmonella Gallinarum porin. To evaluate passive immunity against Salmonella Gallinarum, 200 broiler breeder hens were immunized with either 10 microg of Salmonella Gallinarum porins, 30 microg of Salmonella Gallinarum porins, or PBS without porins as a control group. Anti-Salmonella Gallinarum porin antibodies were detected in broiler breeder serum and in fertile eggs (P < 0.05). Consequently, chickens from immunized broiler breeder hens were protected between 53 to 70% against challenges of 20 to 500 half-maximal lethal dose of Salmonella Gallinarum (P < 0.001) when compared with control hens that were injected with PBS. These results suggest that Salmonella Gallinarum porins, as those of other Salmonella species, participate in the induction of the passive protective immunity, and the humoral immune response may be one of the mechanisms involved in the establishment of this protection.

  12. Mitochondrial porin VDAC 1 seems to be functional in rickettsial cells.

    PubMed

    Emelyanov, Victor V

    2009-05-01

    We have recently shown that Rickettsia prowazekii (typhus group rickettsiae) cells incorporate human mitochondrial porin VDAC1. Here, I report on the import of porin by rickettsiae of spotted fever group. It was shown that rickettsial cells of heavy band of Renografin density gradient, known as permeabilized rickettsiae, contain much more VDAC 1 compared to the cells of light band, that is, non-permeabilized rickettsiae. These data hint at a functionality of mitochondrial porin in rickettsial cells. The cells of Rickettsia canadensis were broken in French-press, and total membranes were fractionated on linear sucrose density gradient. In this way, previous data were confirmed that an outer rickettsial membrane does not contain porin, while fraction of intermediate density, which likely represents Bayer's adhesion zones, is enriched in VDAC 1. This is consistent with earlier results and their interpretation that imported porin seems to localize to contact sites between inner and outer rickettsial membranes. Based on well-known phylogenetic relationship of Rickettsiales and mitochondria, an evolutionary scenario for the origin of protein import machinery is proposed. A dependence of rickettsiae upon essential protein(s), destined to organelle, is also viewed as a nature of their obligate endosymbiotic lifestyle.

  13. Comparative aspects of the diffusion of norfloxacin, cefepime and spermine through the F porin channel of Enterobacter cloacae.

    PubMed Central

    Chevalier, J; Malléa, M; Pagès, J M

    2000-01-01

    In Enterobacteriaceae, the permeability of the outer membrane to hydrophilic antibiotics is associated with the presence of pore-forming proteins. We tested the diffusion of the fluoroquinolone norfloxacin in four Enterobacter cloacae strains: a clinical isolate and three derivatives variously producing or lacking the D and F porins. We analysed the entry of norfloxacin into E. cloacae cells in the presence of either the polyamine spermine or the recently developed cefepime, which are known to penetrate through the Escherichia coli OmpF porin. Uptake of the fluoroquinolone was decreased in both cases; the initial rate of penetration decreased as more spermine blocked the channel. Our results indicate that, like beta-lactam molecules, fluoroquinolones translocate through the outer membrane via the F porin and that cefepime and norfloxacin entries are polyamine-sensitive. This suggests that the closure of the F porin channel by polyamines might modulate the susceptibility of E. cloacae to both fluoroquinolone and cephalosporin antibiotics. PMID:10794735

  14. An adaptive response of Enterobacter aerogenes to imipenem: regulation of porin balance in clinical isolates.

    PubMed

    Lavigne, Jean-Philippe; Sotto, Albert; Nicolas-Chanoine, Marie-Hélène; Bouziges, Nicole; Pagès, Jean-Marie; Davin-Regli, Anne

    2013-02-01

    Imipenem (IPM) is a carbapenem antibiotic frequently used in severe hospital infections. Several reports have mentioned the emergence of resistant isolates exhibiting membrane modifications. A study was conducted between September 2005 and August 2007 to survey infections due to Enterobacter aerogenes in patients hospitalised in a French university hospital. Resistant E. aerogenes clinical isolates obtained from patients treated with IPM and collected during the 3 months following initiation of treatment were phenotypically and molecularly characterised for β-lactamases, efflux pumps activity and outer membrane proteins. Among the 339 patients infected with E. aerogenes during the study period, 41 isolates (12.1%) were resistant to extended-spectrum cephalosporins and 17 patients (5.0%) were treated with IPM. The isolates from these 17 patients presented TEM-24 and basal efflux expression. Following IPM treatment, an IPM-intermediate-susceptible (IPM-I) isolate emerged in 11 patients and an IPM-resistant (IPM-R) isolate in 6 patients. A change in the porin balance (Omp35/Omp36) was observed in IPM-I isolates exhibiting ertapenem resistance. Finally, a porin deficiency (Omp35 and Omp36 absence) was detected in IPM-R isolates associated with efflux pump expression. This study indicates that the alteration in porin expression, including the shift of porin expression and lack of porins, contribute to the E. aerogenes adaptive response to IPM treatment.

  15. 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.

  16. 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

  17. Improved purification of native meningococcal porin PorB and studies on its structure/function.

    PubMed

    Massari, Paola; King, Carol A; MacLeod, Heather; Wetzler, Lee M

    2005-12-01

    The outer membrane protein PorB of Neisseria meningitidis is a pore-forming protein which has various effects on eukaryotic cells. It has been shown to (1) up-regulate the surface expression of the co-stimulatory molecule CD86 and of MHC class II (which are TLR2/MyD88 dependent and related to the porin's immune-potentiating ability), (2) be involved in prevention of apoptosis by modulating the mitochondrial membrane potential, and (3) form pores in eukaryotic cells. As an outer membrane protein, its native trimeric form isolation is complicated by its insoluble nature, requiring the presence of detergent throughout the whole procedure, and by its tight association with other outer membrane components, such as neisserial LOS or lipoproteins. In this study, an improved chromatographic purification method to obtain an homogeneous product free of endotoxin and lipoprotein is described, without loss of any of the above-mentioned properties of the porin. Furthermore, we have investigated the requirement of the native trimeric structure for the porin's activity. Inactivation of functional PorB trimers into non-functional monomers was achieved by incubation on ice. Thus, routine long- and medium-term storage at low temperature may be a cause of porin inactivation.

  18. Variation in the Neisseria lactamica porin, and its relationship to meningococcal PorB.

    PubMed

    Bennett, Julia S; Callaghan, Martin J; Derrick, Jeremy P; Maiden, Martin C J

    2008-05-01

    One potential vaccine strategy in the fight against meningococcal disease involves the exploitation of outer-membrane components of Neisseria lactamica, a commensal bacterium closely related to the meningococcus, Neisseria meningitidis. Although N. lactamica shares many surface structures with the meningococcus, little is known about the antigenic diversity of this commensal bacterium or the antigenic relationships between N. lactamica and N. meningitidis. Here, the N. lactamica porin protein (Por) was examined and compared to the related PorB antigens of N. meningitidis, to investigate potential involvement in anti-meningococcal immunity. Relationships among porin sequences were determined using distance-based methods and F(ST), and maximum-likelihood analyses were used to compare the selection pressures acting on the encoded proteins. These analyses demonstrated that the N. lactamica porin was less diverse than meningococcal PorB and although it was subject to positive selection, this was not as strong as the positive selection pressures acting on the meningococcal porin. In addition, the N. lactamica porin gene sequences and the protein sequences of the loop regions predicted to be exposed to the human immune system were dissimilar to the corresponding sequences in the meningococcus. This suggests that N. lactamica Por, contrary to previous suggestions, may have limited involvement in the development of natural immunity to meningococcal disease and might not be effective as a meningococcal vaccine component.

  19. Isolation and characterization of a conserved porin protein from Helicobacter pylori.

    PubMed Central

    Doig, P; Exner, M M; Hancock, R E; Trust, T J

    1995-01-01

    Helicobacter pylori is a causative agent of gastritis in humans and is correlated with gastric ulcer formation. Infections with this bacterium have proven difficult to treat with antimicrobial agents. To better understand how this bacterium transports compounds such as antimicrobial agents across its outer membrane, identification of porin proteins is important. We have recently identified a family of H. pylori porins (HopA to HopD) (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995). Here, we report on an unrelated porin species (HopE) from this bacterium. This protein had a apparent molecular mass of 31 kDa and was seen to form 50- and 90-kDa aggregates that were designated putative dimeric and trimeric forms, respectively. The protein was purified to homogeneity and, with a model planar lipid membrane system, was shown to act as a nonselective pore with a single channel conductance in 1.0 M KCl of 1.5 nS, similarly to other bacterial nonspecific porins. An internal peptide sequence of HopE shared homology with the P2 porin of Haemophilus influenzae. HopE was also shown to be antigenic in vivo as assessed by sera taken from H. pylori-infected individuals and was immunologically conserved with both patient sera and specific monoclonal antibodies. From these data, it appears that HopE is a major nonselective porin of H. pylori. The implications of these findings are discussed. PMID:7559328

  20. The binding of antibiotics in OmpF porin.

    PubMed

    Ziervogel, Brigitte K; Roux, Benoît

    2013-01-08

    The structure of OmpF porin in complex with three common antibiotics (zwitterionic ampicillin, anionic ertapenem, and di-anionic carbenicillin) was determined using X-ray crystallography. The three antibiotics are found to bind within the extracellular and periplasmic pore vestibules, away from the narrow OmpF constriction zone. Using the X-ray structures as a starting point, nonequilibrium molecular dynamics simulations with an applied membrane voltage show that ionic current through the OmpF channel is blocked with bound ampicillin, but not with bound carbenicillin. The susceptibility of Escherichia coli expressing OmpF mutants to ampicillin and carbenicillin was also experimentally characterized using microbiologic assays. These results show that general diffusion by OmpF porins allows for transfer of molecules with varied charged states and give insights into the design of more efficient antibiotics. A better understanding of this mechanism will shed light on nature's way of devising channels able to enhance the transport of molecules through membranes.

  1. The Binding of Antibiotics in OmpF Porin

    PubMed Central

    Ziervogel, Brigitte K.; Roux, Benoît

    2012-01-01

    The structure of OmpF porin in complex with three common antibiotics (zwitterionic ampicillin, anionic ertapenem, and di-anionic carbenicillin) was determined using X-ray crystallography. The three antibiotics are found to bind within the extracellular and periplasmic pore vestibules, away from the narrow OmpF constriction zone. Using the X-ray structures as a starting point, non-equilibrium MD simulations with an applied membrane voltage show that ionic current through the OmpF channel is blocked with bound ampicillin, but not with bound carbenicillin. The susceptibility of E. coli expressing OmpF mutants to ampicillin and carbenicillin was also experimentally characterized using microbiological assays. These results show that general diffusion by OmpF porins allows for transfer of molecules with varied charged states and give new insights into the design of more efficient antibiotics. A better understanding of this mechanism will shed light on nature's way of devising channels able to enhance the transport of molecules through membranes. PMID:23201272

  2. Genomic analyses of bacterial porin-cytochrome gene clusters

    DOE PAGES

    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. Whole-Cell-Based Assay To Evaluate Structure Permeation Relationships for Carbapenem Passage through the Pseudomonas aeruginosa Porin OprD.

    PubMed

    Iyer, Ramkumar; Sylvester, Mark A; Velez-Vega, Camilo; Tommasi, Ruben; Durand-Reville, Thomas F; Miller, Alita A

    2017-04-14

    The global emergence of antibiotic resistance, especially in Gram-negative bacteria, is an urgent threat to public health. Discovery of novel classes of antibiotics with activity against these pathogens has been impeded by a fundamental lack of understanding of the molecular drivers underlying small molecule uptake. Although it is well-known that outer membrane porins represent the main route of entry for small, hydrophilic molecules across the Gram-negative cell envelope, the structure-permeation relationship for porin passage has yet to be defined. To address this knowledge gap, we developed a sensitive and specific whole-cell approach in Escherichia coli called titrable outer membrane permeability assay system (TOMAS). We used TOMAS to characterize the structure porin-permeation relationships of a set of novel carbapenem analogues through the Pseudomonas aeruginosa porin OprD. Our results show that small structural modifications, especially the number and nature of charges and their position, have dramatic effects on the ability of these molecules to permeate cells through OprD. This is the first demonstration of a defined relationship between specific molecular changes in a substrate and permeation through an isolated porin. Understanding the molecular mechanisms that impact antibiotic transit through porins should provide valuable insights to antibacterial medicinal chemistry and may ultimately allow for the rational design of porin-mediated uptake of small molecules into Gram-negative bacteria.

  4. 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.

  5. Implication of porins in beta-lactam resistance of Providencia stuartii.

    PubMed

    Tran, Que-Tien; Mahendran, Kozhinjampara R; Hajjar, Eric; Ceccarelli, Matteo; Davin-Regli, Anne; Winterhalter, Mathias; Weingart, Helge; Pagès, Jean-Marie

    2010-10-15

    An integrative approach combining biophysical and microbiological methods was used to characterize the antibiotic translocation through the outer membrane of Providencia stuartii. Two novel members of the General Bacterial Porin family of Enterobacteriaceae, named OmpPst1 and OmpPst2, were identified in P. stuartii. In the presence of ertapenem (ERT), cefepime (FEP), and cefoxitin (FOX) in growth media, several resistant derivatives of P. stuartii ATCC 29914 showed OmpPst1-deficiency. These porin-deficient strains showed significant decrease of susceptibility to β-lactam antibiotics. OmpPst1 and OmpPst2 were purified to homogeneity and reconstituted into planar lipid bilayers to study their biophysical characteristics and their interactions with β-lactam molecules. Determination of β-lactam translocation through OmpPst1 and OmpPst2 indicated that the strength of interaction decreased in the order of ertapenem ≫ cefepime > cefoxitin. Moreover, the translocation of these antibiotics through OmpPst1 was more efficient than through OmpPst2. Heterologous expression of OmpPst1 in the porin-deficient E. coli strain BL21(DE3)omp8 was associated with a higher antibiotic susceptibility of the E. coli cells to β-lactams compared with expression of OmpPst2. All our data enlighten the involvement of porins in the resistance of P. stuartii to β-lactam antibiotics.

  6. Rectification Properties and pH-Dependent Selectivity of Meningococcal Class 1 Porin

    PubMed Central

    Cervera, Javier; Komarov, Alexander G.; Aguilella, Vicente M.

    2008-01-01

    We studied the current rectification properties and selectivity of class 1 porin (PorA) from Neisseria meningitidis (strain H44/76 Δ3Δ4) reconstituted in planar lipid membranes varying salt concentrations and pH. PorA channel shows voltage gating with a characteristic time remarkably longer than other porins. Its current-voltage asymmetry, evaluated as the current rectification ratio, changes nonmonotonically with salt concentration. Interestingly, it reaches its maximum value at physiological concentration. Porin selectivity, quantified by reversal potential measurements, is also significantly asymmetric. Depending on the direction of the salt gradient, the channel becomes more or less selective (10:1 vs. 5:1 Na+/Cl−). Besides, the reversal potential measurements suggest that porin inserts directionally following the concentration gradient. Measurements over a wide range of pH show that although PorA is strongly cation selective at pH >6, its selectivity gradually changes to anionic in an acidic medium (pH < 4). We show that a continuum electrodiffusion model quantitatively accounts for conductance and reversal potential measurements at positive and negative applied voltages. PMID:17965131

  7. Novel Mechanism of Escherichia coli Porin Regulation

    PubMed Central

    Castillo-Keller, Maria; Vuong, Phu; Misra, Rajeev

    2006-01-01

    A novel mechanism of Escherichia coli porin regulation was discovered from multicopy suppressors that permitted growth of cells expressing a mutant OmpC protein in the absence of DegP. Analyses of two suppressors showed that both substantially lowered OmpC expression. Suppression activities were confined to a short DNA sequence, which we designated ipeX for inhibition of porin expression, and to DNA containing a 3′-truncated ompR gene. The major effect of ipeX on ompC expression was exerted posttranscriptionally, whereas the truncated OmpR protein reduced ompC transcription. ipeX was localized within an untranslated region of 247 base pairs between the stop codon of nmpC—a remnant porin gene from the cryptic phage qsr′ (DLP12) genome—and its predicted Rho-independent transcriptional terminator. Interestingly, another prophage, PA-2, which encodes a porin similar to NmpC, known as Lc, has sequences downstream from lc identical to that of ipeX. PA-2 lysogenization leads to Lc expression and OmpC inhibition. Our data show that the synthesis of the lc transcript, whose 3′ end contains the corresponding ipeX sequence, inhibits OmpC expression. Overexpression of ipeX RNA inhibited both OmpC and OmpF expression but not that of OmpA. ompC-phoA chimeric gene constructs revealed a 248-bp untranslated region of ompC required for ipeX-mediated inhibition. However, no sequence complementarity was found between ipeX and this region of ompC, indicating that inhibition may not involve simple base pairing between the two RNA molecules. The effect of ipeX on ompC, but not on ompF, was independent of the RNA chaperone Hfq. PMID:16385048

  8. Characterisation and immune responses to meningococcal recombinant porin complexes incorporated into liposomes.

    PubMed

    Sánchez, Sandra; Abel, Ana; Marzoa, Juan; Gorringe, Andrew; Criado, Teresa; Ferreirós, Carlos M

    2009-08-27

    We have analysed the structure of meningococcal outer membrane complexes and found that the main complexes are formed by different combinations of PorA and/or PorB molecules, associated to other proteins such as RmpM. In view of the growing knowledge of the importance of conformational epitopes in the immune responses to many pathogens, our aim in this study was to analyse the interactions of PorA and PorB by reconstitution of both recombinant porins into liposomes and determine the relevance of these interactions for the immune response. Recombinant PorA and PorB incorporated into liposomes associate forming complexes that are homomeric when only one of the porins is present, but heteromeric when both neisserial porins are present, mimicking those found previously in native outer membrane vesicles (OMVs). Association of PorA and PorB to form heterocomplexes modifies the immunogenicity of at least PorB, allowing the production of antibodies that recognise conformational epitopes, and produces new epitopes that react with a 50 kDa outer membrane protein not yet identified.

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

    PubMed Central

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

    2016-01-01

    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

  10. 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

  11. Comparative proteomic analysis of the Haemophilus ducreyi porin-deficient mutant 35000HP::P2AB.

    PubMed

    Davie, Jeremiah J; Campagnari, Anthony A

    2009-04-01

    Haemophilus ducreyi is an obligate human pathogen and the causative agent of the sexually transmitted, genital ulcerative disease chancroid. The genome of strain 35000HP contains two known porin proteins, OmpP2A and OmpP2B. Loss of OmpP2A and OmpP2B expression in the mutant 35000HP::P2AB resulted in no obvious growth defect or phenotype. Comparison of outer membrane profiles indicated increased expression of the 58.5-kDa chaperone, GroEL, in the porin-deficient mutant. A proteomics-based comparison resulted in the identification of 231 proteins present in membrane-associated protein samples, of which a subset of 56 proteins was differentially expressed at a level of 1.5-fold or greater in the porin-deficient strain 35000HP::P2AB relative to that in 35000HP. Twenty of the differentially expressed proteins were selected for real-time PCR, resulting in the validation of 90% of the selected subgroup. Proteins identified in these studies suggested a decreased membrane stability phenotype, which was verified by disk diffusion assay. Loss of OmpP2A and OmpP2B resulted in global protein expression changes which appear to compensate for the absence of porin expression in 35000HP::P2AB.

  12. The Vibrio cholerae ToxR-Regulated Porin OmpU Confers Resistance to Antimicrobial Peptides

    PubMed Central

    Mathur, Jyoti; Waldor, Matthew K.

    2004-01-01

    BPI (bactericidal/permeability-increasing) is a potent antimicrobial protein that was recently reported to be expressed as a surface protein on human gastrointestinal tract epithelial cells. In this study, we investigated the resistance of Vibrio cholerae, a small-bowel pathogen that causes cholera, to a BPI-derived peptide, P2. Unlike in Escherichia coli and Salmonella enterica serovar Typhimurium, resistance to P2 in V. cholerae was not dependent on the BipA GTPase. Instead, we found that ToxR, the master regulator of V. cholerae pathogenicity, controlled resistance to P2 by regulating the production of the outer membrane protein OmpU. Both toxR and ompU mutants were at least 100-fold more sensitive to P2 than were wild-type cells. OmpU also conferred resistance to polymyxin B sulfate, suggesting that this porin may impart resistance to cationic antibacterial proteins via a common mechanism. Studies of stationary-phase cells revealed that the ToxR-repressed porin OmpT may also contribute to P2 resistance. Finally, although the mechanism of porin-mediated resistance to antimicrobial peptides remains elusive, our data suggest that the BPI peptide sensitivity of OmpU-deficient V. cholerae is not attributable to a generally defective outer membrane. PMID:15155667

  13. 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

  14. Polyamines Inhibit Porin-Mediated Fluoroquinolone Uptake in Mycobacteria

    PubMed Central

    Sarathy, Jansy Passiflora; Lee, Edmund; Dartois, Véronique

    2013-01-01

    Polyamines decrease the permeability of the outer membrane of Escherichia coli to fluoroquinolones and β-lactams. In this study, we tested the effect of four polyamines (spermidine, spermine, cadaverine and putrescine) on fluoroquinolone uptake in Mycobacterium bovis BCG. Our results show that polyamines are also capable of reducing the permeability of the mycobacterial outer membrane to fluoroquinolones. Spermidine was most effective and demonstrated reversible dose- and pH-dependent inhibition of ciprofloxacin accumulation. The extent of this inhibition was demonstrated across the fluoroquinolone compound class to varying degrees. Furthermore, we have shown that the addition of spermidine increases the survival of M. bovis BCG after a 5-day exposure to ciprofloxacin by up to 25 times. The treatment of actively-replicating Mycobacterium tuberculosis with spermidine reduced ciprofloxacin accumulation by half while non-replicating nutrient-starved M. tuberculosis cultures lacked similar sensitivity to polyamines. Gene expression studies showed that several outer membrane proteins are significantly down–regulated during the shift to non–replication. Collectively, these characteristics of fluoroquinolone uptake in M. bovis BCG are consistent with facilitated transport by porin-like proteins and suggest that a reduction in intracellular uptake contributes to the phenotypic drug resistance demonstrated by M. tuberculosis in the non-replicating state. PMID:23755283

  15. Topology prediction of Brucella abortus Omp2b and Omp2a porins after critical assessment of transmembrane beta strands prediction by several secondary structure prediction methods.

    PubMed

    Paquet, J Y; Vinals, C; Wouters, J; Letesson, J J; Depiereux, E

    2000-02-01

    In order to propose a reliable model for Brucella porin topology, several structure prediction methods were evaluated in their ability to predict porin topology. Four porins of known structure were selected as test-cases and their secondary structure delineated. The specificity and sensitivity of 11 methods were separately evaluated. Our critical assessment shows that some secondary structure prediction methods (PHD, Dsc, Sopma) originally designed to predict globular protein structure are useful on porin topology prediction. The overall best prediction is obtained by combining these three "generalist" methods with a transmembrane beta strand prediction technique. This "consensus" method was applied to Brucella porins Omp2b and Omp2a, sharing no sequence homology with any other porin. The predicted topology is a 16-stranded antiparallel beta barrel with Omp2a showing a higher number of negatively charged residue in the exposed loops than Omp2b. Experiments are in progress to validate the proposed topology and the functional hypotheses. The ability of the proposed consensus method to predict topology of complex outer membrane protein is briefly discussed.

  16. Salmonella Typhi Porins OmpC and OmpF Are Potent Adjuvants for T-Dependent and T-Independent Antigens.

    PubMed

    Pérez-Toledo, Marisol; Valero-Pacheco, Nuriban; Pastelin-Palacios, Rodolfo; Gil-Cruz, Cristina; Perez-Shibayama, Christian; Moreno-Eutimio, Mario A; Becker, Ingeborg; Pérez-Tapia, Sonia Mayra; Arriaga-Pizano, Lourdes; Cunningham, Adam F; Isibasi, Armando; Bonifaz, Laura C; López-Macías, Constantino

    2017-01-01

    Several microbial components, such as bacterial DNA and flagellin, have been used as experimental vaccine adjuvants because of their inherent capacity to efficiently activate innate immune responses. Likewise, our previous work has shown that the major Salmonella Typhi (S. Typhi) outer membrane proteins OmpC and OmpF (porins) are highly immunogenic protective antigens that efficiently stimulate innate and adaptive immune responses in the absence of exogenous adjuvants. Moreover, S. Typhi porins induce the expression of costimulatory molecules on antigen-presenting cells through toll-like receptor canonical signaling pathways. However, the potential of major S. Typhi porins to be used as vaccine adjuvants remains unknown. Here, we evaluated the adjuvant properties of S. Typhi porins against a range of experimental and clinically relevant antigens. Co-immunization of S. Typhi porins with ovalbumin (OVA), an otherwise poorly immunogenic antigen, enhanced anti-OVA IgG titers, antibody class switching, and affinity maturation. This adjuvant effect was dependent on CD4(+) T-cell cooperation and was associated with an increase in IFN-γ, IL-17A, and IL-2 production by OVA-specific CD4(+) T cells. Furthermore, co-immunization of S. Typhi porins with an inactivated H1N1 2009 pandemic influenza virus experimental vaccine elicited higher hemagglutinating anti-influenza IgG titers, antibody class switching, and affinity maturation. Unexpectedly, co-administration of S. Typhi porins with purified, unconjugated Vi capsular polysaccharide vaccine (Vi CPS)-a T-independent antigen-induced higher IgG antibody titers and class switching. Together, our results suggest that S. Typhi porins OmpC and OmpF are versatile vaccine adjuvants, which could be used to enhance T-cell immune responses toward a Th1/Th17 profile, while improving antibody responses to otherwise poorly immunogenic T-dependent and T-independent antigens.

  17. Salmonella Typhi Porins OmpC and OmpF Are Potent Adjuvants for T-Dependent and T-Independent Antigens

    PubMed Central

    Pérez-Toledo, Marisol; Valero-Pacheco, Nuriban; Pastelin-Palacios, Rodolfo; Gil-Cruz, Cristina; Perez-Shibayama, Christian; Moreno-Eutimio, Mario A.; Becker, Ingeborg; Pérez-Tapia, Sonia Mayra; Arriaga-Pizano, Lourdes; Cunningham, Adam F.; Isibasi, Armando; Bonifaz, Laura C.; López-Macías, Constantino

    2017-01-01

    Several microbial components, such as bacterial DNA and flagellin, have been used as experimental vaccine adjuvants because of their inherent capacity to efficiently activate innate immune responses. Likewise, our previous work has shown that the major Salmonella Typhi (S. Typhi) outer membrane proteins OmpC and OmpF (porins) are highly immunogenic protective antigens that efficiently stimulate innate and adaptive immune responses in the absence of exogenous adjuvants. Moreover, S. Typhi porins induce the expression of costimulatory molecules on antigen-presenting cells through toll-like receptor canonical signaling pathways. However, the potential of major S. Typhi porins to be used as vaccine adjuvants remains unknown. Here, we evaluated the adjuvant properties of S. Typhi porins against a range of experimental and clinically relevant antigens. Co-immunization of S. Typhi porins with ovalbumin (OVA), an otherwise poorly immunogenic antigen, enhanced anti-OVA IgG titers, antibody class switching, and affinity maturation. This adjuvant effect was dependent on CD4+ T-cell cooperation and was associated with an increase in IFN-γ, IL-17A, and IL-2 production by OVA-specific CD4+ T cells. Furthermore, co-immunization of S. Typhi porins with an inactivated H1N1 2009 pandemic influenza virus experimental vaccine elicited higher hemagglutinating anti-influenza IgG titers, antibody class switching, and affinity maturation. Unexpectedly, co-administration of S. Typhi porins with purified, unconjugated Vi capsular polysaccharide vaccine (Vi CPS)—a T-independent antigen—induced higher IgG antibody titers and class switching. Together, our results suggest that S. Typhi porins OmpC and OmpF are versatile vaccine adjuvants, which could be used to enhance T-cell immune responses toward a Th1/Th17 profile, while improving antibody responses to otherwise poorly immunogenic T-dependent and T-independent antigens. PMID:28337196

  18. X-ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from Rhodobacter capsulatus: implications for ion selectivity and single-channel conductance.

    PubMed Central

    Przybylski, M.; Glocker, M. O.; Nestel, U.; Schnaible, V.; Blüggel, M.; Diederichs, K.; Weckesser, J.; Schad, M.; Schmid, A.; Welte, W.; Benz, R.

    1996-01-01

    The role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinylporins determined by mass spectrometry and X-ray crystallography. Molecular weight and peptide mapping analyses using matrix-assisted laser desorption-ionization mass spectrometry identified selective succinylation of three lysine-epsilon-amino groups (Lys-46, Lys-298, Lys-300) and the N-terminal alpha-amino group. The structure of a tetra-succinylated porin (TS-porin) was determined to 2.4 A and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di-succinylated porin (MS-porin) and a TS-porin are localized at the inner channel surface and are solvent-accessible: Lys-46 is located at the channel constriction site, whereas Lys-298, Lys-300, and the N-terminus are all near the periplasmic entrance of the channel. The Lys-46 residue at the central constriction loop was modeled as succinyl-lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS-and TS-porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single-channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure

  19. 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

  20. Porins in the Cell Wall of Mycobacteria

    NASA Astrophysics Data System (ADS)

    Trias, Joaquim; Jarlier, Vincent; Benz, Roland

    1992-11-01

    The cell wall of mycobacteria is an efficient permeability barrier that makes mycobacteria naturally resistant to most antibiotics. Liposome swelling assays and planar bilayer experiments were used to investigate the diffusion process of hydrophilic molecules through the cell wall of Mycobacterium chelonae and identify the main hydrophilic pathway. A 59-kilodalton cell wall protein formed a water-filled channel with a diameter of 2.2 nanometers and an average single-channel conductance equal to 2.7 nanosiemens in 1 M potassium chloride. These results suggest that porins can be found in the cell wall of a Gram-positive bacterium. A better knowledge of the hydrophilic pathways should help in the design of more effective antimycobacterial agents.

  1. 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.

  2. Isolation and characterization of a family of porin proteins from Helicobacter pylori.

    PubMed Central

    Exner, M M; Doig, P; Trust, T J; Hancock, R E

    1995-01-01

    Two-dimensional gel electrophoresis was used to identify heat-modifiable outer membrane proteins, which were candidates for porins, from Helicobacter pylori membrane preparations. Four such proteins with apparent molecular masses of 48, 49, 50, and 67 kDa were isolated. The four proteins copurified together after selective detergent solubilizations followed by anion-exchange chromatography, and each protein was ultimately purified to homogeneity by gel purification. These proteins were then tested for pore-forming ability with a planar lipid bilayer model membrane system. All four proteins appeared to be present as monomers, and they formed pores with low single-channel conductances in 1.0 M KCl of 0.36, 0.36, 0.30, and 0.25 nS, respectively, for the 48-, 49-, 50-, and 67-kDa proteins which we propose to designate HopA, HopB, HopC, and HopD. N-terminal amino acid sequence analyses showed a high degree of homology among all four proteins, and it appears that these proteins constitute a family of related porins in H. pylori. PMID:7534278

  3. Putative porin of Bradyrhizobium sp. (Lupinus) bacteroids induced by glyphosate.

    PubMed

    de María, Nuria; Guevara, Angeles; Serra, M Teresa; García-Luque, Isabel; González-Sama, Alfonso; García de Lacoba, Mario; de Felipe, M Rosario; Fernández-Pascual, Mercedes

    2007-08-01

    Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed.

  4. Toward screening for antibiotics with enhanced permeation properties through bacterial porins.

    PubMed

    Hajjar, Eric; Bessonov, Andrey; Molitor, Alexander; Kumar, Amit; Mahendran, Kozhinjampara R; Winterhalter, Mathias; Pagès, Jean-Marie; Ruggerone, Paolo; Ceccarelli, Matteo

    2010-08-17

    Gram-negative bacteria are protected by an outer membrane barrier, and to reach their periplasmic target, penicillins have to diffuse through outer membrane porins such as OmpF. Here we propose a structure-dynamics-based strategy for improving such antibiotic uptake. Using a variety of experiments (high-resolution single channel recording, Minimum Inhibitory Concentration (MIC), liposome swelling assay) and accelerated molecular simulations, we decipher the subtle balance of interactions governing ampicillin diffusion through the porin OmpF. This suggests mutagenesis of a hot spot residue of OmpF for which additional simulations reveal drastic changes in the molecular and energetic pathway of ampicillin's diffusion. Inverting the problem, we predict and describe how benzylpenicillin diffuses with a lower effective energy barrier by interacting differently with OmpF. The thorough comparison between the theoretical predictions and the three independent experiments, which were set up to measure the kinetics of transport and biological activity, gives insights on how to combine such different investigation techniques with the aim of providing complementary validation. Our study illustrates the importance of microscopic interactions at the constriction region of the biological channel to control the antibiotic flux through it. We conclude by providing a complete inventory of the channel and antibiotic hot spots and discuss the implications in terms of antibacterial screening and design.

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

    PubMed

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

    2015-09-18

    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.

  6. 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

  7. Differential expression of porins OmpP2A and OmpP2B of Haemophilus ducreyi.

    PubMed

    Prather, Derrick T; Bains, Manjeet; Hancock, Robert E W; Filiatrault, Melanie J; Campagnari, Anthony A

    2004-11-01

    Haemophilus ducreyi is a strict human pathogen and the causative agent of the sexually transmitted disease chancroid. The genome of the human-passaged strain of H. ducreyi (35000HP) contains two homologous genes whose protein products have estimated molecular masses of 46 and 43 kDa. A comparative analysis of the deduced amino acid sequences revealed that these proteins share 27 to 33% identity to the outer membrane protein P2 (OmpP2), a major porin of Haemophilus influenzae. Therefore, these proteins have been designated OmpP2A and OmpP2B, respectively. The detection of ompP2A and ompP2B transcripts by reverse transcriptase PCR indicated that these genes were independently transcribed in H. ducreyi 35000HP. Western blot analysis of outer membrane proteins isolated from a geographically diverse collection of H. ducreyi clinical isolates revealed that OmpP2A and OmpP2B were differentially expressed among these strains. Although PCR analysis suggested that ompP2A and ompP2B were conserved among the strains tested, the differential expression observed was due to nucleotide additions and partial gene deletions. Purified OmpP2A and OmpP2B were isolated under nondenaturing conditions, and subsequent analysis demonstrated that these two proteins exhibited porin activity. OmpP2A and OmpP2B are the first porins described for H. ducreyi.

  8. Identification and Localization of Myxococcus xanthus Porins and Lipoproteins

    PubMed Central

    Bhat, Swapna; Zhu, Xiang; Patel, Ricky P.; Orlando, Ron; Shimkets, Lawrence J.

    2011-01-01

    Myxococcus xanthus DK1622 contains inner (IM) and outer membranes (OM) separated by a peptidoglycan layer. Integral membrane, β-barrel proteins are found exclusively in the OM where they form pores allowing the passage of nutrients, waste products and signals. One porin, Oar, is required for intercellular communication of the C-signal. An oar mutant produces CsgA but is unable to ripple or stimulate csgA mutants to develop suggesting that it is the channel for C-signaling. Six prediction programs were evaluated for their ability to identify β-barrel proteins. No program was reliable unless the predicted proteins were first parsed using Signal P, Lipo P and TMHMM, after which TMBETA-SVM and TMBETADISC-RBF identified β-barrel proteins most accurately. 228 β-barrel proteins were predicted from among 7331 protein coding regions, representing 3.1% of total genes. Sucrose density gradients were used to separate vegetative cell IM and OM fractions, and LC-MS/MS of OM proteins identified 54 β-barrel proteins. Another class of membrane proteins, the lipoproteins, are anchored in the membrane via a lipid moiety at the N-terminus. 44 OM proteins identified by LC-MS/MS were predicted lipoproteins. Lipoproteins are distributed between the IM, OM and ECM according to an N-terminal sorting sequence that varies among species. Sequence analysis revealed conservation of alanine at the +7 position of mature ECM lipoproteins, lysine at the +2 position of IM lipoproteins, and no noticable conservation within the OM lipoproteins. Site directed mutagenesis and immuno transmission electron microscopy showed that alanine at the +7 position is essential for sorting of the lipoprotein FibA into the ECM. FibA appears at normal levels in the ECM even when a +2 lysine is added to the signal sequence. These results suggest that ECM proteins have a unique method of secretion. It is now possible to target lipoproteins to specific IM, OM and ECM locations by manipulating the amino acid

  9. Purification procedure and monoclonal antibodies: two instruments for research on vertebrate porins.

    PubMed

    Reymann, S; Kiafard, Z; Rohm, B; Strutz, N; Hesse, D; Kratzin, H D; Zimmermann, B; Thinnes, F P; Hilschmann, N

    1999-10-15

    On Western blots of skeletal muscle preparations of different vertebrate classes, four monoclonal anti-human type 1 porin antibodies recognize one single band of either 30.5 or 31 kDa, respectively. To confirm that it is eukaryotic porin which is labeled by the antibodies, we used a purification procedure developed for human type 1 porin for porins from skeletal muscle of shark, frog, and turkey. Applied to different mammalian species and tissues, this procedure exclusively provides type 1 porin. However, applied to shark skeletal muscle, it provides two porin isotypes in nearly equal amounts. In the case of frog skeletal muscle, the procedure provides mainly type 2 porin and a lower amount of type 1 porin. Applied to turkey skeletal muscle, the method provides exclusively type 2 porin. As demonstrated by two-dimensional Western blots, both shark and frog porin isotypes and the turkey type 2 porin are recognized by our antibodies. Furthermore, we elucidated the entire amino acid sequence of frog type 2 porin. Copyright 1999 Academic Press.

  10. Klebsiella pneumoniae Major Porins OmpK35 and OmpK36 Allow More Efficient Diffusion of β-Lactams than Their Escherichia coli Homologs OmpF and OmpC.

    PubMed

    Sugawara, Etsuko; Kojima, Seiji; Nikaido, Hiroshi

    2016-12-01

    Klebsiella pneumoniae, one of the most important nosocomial pathogens, is becoming a major problem in health care because of its resistance to multiple antibiotics, including cephalosporins of the latest generation and, more recently, even carbapenems. This is largely due to the spread of plasmid-encoded extended-spectrum β-lactamases. However, antimicrobial agents must first penetrate the outer membrane barrier in order to reach their targets, and hydrophilic and charged β-lactams presumably diffuse through the porin channels. Unfortunately, the properties of K. pneumoniae porin channels are largely unknown. In this study, we made clean deletions of K. pneumoniae porin genes ompK35 and ompK36 and examined the antibiotic susceptibilities and diffusion rates of β-lactams. The results showed that OmpK35 and OmpK36 produced larger more permeable channels than their Escherichia coli homologs OmpF and OmpC; OmpK35 especially produced a diffusion channel of remarkably high permeability toward lipophilic (benzylpenicillin) and large (cefepime) compounds. These results were also confirmed by expressing various porins in an E. coli strain lacking major porins and the major multidrug efflux pump AcrAB. Our data explain why the development of drug resistance in K. pneumoniae is so often accompanied by the mutational loss of its porins, especially OmpK35, in addition to the various plasmid-carried genes of antibiotic resistance, because even hydrolysis by β-lactamases becomes inefficient in producing high levels of resistance if the bacterium continues to allow a rapid influx of β-lactams through its wide porin channels. In Gram-negative bacteria, drugs must first enter the outer membrane, usually through porin channels. Thus, the quantitative examination of influx rates is essential for the assessment of resistance mechanisms, yet no such studies exist for a very important nosocomial pathogen, Klebsiella pneumoniae We found that the larger channel porin of this organism

  11. Chronic kidney disease predicts impaired membrane microviscosity of red blood cells in hypertensive and normotensive subjects.

    PubMed

    Tsuda, Kazushi

    2013-01-01

    Current evidence indicates that abnormalities in physical properties of the cell membranes may be strongly linked to hypertension and other circulatory disorders. Recent studies have shown that chronic kidney disease (CKD) might be a risk factor for cardiovascular and cerebrovascular outcomes. The purpose of the present study was to examine the possible relationship between kidney function and membrane fluidity (a reciprocal value of membrane microviscosity) of red blood cells (RBCs) in hypertensive and normotensive subjects using an electron spin resonance (ESR) and spin-labeling method. The order parameter (S) for the ESR spin-label agent (5-nitroxide stearate) in RBC membranes was significantly higher in hypertensive subjects than in normotensive subjects, indicating that membrane fluidity was decreased in hypertension. The order parameter (S) of RBCs was inversely correlated with estimated glomerular filtration rate (eGFR), suggesting that a decreased eGFR value might be associated with reduced membrane fluidity of RBCs. Multivariate regression analysis also demonstrated that, after adjustment for general risk factors, eGFR might be a significant predictor of membrane fluidity of RBCs. The reduced levels of both membrane fluidity of RBCs and eGFR were associated with increased plasma 8-iso-prostaglandin F2α (an index of oxidative stress) and decreased plasma nitric oxide (NO)-metabolites, suggesting that kidney function could be a determinant of membrane microviscosity of RBCs, at least in part, via oxidative stress- and NO-dependent mechanisms. The ESR study suggests that CKD might have a close correlation with impaired rheologic behavior of RBCs and microcirculatory disorders in hypertensive subjects.

  12. Gene envY of Escherichia coli K-12 affects thermoregulation of major porin expression.

    PubMed Central

    Lundrigan, M D; Earhart, C F

    1984-01-01

    The temperature-dependent expression of OmpF and OmpC, the major channel-forming proteins of the Escherichia coli K-12 outer membrane, was studied. In wild-type cells, decreasing growth temperatures resulted in increased amounts of OmpF protein and correspondingly decreased quantities of OmpC protein. Bacteria deleted for the 13-min chromosomal region did not exhibit this temperature-dependent fluctuation in porin proteins. Plasmid pML22, which consists of pBR322 containing a 0.5-megadalton E. coli chromosomal DNA insert, complemented the thermoregulatory defect. The regulatory gene was named envY. In minicells, pML22 directed the synthesis of an envelope polypeptide (EnvY) having an apparent molecular weight of 25,000. The EnvY protein was synthesized in minicells in greater amounts at 27 degrees C than at 37 degrees C, and a reducing agent was necessary in the solubilization buffer for its subsequent detection on polyacrylamide gels. The results describe the initial characterization of a regulatory system which, along with proteins of the ompB operon, the cyclic AMP system, and the tolC gene product, is involved in a complex network affecting major porin expression. Images PMID:6317653

  13. 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.

  14. Putative Porin of Bradyrhizobium sp. (Lupinus) Bacteroids Induced by Glyphosate▿

    PubMed Central

    de María, Nuria; Guevara, Ángeles; Serra, M. Teresa; García-Luque, Isabel; González-Sama, Alfonso; de Lacoba, Mario García; de Felipe, M. Rosario; Fernández-Pascual, Mercedes

    2007-01-01

    Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed. PMID:17557843

  15. Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli.

    PubMed Central

    Hirai, K; Aoyama, H; Irikura, T; Iyobe, S; Mitsuhashi, S

    1986-01-01

    The mechanism of penetration of quinolones through the bacterial outer membrane was studied with lipopolysaccharide-deficient and porin-deficient mutants. The data indicated that the lipopolysaccharide layer might form a permeability barrier for hydrophobic quinolones such as nalidixic acid but not for hydrophilic quinolones such as norfloxacin and ciprofloxacin. The results also showed that quinolones with a low relative hydrophobicity appeared to permeate through OmpF porin, whereas quinolones with a low relative hydrophobicity appeared to permeate through OmpF porin, whereas quinolones with a high relative hydrophobicity appeared to permeate through both OmpF porin and phospholipid bilayers. PMID:3521490

  16. Impairment of cerebral autoregulation in pediatric extracorporeal membrane oxygenation associated with neuroimaging abnormalities.

    PubMed

    Tian, Fenghua; Morriss, Michael Craig; Chalak, Lina; Venkataraman, Ramgopal; Ahn, Chul; Liu, Hanli; Raman, Lakshmi

    2017-10-01

    Extracorporeal membrane oxygenation (ECMO) is a life-supporting therapy for critically ill patients with severe respiratory and/or cardiovascular failure. Cerebrovascular impairment can result in hemorrhagic and ischemic complications commonly seen in the patients supported on ECMO. We investigated the degree of cerebral autoregulation impairment during ECMO as well as whether it is predictive of neuroimaging abnormalities. Spontaneous fluctuations of mean arterial pressure (MAP) and cerebral tissue oxygen saturation ([Formula: see text]) were continuously measured during the ECMO run. The dynamic relationship between the MAP and [Formula: see text] fluctuations was assessed based on wavelet transform coherence (WTC). Neuroimaging was conducted during and/or after ECMO as standard of care, and the abnormalities were evaluated based on a scoring system that had been previously validated among ECMO patients. Of the 25 patients, 8 (32%) had normal neuroimaging, 7 (28%) had mild to moderate neuroimaging abnormalities, and the other 10 (40%) had severe neuroimaging abnormalities. The degrees of cerebral autoregulation impairment quantified based on WTC showed significant correlations with the neuroimaging scores ([Formula: see text]; [Formula: see text]). Evidence that cerebral autoregulation impairment during ECMO was related to the patients' neurological outcomes was provided.

  17. Enhanced water permeability and tunable ion selectivity in subnanometer carbon nanotube porins.

    PubMed

    Tunuguntla, Ramya H; Henley, Robert Y; Yao, Yun-Chiao; Pham, Tuan Anh; Wanunu, Meni; Noy, Aleksandr

    2017-08-25

    Fast water transport through carbon nanotube pores has raised the possibility to use them in the next generation of water treatment technologies. We report that water permeability in 0.8-nanometer-diameter carbon nanotube porins (CNTPs), which confine water down to a single-file chain, exceeds that of biological water transporters and of wider CNT pores by an order of magnitude. Intermolecular hydrogen-bond rearrangement, required for entry into the nanotube, dominates the energy barrier and can be manipulated to enhance water transport rates. CNTPs block anion transport, even at salinities that exceed seawater levels, and their ion selectivity can be tuned to configure them into switchable ionic diodes. These properties make CNTPs a promising material for developing membrane separation technologies. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  18. 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

  19. 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

  20. Structures of the OmpF porin crystallized in the presence of foscholine-12

    PubMed Central

    Kefala, Georgia; Ahn, Chihoon; Krupa, Martin; Esquivies, Luis; Maslennikov, Innokentiy; Kwiatkowski, Witek; Choe, Senyon

    2010-01-01

    The endogenous Escherichia coli porin OmpF was crystallized as an accidental by-product of our efforts to express, purify, and crystallize the E. coli integral membrane protein KdpD in the presence of foscholine-12 (FC12). FC12 is widely used in membrane protein studies, but no crystal structure of a protein that was both purified and crystallized with this detergent has been reported in the Protein Data Bank. Crystallization screening for KdpD yielded two different crystals of contaminating protein OmpF. Here, we report two OmpF structures, the first membrane protein crystal structures for which extraction, purification, and crystallization were done exclusively with FC12. The first structure was refined in space group P21 with cell parameters a = 136.7 Å, b = 210.5 Å, c = 137 Å, and β = 100.5°, and the resolution of 3.8 Å. The second structure was solved at the resolution of 4.4 Å and was refined in the P321 space group, with unit cell parameters a = 215.5 Å, b = 215.5 Å, c = 137.5 Å, and γ = 120°. Both crystal forms show novel crystal packing, in which the building block is a tetrahedral arrangement of four trimers. Additionally, we discuss the use of FC12 for membrane protein crystallization and structure determination, as well as the problem of the OmpF contamination for membrane proteins overexpressed in E. coli. PMID:20196071

  1. Expression of a Gene for a Porin-Like Protein of the OmpA Family from Mycobacterium tuberculosis H37Rv

    PubMed Central

    Senaratne, Ryan H.; Mobasheri, Hamid; Papavinasasundaram, K. G.; Jenner, Peter; Lea, Edward J. A.; Draper, Philip

    1998-01-01

    An open reading frame in the genomic database of Mycobacterium tuberculosis H37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned into Escherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of broken M. tuberculosis cells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed in M. tuberculosis cells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule from M. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell. PMID:9657995

  2. Carbohydrate-reactive, pore-forming outer membrane proteins of Aeromonas hydrophila.

    PubMed Central

    Quinn, D M; Atkinson, H M; Bretag, A H; Tester, M; Trust, T J; Wong, C Y; Flower, R L

    1994-01-01

    Two outer membrane proteins of Aeromonas hydrophila A6, isolated in a one-step affinity chromatography process based on carbohydrate reactivity, were found to be pore-forming molecules in artificial planar bilayer membranes. These carbohydrate-reactive outer membrane proteins (CROMPs; M(r)s, 40,000 and 43,000) were subjected to amino acid analysis. The amino acid profiles for these two outer membrane proteins were almost identical. A partial protein sequence of a 14-amino-acid fragment of the 40,000-Da protein revealed homology with outer membrane porins of Escherichia coli and A. hydrophila. CROMPs were compared with carbohydrate-reactive porins also extracted from outer membranes of A. hydrophila A6. These porins were isolated by using standard porin purification techniques (insolubility in 2% sodium dodecyl sulfate, solubility in 0.4 M NaCl, and Sephacryl S-200 gel filtration), and then Synsorb H type 2 affinity chromatography was done. The physical and functional properties of the carbohydrate-reactive porins and CROMPs were found to be identical. On the basis of pore-forming properties in planar lipid bilayers and channel inhibition with maltotriose solutions, a nonspecific, general diffusion porin and a LamB-like maltoporin were identified in both CROMP and carbohydrate-reactive porin preparations. To our knowledge, the use of carbohydrate reactivity to isolate channel-forming proteins from bacterial outer membranes has not been reported previously. Images PMID:7520425

  3. 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.

  4. Active protection of mice against Salmonella typhi by immunization with strain-specific porins.

    PubMed

    Isibasi, A; Ortiz-Navarrete, V; Paniagua, J; Pelayo, R; González, C R; García, J A; Kumate, J

    1992-01-01

    NIH mice were immunized with between 2.5 and 30 micrograms of two highly purified porins, 34 kDa and 36 kDa, isolated from the virulent strain Salmonella typhi 9,12, Vi:d. Of mice immunized with 10 micrograms of porins, 90% were protected against a challenge with up to 500 LD50 (50% lethal doses) of S. typhi 9,12,Vi:d and only 30% protection was observed in mice immunized with the same dose of porins but challenged with the heterologous strain Salmonella typhimurium. These results demonstrate the utility of porins for the induction of a protective status against S. typhi in mice.

  5. High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells.

    PubMed

    Billis, Puja; Will, Yvonne; Nadanaciva, Sashi

    2014-02-19

    Reactive oxygen species (ROS) are constantly produced in cells as a result of aerobic metabolism. When there is an excessive production of ROS and the cell's antioxidant defenses are overwhelmed, oxidative stress occurs. The superoxide anion is a type of ROS that is produced primarily in mitochondria but is also generated in other regions of the cell including peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. Here, a high-content imaging assay using the dye dihydroethidium is described for identifying compounds that generate superoxide in eukaryotic cells. A high-content imaging assay using the fluorescent dye tetramethylrhodamine methyl ester is also described to identify compounds that impair mitochondrial membrane potential in eukaryotic cells. The purpose of performing both assays is to identify compounds that (1) generate superoxide at lower concentrations than they impair mitochondrial membrane potential, (2) impair mitochondrial membrane potential at lower concentrations than they generate superoxide, (3) generate superoxide and impair mitochondrial function at similar concentrations, and (4) do not generate superoxide or impair mitochondrial membrane potential during the duration of the assays.

  6. Endothelin-1 Impairs Glucose Transporter Trafficking via a Membrane-Based Mechanism

    PubMed Central

    Strawbridge, Andrew B.; Elmendorf, Jeffrey S.

    2008-01-01

    Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction. PMID:16240321

  7. Endothelin-1 impairs glucose transporter trafficking via a membrane-based mechanism.

    PubMed

    Strawbridge, Andrew B; Elmendorf, Jeffrey S

    2006-03-01

    Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction. 2005 Wiley-Liss, Inc.

  8. 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

    2016-09-13

    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.

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

    PubMed Central

    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

  10. 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

  11. Structure of Carbon Nanotube Porins in Lipid Bilayers: An in Situ Small-Angle X-ray Scattering (SAXS) Study [Atomic-level structure of carbon nanotube porins in lipid bilayers: an in-situ small-angle x-ray scattering (SAXS) study

    DOE PAGES

    Tran, Ich C.; Tunuguntla, Ramya H.; Kim, Kyunghoon; ...

    2016-06-20

    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. Furthermore, 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.more » These 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.« less

  12. Structure of Carbon Nanotube Porins in Lipid Bilayers: An in Situ Small-Angle X-ray Scattering (SAXS) Study [Atomic-level structure of carbon nanotube porins in lipid bilayers: an in-situ small-angle x-ray scattering (SAXS) study

    SciTech Connect

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

    2016-06-20

    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. Furthermore, 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. These 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.

  13. In vivo protein interaction network analysis reveals porin-localized antibiotic inactivation in Acinetobacter baumannii strain AB5075.

    PubMed

    Wu, Xia; Chavez, Juan D; Schweppe, Devin K; Zheng, Chunxiang; Weisbrod, Chad R; Eng, Jimmy K; Murali, Ananya; Lee, Samuel A; Ramage, Elizabeth; Gallagher, Larry A; Kulasekara, Hemantha D; Edrozo, Mauna E; Kamischke, Cassandra N; Brittnacher, Mitchell J; Miller, Samuel I; Singh, Pradeep K; Manoil, Colin; Bruce, James E

    2016-11-11

    The nosocomial pathogen Acinetobacter baumannii is a frequent cause of hospital-acquired infections worldwide and is a challenge for treatment due to its evolved resistance to antibiotics, including carbapenems. Here, to gain insight on A. baumannii antibiotic resistance mechanisms, we analyse the protein interaction network of a multidrug-resistant A. baumannii clinical strain (AB5075). Using in vivo chemical cross-linking and mass spectrometry, we identify 2,068 non-redundant cross-linked peptide pairs containing 245 intra- and 398 inter-molecular interactions. Outer membrane proteins OmpA and YiaD, and carbapenemase Oxa-23 are hubs of the identified interaction network. Eighteen novel interactors of Oxa-23 are identified. Interactions of Oxa-23 with outer membrane porins OmpA and CarO are verified with co-immunoprecipitation analysis. Furthermore, transposon mutagenesis of oxa-23 or interactors of Oxa-23 demonstrates changes in meropenem or imipenem sensitivity in strain AB5075. These results provide a view of porin-localized antibiotic inactivation and increase understanding of bacterial antibiotic resistance mechanisms.

  14. Characterization of Ciprofloxacin Permeation Pathways across the Porin OmpC Using Metadynamics and a String Method.

    PubMed

    Prajapati, Jigneshkumar Dahyabhai; Fernández Solano, Carlos José; Winterhalter, Mathias; Kleinekathöfer, Ulrich

    2017-09-12

    The rapid spreading of antimicrobial resistance in Gram-negative bacteria has become a major threat for humans as well as animals. As one of the main factors involved, the permeability of the outer membrane has attracted a great deal of attention recently. However, the knowledge regarding the translocation mechanisms for most available antibiotics is so far rather limited. Here, a theoretical study concerning the diffusion route of ciprofloxacin across the outer membrane porin OmpC from E. coli is presented. To this end, we establish a protocol to characterize meaningful permeation pathways by combining metadynamics with the zero-temperature string method. It was found that the lowest-energy pathway requires a reorientation of ciprofloxacin in the extracellular side of the porin before reaching the constriction region with its carboxyl group ahead. Several affinity sites have been identified, and their metastability has been evaluated using unbiased simulations. Such a detailed understanding is potentially very helpful in guiding the development of next generation antibiotics.

  15. In vivo protein interaction network analysis reveals porin-localized antibiotic inactivation in Acinetobacter baumannii strain AB5075

    PubMed Central

    Wu, Xia; Chavez, Juan D.; Schweppe, Devin K.; Zheng, Chunxiang; Weisbrod, Chad R.; Eng, Jimmy K.; Murali, Ananya; Lee, Samuel A.; Ramage, Elizabeth; Gallagher, Larry A.; Kulasekara, Hemantha D.; Edrozo, Mauna E.; Kamischke, Cassandra N.; Brittnacher, Mitchell J.; Miller, Samuel I.; Singh, Pradeep K.; Manoil, Colin; Bruce, James E.

    2016-01-01

    The nosocomial pathogen Acinetobacter baumannii is a frequent cause of hospital-acquired infections worldwide and is a challenge for treatment due to its evolved resistance to antibiotics, including carbapenems. Here, to gain insight on A. baumannii antibiotic resistance mechanisms, we analyse the protein interaction network of a multidrug-resistant A. baumannii clinical strain (AB5075). Using in vivo chemical cross-linking and mass spectrometry, we identify 2,068 non-redundant cross-linked peptide pairs containing 245 intra- and 398 inter-molecular interactions. Outer membrane proteins OmpA and YiaD, and carbapenemase Oxa-23 are hubs of the identified interaction network. Eighteen novel interactors of Oxa-23 are identified. Interactions of Oxa-23 with outer membrane porins OmpA and CarO are verified with co-immunoprecipitation analysis. Furthermore, transposon mutagenesis of oxa-23 or interactors of Oxa-23 demonstrates changes in meropenem or imipenem sensitivity in strain AB5075. These results provide a view of porin-localized antibiotic inactivation and increase understanding of bacterial antibiotic resistance mechanisms. PMID:27834373

  16. Porin OmpP2 of Haemophilus influenzae shows specificity for nicotinamide-derived nucleotide substrates.

    PubMed

    Andersen, Christian; Maier, Elke; Kemmer, Gabrielle; Blass, Julia; Hilpert, Anna-Karina; Benz, Roland; Reidl, Joachim

    2003-07-04

    Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with KS values of approximately 8 and 4mm, respectively, in 0.1 m KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.

  17. 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

  18. Hemorrhagic shock impairs myocardial cell volume regulation and membrane integrity in dogs

    SciTech Connect

    Horton, J.W.

    1987-06-01

    An in vitro myocardial slice technique was used to quantitate alterations in cell volume regulation and membrane integrity after 2 h or hemorrhagic shock. After in vitro incubation in Krebs-Ringer-phosphate medium containing trace (/sup 14/C)inulin, values (ml H/sub 2/O/g dry wt) for control nonshocked myocardial slices were 4.03 /plus minus/ 0.11 (SE) for total water, 2.16 /plus minus/ 0.07 for inulin impermeable space, and 1.76 /plus minus/ 0.15 for inulin diffusible space. Shocked myocardial slices showed impaired response to cold incubation. After 2 h of in vivo shock, total tissue water, inulin diffusible space, and inulin impermeable space increased significantly for subendocardium, whereas changes in subepicardium parameters were minimal. Shock-induced cellular swelling was accompanied by an increased total tissue sodium, but no change in tissue potassium. Calcium entry blockade in vivo significantly reduced subendocardial total tissue water as compared with shock-untreated dogs. In addition, calcium entry blockade reduced shock-induced increases in inulin diffusible space. In vitro myocardial slice studies confirm alterations in subendocardial membrane integrity after 2 h of in vivo hemorrhagic shock. Shock-induced abnormalities in myocardial cell volume regulation are reduced by calcium entry blockade in vivo.

  19. 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

  20. Pathogen-associated porin turns IL-10 competent B-1a cells toward proinflammatory cytokine response.

    PubMed

    Ghosh, Amlan Kanti; Sinha, Debolina; Biswas, Ratna; Biswas, Tapas

    2016-12-01

    Shigellosis is a major problem in the developing countries causing mortality and morbidity particularly among the children. Shigella spp. harbours the epithelial cells of the human colon to infect the host and spread the disease. We analyzed the response of B-1a cells, the major component of the mucosal immune system to porin of Shigella dysenteriae type 1. We show that porin while proliferating B-1a cells, deplete Siglec-G, the inhibitory molecule present on B-1a cells. Adjuvanticity of porin has been shown to govern innate signaling for promoting host adaptive immune response. Up-regulation of CD69 and CD40 denotes activation of the cells parallel to abrogation of Siglec-G. As a result of cell activation, porin stimulated the inflammatory cytokines of CD5(+) B-1a cells, otherwise rich in IL-10. The work shows B-1a cell responses promote the immunopotentiating activity of porin.

  1. 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.

  2. 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

  3. 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

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

    2014-04-01

    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.

  4. Functional role of ompF and ompC porins in pathogenesis of avian pathogenic Escherichia coli.

    PubMed

    Hejair, Hassan M A; Zhu, Yingchu; Ma, Jiale; Zhang, Yue; Pan, Zihao; Zhang, Wei; Yao, Huochun

    2017-03-14

    Avian pathogenic Escherichia coli is an important pathogen causes systemic infections in avian species and large economic losses in poultry industry worldwide. The functional role of porins during the infection and their mechanisms of interaction with host tissues for adhesion to and invasion are poorly understood. However, whether porins play a role in infection remains unclear. In this study we evaluated the potential of ompF and ompC outer membrane porins in the pathogenesis of avian pathogenic E. coli (APEC) strain TW-XM. The ompF and ompC were deleted to generate a series of mutants. We found that, ΔompF and ΔompC reduced significantly the adherence by 41.3% and 46.1% and invasion capabilities of APEC to mouse brain microvascular endothelial cell (BMEC) bEnd.3 cells in vitro by 51.9% and 49.7% respectively, compared with the wild strain TW-XM. In vivo experiment based on the measurement of the LD50 have also shown that, ΔompF and ΔompC reduced the bacterial virulence by 9.8-fold, 12.3-fold in ducklings and 9-fold, 10.2-fold in mouse models. Animal infection experiments further revealed that, loss of ompF and ompC reduced TW-XM colonization and invasion capacity in brains, lungs and blood compared to wild-type strain TW-XM (P > 0.01). These virulence-related phenotypes were partially recoverable by genetic complementation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) indicated that, the loss of ompF and ompC significantly decreased the expression levels of ompA, fimC and iBeA genes in the mutant strains, compared to wild-type strainTW-XM (P < 0.01). Collectively, our data demonstrate that inactivation of these two porins decreased adhesion, invasion, colonization, proliferation capacities, possibly by reduced expression levels of ompA, fimC and iBeA, which may indicate the involvement of ompF and ompC in APEC pathogenesis.

  5. High-Yield Synthesis and Optical Properties of Carbon Nanotube Porins

    DOE PAGES

    Tunuguntla, Ramya H.; Chen, Xi; Belliveau, Allison; ...

    2017-01-18

    Carbon nanotube porins (CNTPs) are a convenient membrane-based model system for studying nanofluidic transport that replicates a number of key structural features of biological membrane channels. We present a generalized approach for CNTP synthesis using sonochemistry-assisted segmenting of carbon nanotubes. Prolonged tip sonication in the presence of lipid molecules debundles and fragments long carbon nanotube aggregates into stable and water-soluble individual CNTPs with lengths in the range 5–20 nm. We discuss the main parameters that determine the efficiency and the yield of this process, describe the optimized conditions for high-yield CNTP synthesis, and demonstrate that this methodology can be adaptedmore » for synthesis of CNTPs of different diameters. We also present the optical properties of CNTPs and show that a combination of Raman and UV–vis–NIR spectroscopy can be used to monitor the quality of the CNTP synthesis. Altogether, CNTPs represent a versatile nanopore building block for creating higher-order functional biomimetic materials.« less

  6. Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria.

    PubMed

    Gupta, Shelly; Prasad, G V R Krishna; Mukhopadhaya, Arunika

    2015-12-25

    Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.

  7. Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria*

    PubMed Central

    Gupta, Shelly; Prasad, G. V. R. Krishna; Mukhopadhaya, Arunika

    2015-01-01

    Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role. PMID:26559970

  8. Proteomic analysis of human U937 cell line activation mediated by Haemophilus influenzae type b P2 porin and its surface-exposed loop 7.

    PubMed

    Severino, Valeria; Chambery, Angela; Vitiello, Mariateresa; Cantisani, Marco; Galdiero, Stefania; Galdiero, Massimiliano; Malorni, Livia; Di Maro, Antimo; Parente, Augusto

    2010-02-05

    The virulence of Haemophilus influenzae type b (Hib) has been attributed to a variety of potential factors associated with its cell surface, including lipopolysaccharides (LPS) and major outer membrane proteins (OMPs). P2 porin, one of the best-characterized porins in terms of its functional characteristics, is the most abundant OMP in Hib and has also been shown to possess proinflammatory activity. To characterize the role played by bacterial surface components in disease onset and development, the proteomic profiling of human U937 cell line activated by H. influenzae type b P2 porin and its most active surface-exposed loop (L7) was performed by means of two-dimensional electrophoresis and mass spectrometry. The study provided a list of candidate proteins with potential relevance in the host immune and inflammatory response. Most of the differentially expressed proteins are involved in metabolic processes, remodelling of cytoskeleton, stress response and signal transduction pathways. The results constitute the basis for dissecting signal transduction cascades activated by P2 stimulation and gain insights into the molecular events involved in the modulation of pathogen-host cell interactions.

  9. Release of Free DNA by Membrane-Impaired Bacterial Aerosols Due to Aerosolization and Air Sampling

    PubMed Central

    Zhen, Huajun; Han, Taewon; Fennell, Donna E.

    2013-01-01

    We report here that stress experienced by bacteria due to aerosolization and air sampling can result in severe membrane impairment, leading to the release of DNA as free molecules. Escherichia coli and Bacillus atrophaeus bacteria were aerosolized and then either collected directly into liquid or collected using other collection media and then transferred into liquid. The amount of DNA released was quantified as the cell membrane damage index (ID), i.e., the number of 16S rRNA gene copies in the supernatant liquid relative to the total number in the bioaerosol sample. During aerosolization by a Collison nebulizer, the ID of E. coli and B. atrophaeus in the nebulizer suspension gradually increased during 60 min of continuous aerosolization. We found that the ID of bacteria during aerosolization was statistically significantly affected by the material of the Collison jar (glass > polycarbonate; P < 0.001) and by the bacterial species (E. coli > B. atrophaeus; P < 0.001). When E. coli was collected for 5 min by filtration, impaction, and impingement, its ID values were within the following ranges: 0.051 to 0.085, 0.16 to 0.37, and 0.068 to 0.23, respectively; when it was collected by electrostatic precipitation, the ID values (0.011 to 0.034) were significantly lower (P < 0.05) than those with other sampling methods. Air samples collected inside an equine facility for 2 h by filtration and impingement exhibited ID values in the range of 0.30 to 0.54. The data indicate that the amount of cell damage during bioaerosol sampling and the resulting release of DNA can be substantial and that this should be taken into account when analyzing bioaerosol samples. PMID:24096426

  10. Filtering with Electric Field: The Case of E. coli Porins.

    PubMed

    Acosta-Gutierrez, Silvia; Scorciapino, Mariano Andrea; Bodrenko, Igor; Ceccarelli, Matteo

    2015-05-21

    Although the role of general bacterial porins is well established as main pathway for polar antibiotics, the molecular details of their mode-of-action are still under debate. Using molecular dynamics simulations and water as a probe, we demonstrated the strong ordering of water molecules, differently tuned along the axis of diffusion in the transversal direction. Preserved features and important differences were characterized for different channels, allowing to put forward a general model for molecular filtering. The intrinsic electric field, responsible for water ordering, (i) filters those dipolar molecules that can compensate the entropy decrease by dipole alignment in the restricted region and (ii) might create an additional barrier by changing direction when escaping from the restricted region. We tested this model using two antibiotics, cefepime and cefotaxime, through metadynamics free energy calculations. A rational drug design should take this into account for screening molecules with improved permeation properties.

  11. A 3-dimensional trimeric β-barrel model for Chlamydia MOMP contains conserved and novel elements of Gram-negative bacterial porins.

    PubMed

    Feher, Victoria A; Randall, Arlo; Baldi, Pierre; Bush, Robin M; de la Maza, Luis M; Amaro, Rommie E

    2013-01-01

    Chlamydia trachomatis is the most prevalent cause of bacterial sexually transmitted diseases and the leading cause of preventable blindness worldwide. Global control of Chlamydia will best be achieved with a vaccine, a primary target for which is the major outer membrane protein, MOMP, which comprises ~60% of the outer membrane protein mass of this bacterium. In the absence of experimental structural information on MOMP, three previously published topology models presumed a16-stranded barrel architecture. Here, we use the latest β-barrel prediction algorithms, previous 2D topology modeling results, and comparative modeling methodology to build a 3D model based on the 16-stranded, trimeric assumption. We find that while a 3D MOMP model captures many structural hallmarks of a trimeric 16-stranded β-barrel porin, and is consistent with most of the experimental evidence for MOMP, MOMP residues 320-334 cannot be modeled as β-strands that span the entire membrane, as is consistently observed in published 16-stranded β-barrel crystal structures. Given the ambiguous results for β-strand delineation found in this study, recent publications of membrane β-barrel structures breaking with the canonical rule for an even number of β-strands, findings of β-barrels with strand-exchanged oligomeric conformations, and alternate folds dependent upon the lifecycle of the bacterium, we suggest that although the MOMP porin structure incorporates canonical 16-stranded conformations, it may have novel oligomeric or dynamic structural changes accounting for the discrepancies observed.

  12. 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.

  13. Diisopropylfluorophosphate Impairs the Transport of Membrane-Bound Organelles in Rat Cortical Axons.

    PubMed

    Gao, Jie; Naughton, Sean X; Wulff, Heike; Singh, Vikrant; Beck, Wayne D; Magrane, Jordi; Thomas, Bobby; Kaidery, Navneet Ammal; Hernandez, Caterina M; Terry, Alvin V

    2016-03-01

    The extensive use of organophosphates (OPs) is an ongoing environmental health concern due to multiple reports of OP-related neurologic abnormalities. The mechanism of the acute toxicity of OPs has been attributed to inhibition of acetylcholinesterase (AChE), but there is growing evidence that this may not account for all the long-term neurotoxic effects of OPs. In previous experiments (using ex vivo and in vitro model systems) we observed that the insecticide OP chlorpyrifos impaired the movements of vesicles and mitochondria in axons. Here, using a time-lapse imaging technique, we evaluated the OP-nerve agent diisopropylfluorophosphate (DFP) across a wide range of concentrations (subnanomolar to micromolar) for effects on fast axonal transport of membrane-bound organelles (MBOs) that contain the amyloid precursor protein (APP) tagged with the fluorescent marker Dendra2 (APPDendra2). Both 1 and 24 hours of exposure to DFP and a positive control compound, colchicine, resulted in a decrease in the velocity of anterograde and retrograde movements of MBOs and an increase in the number of stationary MBOs. These effects occurred at picomolar (100 pM) to low nanomolar (0.1 nM) concentrations that were not associated with compromised cell viability or cytoskeletal damage. Moreover, the effects of DFP on axonal transport occurred at concentrations that did not inhibit AChE activity, and they were not blocked by cholinergic receptor antagonists. Given the fundamental importance of axonal transport to neuronal function, these observations may explain some of the long-term neurologic deficits that have been observed in humans who have been exposed to OPs. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  14. Diisopropylfluorophosphate Impairs the Transport of Membrane-Bound Organelles in Rat Cortical Axons

    PubMed Central

    Gao, Jie; Naughton, Sean X.; Wulff, Heike; Singh, Vikrant; Beck, Wayne D.; Magrane, Jordi; Thomas, Bobby; Kaidery, Navneet Ammal; Hernandez, Caterina M.

    2016-01-01

    The extensive use of organophosphates (OPs) is an ongoing environmental health concern due to multiple reports of OP-related neurologic abnormalities. The mechanism of the acute toxicity of OPs has been attributed to inhibition of acetylcholinesterase (AChE), but there is growing evidence that this may not account for all the long-term neurotoxic effects of OPs. In previous experiments (using ex vivo and in vitro model systems) we observed that the insecticide OP chlorpyrifos impaired the movements of vesicles and mitochondria in axons. Here, using a time-lapse imaging technique, we evaluated the OP-nerve agent diisopropylfluorophosphate (DFP) across a wide range of concentrations (subnanomolar to micromolar) for effects on fast axonal transport of membrane-bound organelles (MBOs) that contain the amyloid precursor protein (APP) tagged with the fluorescent marker Dendra2 (APPDendra2). Both 1 and 24 hours of exposure to DFP and a positive control compound, colchicine, resulted in a decrease in the velocity of anterograde and retrograde movements of MBOs and an increase in the number of stationary MBOs. These effects occurred at picomolar (100 pM) to low nanomolar (0.1 nM) concentrations that were not associated with compromised cell viability or cytoskeletal damage. Moreover, the effects of DFP on axonal transport occurred at concentrations that did not inhibit AChE activity, and they were not blocked by cholinergic receptor antagonists. Given the fundamental importance of axonal transport to neuronal function, these observations may explain some of the long-term neurologic deficits that have been observed in humans who have been exposed to OPs. PMID:26718240

  15. Ultrastructural organization of chloroplast membranes in mutants of Pisum sativum L. with impaired activity in the photosystems.

    PubMed

    Popov, V I; Matorin, D N; Gostimsky, S V; Tageeva, S V; Allakhverdov, B L

    1981-05-01

    The ultrastructural organization and the photosynthesis reactions of chloroplast membranes were studied in three lethal mutants of Pisum sativum, Chl-1, Chl-19 and Chl-5, all lacking the capacity to evolve oxygen. The rates of 2,6-dichloroindophenol reduction, delayed fluorescence and electron-spin-resonance signal 1 indicate that Chl-1 and Chl-19 have an impaired activity in photosystem II (PS II), while in Chl-5 the electron transport is blocked between PS I and the reactions of CO2 fixation. Ultrathin sectioning demonstrates the presence of giant grana in the chloroplasts of Chl-1 and Chl-19, while the chloroplast structure of the Chl-5 is very similar to that of the wild-type. The grana of the Chl-19 mutant contain large multilamellar regions of tightly packed membranes. When the chloroplast membranes were studied by freeze-fracture, the exoplasmic and protoplasmic fracture faces (EF and PF, respectively) in both stacked and unstacked membranes were found to show large differences in particle concentrations and relative population area (per μm(2)), and also in particle size distribution, between all mutant chloroplast membranes and the wild-type. A close correlation between increasing kmt (ratio of particle concentrations on PF/EF) and PS II activity was observed. The differences in particle concentrations on both fracture faces in different regions of the intact chloroplast membranes of the wild-type are the consequence of a rearrangement of existing membrane components by lateral particle movements since quantitative measurements demonstrate almost complete conservation of intramembrane particles in number and size during the stacking of stroma thylakoid membranes. The results indicating particle movements strongly support the concept that the chloroplast membranes have a highly dynamic structure.

  16. The Sinorhizobium meliloti essential porin RopA1 is a target for numerous bacteriophages.

    PubMed

    Crook, Matthew B; Draper, Alicia L; Guillory, R Jordan; Griffitts, Joel S

    2013-08-01

    The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti harbors a gene, SMc02396, which encodes a predicted outer membrane porin that is conserved in many symbiotic and pathogenic bacteria in the order Rhizobiales. Here, this gene (renamed ropA1) is shown to be required for infection by two commonly utilized transducing bacteriophages (ΦM12 and N3). Mapping of S. meliloti mutations conferring resistance to ΦM12, N3, or both phages simultaneously revealed diverse mutations mapping within the ropA1 open reading frame. Subsequent tests determined that RopA1, lipopolysaccharide, or both are required for infection by all of a larger collection of Sinorhizobium-specific phages. Failed attempts to disrupt or delete ropA1 suggest that this gene is essential for viability. Phylogenetic analysis reveals that ropA1 homologs in many Rhizobiales species are often found as two genetically linked copies and that the intraspecies duplicates are always more closely related to each other than to homologs in other species, suggesting multiple independent duplication events.

  17. 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.

  18. Positive regulation of the Vibrio cholerae porin OmpT by iron and fur.

    PubMed

    Craig, S A; Carpenter, C D; Mey, A R; Wyckoff, E E; Payne, S M

    2011-12-01

    The transcription factor Fur regulates the expression of a number of genes in Vibrio cholerae in response to changes in the level of available iron. Fur usually acts as a repressor, but here we show that Fur positively regulates the expression of ompT, which encodes a major outer membrane porin. OmpT levels increased when the bacteria were grown in medium containing relatively high levels of iron, and this effect required Fur. The level of ompT mRNA also is increased in the presence of iron and Fur. The effect of iron on OmpT levels was independent of the known ompT regulators ToxR and Crp, and it did not require RyhB, which has been shown to be responsible for positive regulation by iron of some V. cholerae genes. Electrophoretic mobility shift assays showed that Fur binds upstream of the ompT transcription start site in a region overlapping known binding sites for ToxR and Crp. These data suggest that Fur and iron positively regulate ompT expression through the direct binding of Fur to the ompT promoter.

  19. 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

  20. Genetic analysis of Arabidopsis mutants impaired in plastid lipid import reveals a role of membrane lipids in chloroplast division.

    PubMed

    Fan, Jilian; Xu, Changcheng

    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. :

  1. 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.

  2. Immunization with major outer membrane proteins in experimental salmonellosis of mice.

    PubMed Central

    Kuusi, N; Nurminen, M; Saxen, H; Valtonen, M; Mäkelä, P H

    1979-01-01

    Porin (outer membrane protein) preparations extracted from a rough (Rb2) mutant of Salmonella typhimurium proved to be good immunogens in mice and rabbits. The antibody response achieved was measured by using enzyme-linked immunosorbent assay techniques. High titers of both antiporin and antilipopolysaccharide were detected in both species. The rabbit antiserum raised against the porins and the porin preparations themselves had a highly significant protective capacity against intraperitoneal Salmonella infection of mice. Absorption of the rabbit antiporin serum with lipopolysaccharide immunosorbent did not change its protective capacity in a passive immunization experiment, suggesting that the antiporin antibody preparations were the active components. Images PMID:387596

  3. Extracellular secretion of the Borrelia burgdorferi Oms28 porin and Bgp, a glycosaminoglycan binding protein.

    PubMed

    Cluss, Robert G; Silverman, Damon A; Stafford, Thomas R

    2004-11-01

    Borrelia burgdorferi, the Lyme disease pathogen, cycles between its Ixodes tick vector and vertebrate hosts, adapting to vastly different biochemical environments. Spirochete gene expression as a function of temperature, pH, growth phase, and host milieu is well studied, and recent work suggests that regulatory networks are involved. Here, we examine the release of Borrelia burgdorferi strain B31 proteins into conditioned medium. Spirochetes intrinsically radiolabeled at concentrations ranging from 10(7) to 10(9) cells per ml secreted Oms28, a previously characterized outer membrane porin, into RPMI medium. As determined by immunoblotting, this secretion was not associated with outer membrane blebs or cytoplasmic contamination. A similar profile of secreted proteins was obtained for spirochetes radiolabeled in mixtures of RPMI medium and serum-free Barbour-Stoenner-Kelly (BSK II) medium. Proteomic liquid chromatography-tandem mass spectrometry analysis of tryptic fragments derived from strain B31 culture supernatants confirmed the identity of the 28-kDa species as Oms28 and revealed a 26-kDa protein as 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs-2), previously described as Bgp, a glycosaminoglycan-binding protein. The release of Oms28 into the culture medium is more selective when the spirochetes are in logarithmic phase of growth compared to organisms obtained from stationary phase. As determined by immunoblotting, stationary-phase spirochetes released OspA, OspB, and flagellin. Oms28 secreted by strains B31, HB19, and N40 was also recovered by radioimmunoprecipitation. This is the first report of B. burgdorferi protein secretion into the extracellular environment. The possible roles of Oms28 and Bgp in the host-pathogen interaction are considered.

  4. Extracellular Secretion of the Borrelia burgdorferi Oms28 Porin and Bgp, a Glycosaminoglycan Binding Protein

    PubMed Central

    Cluss, Robert G.; Silverman, Damon A.; Stafford, Thomas R.

    2004-01-01

    Borrelia burgdorferi, the Lyme disease pathogen, cycles between its Ixodes tick vector and vertebrate hosts, adapting to vastly different biochemical environments. Spirochete gene expression as a function of temperature, pH, growth phase, and host milieu is well studied, and recent work suggests that regulatory networks are involved. Here, we examine the release of Borrelia burgdorferi strain B31 proteins into conditioned medium. Spirochetes intrinsically radiolabeled at concentrations ranging from 107 to 109 cells per ml secreted Oms28, a previously characterized outer membrane porin, into RPMI medium. As determined by immunoblotting, this secretion was not associated with outer membrane blebs or cytoplasmic contamination. A similar profile of secreted proteins was obtained for spirochetes radiolabeled in mixtures of RPMI medium and serum-free Barbour-Stoenner-Kelly (BSK II) medium. Proteomic liquid chromatography-tandem mass spectrometry analysis of tryptic fragments derived from strain B31 culture supernatants confirmed the identity of the 28-kDa species as Oms28 and revealed a 26-kDa protein as 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs-2), previously described as Bgp, a glycosaminoglycan-binding protein. The release of Oms28 into the culture medium is more selective when the spirochetes are in logarithmic phase of growth compared to organisms obtained from stationary phase. As determined by immunoblotting, stationary-phase spirochetes released OspA, OspB, and flagellin. Oms28 secreted by strains B31, HB19, and N40 was also recovered by radioimmunoprecipitation. This is the first report of B. burgdorferi protein secretion into the extracellular environment. The possible roles of Oms28 and Bgp in the host-pathogen interaction are considered. PMID:15501754

  5. Th1 and Th2 cell involvement in immune response to Salmonella typhimurium porins.

    PubMed Central

    Galdiero, M; De Martino, L; Marcatili, A; Nuzzo, I; Vitiello, M; Cipollaro de l'Ero, G

    1998-01-01

    In understanding the regulation of the specific immune response to Salmonella typhimurium, the role of a surface major component (porins) was studied. In this study we demonstrate that purified porins are able to induce a different response to that induced by the porins present on the S. typhimurium cell surface. Porin-treated or orally infected mice show anti-porin antibodies with bactericidal activity. The complete adoptive transfer of resistance to S. typhimurium is achieved only using splenic T cells from survivor mice after experimental infection. After stimulation with specific antigen in vitro CD4+ cells from porin-immunized mice released large amounts of interleukin-4 (IL-4), at a time when CD4+ cells from S. typhimurium-infected mice predominantly secreted interferon-gamma (IFN-gamma). Limiting dilution analysis showed that infection resulted in a higher precursor frequency of IFN-gamma-producing CD4+ T cells and a lower precursor frequency of IL-4-producing CD4+ T cells, while immunization with porins resulted in a higher precursor frequency of IL-4-producing cells and a low frequency of IFN-gamma-producing cells. Analysis of polymerase chain reaction-amplified cDNA from the spleens of infected mice revealed that IFN-gamma, IL-2 and IL-12 p40 mRNA were found 5 days after in vitro challenge and increased after 15 days; IL-10 expression was barely present after both 5 and 15 days, while IL-4 mRNA expression was not detected. In immunized mice, the IL-4 mRNA expression increased after 15 days, IFN-gamma mRNA expression disappeared entirely after 15 days, while IL-2, IL-10 and IL-12 mRNA remained relatively unchanged. Images Figure 1 Figure 8 PMID:9708180

  6. Human airway epithelial cell responses to Neisseria lactamica and purified porin via Toll-like receptor 2-dependent signaling.

    PubMed

    Liu, Xiuping; Wetzler, Lee M; Nascimento, Laura Oliveira; Massari, Paola

    2010-12-01

    The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin's surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.

  7. A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD

    PubMed Central

    Fröhlich, Kathrin S.; Papenfort, Kai; Berger, Allison A.; Vogel, Jörg

    2012-01-01

    A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, σS (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by σS. We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3′-RACE, an experimental approach that promises to be of use in predicting other sRNA–target interactions in bacteria. PMID:22180532

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

    PubMed Central

    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

  9. Impaired Geranylgeranyltransferase-I Regulation Reduces Membrane-Associated Rho-Protein Levels in Aged Mouse Brain

    PubMed Central

    Afshordel, Sarah; Wood, W. Gibson; Igbavboa, Urule; Muller, Walter E.; Eckert, Gunter P.

    2014-01-01

    Synaptic impairment rather than neuronal loss may be the leading cause of cognitive dysfunction in brain aging. Certain small Rho-GTPases are involved in synaptic plasticity, and their dysfunction is associated with brain aging and neurodegeneration. Rho-GTPases undergo prenylation by attachment of geranylgeranylpyrophosphate (GGPP) catalyzed by GGTase-I. We examined age-related changes in the abundance of Rho- and Rab proteins in membrane and cytosolic fractions as well as of GGTase-I in brain tissue of 3 and 23 mo old C57BL/6 mice. We report a shift in the cellular localization of Rho-GTPases towards reduced levels of membrane-associated and enhanced cytosolic levels of those proteins in aged mouse brain as compared with younger mice. The age-related reduction of membrane-associated Rho-proteins was associated with a reduction in GGTase-I levels that regulates binding of GGPP to Rho-GTPases. Proteins prenylated by GGTase-II were not reduced in aged brain indicating a specific targeting of GGTase-I in the aged brain. Inhibition of GGTase-I in vitro modeled the effects of aging we observed in vivo. We demonstrate for the first time a decrease of membrane-associated Rho proteins in aged brain in association with down-regulation of GGTase-I. This down-regulation could be one of the mechanisms causing age-related weakening of synaptic plasticity. PMID:24428713

  10. Trends of the major porin gene (ompF) evolution: insight from the genus Yersinia.

    PubMed

    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.

  11. Lymphocytic proliferative response to outer-membrane proteins isolated from Salmonella.

    PubMed

    González, C R; Isibasi, A; Ortiz-Navarrete, V; Paniagua, J; García, J A; Blanco, F; Kumate, J

    1993-01-01

    Porins isolated from Salmonella typhi have been demonstrated to protect against the challenge with this bacteria in mice. The mechanism has not been clarified, but could be associated with activation of both humoral and cellular immunity. In order to evaluate the induction of specific T cell responses, the lymphocytic proliferation to porins isolated from Salmonella typhimurium, Salmonella typhi and Escherichia coli was examined by 3H-thymidine incorporation assay in mice immunized with three different antigens: acetone-killed S. typhimurium, its porins, or outer-membrane proteins (OMPs) isolated from S. typhi. Higher proliferative responses were observed in mice immunized with porins and OMPs compared with those which received the acetone-killed bacteria. Although cross-reactivity was observed between porins, they were not mitogenic. Moreover, porins were able to activate T lymphocytes isolated from mice immunized with S. typhi OMPs. These results suggest that T cell activation, through the release of lymphokines, may play a role in the induction of protective immunity with porins.

  12. Carbapenem and cefoxitin resistance of Klebsiella pneumoniae strains associated with porin OmpK36 loss and DHA-1 β-lactamase production.

    PubMed

    Shi, Weifeng; Li, Kun; Ji, Yun; Jiang, Qinbo; Wang, Yuyue; Shi, Mei; Mi, Zuhuang

    2013-01-01

    Clinical isolates of carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) strains are being increased worldwide. Five pan-resistant K. pneumoniae strains have been isolated from respiratory and ICU wards in a Chinese hospital, and reveal strong resistance to all β-lactams, fluoroquinolones and aminoglycosides. Totally 27 β-lactamase genes and 2 membrane pore protein (porin) genes in 5 K. pneumoniae strains were screened by polymerase chain reaction (PCR). The results indicated that all of 5 K. pneumoniae strains carried blaTEM-1 and blaDHA-1 genes, as well as base deletion and mutation of OmpK35 or OmpK36 genes. Compared with carbapenem-sensitive isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the resistant isolates markedly lacked the protein band of 34-40 kDa, which might be the outer membrane proteins of OmpK36 according to the electrophoresis mobility. In addition, the conjugation test was confirmed that blaDHA-1 mediated by plasmids could be transferred between resistant and sensitive strains. When reserpine (30 μg/mL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 μg/mL) were added in imipenem and meropenem, the MICs had no change against K. pneumoniae strains. These results suggest that both DHA-1 β-lactamase and loss or deficiency of porin OmpK36 may be the main reason for the cefoxitin and carbapenem resistance in K. pneumoniae strains in our hospital.

  13. Characterizing pathology in erythrocytes using morphological and biophysical membrane properties: Relation to impaired hemorheology and cardiovascular function in rheumatoid arthritis.

    PubMed

    Olumuyiwa-Akeredolu, Oore-Ofe O; Soma, Prashilla; Buys, Antoinette V; Debusho, Legesse Kassa; Pretorius, Etheresia

    2017-09-14

    The inflammatory burden of the complex Rheumatoid Arthritis (RA) disease affects several organ-systems, including rheological properties of blood and its formed elements. Red blood cells (RBCs) are constantly exposed to circulating dysregulated inflammatory molecules that are co-transported within the vasculature; and their membranes may be particularly vulnerable to the accompanying oxidative stress. In the current study, we investigate biophysical and ultrastructural characteristics of RBCs obtained from a cohort of patients using atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy (CM). Statistical analyses of AFM data showed that RA RBCs possessed significantly reduced membrane elasticity relative to that of RBCs from healthy individuals (P-value <0.0001). SEM imaging of RA RBCs revealed increased anisocytes and poikilocytes. Poikilocytes included knizocytes, stomatocytes, dacryocytes, irregularly contracted cells, and knot cells. CM imaging of several RA RBCs, spectrin, and band 3 protein networks portrayed the similar morphological profiles. Analyses of CM images confirmed changes to distribution of band-3 skeletal protein, a protein critical for gaseous exchange functions of the RBC and preventing membrane surface loss. Decreased membrane deformability impairs the RBC's capacity to adequately adapt its shape to navigate blood vessels, especially microvasculature, and this decrease is also reflected in the cell's morphology. Changes to morphology and deformability may also indicate loss of functional domains and/or pathological protein and lipid associations. These findings suggest that RA disease and/or its concomitant factors impact on the RBC and its membrane integrity with potential for exacerbating pathological cellular function, hemorheology, and cardiovascular function. Copyright © 2017. Published by Elsevier B.V.

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

    PubMed

    Sharbati, Soroush; Schramm, Kira; Rempel, Sonja; Wang, Hwa; Andrich, Ronny; Tykiel, Verena; Kunisch, Ralph; Lewin, Astrid

    2009-02-09

    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. 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. This study demonstrates the important role of porin expression in growth as well as antibiotic susceptibility of the opportunistic bacterium M. fortuitum.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Study of the protein complex, pore diameter, and pore-forming activity of the Borrelia burgdorferi P13 porin.

    PubMed

    Bárcena-Uribarri, Iván; Thein, Marcus; Barbot, Mariam; Sans-Serramitjana, Eulalia; Bonde, Mari; Mentele, Reinhard; Lottspeich, Friedrich; Bergström, Sven; Benz, Roland

    2014-07-04

    P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 m KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ±100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Study of the Protein Complex, Pore Diameter, and Pore-forming Activity of the Borrelia burgdorferi P13 Porin*

    PubMed Central

    Bárcena-Uribarri, Iván; Thein, Marcus; Barbot, Mariam; Sans-Serramitjana, Eulalia; Bonde, Mari; Mentele, Reinhard; Lottspeich, Friedrich; Bergström, Sven; Benz, Roland

    2014-01-01

    P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 m KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ±100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi. PMID:24825899

  4. Ethylhexylglycerin Impairs Membrane Integrity and Enhances the Lethal Effect of Phenoxyethanol

    PubMed Central

    Langsrud, Solveig; Steinhauer, Katrin; Lüthje, Sonja; Weber, Klaus; Goroncy-Bermes, Peter; Holck, Askild L.

    2016-01-01

    Preservatives are added to cosmetics to protect the consumers from infections and prevent product spoilage. The concentration of preservatives should be kept as low as possible and this can be achieved by adding potentiating agents. The aim of the study was to investigate the mechanisms behind potentiation of the bactericidal effect of a commonly used preservative, 2-phenoxyethanol (PE), by the potentiating agent ethylhexylglycerin (EHG). Sub-lethal concentrations of EHG (0.075%) and PE (0.675%) in combination led to rapid killing of E. coli (> 5 log reduction of cfu after 30 min), leakage of cellular constituents, disruption of the energy metabolism, morphological deformities of cells and condensation of DNA. Used alone, EHG disrupted the membrane integrity even at low concentrations. In conclusion, sub-lethal concentrations of EHG potentiate the effect of PE through damage of the cell membrane integrity. Thus, adding EHG to PE in a 1:9 ratio has a similar effect on membrane damage and bacterial viability as doubling the concentration of PE. This study provides insight about the mechanism of action of a strong potentiating agent, EHG, which is commonly used in cosmetics together with PE. PMID:27783695

  5. 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.

  6. 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

  7. Effectiveness of natural and synthetic complexes of porin and O polysaccharide as vaccines against Brucella abortus in mice.

    PubMed Central

    Winter, A J; Rowe, G E; Duncan, J R; Eis, M J; Widom, J; Ganem, B; Morein, B

    1988-01-01

    A single vaccination of mice with a complex of porin and smooth lipopolysaccharide (porin-S-LPS) extracted from virulent Brucella abortus 2308 provided significant protection (P less than 0.01 to P less than 0.001) against challenge with the same strain, equivalent to that achieved by vaccination with living attenuated B. abortus 19. The porin-S-LPS vaccine given without adjuvant or in several adjuvants (trehalose dimycolate and muramyl dipeptide; the pluronic polymer L-121 and muramyl dipeptide; or complexed with Quil A in immunostimulating complexes) provided equivalent protection. In contrast, one vaccination with porin complexed with rough LPS (porin-R-LPS) from a rough mutant of strain 2308 provided no protection with any adjuvant tested. In one experiment, two inoculations with the porin-R-LPS resulted in a low level of protection, probably owing to priming of the animals for production of O-polysaccharide-specific antibodies. However, one vaccination with rough-strain porin covalently bound to purified O polysaccharide conferred protection equal to that obtained with natural complexes of porin-S-LPS or with living strain 19. A synthetic vaccine containing long chains of O polysaccharide was more effective than one prepared with short chains. Protective vaccines caused the formation of increased concentrations of circulating O-polysaccharide-specific antibodies, although there were individual exceptions to the quantitative association between O-polysaccharide-specific antibodies and protection. Antibodies specific for porin or R-LPS were found in negligible quantities in vaccinated mice. These results provide additional evidence that the O polysaccharide will constitute an essential component of an effective subcellular vaccine against B. abortus and that O-polysaccharide-specific antibodies play an important role in protective immunity in brucellosis. PMID:2844673

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

    PubMed

    Norkin, L C

    1977-03-01

    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.

  9. 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

  10. Rabies virus infection selectively impairs membrane receptor functions in neuronal model cells.

    PubMed

    Koschel, K; Halbach, M

    1979-03-01

    A persistent infection with rabies virus (HEP-Flury) was established in the CNS-derived hybrid cell line 108CC15 which possesses specific membrane receptors for prostaglandins, catecholamines and acetylcholine. We report a differential virus influence on the specific receptor response to PGE, isoproterenol and acetycholine as indicated by typical changes of the intracellular cyclic AMP levels. As the adenylate cyclase activity was unchanged in infected cells in vitro, a selective virus influence on specific receptors themselves or their coupling to the cAMP synthesizing system must be considered.

  11. Interaction of the antibiotic minocycline with liver mitochondria - role of membrane permeabilization in the impairment of respiration.

    PubMed

    Schönfeld, Peter; Siemen, Detlef; Kreutzmann, Peter; Franz, Claudia; Wojtczak, Lech

    2013-12-01

    Several studies have proposed that the antibiotic minocycline (MC) has cytoprotective activities. Nevertheless, when cells have been exposed to MC at micromolar concentrations, detrimental effects have been also observed. Despite the known inhibitory activity of MC on ATP synthesis and the Ca(2+) retention capacity of isolated rat liver and brain mitochondria, the underlying mechanism is still debated. Here, we present further arguments supporting our concept that MC acting on rat liver mitochondria suspended in KCl medium permeabilizes the inner membrane. Supplementation of the medium with cytochrome c and NAD(+) strongly enhanced the respiration of MC-treated mitochondria, thus partly preventing or reversing the inhibitory effect of MC on state 3 or uncoupled respiration. These results indicate that MC produced depletion of mitochondrial cytochrome c and NAD(+) , thus impairing mitochondrial respiration. In addition, NADH oxidation by alamethicin-permeabilized mitochondria supplemented with cytochrome c was insensitive to 200 μm MC, arguing against direct impairment of respiratory chain complexes by MC. Finally, a surprising feature of MC was its accumulation or binding by intact rat liver mitochondria, but not by mitochondria permeabilized with alamethicin or disrupted by freezing and thawing. © 2013 FEBS.

  12. 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

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

    PubMed

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

    2011-12-01

    Mitochondrial dysfunction plays an important role in mediating ischemic brain damage. Immp2l is an inner mitochondrial membrane peptidase that processes mitochondrial protein cytochrome c1 (Cyc1). Homozygous mutation of Immp2l (Immp2l(Tg(Tyr)979Ove) or Immp2l(-/-)) elevates mitochondrial membrane potential, increases superoxide (O(2)(-)) production in the brain and impairs fertility. The objectives of this study are to explore the effects of heterozygous mutation of Immp2l (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 O(2)(-) 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 Immp2l deficiency increases ischemic brain damage by enhancing O(2)(-) production and damaging mitochondrial functional performance. Published by Elsevier Inc.

  14. Brucella suis-Impaired Specific Recognition of Phagosomes by Lysosomes due to Phagosomal Membrane Modifications

    PubMed Central

    Naroeni, Aroem; Jouy, Nicolas; Ouahrani-Bettache, Safia; Liautard, Jean-Pierre; Porte, Françoise

    2001-01-01

    Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments. PMID:11119541

  15. 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.

  16. Cell membrane damage is involved in the impaired survival of bone marrow stem cells by oxidized low-density lipoprotein.

    PubMed

    Li, Xin; Xiao, Yuan; Cui, Yuqi; Tan, Tao; Narasimhulu, Chandrakala A; Hao, Hong; Liu, Lingjuan; Zhang, Jia; He, Guanglong; Verfaillie, Catherine M; Lei, Minxiang; Parthasarathy, Sampath; Ma, Jianjie; Zhu, Hua; Liu, Zhenguo

    2014-12-01

    Cell therapy with bone marrow stem cells (BMSCs) remains a viable option for tissue repair and regeneration. A major challenge for cell therapy is the limited cell survival after implantation. This study was to investigate the effect of oxidized low-density lipoprotein (ox-LDL, naturally present in human blood) on BMSC injury and the effect of MG53, a tissue repair protein, for the improvement of stem cell survival. Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox-LDL, which caused significant cell death as reflected by the increased LDH release to the media. Exposure of MAPCs to ox-LDL led to entry of fluorescent dye FM1-43 measured under confocal microscope, suggesting damage to the plasma membrane. Ox-LDL also generated reactive oxygen species (ROS) as measured with electron paramagnetic resonance spectroscopy. While antioxidant N-acetylcysteine completely blocked ROS production from ox-LDL, it failed to prevent ox-LDL-induced cell death. When MAPCs were treated with the recombinant human MG53 protein (rhMG53) ox-LDL induced LDH release and FM1-43 dye entry were significantly reduced. In the presence of rhMG53, the MAPCs showed enhanced cell survival and proliferation. Our data suggest that membrane damage induced by ox-LDL contributed to the impaired survival of MAPCs. rhMG53 treatment protected MAPCs against membrane damage and enhanced their survival which might represent a novel means for improving efficacy for stem cell-based therapy for treatment of diseases, especially in setting of hyperlipidemia.

  17. Loss of gamma-secretase function impairs endocytosis of lipoprotein particles and membrane cholesterol homeostasis.

    PubMed

    Tamboli, Irfan Y; Prager, Kai; Thal, Dietmar R; Thelen, Karin M; Dewachter, Ilse; Pietrzik, Claus U; St George-Hyslop, Peter; Sisodia, Sangram S; De Strooper, Bart; Heneka, Michael T; Filippov, Mikhail A; Müller, Ulrike; van Leuven, Fred; Lütjohann, Dieter; Walter, Jochen

    2008-11-12

    Presenilins (PSs) are components of the gamma-secretase complex that mediates intramembranous cleavage of type I membrane proteins. We show that gamma-secretase is involved in the regulation of cellular lipoprotein uptake. Loss of gamma-secretase function decreased endocytosis of low-density lipoprotein (LDL) receptor. The decreased uptake of lipoproteins led to upregulation of cellular cholesterol biosynthesis by increased expression of CYP51 and enhanced metabolism of lanosterol. Genetic deletion of PS1 or transgenic expression of PS1 mutants that cause early-onset Alzheimer's disease led to accumulation of gamma-secretase substrates and mistargeting of adaptor proteins that regulate endocytosis of the LDL receptor. Consistent with decreased endocytosis of these receptors, PS1 mutant mice have elevated levels of apolipoprotein E in the brain. Thus, these data demonstrate a functional link between two major genetic factors that cause early-onset and late-onset Alzheimer's disease.

  18. [Fetal lung development on MRT. Normal course and impairment due to premature rupture of membranes].

    PubMed

    Kasprian, G; Brugger, P C; Helmer, H; Langer, M; Balassy, C; Prayer, D

    2006-02-01

    A well-organized interplay between many molecular factors as well as mechanical forces influence fetal lung development. At the end of this complex process a sufficiently sized and structurally mature organ should ensure the postnatal survival of the newborn. Besides prenatal ultrasonography, magnetic resonance imaging (MRI) can now be used to investigate normal and pathological human lung growth in utero. Oligohydramnios, due to premature rupture of membranes (PROM), is an important risk factor for compromised fetal lung growth. In these situations MR volumetry can be used to measure the size of the fetal lung quite accurately. Together with the evaluation of lung signal intensities on T2-weighted sequences, fetuses with pulmonary hypoplasia can be readily detected.

  19. Antigenic determinants of the OmpC porin from Salmonella typhimurium.

    PubMed Central

    Singh, S P; Singh, S R; Williams, Y U; Jones, L; Abdullah, T

    1995-01-01

    The antigenic determinants of Salmonella typhimurium OmpC were investigated by the analysis of cyanogen bromide (CNBr)-generated porin peptides with antiporin monoclonal antibodies (MAbs). We identified six bands (f1 to f6) with estimated molecular masses of 35.5, 31.0, 25.0, 22.5, 13.8, and 10.0 kDa, respectively. In addition, two small fragments (f7 and f8; 3.0 to 6.0 kDa) were detected only infrequently. The OmpC monomer or its CNBr-generated peptides were electrophoretically transferred to a polyvinylidene difluoride membrane and then subjected to amino acid composition analysis and N-terminal sequencing. A comparison of the amino acid composition data with known compositions of Escherichia coli and Salmonella typhi OmpC showed some differences; however, the amino acid sequences of 71 residues identified in S. typhimurium showed 88 and 98% identity with OmpC from E. coli and S. typhi, respectively. The screening of CNBr peptides with the 12 anti-(S. typhimurium) OmpC MAbs by Western blot (immunoblot), in conjunction with the prediction of the OmpC folding pattern based on the known three-dimensional structure of E. coli OmpF, showed that four MAbs reacted with surface-exposed epitopes on loops L2, L8, and L4 to L7, four MAbs reacted with a region in the eyelet structure on loop L3, and four MAbs reacted with the buried epitopes on transmembrane beta strands. The MAbs reacting with surface-exposed loops showed no cross-reaction with E. coli OmpC, whose sequence has diverged extensively from that of S. typhi and (probably) S. typhimurium OmpC only in regions of the externally exposed loops. In contrast, MAbs reacting with transmembrane beta strands, whose sequence is strongly conserved, showed strong cross-reaction with E. coli OmpC. These results show that comparison with the E. coli OmpF structure predicts the folding pattern of S. typhimurium OmpC rather accurately and that evolutionary divergence in sequences is confined to the external loops. The possible

  20. Role of Porins in the Susceptibility of Mycobacterium smegmatis and Mycobacterium chelonae to Aldehyde-Based Disinfectants and Drugs ▿

    PubMed Central

    Svetlíková, Zuzana; Škovierová, Henrieta; Niederweis, Michael; Gaillard, Jean-Louis; McDonnell, Gerald; Jackson, Mary

    2009-01-01

    Nosocomial outbreaks attributable to glutaraldehyde-resistant, rapidly growing mycobacteria are increasing. Here, evidence is provided that defects in porin expression dramatically increase the resistance of Mycobacterium smegmatis and Mycobacterium chelonae to glutaraldehyde and another aldehyde disinfectant, ortho-phthalaldehyde. Since defects in porin activity also dramatically increased the resistance of M. chelonae to drugs, there is thus some concern that the widespread use of glutaraldehyde and ortho-phthalaldehyde in clinical settings may select for drug-resistant bacteria. PMID:19581465

  1. Multiple kidney cysts in thin basement membrane disease with proteinuria and kidney function impairment

    PubMed Central

    Sevillano, Angel M.; Gutierrez, Eduardo; Morales, Enrique; Hernandez, Eduardo; Molina, Maria; Gonzalez, Ester; Praga, Manuel

    2014-01-01

    Background Some patients with thin basement membrane disease (TBMD) develop proteinuria, hypertension and different degrees of CKD, besides the persistent microhaematuria characteristic of the disease. Little is known about factors associated with this unfavourable outcome. Methods We reviewed clinical, pathological and radiological features of 32 patients with biopsy-proven TBMD. Patients were divided in two groups: those with persistent normal kidney function and negative or minimal proteinuria (n = 16) and those with persistent proteinuria >0.5 g/day (n = 16). Results Patients with proteinuria had a worse kidney function at baseline than those with negative proteinuria. Global or segmental glomerulosclerosis, together with interstitial fibrosis, was found in 37% of patients with proteinuria. All proteinuric patients were treated with renin–angiotensin system blockers. At the end of follow-up (198 months in proteinuric patients and 210 months in patients with negative proteinuria) the prevalence of hypertension was 68% in proteinuric patients (12% at baseline), compared with 12 and 6%, respectively, in non-proteinuric patients. A slow decline of renal function was observed in proteinuric patients, although no patient developed end-stage kidney disease. Ultrasound studies showed bilateral kidney cysts in nine patients (56%) with proteinuria. Cysts were bilateral and countless in six patients, and bilateral but with a limited number of cysts in the three remaining patients. No cysts were found in patients with negative proteinuria. Conclusions Some patients with TBMD develop hypertension, proteinuria and CKD. Multiple bilateral kidney cysts were found in a majority (56%) of these patients. Further studies are needed to investigate the pathogenesis and the influence on long-term outcome of this TBMD-associated multiple kidney cysts. PMID:25852885

  2. Association of resistin with impaired membrane fluidity of red blood cells in hypertensive and normotensive men: an electron paramagnetic resonance study.

    PubMed

    Tsuda, Kazushi

    2016-10-01

    Abnormalities in physical properties of the cell membranes may strongly be linked to hypertension. Recent evidence indicates that resistin may actively participate in the pathophysiology of insulin resistance, diabetes mellitus, hypertension and other circulatory disorders. The present study was undertaken to investigate the possible relationships among plasma resistin, oxidative stress and membrane fluidity (a reciprocal value of membrane microviscosity) in hypertension. We measured the membrane fluidity of red blood cells (RBCs) in hypertensive and normotensive men using an electron paramagnetic resonance (EPR) and spin-labeling method. The order parameter (S) for the spin-label agents (5-nitroxide stearate) in EPR spectra of red blood cell (RBC) membranes was significantly higher in hypertensive men than in normotensive men, indicating that membrane fluidity was decreased in hypertension. Plasma resistin levels were correlated with systolic blood pressure and 8-iso-prostaglandin F2α levels (an index of oxidative stress). Furthermore, the order parameter (S) of RBCs significantly correlated with plasma resistin and plasma 8-isoPG F2α, suggesting that reduced membrane fluidity of RBCs might be associated with hyperresistinemia and increased oxidative stress. Multivariate regression analysis showed that, after adjustment for confounding factors, plasma resistin might be an independent determinant of membrane fluidity of RBCs. The EPR study suggests that resistin might have a close correlation with impaired rheologic behavior of RBCs and microcirculatory dysfunction in hypertension, at least in part, via an oxidative stress-dependent mechanism.

  3. Haemophilus influenzae Porin Contributes to Signaling of the Inflammatory Cascade in Rat Brain

    PubMed Central

    Galdiero, Massimiliano; D'Amico, Michele; Gorga, Fernanda; Di Filippo, Clara; D'Isanto, Marina; Vitiello, Mariateresa; Longanella, Anna; Tortora, Annalisa

    2001-01-01

    In the present study we observed that the Haemophilus influenzae type b (Hib) porin, among the different surface bacterial components, is involved in the pathophysiology of bacterial meningitis. This study demonstrates that inoculation of Hib porin into the fourth cerebral ventricle causes the simultaneous expression of interleukin-1α (IL-1α), tumor necrosis factor alpha (TNF-α), and macrophage inflammatory protein 2 (MIP-2) at 6 h after inoculation. At 24 h, the expression of MIP-2 decreases while the expression of IL-1α and TNF-α increases. The mRNA expression of IL-1α, TNF-α, and MIP-2 is correlated with injury to the blood-brain barrier as demonstrated by the appearance of serum proteins and leukocytes in cerebrospinal fluid and by the increase in brain water content. PMID:11119509

  4. Characterization of a Novel Porin Involved in Systemic Yersinia enterocolitica Infection

    PubMed Central

    Mildiner-Earley, Shirly; Miller, Virginia L.

    2006-01-01

    Yersinia enterocolitica is an enteric pathogen capable of causing systemic disease in a murine model. We have identified a novel protein, systemic factor protein A (SfpA), conserved in other pathogenic bacteria that is involved in systemic disease. Analysis of bacterial colonization revealed that a ΔsfpA strain is defective in mesenteric lymph node colonization. Bioinformatics and functional studies suggest that SfpA is a porin. PMID:16790812

  5. 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.

  6. 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.

  7. The plasma membrane redox system is impaired by amyloid β-peptide and in the hippocampus and cerebral cortex of 3xTgAD mice.

    PubMed

    Hyun, Dong-Hoon; Mughal, Mohamed R; Yang, Hyunwon; Lee, Ji Hyun; Ko, Eun Joo; Hunt, Nicole D; de Cabo, Rafael; Mattson, Mark P

    2010-10-01

    Membrane-associated oxidative stress has been implicated in the synaptic dysfunction and neuronal degeneration that occurs in Alzheimer's disease (AD), but the underlying mechanisms are unknown. Enzymes of the plasma membrane redox system (PMRS) provide electrons for energy metabolism and recycling of antioxidants. Here, we show that activities of several PMRS enzymes are selectively decreased in plasma membranes from the hippocampus and cerebral cortex of 3xTgAD mice, an animal model of AD. Our results that indicate the decreased PMRS enzyme activities are associated with decreased levels of coenzyme Q(10) and increased levels of oxidative stress markers. Neurons overexpressing the PMRS enzymes (NQO1 or cytochrome b5 reductase) exhibit increased resistance to amyloid β-peptide (Aβ). If and to what extent Aβ is the cause of the impaired PMRS enzymes in the 3xTgAD mice is unknown. Because these mice also express mutant tau and presenilin-1, it is possible that one or more of the PMRS could be adversely affected by these mutations. Nevertheless, the results of our cell culture studies clearly show that exposure of neurons to Aβ1-42 is sufficient to impair PMRS enzymes. The impairment of the PMRS in an animal model of AD, and the ability of PMRS enzyme activities to protect neurons against Aβ-toxicity, suggest enhancement PMRS function as a novel approach for protecting neurons against oxidative damage in AD and related disorders. Copyright © 2010 Elsevier Inc. All rights reserved.

  8. Long term potentiation is impaired in membrane glycoprotein CD200-deficient mice: a role for Toll-like receptor activation.

    PubMed

    Costello, Derek A; Lyons, Anthony; Denieffe, Stephanie; Browne, Tara C; Cox, F Fionnuala; Lynch, Marina A

    2011-10-07

    The membrane glycoprotein CD200 is expressed on several cell types, including neurons, whereas expression of its receptor, CD200R, is restricted principally to cells of the myeloid lineage, including microglia. The interaction between CD200 and CD200R maintains microglia and macrophages in a quiescent state; therefore, CD200-deficient mice express an inflammatory phenotype exhibiting increased macrophage or microglial activation in models of arthritis, encephalitis, and uveoretinitis. Here, we report that lipopolysaccharide (LPS) and Pam(3)CysSerLys(4) exerted more profound effects on release of the proinflammatory cytokines, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNFα), in glia prepared from CD200(-/-) mice compared with wild type mice. This effect is explained by the loss of CD200 on astrocytes, which modulates microglial activation. Expression of Toll-like receptors 4 and 2 (TLR4 and -2) was increased in glia prepared from CD200(-/-) mice, and the evidence indicates that microglial activation, assessed by the increased numbers of CD11b(+) cells that stained positively for both MHCII and CD40, was enhanced in CD200(-/-) mice compared with wild type mice. These neuroinflammatory changes were associated with impaired long term potentiation (LTP) in CA1 of hippocampal slices prepared from CD200(-/-) mice. One possible explanation for this is the increase in TNFα in hippocampal tissue prepared from CD200(-/-) mice because TNFα application inhibited LTP in CA1. Significantly, LPS and Pam(3)CysSerLys(4), at concentrations that did not affect LTP in wild type mice, inhibited LTP in slices prepared from CD200(-/-) mice, probably due to the accompanying increase in TLR2 and TLR4. Thus, the neuroinflammatory changes that result from CD200 deficiency have a negative impact on synaptic plasticity.

  9. Effects of DHA-phospholipids, melatonin and tryptophan supplementation on erythrocyte membrane physico-chemical properties in elderly patients suffering from mild cognitive impairment.

    PubMed

    Cazzola, Roberta; Rondanelli, Mariangela; Faliva, Milena; Cestaro, Benvenuto

    2012-12-01

    A randomized, double-blind placebo-controlled clinical trial was carried out to assess the efficacy of a docosahexenoic acid (DHA)-phospholipids, melatonin and tryptophan supplemented diet in improving the erythrocyte oxidative stress, membrane fluidity and membrane-bound enzyme activities of elderly subjects suffering from mild cognitive impairment (MCI). These subjects were randomly assigned to the supplement group (11 subjects, 9F and 2M; age 85.3±5.3y) or placebo group (14-matched subjects, 11F and 3M; 86.1±6.5). The duration of the treatment was 12weeks. The placebo group showed no significant changes in erythrocyte membrane composition and function. The erythrocyte membranes of the supplement group showed a significant increase in eicosapentenoic acid, docosapentenoic acid and DHA concentrations and a significant decrease in arachidonic acid, malondialdehyde and lipofuscin levels. These changes in membrane composition resulted in an increase in the unsaturation index, membrane fluidity and acetylcholine esterase activity. Moreover, a significant increase in the ratio between reduced and oxidized glutathione was observed in the erythrocyte of the supplement group. Although this study is a preliminary investigation, we believe these findings to be of great speculative and interpretative interest to better understand the complex and multi-factorial mechanisms behind the possible links between diets, their functional components and possible molecular processes that contribute to increasing the risk of developing MCI and Alzheimer's.

  10. Separation of the outer membrane and identification of major outer membrane proteins from Porphyromonas gingivalis.

    PubMed

    Murakami, Yukitaka; Imai, Masashi; Nakamura, Hiroshi; Yoshimura, Fuminobu

    2002-04-01

    The outer membrane of Porphyromonas gingivalis, an oral strict anaerobe, was isolated by sucrose density gradient centrifugation. The outer membrane obtained by the differential detergent extraction method, previously reported, showed an essentially similar protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), confirming that the latter method is suitable for the study of outer membrane proteins in this organism. N-terminal amino acid sequence analysis revealed that major outer membrane proteins in this organism included Arg-gingipain, Lys-gingipain, RagA (a TonB-linked receptor), and putative porins that were homologous to Escherichia coli OmpA.

  11. A porin-like protein from oral secretions of Spodoptera littoralis larvae induces defense-related early events in plant leaves.

    PubMed

    Guo, Huijuan; Wielsch, Natalie; Hafke, Jens B; Svatoš, Aleš; Mithöfer, Axel; Boland, Wilhelm

    2013-09-01

    Insect herbivory on plants is a complex incident consisting of at least two different aspects, namely mechanical damage and chemical challenge, as feeding insects introduce oral secretions (OS) into the wounded tissue of the attacked plant. Mechanical wounding alone is sufficient to induce a set of defense-related reactions in host plants, but some early events such as membrane potential (Vm) changes and cytosolic Ca²⁺-elevations can be triggered only by herbivores suggesting that OS-derived molecules are involved in those processes. Following an assay-guided purification based on planar lipid bilayer membrane technique in combination with proteomic analysis, a porin-like protein (PLP) of most likely bacterial origin was determined from collected OS of Spodoptera littoralis larvae. PLP exhibited channel-forming activity. Further, early defense-related events in plant-insect interaction were evaluated by using a purified fraction and α-hemolysin (α-HL) as a commercial pore-forming compound. Both up-regulated the calmodulin-like CML42 in Arabidopsis thaliana, which only responds to oral secretion and not to wounding. An elevation of in vivo [Ca²⁺](cyt) was not observed. Because membrane channel formation is a widespread phenomenon in plant-insect interactions, this PLP might represent an example for microbial compounds from the insect gut which are initially involved in plant-insect interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin

    PubMed Central

    2004-01-01

    In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei) and BthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expression vector and Escherichia coli host strain Origami(DE3). The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refolding experiments. As seen on SDS/PAGE, the 38 kDa band completely migrated to ∼110 kDa when the purified monomeric proteins were refolded in a buffer system containing 10% (w/v) Zwittergent® 3-14, together with a subsequent heating to 95 °C for 5 min. CD spectroscopy revealed that the 110 kDa proteins contained a predominant β-sheet structure, which corresponded completely to the structure of the Omp38 proteins isolated from B. pseudomallei and B. thailandensis. Immunoblot analysis using anti-BpsOmp38 polyclonal antibodies and peptide mass analysis by MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS confirmed that the expressed proteins were BpsOmp38 and BthOmp38. The anti-BpsOmp38 antibodies considerably exhibited the inhibitory effects on the permeation of small sugars through the Omp38-reconstituted liposomes. A linear relation between relative permeability rates and Mr of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin. PMID:15329048

  13. 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.

  14. Platelet Membrane β-Secretase Activity in Mild Cognitive Impairment and Conversion to Dementia: a Longitudinal Study

    PubMed Central

    McGuinness, Bernadette; Fuchs, Marc; Barrett, Suzanne L.; Passmore, A. Peter; Johnston, Janet A.

    2015-01-01

    A blood-based biomarker to complement the clinical and neuropsychological assessments used to evaluate the risk of individuals with mild cognitive impairment (MCI) developing Alzheimer’s disease (AD) would be invaluable. Previous pilot studies by our group identified elevated platelet membrane β-secretase activity in patients with AD and MCI, as compared to controls, and this activity was influenced by membrane cholesterol levels. The present study investigated baseline platelet membrane β-secretase activity and cholesterol levels in 97 MCI participants and 85 controls and explored whether these parameters differed in individuals with stable MCI, as compared to those who subsequently developed AD. To evaluate signal specificity, β-secretase activity assays were conducted in the presence and absence of beta-site amyloid-β protein precursor-cleaving enzyme (BACE) inhibitors. Baseline platelet membrane β-secretase activity did not differ significantly in MCI participants, as compared to controls, and platelet membrane cholesterol levels were significantly lower in the MCI group. The longitudinal study indicated that the activities inhibited by two different BACE inhibitors did not predict conversion to AD; however, the activity that was not affected by BACE inhibitors was significantly (40%) higher in individuals with stable MCI, as compared with those who subsequently developed AD. These findings indicated that further research into the source of this activity could contribute to a measure facilitating prediction of the risk of conversion from MCI to AD. PMID:26639974

  15. Neuroinflammation increases GABAergic tone and impairs cognitive and motor function in hyperammonemia by increasing GAT-3 membrane expression. Reversal by sulforaphane by promoting M2 polarization of microglia.

    PubMed

    Hernandez-Rabaza, Vicente; Cabrera-Pastor, Andrea; Taoro-Gonzalez, Lucas; Gonzalez-Usano, Alba; Agusti, Ana; Balzano, Tiziano; Llansola, Marta; Felipo, Vicente

    2016-04-18

    Hyperammonemia induces neuroinflammation and increases GABAergic tone in the cerebellum which contributes to cognitive and motor impairment in hepatic encephalopathy (HE). The link between neuroinflammation and GABAergic tone remains unknown. New treatments reducing neuroinflammation and GABAergic tone could improve neurological impairment. The aims were, in hyperammonemic rats, to assess whether: (a) Enhancing endogenous anti-inflammatory mechanisms by sulforaphane treatment reduces neuroinflammation and restores learning and motor coordination. (b) Reduction of neuroinflammation by sulforaphane normalizes extracellular GABA and glutamate-NO-cGMP pathway and identify underlying mechanisms. (c) Identify steps by which hyperammonemia-induced microglial activation impairs cognitive and motor function and how sulforaphane restores them. We analyzed in control and hyperammonemic rats, treated or not with sulforaphane, (a) learning in the Y maze; (b) motor coordination in the beam walking; (c) glutamate-NO-cGMP pathway and extracellular GABA by microdialysis; (d) microglial activation, by analyzing by immunohistochemistry or Western blot markers of pro-inflammatory (M1) (IL-1b, Iba-1) and anti-inflammatory (M2) microglia (Iba1, IL-4, IL-10, Arg1, YM-1); and (e) membrane expression of the GABA transporter GAT-3. Hyperammonemia induces activation of astrocytes and microglia in the cerebellum as assessed by immunohistochemistry. Hyperammonemia-induced neuroinflammation is associated with increased membrane expression of the GABA transporter GAT-3, mainly in activated astrocytes. This is also associated with increased extracellular GABA in the cerebellum and with motor in-coordination and impaired learning ability in the Y maze. Sulforaphane promotes polarization of microglia from the M1 to the M2 phenotype, reducing IL-1b and increasing IL-4, IL-10, Arg1, and YM-1 in the cerebellum. This is associated with astrocytes deactivation and normalization of GAT-3 membrane

  16. Diabetes-induced impairments of the exocytosis process and the effect of gabapentin: the link with cholesterol level in neuronal plasma membranes.

    PubMed

    Trikash, Irene; Gumenyuk, Vitaliy; Kuchmerovska, Tamara

    2015-04-01

    Diabetic neuropathy represents one of the most prevalent complications of diabetes mellitus. The aim of this study was to investigate the effect of diabetes-induced disturbances in neurons on the Ca(2+)-triggered membrane fusion process in cell-free system in relation to plasmalemma cholesterol level. The gabapentin therapy on the exocytosis process was also studied. The diabetes in rats was induced by streptozotocin (60 mg/kg of body weight, i.p.). After 4 weeks of diabetes induction the one group of diabetic rats was treated with gabapentin (50 mg/kg, i.p.) during 1 month. Fusion experiments were performed in the cell-free model system using fluorescent dye octadecylrhodamine B. The [2-(14)C]serotonin preloaded synaptosomes were used for assay of stimulated neurotransmitter release. The synaptosomal plasma membrane cholesterol level in diabetic rats was on 12 % higher than in control and was decreased on 5 % after gabapentin therapy. The rate of synaptic vesicles fusion with plasma membranes in the presence of Ca(2+) and synaptosomal cytosolic proteins was decreased to 14.5 % in diabetic rats as compared to control (23 %) and after gabapentin administration to diabetic rats was raised to 18 %. At diabetes the stimulated synaptosomal serotonin release was increased in 1.7-2 folds and was partially normalized by gabapentin therapy. Together, these findings suggest that elevated cholesterol content in neuronal plasma membranes at diabetes impairs the membrane fusion process in neurons that can induce the development of neuropathy. Diabetes-evoked impairments of the exocytotic process can be attenuated by gabapentin therapy.

  17. Deletion of the glycosyltransferase bgsB of Enterococcus faecalis leads to a complete loss of glycolipids from the cell membrane and to impaired biofilm formation

    PubMed Central

    2011-01-01

    Background Deletion of the glycosyltransferase bgsA in Enterococcus faecalis leads to loss of diglucosyldiacylglycerol from the cell membrane and accumulation of its precursor monoglucosyldiacylglycerol, associated with impaired biofilm formation and reduced virulence in vivo. Here we analyzed the function of a putative glucosyltransferase EF2890 designated biofilm-associated glycolipid synthesis B (bgsB) immediately downstream of bgsA. Results A deletion mutant was constructed by targeted mutagenesis in E. faecalis strain 12030. Analysis of cell membrane extracts revealed a complete loss of glycolipids from the cell membrane. Cell walls of 12030ΔbgsB contained approximately fourfold more LTA, and 1H-nuclear magnetic resonance (NMR) spectroscopy suggested that the higher content of cellular LTA was due to increased length of the glycerol-phosphate polymer of LTA. 12030ΔbgsB was not altered in growth, cell morphology, or autolysis. However, attachment to Caco-2 cells was reduced to 50% of wild-type levels, and biofilm formation on polystyrene was highly impaired. Despite normal resistance to cationic antimicrobial peptides, complement and antibody-mediated opsonophagocytic killing in vitro, 12030ΔbgsB was cleared more rapidly from the bloodstream of mice than wild-type bacteria. Overall, the phenotype resembles the respective deletion mutant in the bgsA gene. Our findings suggest that loss of diglucosyldiacylglycerol or the altered structure of LTA in both mutants account for phenotypic changes observed. Conclusions In summary, BgsB is a glucosyltransferase that synthesizes monoglucosyldiacylglycerol. Its inactivation profoundly affects cell membrane composition and has secondary effects on LTA biosynthesis. Both cell-membrane amphiphiles are critical for biofilm formation and virulence of E. faecalis. PMID:21470413

  18. Impairment of the class IIa bacteriocin receptor function and membrane structural changes are associated to enterocin CRL35 high resistance in Listeria monocytogenes.

    PubMed

    Masias, Emilse; Dupuy, Fernando G; da Silva Sanches, Paulo Ricardo; Farizano, Juan Vicente; Cilli, Eduardo; Bellomio, Augusto; Saavedra, Lucila; Minahk, Carlos

    2017-07-01

    Enterocin CRL35 is a class IIa bacteriocin with anti-Listeria activity. Resistance to these peptides has been associated with either the downregulation of the receptor expression or changes in the membrane and cell walls. The scope of the present work was to characterize enterocin CRL35 resistant Listeria strains with MICs more than 10,000 times higher than the MIC of the WT sensitive strain. Listeria monocytogenes INS7 resistant isolates R2 and R3 were characterized by 16S RNA gene sequencing and rep-PCR. Bacterial growth kinetic was studied in different culture media. Plasma membranes of sensitive and resistant bacteria were characterized by FTIR and Langmuir monolayer techniques. The growth kinetic of the resistant isolates was slower as compared to the parental strain in TSB medium. Moreover, the resistant isolates barely grew in a glucose-based synthetic medium, suggesting that these cells had a major alteration in glucose transport. Resistant bacteria also had alterations in their cell wall and, most importantly, membrane lipids. In fact, even though enterocin CRL35 was able to bind to the membrane-water interface of both resistant and parental sensitive strains, this peptide was only able to get inserted into the latter membranes. These results indicate that bacteriocin receptor is altered in combination with membrane structural modifications in enterocin CRL35-resistant L. monocytogenes strains. Highly enterocin CRL35-resistant isolates derived from Listeria monocytogenes INS7 have not only an impaired glucose transport but also display structural changes in the hydrophobic core of their plasma membranes. Copyright © 2017. Published by Elsevier B.V.

  19. 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.

  20. Energy-Dependent Accumulation of Norfloxacin and Porin Expression in Clinical Isolates of Klebsiella pneumoniae and Relationship to Extended-Spectrum β-Lactamase Production

    PubMed Central

    Martínez-Martínez, Luis; Pascual, Alvaro; Conejo, María del Carmen; García, Isabel; Joyanes, Providencia; Doménech-Sánchez, Antonio; Benedí, Vicente Javier

    2002-01-01

    The relationships between porin deficiency, active efflux of fluoroquinolones, and extended-spectrum β-lactamase (ESBL) production were determined for 53 clinical isolates of Klebsiella pneumoniae. Thirty-two ESBL-positive strains (including 22 strains expressing porins and 10 strains lacking porins) and 21 ESBL-negative strains were evaluated. Active efflux of norfloxacin was defined as a ≥50% increase in the accumulation of norfloxacin in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) in comparison with the corresponding basal value in the absence of CCCP. The quinolone resistance-determining regions of both gyrA and parC from 13 strains, representing all isolates with different porin profiles and with or without active efflux, were determined. Porin loss was significantly more common among ESBL-positive strains (10 of 32 [31.2%]) than among ESBL-negative strains (0 of 2 [0%]) (P < 0.01). Active efflux was observed in 7 of 10 (70%) strains lacking porins and in 4 of 43 (9.3%) strains producing porins (P < 0.001). The 11 strains showing active efflux corresponded to 3 of 21 (14.3%) ESBL-negative strains and 8 of 32 (25.5%) ESBL-positive strains (P > 0.05). Basal values of norfloxacin accumulation were higher in strains lacking active efflux than in those that had this mechanism (P < 0.05). In the absence of topoisomerase changes, the contribution of either porin loss or active efflux to fluoroquinolone resistance in K. pneumoniae was negligible. It is concluded that among K. pneumoniae strains of clinical origin, porin loss was observed only in those producing ESBL, and that a significant number of porin-deficient strains also expressed active efflux of norfloxacin. In terms of fluoroquinolone resistance, both mechanisms are significant only in the presence of topoisomerase modifications. PMID:12435697

  1. Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. I. Impairment of resealing and formation of aqueous pores in the ghost membrane after modification of SH groups.

    PubMed

    Klonk, S; Deuticke, B

    1992-04-29

    Resealed human erythrocyte ghosts prepared by a two-step procedure were shown to have small residual barrier defects with the properties of aqueous pores, such as size discrimination of hydrophilic nonelectrolytes (erythritol to sucrose), indicative of an apparent pore radius of about 0.7 nm, and a low activation energy (about 12-20 kJ/mol (mannitol, sucrose)) of the leak fluxes. As in other cases (Deuticke et al. (1991) Biochim. Biophys. Acta 1067, 111-122) these leak fluxes can be inhibited by phloretin. Treatment of such resealed ghosts with the mild SH oxidizing agent, diamide, induces additional membrane leaks to the same extent and with the same properties as in native erythrocytes (Deuticke et al. (1983) Biochim. Biophys. Acta 731, 196-210), including reversibility of the leak by SH reducing agents, inhibition by phloretin and stimulation by alkanols. In contrast, resealed ghosts prepared either from diamide-treated erythrocytes or by adding diamide to the 'open' membranes prior to reconstitution of high ionic strength and raising the temperature, exhibit a state of greater leakiness. This leakiness is somewhat different in its origin from the former class of leaks, since it can also be produced by N-ethylmaleimide, which is essentially ineffective when added to the membrane in its 'tight' state. The leaks induced in the 'open' state of the membrane, which can be regarded as a consequence of an impaired resealing, are nevertheless reversible by reducing agents added after resealing and are comparable in many, but not all their characteristics to leaks induced in the 'tight' state of the membrane. Resealing in the presence of the isothiocyanostilbenes DIDS or SITS mimicks the leak forming effect of diamide by modifying a small population of SH groups, while amino groups seem not to be involved. The findings indicate and substantiate an important role of the redox state of membrane skeletal protein sulfhydryls in the maintenance and the re-establishment of the

  2. Carbapenem resistance in a clinical isolate of Enterobacter aerogenes is associated with decreased expression of OmpF and OmpC porin analogs.

    PubMed

    Yigit, Hesna; Anderson, Gregory J; Biddle, James W; Steward, Christine D; Rasheed, J Kamile; Valera, Lourdes L; McGowan, John E; Tenover, Fred C

    2002-12-01

    We investigated the mechanism of imipenem resistance in Enterobacter aerogenes strain 810, a clinical isolate from the United States for which the imipenem MIC was 16 micro g/ml and the meropenem MIC was 8 micro g/ml. An imipenem-susceptible revertant, strain 810-REV, was obtained after multiple passages of the strain on nonselective media. For the revertant, the imipenem MIC was /=128 micro g/ml), cefoxitin (>/=32 micro g/ml), and cefotaxime (>/=64 micro g/ml) remained the same. The beta-lactamase and porin profiles of the parent, the revertant, and carbapenem-susceptible type strain E. aerogenes ATCC 13048 were determined. Strains 810 and 810-REV each produced two beta-lactamases with pIs of 8.2 and 5.4. The beta-lactamase activities of the parent and revertant were similar, even after induction with subinhibitory concentrations of imipenem. While 810-REV produced two major outer membrane proteins of 42 and 39 kDa that corresponded to Escherichia coli porins OmpC and OmpF, respectively, the parent strain appeared to produce similar quantities of the 39-kDa protein (OmpF) but decreased amounts of the 42-kDa protein (OmpC). When the parent strain was grown in the presence of imipenem, the 42-kDa protein was not detectable by gel electrophoresis. However, Western blot analysis of the outer membrane proteins of the parent and revertant with polyclonal antisera raised to the OmpC and OmpF analogs of Klebsiella pneumoniae (anti-OmpK36 and anti-OmpK35, respectively) showed that strain 810 expressed only the 42-kDa OmpC analog in the absence of imipenem (the 39-kDa protein was not recognized by the anti-OmpF antisera) and neither the OmpC nor the OmpF analog in the presence of imipenem. The OmpC analog is apparently down-regulated in the presence of imipenem; however, 810-REV expressed both OmpC and OmpF analogs. These data

  3. High-Level Carbapenem Resistance in a Klebsiella pneumoniae Clinical Isolate Is Due to the Combination of blaACT-1 β-Lactamase Production, Porin OmpK35/36 Insertional Inactivation, and Down-Regulation of the Phosphate Transport Porin PhoE

    PubMed Central

    Kaczmarek, Frank M.; Dib-Hajj, Fadia; Shang, Wenchi; Gootz, Thomas D.

    2006-01-01

    Clinical isolates of Klebsiella pneumoniae resistant to carbapenems and essentially all other antibiotics (multidrug resistant) are being isolated from some hospitals in New York City with increasing frequency. A highly related pair of K. pneumoniae strains isolated on the same day from one patient in a hospital in New York City were studied for antibiotic resistance. One (KP-2) was resistant to imipenem, meropenem, and sulopenem (MICs of 16 to 32 μg/ml) while the other (KP-1) was susceptible (MIC of 0.5 μg/ml); both contained the blaACT-1, blaSHV-1, and blaTEM-1 β-lactamases. blaACT-1 in both strains was encoded on a large ∼150-kb plasmid. Both isolates contained an identical class 1 integron encoding resistance to aminoglycosides and chloramphenicol. They each had identical insertions in ompK35 and ompK36, resulting in disruption of these key porin genes. The carbapenem-resistant and -susceptible isolates were extensively studied for differences in the structural and regulatory genes for the operons acrRAB, marORAB, romA-ramA, soxRS, micF, micC, phoE, phoBR, rpoS, and hfq. No changes were detected between the isolates except for a significant down-regulation of ompK37, phoB, and phoE in KP-2 as deduced from reverse transcription-PCR analysis of mRNA and polyacrylamide gel electrophoresis separation of outer membrane proteins. Backcross analysis was conducted using the wild-type phoE gene cloned into the vector pGEM under regulation of its native promoter as well as the lacZ promoter following transformation into the resistant KP-2 isolate. The wild-type gene reversed carbapenem resistance only when under control of the heterologous lacZ promoter. In the background of ompK35-ompK36 gene disruption, the up-regulation of phoE in KP-1 apparently compensated for porin loss and conferred carbapenem susceptibility. Down-regulation of phoE in KP-2 may represent the normal state of this gene, or it may have been selected from KP-1 in vivo under antibiotic pressure

  4. High-level carbapenem resistance in a Klebsiella pneumoniae clinical isolate is due to the combination of bla(ACT-1) beta-lactamase production, porin OmpK35/36 insertional inactivation, and down-regulation of the phosphate transport porin phoe.

    PubMed

    Kaczmarek, Frank M; Dib-Hajj, Fadia; Shang, Wenchi; Gootz, Thomas D

    2006-10-01

    Clinical isolates of Klebsiella pneumoniae resistant to carbapenems and essentially all other antibiotics (multidrug resistant) are being isolated from some hospitals in New York City with increasing frequency. A highly related pair of K. pneumoniae strains isolated on the same day from one patient in a hospital in New York City were studied for antibiotic resistance. One (KP-2) was resistant to imipenem, meropenem, and sulopenem (MICs of 16 to 32 microg/ml) while the other (KP-1) was susceptible (MIC of 0.5 microg/ml); both contained the bla(ACT-1), bla(SHV-1), and bla(TEM-1) beta-lactamases. bla(ACT-1) in both strains was encoded on a large approximately 150-kb plasmid. Both isolates contained an identical class 1 integron encoding resistance to aminoglycosides and chloramphenicol. They each had identical insertions in ompK35 and ompK36, resulting in disruption of these key porin genes. The carbapenem-resistant and -susceptible isolates were extensively studied for differences in the structural and regulatory genes for the operons acrRAB, marORAB, romA-ramA, soxRS, micF, micC, phoE, phoBR, rpoS, and hfq. No changes were detected between the isolates except for a significant down-regulation of ompK37, phoB, and phoE in KP-2 as deduced from reverse transcription-PCR analysis of mRNA and polyacrylamide gel electrophoresis separation of outer membrane proteins. Backcross analysis was conducted using the wild-type phoE gene cloned into the vector pGEM under regulation of its native promoter as well as the lacZ promoter following transformation into the resistant KP-2 isolate. The wild-type gene reversed carbapenem resistance only when under control of the heterologous lacZ promoter. In the background of ompK35-ompK36 gene disruption, the up-regulation of phoE in KP-1 apparently compensated for porin loss and conferred carbapenem susceptibility. Down-regulation of phoE in KP-2 may represent the normal state of this gene, or it may have been selected from KP-1 in vivo

  5. Adaptive and mutational resistance: role of porins and efflux pumps in drug resistance.

    PubMed

    Fernández, Lucía; Hancock, Robert E W

    2012-10-01

    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.

  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. 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.

  8. Epstein Barr-Virus Latent Membrane Protein-2A-Induced ΔNp63α Expression is Associated with Impaired Epithelial Cell Differentiation

    PubMed Central

    Fotheringham, Julie A.; Mazzucca, Stephanie; Raab-Traub, Nancy

    2010-01-01

    Epstein-Barr virus (EBV) is an oncogenic γ-herpes virus associated with malignancies that develop in both lymphoid and epithelial cells including nasopharyngeal carcinoma (NPC). The EBV protein latent membrane protein 2A (LMP2A) is expressed in NPC and can modulate epithelial proliferation, transformation, and differentiation, and as such, may promote malignancy. A key regulator of epithelial cell differentiation is the transcription factor p63, a member of the p53 family. This study examines the potential contribution of p63 to LMP2A-mediated inhibition of epithelial differentiation. Stable expression of LMP2A increased the protein level and stability of the ΔNp63α isoform, and in two epithelial cell lines, LMP2A interacted with ΔNp63α under stable and transient expression systems. LMP2A and ΔNp63α were localized to the cytoplasm and nuclear membrane and co-immunoprecipitated in the same fractions. Following induction of epithelial cell differentiation by calcium, expression of differentiation markers was impaired in both ΔNp63α and LMP2 expressing cells. Induction of p63α, association of p63α with LMP2A, and impairment of differentiation required the PY and ITAM signaling motif of LMP2A. By associating with and being regulated by LMP2A,ΔNp63α may function as a unique regulator of LMP2A effects on epithelial differentiation and contribute to EBV-associated epithelial cancers. PMID:20498633

  9. Porins from Salmonella enterica Serovar Typhimurium Activate the Transcription Factors Activating Protein 1 and NF-κB through the Raf-1-Mitogen-Activated Protein Kinase Cascade

    PubMed Central

    Galdiero, Massimiliano; Vitiello, Mariateresa; Sanzari, Emma; D’Isanto, Marina; Tortora, Annalisa; Longanella, Anna; Galdiero, Stefania

    2002-01-01

    In this study we examined the ability of Salmonella enterica serovar Typhimurium porins to activate activating protein 1 (AP-1) and nuclear factor κB (NF-κB) through the mitogen-activated protein kinase (MAPK) cascade, and we identified the AP-1-induced protein subunits. Our results demonstrate that these enzymes may participate in cell signaling pathways leading to AP-1 and NF-κB activation following porin stimulation of cells. Raf-1 was phosphorylated in response to the treatment of U937 cells with porins; moreover, the porin-mediated increase in Raf-1 phosphorylation is accompanied by the phosphorylation of MAPK kinase 1/2 (MEK1/2), p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase. We used three different inhibitors of phosphorylation pathways: 2′-amino-3′-methoxyflavone (PD-098059), a selective inhibitor of MEK1 activator and the MAPK cascade; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of the p38 pathway; and 7β-acetoxy-1α,6β,9α-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), an inhibitor at the level of Raf-1 kinase. PD-098059 pretreatment of cells decreases AP-1 and NF-κB activation by lipopolysaccharide (LPS) but not by porins, and SB203580 pretreatment of cells decreases mainly AP-1 and NF-κB activation by porins; in contrast, forskolin pretreatment of cells does not affect AP-1 and NF-κB activation following either porin or LPS stimulation. Our data suggest that the p38 signaling pathway mainly regulates AP-1 and NF-κB activation in cells treated with S. enterica serovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only after porin stimulation

  10. Porins from Salmonella enterica serovar Typhimurium activate the transcription factors activating protein 1 and NF-kappaB through the Raf-1-mitogen-activated protein kinase cascade.

    PubMed

    Galdiero, Massimiliano; Vitiello, Mariateresa; Sanzari, Emma; D'Isanto, Marina; Tortora, Annalisa; Longanella, Anna; Galdiero, Stefania

    2002-02-01

    In this study we examined the ability of Salmonella enterica serovar Typhimurium porins to activate activating protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) through the mitogen-activated protein kinase (MAPK) cascade, and we identified the AP-1-induced protein subunits. Our results demonstrate that these enzymes may participate in cell signaling pathways leading to AP-1 and NF-kappaB activation following porin stimulation of cells. Raf-1 was phosphorylated in response to the treatment of U937 cells with porins; moreover, the porin-mediated increase in Raf-1 phosphorylation is accompanied by the phosphorylation of MAPK kinase 1/2 (MEK1/2), p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase. We used three different inhibitors of phosphorylation pathways: 2'-amino-3'-methoxyflavone (PD-098059), a selective inhibitor of MEK1 activator and the MAPK cascade; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of the p38 pathway; and 7beta-acetoxy-1alpha,6beta,9alpha-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), an inhibitor at the level of Raf-1 kinase. PD-098059 pretreatment of cells decreases AP-1 and NF-kappaB activation by lipopolysaccharide (LPS) but not by porins, and SB203580 pretreatment of cells decreases mainly AP-1 and NF-kappaB activation by porins; in contrast, forskolin pretreatment of cells does not affect AP-1 and NF-kappaB activation following either porin or LPS stimulation. Our data suggest that the p38 signaling pathway mainly regulates AP-1 and NF-kappaB activation in cells treated with S. enterica serovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only

  11. 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

  12. Type-1 Cannabinoid Receptors Reduce Membrane Fluidity of Capacitated Boar Sperm by Impairing Their Activation by Bicarbonate

    PubMed Central

    Barboni, Barbara; Bernabò, Nicola; Palestini, Paola; Botto, Laura; Pistilli, Maria Gabriella; Charini, Marco; Tettamanti, Enzo; Battista, Natalia

    2011-01-01

    Background Mammalian spermatozoa acquire their full fertilizing ability (so called capacitation) within the female genital tract, where they are progressively exposed to inverse gradients of inhibiting and stimulating molecules. Methodology/Principal Findings In the present research, the effect on this process of anandamide, an endocannabinoid that can either activate or inhibit cannabinoid receptors depending on its concentration, and bicarbonate, an oviductal activatory molecule, was assessed, in order to study the role exerted by the type 1 cannabinoid receptor (CB1R) in the process of lipid membrane remodeling crucial to complete capacitation. To this aim, boar sperm were incubated in vitro under capacitating conditions (stimulated by bicarbonate) in the presence or in the absence of methanandamide (Met-AEA), a non-hydrolysable analogue of anandamide. The CB1R involvement was studied by using the specific inhibitor (SR141716) or mimicking its activation by adding a permeable cAMP analogue (8Br-cAMP). By an immunocytochemistry approach it was shown that the Met-AEA inhibits the bicarbonate-dependent translocation of CB1R from the post-equatorial to equatorial region of sperm head. In addition it was found that Met-AEA is able to prevent the bicarbonate-induced increase in membrane disorder and the cholesterol extraction, both preliminary to capacitation, acting through a CB1R-cAMP mediated pathway, as indicated by MC540 and filipin staining, EPR spectroscopy and biochemical analysis on whole membranes (CB1R activity) and on membrane enriched fraction (C/P content and anisotropy). Conclusions/Significance Altogether, these data demonstrate that the endocannabinoid system strongly inhibits the process of sperm capacitation, acting as membrane stabilizing agent, thus increasing the basic knowledge on capacitation-related signaling and potentially opening new perspectives in diagnostics and therapeutics of male infertility. PMID:21829686

  13. The nuclear membrane and mechanotransduction: impaired nuclear mechanics and mechanotransduction in lamin A/C deficient cells.

    PubMed

    Lammerding, Jan; Lee, Richard T

    2005-01-01

    Mutations in the lamin A/C gene cause a variety of human diseases including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy and Hutchinson-Gilford progeria syndrome. The tissue specific effects of lamin mutations are unclear, in part because the function of lamin A/C is incompletely defined, but the many muscle specific phenotypes suggest that defective lamin A/C could increase cellular mechanical sensitivity. Lamin A/C deficient fibroblasts were subjected to mechanical strain to measure nuclear mechanical properties and strain-induced signalling. We found that lamin A/C deficient fibroblasts are characterized by impaired nuclear mechanics and mechanotransduction, reflected by increased nuclear deformations, increased nuclear fragility, attenuated expression of mechanosensitive genes, and impaired transcriptional activation, leading to impaired viability of mechanically strained cells. Lamins and other nuclear envelope proteins can thus affect several levels of the mechanotransduction cascade, altering nuclear and cytoskeletal mechanics as well as playing an important role in mechanically activated gene regulation. Individual mutations in the lamin A/C gene could potentially selectively interfere with any of these functions, explaining the tissue-specific effects observed in the laminopathies.

  14. Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum.

    PubMed

    Issa, Hanane; Huc-Claustre, Emilie; Reddad, Thamila; Bonadé Bottino, Nolwenn; Tropis, Maryelle; Houssin, Christine; Daffé, Mamadou; Bayan, Nicolas; Dautin, Nathalie

    2017-01-01

    Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated.

  15. Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum

    PubMed Central

    Issa, Hanane; Huc-Claustre, Emilie; Reddad, Thamila; Bonadé Bottino, Nolwenn; Tropis, Maryelle; Houssin, Christine; Daffé, Mamadou; Bayan, Nicolas

    2017-01-01

    Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated. PMID:28199365

  16. C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles.

    PubMed

    Lee, Kyung-Ha; Zhang, Peipei; Kim, Hong Joo; Mitrea, Diana M; Sarkar, Mohona; Freibaum, Brian D; Cika, Jaclyn; Coughlin, Maura; Messing, James; Molliex, Amandine; Maxwell, Brian A; Kim, Nam Chul; Temirov, Jamshid; Moore, Jennifer; Kolaitis, Regina-Maria; Shaw, Timothy I; Bai, Bing; Peng, Junmin; Kriwacki, Richard W; Taylor, J Paul

    2016-10-20

    Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.

  17. 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

  18. Epstein-Barr virus latent membrane protein-2A-induced DeltaNp63alpha expression is associated with impaired epithelial-cell differentiation.

    PubMed

    Fotheringham, J A; Mazzucca, S; Raab-Traub, N

    2010-07-29

    Epstein-Barr virus (EBV) is an oncogenic gamma-herpes virus associated with malignancies that develop in both lymphoid and epithelial cells including nasopharyngeal carcinoma (NPC). The EBV protein, latent membrane protein 2A (LMP2A), is expressed in NPC and can modulate epithelial proliferation, transformation and differentiation, and as such may promote malignancy. A key regulator of epithelial-cell differentiation is the transcription factor p63, a member of the p53 family. This study examines the potential contribution of p63 to LMP2A-mediated inhibition of epithelial-cell differentiation. Stable expression of LMP2A increased the protein level and stability of the DeltaNp63alpha isoform and in two epithelial cell lines, LMP2A interacted with DeltaNp63alpha under stable- and transient-expression systems. LMP2A and DeltaNp63alpha were localized to the cytoplasm and nuclear membrane and co-immunoprecipitated in the same fractions. Following induction of epithelial-cell differentiation by calcium, expression of differentiation markers was impaired in both DeltaNp63alpha- and LMP2A-expressing cells. Induction of p63alpha, association of p63alpha with LMP2A and impairment of differentiation required the PY and immunoreceptor tyrosine-based activation motif (ITAM) signaling motif of LMP2A. By associating with and being regulated by LMP2A, DeltaNp63alpha may function as a unique regulator of LMP2A effects on epithelial differentiation and contribute to EBV-associated epithelial cancers.

  19. 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

  20. Amphipols: Polymers that keep membrane proteins soluble in aqueous solutions

    PubMed Central

    Tribet, Christophe; Audebert, Roland; Popot, Jean-Luc

    1996-01-01

    Amphipols are a new class of surfactants that make it possible to handle membrane proteins in detergent-free aqueous solution as though they were soluble proteins. The strongly hydrophilic backbone of these polymers is grafted with hydrophobic chains, making them amphiphilic. Amphipols are able to stabilize in aqueous solution under their native state four well-characterized integral membrane proteins: (i) bacteriorhodopsin, (ii) a bacterial photosynthetic reaction center, (iii) cytochrome b6f, and (iv) matrix porin. PMID:8986761

  1. Institution of Veno-arterial Extracorporeal Membrane Oxygenation Does Not Lead to Increased Wall Stress in Patients with Impaired Myocardial Function.

    PubMed

    Koth, Andrew M; Axelrod, David M; Reddy, Sushma; Roth, Stephen J; Tacy, Theresa A; Punn, Rajesh

    2017-03-01

    The effect of veno-arterial extracorporeal membrane oxygenation (VA ECMO) on wall stress in patients with cardiomyopathy, myocarditis, or other cardiac conditions is unknown. We set out to determine the circumferential and meridional wall stress (WS) in patients with systemic left ventricles before and during VA ECMO. We established a cohort of patients with impaired myocardial function who underwent VA ECMO therapy from January 2000 to November 2013. Demographic and clinical data were collected and inotropic score calculated. Measurements were taken on echocardiograms prior to the initiation of VA ECMO and while on full-flow VA ECMO, in order to derive wall stress (circumferential and meridional), VCFc, ejection fraction, and fractional shortening. A post hoc sub-analysis was conducted, separating those with pulmonary hypertension (PH) and those with impaired systemic output. Thirty-three patients met inclusion criteria. The patients' median age was 0.06 years (range 0-18.7). Eleven (33%) patients constituted the organ failure group (Gr2), while the remaining 22 (66%) patients survived to discharge (Gr1). WS and all other echocardiographic measures were not different when comparing patients before and during VA ECMO. Ejection and shortening fraction, WS, and VCFc were not statistically different comparing the survival and organ failure groups. The patients' position on the VCFc-WS curve did not change after the initiation of VA ECMO. Those with PH had decreased WS as well as increased EF after ECMO initiation, while those with impaired systemic output showed no difference in those parameters with initiation of ECMO. The external workload on the myocardium as indicated by WS is unchanged by the institution of VA ECMO support. Furthermore, echocardiographic measures of cardiac function do not reflect the changes in ventricular performance inherent to VA ECMO support. These findings are informative for the interpretation of echocardiograms in the setting of VA ECMO

  2. The HIV-1 Pr55 gag polyprotein binds to plastidial membranes and leads to severe impairment of chloroplast biogenesis and seedling lethality in transplastomic tobacco plants.

    PubMed

    Scotti, N; Sannino, L; Idoine, A; Hamman, P; De Stradis, A; Giorio, P; Maréchal-Drouard, L; Bock, R; Cardi, T

    2015-04-01

    Chloroplast genetic engineering has long been recognised as a powerful technology to produce recombinant proteins. To date, however, little attention has been given to the causes of pleiotropic effects reported, in some cases, as consequence of the expression of foreign proteins in transgenic plastids. In this study, we investigated the phenotypic alterations observed in transplastomic tobacco plants accumulating the Pr55(gag) polyprotein of human immunodeficiency virus (HIV-1). The expression of Pr55(gag) at high levels in the tobacco plastome leads to a lethal phenotype of seedlings grown in soil, severe impairment of plastid development and photosynthetic activity, with chloroplasts largely resembling undeveloped proplastids. These alterations are associated to the binding of Pr55(gag) to thylakoids. During particle assembly in HIV-1 infected human cells, the binding of Pr55(gag) to a specific lipid [phosphatidylinositol-(4-5) bisphosphate] in the plasma membrane is mediated by myristoylation at the amino-terminus and the so-called highly basic region (HBR). Surprisingly, the non-myristoylated Pr55(gag) expressed in tobacco plastids was likely able, through the HBR motif, to bind to nonphosphorous glycerogalactolipids or other classes of lipids present in plastidial membranes. Although secondary consequences of disturbed chloroplast biogenesis on expression of nuclear-encoded plastid proteins cannot be ruled out, results of proteomic analyses suggest that their altered accumulation could be due to retrograde control in which chloroplasts relay their status to the nucleus for fine-tuning of gene expression.

  3. Tuning the affinity of anion binding sites in porin channels with negatively charged residues: molecular details for OprP.

    PubMed

    Modi, Niraj; Bárcena-Uribarri, Iván; Bains, Manjeet; Benz, Roland; Hancock, Robert E W; Kleinekathöfer, Ulrich

    2015-02-20

    The cell envelope of the Gram negative opportunistic pathogen Pseudomonas aeruginosa is poorly permeable to many classes of hydrophilic molecules including antibiotics due to the presence of the narrow and selective porins. Here we focused on one of the narrow-channel porins, that is, OprP, which is responsible for the high-affinity uptake of phosphate ions. Its two central binding sites for phosphate contain a number of positively charged amino acids together with a single negatively charged residue (D94). The presence of this negatively charged residue in a binding site for negatively charged phosphate ions is highly surprising due to the potentially reduced binding affinity. The goal of this study was to better understand the role of D94 in phosphate binding, selectivity, and transport using a combination of mutagenesis, electrophysiology, and free-energy calculations. The presence of a negatively charged residue in the binding site is critical for this specific porin OprP as emphasized by the evolutionary conservation of such negatively charged residue in the binding site of several anion-selective porins. Mutations of D94 in OprP to any positively charged or neutral residue increased the binding affinity of phosphate for OprP. Detailed analysis indicated that this anionic residue in the phosphate binding site of OprP, despite its negative charge, maintained energetically favorable phosphate binding sites in the central region of the channel and at the same time decreased residence time thus preventing excessively strong binding of phosphate that would oppose phosphate flux through the channel. Intriguingly mutations of D94 to positively charged residues, lysine and arginine, resulted in very different binding affinities and free energy profiles, indicating the importance of side chain conformations of these positively charged residues in phosphate binding to OprP.

  4. Impaired binding of the age-related macular degeneration-associated complement factor H 402H allotype to Bruch's membrane in human retina.

    PubMed

    Clark, Simon J; Perveen, Rahat; Hakobyan, Svetlana; Morgan, B Paul; Sim, Robert B; Bishop, Paul N; Day, Anthony J

    2010-09-24

    Age-related macular degeneration (AMD) is the predominant cause of blindness in the industrialized world where destruction of the macula, i.e. the central region of the retina, results in loss of vision. AMD is preceded by the formation of deposits in the macula, which accumulate between the Bruch's membrane and the retinal pigment epithelium (RPE). These deposits are associated with complement-mediated inflammation and perturb retinal function. Recent genetic association studies have demonstrated that a common allele (402H) of the complement factor H (CFH) gene is a major risk factor for the development of AMD; CFH suppresses complement activation on host tissues where it is believed to bind via its interaction with polyanionic structures. We have shown previously that this coding change (Y402H; from a tyrosine to histidine residue) alters the binding of the CFH protein to sulfated polysaccharides. Here we demonstrate that the AMD-associated polymorphism profoundly affects CFH binding to sites within human macula. Notably, the AMD-associated 402H variant binds less well to heparan sulfate and dermatan sulfate glycosaminoglycans within Bruch's membrane when compared with the 402Y form; both allotypes exhibit a similar level of binding to the RPE. We propose that the impaired binding of the 402H variant to Bruch's membrane results in an overactivation of the complement pathway leading to local chronic inflammation and thus contributes directly to the development and/or progression of AMD. These studies therefore provide a putative disease mechanism and add weight to the genetic association studies that implicate the 402H allele as an important risk factor in AMD.

  5. 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

  6. Impairment of pH Gradient and Membrane Potential Mediates Redox Dysfunction in the Mitochondria of the Post-ischemic Heart

    PubMed Central

    Kang, Patrick T.; Chen, Chwen-Lih; Lin, Paul; Chilian, William M.; Chen, Yeong-Renn

    2017-01-01

    The mitochondrial electrochemical gradient (Δp), which comprises the pH gradient (ΔpH) and the membrane potential (ΔΨ), is crucial in controlling energy transduction. During myocardial ischemia and reperfusion (IR), mitochondrial dysfunction mediates superoxide (•O2−) and H2O2 overproduction leading to oxidative injury. However, the role of ΔpH and ΔΨ in post-ischemic injury is not fully established. Here we studied mitochondria from the risk region of rat hearts subjected to 30 min of coronary ligation and 24 h of reperfusion in vivo. In the presence of glutamate, malate and ADP, normal mitochondria (mitochondria of non-ischemic region, NR) exhibited a heightened state 3 oxygen consumption rate (OCR) and reduced •O2− and H2O2 production when compared to state 2 conditions. Oligomycin (increases ΔpH by inhibiting ATP synthase) increased •O2− and H2O2 production in normal mitochondria, but not significantly in the mitochondria of the risk region (IR mitochondria or post-ischemic mitochondria), indicating that normal mitochondrial •O2− and H2O2 generation is dependent on ΔpH and that IR impaired the ΔpH of normal mitochondria. Conversely, nigericin (dissipates ΔpH) dramatically reduced •O2− and H2O2 generation by normal mitochondria under state 4 conditions, and this nigericin quenching effect was less pronounced in IR mitochondria. Nigericin also increased mitochondrial OCR, and predisposed normal mitochondria to a more oxidized redox status assessed by increased oxidation of cyclic hydroxylamine, CM-H. IR mitochondria, although more oxidized than normal mitochondria, were not responsive to nigericin-induced CM-H oxidation, which is consistent with the result that IR induced ΔpH impairment in normal mitochondria. Valinomycin, a K+ ionophore used to dissipate ΔΨ, drastically diminished •O2− and H2O2 generation by normal mitochondria, but less pronounced effect on IR mitochondria under state 4 conditions, indicating that ΔΨ also

  7. Genetic Variants of the FADS Gene Cluster Are Associated with Erythrocyte Membrane LC PUFA Levels in Patients with Mild Cognitive Impairment.

    PubMed

    Schuchardt, J P; Köbe, T; Witte, V; Willers, J; Gingrich, A; Tesky, V; Pantel, J; Rujescu, D; Illig, T; Flöel, A; Hahn, A

    2016-01-01

    Long-chain (> 20 C-atoms) polyunsaturated fatty acids (LC PUFAs) of both the omega-6 (n-6) and omega-3 (n-3) series are important for the functional integrity of brain and thereby cognition, memory and mood. Clinical studies observed associations between altered LC PUFA levels and neurodegenerative diseases such as Alzheimer´s disease and its prodromal stage, mild cognitive impairment (MCI). The present study examined the LC PUFA status of MCI patients with specific view on the relative LC n-3 PUFA levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in erythrocyte membranes (omega-3 index). 12 single nucleotide polymorphisms (SNPs) of the FADS1, FADS2, and FADS3 gene clusters were genotyped in 111 MCI patients and evaluated associations with PUFA levels in erythrocyte membranes (primary outcome). In addition, the associations between FADS SNPs and LC PUFA levels with serum lipid levels as well as depressive symptoms were examined (secondary outcomes). Minor allele carrier of rs174546, rs174548 (FADS1), rs3834458, rs1535, rs174574, rs174575, rs174576, and rs174578 (FADS2) showed significant higher n-6 and n-3 precursor PUFA levels (linoleic acid, and alpha-linolenic acid, respectively) and lower arachidonic acid (AA) levels in erythrocyte membranes compared to the major allele carriers. Differences in EPA and DHA levels were not significant. Minor allele carriers of rs174574, rs174576 and rs174578 (FADS2) and rs174455 (FADS3) exhibited significant higher triglyceride levels, whereas minor allele carriers for rs174449 and rs174455 (FADS3) exhibited significant higher total- and LDL-cholesterol levels compared to the more common variant. The mean omega-3 index of the study cohort was 6.19 ± 1.55 %. In more than 85 % of the patients, the omega-3 index was below 8 % and in 23 % below 5 %. Moreover, it was shown that a low DHA status and omega-3 index was associated with depressive symptoms (Beck's depression-inventory). These findings indicate an

  8. A kinetic Monte Carlo approach to investigate antibiotic translocation through bacterial porins

    NASA Astrophysics Data System (ADS)

    Ceccarelli, Matteo; Vargiu, Attilio V.; Ruggerone, Paolo

    2012-03-01

    Many relevant biological processes take place on time scales not reachable by standard all-atom computer simulations. The translocation of antibiotics through non-specific bacterial porins is an example. Microscopic effects compete to determine penetration routes and, consequently, free energy barriers to be overcome. Since bacteria can develop resistance to treatment also by reducing their antibiotic permeability, to understand the microscopic aspects of antibiotic translocation is an important step to rationalize drug design. Here, to investigate the translocation we propose a complete numerical model that combines the diffusion-controlled rate theory and a kinetic Monte Carlo scheme based on both experimental data and microscopically well-founded all-atom simulations. Within our model, an antibiotic translocating through an hour-glass-shaped channel can be described as a molecule moving on a potential of mean force featuring several affinity sites and a high central barrier. The implications of our results for the characterization of antibiotic translocation at in vivo concentrations are discussed. The presence of an affinity site close to the mouth of the channel seems to favor the translocation of antibiotics, the affinity site acting as a particle reservoir. Possible connections between results and the appearance of mutations in clinical strains are also outlined.

  9. Fusion of phospholipid vesicles with a planar membrane depends on the membrane permeability of the solute used to create the osmotic pressure

    PubMed Central

    1989-01-01

    Phospholipid vesicles fuse with a planar membrane when they are osmotically swollen. Channels in the vesicle membrane are required for swelling to occur when the vesicle-containing compartment is made hyperosmotic by adding a solute (termed an osmoticant). We have studied fusion using two different channels, porin, a highly permeable channel, and nystatin, a much less permeable channel. We report that an osmoticant's ability to support fusion (defined as the magnitude of osmotic gradient necessary to obtain sustained fusion) depends on both its permeability through lipid bilayer as well as its permeability through the channel by which it enters the vesicle interior. With porin as the channel, formamide requires an osmotic gradient about ten times that required with urea, which is approximately 1/40th as permeant as formamide through bare lipid membrane. When nystatin is the channel, however, fusion rates sustained by osmotic gradients of formamide are within a factor of two of those obtained with urea. Vesicles containing a porin-impermeant solute can be induced to swell and fuse with a planar membrane when the impermeant bathing the vesicles is replaced by an isosmotic quantity of a porin-permeant solute. With this method of swelling, formamide is as effective as urea in obtaining fusion. In addition, we report that binding of vesicles to the planar membrane does not make the contact region more permeable to the osmoticant than is bare lipid bilayer. In the companion paper, we quantitatively account for the observation that the ability of a solute to promote fusion depends on its permeability properties and the method of swelling. We show that the intravesicular pressure developed drives fusion. PMID:2539429

  10. Brattleboro rats have impaired apical membrane water permeability regulation in the outer medullary collecting duct principal cells.

    PubMed

    Baturina, Galina S; Katkova, Liubov E; Zarogiannis, Sotirios G; Solenov, Evgeniy I

    2016-12-01

    Vasopressin (AVP) regulates the body salt-water balance. Brattleboro rats carry an AVP gene mutation resulting in a recessive form of central diabetes insipidus, being ideal for AVP deficiency studies. Herein, we studied the water permeability of the apical and basolateral sides of outer medullary collecting duct (OMCD) principal cells in response to dDAVP (a V2 receptor agonist) administration in Wistar and Brattleboro rats. Biophysical measurements of the water permeability (Pf ) of isolated OMCD principal cells were performed with the calcein quenching method with/without dDAVP (10(-8)  mol/L). mRNA transcripts and protein levels of AQP2, AQP3 and AQP4 were assessed by RT-PCR and western blot respectively. dDAVP increased the apical and basolateral Pf of OMCD principal cells in Wistar rats, while in Brattleboro rats this effect was present basolaterally. Long-term dDAVP administration in both strains resulted in a significant increase in mRNA expression of all assessed AQP's while only the protein levels of AQP2 and AQP3 were significantly increased. Short-term (20 minutes) dDAVP treatment of isolated OMCD fragments resulted in significantly increased plasma membrane expression of AQP2 in Wistar rats and of AQP2 and AQP3 in Brattleboro rats. In summary, dDAVP induces different expression of AQP2, AQP3 and AQP4 in Wistar and Brattleboro rats during short- and long-term treatment. In Wistar rats dDAVP mainly increased AQP2 expression while in Brattleboro rats it increased functional water permeability mainly by AQP3 expression. © 2016 John Wiley & Sons Australia, Ltd.

  11. Exercise affects memory acquisition, anxiety-like symptoms and activity of membrane-bound enzyme in brain of rats fed with different dietary fats: impairments of trans fat.

    PubMed

    Teixeira, A M; Pase, C S; Boufleur, N; Roversi, K; Barcelos, R C S; Benvegnú, D M; Segat, H J; Dias, V T; Reckziegel, P; Trevizol, F; Dolci, G S; Carvalho, N R; Soares, F A A; Rocha, J B T; Emanuelli, T; Bürger, M E

    2011-11-10

    Here we evaluated the influence of physical exercise on behavior parameters and enzymatic status of rats supplemented with different dietary fatty acids (FA). Male Wistar rats fed diets enriched with soybean oil (SO), lard (L), or hydrogenated vegetable fat (HVF) for 48 weeks were submitted to swimming (30 min/d, five times per week) for 90 days. Dietary FA per se did not cause anxiety-like symptoms in the animals, but after physical exercise, SO group showed a better behavioral performance than L and the HVF groups in elevated plus maze (EPM). In Barnes maze, HVF group showed impaired memory acquisition as compared to L group, and exercise reversed this effect. SO-fed rats showed an improvement in memory acquisition after 1 day of training, whereas lard caused an improvement of memory only from day 4. HVF-fed rats showed no improvement of memory acquisition, but this effect was reversed by exercise in all training days. A lower activity of the Na(+)K(+)-ATPase in brain cortex of rats fed lard and HVF was observed, and this effect was maintained after exercise. Similarly, the HVF diet was related to lower activity of hippocampal Na(+)K(+)-ATPase, and exercise reduced activity of this enzyme in the SO and L groups. Our findings show influences of dietary FA on memory acquisition, whereas regular exercise improved this function and was beneficial on anxiety-like symptoms. As FA are present in neuronal membrane phospholipids and play a critical role in brain function, our results suggest that low incorporation of trans FA in neuronal membranes may act on cortical and hippocampal Na(+)K(+)-ATPase activity, but this change appears to be unrelated to the behavioral parameters primarily harmed by consumption of trans and less so by saturated FA, which were reversed by exercise.

  12. β cell membrane remodelling and procoagulant events occur in inflammation-driven insulin impairment: a GLP-1 receptor dependent and independent control.

    PubMed

    Gleizes, Céline; Kreutter, Guillaume; Abbas, Malak; Kassem, Mohamad; Constantinescu, Andrei Alexandru; Boisramé-Helms, Julie; Yver, Blandine; Toti, Florence; Kessler, Laurence

    2016-02-01

    Inflammation and hyperglycaemia are associated with a prothrombotic state. Cell-derived microparticles (MPs) are the conveyors of active procoagulant tissue factor (TF) and circulate at high concentration in diabetic patients. Liraglutide, a glucagon-like peptide (GLP)-1 analogue, is known to promote insulin secretion and β-cell preservation. In this in vitro study, we examined the link between insulin impairment, procoagulant activity and plasma membrane remodelling, under inflammatory conditions. Rin-m5f β-cell function, TF activity mediated by MPs and their modulation by 1 μM liraglutide were examined in a cell cross-talk model. Methyl-β-cyclodextrine (MCD), a cholesterol depletor, was used to evaluate the involvement of raft on TF activity, MP shedding and insulin secretion as well as Soluble N-éthylmaleimide-sensitive-factor Attachment protein Receptor (SNARE)-dependent exocytosis. Cytokines induced a two-fold increase in TF activity at MP surface that was counteracted by liraglutide. Microparticles prompted TF activity on the target cells and a two-fold decrease in insulin secretion via protein kinase A (PKA) and p38 signalling, that was also abolished by liraglutide. Large lipid raft clusters were formed in response to cytokines and liraglutide or MCD-treated cells showed similar patterns. Cells pre-treated by saturating concentration of the GLP-1r antagonist exendin (9-39), showed a partial abolishment of the liraglutide-driven insulin secretion and liraglutide-decreased TF activity. Measurement of caspase 3 cleavage and MP shedding confirmed the contribution of GLP-1r-dependent and -independent pathways. Our results confirm an integrative β-cell response to GLP-1 that targets receptor-mediated signalling and membrane remodelling pointing at the coupling of insulin secretion and inflammation-driven procoagulant events. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and

  13. Membrane lipid defects are responsible for the generation of reactive oxygen species in peripheral blood mononuclear cells from vitiligo patients.

    PubMed

    Dell'Anna, Maria Lucia; Ottaviani, Monica; Bellei, Barbara; Albanesi, Veronica; Cossarizza, Andrea; Rossi, Luisa; Picardo, Mauro

    2010-04-01

    The pathogenesis of vitiligo, an acquired depigmenting disease of the skin, involves oxidative stress. Based on that, the generation of reactive oxygen species (ROS) by the mitochondria may be relevant in the pathogenesis of vitiligo. Here, we evaluate the biochemical and functional alterations involved in the defective activity that has been previously described both in melanocytes and peripheral blood mononuclear cells (PBMC) from vitiligo patients. Moreover, we used a freeze-thaw test as a mild stress stimulus to disclose any latent defects in the assembly of membrane lipids that may compromise the functionality of the membrane itself. We show that the lipid constitution of the membrane is altered in vitiligo. Specifically, the cardiolipin (CL) level in the mitochondrial inner membrane is reduced and the level of cholesterol is increased. Furthermore, an increase in the expression level of 3-hydroxy-3methyl-glutaryl-CoenzymeA-reductase (HMG-CoA reductase), the rate-limiting enzyme for cholesterol biosynthesis, was also seen. Associated with that, the expression of electron transport chain (ETC) lipid-dependent subunits was also modified, and their expression was further affected by the freeze-thaw stress. The expression of CL-independent mitochondrial proteins, such as porin and Bcl2, were unaffected in vitiligo PBMC. These data confirm that ETC protein expression mainly correlates with lipid arrangement and that loss of their expression is not due to generalized or random oxidative-mediated damage. We suggest that the modification of membrane lipid components in vitiligo cells may be the biochemical basis for the mitochondrial impairment and the subsequent production of intracellular ROS following the exposure to a mild stress. J. Cell. Physiol. 223: 187-193, 2010. (c) 2009 Wiley-Liss, Inc.

  14. Sodium chlorate, a major water disinfection byproduct, alters brush border membrane enzymes, carbohydrate metabolism and impairs antioxidant system of Wistar rat intestine.

    PubMed

    Ali, Shaikh Nisar; Ansari, Fariheen Aisha; Khan, Aijaz Ahmed; Mahmood, Riaz

    2017-05-01

    Sodium chlorate (NaClO3 ) is a widely used nonselective herbicide. It is also generated as a by-product during disinfection of drinking water by chlorine dioxide. The purpose of this study was to evaluate the effect of NaClO3 on rat intestine. Adult male rats were randomly divided into five groups: control and remaining four groups were administered orally different doses of NaClO3 and sacrificed 24 h after the treatment. The administration of NaClO3 produced acute oxidative stress in the intestine, which manifested in the form of markedly enhanced malondialdehyde levels and carbonyl content and lowered total sulfhydryl groups and glutathione levels. The activities of several brush border membrane (BBM) enzymes were greatly reduced as compared to control. There were alterations in the activities of various enzymes of carbohydrate metabolism and those involved in maintaining the antioxidant defense system. Histological studies support the biochemical results showing NaClO3 dose-dependent increase in tissue damage. Thus, the present study shows that oral administration of NaClO3 decreases the activities of BBM enzymes, induces oxidative stress, alters metabolic pathways, and impairs the antioxidant system of rat intestine. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1607-1616, 2017. © 2017 Wiley Periodicals, Inc.

  15. Juglone disrupts root plasma membrane H+-ATPase activity and impairs water uptake, root respiration, and growth in soybean (Glycine max) and corn (Zea mays).

    PubMed

    Hejl, Angela M; Koster, Karen L

    2004-02-01

    Juglone is phytotoxic, but the mechanisms of growth inhibition have not been fully explained. Previous studies have proposed that disruption of electron transport functions in mitochondria and chloroplasts contribute to observed growth reduction in species exposed to juglone. In studies reported here, corn and soybean seedlings grown in nutrient solution amended with 10, 50, or 100 microM juglone showed significant decreases in root and shoot dry weights and lengths with increasing concentrations. However, no significant differences in leaf chlorophyll fluorescence or CO2-dependent leaf oxygen evolution were observed, even in seedlings that were visibly affected. Disruption of root oxygen uptake was positively correlated with increasing concentrations of juglone, suggesting that juglone may reach mitochondria in root cells. Water uptake and acid efflux also decreased for corn and soybean seedlings treated with juglone, suggesting that juglone may affect metabolism of root cells by disrupting root plasma membrane function. Therefore, the effect of juglone on H+-ATPase activity in corn and soybean root microsomes was tested. Juglone treatments from 10 to 1000 microM significantly reduced H+-ATPase activity compared to controls. This inhibition of H+-ATPase activity and observed reduction of water uptake offers a logical explanation for previously documented phytotoxicity of juglone. Impairment of this enzyme's activity could affect plant growth in a number of ways because proton-pumping in root cells drives essential plant processes such as solute uptake and, hence, water uptake.

  16. A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin-resistant diabetes.

    PubMed Central

    Accili, D; Frapier, C; Mosthaf, L; McKeon, C; Elbein, S C; Permutt, M A; Ramos, E; Lander, E; Ullrich, A; Taylor, S I

    1989-01-01

    Insulin binds to a receptor on the cell surface, thereby triggering a biological response within the target cell. Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a family in which two sisters have a genetic form of insulin-resistant diabetes mellitus. The technique of homozygosity mapping has been used to demonstrate that the mutation causing diabetes in this consanguineous family is genetically linked to the insulin receptor gene. The two insulin-resistant sisters are homozygous for a mutation encoding substitution of valine for phenylalanine at position 382 in the alpha-subunit of the insulin receptor. Transfection of mutant insulin receptor cDNA into NIH3T3 cells demonstrated that the Val382 mutation impaired post-translational processing and retarded transport of the insulin receptor to the plasma membrane. Thus, the mutation causes insulin resistance by decreasing the number of insulin receptors on the surface of the patients' cells. Images PMID:2573522

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

    PubMed

    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

    2013-08-01

    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.

  18. 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

  19. Diffusion of beta-lactam antibiotics through the porin channels of Escherichia coli K-12.

    PubMed Central

    Yoshimura, F; Nikaido, H

    1985-01-01

    Diffusion rates of various beta-lactam antibiotics through the OmpF and OmpC porin channels of Escherichia coli K-12 were measured by the use of reconstituted proteoliposomes. The results can be interpreted on the basis of the gross physicochemical properties of the antibiotics along the following lines. (i) As noted previously (Nikaido et al., J. Bacteriol., 153:232-240, 1983), there was a monotonous dependence of the penetration rate on the hydrophobicity of the molecule among the classical monoanionic beta-lactams, and a 10-fold increase in the octanol-water partition coefficient of the uncharged molecule decreased the penetration rate by a factor of 5 to 6. (ii) Compounds with exceptionally bulky side chains, such as mezlocillin, piperacillin, and cefoperazone, showed much slower penetration rates than expected from their hydrophobicity. (iii) The substituted oxime side chain on the alpha-carbon of the substituent group at position 7 of the cephem nucleus decreased the penetration rate almost by an order of magnitude; this appears to be largely due to the steric effect. (iv) The presence of a methoxy group at position 7 of the cephalosporins also reduced the penetration rate by 20%, probably also due to the steric hindrance. (v) Zwitterionic compounds penetrated very rapidly, and the correlation between the rate and hydrophobicity appeared to be much weaker than with the monoanionic compounds. Imipenem showed the highest permeability among the compounds tested, presumably due, at least in part, to its compact molecular structure. (vi) Compounds with two negative charges penetrated more slowly than did analogs with only one negatively charged group. Among them, only moxalactam, ceftriaxone, and azthreonam showed penetration rates corresponding to, or higher than, 10% of that of imipenem. PMID:2580479

  20. Restoration of antibody binding to blotted meningococcal outer membrane proteins using various detergents.

    PubMed

    Wedege, E; Bryn, K; Frøholm, L O

    1988-10-04

    Restoration of IgG antibody binding to heat-denatured meningococcal outer membrane proteins has been studied on immunoblots with a series of 14 detergents. Nitrocellulose strips with the blotted proteins were incubated with the detergents and sera from human volunteers vaccinated with meningococcal membrane proteins. Zwitterionic and ionic detergents, containing substituted quarternary ammonium or amino groups with a minimum of 10 C atoms in the alkyl chain, restored the antigenicity of the serotype-specific class 2 porin protein. The concentrations of the Zwittergent detergents necessary for activation decreased with increasing alkyl chain length of the homologues. Only zwitterionic detergents renatured the class 1 protein. Both proteins were weakly antigenic in the presence of the nonionic detergents Triton X-100 and Tween 20. Meningococcal lipopolysaccharide restored antibody binding to the porin, but not to the class 1 protein. Similar concentrations of lipopolysaccharides from two other gram-negative bacteria had no effect.

  1. Activation of the Complement Classical Pathway (C1q Binding) by Mesophilic Aeromonas hydrophila Outer Membrane Protein

    PubMed Central

    Merino, Susana; Nogueras, Maria Mercedes; Aguilar, Alicia; Rubires, Xavier; Albertí, Sebastian; Benedí, Vicente Javier; Tomás, Juan M.

    1998-01-01

    The mechanism of killing of Aeromonas hydrophila serum-sensitive strains in nonimmune serum by the complement classical pathway has been studied. The bacterial cell surface component that binds C1q more efficiently was identified as a major outer membrane protein of 39 kDa, presumably the porin II described by D. Jeanteur, N. Gletsu, F. Pattus, and J. T. Buckley (Mol. Microbiol. 6:3355–3363, 1992), of these microorganisms. We have demonstrated that the purified form of porin II binds C1q and activates 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 of factor D). Binding of C1q to other components of the bacterial outer membrane, in particular to rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the outer membrane protein. The strains possessing O-antigen lipopolysaccharide bind less C1q than the serum-sensitive strains, because the outer membrane protein is less accessible, and are resistant to complement-mediated killing. Finally, a similar or identical outer membrane protein (presumably porin II) that binds C1q was shown to be present in strains from the most common mesophilic Aeromonas O serogroups. PMID:9673268

  2. Inhibition of Latent Membrane Protein 1 Impairs the Growth and Tumorigenesis of Latency II Epstein-Barr Virus-Transformed T Cells

    PubMed Central

    Ndour, Papa Alioune; Brocqueville, Guillaume; Ouk, Tan-Sothéa; Goormachtigh, Gautier; Morales, Olivier; Mougel, Alexandra; Bertout, Julie; Melnyk, Oleg; Fafeur, Véronique; Feuillard, Jean; Coll, Jean

    2012-01-01

    Epstein-Barr virus (EBV) is a common human herpesvirus. Infection with EBV is associated with several human malignancies in which the virus expresses a set of latent proteins, among which is latent membrane protein 1 (LMP1). LMP1 is able to transform numerous cell types and is considered the main oncogenic protein of EBV. The mechanism of action is based on mimicry of activated members of the tumor necrosis factor (TNF) receptor superfamily, through the ability of LMP1 to bind similar adapters and to activate signaling pathways. We previously generated two unique models: a monocytic cell line and a lymphocytic (NC5) cell line immortalized by EBV that expresses the type II latency program. Here we generated LMP1 dominant negative forms (DNs), based on fusion between green fluorescent protein (GFP) and transformation effector site 1 (TES1) or TES2 of LMP1. Then we generated cell lines conditionally expressing these DNs. These DNs inhibit NF-κB and Akt pathways, resulting in the impairment of survival processes and increased apoptosis in these cell lines. This proapoptotic effect is due to reduced interaction of LMP1 with specific adapters and the recruitment of these adapters to DNs, which enable the generation of an apoptotic complex involving TRADD, FADD, and caspase 8. Similar results were obtained with cell lines displaying a latency III program in which LMP1-DNs decrease cell viability. Finally, we prove that synthetic peptides display similar inhibitory effects in EBV-infected cells. DNs derived from LMP1 could be used to develop therapeutic approaches for malignant diseases associated with EBV. PMID:22258264

  3. Inhibition of latent membrane protein 1 impairs the growth and tumorigenesis of latency II Epstein-Barr virus-transformed T cells.

    PubMed

    Ndour, Papa Alioune; Brocqueville, Guillaume; Ouk, Tan-Sothéa; Goormachtigh, Gautier; Morales, Olivier; Mougel, Alexandra; Bertout, Julie; Melnyk, Oleg; Fafeur, Véronique; Feuillard, Jean; Coll, Jean; Adriaenssens, Eric

    2012-04-01

    Epstein-Barr virus (EBV) is a common human herpesvirus. Infection with EBV is associated with several human malignancies in which the virus expresses a set of latent proteins, among which is latent membrane protein 1 (LMP1). LMP1 is able to transform numerous cell types and is considered the main oncogenic protein of EBV. The mechanism of action is based on mimicry of activated members of the tumor necrosis factor (TNF) receptor superfamily, through the ability of LMP1 to bind similar adapters and to activate signaling pathways. We previously generated two unique models: a monocytic cell line and a lymphocytic (NC5) cell line immortalized by EBV that expresses the type II latency program. Here we generated LMP1 dominant negative forms (DNs), based on fusion between green fluorescent protein (GFP) and transformation effector site 1 (TES1) or TES2 of LMP1. Then we generated cell lines conditionally expressing these DNs. These DNs inhibit NF-κB and Akt pathways, resulting in the impairment of survival processes and increased apoptosis in these cell lines. This proapoptotic effect is due to reduced interaction of LMP1 with specific adapters and the recruitment of these adapters to DNs, which enable the generation of an apoptotic complex involving TRADD, FADD, and caspase 8. Similar results were obtained with cell lines displaying a latency III program in which LMP1-DNs decrease cell viability. Finally, we prove that synthetic peptides display similar inhibitory effects in EBV-infected cells. DNs derived from LMP1 could be used to develop therapeutic approaches for malignant diseases associated with EBV.

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

    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.

  5. 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.

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

    PubMed

    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

    2014-10-30

    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.

  7. Isolation and Characterization of the Outer Membrane of Borrelia hermsii

    PubMed Central

    Shang, Ellen S.; Skare, Jonathan T.; Exner, Maurice M.; Blanco, David R.; Kagan, Bruce L.; Miller, James N.; Lovett, Michael A.

    1998-01-01

    The outer membrane of Borrelia hermsii has been shown by freeze-fracture analysis to contain a low density of membrane-spanning outer membrane proteins which have not yet been isolated or identified. In this study, we report the purification of outer membrane vesicles (OMV) from B. hermsii HS-1 and the subsequent identification of their constituent outer membrane proteins. The B. hermsii outer membranes were released by vigorous vortexing of whole organisms in low-pH, hypotonic citrate buffer and isolated by isopycnic sucrose gradient centrifugation. The isolated OMV exhibited porin activities ranging from 0.2 to 7.2 nS, consistent with their outer membrane origin. Purified OMV were shown to be relatively free of inner membrane contamination by the absence of measurable β-NADH oxidase activity and the absence of protoplasmic cylinder-associated proteins observed by Coomassie blue staining. Approximately 60 protein spots (some of which are putative isoelectric isomers) with 25 distinct molecular weights were identified as constituents of the OMV enrichment. The majority of these proteins were also shown to be antigenic with sera from B. hermsii-infected mice. Seven of these antigenic proteins were labeled with [3H]palmitate, including the surface-exposed glycerophosphodiester phosphodiesterase, the variable major proteins 7 and 33, and proteins of 15, 17, 38, 42, and 67 kDa, indicating that they are lipoprotein constituents of the outer membrane. In addition, immunoblot analysis of the OMV probed with antiserum to the Borrelia garinii surface-exposed p66/Oms66 porin protein demonstrated the presence of a p66 (Oms66) outer membrane homolog. Treatment of intact B. hermsii with proteinase K resulted in the partial proteolysis of the Oms66/p66 homolog, indicating that it is surface exposed. This identification and characterization of the OMV proteins should aid in further studies of pathogenesis and immunity of tick-borne relapsing fever. PMID:9488399

  8. 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

    2014-10-03

    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.

  9. Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation

    PubMed Central

    Kwon, Tackmin

    2016-01-01

    The efficiency of Agrobacterium-mediated transformation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection. PMID:27643450

  10. Membrane permeability, a pivotal function involved in antibiotic resistance and virulence in Enterobacter aerogenes clinical isolates.

    PubMed

    Lavigne, J-P; Sotto, A; Nicolas-Chanoine, M-H; Bouziges, N; Bourg, G; Davin-Regli, A; Pagès, J-M

    2012-06-01

    Imipenem-susceptible E. aerogenes isolates exhibiting extended spectrum β-lactamases, target mutations and a basal efflux expression, were identified in five patients. After imipenem treatment, imipenem-intermediate susceptible (IMI-I) or resistant (IMI-R) isolates emerged in these patients. Alteration in porin synthesis and increase in efflux expression were observed in the IMI-I isolates whereas complete loss of the porins, LPS alteration and efflux overexpression were observed in the IMI-R isolates. Bacterial virulence of the strains was investigated by the Caenorhabditis elegans model. The IMI-R isolates were shown to be significantly less virulent than the IMI-susceptible or IMI-I isolates. The pleiotropic membrane alteration and its associated fitness burden exhibited by E. aerogenes isolates influence their antibiotic resistance and their virulence behaviour. These findings highlight the balance between the low permeability-related resistance and virulence and their relationships with the treatment of resistant pathogens.

  11. Studies on the expression of outer membrane protein 2 in escherichia coli.

    PubMed

    Fralick, J A; Diedrich, D L

    1982-01-01

    The relative level of protein 2 expressed in the outer membrane of strains of Escherichia coli K-12 lysogenized with bacteriophage PA-2 was found to be influenced by both the growth temperature and lc+ gene dosage. An increase in either of these parameters was accompanied by an increase in the level of protein 2 up to an apparent saturation level. Any increase in the amount of protein 2 was accompanied by a concomittant decrease in the amount of OmpF and OmpC porins. This inverse relationship led to the maintenance of an approximately constant protein mass per unit of peptidoglycan. Our results are discussed in light of recent genetic studies on the regulation of the OmpF and OmpC porins and can be explained through the competition of these three matrix proteins for a common export or insertion site.

  12. Identification of Neisseria meningitidis outer membrane vesicle complexes using 2-D high resolution clear native/SDS-PAGE.

    PubMed

    Marzoa, Juan; Sánchez, Sandra; Ferreirós, Carlos M; Criado, María Teresa

    2010-01-01

    The identification and characterization of meningococcal outer membrane vesicle complexes can be important for gaining an in-depth understaining of their structure and functionality. Analysis of the vesicle complexome by 'traditional' 2-D analysis, in which isoelectrofocusing is used for separation in the first dimension, is hampered by the high hydrophobicity and extreme isoelectric points of many relevant proteins. Analysis of the meningococcal outer membrane vesicle complexome using Blue Native (nondenaturing) electrophoresis instead of isoelectrofocusing in the first dimension showed several porin complexes, but their composition could not be clearly resolved after separation by SDS-PAGE in the second dimension. In this work, using a recently described native separation technique -high resolution Clear Native Electrophoresis-and different bidimensional approaches, we were able to demonstrate the presence of relevant outer membrane complexes which could be resolved with a higher resolution than in previous analysis. The most relevant were nine porin complexes formed by different combinations of the meningococcal PorA, PorB and RmpM proteins, and comparison with the complexes formed in specific knockout mutants allowed us to infer the relevance of each porin in the formation of each complex.

  13. 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.

  14. 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

  15. Peroxisome biogenesis disorders: molecular basis for impaired peroxisomal membrane assembly: in metabolic functions and biogenesis of peroxisomes in health and disease.

    PubMed

    Fujiki, Yukio; Yagita, Yuichi; Matsuzaki, Takashi

    2012-09-01

    Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS). Gene defects of peroxins required for both membrane assembly and matrix protein import are identified: ten mammalian pathogenic peroxins for ten complementation groups of PBDs, are required for matrix protein import; three, Pex3p, Pex16p and Pex19p, are shown to be essential for peroxisome membrane assembly and responsible for the most severe ZS in PBDs of three complementation groups 12, 9, and 14, respectively. Patients with severe ZS with defects of PEX3, PEX16, and PEX19 tend to carry severe mutation such as nonsense mutations, frameshifts and deletions. With respect to the function of these three peroxins in membrane biogenesis, two distinct pathways have been proposed for the import of peroxisomal membrane proteins in mammalian cells: a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex16p-dependent class II pathway. In class II pathway, Pex19p also forms a soluble complex with newly synthesized Pex3p as the chaperone for Pex3p in the cytosol and directly translocates it to peroxisomes. Pex16p functions as the peroxisomal membrane receptor that is specific to the Pex3p-Pex19p complexes. A model for the import of peroxisomal membrane proteins is suggested, providing new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p. Another model suggests that in Saccharomyces cerevisiae peroxisomes likely emerge from the endoplasmic reticulum. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. 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.

  17. The PorB porin from commensal Neisseria lactamica induces Th1 and Th2 immune responses to ovalbumin in mice and is a potential immune adjuvant.

    PubMed

    Liu, Xiuping; Wetzler, Lee M; Massari, Paola

    2008-02-06

    Porins from pathogenic Neisseriae are among several bacterial products with immune adjuvant activity. Neisseria meningitidis (Nme) PorB, has been shown to induce immune cells activation in a TLR2-dependent manner and acts as a vaccine immune adjuvant. The PorB porin from Neisseria lactamica (Nlac), a common nasopharyngeal commensal, shares significant structural and functional similarities with Nme PorB. In this work we ask whether the immune adjuvant ability of porins from pathogenic Neisserial strains is a characteristic shared with porins from non-pathogenic Neisserial species or whether it is unique for bacterial products derived from microorganisms capable of inducing inflammation and disease. We evaluated the potential immune adjuvant effect of Nlac PorB in mice using ovalbumin (OVA) as a prototype antigen. Immunization with Nlac PorB/OVA induced high OVA-specific IgG and IgM titers compared to OVA alone, similar to other adjuvants such as Nme PorB and alum. High titers of IgG1 and IgG2b were detected as well as production of IL-4, IL-10, IL-12 and INF-gamma in response to Nlac PorB, consistent with induction of both a Th1-type and a Th2-type immune response. OVA-specific proliferation was also determined in splenocytes from Nlac PorB/OVA-immunized mice. In addition, B cell activation in vitro and cytokine production in response to Nlac PorB was found to be mediated by TLR2, in a similar manner to Nme PorB.

  18. High prevalence of non-clonal imipenem-nonsusceptible Enterobacter spp. isolates in Korea and their association with porin down-regulation.

    PubMed

    Lee, Ji-Young; Hong, Yoon-Kyoung; Lee, Haejeong; Ko, Kwan Soo

    2017-01-01

    We investigated the prevalence and clonal distribution of imipenem-nonsusceptible Enterobacter clinical isolates from hospitals in Korea and the contributions of various mechanisms to imipenem nonsusceptibility. The in vitro antimicrobial susceptibility to imipenem of 357 non-duplicated Enterobacter isolates obtained from eight geographically distant tertiary care hospitals in Korea was evaluated. Imipenem-nonsusceptible Enterobacter isolates were genotyped. Additionally, β-lactamase genes were screened using PCR, and the expression of efflux pump and porin genes was investigated using quantitative RT-PCR. A total of 31 isolates (8.7%) were not susceptible to imipenem. Clonal diversity of 17 imipenem-nonsusceptible E. cloacae isolates was demonstrated by multilocus sequence typing. Fourteen imipenem-nonsusceptible E. aerogenes isolates were found to be distantly genetically related by an ERIC-PCR analysis. Expression levels of porin ompD and ompK35 genes were decreased in all imipenem-nonsusceptible E. cloacae and E. aerogenes isolates. However, only two isolates were found positive for blaIMP and blaVIM genes, and expression of the efflux pump gene, acrB, was not associated with reduced imipenem susceptibility. Imipenem resistance seems to have occurred independently in most of the imipenem-nonsusceptible isolates in this study, and decreased porin expression was found to be the main mechanism underlying this reduced susceptibility to imipenem. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. [Purification of Salmonella Typhimurium OmpD porin induces long-term high levels of antibodies: implications on the development of vaccines against non-typhoid salmonella].

    PubMed

    Pérez-Toledo, Marisol; Martínez-Amador, Paola A; Pastelin-Palacios, Rodolfo; Isibasi, Armando; Cunningham, Adam F; López-Macías, Constantino

    2016-10-01

    In the present work, we report, for the first time, on the purification of the Salmonella Typhimurium OmpD porin. We assessed the integrity and purity of the protein and evaluated the immunogenicity of the protein and its ability to induce antibody without exogenous adjuvant. We observed that 10 μg OmpD induced high antibody levels of IgM and IgG, which were maintained for more than 260 days after immunization. Immunization with OmpD induced multiple IgG antibody isotypes including IgG1, IgG2a, IgG2b, and IgG3 subclasses. Furthermore, these antibodies were able to recognize and bind to the bacterial surface. Our results demonstrate the high immunogenicity of S. Typhimurium OmpD porin, which induces long-lasting antibodies which may be and important target of the immune response against Salmonella infection. In conclusion, we propose the OmpD porin could be used within novel subunit vaccine formulations that do not need additional adjuvant and that confer long lasting humoral immunity against Salmonella infections.

  20. The Fusobacterium nucleatum major outer-membrane protein (FomA) forms trimeric, water-filled channels in lipid bilayer membranes.

    PubMed

    Kleivdal, H; Benz, R; Jensen, H B

    1995-10-01

    The pore-forming activity of the major outer-membrane protein FomA of the anaerobic Fusobacterium nucleatum was studied in artificial lipid bilayer membranes. FomA was isolated from F. nucleatum strains Fev1, ATCC 10953, and ATCC 25586 by extraction with lithium dodecyl sulfate and lithium chloride and had an apparent molecular mass of about 40 kDa. When solubilized at low temperatures, the protein ran with an apparent molecular mass of about 62 kDa on SDS/PAGE. Cross-linking experiments and two-dimensional SDS/PAGE gave evidence that the 62-kDa protein band represented the trimeric form of FomA. The protein trimers were susceptible to SDS and temperature. The stability of the porin trimers varied among the strains. The properties of the FomA channels were studied in reconstitution experiments with black lipid bilayer membranes. The F. nucleatum porins formed channels with single-channel conductances in the range 0.66-1.30 nS in M KCl. The single-channel conductance was a function of the mobilities of the ions present in the aqueous solution bathing the bilayer membrane. This means that FomA forms general diffusion channels since (a) the conductance showed a linear dependence on the salt concentration, (b) the ion selectivity was small and varied for the three strains, and (c) the channels did not exhibit any binding site for maltotriose or triglycine. The water-filled channel was voltage dependent, and conductance decrements were observed at transmembrane potentials of +/- 50 mV. The conductance decrement steps were about one-third of the total conductance of a functional unit in its fully 'open' state. This strongly suggests that the trimer is the functional unit of the porin.

  1. 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

  2. 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.

  3. Phenotypic and Genotypic Analyses of Neisseria gonorrhoeae Isolates That Express Frequently Recovered PorB PIA Variable Region Types Suggest that Certain P1a Porin Sequences Confer a Selective Advantage for Urogenital Tract Infection▿

    PubMed Central

    Garvin, Lotisha E.; Bash, Margaret C.; Keys, Christine; Warner, Douglas M.; Ram, Sanjay; Shafer, William M.; Jerse, Ann E.

    2008-01-01

    Typing of the porB variable region (VR) is an epidemiological tool that classifies gonococcal strains based on sequence differences in regions of the porB gene that encode surface-exposed loops. The frequent isolation of certain porB VR types suggests that some porin sequences confer a selective advantage during infection and/or transmission. Alternatively, certain porin types may be markers of strains that are successful due to factors unrelated to porin. In support of the first hypothesis, here we show urogenital tract isolates representing the most common PIA VR types identified in an urban clinic in Baltimore, MD, over a 10-year period belonged to several different clonal types, as determined by pulsed-field gel electrophoresis (PFGE). Serum resistance, which was confirmed by factor H and C4b-binding protein binding studies, was more often associated with gonococcal the most common VR types. In contrast, three porin-independent phenotypes, namely, lactoferrin utilization, β-lactamase production, and multiple transferable resistance (Mtr), were segregated with the PFGE cluster and not with the VR type. Data combined with another PIA strain collection showed a strong correlation between serum resistance and the most common VR types. A comparison of VR typing hybridization patterns and nucleotide sequences of 12 porB1a genes suggests that certain porin loop 1, 3, 6, and/or 7 sequences may play a role in the serum resistance phenotype. We conclude that some PorB PIA sequences confer a survival or transmission advantage in the urogenital tract, perhaps via increased resistance to complement-mediated killing. The capacity of some porin types to evade a porin-specific adaptive immune response must also be considered. PMID:18541655

  4. Negative osmoregulation of the Salmonella ompS1 porin gene independently of OmpR in an hns background.

    PubMed

    Flores-Valdez, Mario Alberto; Puente, José Luis; Calva, Edmundo

    2003-11-01

    The ompS1 gene encodes a quiescent porin in Salmonella enterica serovars Typhi and Typhimurium. By using random mariner transposon mutagenesis, mutations that caused derepression of ompS1 expression were isolated, one in S. enterica serovar Typhi and two in S. enterica serovar Typhimurium. All of them mapped in the hns gene in the region coding for the carboxy terminus of the H-NS nucleoid protein. The derepressed ompS1 expression was subject to negative regulation at high osmolarity, both in the presence and in the absence of OmpR. This observation was possible due to the fact that there are two promoters: P1, which is OmpR dependent, and P2, which does not require OmpR for activation (rather, OmpR represses P2). The sequences upstream from position -88, a region previously shown to be involved in the negative regulation of ompS1, can form a static bend, and the integrity of this region was required for function and binding of H-NS and for osmoregulation, as determined with gene reporter fusions of different lengths and with a 31-bp deletion mutant. This is consistent with the notion that this region determines a structure required for repression. Hence, ompS1 shares negative regulation by H-NS with other loci, such as the bgl operon and the ade gene.

  5. Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA.

    PubMed

    Corcoran, Colin P; Podkaminski, Dimitri; Papenfort, Kai; Urban, Johannes H; Hinton, Jay C D; Vogel, Jörg

    2012-05-01

    MicF is a textbook example of a small regulatory RNA (sRNA) that acts on a trans-encoded target mRNA through imperfect base pairing. Discovery of MicF as a post-transcriptional repressor of the major Escherichia coli porin OmpF established the paradigm for a meanwhile common mechanism of translational inhibition, through antisense sequestration of a ribosome binding site. However, whether MicF regulates additional genes has remained unknown for almost three decades. Here, we have harnessed the new superfolder variant of GFP for reporter-gene fusions to validate newly predicted targets of MicF in Salmonella. We show that the conserved 5' end of MicF acts by seed pairing to repress the mRNAs of global transcriptional regulator Lrp, and periplasmic protein YahO, while a second targeting region is also required to regulate the mRNA of the lipid A-modifying enzyme LpxR. Interestingly, MicF targets lpxR at both the ribosome binding site and deep within the coding sequence. MicF binding in the coding sequence of lpxR decreases mRNA stability through exacerbating the use of a native RNase E site proximal to the short MicF-lpxR duplex. Altogether, this study assigns the classic MicF sRNA to the growing class of Hfq-associated regulators that use diverse mechanisms to impact multiple loci.

  6. Elucidating in Vivo Structural Dynamics in Integral Membrane Protein by Hydroxyl Radical Footprinting*

    PubMed Central

    Zhu, Yi; Guo, Tiannan; Park, Jung Eun; Li, Xin; Meng, Wei; Datta, Arnab; Bern, Marshall; Lim, Sai Kiang; Sze, Siu Kwan

    2009-01-01

    We describe here a novel footprinting technique to probe the in vivo structural dynamics of membrane protein. This method utilized in situ generation of hydroxyl radicals to oxidize and covalently modify biomolecules on living Escherichia coli cell surface. After enriching and purifying the membrane proteome, the modified amino acid residues of the protein were identified with tandem mass spectrometry to map the solvent-accessible surface of the protein that will form the footprint of in vivo structure of the protein. Of about 100 outer membrane proteins identified, we investigated the structure details of a typical β-barrel structure, the porin OmpF. We found that six modified tryptic peptides of OmpF were reproducibly detected with 19 amino acids modified under the physiological condition. The modified amino acid residues were widely distributed in the external loop area, β-strands, and periplasmic turning area, and all of them were validated as solvent-accessible according to the crystallography data. We further extended this method to study the dynamics of the voltage gating of OmpF in vivo using mimic changes of physiological circumstance either by pH or by ionic strength. Our data showed the voltage gating of porin OmpF in vivo for the first time and supported the proposed mechanism that the local electrostatic field changes in the eyelet region may alter the porin channels to switch. Thus, this novel method can be a potentially efficient method to study the structural dynamics of the membrane proteins of a living cell. PMID:19473960

  7. Mutant PrP suppresses glutamatergic neurotransmission in cerebellar granule neurons by impairing membrane delivery of VGCC α(2)δ-1 Subunit.

    PubMed

    Senatore, Assunta; Colleoni, Simona; Verderio, Claudia; Restelli, Elena; Morini, Raffaella; Condliffe, Steven B; Bertani, Ilaria; Mantovani, Susanna; Canovi, Mara; Micotti, Edoardo; Forloni, Gianluigi; Dolphin, Annette C; Matteoli, Michela; Gobbi, Marco; Chiesa, Roberto

    2012-04-26

    How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α(2)δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α(2)δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.

  8. Mutant PrP Suppresses Glutamatergic Neurotransmission in Cerebellar Granule Neurons by Impairing Membrane Delivery of VGCC α2δ-1 Subunit

    PubMed Central

    Senatore, Assunta; Colleoni, Simona; Verderio, Claudia; Restelli, Elena; Morini, Raffaella; Condliffe, Steven B.; Bertani, Ilaria; Mantovani, Susanna; Canovi, Mara; Micotti, Edoardo; Forloni, Gianluigi; Dolphin, Annette C.; Matteoli, Michela; Gobbi, Marco; Chiesa, Roberto

    2012-01-01

    Summary How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α2δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α2δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies. PMID:22542184

  9. 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.

  10. A mutation in the inner mitochondrial membrane peptidase 2-like gene (Immp2l) affects mitochondrial function and impairs fertility in mice.

    PubMed

    Lu, Baisong; Poirier, Christophe; Gaspar, Tamas; Gratzke, Christian; Harrison, Wilbur; Busija, David; Matzuk, Martin M; Andersson, Karl-Erik; Overbeek, Paul A; Bishop, Colin E

    2008-04-01

    The mitochondrion is involved in energy generation, apoptosis regulation, and calcium homeostasis. Mutations in genes involved in mitochondrial processes often result in a severe phenotype or embryonic lethality, making the study of mitochondrial involvement in aging, neurodegeneration, or reproduction challenging. Using a transgenic insertional mutagenesis strategy, we generated a mouse mutant, Immp2lTg(Tyr)979Ove, with a mutation in the inner mitochondrial membrane peptidase 2-like (Immp2l) gene. The mutation affected the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The inefficient processing of mitochondrial membrane proteins perturbed mitochondrial function so that mitochondria from mutant mice manifested hyperpolarization, higher than normal superoxide ion generation, and higher levels of ATP. Homozygous Immp2lTg(Tyr)979Ove females were infertile due to defects in folliculogenesis and ovulation, whereas mutant males were severely subfertile due to erectile dysfunction. The data suggest that the high superoxide ion levels lead to a decrease in the bioavailability of nitric oxide and an increase in reactive oxygen species stress, which underlies these reproductive defects. The results provide a novel link between mitochondrial dysfunction and infertility and suggest that superoxide ion targeting agents may prove useful for treating infertility in a subpopulation of infertile patients.

  11. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

    PubMed Central

    Findlay, Heather E; McClafferty, Heather; Ashley, Richard H

    2005-01-01

    Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide

  12. 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

    2015-11-04

    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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins.

    PubMed

    Mellgren, Ronald L

    2008-04-24

    HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside GM1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A-C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein

  1. 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.

  2. 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

  3. 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 Central

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

    2015-01-01

    The particular importance of Ca2+ signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca2+ ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca2+. 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 Ca2+ ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca2+ transients generated by cell stimulation and impairs its Ca2+ extrusion function under conditions of low resting cytosolic Ca2+ 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 Ca2+-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 Ca2+ homeostasis and the previous finding that PMCAs act as digenic modulators in Ca2+-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype. PMID:25953895

  4. NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes

    PubMed Central

    Reghellin, V.; Donnici, L.; Fenu, S.; Berno, V.; Calabrese, V.; Pagani, M.; Abrignani, S.; Peri, F.

    2014-01-01

    The hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIIIα complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIIIα-targeting inhibitor. In addition, both the NS5A and PI4KIIIα classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIIIα. Because of the similarities between the effects induced by treatment with PI4KIIIα or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIIIα complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIIIα complex. PMID:25224012

  5. 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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. 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

  7. The influence of different cucumariosides on immunogenicity of OmpF porin from Yersinia pseudotuberulosis as a model protein antigen of tubular immunostimulating complex

    NASA Astrophysics Data System (ADS)

    Sanina, N. M.; Chopenko, N. S.; Davydova, L. A.; Mazeika, A. N.; Portnyagina, O. Yu.; Kim, N. Yu.; Golotin, V. A.; Kostetsky, E. Y.; Shnyrov, V. L.

    2017-09-01

    Nanoparticulate tubular immunostimulating complex (TI-complex) is a novel promising adjuvant carrier of antigens allowing to create safe and effective vaccines of new generation. The adjuvant activity of TI-complexes based on monogalactosyldyacylglycerol (MGDG) from the sea alga Ulva lactuca and different triterpene glycosides cucumariosides (CDs) from marine invertebrate Cucumaria japonica and their fractions was studied to assess effects of different CDs on the immunogenicity of porin OmpF from Yersinia pseudotuberculosis (YOmpF). TI-complexes with cucumarioside A2-2 (CDA2-2) maximally stimulated anti-porin antibody production. Studies of protein intrinsic fluorescence showed that all CDs had a relaxing effect on the conformation of YOmpF, loosening peripheral region of protein and promoting exposure of the protein antigenic determinants to the water environment. The greatest immunostimulating effect of TI-complexes comprising CDA2-2 was accompanied by mild effect of this CD on the tertiary structure of protein antigen YOmpF, whereas cucumarioside E (CDE) and cucumarioside A2-4 (CDA2-4) caused especially sharp redistribution of spectral form of the YOmpF corresponding to the emission of an intrinsic protein fluorophore tryptophan.

  8. Participation of the Salmonella OmpD porin in the infection of RAW264.7 macrophages and BALB/c mice.

    PubMed

    Ipinza, Francisco; Collao, Bernardo; 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.

  9. Factor H-Dependent Alternative Pathway Inhibition Mediated by Porin B Contributes to Virulence of Neisseria meningitidis

    PubMed Central

    Lewis, Lisa A.; Vu, David M.; Vasudhev, Shreekant; Shaughnessy, Jutamas; Granoff, Dan M.; Ram, Sanjay

    2013-01-01

    ABSTRACT The identification of “factor H binding protein (fHbp)-null” invasive meningococcal isolates and the realization that widespread use of fHbp-based vaccines could herald selection of such strains prompted us to characterize novel mechanisms of alternative pathway (AP) inhibition on meningococci. Of seven strains engineered to lack four known AP-inhibiting molecules, capsular polysaccharide, lipooligosaccharide sialic acid, fHbp, and neisserial surface protein A (quadruple mutants), four strains inhibited human AP-mediated C3 deposition. All four expressed the porin B2 (PorB2) molecule, and three strains belonged to the hypervirulent ST-11 lineage. Consistent with reduced C3 deposition, the rate of C3a generation by a PorB2 isolate was lower than that by a PorB3 strain. Allelic replacement of PorB3 with PorB2, in both encapsulated and unencapsulated strains, confirmed the role of PorB2 in AP inhibition. Expression of PorB2 increased resistance to complement-dependent killing relative to that seen in an isogenic PorB3-expressing strain. Adult rabbit and mouse APs were unimpeded on all mutants, and human fH inhibited nonhuman C3 deposition on PorB2-expressing strains, which provided functional evidence for human fH-dependent AP regulation by PorB2. Low-affinity binding of full-length human fH to quadruple mutants expressing PorB2 was demonstrated. fH-like protein 1 (FHL-1; contains fH domains 1 through 7) and fH domains 6 and 7 fused to IgG Fc bound to one PorB2-expressing quadruple mutant, which suggested that fH domains 6 and 7 may interact with PorB2. These results associate PorB2 expression with serum resistance and presage the appearance of fHbp-null and hypervirulent ST-11 isolates that may evade killing by fHbp-based vaccines. PMID:24129254

  10. A new strategy for inhibition of the spoilage yeasts Saccharomyces cerevisiae and Zygosaccharomyces bailii based on combination of a membrane-active peptide with an oligosaccharide that leads to an impaired glycosylphosphatidylinositol (GPI)-dependent yeast wall protein layer.

    PubMed

    Bom, I J; Klis, F M; de Nobel, H; Brul, S

    2001-12-01

    Glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in yeast are connected to the beta-1,3-glucan network via a beta-1,6-glucan moiety. Addition of gentiobiose or beta-1,6-glucan oligomers to growing cells affected the construction of a normal layer of GPI-dependent cell wall proteins at the outer rim of the Saccharomyces cerevisiae cell wall. Treated S. cerevisiae cells secreted significant amounts of cell wall protein 2, were much more sensitive to the lytic action of zymolyase 20T and displayed a marked increase in sensitivity to the small amphipathic antimicrobial peptide MB-21. Similar results in terms of sensitization of yeast cells to the antimicrobial peptide were obtained with the notorious food spoilage yeast Zygosaccharomyces bailii. Our results indicate that treating cells with a membrane-perturbing compound together with compounds that lead to an impaired construction of a normal GPI-dependent yeast wall protein layer represents an effective strategy to prevent the growth of major food spoilage yeasts.

  11. 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.

  12. 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

  13. Novel channels of the inner mitochondrial membrane.

    PubMed

    Zoratti, Mario; De Marchi, Umberto; Gulbins, Erich; Szabò, Ildikò

    2009-05-01

    Along with a large number of carriers, exchangers and "pumps", the inner mitochondrial membrane contains ion-conducting channels which endow it with controlled permeability to small ions. Some have been shown to be the mitochondrial counterpart of channels present also in other cellular membranes. The manuscript summarizes the current state of knowledge on the major inner mitochondrial membrane channels, properties, identity and proposed functions. Considerable attention is currently being devoted to two K(+)-selective channels, mtK(ATP) and mtBK(Ca). Their activation in "preconditioning" is considered by many to underlie the protection of myocytes and other cells against subsequent ischemic damage. We have recently shown that in apoptotic lymphocytes inner membrane mtK(V)1.3 interacts with the pro-apoptotic protein Bax after the latter has inserted into the outer mitochondrial membrane. Whether the just-discovered mtIK(Ca) has similar cellular role(s) remains to be seen. The Ca(2+) "uniporter" has been characterized electrophysiologically, but still awaits a molecular identity. Chloride-selective channels are represented by the 107 pS channel, the first mitochondrial channel to be observed by patch-clamp, and by a approximately 400 pS pore we have recently been able to fully characterize in the inner membrane of mitochondria isolated from a colon tumour cell line. This we propose to represent a component of the Permeability Transition Pore. The available data exclude the previous tentative identification with porin, and indicate that it coincides instead with the still molecularly unidentified "maxi" chloride channel.

  14. Efficacy of Cefepime and Imipenem in Experimental Murine Pneumonia Caused by Porin-Deficient Klebsiella pneumoniae Producing CMY-2 β-Lactamase

    PubMed Central

    Pichardo, Cristina; Rodríguez-Martínez, José Manuel; Pachón-Ibañez, María E.; Conejo, Carmen; Ibáñez-Martínez, José; Martínez-Martínez, Luis; Pachón, Jerónimo; Pascual, Álvaro

    2005-01-01

    Previous studies have shown decreased in vitro activity of zwitterionic cephalosporins and carbapenems against porin-deficient Klebsiella pneumoniae expressing a plasmid-mediated AmpC-type β-lactamase (PACBL). The in vitro and in vivo activities of cefepime and imipenem were evaluated against the porin-deficient strain K. pneumoniae C2 and its CMY-2-producing derivative [K. pneumoniae C2(pMG248)]. The MICs (in micrograms/milliliter) of cefepime and imipenem against K. pneumoniae C2 were 0.125 and 0.25, respectively, while the corresponding values against K. pneumoniae C2(pMG248) were 8 and 16. Cefepime showed a greater inoculum effect than imipenem against both strains. Imipenem showed a significant postantibiotic effect (>2 h) against K. pneumoniae C2(pMG248) at 1×, 2×, 4×, 6×, and 8× MIC. The maximum concentrations of drug in serum of cefepime and imipenem in a pneumonia model using mice were 124.1 and 16.9 μg/ml, respectively. ΔT/MIC for K. pneumoniae C2 and C2(pMG248) were 1.29 h and 0.34 h for imipenem and 2.96 h and 1.27 h for cefepime. Both imipenem (30 mg/kg of body weight every 3 h) and cefepime (60 mg/kg every 4 h), administered for 72 h, increased the survival rate (86.6% and 100%) compared with untreated control animals (26.6%, P < 0.003) infected with K. pneumoniae C2. For the CMY-2-producing strain, imipenem, but not cefepime, increased the survival rate compared to the controls (86.6% and 40% versus 40%, P < 0.01). Bacterial concentration of the lungs was significantly decreased by both antimicrobials. In conclusion, imipenem was more active in terms of survival than cefepime for the treatment of murine pneumonia caused by a porin-deficient K. pneumoniae expressing PACBL CMY-2. PMID:16048941

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

    PubMed

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

    1989-07-01

    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.

  17. Conserved outer membrane protein of Neisseria meningitidis involved in capsule expression.

    PubMed Central

    Frosch, M; Müller, D; Bousset, K; Müller, A

    1992-01-01

    In Neisseria meningitidis, translocation of capsular polysaccharides to the cell surface is mediated by a transport system that fits the characteristics of ABC (ATP-binding cassette) transporters. One protein of this transport system, termed CtrA, is located in the outer membrane. By use of a CtrA-specific monoclonal antibody, we could demonstrate that CtrA occurs exclusively in N. meningitidis and not in other pathogenic or nonpathogenic Neisseria species. Nucleotide sequence comparison of the ctrA gene from different meningococcal serogroups indicated that CtrA is strongly conserved in all meningococcal serogroups, independent of the chemical composition of the capsular polysaccharide. Secondary structure analysis revealed that CtrA is anchored in the outer membrane by eight membrane-spanning amphipathic beta strands, a structure of proteins that function as porins. Images PMID:1371768

  18. Structure and properties of the outer membranes of Brucella abortus and Brucella melitensis.

    PubMed

    Moriyón, I; López-Goñi, I

    1998-03-01

    The brucellae are Gram-negative bacteria characteristically able to multiply facultatively within phagocytic cells and which cause a zoonosis of world-wide importance. This article reviews the structure and topology of the main components (lipopolysaccharide, native hapten polysaccharide, free lipids and proteins) of the outer membranes of Brucella abortus and B. melitensis, as well as some distinctive properties (permeability and interactions with cationic peptides) of these membranes. On these data, an outer membrane model is proposed in which, as compared to other Gram-negatives, there is a stronger hydrophobic anchorage for the lipopolysaccharide, free lipids, porin proteins and lipoproteins, and a reduced surface density of anionic groups, which could be partially or totally neutralized by ornithine lipids. This model accounts for the permeability of Brucella to hydrophobic permeants and for its resistance to the bactericidal oxygen-independent systems of phagocytes.

  19. Taste - impaired

    MedlinePlus

    ... longer. Causes of impaired taste include: Bell's palsy Common cold Flu and other viral infections Nasal infection, nasal ... your diet. For taste problems due to the common cold or flu, normal taste should return when the ...

  20. Impaired Driving

    MedlinePlus

    Impaired driving is dangerous. It's the cause of more than half of all car crashes. It means operating a ... texting Having a medical condition which affects your driving For your safety and the safety of others, ...

  1. Impaired Driving

    MedlinePlus

    ... people were killed in alcohol-impaired driving crashes, accounting for nearly one-third (31%) of all traffic- ... promotion efforts into practice that influence economic, organizational, policy, and school/community action. 13,14 Using community- ...

  2. Mutations in genes cpxA and cpxB alter the protein composition of Escherichia coli inner and outer membranes.

    PubMed Central

    McEwen, J; Silverman, P M

    1982-01-01

    Mutations in chromosomal genes cpxA and cpxB altered the protein composition of the inner and outer bacterial membranes. Electrophoretic analyses of membrane proteins from isogenic strains differing only at their cpx loci and of spontaneous cpxA+ revertants of a cpxA cpxB double mutant showed that the alterations define a pattern that is uniquely attributable to the cpx mutations. Two major outer membrane proteins, the OmpF matrix porin and the murein lipoprotein, were deficient or absent from the outer membrane of mutant cells, whereas the quantities of two other major outer membrane proteins, the OmpC matrix porin and the OmpA protein, were not significantly altered. The cpx mutations did not generally alter the functional or chemical properties of the cell envelope. In the electron microscope, mutant cells appeared ovoid, but individual cells showed no surface irregularities to suggest gross defects in the cell envelope. These observations suggest that the primary effect of the mutations is to alter selectively the synthesis or translocation of certain envelope proteins. Images PMID:7050092

  3. Resistance of Escherichia coli to nourseothricin (streptothricin): sensitization of resistant strains by abolition of its outer membrane resistance.

    PubMed

    Seltmann, G

    1992-01-01

    The polycationic antibiotic, nourseothricin, represents a mixture of several streptothricins, mainly D and F. The molecular weight of the latter compound amounts to 486. Obviously, although very slowly, it can pass the outer membrane via the porin pores. It has been shown earlier that nourseothricin is able to generate some kind of channels into the outer membrane through which it can pass the cell wall. On the other hand, there were indications that resistant strains containing a streptothricin-inactivating acetyl transferase possess an additional protecting system, namely a reduced penetrability of the outer membrane. In this study, it could be shown that such strains indeed could be rendered sensitive by damaging the barrier function of the outer membrane.

  4. Physical Impairment

    NASA Astrophysics Data System (ADS)

    Trewin, Shari

    Many health conditions can lead to physical impairments that impact computer and Web access. Musculoskeletal conditions such as arthritis and cumulative trauma disorders can make movement stiff and painful. Movement disorders such as tremor, Parkinsonism and dystonia affect the ability to control movement, or to prevent unwanted movements. Often, the same underlying health condition also has sensory or cognitive effects. People with dexterity impairments may use a standard keyboard and mouse, or any of a wide range of alternative input mechanisms. Examples are given of the diverse ways that specific dexterity impairments and input mechanisms affect the fundamental actions of Web browsing. As the Web becomes increasingly sophisticated, and physically demanding, new access features at the Web browser and page level will be necessary.

  5. 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.

  6. Hearing Impairment

    MedlinePlus

    ... may include inserting an object such as a cotton swab too far into the ear, a sudden explosion or other loud noise, a sudden change in air pressure, a head injury, or repeated ear infections. Sensorineural hearing impairment results from problems with or damage ...

  7. 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.

  8. 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

  9. Evaluation of immunological responses to recombinant Porin A protein (rPoA) from native strains of Neisseria meningitidis serogroups A and B using OMV as an adjuvant in BALB/c mice.

    PubMed

    Afrough, Parviz; Bouzari, Saeid; Mousavi, Seyed Fazlollah; Asadi Karam, Mohammad Reza; Vaziri, Farzam; Fateh, Abolfazl; Behrouzi, Ava; Malekan, Mohammadali; Siadat, Seyed Davar

    2017-09-20

    Neisseria meningitidis is one of the main causes of sepsis and meningitis, which are two serious life-threatening diseases in both children and adolescents. Porin A (porA) from both serogroup A and B were cloned into the pET28a plasmid and expressed in E. coli BL21 (DE3). The protein was expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. BALB/c mice were subcutaneously injected three times with 25 μg of the recombinant PorA. Specific total IgG antibodies and isotypes were evaluated using ELISA assay. Opsonophagocytic assay (OPA) and Serum Bactericidal assay (SBA) were performed. Results showed that vaccinated mice exhibited higher levels of anti-Porin A (p < 0.05) with a predominant IgG1 response compared to the control group. Results from in vitro experiments indicated that N. meningitidis was opsonized with immunized-mice sera, and compared to non-immunized mice, immunized mice displayed significantly increased phagocytic uptake and effective intracellular killing. In this study, serogroup B N. meningitidis OMV of strain CSBPI G-245 and complete and incomplete Freund's adjuvant were used. Results demonstrated that Porin A could be a valuable target for the development of immunotherapeutic strategies against N. meningitidis. Copyright © 2017. Published by Elsevier Ltd.

  10. The role of outer membrane in Serratia marcescens intrinsic resistance to antibiotics.

    PubMed

    Sánchez, L; Ruiz, N; Leranoz, S; Viñas, M; Puig, M

    1997-09-01

    Three different porins from Serratia marcescens were described. They were named Omp1, Omp2 and Omp3 and their molecular weights were 42, 40 and 39 kDa respectively. Omp2 and Omp3 showed osmoregulation and thermoregulation in a similar way to OmpC and OmpF of Escherichia coli. Permeability coefficients of the outer membrane of this species were calculated following the Zimmermann and Rosselet method. P values were similar to those obtained in Escherichia coli, which suggests that the chromosomal beta-lactamase would play a major role in the resistance of Serratia marcescens to beta-lactam antibiotics. Both MIC values and permeabilities were modified by salycilates and acetylsalycilate. Synergism between the outer membrane and the beta-lactamase was also evaluated. When bacteria grew in the presence of a beta-lactam in the medium, the beta-lactamase accounted for most of the resistance.

  11. Analysis of Moraxella catarrhalis outer membrane antigens cross-reactive with Neisseria meningitidis and Neisseria lactamica.

    PubMed

    Troncoso, Gemma; Sánchez, Sandra; Criado, María Teresa; Ferreirós, Carlos

    2004-01-15

    Mouse sera against outer membrane proteins from Moraxella catarrhalis, Neisseria meningitidis and Neisseria lactamica, and human sera from both healthy individuals and patients convalescing from meningococcal meningitis were used to identify cross-reactive antigens. Mouse anti-N. meningitidis and anti-N. lactamica sera recognized 77, 62 and 32 kDa outer membrane antigens in M. catarrhalis strains; on the contrary, the meningococcal porin PorB (38-42 kDa) was recognized by one of the two anti-M. catarrhalis sera. Human sera from both healthy individuals and patients convalescing from meningococcal meningitis also showed cross-reactive antibodies against these proteins. The existence of cross-reactive antigens in M. catarrhalis and N. meningitidis (as well as in N. lactamica) could favor the development of natural immunization against both pathogens.

  12. Isolation, sequencing and expression of Bartonella henselae omp43 and predicted membrane topology of the deduced protein.

    PubMed

    Burgess, A W; Paquet, J Y; Letesson, J J; Anderson, B E

    2000-08-01

    The infection of and interaction of human endothelial cells with Bartonella henselae is one of the most interesting aspects of Bartonella -associated disease. The gene encoding the 43 kDa B. henselae outer membrane protein (Omp43) that binds endothelial cells was cloned and sequenced. Sequence analysis revealed an open reading frame of 1206 nucleotides coding for a protein of 402 amino acids. Analysis of the deduced amino acid sequence shows 38% identity over the entire sequence to the Brucella spp. In addition to this Omp2b porin also shows a signal sequence and peptidase cleavage site. Cleavage of the signal peptide results in a mature 380 amino acid polypeptide with a predicted molecular weight of 42 kDa. Omp43 was expressed in Escherichia coli as a fusion protein. Purified recombinant Omp43 at concentrations of 11 and 2.75 microg/ml bound to intact human umbilical vein endothelial cells. Membrane topology analysis predicts that Omp43 exists as a 16 stranded beta barrel protein, similar to that predicted for the Omp2b Brucella abortus porin. Characterization and expression of the gene encoding Omp43 should provide a tool for further investigation of the role of adherence to endothelial cells in the pathogenesis of B. henselae. Copyright 2000 Academic Press.

  13. 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

  14. Projection maps of three members of the KdgM outer membrane protein family.

    PubMed

    Signorell, Gian A; Chami, Mohamed; Condemine, Guy; Schenk, Andreas D; Philippsen, Ansgar; Engel, Andreas; Remigy, Herve W

    2007-12-01

    We present the projection structures of the three outer membrane porins KdgM and KdgN from Erwinia chrysanthemi and NanC from Escherichia coli, based on 2D electron crystallography. A wide screening of 2D crystallization conditions yielded tubular crystals of a suitable size and quality to perform high-resolution electron microscopy. Data processing of untilted samples allowed us to separate the information of the two crystalline layers and resulted in projection maps to a resolution of up to 7A. All three proteins exhibit a similar putative beta-barrel structure and the three crystal forms have the same symmetry. However, there are differences in the packing arrangements of the monomers as well as the densities of the projections. To interpret these projections, secondary structure prediction was performed using beta-barrel specific prediction algorithms. The predicted transmembrane beta-barrels have a high similarity in the arrangement of the putative beta-strands and the loops, but do not match those of OmpG, a related protein porin whose structure was solved.

  15. 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.

  16. 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.

  17. All Vision Impairment

    MedlinePlus

    ... USAJobs 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. ...

  18. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. The DNA static curvature has a role in the regulation of the ompS1 porin gene in Salmonella enterica serovar Typhi.

    PubMed

    De la Cruz, Miguel Angel; Merino, Enrique; Oropeza, Ricardo; Téllez, Juan; Calva, Edmundo

    2009-07-01

    The DNA static curvature has been described to play a key role as a regulatory element in the transcription process of several bacterial genes. Here, the role of DNA curvature in the expression of the ompS1 porin gene in Salmonella enterica serovar Typhi is described. The web server mutacurve was used to predict mutations that diminished or restored the extent of DNA curvature in the 5' regulatory region of ompS1. Using these predictions, curvature was diminished by site-directed mutagenesis of only two residues, and curvature was restored by further mutagenesis of the same two residues. Lowering the extent of DNA curvature resulted in an increase in ompS1 expression and in the diminution of the affinity of the silencer proteins H-NS and StpA for the ompS1 5' regulatory region. These mutations were in a region shown not to contain the H-NS nucleation site, consistent with the notion that the effect on expression was due to changes in DNA structural topology.

  20. 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

    Goessens, Wil H F; van der Bij, Akke K; van Boxtel, Ria; Pitout, Johann D D; van Ulsen, Peter; Melles, Damian C; Tommassen, Jan

    2013-08-01

    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 bla(CTX-M-15) and bla(OXA-1). The CR E. coli strain additionally harbored bla(CMY-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.

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

    PubMed

    Khalili-Araghi, Fatemeh; Ziervogel, Brigitte; Gumbart, James C; Roux, Benoît

    2013-10-01

    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.

  2. 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.

  3. 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.

  4. 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

  5. Molecular origin of the cation selectivity in OmpF porin: single channel conductances vs. free energy calculation.

    PubMed

    Danelon, Christophe; Suenaga, Atsushi; Winterhalter, Mathias; Yamato, Ichiro

    2003-07-01

    Ion current through single outer membrane protein F (OmpF) trimers was recorded and compared to molecular dynamics simulation. Unidirectional insertion was revealed from the asymmetry in channel conductance. Single trimer conductance showed particularly high values at low symmetrical salt solution. The conductance values of various alkali metal ion solutions were proportional to the monovalent cation mobility values in the bulk phase, LiCl

  6. 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

  7. Efflux pumps expression and its association with porin down-regulation and β-lactamase production among Pseudomonas aeruginosa causing bloodstream infections in Brazil

    PubMed Central

    2010-01-01

    Background Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile. Results Aztreonam exhibited the highest in vitro activity against the P. aeruginosa isolates studied (64.4% susceptibility), whereas susceptibility rates of imipenem and meropenem were both 47.5%. The MexXY-OprM and MexAB-OprM efflux systems were overexpressed in 50.8% and 27.1% of isolates studied, respectively. Overexpression of the MexEF-OprN and MexCD-OprJ systems was not observed. AmpC β-lactamase was overexpressed in 11.9% of P. aeruginosa isolates. In addition, decreased oprD expression was also observed in 69.5% of the whole collection, and in 87.1% of the imipenem non-susceptible P. aeruginosa clinical isolates. The MBL-encoding genes blaSPM-1 and blaIMP-1 were detected in 23.7% and 1.7% P. aeruginosa isolates, respectively. The blaGES-1 was detected in 5.1% of the isolates, while blaGES-5 and blaCTX-M-2 were observed in 1.7% of the isolates evaluated. In the present study, we have observed that efflux systems represent an adjuvant mechanism for antimicrobial resistance. Conclusions Efflux systems in association of distinct mechanisms such as the porin down-regulation, AmpC overproduction and secondary β-lactamases play also an important role in the multi-drug resistance phenotype among P. aeruginosa clinical isolates. PMID:20704733

  8. Contribution of β-lactamases and porin proteins OmpK35 and OmpK36 to carbapenem resistance in clinical isolates of KPC-2-producing Klebsiella pneumoniae.

    PubMed

    Zhang, Ying; Jiang, Xiaofei; Wang, Yanyan; Li, Gang; Tian, Yueru; Liu, Hong; Ai, Fuqi; Ma, Yiming; Wang, Bei; Ruan, Feiyi; Rajakumar, Kumar

    2014-01-01

    Fifty-seven carbapenem-resistant Klebsiella pneumoniae isolates belonging to ST11 (50 isolates), ST423 (5 isolates), and two other sequence types were studied. All were positive for blaKPC-2, blaTEM-1, and blaCTX-M-14. SDS-PAGE analysis of six representative isolates demonstrated varied porin expression. Nevertheless, when blaKPC-2 was deleted, carbapenem resistance was markedly reduced. Additionally, SHV-12, DHA-1, and/or VIM-1 appeared to contribute to accessory carbapenemase activity. In contrast, OmpK35 and/or OmpK36 deficiency seemed to serve only as a minor cooperative factor.

  9. Expression of a Mitochondrial Progesterone Receptor (PR-M) in Leiomyomata and Association With Increased Mitochondrial Membrane Potential

    PubMed Central

    Feng, Quanling; Crochet, John R.; Dai, Qunsheng; Leppert, Phyllis C.

    2014-01-01

    Context: Clinical evidence supports a role for progestins in the growth of leiomyomata (fibroids). The mechanism(s) for this is thought to involve gene regulation via the nuclear progesterone receptors. Recently a mitochondrial progesterone receptor (PR-M) has been identified with evidence of a progesterone/progestin-dependent increase in cellular respiration. This observation raises a possible new mechanism whereby progesterone/progestin may affect the growth of fibroids. Objective: The goals of this research were to determine differential expression of PR-M in normal myometrium compared with the edge of a fibroid within the same uterus, to demonstrate a progestin-dependent increase in mitochondria membrane potential using an immortalized human myometrial cell line and to examine mitochondrial membrane potential in transfected cells expressing the complete coding sequence of PR-M. Design: Protein levels of PR-M, PR-B, PR-A, mitochondrial porin, and glyceraldehyde-3-phosphate dehydrogenase were determined in the myometrium and adjacent edge of a fibroid in 10 subjects undergoing hysterectomy for benign indications. Mitochondrial membrane potential was determined by fluorescent emission of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolecarbocyanide iodine in hTERT-HM cells treated with R5020 and in transfected hTERT-HM cells determined by the fluorescent emission of tetramethylrhodamine methyl ester. Results: Higher levels of PR-M and mitochondrial porin were found in the fibroid edge compared with adjacent myometrium. Progestin increased mitochondrial membrane potential in hTERT-HM cells, which was not affected by a translation inhibitor. This effect was exaggerated in hTERT-HM cells expressing PR-M after transient transfection. Conclusion: These studies suggest a mechanism whereby progesterone/progestin may affect the growth of fibroids by altering mitochondrial activity. PMID:24423317

  10. In vitro antibacterial activities of tosufloxacin against and uptake of tosufloxacin by outer membrane mutants of Escherichia coli, Proteus mirabilis, and Salmonella typhimurium.

    PubMed Central

    Mitsuyama, J; Itoh, Y; Takahata, M; Okamoto, S; Yasuda, T

    1992-01-01

    The antibacterial activities of tosufloxacin and other quinolones against and apparent uptakes of tosufloxacin and other quinolones by outer membrane mutants of Escherichia coli, Proteus mirabilis, and Salmonella typhimurium were studied. The hydrophobicity of tosufloxacin was nearly equal to that of ofloxacin or lower than those of sparfloxacin and nalidixic acid. OmpF- and OmpC-deficient E. coli and 40-kDa porin-deficient P. mirabilis mutants were twofold more susceptible to tosufloxacin and sparfloxacin but two- to fourfold less susceptible to other quinolones than their parent strains. In S. typhimurium lipopolysaccharide-deficient (rough) mutants, the differences in susceptibility to tosufloxacin were similar to those to sparfloxacin and nalidixic acid. The apparent uptake of tosufloxacin by intact cells was increased in porin-deficient mutants compared with that by their parent strain. These results suggest that the permeation route of tosufloxacin across the outer membrane is different from that of other fluoroquinolones and that tosufloxacin may permeate mainly through the nonporin pathway, presumably phospholipid bilayers. However, this characteristic is independent of the hydrophobicity of the molecule. Images PMID:1329639

  11. Proteomic analysis of Neisseria lactamica and N eisseria meningitidis outer membrane vesicle vaccine antigens.

    PubMed

    Vaughan, Thomas E; Skipp, Paul J; O'Connor, C David; Hudson, Michael J; Vipond, Richard; Elmore, Michael J; Gorringe, Andrew R

    2006-06-19

    Vaccines to prevent meningococcal disease have been developed from the outer membrane vesicles (OMVs) of Neisseria meningitidis and the related commensal organism Neisseria lactamica. In addition to lipopolysaccharide and the major porins, these vaccines contain a large number of proteins that are incompletely characterised. Here we describe comparative proteomic analyses of the N. lactamica OMV vaccine and OMVs from a serogroup B strain of N. meningitidis. Tandem mass-spectrometry data for trypsinised N. lactamica OMV vaccine were matched to an incompletely assembled genome sequence from the same strain to give 65 robust protein identifications and a further 122 single- or two-peptide matches. Fifty-seven N. meningitidis K454 proteins were identified robustly (and a further 68 from single- or two-peptide matches) by inference from the N. meningitidis MC58 genome. The results suggest that OMVs have a hitherto unappreciated complexity and pinpoint novel candidate antigens for further characterisation.

  12. Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

    PubMed

    Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

    To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function.

  13. Cortical Visual Impairment

    MedlinePlus

    ... Frequently Asked Questions Español Condiciones Chinese Conditions Cortical Visual Impairment En Español Read in Chinese What is cortical visual impairment? Cortical visual impairment (CVI) is a decreased ...

  14. 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.

  15. Membrane Proteins in Four Acts: Function Precedes Structure Determination

    PubMed Central

    Cramer, W. A.; Zakharov, S. D.; Hasan, S. Saif; Zhang, H.; Baniulis, D.; Zhalnina, M. V.; Soriano, G. M.; Sharma, O.; Rochet, J. C.; Ryan, C.; Whitelegge., J.; Kurisu, G.; Yamashita, E.

    2011-01-01

    Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/ proton translocation). (1) Crystal structures of the eight subunit heterooligomeric trans-membrane dimeric cytochrome b6f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of seventeen monotopic and polytopic hetero-subunits. (II) β-barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B12 binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins (1). A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a “fishing pole” model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83 Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained (2). A crystal structure of the N-terminal translocation domain of colicin E3

  16. The Core Components of Organelle Biogenesis and Membrane Transport in the Hydrogenosomes of Trichomonas vaginalis

    PubMed Central

    Rada, Petr; Doležal, Pavel; Jedelský, Petr L.; Bursac, Dejan; Perry, Andrew J.; Šedinová, Miroslava; Smíšková, Kateřina; Novotný, Marian; Beltrán, Neritza Campo; Hrdý, Ivan; Lithgow, Trevor; Tachezy, Jan

    2011-01-01

    Trichomonas vaginalis is a parasitic protist of the Excavata group. It contains an anaerobic form of mitochondria called hydrogenosomes, which produce hydrogen and ATP; the majority of mitochondrial pathways and the organellar genome were lost during the mitochondrion-to-hydrogenosome transition. Consequently, all hydrogenosomal proteins are encoded in the nucleus and imported into the organelles. However, little is known about the membrane machineries required for biogenesis of the organelle and metabolite exchange. Using a combination of mass spectrometry, immunofluorescence microscopy, in vitro import assays and reverse genetics, we characterized the membrane proteins of the hydrogenosome. We identified components of the outer membrane (TOM) and inner membrane (TIM) protein translocases include multiple paralogs of the core Tom40-type porins and Tim17/22/23 channel proteins, respectively, and uniquely modified small Tim chaperones. The inner membrane proteins TvTim17/22/23-1 and Pam18 were shown to possess conserved information for targeting to mitochondrial inner membranes, but too divergent in sequence to support the growth of yeast strains lacking Tim17, Tim22, Tim23 or Pam18. Full complementation was seen only when the J-domain of hydrogenosomal Pam18 was fused with N-terminal region and transmembrane segment of the yeast homolog. Candidates for metabolite exchange across the outer membrane were identified including multiple isoforms of the β-barrel proteins, Hmp35 and Hmp36; inner membrane MCF-type metabolite carriers were limited to five homologs of the ATP/ADP carrier, Hmp31. Lastly, hydrogenosomes possess a pathway for the assembly of C-tail-anchored proteins into their outer membrane with several new tail-anchored proteins being identified. These results show that hydrogenosomes and mitochondria share common core membrane components required for protein import and metabolite exchange; however, they also reveal remarkable differences that reflect the

  17. 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.

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

    PubMed

    Pottosin, Igor; Shabala, Sergey

    2016-03-07

    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. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  19. 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.

  20. Tropomyosin modulates erythrocyte membrane stability

    PubMed Central

    An, Xiuli; Salomao, Marcela; Guo, Xinhua; Gratzer, Walter; Mohandas, Narla

    2007-01-01

    The ternary complex of spectrin, actin, and 4.1R (human erythrocyte protein 4.1) defines the nodes of the erythrocyte membrane skeletal network and is inseparable from membrane stability under mechanical stress. These junctions also contain tropomyosin (TM) and the other actin-binding proteins, adducin, protein 4.9, tropomodulin, and a small proportion of capZ, the functions of which are poorly defined. Here, we have examined the consequences of selective elimination of TM from the membrane. We have shown that the mechanical stability of the membranes of resealed ghosts devoid of TM is grossly, but reversibly, impaired. That the decreased membrane stability of TM-depleted membranes is the result of destabilization of the ternary complex of the network junctions is demonstrated by the strongly facilitated entry into the junctions in situ of a β-spectrin peptide, containing the actin- and 4.1R-binding sites, after extraction of the TM. The stabilizing effect of TM is highly specific, in that it is only the endogenous isotype, and not the slightly longer muscle TM that can bind to the depleted membranes and restore their mechanical stability. These findings have enabled us identify a function for TM in elevating the mechanical stability of erythrocyte membranes by stabilizing the spectrin-actin-4.1R junctional complex. PMID:17008534

  1. [Epiretinal membranes].

    PubMed

    Dupas, B; Tadayoni, R; Gaudric, A

    2015-11-01

    Idiopathic epiretinal membranes represent a common condition, and are present in approximately 10% of people over the age of 70 years. They are idiopathic in 80% of cases, or may be secondary to various conditions such as a prior retinal detachment, or vascular or inflammatory retinal diseases. The main symptoms are visual loss and metamorphopsia. The diagnosis of epiretinal membrane is currently facilitated by OCT, which provides prognostic and therapeutic decision-making assistance. Surgery for epiretinal membranes is currently well codified through sutureless vitrectomy and dyes. Dissection of the membrane (with or without associated peeling of the internal limiting membrane) ensures good anatomical and functional results, while being relatively minimally invasive. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  2. 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

    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

    2011-12-02

    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.

  3. 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.

  4. Metaxin deficiency alters mitochondrial membrane permeability and leads to resistance to TNF-induced cell killing.

    PubMed

    Ono, Koh; Wang, Xiaofei; Kim, Sung Ouk; Armstrong, Lucas C; Bornstein, Paul; Han, Jiahuai

    2010-02-01

    Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-X(L) overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxin-deficient (Met(mut)) and Bcl-X(L)-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaΔTM/C) or Bcl-X(L) in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-X(L) overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-X(L) appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-X(L) may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-X(L) overexpression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.

  5. High-level resistance to meropenem in clinical isolates of Pseudomonas aeruginosa in the absence of carbapenemases: role of active efflux and porin alterations.

    PubMed

    Chalhoub, Hussein; Sáenz, Yolanda; Rodriguez-Villalobos, Hector; Denis, Olivier; Kahl, Barbara C; Tulkens, Paul M; Van Bambeke, Françoise

    2016-12-01

    High-level carbapenem resistance is worryingly increasing in clinical isolates and is often attributed to carbapenemase expression. This study aimed to determine the mechanisms leading to high-level meropenem resistance in six carbapenemase-negative Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients and seven carbapenemase-positive isolates from patients suffering from hospital-acquired pneumonia (HAP). MICs were determined in the absence or presence of l-arginine or glycine-glutamate as competitive substrates for OprD (OccD1) or OpdP (OccD3), respectively, or the efflux pump inhibitor Phe-Arg β-naphthylamide (PAβN). β-Lactamases were screened by phenotypic tests and/or PCR. The oprD gene and its promoter were sequenced; protein expression was evidenced by SDS-PAGE. mexA, mexX, mexC and mexE transcripts were evaluated by real-time and semiquantitative PCR. Meropenem/imipenem MICs were 64-128/16-32 mg/L and 128/128-256 mg/L in CF and HAP isolates, respectively; PAβN reduced meropenem MICs to 4-16 mg/L only and specifically in CF isolates; porin competitors had no effect on MICs. All isolates showed an increase in transcription levels of mexA, mexX and/or mexC and mutations in oprD leading to production of truncated proteins. AmpC-type cephalosporinases were overexpressed in CF isolates and VIM-2 was expressed in HAP isolates. Antibiotic exclusion from bacteria by concomitant efflux and reduced uptake is sufficient to confer high-level resistance to meropenem in isolates overexpressing AmpC-type cephalosporinases. As efflux is preponderant in these isolates, it confers a paradoxical phenotype where meropenem is less active than imipenem. Concomitant susceptibility testing of both carbapenems and rapid elucidation of the most probable resistance mechanisms is thus warranted. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  6. Activity of ciprofloxacin and levofloxacin in experimental pneumonia caused by Klebsiella pneumoniae deficient in porins, expressing active efflux and producing QnrA1.

    PubMed

    Rodríguez-Martínez, J M; Pichardo, C; García, I; Pachón-Ibañez, M E; Docobo-Pérez, F; Pascual, A; Pachón, J; Martínez-Martínez, L

    2008-07-01

    The objective of this study was to evaluate the activities of ciprofloxacin and levofloxacin in a murine model of pneumonia caused by Klebsiella pneumoniae C2 (with altered GyrA, deficient in porins and expressing active efflux of quinolones) and the transconjugant C2pMG252 derived from it and expressing the qnrA1 determinant. MICs and MBCs of the two quinolones were determined according to CLSI guidelines. Time-kill curves (at 1x and 4x MIC) were also performed to assess bactericidal activity. An experimental model of pneumonia in mice was evaluated. Groups of 15 mice were infected with either strain and treated with ciprofloxacin (80 mg/kg/day) or levofloxacin (100 mg/kg/day). Control non-treated animals were also evaluated. In the case of strain C2, log(10) CFU/g of lung in non-treated animals was 9.16 +/- 2.16. This value was reduced to 3.53 +/- 1.04 (p <0.001) and 3.38 +/- 0.46 (p <0.001) in animals treated with ciprofloxacin or levofloxacin, respectively. Percentages of surviving mice were 26.7% (control group) and 100% (both ciprofloxacin and levofloxacin; p <0.001 vs. controls). Bacterial counts (log(10) CFU/g) in lungs of animals infected with strain C2pMG252 were 9.65 +/- 2.49 in non-treated animals and 7.74 +/- 2.67 and 7.57 +/- 3.84 for those treated with ciprofloxacin or levofloxacin, respectively (p >0.05 vs. control group). Of non-treated animals infected with strain C2pMG252, 14.3% survived. Ciprofloxacin and levofloxacin improved the survival in these mice (53.3% for both antimicrobials, p 0.03). In conclusion, the expression of qnrA1 in K. pneumoniae with additional mechanisms of resistance causes decreased efficacy of fluoroquinolones in a pneumonia model in mice.

  7. 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.

  8. 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

  9. Visual impairment in the hearing impaired students.

    PubMed

    Gogate, Parikshit; Rishikeshi, Nikhil; Mehata, Reshma; Ranade, Satish; Kharat, Jitesh; Deshpande, Madan

    2009-01-01

    Ocular problems are more common in children with hearing problems than in normal children. Neglected visual impairment could aggravate educational and social disability. To detect and treat visual impairment, if any, in hearing-impaired children. Observational, clinical case series of hearing-impaired children in schools providing special education. Hearing-impaired children in selected schools underwent detailed visual acuity testing, refraction, external ocular examination and fundoscopy. Ocular motility testing was also performed. Teachers were sensitized and trained to help in the assessment of visual acuity using Snellen's E charts. Refractive errors and squint were treated as per standard practice. Excel software was used for data entry and SSPS for analysis. The study involved 901 hearing-impaired students between four and 21 years of age, from 14 special education schools. A quarter of them (216/901, 24%) had ocular problems. Refractive errors were the most common morbidity 167(18.5%), but only 10 children were using appropriate spectacle correction at presentation. Fifty children had visual acuity less than 20/80 at presentation; after providing refractive correction, this number reduced to three children, all of whom were provided low-vision aids. Other common conditions included strabismus in 12 (1.3%) children, and retinal pigmentary dystrophy in five (0.6%) children. Ocular problems are common in hearing-impaired children. Screening for ocular problems should be made mandatory in hearing-impaired children, as they use their visual sense to compensate for the poor auditory sense.

  10. 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.

  11. Outer membrane protein A and OprF – Versatile roles in Gram-negative bacterial infections

    PubMed Central

    Krishnan, Subramanian; Prasadarao, Nemani V.

    2012-01-01

    Outer membrane protein A (OmpA) is an abundant protein of Escherichia coli and other enterobacteria with a multitude of functions. Although the structural features and porin function of OmpA were well studied, its role in the pathogenesis of various bacterial infections has been emerging for the past decade. The four extracellular loops of OmpA interact with a variety of host tissues for adhesion, invasion and evasion of host-defense mechanisms. This review describes how various regions present in the extracellular loops of OmpA contribute to the pathogenesis of neonatal meningitis induced by E. coli K1 and for many other functions. In addition, the function of OmpA like proteins such as OprF of Pseudomonas aeruginosa is also discussed herein. PMID:22240162

  12. Impaired Waters and TMDLs

    EPA Pesticide Factsheets

    The 303(d) program provides guidance and technical resources to assist States in submitting lists of impaired waterbodies and the development of Total Maximum Daily Loads of the pollutant causing the impairment.

  13. Impaired Driving - Multiple Languages

    MedlinePlus

    ... Are Here: Home → Multiple Languages → All Health Topics → Impaired Driving URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Impaired Driving - Multiple Languages To use the sharing features on ...

  14. 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.

  15. Deciphering the function of the outer membrane protein OprD homologue of Acinetobacter baumannii.

    PubMed

    Catel-Ferreira, Manuella; Nehmé, Rony; Molle, Virginie; Aranda, Jesús; Bouffartigues, Emeline; Chevalier, Sylvie; Bou, Germán; Jouenne, Thierry; Dé, Emmanuelle

    2012-07-01

    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 Fe(3+) and Mg(2+), 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.

  16. 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.…

  17. Metamaterial membranes

    NASA Astrophysics Data System (ADS)

    Restrepo-Flórez, Juan Manuel; Maldovan, Martin

    2017-01-01

    We introduce a new class of metamaterial device to achieve separation of compounds by using coordinate transformations and metamaterial theory. By rationally designing the spatial anisotropy for mass diffusion, we simultaneously concentrate different compounds in different spatial locations, leading to separation of mixtures across a metamaterial membrane. The separation of mixtures into their constituent compounds is critically important in biophysics, biomedical, and chemical applications. We present a practical case where a mixture of oxygen and nitrogen diffusing through a polymeric planar matrix is separated. This work opens doors to new paradigms in membrane separations via coordinate transformations and metamaterials by introducing novel properties and unconventional mass diffusion phenomena.

  18. Antibiotic Resistance and Regulation of the Gram-Negative Bacterial Outer Membrane Barrier by Host Innate Immune Molecules

    PubMed Central

    2016-01-01

    ABSTRACT The Gram-negative outer membrane is an important barrier that provides protection against toxic compounds, which include antibiotics and host innate immune molecules such as cationic antimicrobial peptides. Recently, significant research progress has been made in understanding the biogenesis, regulation, and functioning of the outer membrane, including a recent paper from the laboratory of Dr. Brett Finlay at the University of British Columbia (J. van der Heijden et al., mBio 7:e01238-16, 2016, http://dx.doi.org/10.1128/mBio.01541-16). These investigators demonstrate that toxic oxygen radicals, such as those found in host tissues, regulate outer membrane permeability by altering the outer membrane porin protein channels to regulate the influx of oxygen radicals as well as β-lactam antibiotics. This commentary provides context about this interesting paper and discusses the prospects of utilizing increased knowledge of outer membrane biology to develop new antibiotics for antibiotic-resistant Gram-negative bacteria. PMID:27677793

  19. Docosahexaenoic acid-induced amelioration on impairment of memory learning in amyloid beta-infused rats relates to the decreases of amyloid beta and cholesterol levels in detergent-insoluble membrane fractions.

    PubMed

    Hashimoto, Michio; Hossain, Shahdat; Agdul, Haqu; Shido, Osamu

    2005-12-30

    We investigated the effects of dietary administration of docosahexaenoic acid (DHA; C22:6n-3) on the levels of amyloid beta (A beta) peptide (1-40) and cholesterol in the nonionic detergent Triton 100 x-insoluble membrane fractions (DIFs) of the cerebral cortex and, also, on learning-related memory in an animal model of Alzheimer's disease (AD) rats infused with A beta peptide (1-40) into the cerebral ventricle. The infusion increased the levels of A beta peptide and cholesterol in the DIFs concurrently with a significant increase in reference memory errors (measured by eight-arm radial-maze tasks) compared with those of vehicle rats. Conversely, the dietary administration of DHA to AD-model rats decreased the levels of A beta peptide and cholesterol in the DIFs, with the decrease being more prominent in the DHA-administered rats. Regression analysis revealed a significant positive correlation between A beta peptide and each of cholesterol, palmitic acid and stearic acid, and between the number of reference memory errors and each of cholesterol, palmitic, stearic and oleic acid; moreover, a significant negative correlation was observed between the number of reference memory errors and the molar ratio of DHA to palmitic plus stearic acid. These results suggest that DHA-induced protection of memory deficits in AD-model rats is related to the interactions of cholesterol, palmitic acid or stearic acid with A beta peptides in DIFs where DHA ameliorates these interactions.

  20. 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

  1. A novel Geobacteraceae-specific outer membrane protein J (OmpJ) is essential for electron transport to Fe (III) and Mn (IV) oxides in Geobacter sulfurreducens

    PubMed Central

    Afkar, Eman; Reguera, Gemma; Schiffer, Marianne; Lovley, Derek R

    2005-01-01

    Background Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III) reduction in the Geobacter species that are the predominant Fe(III) reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration. Results When outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J), was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe (III) and insoluble Mn (IV) oxide, as the electron acceptor. The heme c content in the mutant strain was ca. 50% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal reduction. Conclusion Omp

  2. Membranous nephropathy, antitubular basement membrane antibodies and alveolar hemorrhage in a diabetic child.

    PubMed

    Gallego, N; Olivares, F; Mampaso, F; Gonzalo, A; Barrio, R; Estepa, R; Ortuño, J

    1990-01-01

    We describe an 8-year-old boy who was diagnosed as having diabetes mellitus at the age of 3 months. During the follow-up the diabetes was uncontrolled, and he presented nephrotic syndrome with renal function impairment, a renal biopsy showing a membranous nephropathy. Subsequently he had episodes of anemia and dyspnea, due to alveolar hemorrhage, and he also developed Fanconi's syndrome. A later renal biopsy showed membranous glomerulonephritis and interstitial nephritis. The presence of antitubular basement membrane antibodies was noted but antialveolar basement membrane antibodies were not detected. We do not believe that this unusual clinical picture was a coincidence, and we speculate about a possible explanation.

  3. 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.

  4. 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,…

  5. Visual impairment in the hearing impaired students

    PubMed Central

    Gogate, Parikshit; Rishikeshi, Nikhil; Mehata, Reshma; Ranade, Satish; Kharat, Jitesh; Deshpande, Madan

    2009-01-01

    Background: Ocular problems are more common in children with hearing problems than in normal children. Neglected visual impairment could aggravate educational and social disability. Aim: To detect and treat visual impairment, if any, in hearing-impaired children. Setting and Design: Observational, clinical case series of hearing-impaired children in schools providing special education. Materials and Methods: Hearing-impaired children in selected schools underwent detailed visual acuity testing, refraction, external ocular examination and fundoscopy. Ocular motility testing was also performed. Teachers were sensitized and trained to help in the assessment of visual acuity using Snellen's E charts. Refractive errors and squint were treated as per standard practice. Statistical Analysis: Excel software was used for data entry and SSPS for analysis. Results: The study involved 901 hearing-impaired students between four and 21 years of age, from 14 special education schools. A quarter of them (216/901, 24%) had ocular problems. Refractive errors were the most common morbidity 167(18.5%), but only 10 children were using appropriate spectacle correction at presentation. Fifty children had visual acuity less than 20/80 at presentation; after providing refractive correction, this number reduced to three children, all of whom were provided low-vision aids. Other common conditions included strabismus in 12 (1.3%) children, and retinal pigmentary dystrophy in five (0.6%) children. Conclusion: Ocular problems are common in hearing-impaired children. Screening for ocular problems should be made mandatory in hearing-impaired children, as they use their visual sense to compensate for the poor auditory sense. PMID:19861747

  6. 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,…

  7. Immunogenicity of Pseudomonas aeruginosa outer membrane antigens examined by crossed immunoelectrophoresis.

    PubMed Central

    Lam, J S; Mutharia, L M; Hancock, R E; Høiby, N; Lam, K; Baek, L; Costerton, J W

    1983-01-01

    By crossed immunoelectrophoresis 36 different anode-migrating antigens were demonstrated in sonicated antigen preparations of Pseudomonas aeruginosa. We numbered these antigens to establish a reference precipitin pattern. Antigen no. 31 was identified as the lipopolysaccharide (LPS) antigen, because it was found to be responsible for the O-group specificity and because it reacted with anti-LPS monoclonal antibodies and with Limulus amoebocyte lysate. Purified outer membrane proteins F (porin), H2, and I used as antigens formed precipitins with the reference antibodies, thus establishing their antigenicity. LPS that copurified with protein F and slightly contaminated protein H2 was detectable as an extra precipitin (antigen no. 31). The use of monoclonal antibodies specific for smooth LPS and rough LPS revealed different antigenic determinants in the LPS molecule and suggested that antigen no. 5 could be the core region of the LPS which is equivalent to the rough LPS. Antibodies against these outer membrane antigens were detected in patients with chronic P. aeruginosa pneumonia and in patients with acute P. aeruginosa bacteremia. Antibodies with the same specificity were also found in rats chronically infected with P. aeruginosa 7 days postinfection. This demonstrates the surface accessibility and antigenic reactivity of outer membrane antigens. Images PMID:6194119

  8. 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

  9. 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 Central

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

    2016-01-01

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

  10. 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

  11. Organic Electrolyte Permselective Membranes.

    DTIC Science & Technology

    ORGANIC SOLVENTS), (*STORAGE BATTERIES, (*BATTERY SEPARATORS, MEMBRANES ), (* MEMBRANES , TRANSPORT PROPERTIES), LITHIUM, COPPER COMPOUNDS, DIFFUSION, CHLORIDES, IONS, ELECTRODIALYSIS , ION EXCHANGE RESINS.

  12. 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

  13. 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.

  14. Novel cationic peptide TP359 down-regulates the expression of outer membrane biogenesis genes in Pseudomonas aeruginosa: a potential TP359 anti-microbial mechanism.

    PubMed

    Dosunmu, Ejovwoke F; Chaudhari, Atul A; Bawage, Swapnil; Bakeer, Mona K; Owen, Donald R; Singh, Shree R; Dennis, Vida A; Pillai, Shreekumar R

    2016-08-22

    Antimicrobial peptides (AMPs) are a class of antimicrobial agents with broad-spectrum activities. Several reports indicate that cationic AMPs bind to the negatively charged bacterial membrane causing membrane depolarization and damage. However, membrane depolarization and damage may be insufficient to elicit cell death, thereby suggesting that other mechanism(s) of action could be involved in this phenomenon. In this study, we investigated the antimicrobial activity of a novel antimicrobial peptide, TP359, against two strains of Pseudomonas aeruginosa, as well as its possible mechanisms of action. TP359 proved to be bactericidal against P. aeruginosa as confirmed by the reduced bacteria counts, membrane damage and cytoplasmic membrane depolarization. In addition, it was non-toxic to mouse J774 macrophages and human lung A549 epithelial cells. Electron microscopy analysis showed TP359 bactericidal effects by structural changes of the bacteria from viable rod-shaped cells to those with cell membrane damages, proceeding into the efflux of cytoplasmic contents and emergence of ghost cells. Gene expression analysis on the effects of TP359 on outer membrane biogenesis genes underscored marked down-regulation, particularly of oprF, which encodes a major structural and outer membrane porin (OprF) in both strains studied, indicating that the peptide may cause deregulation of outer membrane genes and reduced structural stability which could lead to cell death. Our data shows that TP359 has potent antimicrobial activity against P aeruginosa. The correlation between membrane damage, depolarization and reduced expression of outer membrane biogenesis genes, particularly oprF may suggest the bactericidal mechanism of action of the TP359 peptide.

  15. 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

  16. 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…

  17. 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.

  18. 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…

  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. Membrane proteins in four acts: function precedes structure determination.

    PubMed

    Cramer, W A; Zakharov, S D; Saif Hasan, S; Zhang, H; Baniulis, D; Zhalnina, M V; Soriano, G M; Sharma, O; Rochet, J C; Ryan, C; Whitelegge, J; Kurisu, G; Yamashita, E

    2011-12-01

    Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b(6)f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) β-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B(12) binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a "fishing pole" model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to

  1. Modification of Salmonella Lipopolysaccharides Prevents the Outer Membrane Penetration of Novobiocin.

    PubMed

    Nobre, Thatyane M; Martynowycz, Michael W; Andreev, Konstantin; Kuzmenko, Ivan; Nikaido, Hiroshi; Gidalevitz, David

    2015-12-15

    Small hydrophilic antibiotics traverse the outer membrane of Gram-negative bacteria through porin channels. Large lipophilic agents traverse the outer membrane through its bilayer, containing a majority of lipopolysaccharides in its outer leaflet. Genes controlled by the two-component regulatory system PhoPQ modify lipopolysaccharides. We isolate lipopolysaccharides from isogenic mutants of Salmonella sp., one lacking the modification, the other fully modified. These lipopolysaccharides were reconstituted as monolayers at the air-water interface, and their properties, as well as their interaction with a large lipophilic drug, novobiocin, was studied. X-ray reflectivity showed that the drug penetrated the monolayer of the unmodified lipopolysaccharides reaching the hydrophobic region, but was prevented from this penetration into the modified lipopolysaccharides. Results correlate with behavior of bacterial cells, which become resistant to antibiotics after PhoPQ-regulated modifications. Grazing incidence x-ray diffraction showed that novobiocin produced a striking increase in crystalline coherence length, and the size of the near-crystalline domains.

  2. Purification, Refolding, and Crystallization of the Outer Membrane Protein OmpG from Escherichia coli.

    PubMed

    Köster, Stefan; van Pee, Katharina; Yildiz, Özkan

    2015-01-01

    OmpG is a pore-forming protein from E. coli outer membranes. Unlike the classical outer membrane porins, which are trimers, the OmpG channel is a monomeric β-barrel made of 14 antiparallel β-strands with short periplasmic turns and longer extracellular loops. The channel activity of OmpG is pH dependent and the channel is gated by the extracellular loop L6. At neutral/high pH, the channel is open and permeable for substrate molecules with a size up to 900 Da. At acidic pH, loop L6 folds across the channel and blocks the pore. The channel blockage at acidic pH appears to be triggered by the protonation of a histidine pair on neighboring β-strands, which repel one another, resulting in the rearrangement of loop L6 and channel closure. OmpG was purified by refolding from inclusion bodies and crystallized in two and three dimensions. Crystallization and analysis by electron microscopy and X-ray crystallography revealed the fundamental mechanisms essential for the channel activity.

  3. Association of the outer membrane protein Omp33 with fitness and virulence of Acinetobacter baumannii.

    PubMed

    Smani, Younes; Dominguez-Herrera, Juan; Pachón, Jerónimo

    2013-11-15

    Outer membrane protein 33 (Omp33) is an outer membrane porin of Acinetobacter baumannii associated with carbapenem resistance. However, the role of Omp33 in the fitness and virulence of A. baumannii remains unknown. In the present study, we investigated the role of Omp33 in fitness and virulence of A. baumannii by using an isogenic knockout strain deficient in the omp33 gene (JPAB02), derived from the ATCC 17978 wild-type (wt). Both in vitro and in vivo defect in the growth rate was found in the JPAB02 strain in competition with the ATCC 17978 wt, highlighting the effect of Omp33 on the metabolic fitness. A significant reduction was observed both in adherence and invasion of human lung epithelial cells and in cytotoxicity of these cells and macrophages with JPAB02. In a murine peritoneal sepsis model, the JPAB02 strain exhibited lower lethal dose 0 (LD0), LD50, and LD100, and dissemination in mice, with reduced bacterial concentration in spleen and lungs. From these data, we concluded that Omp33 plays an important role for fitness and virulence of A. baumannii.

  4. Modification of Salmonella Lipopolysaccharides Prevents the Outer Membrane Penetration of Novobiocin

    PubMed Central

    Nobre, Thatyane M.; Martynowycz, Michael W.; Andreev, Konstantin; Kuzmenko, Ivan; Nikaido, Hiroshi; Gidalevitz, David

    2015-01-01

    Small hydrophilic antibiotics traverse the outer membrane of Gram-negative bacteria through porin channels. Large lipophilic agents traverse the outer membrane through its bilayer, containing a majority of lipopolysaccharides in its outer leaflet. Genes controlled by the two-component regulatory system PhoPQ modify lipopolysaccharides. We isolate lipopolysaccharides from isogenic mutants of Salmonella sp., one lacking the modification, the other fully modified. These lipopolysaccharides were reconstituted as monolayers at the air-water interface, and their properties, as well as their interaction with a large lipophilic drug, novobiocin, was studied. X-ray reflectivity showed that the drug penetrated the monolayer of the unmodified lipopolysaccharides reaching the hydrophobic region, but was prevented from this penetration into the modified lipopolysaccharides. Results correlate with behavior of bacterial cells, which become resistant to antibiotics after PhoPQ-regulated modifications. Grazing incidence x-ray diffraction showed that novobiocin produced a striking increase in crystalline coherence length, and the size of the near-crystalline domains. PMID:26682812

  5. Vascular Cognitive Impairment.

    PubMed

    Dichgans, Martin; Leys, Didier

    2017-02-03

    Cerebrovascular disease typically manifests with stroke, cognitive impairment, or both. Vascular cognitive impairment refers to all forms of cognitive disorder associated with cerebrovascular disease, regardless of the specific mechanisms involved. It encompasses the full range of cognitive deficits from mild cognitive impairment to dementia. In principle, any of the multiple causes of clinical stroke can cause vascular cognitive impairment. Recent work further highlights a role of microinfarcts, microhemorrhages, strategic white matter tracts, loss of microstructural tissue integrity, and secondary neurodegeneration. Vascular brain injury results in loss of structural and functional connectivity and, hence, compromise of functional networks within the brain. Vascular cognitive impairment is common both after stroke and in stroke-free individuals presenting to dementia clinics, and vascular pathology frequently coexists with neurodegenerative pathology, resulting in mixed forms of mild cognitive impairment or dementia. Vascular dementia is now recognized as the second most common form of dementia after Alzheimer's disease, and there is increasing awareness that targeting vascular risk may help to prevent dementia, even of the Alzheimer type. Recent advances in neuroimaging, neuropathology, epidemiology, and genetics have led to a deeper understanding of how vascular disease affects cognition. These new findings provide an opportunity for the present reappraisal of vascular cognitive impairment. We further briefly address current therapeutic concepts.

  6. A novel Geobacteraceae-specific outer membrane protein J (OmpJ) is essential for electron transport to Fe(III) and Mn(IV) oxides in Geobacter sulfurreducens.

    PubMed

    Afkar, Eman; Reguera, Gemma; Schiffer, Marianne; Lovley, Derek R

    2005-07-06

    Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III) reduction in the Geobacter species that are the predominant Fe(III) reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration. When outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J), was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe(III) and insoluble Mn(IV) oxide, as the electron acceptor. The heme c content in the mutant strain was ca. 50% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal reduction. OmpJ is a putative porin found in the

  7. 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.

  8. 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…

  9. 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…

  10. 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)

  11. 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.

  12. 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. ...

  13. Kids' Quest: Vision Impairment

    MedlinePlus

    ... most important job. 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 ...

  14. Speech impairment (adult)

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003204.htm Speech impairment (adult) To use the sharing features on ... 2017, A.D.A.M., Inc. Duplication for commercial use must be authorized in writing by ADAM ...

  15. Evaluation of hearing impairment.

    PubMed

    Zadeh, M H; Selesnick, S H

    2001-01-01

    Hearing impairment is among the most common medical condition presenting to health care professionals. Ear anatomy, physiology, and pathology resulting in hearing loss are discussed. A systematic approach to evaluation, diagnosis, and treatment is presented.

  16. Imipenem Resistance of Enterobacter aerogenes Mediated by Outer Membrane Permeability

    PubMed Central

    Bornet, Charléric; Davin-Regli, Anne; Bosi, Claude; Pages, Jean-Marie; Bollet, Claude

    2000-01-01

    Multidrug-resistant Enterobacter aerogenes strains are increasingly isolated in Europe and especially in France. Treatment leads to imipenem resistance, because of a lack of porin. We studied the evolution of resistance in 29 strains isolated from four patients during their clinical course. These strains belonged to the prevalent epidemiological type observed in France in previous studies (C. Bosi, et al., J. Clin. Microbiol. 37:2165–2169, 1999; A. Davin-Regli et al., J. Clin. Microbiol. 34:1474–1480, 1996). They also harbored a TEM-24 extended-spectrum β-lactamase-coding gene. Thirteen strains were susceptible to gentamicin and resistant to imipenem and cefepime. All of the patients showed E. aerogenes strains with this resistance after an imipenem treatment. One patient showed resistance to imipenem after a treatment with cefpirome. Twelve of these 13 strains showed a lack of porin. Cessation of treatment with imipenem for three patients was followed by reversion of susceptibility to this antibiotic and the reappearance of porins, except in one case. For one patient, we observed three times in the same day the coexistence of resistant strains lacking porin and susceptible strains possessing porin. The emergence of multidrug-resistant E. aerogenes strains is very disquieting. In our study, infection by E. aerogenes increased the severity of the patients' illnesses, causing a 100% fatality rate. PMID:10698994

  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. Profiling posttraumatic functional impairment.

    PubMed

    Norman, Sonya B; Stein, Murray B; Davidson, Jonathan R T

    2007-01-01

    Many individuals who have been exposed to psychological trauma suffer from impaired functioning, regardless of whether they have PTSD. Our purpose was to identify a subset of PTSD symptoms linked to functional impairment to a) improve the likelihood that individuals with posttraumatic impairment receive treatment, and b) offer a method to assess cost-burden of trauma history in epidemiological studies. We examined patterns of trauma-related symptoms in two independent community surveys (N=1002 and 630). Rank ordering of symptoms and their associations with impairment guided construction of an impairment-related profile in the first data set. The profile was then tested in the second data set. The derived symptom profile, consisting of intense recollections and/or emotional symptoms upon exposure to reminders, plus one or more of numbing/detachment, avoidance, sleep problems, concentration problems, or hypervigilance, detected the majority (88% and 74%) of persons with posttraumatic functional impairment. The symptom profile can help identify traumatized individuals who may benefit from treatment but do not necessarily meet criteria for PTSD.

  19. 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

  20. 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.

  1. 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.

  2. ISPa46, a novel insertion sequence in the oprD porin gene of an imipenem-resistant Pseudomonas aeruginosa isolate from a cystic fibrosis patient in Marseille, France.

    PubMed

    Diene, Seydina M; L'homme, Tiphanie; Bellulo, Sophia; Stremler, Nathalie; Dubus, Jean-Christophe; Mely, Laurent; Leroy, Sylvie; Degand, Nicolas; Rolain, Jean-Marc

    2013-09-01

    Clinical isolates of Pseudomonas aeruginosa exhibiting high-level resistance to carbapenems were recovered from a French patient with cystic fibrosis (CF) who had not received carbapenem therapy. This study was conducted to investigate the molecular mechanism conferring the carbapenem-resistant phenotype in clinical isolates of P. aeruginosa recovered from the same CF patient chronically colonised since 2005. Investigation of imipenem resistance of P. aeruginosa strain_02 isolated in May 2011 showed no carbapenemase activity. However, amplification and sequencing of the oprD porin gene revealed disruption of this gene by an insertion sequence (IS) element of 1337 bp that contained a novel transposase of 1227 bp (ISPa46) bordered by two terminal imperfect inverted repeats of 28 bp, which was associated with carbapenem resistance. Retrospective analysis of five additional strains of P. aeruginosa isolated before May 2011 from the same patient revealed that all isolates were likely to be the same clone by multilocus sequence typing analysis (ST540/551), but one of the five isolates was imipenem-susceptible. Although it was possible to demonstrate the presence of ISPa46 in all strains by PCR, this IS was transposed in the oprD gene only for imipenem-resistant isolates. Therefore, this study reports a novel IS element (ISPa46) in P. aeruginosa clinical isolates of a CF patient in Marseille, France, that was associated with carbapenem resistance and was selected in the absence of carbapenem treatment.

  3. Synthetic Effect between Envelope Stress and Lack of Outer Membrane Vesicle Production in Escherichia coli

    PubMed Central

    Schwechheimer, Carmen

    2013-01-01

    Outer membrane vesicles (OMVs) are composed of outer membrane and periplasmic components and are ubiquitously secreted by Gram-negative bacteria. OMVs can disseminate virulence factors for pathogenic bacteria as well as serve as an envelope stress response. From a transposon mutant screen for OMV phenotypes, it was discovered that an nlpA mutant of Escherichia coli produces fewer OMVs than the wild type, whereas a degP mutant produces higher levels of OMVs. NlpA is an inner-membrane-anchored lipoprotein that has a minor role in methionine import. DegP is a periplasmic chaperone/protease for misfolded envelope proteins that is critical when cells are heat shocked. To reveal how these proteins contribute to OMV production, the mutations were combined and the double mutant analyzed. The ΔnlpA ΔdegP strain displayed a high-temperature growth defect that corresponded to the production of fewer OMVs than produced by the ΔdegP strain. This phenotype also pertained to other undervesiculation mutations in a ΔdegP background. The hypovesiculation phenotype of ΔnlpA in the wild-type strain as well as in the degP deletion strain was found to be a stationary-phase phenomenon. The periplasm of the ΔnlpA ΔdegP strain was determined to contain significantly more protein in stationary phase than the wild type. Additionally, misfolded DegP substrate outer membrane porins were detected in ΔdegP mutant-derived OMVs. These data suggest that an accumulation of envelope proteins resulting from decreased vesiculation was toxic and contributed to the growth defect. We conclude that OMV production contributes to relieve the envelope of accumulated toxic proteins and that NlpA plays an important role in the production of vesicles in stationary phase. PMID:23852867

  4. Synthetic effect between envelope stress and lack of outer membrane vesicle production in Escherichia coli.

    PubMed

    Schwechheimer, Carmen; Kuehn, Meta J

    2013-09-01

    Outer membrane vesicles (OMVs) are composed of outer membrane and periplasmic components and are ubiquitously secreted by Gram-negative bacteria. OMVs can disseminate virulence factors for pathogenic bacteria as well as serve as an envelope stress response. From a transposon mutant screen for OMV phenotypes, it was discovered that an nlpA mutant of Escherichia coli produces fewer OMVs than the wild type, whereas a degP mutant produces higher levels of OMVs. NlpA is an inner-membrane-anchored lipoprotein that has a minor role in methionine import. DegP is a periplasmic chaperone/protease for misfolded envelope proteins that is critical when cells are heat shocked. To reveal how these proteins contribute to OMV production, the mutations were combined and the double mutant analyzed. The ΔnlpA ΔdegP strain displayed a high-temperature growth defect that corresponded to the production of fewer OMVs than produced by the ΔdegP strain. This phenotype also pertained to other undervesiculation mutations in a ΔdegP background. The hypovesiculation phenotype of ΔnlpA in the wild-type strain as well as in the degP deletion strain was found to be a stationary-phase phenomenon. The periplasm of the ΔnlpA ΔdegP strain was determined to contain significantly more protein in stationary phase than the wild type. Additionally, misfolded DegP substrate outer membrane porins were detected in ΔdegP mutant-derived OMVs. These data suggest that an accumulation of envelope proteins resulting from decreased vesiculation was toxic and contributed to the growth defect. We conclude that OMV production contributes to relieve the envelope of accumulated toxic proteins and that NlpA plays an important role in the production of vesicles in stationary phase.

  5. The influence of poly-(L-lysine) and porin on the domain structure of mixed vesicles composed of lipopolysaccharide and phospholipid: an infrared spectroscopic study.

    PubMed Central

    Lasch, P; Schultz, C P; Naumann, D

    1998-01-01

    Fourier transform infrared (FTIR) spectroscopy has been used to study the thermotropic phase behavior of binary lipid mixtures composed of deuterated phospholipids (PLs) and lipopolysaccharides (LPSs). Furthermore, the influence of an extrinsic high-molecular, polycationic polypeptide (poly-(L-lysine), PLL(500)) and an intrinsic membrane protein (outer membrane protein F, OmpF) on these binary mixtures was investigated by FTIR spectroscopy. "Deep rough" mutant LPS (ReLPS), isolated from Salmonella minnesota R595, and perdeuterated 1,2-dimyristoylphosphatidylethanolamine (DMPEd54) were used as model lipids. Deuteration of one of the lipids permitted the detection of lipid protein interaction with each lipid component separately. For this purpose, the symmetric >CH2 and >CD2 stretching bands were utilized as specific monitors to scrutinize the state of order of the membranes. From the individual phase transition temperatures Tm and the shape of the phase transition profiles, it is established that ReLPS and DMPEd54 are molecularly immiscible. In addition to the two domains of the pure lipid components, a third, domain-like structure is detected that may coexist with these pure domains. This domain-like structure undergoes a gel to liquid-crystalline L1 (beta <--> alpha) phase transition at temperatures distinctly different from that of the respective pure lipid domains. The nature of this type of domain is discussed in terms of a "border region" model that adequately explains the experimentally observed complex phase transition profiles. It is further demonstrated that the extrinsic polycationic polypeptide PLL(500) and the intrinsic, pore-forming protein OmpF isolated from Escherichia coli interact preferentially and highly specifically with the negatively charged ReLPS. Both the synthetic polypeptide and the pore-forming protein increased the tendency of ReLPS and DMPEd54 to segregate into distinct, well-separated domains. Whereas the transition profiles of the

  6. Mainstreaming the Hearing Impaired Student.

    ERIC Educational Resources Information Center

    Szelazkiewicz, Sara

    This review of the literature on mainstreaming students with hearing impairments begins by defining "hearing impairment,""deafness," and "hard of hearing". The paper briefly considers causes of hearing impairments and types of hearing impairments. Suggestions for preparing a class for a student with hearing loss includes having a visible class…

  7. Trainable Mentally Impaired/Severely Multiply Impaired/Autistic Impaired/Severely Mentally Impaired. Product Evaluation Report 1989-1990.

    ERIC Educational Resources Information Center

    Claus, Richard N.; And Others

    The evaluation report describes special education services provided to trainable mentally impaired (TMI), autistic impaired (AI), severely multiply impaired (SXI), and severely mentally impaired (SMI) students at and through the Melvin G. Millet Learning Center (Bridgeport, Michigan). The eight program components are described individually and…

  8. Experimenting with Liquid Membranes.

    ERIC Educational Resources Information Center

    Lamb, J. D.; And Others

    1980-01-01

    Outlined are two experiments using liquid membranes that illustrate carrier-facilitated transport, where chemical species are ushered across the membrane by selective "carrier" molecules residing in the membrane. The use of liquid membranes as models for studying and describing biological transport mechanisms is explored. (CS)

  9. 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.

  10. Social communication impairments: pragmatics.

    PubMed

    Russell, Robert L

    2007-06-01

    Social communication or pragmatic impairments are characterized and illustrated as involving inappropriate or ineffective use of language and gesture in social contexts. Three clinical vignettes illustrate different pragmatic impairments and the wealth of diagnostic information that can be garnered from observation of a child's social communication behavior. Definitions of, and developmental milestones in, domains of pragmatic competence are provided. Several screening instruments are suggested for use in assessing pragmatic competence within the time-frame of a pediatric examination. Frequent comorbid psychiatric conditions are described and a sample of current neurobiologic research is briefly summarized.

  11. Membrane position control

    NASA Technical Reports Server (NTRS)

    Su, Ji (Inventor); Harrison, Joycelyn S. (Inventor)

    2004-01-01

    A membrane structure includes at least one electroactive bending actuator fixed to a supporting base. Each electroactive bending actuator is operatively connected to the membrane for controlling membrane position. Any displacement of each electroactive bending actuator effects displacement of the membrane. More specifically, the operative connection is provided by a guiding wheel assembly and a track, wherein displacement of the bending actuator effects translation of the wheel assembly along the track, thereby imparting movement to the membrane.

  12. Nanoporous Membrane Immunosensor

    DTIC Science & Technology

    2000-01-01

    Another aspect of the invention is a method for detecting an analyte in a test sample, having 5 the steps: (a) modifying a side of a semipermeable... side of the membrane with the membrane modifiers; (c) drawing the test sample through the membrane, osmotically or with the application of...immunoassay labels on the side of the membrane with the membrane modifiers, where these labels have label binding ligands where these label binding

  13. 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

  14. Anarthria impairs subvocal counting.

    PubMed

    Cubelli, R; Nichelli, P; Pentore, R

    1993-12-01

    We studied subvocal counting in two pure anarthric patients. Analysis showed that they performed definitively worse than normal subjects free to articulate subvocally and their scores were in the lower bounds of the performances of subjects suppressing articulation. These results suggest that subvocal counting is impaired after anarthria.

  15. Aids for visual impairment.

    PubMed Central

    Dudley, N J

    1990-01-01

    This article provides only a flavour of the type and range of aids available to the visually impaired person. Many other aids for leisure, learning, and daily living are illustrated in the RNIB equipment and games catalogue. Images FIG 1 FIG 3 FIG 4 FIG 5 FIG 6 FIG 7 FIG 8 FIG 9 FIG 10 PMID:2252929

  16. Emotionally Impaired Elementary Curriculum.

    ERIC Educational Resources Information Center

    Taulbee, Dianne R.; And Others

    A curriculum is presented for teaching emotionally impaired elementary students. The curriculum document describes program management techniques, strategies for developing and maintaining teacher-student relationships, and therapy/change systems. It outlines referral and eligibility procedures and exit criteria. It contains job descriptions for…

  17. Emotionally Impaired Elementary Curriculum.

    ERIC Educational Resources Information Center

    Taulbee, Dianne R.; And Others

    The Jackson County (Michigan) Intermediate School District curriculum for teaching emotionally impaired elementary students is presented. The curriculum document describes program management techniques, strategies for developing and maintaining teacher-student relationships, and therapy/change systems. It outlines referral and eligibility…

  18. Membranes and membrane plates used in ELISPOT.

    PubMed

    Weiss, Alan J

    2005-01-01

    Membrane-bottomed, 96-well plates constitute the format in which the overwhelming majority of enzyme-linked immunospot (ELISPOT) assays are performed. The membranes in these plates are made from either nitrocellulose or polyvinylidene fluoride. These membranes are well suited for ELISPOT because they have high antibody binding capacities and because their white color provides an excellent backdrop for ELISPOT enumeration. These two membranes and, ultimately, the 96-well plates used in ELISPOT assays were commercialized for filtration applications and later optimized for deoxyribonucleic acid hybridization and protein chemistry applications. In this chapter, an overview of the development and biotechnology applications of nitrocellulose and polyvinylidene fluoride membrane is provided and characteristics and attributes of each of the membranes that are relevant to ELISPOT are summarized.

  19. Comparing the Temperature-Dependent Conductance of the Two Structurally Similar E. coli Porins OmpC and OmpF

    PubMed Central

    Biró, István; Pezeshki, Soroosh; Weingart, Helge; Winterhalter, Mathias; Kleinekathöfer, Ulrich

    2010-01-01

    Abstract The temperature-dependent ion conductance of OmpC, a major outer membrane channel of Escherichia coli, is predicted using all-atom molecular dynamics simulations and experimentally verified. To generalize previous results, OmpC is compared to its structural homolog OmpF at different KCl concentrations, pH values, and a broad temperature range. At low salt concentrations and up to room temperature, the molecular modeling predicts the experimental conductance accurately. At high salt concentrations above 1 M KCl and above room temperature, the simulations underestimate the conductance. Moreover, the temperature dependence of the channel conductance is different from that of the bulk, both in experiment and simulation, indicating a strong contribution of surface effects to the ion conductance. With respect to OmpC, subconductance levels can be observed in experiments only. Subconductance and gating levels can be clearly distinguished by their differences in conductance values and temperature-dependent behavior. With increasing temperature, the probability of a subconductance state to occur, increases, while the dwell time is decreased. The open probability, frequency, and dwell time of such states is largely pH- and KCl concentration-independent, while their amplitudes show a lower increase with increasing salt concentration than gating amplitudes. Voltage dependence of subconductance has been found to be negligible within the uncertainty of the measurements. PMID:20441746

  20. Vestibular Impairment in Dementia

    PubMed Central

    Harun, Aisha; Oh, Esther S.; Bigelow, Robin T.; Studenski, Stephanie; Agrawal, Yuri

    2017-01-01

    Objective Recent studies suggest an association between vestibular and cognitive function. The goal of the study was to investigate whether vestibular function was impaired in individuals with mild cognitive impairment (MCI) and Alzheimer’s disease (AD) compared to cognitively normal individuals. Study Design Cross-sectional study. Setting Outpatient memory clinic and longitudinal observational study unit. Patients Older individuals ≥ 55 years with MCI or AD. Age, gender and education-matched normal controls were drawn from the Baltimore Longitudinal Study of Aging (BLSA). Intervention Saccular and utricular function was assessed with cervical and ocular vestibular-evoked myogenic potentials (c- and oVEMPs) respectively, and horizontal semicircular canal function was assessed with video head impulse testing. Main Outcome Measures Presence or absence of VEMP responses, VEMP amplitude and vestibular ocular reflex (VOR) gain were measured. Results Forty-seven individuals with cognitive impairment (MCI N=15 and AD N=32) underwent testing and were matched with 94 controls. In adjusted analyses, bilaterally absent cVEMPs were associated with an over three-fold odds of AD (OR 3.42, 95% CI 1.33–8.91, p=0.011). One microvolt increases in both cVEMP and oVEMP amplitudes were associated with decreased odds of AD (OR 0.28, 95% CI 0.09–0.93, p=0.038 and OR 0.92, 95% CI 0.85–0.99, p=0.036, respectively). There was no significant difference in VOR gain between the groups. Conclusions These findings confirm and extend emerging evidence of an association between vestibular dysfunction and cognitive impairment. Further investigation is needed to determine the causal direction for the link between peripheral vestibular loss and cognitive impairment. PMID:27466890

  1. Vestibular Impairment in Dementia.

    PubMed

    Harun, Aisha; Oh, Esther S; Bigelow, Robin T; Studenski, Stephanie; Agrawal, Yuri

    2016-09-01

    Recent studies suggest an association between vestibular and cognitive function. The goal of the study was to investigate whether vestibular function was impaired in individuals with mild cognitive impairment (MCI) and Alzheimer's disease (AD) compared with cognitively normal individuals. Cross-sectional study. Outpatient memory clinic and longitudinal observational study unit. Older individuals ≥55 years with MCI or AD. Age, sex, and education-matched normal controls were drawn from the Baltimore Longitudinal Study of Aging (BLSA). Saccular and utricular function was assessed with cervical and ocular vestibular-evoked myogenic potentials (c- and oVEMPs) respectively, and horizontal semicircular canal function was assessed with video head impulse testing. Presence or absence of VEMP responses, VEMP amplitude, and vestibular ocular reflex (VOR) gain were measured. Forty-seven individuals with cognitive impairment (MCI N = 15 and AD N = 32) underwent testing and were matched with 94 controls. In adjusted analyses, bilaterally absent cVEMPs were associated with an over three-fold odds of AD (OR 3.42, 95% CI 1.33-8.91, p = 0.011). One microvolt increases in both cVEMP and oVEMP amplitudes were associated with decreased odds of AD (OR 0.28, 95% CI 0.09-0.93, p = 0.038 and OR 0.92, 95% CI 0.85-0.99, p = 0.036, respectively). There was no significant difference in VOR gain between the groups. These findings confirm and extend emerging evidence of an association between vestibular dysfunction and cognitive impairment. Further investigation is needed to determine the causal direction for the link between peripheral vestibular loss and cognitive impairment.

  2. 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

  3. 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.

  4. Function and Expression of an N-Acetylneuraminic Acid-Inducible Outer Membrane Channel in Escherichia coli

    PubMed Central

    Condemine, Guy; Berrier, Catherine; Plumbridge, Jacqueline; Ghazi, Alexandre

    2005-01-01

    The Escherichia coli yjhA (renamed nanC) gene encodes a protein of the KdgM family of outer membrane-specific channels. It is transcribed divergently from fimB, a gene involved in the site-specific inversion of the region controlling transcription of the fimbrial structural genes but is separated from it by one of the largest intergenic regions in E. coli. We show that nanC expression is induced by N-acetylneuraminic acid and modulated by N-acetylglucosamine. This regulation occurs via the NanR and NagC regulators, which also control fimB expression. nanC expression is also activated by the regulators cyclic AMP-catabolite activator protein, OmpR, and CpxR. When the NanC protein was reconstituted into liposomes, it formed channels with a conductance of 450 pS at positive potential and 300 to 400 pS at negative potential in 800 mM KCl. The channels had a weak anionic selectivity. In an ompR background, where the general porins OmpF and OmpC are absent, NanC is required for growth of E. coli on N-acetylneuraminic acid as the sole carbon source. All these results suggest that NanC is an N-acetylneuraminic acid outer membrane channel protein. PMID:15743943

  5. Proteomic studies highlight outer-membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41.

    PubMed

    Ritter, Andrés; Com, Emmanuelle; Bazire, Alexis; Goncalves, Marina Dos Santos; Delage, Ludovic; Le Pennec, Gaël; Pineau, Charles; Dreanno, Catherine; Compère, Chantal; Dufour, Alain

    2012-11-01

    Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer-membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer-membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB-dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT-PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Pseudomonas aeruginosa outer membrane lipoprotein I gene: molecular cloning, sequence, and expression in Escherichia coli.

    PubMed Central

    Duchêne, M; Barron, C; Schweizer, A; von Specht, B U; Domdey, H

    1989-01-01

    Lipoprotein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. Like porin protein F (OprF), it is a vaccine candidate because it antigenically cross-reacts with all serotype strains of the International Antigenic Typing Scheme. Since lipoprotein I was expressed in Escherichia coli under the control of its own promoter, we were able to isolate the gene by screening a lambda EMBL3 phage library with a mouse monoclonal antibody directed against lipoprotein I. The monocistronic OprI mRNA encodes a precursor protein of 83 amino acid residues including a signal peptide of 19 residues. The mature protein has a molecular weight of 6,950, not including bound glycerol and lipid. Although the amino acid sequences of protein I of P. aeruginosa and Braun's lipoprotein of E. coli differ considerably (only 30.1% identical amino acid residues), peptidoglycan in E. coli, are identical. Using lipoprotein I expressed in E. coli, it can now be tested whether this protein alone, without P. aeruginosa lipopolysaccharide contaminations, has a protective effect against P. aeruginosa infections. Images PMID:2502533

  7. 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.

  8. Bacterial Nanobioreactors--Directing Enzyme Packaging into Bacterial Outer Membrane Vesicles.

    PubMed

    Alves, Nathan J; Turner, Kendrick B; Daniele, Michael A; Oh, Eunkeu; Medintz, Igor L; Walper, Scott A

    2015-11-11

    All bacteria shed outer membrane vesicles (OMVs) loaded with a diverse array of small molecules, proteins, and genetic cargo. In this study we sought to hijack the bacterial cell export pathway to simultaneously produce, package, and release an active enzyme, phosphotriesterase (PTE). To accomplish this goal the SpyCatcher/SpyTag (SC/ST) bioconjugation system was utilized to produce a PTE-SpyCatcher (PTE-SC) fusion protein and a SpyTagged transmembrane porin protein (OmpA-ST), known to be abundant in OMVs. Under a range of physiological conditions the SpyTag and SpyCatcher domains interact with one another and form a covalent isopeptide bond driving packaging of PTE into forming OMVs. The PTE-SC loaded OMVs are characterized for size distribution, number of vesicles produced, cell viability, packaged PTE enzyme kinetics, OMV loading efficiency, and enzyme stability following iterative cycles of freezing and thawing. The PTE-loaded OMVs exhibit native-like enzyme kinetics when assayed with paraoxon as a substrate. PTE is often toxic to expression cultures and has a tendency to lose activity with improper handling. The coexpression of OmpA-ST with PTE-SC, however, greatly improved the overall PTE production levels by mitigating toxicity through exporting of the PTE-SC and greatly enhanced packaged enzyme stability against iterative cycles of freezing and thawing.

  9. Water diffusion through a membrane protein channel: a first passage time approach.

    PubMed

    van Hijkoop, Vincent J; Dammers, Anton J; Malek, Kourosh; Coppens, Marc-Olivier

    2007-08-28

    Water diffusion through OmpF, a porin in the outer membrane of Escherichia coli, is studied by molecular dynamics simulation. A first passage time approach allows characterizing the diffusive properties of a well-defined region of this channel. A carbon nanotube, which is considerably more homogeneous, serves as a model to validate the methodology. Here we find, in addition to the expected regular behavior, a gradient of the diffusion coefficient at the channel ends, witness of the transition from confinement in the channel to bulk behavior in the connected reservoirs. Moreover, we observe the effect of a kinetic boundary layer, which is the counterpart of the initial ballistic regime in a mean square displacement analysis. The overall diffusive behavior of water in OmpF shows remarkable similarity with that in a homogeneous channel. However, a small fraction of the water molecules appears to be trapped by the protein wall for considerable lengths of time. The distribution of trapping times exhibits a broad power law distribution psi(tau) approximately tau (-2.4), up to tau=10 ns, a bound set by the length of the simulation run. We discuss the effect of this distribution on the dynamic properties of water in OmpF in terms of incomplete sampling of phase space.

  10. A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition

    PubMed Central

    Lin, Jinshui; Zhang, Weipeng; Cheng, Juanli; Yang, Xu; Zhu, Kaixiang; Wang, Yao; Wei, Gehong; Qian, Pei-Yuan; Luo, Zhao-Qing; Shen, Xihui

    2017-01-01

    Iron sequestration by host proteins contributes to the defence against bacterial pathogens, which need iron for their metabolism and virulence. A Pseudomonas aeruginosa mutant lacking all three known iron acquisition systems retains the ability to grow in media containing iron chelators, suggesting the presence of additional pathways involved in iron uptake. Here we screen P. aeruginosa mutants defective in growth in iron-depleted media and find that gene PA2374, proximal to the type VI secretion system H3 (H3-T6SS), functions synergistically with known iron acquisition systems. PA2374 (which we have renamed TseF) appears to be secreted by H3-T6SS and is incorporated into outer membrane vesicles (OMVs) by directly interacting with the iron-binding Pseudomonas quinolone signal (PQS), a cell–cell signalling compound. TseF facilitates the delivery of OMV-associated iron to bacterial cells by engaging the Fe(III)-pyochelin receptor FptA and the porin OprF. Our results reveal links between type VI secretion, cell–cell signalling and classic siderophore receptors for iron acquisition in P. aeruginosa. PMID:28348410

  11. 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.

  12. Activity of KB-5246 against outer membrane mutants of Escherichia coli and Salmonella typhimurium.

    PubMed Central

    Kotera, Y; Inoue, M; Mitsuhashi, S

    1990-01-01

    The inhibitory activity of KB-5246 against Escherichia coli DNA gyrase and the antibacterial activity and apparent uptake in E. coli and Salmonella typhimurium outer membrane mutants of KB-5246 were measured. The 50% inhibitory concentrations of KB-5246, ciprofloxacin, oflaxacin, and norfloxacin for E. coli KL-16 DNA gyrase were 0.72, 0.62, 0.84, and 1.16 micrograms/ml, respectively. The activity of KB-5246 was twofold lower against an OmpF-deficient mutant and twofold higher against a mutant which produced OmpF constitutively than against the parent with osmoregulated OmpF production. KB-5246 had twofold-higher activity against a deep rough mutant of S. typhimurium than against the parent. The apparent uptake of KB-5246 in the OmpF-deficient mutant was decreased and its uptake in the deep rough mutant was increased when compared with those in the parents. These results suggest that KB-5246 is taken up by porin and nonporin pathways and has strong inhibitory activity against DNA gyrase, resulting in potent antibacterial activity. PMID:2167038

  13. Geometry of membrane fission.

    PubMed

    Frolov, Vadim A; Escalada, Artur; Akimov, Sergey A; Shnyrova, Anna V

    2015-01-01

    Cellular membranes define the functional geometry of intracellular space. Formation of new membrane compartments and maintenance of complex organelles require division and disconnection of cellular membranes, a process termed membrane fission. Peripheral membrane proteins generally control membrane remodeling during fission. Local membrane stresses, reflecting molecular geometry of membrane-interacting parts of these proteins, sum up to produce the key membrane geometries of fission: the saddle-shaped neck and hour-glass hemifission intermediate. Here, we review the fundamental principles behind the translation of molecular geometry into membrane shape and topology during fission. We emphasize the central role the membrane insertion of specialized protein domains plays in orchestrating fission in vitro and in cells. We further compare individual to synergistic action of the membrane insertion during fission mediated by individual protein species, proteins complexes or membrane domains. Finally, we describe how local geometry of fission intermediates defines the functional design of the protein complexes catalyzing fission of cellular membranes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  14. 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

  15. 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.

  16. 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)

  17. Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

    PubMed Central

    Fraldi, Alessandro; Annunziata, Fabio; Lombardi, Alessia; Kaiser, Hermann-Josef; Medina, Diego Luis; Spampanato, Carmine; Fedele, Anthony Olind; Polishchuk, Roman; Sorrentino, Nicolina Cristina; Simons, Kai; Ballabio, Andrea

    2010-01-01

    The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol-enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders. PMID:20871593

  18. Mechanisms of Membrane Preparation and Membrane Assays.

    DTIC Science & Technology

    The course of the work for the report period involved: Evaluating and developing testing procedures for membrane filters, and the construction, procurement of equipment for such testing; and Numerous sol preparations and castings of membranes using the acetate-butyrate esters of cellulose in lieu of the cellulose acetate in conjunction with the cellulose nitrate base of formulation. (Author)

  19. Impaired Consciousness in Epilepsy

    PubMed Central

    Blumenfeld, Hal

    2013-01-01

    Consciousness is essential to normal human life. In epileptic seizures consciousness is often transiently lost making it impossible for the individual to experience or respond. This has huge consequences for safety, productivity, emotional health and quality of life. To prevent impaired consciousness in epilepsy it is necessary to understand the mechanisms leading to brain dysfunction during seizures. Normally the “consciousness system”—a specialized set of cortical-subcortical structures—maintains alertness, attention and awareness. Recent advances in neuroimaging, electrophysiology and prospective behavioral testing have shed new light on how epileptic seizures disrupt the consciousness system. Diverse seizure types including absence, generalized tonic-clonic and complex partial seizures converge on the same set of anatomical structures through different mechanisms to disrupt consciousness. Understanding these mechanisms may lead to improved treatment strategies to prevent impaired consciousness and improve quality of life in people with epilepsy. PMID:22898735

  20. Age-Related Sensory Impairments and Risk of Cognitive Impairment.

    PubMed

    Fischer, Mary E; Cruickshanks, Karen J; Schubert, Carla R; Pinto, Alex A; Carlsson, Cynthia M; Klein, Barbara E K; Klein, Ronald; Tweed, Ted S

    2016-10-01

    To evaluate the associations between sensory impairments and 10-year risk of cognitive impairment. The Epidemiology of Hearing Loss Study (EHLS), a longitudinal, population-based study of aging in the Beaver Dam, Wisconsin community. Baseline examinations were conducted in 1993 and follow-up examinations have been conducted every 5 years. General community. EHLS members without cognitive impairment at EHLS-2 (1998-2000). There were 1,884 participants (mean age 66.7) with complete EHLS-2 sensory data and follow-up information. Cognitive impairment was defined as a Mini-Mental State Examination score of <24 or history of dementia or Alzheimer's disease. Hearing impairment was a pure-tone average of hearing thresholds (0.5, 1, 2, 4 kHz) of >25 dB hearing level in either ear, visual impairment was a Pelli-Robson contrast sensitivity of <1.55 log units in the better eye, and olfactory impairment was a San Diego Odor Identification Test score of <6. Hearing, visual, and olfactory impairment were independently associated with cognitive impairment risk (hearing: hazard ratio (HR) = 1.90, 95% confidence interval (CI) = 1.11-3.26; vision: HR = 2.05, 95% CI = 1.24-3.38; olfaction: HR = 3.92, 95% CI = 2.45-6.