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. Copyright © 2012 Wiley Periodicals, Inc.

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.

Porin Deficiency in Carbapenem-Resistant Enterobacter aerogenes Strains.

PubMed

Hao, Min; Ye, Meiping; Shen, Zhen; Hu, Fupin; Yang, Yang; Wu, Shi; Xu, Xiaogang; Zhu, Sihui; Qin, Xiaohua; Wang, Minggui

2018-03-13

The more frequent reports of carbapenem-resistant Enterobacteriaceae have raised the alarm for public health. Apart from the production of carbapenemases, deficiency (decreased or loss of expression) of outer membrane proteins (OMPs) has been proposed as a potentially important mechanism of carbapenem resistance. The aim of the present study was to evaluate the contribution of the major OMPs to carbapenem resistance in Enterobacter aerogenes (CREA) isolates and also investigate the role of small RNAs (sRNAs) in inducing porin-associated permeability defects. The differential expression of OMPs was analyzed in four clinical CREA isolates. omp35 and omp36 genes were further investigated by whole-genome sequencing, induction of meropenem resistance, sRNA overexpression, OMP complementation assays, and reverse transcription-quantitative PCR. All four isolates examined were deficient in omp35 and omp36. Functional restoration of these two genes confirmed their contribution to carbapenem resistance. The meropenem induction assay further revealed that porin deficiency plays a role in carbapenem resistance under antibiotic selection pressure. Single-point mutations in omp36 leading to premature stop codons were detected in two of the isolates. Elevated expression levels of the sRNAs micF and micC were detected in the other two porin-deficient isolates, which were predicted to be potential porin regulators from whole-genome sequencing. Overexpression of micF and micC downregulated the expression of Omp35 and Omp36, respectively. Porin deficiency plays an important role in carbapenem resistance among clinical E. aerogenes isolates under regulation of the sRNAs micC and micF. Furthermore, overexpression of micC and micF had a minor to no impact on carbapenem minimum inhibitory concentrations, and thus, the regulatory mechanism is likely to be complex.

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. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Channel specificity and secondary structure of the glucose-inducible porins of Pseudomonas spp.

PubMed

Adewoye, L O; Tschetter, L; O'Neil, J; Worobec, E A

1998-06-01

The OprB porin-mediated glucose transport system was investigated in Pseudomonas chlororaphis, Burkholderia cepacia, and Pseudomonas fluorescens. Kinetic studies of [U-14C]glucose uptake revealed an inducible system of low Km values (0.3-5 microM) and high specificity for glucose. OprB homologs were purified and reconstituted into proteoliposomes. The porin function and channel preference for glucose were demonstrated by liposome swelling assays. Examination of the periplasmic glucose-binding protein (GBP) components by Western immunoblotting using P. aeruginosa GBP-specific antiserum revealed some homology between P. aeruginosa GBP and periplasmic proteins from P. fluorescens and P. chlororaphis but not B. cepacia. Circular dichroism spectropolarimetry of purified OprB-like porins from the three species revealed beta sheet contents of 31-50% in agreement with 40% beta sheet content for the P. aeruginosa OprB porin. These findings suggest that the high-affinity glucose transport system is primarily specific for glucose and well conserved in the genus Pseudomonas although its outer membrane component may differ in channel architecture and specificity for other carbohydrates.

Global survey of Klebsiella pneumoniae major porins from ertapenem non-susceptible isolates lacking carbapenemases.

PubMed

Wise, Mark G; Horvath, Elizabeth; Young, Katherine; Sahm, Daniel F; Kazmierczak, Krystyna M

2018-03-01

To understand the diversity of porin disruption in Klebsiella pneumoniae, the major outer membrane protein (OMP) porins, OmpK35 and OmpK36, were examined in a set of isolates that did not harbour traditional carbapenem-hydrolysing enzymes, but nevertheless tested non-susceptible to ertapenem. A world-wide collection of Klebsiella pneumoniae isolates that were part of the Study for Monitoring Antimicrobial Resistance Trends (SMART) surveillance project over the years 2008-2014 were characterised with regard to their β-lactamase gene carriage and potential permeability defects. Four hundred and eighty-seven isolates that did not carry carbapenemase genes, but were non-susceptible to ertapenem, were investigated by sequence analysis of the genes encoding OmpK35 and OmpK36. Isolates without obvious genetic lesions in either major porin gene were further examined by outer membrane protein SDS-PAGE. The majority of isolates, 83.0 % (404/487), exhibited clear genetic disruption in either or both of the ompK35 and ompK36 genes. Among the proportion of the collection with the highest ertapenem MIC value (>4 mg l -1 ), 60.5 % (115/190) showed mutation in both porin genes. Isolates without obvious genetic mutations were examined by SDS-PAGE, and 90.4 % (75/83) were found to lack or show altered expression of at least one of the major OMPs when compared to an ertapenem sensitive control strain. This study illustrates that porin deficiency in Klebsiella pneumoniae is a widespread phenomenon, and in combination with ESBLs and/or AmpC enzymes, likely accounts for the elevated ertapenem MICs observed in this study.

Characterization of the porins of Campylobacter jejuni and Campylobacter coli and implications for antibiotic susceptibility.

PubMed Central

Page, W J; Huyer, G; Huyer, M; Worobec, E A

1989-01-01

The major outer membrane protein was extracted from Campylobacter coli by Triton X-100/EDTA fractionation of cell envelopes. This heat-modifiable protein was shown to have pore-forming activity in black lipid bilayers. The C. coli porin formed a relatively small cation-selective pore with a mean single-channel conductance of 0.53 +/- 0.16 nS in 1.0 M KCl. There was no evidence of oligomer formation, which suggested that each protein monomer formed a pore. Pore-forming activity of the C. coli porin and similarly prepared Campylobacter jejuni porin was also measured in liposome-swelling assays. These results confirmed the cation selectivity of both pores. The C. coli porin formed a small pore, which hindered the penetration of solutes with a molecular weight of 262, and a larger pore, which hindered the penetration of solutes with a molecular weight of 340, in a protein-concentration-dependent manner. C. jejuni formed one size of pore that was slightly larger than the C. coli pore and just permitted the passage of solutes, with a molecular weight of 340. A review of the literature concerning in vitro screening of antimicrobial agents tended to confirm the low permeability of the C. jejuni outer membrane to hydrophilic antimicrobial agents except when the molecules had molecular weights of less than 360. The porins of C. jejuni and C. coli may contribute to intrinsic resistance to antimicrobial agents, whereas alternative (nonporin) routes of antimicrobial agent uptake may be more important determinants of susceptibility to antimicrobial agents. Images PMID:2543277

Characterization of the porins of Campylobacter jejuni and Campylobacter coli and implications for antibiotic susceptibility.

PubMed

Page, W J; Huyer, G; Huyer, M; Worobec, E A

1989-03-01

The major outer membrane protein was extracted from Campylobacter coli by Triton X-100/EDTA fractionation of cell envelopes. This heat-modifiable protein was shown to have pore-forming activity in black lipid bilayers. The C. coli porin formed a relatively small cation-selective pore with a mean single-channel conductance of 0.53 +/- 0.16 nS in 1.0 M KCl. There was no evidence of oligomer formation, which suggested that each protein monomer formed a pore. Pore-forming activity of the C. coli porin and similarly prepared Campylobacter jejuni porin was also measured in liposome-swelling assays. These results confirmed the cation selectivity of both pores. The C. coli porin formed a small pore, which hindered the penetration of solutes with a molecular weight of 262, and a larger pore, which hindered the penetration of solutes with a molecular weight of 340, in a protein-concentration-dependent manner. C. jejuni formed one size of pore that was slightly larger than the C. coli pore and just permitted the passage of solutes, with a molecular weight of 340. A review of the literature concerning in vitro screening of antimicrobial agents tended to confirm the low permeability of the C. jejuni outer membrane to hydrophilic antimicrobial agents except when the molecules had molecular weights of less than 360. The porins of C. jejuni and C. coli may contribute to intrinsic resistance to antimicrobial agents, whereas alternative (nonporin) routes of antimicrobial agent uptake may be more important determinants of susceptibility to antimicrobial agents.

Influence of Culture Conditions on Expression of the 40-Kilodalton Porin Protein of Vibrio anguillarum Serotype O2

PubMed Central

Davey, Michelle L.; Hancock, Robert E. W.; Mutharia, Lucy M.

1998-01-01

Vibrio anguillarum serotype O2 strains express a 40-kDa outer membrane porin protein. Immunoblot analysis revealed that antigenic determinants of the V. anguillarum O2 40-kDa porin were conserved within bacterial species of the genus Vibrio. The relative amounts of the V. anguillarum O2 40-kDa porin were enhanced by growth of V. anguillarum O2 in CM9 medium containing 5 to 10% sucrose or 0.1 to 0.5 M NaCl. In contrast, the levels of the porin were significantly reduced when cells were grown at 37°C, and a novel 60-kDa protein was also observed. However, the osmolarity or ionic concentration of the growth medium did not influence expression of the 60-kDa protein. Growth in medium containing greater than 0.6 mM EDTA reduced production of the V. anguillarum O2 40-kDa porin and enhanced levels of a novel 19-kDa protein. Thus, expression of the V. anguillarum O2 40-kDa porin was osmoregulated and possibly coregulated by temperature. The N-terminal amino acid sequence of the V. anguillarum O2 40-kDa protein and the effect of environmental factors on the cellular levels of the porin suggested that the V. anguillarum O2 40-kDa porin was functionally similar to the OmpC porin of Escherichia coli. However, pore conductance assays revealed that the V. anguillarum O2 40-kDa porin was a general diffusion porin with a pore size in the range of that of the OmpF porin of E. coli. PMID:9435071

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

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

PubMed

Wylie, J L; Worobec, E A

1995-06-01

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

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

PubMed Central

Wylie, J L; Worobec, E A

1995-01-01

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

Engineering sucrose metabolism in Pseudomonas putida highlights the importance of porins.

PubMed

Löwe, Hannes; Sinner, Peter; Kremling, Andreas; Pflüger-Grau, Katharina

2018-05-28

Using agricultural wastes as a substrate for biotechnological processes is of great interest in industrial biotechnology. A prerequisite for using these wastes is the ability of the industrially relevant microorganisms to metabolize the sugars present therein. Therefore, many metabolic engineering approaches are directed towards widening the substrate spectrum of the workhorses of industrial biotechnology like Escherichia coli, yeast or Pseudomonas putida. For instance, neither xylose or arabinose from cellulosic residues, nor sucrose, the main sugar in waste molasses, can be metabolized by most E. coli and P. putida wild types. We evaluated a new, so far uncharacterized gene cluster for sucrose metabolism from Pseudomonas protegens Pf-5 and showed that it enables P. putida to grow on sucrose as the sole carbon and energy source. Even when integrated into the genome of P. putida, the resulting strain grew on sucrose at rates similar to the rate of the wild type on glucose - making it the fastest growing, plasmid-free P. putida strain known so far using sucrose as substrate. Next, we elucidated the role of the porin, an orthologue of the sucrose porin ScrY, in the gene cluster and found that in P. putida, a porin is needed for sucrose transport across the outer membrane. Consequently, native porins were not sufficient to allow unlimited growth on sucrose. Therefore, we concluded that the outer membrane can be a considerable barrier for substrate transport, depending on strain, genotype and culture conditions, all of which should be taken into account in metabolic engineering approaches. We additionally showed the potential of the engineered P. putida strains by growing them on molasses with efficiencies twice as high as obtained with the wild-type P. putida. This can be seen as a further step towards the production of low-value chemicals and biofuels with P. putida from alternative and more affordable substrates in the future. © 2018 The Authors

Multiple environmental factors regulate the expression of the carbohydrate-selective OprB porin of Pseudomonas aeruginosa.

PubMed

Adewoye, L O; Worobec, E A

1999-12-01

In response to low extracellular glucose concentration, Pseudomonas aeruginosa induces the expression of the outer membrane carbohydrate-selective OprB porin. The promoter region of the oprB gene was cloned into a lacZ transcriptional fusion vector, and the construct was mobilized into P. aeruginosa OprB-deficient strain, WW100, to evaluate additional environmental factors that influence OprB porin gene expression. Growth temperature, pH of the growth medium, salicylate concentration, and carbohydrate source were found to differentially influence porin expression. This expression pattern was compared to those of whole-cell [14C]glucose uptake under conditions of high osmolarity, ionicity, variable pH, growth temperatures, and carbohydrate source. These studies revealed that the high-affinity glucose transport genes are down-regulated by salicylic acid, differentially regulated by pH and temperature, and are specifically responsive to exogenous glucose induction.

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 Central

Kowata, Hikaru; Tochigi, Saeko; Takahashi, Hideyuki

2017-01-01

ABSTRACT 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

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.

Direct Observation of Gold Nanoparticle Assemblies with the Porin MspA on Mica

PubMed Central

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

2009-01-01

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

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

Purification, characterization and sequence analysis of Omp50,a new porin isolated from Campylobacter jejuni.

PubMed Central

Bolla, J M; Dé, E; Dorez, A; Pagès, J M

2000-01-01

A novel pore-forming protein identified in Campylobacter was purified by ion-exchange chromatography and named Omp50 according to both its molecular mass and its outer membrane localization. We observed a pore-forming ability of Omp50 after re-incorporation into artificial membranes. The protein induced cation-selective channels with major conductance values of 50-60 pS in 1 M NaCl. N-terminal sequencing allowed us to identify the predicted coding sequence Cj1170c from the Campylobacter jejuni genome database as the corresponding gene in the NCTC 11168 genome sequence. The gene, designated omp50, consists of a 1425 bp open reading frame encoding a deduced 453-amino acid protein with a calculated pI of 5.81 and a molecular mass of 51169.2 Da. The protein possessed a 20-amino acid leader sequence. No significant similarity was found between Omp50 and porin protein sequences already determined. Moreover, the protein showed only weak sequence identity with the major outer-membrane protein (MOMP) of Campylobacter, correlating with the absence of antigenic cross-reactivity between these two proteins. Omp50 is expressed in C. jejuni and Campylobacter lari but not in Campylobacter coli. The gene, however, was detected in all three species by PCR. According to its conformation and functional properties, the protein would belong to the family of outer-membrane monomeric porins. PMID:11104668

Site-directed mutagenesis studies to probe the role of specific residues in the external loop (L3) of OmpF and OmpC porins in susceptibility of Serratia marcescens to antibiotics.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2007-06-01

Serratia marcescens is a nosocomial bacterium with natural resistance to a broad spectrum of antibiotics, making treatment challenging. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, controlled in part by OmpF and OmpC porin proteins. To investigate the direct role of these porins in the diffusion of antibiotics across the outer membrane, we have created an ompF-ompC porin-deficient strain of S. marcescens. A considerable similarity between the S. marcescens porins and those from other members of Enterobacteriaceae was detected by sequence alignment, with the exception of a change in a conserved region of the third external loop (L3) of the S. marcescens OmpC protein. Serratia marcescens OmpC has aspartic acid instead of glycine in position 112, methionine instead of aspartic acid in position 114, and glutamine in position 124, while in S. marcescens OmpF this is a glycine at position 124. To investigate the role of amino acid positions 112, 114, and 124 and how the observed changes within OmpC porin may play a part in pore permeability, 2 OmpC sites were altered in the Enterobacteriaceae consensus (D112G and M114D) through site-directed mutagenesis. Also, Q124G in OmpC, G124Q in OmpF, and double mutants of these amino acid residues were constructed. Antibiotic accumulation assays and minimal inhibitory concentrations of the strains harboring the mutated porins were performed, while liposome swelling experiments were performed on purified porins. Our results demonstrate that the amino acid at position 114 is not responsible for either antibiotic size or ionic selection, the amino acid at position 112 is responsible for size selection only, and position 124 is involved in both size and ionic selection.

Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins.

PubMed

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

2002-09-24

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

New functions of an old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC).

PubMed

De Pinto, Vito; Messina, Angela; Accardi, Rosita; Aiello, Rita; Guarino, Francesca; Tomasello, Marianna Flora; Tommasino, Massimo; Tasco, Gianluca; Casadio, Rita; Benz, Roland; De Giorgi, Francesca; Ichas, François; Baker, Mark; Lawen, Alfons

2003-03-01

Mitochondrial porin or VDAC (Voltage Dependent Anion selective Channels) was identified for the first time in 1976, on the basis of the evolutionary similarity between the gram negative and mitochondrial outer membranes. Since this achievement VDAC has been extensively investigated: its functional features have been sharply defined upon reconstitution in artificial membranes and its sequence has been determined in many genomes. Unfortunately the tertiary structure has not yet been solved, mainly because it proved to be very difficult to get suitable crystals. Despite this established knowledge, in the last few years this protein has attracted renewed interest. There are two main reasons for this interest: the discovery, in most eukaryotes, of a family of genes encoding VDAC isoforms and the claims of VDAC involvement in the intrinsic pathway of apoptosis and in particular in the mechanism of cytochrome c release from mitochondria. We can affirm that nowadays the eukaryotic porin (or VDAC) is studied in a more general cellular contest, looking at the interactions and integration with other molecules, since VDAC is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. In this minireview we will briefly focus our attention onto the following topics: 1) recent advances about the structure of VDAC; 2) the VDAC-related multigene families; 3) the presence, targeting and function of VDAC in various cell membranes.

The Role of OmpK35, OmpK36 Porins, and Production of β-Lactamases on Imipenem Susceptibility in Klebsiella pneumoniae Clinical Isolates, Cairo, Egypt.

PubMed

Wassef, Mona; Abdelhaleim, Mona; AbdulRahman, Eiman; Ghaith, Doaa

2015-12-01

OmpK35 and OmpK36 are the major outer membrane porins of Klebsiella pneumoniae. We aimed to study the effect of combined porin loss and production of extended-spectrum β-lactamases (ESBLs) on imipenem susceptibility among K. pneumoniae clinical isolates. This study included 91 suspected ESBL-producing K. pneumoniae clinical isolates, isolated from different patient specimens at the Cairo University hospital from January to June 2010. All isolates were subjected to genotypic analysis of the outer membrane protein gene expression using reverse transcription-PCR (RT-PCR) and analysis of OmpK35/36 of 38 isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). By RT-PCR, loss of Omp35 was detected in 78 (85.7%) isolates, loss of Omp36 was detected in 64 (70.32%), and loss of both porins was detected in 62 (68.1%). Out of 91 isolates, 45 (49.5%) were resistant to cefoxitin, and 17 (18.7%) were confirmed as derepressed AmpC producers. Omp35 was lost in all FOX-resistant isolates, whereas Omp36 was lost in 42 (93.3%) (p-value 0.002). The mean of ceftazidime inhibition zone diameter was significantly decreased among ESBL-producing isolates with loss of Omp35/36 (p-value 0.041 and 0.006), respectively. The mean of imipenem minimal inhibitory concentration (MIC) was markedly increased to 8.55 μg/ml among AmpC-producing isolates with Omp35/36 loss, while the mean of imipenem MIC among the 66 confirmed ESBL producers was 0.32 μg/ml. Imipenem MIC was markedly increased among K. pneumoniae isolates showing AmpC production with loss of both porins OmpK35/36. Meanwhile, the association of porin OmpK35/36 loss with ESBL production was not a direct cause of resistance to imipenem.

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

Biophysical characterization of OprB, a glucose-inducible porin of Pseudomonas aeruginosa.

PubMed

Wylie, J L; Bernegger-Egli, C; O'Neil, J D; Worobec, E A

1993-10-01

OprB, a glucose-inducible porin of P. aeruginosa, was characterized by black lipid bilayer analysis and circular dichroism spectroscopy. Black lipid bilayer analysis of OprB revealed a single-channel conductance of 25 pS, the presence of a glucose binding site with a Ks for glucose of 380 +/- 40 mM, and the formation of channels with a strong selection for anions. Analysis of P. aeruginosa OprB circular dichroism spectra revealed a high beta sheet content (40%) which is within the range of that determined for other porins. Values obtained from black lipid bilayer analysis were compared to those previously obtained for OprB of P. putida [Saravolac et al. (1991). J. Bacteriol. 173, 4970-4976] and indicated extensive similarities in the single-channel conductance and glucose-binding properties of these two porins. Immunological and amino terminal sequence analysis revealed a high degree of homology. Of the first 14 amino terminal residues, 12 were identical. A major difference between the two porins was found in their ion selectivity. Whereas P. aeruginosa OprB is anion selective, P. putida OprB and other carbohydrate selective porins are known to be cation selective.

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

Molecular characterization of a 40 kDa OmpC-like porin from Serratia marcescens.

PubMed

Hutsul, J A; Worobec, E

1994-02-01

An oligonucleotide that encodes the N-terminal portion of a 41 kDa porin of Serratia marcescens was used to probe S. marcescens UOC-51 genomic DNA. An 11 kb EcoRI fragment which hybridized with the oligonucleotide was subcloned into Escherichia coli, examined for expression, and sequenced. The product expressed by the cloned gene was 40 kDa. The nucleotide sequence has an ORF of 1.13 kb. When the deduced amino acid sequence was aligned and compared to other enterobacterial porins the cloned S. marcescens porin most closely resembled E. coli OmpC. Although we did not detect osmoregulation or thermoregulation of any porins in S. marcescens UOC-51, sequences analogous to the E. coli osmoregulator OmpR-binding regions are seen upstream to the cloned gene. We examined the regulation of the S. marcescens porin in E. coli and found that its expression increased in a high salt environment. A micF gene, whose transcriptional product functions to inhibit synthesis of OmpF by hybridizing with the ompF transcript, was also seen upstream of the S. marcescens ompC. An alignment with the E. coli micF gene revealed that the functional region of the S. marcescens micF gene is conserved. Based on the results obtained we have determined that S. marcescens UOC-51 produces a 40 kDa porin similar to the E. coli OmpC porin.

Large-scale purification and biochemical characterization of crystallization-grade porin protein P from Pseudomonas aeruginosa.

PubMed

Worobec, E A; Martin, N L; McCubbin, W D; Kay, C M; Brayer, G D; Hancock, R E

1988-04-07

A large-scale purification scheme was developed for lipopolysaccharide-free protein P, the phosphate-starvation-inducible outer-membrane porin from Pseudomonas aeruginosa. This highly purified protein P was used to successfully form hexagonal crystals in the presence of n-octyl-beta-glucopyranoside. Amino-acid analysis indicated that protein P had a similar composition to other bacterial outer membrane proteins, containing a high percentage (50%) of hydrophilic residues. The amino-terminal sequence of this protein, although not homologous to either outer membrane protein, PhoE or OmpF, of Escherichia coli, was found to have an analogous protein-folding pattern. Protein P in the native trimer form was capable of maintaining a stable functional trimer after proteinase cleavage. This suggested the existence of a strongly associated tertiary and quaternary structure. Circular dichroism studies confirmed these results in that a large proportion of the protein structure was determined to be beta-sheet and resistant to acid pH and heating in 0.1% sodium dodecyl sulphate.

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

PubMed Central

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

2011-01-01

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

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

PubMed Central

Siehnel, R J; Worobec, E A; Hancock, R E

1988-01-01

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes. Images PMID:2834340

Functional assay of Salmonella typhi OmpC using reconstituted large unilamellar vesicles: a general method for characterization of outer membrane proteins.

PubMed

Sundara Baalaji, N; Mathew, M K; Krishnaswamy, S

2006-10-01

The immunodominant trimeric beta-barrel outer membrane protein OmpC from Salmonella typhi, the causative agent of typhoid, has been functionally characterized here. The activity in the vesicle environment was studied in vitro using OmpC reconstituted into proteoliposomes. Passage of polysaccharides and polyethyleneglycols through OmpC has been examined to determine the permeability properties. The relative rate of neutral solute flux yields a radius of 1.1 nm for the S. typhi OmpC pore. This is almost double the pore size of Escherichia coli. This provides an example of large pore size present in the porins that form trimers as in the general bacterial porin family. The method used in this study provides a good membrane model for functional studies of porins.

Influence of the surrounding environment in re-naturalized β-barrel membrane proteins.

PubMed

Lopes-Rodrigues, Maximilien; Triguero, Jordi; Torras, Juan; Perpète, Eric A; Michaux, Catherine; Zanuy, David; Alemán, Carlos

2018-03-01

Outer-membrane porins are currently being used to prepare bioinspired nanomembranes for selective ion transport by immobilizing them into polymeric matrices. However, the fabrication of these protein-integrated devices has been found to be strongly influenced by the instability of the β-barrel porin structure, which depends on surrounding environment. In this work, molecular dynamics simulations have been used to investigate the structural stability of a representative porin, OmpF, in three different environments: (i) aqueous solution at pH=7; (ii) a solution of neutral detergent in a concentration similar to the critical micelle concentration; and (iii) the protein embedded into a neutral detergent bilayer. The results indicate that the surrounding environment not only alters the stability of the β-barrel but affects the internal loop responsible of the ions transport, as well as the tendency of the porin proteins to aggregate into trimers. The detergent bilayer preserves the structure of OmpF protein as is found bacteria membranes, while pure aqueous solution induces a strong destabilization of the protein. An intermediate situation occurs for detergent solution. Our results have been rationalized in terms of protein⋯water and protein⋯detergent interactions, which makes them extremely useful for the future design of new generation of bioinspired protein-integrated devices. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

The fitness costs and trade-off shapes associated with the exclusion of nine antibiotics by OmpF porin channels.

PubMed

Phan, Katherine; Ferenci, Thomas

2017-06-01

The trade-off relationship between antibiotic exclusion and nutrient access across the Gram-negative outer membrane is determined by structural constraints in porin channels. The precise nutritional cost of exclusion is unknown for different antibiotics, as are the shapes of the nutrition-susceptibility trade-off. Using a library of 10 engineered isogenic Escherichia coli strains with structural modifications of OmpF porin expressed at a constant level, susceptibilities were measured for nine antibiotics and the nutritional fitness costs estimated by competitions in chemostats. Different antibiotics exhibited a remarkably varied range of geometries in the nutrition-susceptibility trade-off, including convex, concave and sigmoidal trade-off shapes. The trade-off patterns predict the possibility of adaptations in contributing to antibiotic resistance; exclusion of amoxicillin or trimethoprim in ompF mutants can occur with little loss of fitness whereas kanamycin and streptomycin exclusion has a high cost. Some individual OmpF changes even allow positive correlations (trade-ups), resulting in increased fitness and decreased susceptibility specifically to cephalexin or ciprofloxacin. The surprising plasticity of the nutrition-exclusion relationship means that there are no generalisable rules that apply to decreasing susceptibility for all antibiotics. The protein changes are exquisitely specific in determining nutritional fitness and adaptive outcomes in a structural constraint trade-off.

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

Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis

PubMed Central

Elgass, Kirstin D.; Gabriel, Kipros; Dougan, Gordon; Lithgow, Trevor; Heinz, Eva

2018-01-01

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity. PMID:29601598

Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant.

PubMed

Maezawa, S; Hayashi, Y; Nakae, T; Ishii, J; Kameyama, K; Takagi, T

1983-09-28

An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry. It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane. The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.

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

PubMed

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

2009-02-24

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

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

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.

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

Enlightening mineral iron sensing in Pseudomonas fluorescens by surface active maghemite nanoparticles: Involvement of the OprF porin.

PubMed

Magro, Massimiliano; Fasolato, Luca; Bonaiuto, Emanuela; Andreani, Nadia Andrea; Baratella, Davide; Corraducci, Vittorino; Miotto, Giovanni; Cardazzo, Barbara; Vianello, Fabio

2016-10-01

Mineral iron(III) recognition by bacteria is considered a matter of debate. The peculiar surface chemistry of novel naked magnetic nanoparticles, called SAMNs (surface active maghemite nanoparticles) characterized by solvent exposed Fe(3+) sites on their surface, was exploited for studying mineral iron sensing in Pseudomonas fluorescens. SAMNs were applied for mimicking Fe(3+) ions in solution, acting as magnetically drivable probes to evaluate putative Fe(3+) recognition sites on the microorganism surface. Culture broths and nano-bio-conjugates were characterized by UV-Vis spectroscopy and mass spectrometry. The whole heritage of a membrane porin (OprF) of P. fluorescens Ps_22 cells was recognized and firmly bound by SAMNs. The binding of nanoparticles to OprF porin was correlated to a drastic inhibition of a siderophore (pyoverdine) biosynthesis and to the stimulation of the production and rate of formation of a secondary siderophore. The analysis of metabolic pathways, based on P. fluorescens Ps_22 genomic information, evidenced that this putative secondary siderophore does not belong to a selection of the most common siderophores. In the scenario of an adhesion mechanism, it is plausible to consider OprF as the biological component deputed to the mineral iron sensing in P. fluorescens Ps_22, as well as one key of siderophore regulation. The present work sheds light on mineral iron sensing in microorganisms. Peculiar colloidal naked iron oxide nanoparticles offer a useful approach for probing the adhesion of bacterial surface on mineral iron for the identification of the specific recognition site for this iron uptake regulation in microorganisms. Copyright © 2016 Elsevier B.V. All rights reserved.

Dual Regulation of the Small RNA MicC and the Quiescent Porin OmpN in Response to Antibiotic Stress in Escherichia coli

PubMed Central

Dam, Sushovan; Pagès, Jean-Marie

2017-01-01

Antibiotic resistant Gram-negative bacteria are a serious threat for public health. The permeation of antibiotics through their outer membrane is largely dependent on porin, changes in which cause reduced drug uptake and efficacy. Escherichia coli produces two major porins, OmpF and OmpC. MicF and MicC are small non-coding RNAs (sRNAs) that modulate the expression of OmpF and OmpC, respectively. In this work, we investigated factors that lead to increased production of MicC. micC promoter region was fused to lacZ, and the reporter plasmid was transformed into E. coli MC4100 and derivative mutants. The response of micC–lacZ to antimicrobials was measured during growth over a 6 h time period. The data showed that the expression of micC was increased in the presence of β-lactam antibiotics and in an rpoE depleted mutant. Interestingly, the same conditions enhanced the activity of an ompN–lacZ fusion, suggesting a dual transcriptional regulation of micC and the quiescent adjacent ompN. Increased levels of OmpN in the presence of sub-inhibitory concentrations of chemicals could not be confirmed by Western blot analysis, except when analyzed in the absence of the sigma factor σE. We suggest that the MicC sRNA acts together with the σE envelope stress response pathway to control the OmpC/N levels in response to β-lactam antibiotics. PMID:29211019

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.

Helicobacter pylori HopE and HopV porins present scarce expression among clinical isolates

PubMed Central

Lienlaf, Maritza; Morales, Juan Pablo; Díaz, María Inés; Díaz, Rodrigo; Bruce, Elsa; Siegel, Freddy; León, Gloria; Harris, Paul R; Venegas, Alejandro

2010-01-01

AIM: To evaluate how widely Helicobacter pylori (H. pylori) HopE and HopV porins are expressed among Chilean isolates and how seroprevalent they are among infected patients in Chile. METHODS: H. pylori hopE and hopV genes derived from strain CHCTX-1 were cloned by polymerase chain reaction (PCR), sequenced and expressed in Escherichia coli AD494 (DE3). Gel-purified porins were used to prepare polyclonal antibodies. The presence of both genes was tested by PCR in a collection of H. pylori clinical isolates and their expression was detected in lysates by immunoblotting. Immune responses against HopE, HopV and other H. pylori antigens in sera from infected and non-infected patients were tested by Western blotting using these sera as first antibody on recombinant H. pylori antigens. RESULTS: PCR and Western blotting assays revealed that 60 and 82 out of 130 Chilean isolates carried hopE and hopV genes, respectively, but only 16 and 9, respectively, expressed these porins. IgG serum immunoreactivity evaluation of 69 H. pylori-infected patients revealed that HopE and HopV were infrequently recognized (8.7% and 10.1% respectively) compared to H. pylori VacA (68.1%) and CagA (59.5%) antigens. Similar values were detected for IgA serum immunoreactivity against HopE (11.6%) and HopV (10.5%) although lower values for VacA (42%) and CagA (17.4%) were obtained when compared to the IgG response. CONCLUSION: A scarce expression of HopE and HopV among Chilean isolates was found, in agreement with the infrequent seroconversion against these antigens when tested in infected Chilean patients. PMID:20082477

Regulation of Serratia marcescens ompF and ompC porin genes in response to osmotic stress, salicylate, temperature and pH.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2006-02-01

Serratia marcescens is a Gram-negative enterobacterium that has become an important opportunistic pathogen, largely due to its high degree of natural antibiotic resistance. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, which is controlled in part by OmpC and OmpF porin proteins. OmpF expression is regulated by micF, an RNA transcript encoded upstream of the ompC gene, which hybridizes with the ompF transcript to inhibit its translation. Regulation of S. marcescens porin gene expression, as well as that of micF, was investigated using beta-galactosidase reporter gene fusions in response to 5, 8 and 10 % sucrose, 1, 5 and 8 mM salicylate, and different pH and temperature values. beta-Galactosidase activity assays revealed that a lower growth temperature (28 degrees C), a more basic pH (pH 8), and an absence of sucrose and salicylate induce the transcription of the ompF gene, whereas the induction of ompC is stimulated at a higher growth temperature (42 degrees C), acidic pH (pH 6), and maximum concentrations of sucrose (10 %) and salicylate (8 mM). In addition, when multiple conditions were tested, temperature had the predominant effect, followed by pH. In this study, it was found that the MicF regulatory mechanism does not play a role in the osmoregulation of the ompF and ompC genes, whereas MicF does repress OmpF expression in the presence of salicylate and high growth temperature, and under low pH conditions.

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

Exchangeability of N termini in the ligand-gated porins of Escherichia coli.

PubMed

Scott, D C; Cao, Z; Qi, Z; Bauler, M; Igo, J D; Newton, S M; Klebba, P E

2001-04-20

The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest a unique architecture: Their N termini fold into a globular domain that lodges within, and physically obstructs, a transmembrane porin beta-barrel formed by their C termini. We exchanged and deleted the N termini of two such siderophore receptors, FepA and FhuA, which recognize and transport ferric enterobactin and ferrichrome, respectively. The resultant chimeric proteins and empty beta-barrels avidly bound appropriate ligands, including iron complexes, protein toxins, and viruses. Thus, the ability to recognize and discriminate these molecules fully originates in the transmembrane beta-barrel domain. Both the hybrid and the deletion proteins also transported the ferric siderophore that they bound. The FepA constructs showed less transport activity than wild type receptor protein, but the FhuA constructs functioned with turnover numbers that were equivalent to wild type. The mutant proteins displayed the full range of transport functionalities, despite their aberrant or missing N termini, confirming (Braun, M., Killmann, H., and Braun, V. (1999) Mol. Microbiol. 33, 1037-1049) that the globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake. These and other data suggest a transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the channel concomitant with solute internalization.

Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import.

PubMed Central

Hönlinger, A; Bömer, U; Alconada, A; Eckerskorn, C; Lottspeich, F; Dietmeier, K; Pfanner, N

1996-01-01

The preprotein translocase of the outer mitochondrial membrane is a multi-subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re-establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase. Images PMID:8641278

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 bla IMP and bla VIM 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.

Gonococcal Resistance to β-Lactams and Tetracycline Involves Mutation in Loop 3 of the Porin Encoded at the penB Locus

PubMed Central

Gill, M. J.; Simjee, S.; Al-Hattawi, K.; Robertson, B. D.; Easmon, C. S. F.; Ison, C. A.

1998-01-01

penB is a chromosomal mutation that confers resistance to β-lactams and tetracyclines and reduced susceptibility to quinolones in Neisseria gonorrhoeae. It is linked to the porin gene (por) and requires the increased expression of an efflux pump due to mtr. Transformation of a susceptible gonococcus (strain H1) with chromosomal DNA from strain FA140 (penA mtr penB; porin serovar IB1) and conjugal transfer of a β-lactamase-expressing plasmid was used to produce isogenic strains for determination of equilibrium periplasmic penicillin concentrations by the method of Zimmermann and Rosselet (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368–372, 1977). In transformants with the Mtr and PenB phenotypes, equilibrium concentrations of penicillin were reduced. DNA sequence analysis of por from isogenic penB and penB+ transformants revealed 14 sequence differences; nine of these differences resulted in amino acid changes. Three amino acid changes were found in the putative gonococcal equivalent of the pore-constricting loop 3 of Escherichia coli OmpF. Two of these changes (Gly-101–Ala-102→Asp-Asp) result in an increased negative charge at this position in por loop 3. PCR products comprising the complete por gene from strain FA140 were transformed into strain H1-2 (penA mtr; porin serovar IB-3), with the resulting transformants having the antibiotic susceptibility phenotype associated with penB. penB-like mutations were found in loop 3 of clinical isolates of gonococci with chromosomally mediated resistance to penicillin. We conclude that penB is a mutation in loop 3 of por that reduces porin permeability to hydrophilic antibiotics and plays an important role in the development of chromosomally mediated resistance to penicillin and tetracycline in gonococci. PMID:9797206

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.

Outer membrane proteins from rough strains of four Brucella species.

PubMed Central

Santos, J M; Verstreate, D R; Perera, V Y; Winter, A J

1984-01-01

Outer membrane proteins from 15 rough strains of Brucella abortus, B. ovis, B. canis, and B. melitensis were extracted with a dipolar detergent, and outer membrane proteins from selected strains were purified by anion exchange chromatography and gel filtration (Verstreate et al., Infect. Immun. 35:979-989, 1982). Outer membrane proteins produced two types of profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One type, demonstrated by B. abortus, B. ovis, and B. canis strains, contained the three predominant protein groups present in smooth B. abortus strains (Verstreate et al., Infect. Immun. 35:979-989, 1982): groups 1, 2 (porin [Douglas et al., Infect. Immun. 44:16-21]), and 3. B. melitensis strains demonstrated the second profile type, in which there was an additional band between groups 1 and 2. The relative proportion of porin was considerably lower in B. ovis, B. canis, and B. melitensis than in B. abortus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles could be used to distinguish B. abortus and B. melitensis from each other and from B. canis and B. ovis. The amino acid compositions of groups 2 and 3 from rough strains of B. abortus, B. canis, and B. melitensis were similar to those of corresponding proteins from smooth B. abortus strains. Zwittergent-soluble fractions from most rough strains contained antigen [b], which cross-reacted with group 2 from smooth B. abortus strains, and antigens [c] and [d], which cross-reacted with group 3 from smooth B. abortus strains. Antigen [a], shared by groups 2 and 3 (D. R. Verstreate and A. J. Winter, Infect. Immun. 46:182-187, 1984), was detected in most rough strains. None of these antigens were related to either rough or smooth lipopolysaccharide. Images PMID:6480106

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. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Synthesis and characterization of tethered lipid assemblies for membrane protein reconstitution (Review).

PubMed

Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Brenner, Catherine; Ladant, Daniel; Chopineau, Joël

2017-09-28

-coated gold and glass were optimized for protein-free vesicles. This biomimetic membrane delimits an inside "trans" compartment separated from an outside reservoir "cis." Using this tBLM construction, the authors were interested in deciphering two complex molecular mechanisms involving membrane-associated proteins. The first one concerns two mitochondrial proteins, i.e., the porin voltage dependent anion channel (VDAC) embedded in the outer membrane and the nucleotide transporter (adenine nucleotide translocase) that interacts dynamically during mitochondrial pathophysiology. The purified VDAC porin was first reconstituted in proteoliposomes that were subsequently assembled on an amino coated support to form a biomimetic membrane. As a major result, VDAC was reconstituted in this tBLM and calcium channeling was demonstrated across the lipid bilayer. The same two-compartment biomimetic membrane design was further engineered to study the translocation mechanism of a bacterial toxin, the adenylate cyclase toxin, CyaA, from Bordetella pertussis. As a result, the authors developed an elegant in vitro translocation toolkit applicable to potentially a large panel of proteins transported across membranes.

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

PubMed Central

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

2014-01-01

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

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

Molecular mechanisms of membrane impermeability in clinical isolates of Enterobacteriaceae exposed to imipenem selective pressure.

PubMed

Pavez, Monica; Vieira, Camila; de Araujo, Maria Rita; Cerda, Alvaro; de Almeida, Lara Mendes; Lincopan, Nilton; Mamizuka, Elsa Masae

2016-07-01

Intrinsic mechanisms leading to carbapenem-induced membrane impermeability and multidrug resistance are poorly understood in clinical isolates of Enterobacteriaceae. In this study, molecular behaviours during the establishment of membrane impermeability in members of the Enterobacteriaceae family under imipenem selective pressure were investigated. Clinical isolates (n = 22) exhibiting susceptibility to multiple antibiotics or characterised as extended-spectrum β-lactamase (ESBL)- or AmpC-producers were submitted to progressive passages on Mueller-Hinton agar plates containing subclinical concentrations of imipenem [0.5 × the minimum inhibitory concentration (MIC)]. Changes in outer membrane permeability were evaluated by determination of antimicrobial MICs, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and gene expression analysis related to membrane permeability (i.e. omp35-like, omp36-like and acrA) and regulatory mechanisms (i.e. marA and ompR) by quantitative reverse transcription PCR. Following imipenem induction, 73% of isolates showed increased carbapenem MICs by ≥2 doubling dilutions. At an early stage of treatment, imipenem modulated the expression of porins and efflux pump genes, represented by a reduction of 78% in omp36-like and a two-fold increase in acrA expression. Transcriptional factors marA and ompR were also affected by imipenem induction, increasing mRNA expression by 14- and 4-fold, respectively. High marA expression levels were associated with higher values of acrA expression. These results suggest that imipenem is an important factor in the development of an adaptive response to carbapenems by regulating key genes involved in the control of efflux pumps and porins, which could lead to a multidrug-resistant profile in clinical isolates, contributing to possible treatment failure. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Neonatal extracorporeal membrane oxygenation: impaired health at 5 years of age.

PubMed

Madderom, Marlous J; Gischler, Saskia J; Duivenvoorden, Hugo; Tibboel, Dick; Ijsselstijn, Hanneke

2013-02-01

Children treated with neonatal extracorporeal membrane oxygenation may show physical and mental morbidity at a later age. We compared the health-related quality of life of these children with normative data. Prospective longitudinal follow-up study. Outpatient clinic of a level III university hospital. Ninety-five 5-yr-old children who had received neonatal extracorporeal membrane oxygenation support between January 1999 and December 2005. None. The pediatric quality of life inventory was administered at 5 yrs of age. The mothers (n = 74) as proxy-reporters assigned significantly lower health-related quality of life scores for their children than did the parents in the healthy reference group for the total functioning scale of the pediatric quality of life inventory (mean difference: 8.1; p < 0.001). Mothers' scores for 31 children (42%) were indicative of impaired health-related quality of life (≥-1 SD below the reference norm). The children (n = 78) themselves scored significantly lower than did their healthy peers on total functioning (mean difference: 11.0; p < 0.001). Thirty-two children (41%) indicated an impaired health-related quality of life themselves. For the mother proxy- reports, the duration of extracorporeal membrane oxygenation support (R = 0.009; p = 0.010) and the presence of chronic lung disease (R = 0.133; p = 0.002) were negatively related to total functioning. Children with a disabled health status for neuromotor functioning, maximum exercise capacity, behavior, and cognitive functioning at 5 yrs of age had a higher odds ratio of also having a lower health-related quality of life. Health status had no influence on reported emotional functioning. Overall, children treated with extracorporeal membrane oxygenation in the neonatal period reported low health-related quality of life at 5 yrs of age. Because only emotional health-related quality of life was not associated with health status, the pediatric quality of life inventory might be a

Clonal background, resistance gene profile, and porin gene mutations modulate in vitro susceptibility to imipenem/relebactam in diverse Enterobacteriaceae.

PubMed

Gomez-Simmonds, Angela; Stump, Stephania; Giddins, Marla J; Annavajhala, Medini K; Uhlemann, Anne-Catrin

2018-06-11

Treatment options for carbapenem-resistant Enterobacteriaceae (CRE) are limited. While Klebsiella pneumoniae harboring bla KPC account for most CRE, recent evidence points to increasing diversification of CRE. We determined whether CRE species and antibiotic resistance genotype influence response to relebactam (REL), a novel beta-lactamase inhibitor with class A/C activity, combined with imipenem(IMI)/cilastatin. We carried out broth microdilution testing to IMI alone or in the presence of 4 μg/mL REL in 154 clinical isolates collected at a New York City hospital with high prevalence of bla KPC including Enterobacter spp. (n=96), K. pneumoniae (n=44), Escherichia coli (n=1), Serratia marcescens (n=9) and Citrobacter spp. (n=4). Resistance gene profiles and presence of major porin gene disruptions were ascertained by whole genome sequencing. Addition of REL decreased the IMI MIC to the susceptible range (≤1 μg/mL) in 88% of isolates. However, S. marcescens IMI/REL MICs were 4 to 8-fold higher than those of other organisms. Most bla KPC -positive isolates had IMI/REL MICs ≤1 μg/mL (88%), including E. cloacae ST171 (93%) and K. pneumoniae ST258 (82%). Nineteen isolates had IMI/REL MICs ≥2 μg/mL, of which 84% harbored bla KPC and one was bla NDM-1 -positive. Isolates with IMI/REL MICs ≥2 μg/mL versus ≤1 μg/mL were significantly more likely to demonstrate disruption of at least one porin gene (42% versus 19%, p =0.04), although most S. marcescens isolates (67%) had intact porin genes. In conclusion, while REL reduced IMI MICs in a majority of diverse CRE isolates including high-risk clones, chromosomal factors impacted IMI/REL susceptibilities and may contribute to elevated MICs in S. marcescens. Copyright © 2018 American Society for Microbiology.

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

2017-06-01

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. © 2016 BSPP AND JOHN WILEY & SONS LTD.

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

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

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.

Effect of Porins and blaKPC Expression on Activity of Imipenem with Relebactam in Klebsiella pneumoniae: Can Antibiotic Combinations Overcome Resistance?

PubMed

Balabanian, Gregory; Rose, Michael; Manning, Nyla; Landman, David; Quale, John

2018-05-21

Imipenem with relebactam is a novel β-lactam-β-lactamase inhibitor that has activity against most KPC-producing Enterobacteriaceae. Using 10 isolates of KPC-possessing Klebsiella pneumoniae, we assessed the relationship between imipenem-relebactam minimum inhibitory concentrations (MICs) and mechanisms known to contribute to antimicrobial resistance. The effect of adding a second agent was assessed by time-kill experiments. Mutations affecting the genes encoding porins ompK35 and ompK36 and identification of β-lactamases were assessed by PCR. Expression of bla KPC and acrB was assessed by real-time reverse-transcriptase (RT)-PCR, and production of OmpK36 by SDS-PAGE. Time-kill studies were performed using combinations of imipenem-relebactam with polymyxin B, amikacin, or tigecycline. Seven isolates having a disruption in a single porin, or in neither porin, remained susceptible to imipenem-relebactam. The addition of a second agent did not improve the activity of imipenem-relebactam for these isolates, although the addition of tigecycline was antagonistic for three isolates. Three isolates had major disruptions in both ompK35 and ompK36 that correlated with reduced activity of imipenem-relebactam (MICs 2/4, 8/4, and 512/4 μg/mL). Two of these isolates also had overexpression of bla KPC , including the isolate with the highest MIC. These isolates were also resistant to polymyxin B and amikacin. The addition of amikacin provided both synergistic and bactericidal activity for the two more resistant isolates. The activity of imipenem-relebactam against K. pneumoniae is affected by major disruptions of both ompK35 and ompK36 and by expression of the KPC gene. Combining imipenem-relebactam with an aminoglycoside may be a promising approach for isolates with reduced susceptibility to imipenem-relebactam.

A molecular dynamics study of the pores formed by Escherichia coli OmpF porin in a fully hydrated palmitoyloleoylphosphatidylcholine bilayer.

PubMed

Tieleman, D P; Berendsen, H J

1998-06-01

In this paper we study the properties of pores formed by OmpF porin from Escherichia coli, based on a molecular dynamics simulation of the OmpF trimer, 318 palmitoyl-oleoyl-phosphatidylethanolamine lipids, 27 Na+ ions, and 12,992 water molecules. After equilibration and a nanosecond production run, the OmpF trimer exhibits a C-alpha root mean square deviation from the crystal structure of 0.23 nm and a stable secondary structure. No evidence is found for large-scale motions of the L3 loop. We investigate the pore dimensions, conductance, and the properties of water inside the pore. This water forms a complicated pattern, even when averaged over 1 ns of simulation time. Around the pore constriction zone the water dipoles are highly structured in the plane of the membrane, oriented by the strong transversal electric field. In addition, there is a net orientation along the pore axis pointing from the extracellular to the intracellular side of the bilayer. The diffusion coefficients of water inside the pore are greatly reduced compared to bulk. We compare our results to results from model pores (Breed et al., 1996. Biophys. J. 70:1 643-1 661; Sansom et al. 1997. Biophys. J. 73:2404-241 5) and discuss implications for further theoretical work.

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mo, Yiming; Lee, Byung-Kwon; Ankner, John Francis

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), themore » 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.« less

Detergent-associated solution conformations of helical and beta-barrel membrane proteins.

PubMed

Mo, Yiming; Lee, Byung-Kwon; Ankner, John F; Becker, Jeffrey M; Heller, William T

2008-10-23

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 beta-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.

Novel efficient promoter of the mitochondrial porin, voltage-dependent anion channel (VDAC), in the genome of the Yarrowia lipolytica yeast.

PubMed

Kulanbaewa, F F; Sekova, V Yu; Isakova, E P; Deryabina, Y I; Nikolaev, A V

2016-09-01

This article presents the characteristics of the highly inducible promoter of the gene encoding the mitochondrial porin, the voltage-dependent anion channel (VDAC). This promoter is recommended for use in new genetic constructs both in basic research for assessing the adaptive strategy of lower eukaryotes under adverse conditions and in designing new highly competitive transformants producing economically important compounds (proteins, lipids, and organic acids) on its basis.

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.

Modification of Salmonella Lipopolysaccharides Prevents the Outer Membrane Penetration of Novobiocin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nobre, Thatyane M.; Martynowycz, Michael W.; Andreev, Konstantin

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 asmonolayers at the air-water interface, and their properties, aswell 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,butwasmore » prevented fromthis penetration intothemodified lipopolysaccharides.Results correlatewith 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.« less

Transport across the outer membrane porin of mycolic acid containing actinomycetales: Nocardia farcinica.

PubMed

Singh, Pratik Raj; Bajaj, Harsha; Benz, Roland; Winterhalter, Mathias; Mahendran, Kozhinjampara R

2015-02-01

The role of the outer-membrane channel from a mycolic acid containing Gram-positive bacteria Nocardia farcinica, which forms a hydrophilic pathway across the cell wall, was characterized. Single channel electrophysiology measurements and liposome swelling assays revealed the permeation of hydrophilic solutes including sugars, amino acids and antibiotics. The cation selective N. farcinica channel exhibited strong interaction with the positively charged antibiotics; amikacin and kanamycin, and surprisingly also with the negatively charged ertapenem. Voltage dependent kinetics of amikacin and kanamycin interactions were studied to distinguish binding from translocation. Moreover, the importance of charged residues inside the channel was investigated using mutational studies that revealed rate limiting interactions during the permeation. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2005-01-01

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

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Jingquan; Rouse, Sarah L.; Li, Dianfan

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 gatemore » (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.« less

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.

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

PubMed Central

Oppezzo, O J; Avanzati, B; Antón, D N

1991-01-01

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

Entry of Porphyromonas gingivalis outer membrane vesicles into epithelial cells causes cellular functional impairment.

PubMed

Furuta, Nobumichi; Takeuchi, Hiroki; Amano, Atsuo

2009-11-01

Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including proteases termed gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]). We recently showed that P. gingivalis MVs swiftly enter host epithelial cells via an endocytosis pathway and are finally sorted to lytic compartments. However, it remains unknown whether MV entry impairs cellular function. Herein, we analyzed cellular functional impairment following entry of P. gingivalis into epithelial cells, including HeLa and immortalized human gingival epithelial (IHGE) cells. After being taken up by endocytic vacuoles, MVs degraded the cellular transferrin receptor (TfR) and integrin-related signaling molecules, such as paxillin and focal adhesion kinase (FAK), which resulted in depletion of intracellular transferrin and inhibition of cellular migration. Few Rgp-null MVs entered the cells, and these negligibly degraded TfR, whereas paxillin and FAK degradation was significant. In contrast, Kgp-null MVs clearly entered the cells and degraded TfR, while they scarcely degraded paxillin and FAK. In addition, both wild-type and Kgp-null MVs significantly impaired cellular migration, whereas the effect of Rgp-null MVs was limited. Our findings suggest that, following entry of P. gingivalis MVs into host cells, MV-associated gingipains degrade cellular functional molecules such as TfR and paxillin/FAK, resulting in cellular impairment, indicating that P. gingivalis MVs are potent vehicles for transmission of virulence factors into host cells and are involved in the etiology of periodontitis.

Characterization of the Leptospiral Outer Membrane and Description of Three Novel Leptospiral Membrane Proteins

PubMed Central

Haake, David A.; Matsunaga, James

2002-01-01

The outer membrane (OM) of the mammalian pathogen Leptospira kirschneri was isolated in the form of membrane vesicles by alkaline plasmolysis and separated from the protoplasmic cylinder by sucrose density gradient ultracentrifugation. All four components of the alkaline plasmolysis buffer, including 1.0 M NaCl, 27% sucrose (wt/vol), 2 mM EDTA, and 10 mM Tris (pH 9), were required for efficient OM release, as judged by recovery of leptospiral lipopolysaccharide. Two populations of OM vesicles (OMVs) were recovered, with peak concentrations found in the sucrose gradient at densities of 1.16 and 1.18 g/ml. Transmission electron microscopy revealed that the more buoyant OMV population was smaller (<0.1 μm in diameter) than the denser OMV population (0.2 to 0.3 μm in diameter). The densities of both populations of OMVs were distinct from that of the protoplasmic-cylinder material, which was found in the sucrose gradient at a density of 1.20 g/ml. The OMV fractions were free of protoplasmic-cylinder material, as judged by immunoblotting with antibodies to the endoflagellar sheath protein, heat shock protein GroEL, and two novel cytoplasmic membrane proteins, lipoprotein LipL31 and transmembrane protein ImpL63. The protein components of the OMVs were characterized by one- and two-dimensional immunoblotting and found to include previously described OM proteins (OMPs), including the porin OmpL1; the lipoproteins LipL32, LipL36, and LipL41; and the peripheral membrane protein P31LipL45. A number of less well-characterized OMPs were also identified, including those with molecular masses of 16, 21, 21.5, 22, 31, 36, 44, 48, 90, and 116 kDa. The 48-kDa OMP was identified as a novel OM lipoprotein designated LipL48. The use of membrane-specific markers in OM isolation techniques facilitates an accurate description of the leptospiral OM and its components. PMID:12183539

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

A Deficiency in Arabinogalactan Biosynthesis Affects Corynebacterium glutamicum Mycolate Outer Membrane Stability▿

PubMed Central

Bou Raad, Roland; Méniche, Xavier; de Sousa-d'Auria, Celia; Chami, Mohamed; Salmeron, Christophe; Tropis, Marielle; Labarre, Cecile; Daffé, Mamadou; Houssin, Christine; Bayan, Nicolas

2010-01-01

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The ultrastructure of the cell envelope is very atypical. It is composed of a heteropolymer of peptidoglycan and arabinogalactan (AG) covalently associated to an outer membrane. Five arabinosyltransferases are involved in the biosynthesis of AG in C. glutamicum. AftB catalyzes the transfer of Araf (arabinofuranosyl) onto the arabinan domain of the arabinogalactan to form terminal β(1 → 2)-linked Araf residues. Here we show that ΔaftB cells lack half of the arabinogalactan mycoloylation sites but are still able to assemble an outer membrane. In addition, we show that a ΔaftB mutant grown on a rich medium has a perturbed cell envelope and sheds a significant amount of membrane fragments in the external culture medium. These fragments contain mono- and dimycolate of trehalose and PorA/H, the major porin of C. glutamicum, but lack conventional phospholipids that typify the plasma membrane, suggesting that they are derived from the atypical mycolate outer membrane of the cell envelope. This is the first report of outer membrane destabilization in the Corynebacterineae, and it suggests that a strong interaction between the mycolate outer membrane and the underlying polymer is essential for cell envelope integrity. The presence of outer membrane-derived fragments (OMFs) in the external medium of the ΔaftB mutant is also a very promising tool for outer membrane characterization. Indeed, fingerprint analysis of major OMF-associated proteins has already led to the identification of 3 associated mycoloyltransferases and an unknown protein with a C-terminal hydrophobic anchoring domain reminiscent of that found for the S-layer protein PS2 of C. glutamicum. PMID:20363942

Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

PubMed

Klug, Yoel A; Ashkenazi, Avraham; Viard, Mathias; Porat, Ziv; Blumenthal, Robert; Shai, Yechiel

2014-07-15

Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

The ropAe gene encodes a porin-like protein involved in copper transit in Rhizobium etli CFN42.

PubMed

González-Sánchez, Antonio; Cubillas, Ciro A; Miranda, Fabiola; Dávalos, Araceli; García-de Los Santos, Alejandro

2017-12-27

Copper (Cu) is an essential micronutrient for all aerobic forms of life. Its oxidation states (Cu + /Cu 2+ ) make this metal an important cofactor of enzymes catalyzing redox reactions in essential biological processes. In gram-negative bacteria, Cu uptake is an unexplored component of a finely regulated trafficking network, mediated by protein-protein interactions that deliver Cu to target proteins and efflux surplus metal to avoid toxicity. Rhizobium etliCFN42 is a facultative symbiotic diazotroph that must ensure its appropriate Cu supply for living either free in the soil or as an intracellular symbiont of leguminous plants. In crop fields, rhizobia have to contend with copper-based fungicides. A detailed deletion analysis of the pRet42e (505 kb) plasmid from an R. etli mutant with enhanced CuCl 2 tolerance led us to the identification of the ropAe gene, predicted to encode an outer membrane protein (OMP) with a β-barrel channel structure that may be involved in Cu transport. In support of this hypothesis, the functional characterization of ropAe revealed that: (I) gene disruption increased copper tolerance of the mutant, and its complementation with the wild-type gene restored its wild-type copper sensitivity; (II) the ropAe gene maintains a low basal transcription level in copper overload, but is upregulated when copper is scarce; (III) disruption of ropAe in an actP (copA) mutant background, defective in copper efflux, partially reduced its copper sensitivity phenotype. Finally, BLASTP comparisons and a maximum likelihood phylogenetic analysis highlight the diversification of four RopA paralogs in members of the Rhizobiaceae family. Orthologs of RopAe are highly conserved in the Rhizobiales order, poorly conserved in other alpha proteobacteria and phylogenetically unrelated to characterized porins involved in Cu or Mn uptake. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

The voltage-dependent anion channel as a biological transistor: theoretical considerations.

PubMed

Lemeshko, V V; Lemeshko, S V

2004-07-01

The voltage-dependent anion channel (VDAC) is a porin of the mitochondrial outer membrane with a bell-shaped permeability-voltage characteristic. This porin restricts the flow of negatively charged metabolites at certain non-zero voltages, and thus might regulate their flux across the mitochondrial outer membrane. Here, we have developed a mathematical model illustrating the possibility of interaction between two steady-state fluxes of negatively charged metabolites circulating across the VDAC in a membrane. The fluxes interact by contributing to generation of the membrane electrical potential with subsequent closure of the VDAC. The model predicts that the VDAC might function as a single-molecule biological transistor and amplifier, because according to the obtained calculations a small change in the flux of one pair of different negatively charged metabolites causes a significant modulation of a more powerful flux of another pair of negatively charged metabolites circulating across the same membrane with the VDAC. Such transistor-like behavior of the VDAC in the mitochondrial outer membrane might be an important principle of the cell energy metabolism regulation under some physiological conditions.

Characterization of nanostructured surfaces generated by reconstitution of the porin MspA from Mycobacterium smegmatis.

PubMed

Wörner, Michael; Lioubashevski, Oleg; Basel, Matthew T; Niebler, Sandra; Gogritchiani, Eliso; Egner, Nicole; Heinz, Christian; Hoferer, Jürgen; Cipolloni, Michela; Janik, Katharine; Katz, Evgeny; Braun, Andre M; Willner, Itamar; Niederweis, Michael; Bossmann, Stefan H

2007-06-01

Nanostructures with long-term stability at the surface of gold electrodes are generated by reconstituting the porin MspA from Mycobacterium smegmatis into a specially designed monolayer of long-chain lipid surfactant on gold. Tailored surface coverage of gold electrodes with long-chain surfactants is achieved by electrochemically assisted deposition of organic thiosulfates (Bunte salts). The subsequent reconstitution of the octameric-pore MspA is guided by its extraordinary self-assembling properties. Importantly, electrochemical reduction of copper(II) yields copper nanoparticles within the MspA nanopores. Electrochemical impedance spectroscopy, reflection electron microscopy, and atomic force microscopy (AFM) show that: 1) the MspA pores within the self-assembled monolayer (SAM) are monodisperse and electrochemically active, 2) MspA reconstitutes in SAMs and with a 10-nm thickness, 3) AFM is a suitable method to detect pores within SAMs, and 4) the electrochemical reduction of Cu2+ to Cu0 under overpotential conditions starts within the MspA pores.

Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I

PubMed Central

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

2015-01-01

We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprCN and TprCC) orthologous to regions in the major outer sheath protein (MOSPN and MOSPC) of Treponema denticola and that TprCC 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 MOSPC-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSPN-like domains are tethered within the periplasm. TprF, which does not contain a MOSPC-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 MOSPN and MOSPC-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 MOSPN-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP. PMID:25805501

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

PubMed

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

2014-05-01

Molecular typing and mechanisms of carbapenem resistance such as alterations in porin OprD and presence of metallo-beta-lactamases (MBLs), as well as integrons have been studied in a collection of carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from a Spanish hospital. One hundred and twenty-three CRPA isolates were recovered from different samples of 80 patients. Clonal relationship among CRPA was analyzed by SpeI-PFGE. Susceptibility testing to 11 antibiotics and MBL phenotype was determined by microdilution, IP/IPI E-test and double disc method. The oprD gene was studied by PCR and sequencing, and mutations were determined comparing with P. aeruginosa PAO1 sequence. Characterization of MBLs, and class 1 and 2 integrons were studied by PCR and sequencing. SDS-PAGE analysis of outer membrane proteins of selected strains was performed. Seventy-four-per-cent of patients with CRPA were hospitalised in the ICU setting and 50% had long hospitalization stays. Sixty-four different PFGE patterns were detected, and 87 CRPA strains were further analyzed. MBL phenotype was detected in 43 of 87 strains (49.4%), which contained blaVIM-2 gene inside class 1 integrons. VIM-2-producing strains belonged to lineages ST175, ST235, and ST973. A great diversity of nucleotide insertions, deletions, and mutations in oprD gene, and the presence of a new insertion sequence (ISPa45) truncating oprD were identified among CRPA strains. Class 1 integrons were detected in 75% of CRPA strains, blaVIM-2 and the new arrangement aac(3)-Ia+ISPa34+aadA1 (named as In661) being the most frequent gene-cassette arrays detected. Other gene cassettes detected in integrons were: aadB, aadA6, aadA7, aac(6')-Ib', and blaOXA-46. Copyright © 2014 Elsevier GmbH. All rights reserved.

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

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. Published by Elsevier Masson SAS.

Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Piao, Shunfu; Xu, Yongbin; Ha, Nam-Chul, E-mail: hnc@pusan.ac.kr

2008-05-01

A periplasmic membrane-fusion protein MacA from Actinobacillus actinomycetemcomitans, an essential component of the multidrug efflux pump in Gram-negative bacteria, was crystallized. Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange andmore » gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0 Å at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 Å, α = β = 90, γ = 120°, and contains one molecule in the asymmetric unit.« less

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

PubMed Central

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

2012-01-01

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

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. Copyright © 2012 Elsevier Inc. All rights reserved.

Functional microdomains in bacterial membranes.

PubMed

López, Daniel; Kolter, Roberto

2010-09-01

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

Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa.

PubMed

Adewoye, L O; Worobec, E A

2000-08-08

The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins.

Constitutive expression of the maltoporin LamB in the absence of OmpR damages the cell envelope.

PubMed

Reimann, Sylvia A; Wolfe, Alan J

2011-02-01

Cells experience multiple environmental stimuli simultaneously. To survive, they must respond accordingly. Unfortunately, the proper response to one stress easily could make the cell more susceptible to a second coexistent stress. To deal with such a problem, a cell must possess a mechanism that balances the need to respond simultaneously to both stresses. Our recent studies of ompR malT(Con) double mutants show that elevated expression of LamB, the outer membrane porin responsible for maltose uptake, causes cell death when the osmoregulator OmpR is disabled. To obtain insight into the nature of the death experienced by ompR malT(Con) mutants, we described the death process. On the basis of microscopic and biochemical approaches, we conclude that death results from a loss of membrane integrity. On the basis of an unbiased genome-wide search for suppressor mutations, we conclude that this loss of membrane integrity results from a LamB-induced envelope stress that the cells do not sufficiently perceive and thus do not adequately accommodate. Finally, we conclude that this envelope stress involves an imbalance in the lipopolysaccharide/porin composition of the outer membrane and an increased requirement for inorganic phosphate.

Constitutive Expression of the Maltoporin LamB in the Absence of OmpR Damages the Cell Envelope▿ †

PubMed Central

Reimann, Sylvia A.; Wolfe, Alan J.

2011-01-01

Cells experience multiple environmental stimuli simultaneously. To survive, they must respond accordingly. Unfortunately, the proper response to one stress easily could make the cell more susceptible to a second coexistent stress. To deal with such a problem, a cell must possess a mechanism that balances the need to respond simultaneously to both stresses. Our recent studies of ompR malT(Con) double mutants show that elevated expression of LamB, the outer membrane porin responsible for maltose uptake, causes cell death when the osmoregulator OmpR is disabled. To obtain insight into the nature of the death experienced by ompR malT(Con) mutants, we described the death process. On the basis of microscopic and biochemical approaches, we conclude that death results from a loss of membrane integrity. On the basis of an unbiased genome-wide search for suppressor mutations, we conclude that this loss of membrane integrity results from a LamB-induced envelope stress that the cells do not sufficiently perceive and thus do not adequately accommodate. Finally, we conclude that this envelope stress involves an imbalance in the lipopolysaccharide/porin composition of the outer membrane and an increased requirement for inorganic phosphate. PMID:21131484

Disturbed vesicular trafficking of membrane proteins in prion disease.

PubMed

Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

2013-01-01

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

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.

The OmpL porin does not modulate redox potential in the periplasmic space of Escherichia coli.

PubMed

Sardesai, Abhijit A; Genevaux, Pierre; Schwager, Françoise; Ang, Debbie; Georgopoulos, Costa

2003-04-01

The Escherichia coli DsbA protein is the major oxidative catalyst in the periplasm. Dartigalongue et al. (EMBO J., 19, 5980-5988, 2000) reported that null mutations in the ompL gene of E.coli fully suppress all phenotypes associated with dsbA mutants, i.e. sensitivity to the reducing agent dithiothreitol (DTT) and the antibiotic benzylpenicillin, lack of motility, reduced alkaline phosphatase activity and mucoidy. They showed that OmpL is a porin and hypothesized that ompL null mutations exert their suppressive effect by preventing efflux of a putative oxidizing-reducing compound into the medium. We have repeated these experiments using two different ompL null alleles in at least three different E.coli K-12 genetic backgrounds and have failed to reproduce any of the ompL suppressive effects noted above. Also, we show that, contrary to earlier results, ompL null mutations alone do not result in partial DTT sensitivity or partial motility, nor do they appreciably affect bacterial growth rates or block propagation of the male-specific bacteriophage M13. Thus, our findings clearly demonstrate that ompL plays no perceptible role in modulating redox potential in the periplasm of E.coli.

Protective capacity of antibodies to outer-membrane components of Escherichia coli in a systemic mouse peritonitis model.

PubMed

Vuopio-Varkila, J; Karvonen, M; Saxén, H

1988-02-01

Antibody-mediated protection was studied in an experimental murine model of peritonitis-septicaemia with Escherichia coli O18:K1. Protection from lethal intraperitoneal challenge was achieved by passive immunisation with horse anti-K1 capsular antiserum (H46) or rabbit antiserum to the homologous O18 antigen. The maximum increase in LD50 achieved with anti-K1 and anti-O18 antibodies was 10- and 5-fold, respectively. The protective capacity of the anti-O serum was found to be in the IgG fraction. Rabbits were also immunised with various semi-purified or purified outer-membrane-protein preparations (porins and OmpA protein) from rough E. coli or Salmonella strains or with whole E. coli J5 bacteria. Although this immunisation resulted in high antibody titres to homologous and, to a lesser extent, also to heterologous antigens, none of the antisera protected against challenge with the capsulate E. coli O18:K1 bacteria.

Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion

PubMed Central

Wang, Yanfeng; Zhao, Liang; Suzuki, Aae; Lian, Lurong; Min, Sang H.; Wang, Ziqian; Litvinov, Rustem I.; Stalker, Timothy J.; Yago, Tadayuki; Klopocki, Arkadiusz G.; Schmidtke, David W.; Yin, Helen; Choi, John K.; McEver, Rodger P.; Weisel, John W.; Hartwig, John H.; Abrams, Charles S.

2013-01-01

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIβ, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion. PMID:23372168

Structural basis for maintenance of bacterial outer membrane lipid asymmetry.

PubMed

Abellón-Ruiz, Javier; Kaptan, Shreyas S; Baslé, Arnaud; Claudi, Beatrice; Bumann, Dirk; Kleinekathöfer, Ulrich; van den Berg, Bert

2017-12-01

The Gram-negative bacterial outer membrane (OM) is a unique bilayer that forms an efficient permeation barrier to protect the cell from noxious compounds 1 , 2 . The defining characteristic of the OM is lipid asymmetry, with phospholipids comprising the inner leaflet and lipopolysaccharides comprising the outer leaflet 1-3 . This asymmetry is maintained by the Mla pathway, a six-component system that is widespread in Gram-negative bacteria and is thought to mediate retrograde transport of misplaced phospholipids from the outer leaflet of the OM to the cytoplasmic membrane 4 . The OM lipoprotein MlaA performs the first step in this process via an unknown mechanism that does not require external energy input. Here we show, using X-ray crystallography, molecular dynamics simulations and in vitro and in vivo functional assays, that MlaA is a monomeric α-helical OM protein that functions as a phospholipid translocation channel, forming a ~20-Å-thick doughnut embedded in the inner leaflet of the OM with a central, amphipathic pore. This architecture prevents access of inner leaflet phospholipids to the pore, but allows outer leaflet phospholipids to bind to a pronounced ridge surrounding the channel, followed by diffusion towards the periplasmic space. Enterobacterial MlaA proteins form stable complexes with OmpF/C 5,6 , but the porins do not appear to play an active role in phospholipid transport. MlaA represents a lipid transport protein that selectively removes outer leaflet phospholipids to help maintain the essential barrier function of the bacterial OM.

Evaluation of recombinant porin (rOmp2a) protein as a potential antigen candidate for serodiagnosis of Human Brucellosis.

PubMed

Pathak, Prachi; Kumar, Ashu; Thavaselvam, Duraipandian

2017-07-11

Brucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay). Omp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23). The rOmp2a was found to be

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

Cloning and nucleotide sequence of the Pseudomonas aeruginosa glucose-selective OprB porin gene and distribution of OprB within the family Pseudomonadaceae.

PubMed

Wylie, J L; Worobec, E A

1994-03-01

OprB is a glucose-selective porin known to be produced by Pseudomonas aeruginosa and Pseudomonas putida. We have cloned and sequenced the oprB gene of P. aeruginosa and obtained expression of OprB in Escherichia coli. The mature protein consists of 423 amino acid residues with a deduced molecular mass of 47597 Da. Several clusters of amino acid residues, potentially involved in the structure or function of the protein, were identified. An area of regional homology with E. coli LamB was also identified. Carbohydrate-inducible proteins, potentially homologous to OprB, were identified in several rRNA homology-group-I pseudomonads by sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis, Western immunoblotting and N-terminal amino acid sequencing. These species also contained DNA that hybridized to a P. aeruginosa oprB gene probe.

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

Pseudomonas aeruginosa reveals high intrinsic resistance to penem antibiotics: penem resistance mechanisms and their interplay.

PubMed

Okamoto, K; Gotoh, N; Nishino, T

2001-07-01

Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC beta-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encoded Escherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC beta-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC beta-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8- and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC beta-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC beta-lactamase but not from the lower affinity of PBPs for penems.

Pseudomonas aeruginosa Reveals High Intrinsic Resistance to Penem Antibiotics: Penem Resistance Mechanisms and Their Interplay

PubMed Central

Okamoto, Kiyomi; Gotoh, Naomasa; Nishino, Takeshi

2001-01-01

Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC β-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encoded Escherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC β-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC β-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8- and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC β-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC β-lactamase but not from the lower affinity of PBPs for penems. PMID:11408209

Deletion of degQ gene enhances outer membrane vesicle production of Shewanella oneidensis cells.

PubMed

Ojima, Yoshihiro; Mohanadas, Thivagaran; Kitamura, Kosei; Nunogami, Shota; Yajima, Reiki; Taya, Masahito

2017-04-01

Shewanella oneidensis is a Gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration. In this study, S. oneidensis degQ gene, encoding a putative periplasmic serine protease, was cloned and expressed. The activity of purified DegQ was inhibited by diisopropyl fluorophosphate, a typical serine protease-specific inhibitor, indicating that DegQ is a serine protease. In-frame deletion and subsequent complementation of the degQ were carried out to examine the effect of envelope stress on the production of outer membrane vesicles (OMVs). Analysis of periplasmic proteins from the resulting S. oneidensis strain showed that deletion of degQ induced protein accumulation and resulted in a significant decrease in protease activity within the periplasmic space. OMVs from the wild-type and mutant strains were purified and observed by transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the OMVs showed a prominent band at ~37 kDa. Nanoliquid chromatography-tandem mass spectrometry analysis identified three outer membrane porins (SO3896, SO1821, and SO3545) as dominant components of the band, suggesting that these proteins could be used as indices for comparing OMV production by S. oneidensis strains. Quantitative evaluation showed that degQ-deficient cells had a fivefold increase in OMV production compared with wild-type cells. Thus, the increased OMV production following the deletion of DegQ in S. oneidensis may be responsible for the increase in envelope stress.

Mutations in BIN1 Associated with Centronuclear Myopathy Disrupt Membrane Remodeling by Affecting Protein Density and Oligomerization

PubMed Central

Wu, Tingting; Shi, Zheng; Baumgart, Tobias

2014-01-01

The regulation of membrane shapes is central to many cellular phenomena. Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are key players for membrane remodeling during endocytosis, cell migration, and endosomal sorting. BIN1, which contains an N-BAR domain, is assumed to be essential for biogenesis of plasma membrane invaginations (T-tubules) in muscle tissues. Three mutations, K35N, D151N and R154Q, have been discovered so far in the BAR domain of BIN1 in patients with centronuclear myopathy (CNM), where impaired organization of T-tubules has been reported. However, molecular mechanisms behind this malfunction have remained elusive. None of the BIN1 disease mutants displayed a significantly compromised curvature sensing ability. However, two mutants showed impaired membrane tubulation both in vivo and in vitro, and displayed characteristically different behaviors. R154Q generated smaller membrane curvature compared to WT N-BAR. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appeared to be the primary reason for the observation of impaired deformation capacity. The D151N mutant was unable to tubulate liposomes under certain experimental conditions. At medium protein concentrations we found ‘budding’ structures on liposomes that we hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutation impaired protein oligomerization upon membrane binding. Although we found an insignificant difference between WT and K35N N-BAR in in vitro assays, depolymerizing actin in live cells allowed tubulation of plasma membranes through the K35N mutant. Our results provide insights into the membrane-involved pathophysiological mechanisms leading to human disease. PMID:24755653

Rapamycin mitigates erythrocyte membrane transport functions and oxidative stress during aging in rats.

PubMed

Singh, Abhishek Kumar; Singh, Sandeep; Garg, Geetika; Rizvi, Syed Ibrahim

2018-02-01

Erythrocyte membrane is a suitable model to study various metabolic and physiological functions as it undergoes variety of biochemical changes during aging. An age-dependent modulatory effect of rapamycin on erythrocyte membrane functions is completely unknown. Therefore, the present study was undertaken to investigate the effect of rapamycin on age-dependent impaired activities of transporters/exchangers, altered levels of redox biomarkers, viz. protein carbonyl (PC), lipid hydroperoxides (LHs), total thiol (-SH), sialic acid (SA) and intracellular calcium ion [Ca 2+ ]i, and osmotic fragility of erythrocyte membrane. A significant reduction in membrane-bound activities of Na + /K + -ATPase (NKA) and Ca 2+ -ATPase (PMCA), and levels of -SH and SA was observed along with a simultaneous induction in Na + /H + exchanger (NHE) activity and levels of [Ca 2+ ]i, PC, LH and osmotic fragility in old-aged rats. Rapamycin was found to be a promising age-delaying drug that significantly reversed the aging-induced impaired activities of membrane-bound ATPases and altered levels of redox biomarkers.

Hyperammonemia induces glial activation, neuroinflammation and alters neurotransmitter receptors in hippocampus, impairing spatial learning: reversal by sulforaphane.

PubMed

Hernández-Rabaza, Vicente; Cabrera-Pastor, Andrea; Taoro-González, Lucas; Malaguarnera, Michele; Agustí, Ana; Llansola, Marta; Felipo, Vicente

2016-02-16

Patients with liver cirrhosis and minimal hepatic encephalopathy (MHE) show mild cognitive impairment and spatial learning dysfunction. Hyperammonemia acts synergistically with inflammation to induce cognitive impairment in MHE. Hyperammonemia-induced neuroinflammation in hippocampus could contribute to spatial learning impairment in MHE. Two main aims of this work were: (1) to assess whether chronic hyperammonemia increases inflammatory factors in the hippocampus and if this is associated with microglia and/or astrocytes activation and (2) to assess whether hyperammonemia-induced neuroinflammation in the hippocampus is associated with altered membrane expression of glutamate and GABA receptors and spatial learning impairment. There are no specific treatments for cognitive alterations in patients with MHE. A third aim was to assess whether treatment with sulforaphane enhances endogenous the anti-inflammatory system, reduces neuroinflammation in the hippocampus of hyperammonemic rats, and restores spatial learning and if normalization of receptor membrane expression is associated with learning improvement. We analyzed the following in control and hyperammonemic rats, treated or not with sulforaphane: (1) microglia and astrocytes activation by immunohistochemistry, (2) markers of pro-inflammatory (M1) (IL-1β, IL-6) and anti-inflammatory (M2) microglia (Arg1, YM-1) by Western blot, (3) membrane expression of GABA, AMPA, and NMDA receptors using the BS3 cross-linker, and (4) spatial learning using the radial maze. The results reported show that hyperammonemia induces astrocytes and microglia activation in the hippocampus, increasing pro-inflammatory cytokines IL-1β and IL-6. This is associated with altered membrane expression of AMPA, NMDA, and GABA receptors which would be responsible for altered neurotransmission and impairment of spatial learning in the radial maze. Treatment with sulforaphane promotes microglia differentiation from pro-inflammatory M1 to anti

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

Topology of the membrane protein LamB by epitope tagging and a comparison with the X-ray model.

PubMed

Newton, S M; Klebba, P E; Michel, V; Hofnung, M; Charbit, A

1996-06-01

We previously developed a genetic approach to study, with a single antibody, the topology of the outer membrane protein LamB, an Escherichia coli porin with specificity towards maltodextrins and a receptor for bacteriophage lambda. Our initial procedure consisted of inserting at random the same reporter epitope (the C3 neutralization epitope from poliovirus) into permissive sites of LamB (i.e., sites which tolerate insertions without deleterious effects on the protein activities or the cell). A specific monoclonal antibody was then used to examine the position of the inserted epitope with respect to the protein and the membrane. In the present work, we set up a site-directed procedure to insert the C3 epitope at new sites in order to distinguish between two-dimensional folding models. This allowed us to identify two new surface loops of LamB and to predict another periplasmic exposed region. The results obtained by random and directed epitope tagging are analyzed in light of the recently published X-ray structure of the LamB protein. Study of 23 hybrid LamB-C3 proteins led to the direct identification of five of the nine external loops (L4, L5, L6, L7, and L9) and led to the prediction of four periplasmic loops (I1, I4, I5, and I8) of LamB. Nine of the hybrid proteins did not lead to topological conclusions, and none led to the wrong predictions or conclusions. The comparison indicates that parts of models based on secondary structure predictions alone are not reliable and points to the importance of experimental data in the establishment of outer membrane protein topological models. The advantages and limitations of genetic foreign epitope insertion for the study of integral membrane proteins are discussed.

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis

PubMed Central

Molino, Diana; Nola, Sébastien; Lam, Sin Man; Verraes, Agathe; Proux-Gillardeaux, Véronique; Boncompain, Gaëlle; Perez, Franck; Wenk, Markus; Shui, Guanghou; Danglot, Lydia; Galli, Thierry

2015-01-01

Biological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. Molecular mechanisms responsible for the transport and the organization of these membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI–anchored proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis. PMID:26196023

A role for the membrane Golgi protein Ema in autophagy.

PubMed

Kim, Sungsu; DiAntonio, Aaron

2012-08-01

Autophagy is a cellular homeostatic response that involves degradation of self-components by the double-membraned autophagosome. The biogenesis of autophagosomes has been well described, but the ensuing processes after autophagosome formation are not clear. In our recent study, we proposed a model in which the Golgi complex contributes to the growth of autophagic structures, and that the Drosophila melanogaster membrane protein Ema promotes this process. In fat body cells of the D. melanogaster ema mutant, the recruitment of the Golgi complex protein Lava lamp (Lva) to autophagic structures is impaired and autophagic structures are very small. In addition, in the ema mutant autophagic turnover of SQSTM1/p62 and mitophagy are impaired. Our study not only identifies a role for Ema in autophagy, but also supports the hypothesis that the Golgi complex may be a potential membrane source for the biogenesis and development of autophagic structures.

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

An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.

PubMed

Ferguson, A D; Breed, J; Diederichs, K; Welte, W; Coulton, J W

1998-07-01

FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).

Comparative effects of overproducing the AraC-type transcriptional regulators MarA, SoxS, RarA and RamA on antimicrobial drug susceptibility in Klebsiella pneumoniae.

PubMed

Jiménez-Castellanos, Juan-Carlos; Wan Ahmad Kamil, Wan Nur Ismah; Cheung, Ching Hei Phoebe; Tobin, Maryann S; Brown, James; Isaac, Sophie G; Heesom, Kate J; Schneiders, Thamarai; Avison, Matthew B

2016-07-01

In Klebsiella pneumoniae, overproduction of RamA and RarA leads to increased MICs of various antibiotics; MarA and SoxS are predicted to perform a similar function. We have compared the relative effects of overproducing these four AraC-type regulators on envelope permeability (a combination of outer membrane permeability and efflux), efflux pump and porin production, and antibiotic susceptibility in K. pneumoniae. Regulators were overproduced using a pBAD expression vector. Antibiotic susceptibility was measured using disc testing. Envelope permeability was estimated using a fluorescent dye accumulation assay. Porin and efflux pump production was quantified using proteomics and validated using real-time quantitative RT-PCR. Envelope permeability and antibiotic disc inhibition zone diameters both reduced during overproduction of RamA and to a lesser extent RarA or SoxS, but did not change following overproduction of MarA. These effects were associated with overproduction of the efflux pumps AcrAB (for RamA and SoxS) and OqxAB (for RamA and RarA) and the outer membrane protein TolC (for all regulators). Effects on porin production were strain specific. RamA is the most potent regulator of antibiotic permeability in K. pneumoniae, followed by RarA then SoxS, with MarA having very little effect. This observed relative potency correlates well with the frequency at which these regulators are reportedly overproduced in clinical isolates. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

NagA-dependent uptake of N-acetyl-glucosamine and N-acetyl-chitin oligosaccharides across the outer membrane of Caulobacter crescentus.

PubMed

Eisenbeis, Simone; Lohmiller, Stefanie; Valdebenito, Marianne; Leicht, Stefan; Braun, Volkmar

2008-08-01

Among the 67 predicted TonB-dependent outer membrane transporters of Caulobacter crescentus, NagA was found to be essential for growth on N-acetyl-beta-D-glucosamine (GlcNAc) and larger chitin oligosaccharides. NagA (93 kDa) has a predicted typical domain structure of an outer membrane transport protein: a signal sequence, the TonB box EQVVIT, a hatch domain of 147 residues, and a beta-barrel composed of 22 antiparallel beta-strands linked by large surface loops and very short periplasmic turns. Mutations in tonB1 and exbBD, known to be required for maltose transport via MalA in C. crescentus, and in two additional predicted tonB genes (open reading frames cc2327 and cc3508) did not affect NagA-mediated GlcNAc uptake. nagA is located in a gene cluster that encodes a predicted PTS sugar transport system and two enzymes that convert GlcNAc-6-P to fructose-6-P. Since a nagA insertion mutant did not grow on and transport GlcNAc, diffusion of GlcNAc through unspecific porins in the outer membrane is excluded. Uptake of GlcNAc into tonB and exbBD mutants and reduction but not abolishment of GlcNAc transport by agents which dissipate the electrochemical potential of the cytoplasmic membrane (0.1 mM carbonyl cyanide 3-chlorophenylhydrazone and 1 mM 2,4-dinitrophenol) suggest diffusion of GlcNAc through a permanently open pore of NagA. Growth on (GlcNAc)(3) and (GlcNAc)(5) requires ExbB and ExbD, indicating energy-coupled transport by NagA. We propose that NagA forms a small pore through which GlcNAc specifically diffuses into the periplasm and functions as an energy-coupled transporter for the larger chitin oligosaccharides.

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao Haiyan, E-mail: zhaohy@ku.ed; Sequeira, Reuben D., E-mail: sequen@ku.ed; Galeva, Nadezhda A., E-mail: galeva@ku.ed

2011-01-20

Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus maymore » be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.« less

Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells.

PubMed

Leuzzi, Rosanna; Nesta, Barbara; Monaci, Elisabetta; Cartocci, Elena; Serino, Laura; Soriani, Marco; Rappuoli, Rino; Pizza, Mariagrazia

2013-11-09

Protein PIII is one of the major outer membrane proteins of Neisseria gonorrhoeae, 95% identical to RmpM (reduction modifiable protein M) or class 4 protein of Neisseria meningitidis. RmpM is known to be a membrane protein associated by non-covalent bonds to the peptidoglycan layer and interacting with PorA/PorB porin complexes resulting in the stabilization of the bacterial membrane. The C-terminal domain of PIII (and RmpM) is highly homologous to members of the OmpA family, known to have a role in adhesion/invasion in many bacterial species. The contribution of PIII in the membrane architecture and its role in the interaction with epithelial cells has never been investigated. We generated a ΔpIII knock-out mutant strain and evaluated the effects of the loss of PIII expression on bacterial morphology and on outer membrane composition. Deletion of the pIII gene does not cause any alteration in bacterial morphology or sensitivity to detergents. Moreover, the expression profile of the main membrane proteins remains the same for the wild-type and knock-out strains, with the exception of the NG1873 which is not exported to the outer membrane and accumulates in the inner membrane in the ΔpIII knock-out mutant strain.We also show that purified PIII protein is able to bind human cervical and urethral cells and that the ΔpIII knock-out mutant strain has a lower ability to adhere to human cervical and urethral cells. Here we demonstrated that the PIII protein does not play a key structural role in the membrane organization of gonococcus and does not induce major effects on the expression of the main outer membrane proteins. However, in the PIII knock-out strain, the NG1873 protein is not localized in the outer membrane as it is in the wild-type strain suggesting a possible interaction of PIII with NG1873. The evidence that PIII binds to human epithelial cells derived from the female and male genital tract highlights a possible role of PIII in the virulence of gonococcus

In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem

PubMed Central

Santini, Sébastien; Pinet, Elizabeth; Claverie, Jean-Michel; Davin-Régli, Anne-Véronique; Pagès, Jean-Marie; Masi, Muriel

2015-01-01

Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen. PMID:26398358

In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem.

PubMed

Philippe, Nadège; Maigre, Laure; Santini, Sébastien; Pinet, Elizabeth; Claverie, Jean-Michel; Davin-Régli, Anne-Véronique; Pagès, Jean-Marie; Masi, Muriel

2015-01-01

Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen.

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

Structural inhibition of dynamin-mediated membrane fission by endophilin

PubMed Central

Galli, Valentina; Shen, Peter S; Humbert, Frédéric; De Camilli, Pietro

2017-01-01

Dynamin, which mediates membrane fission during endocytosis, binds endophilin and other members of the Bin-Amphiphysin-Rvs (BAR) protein family. How endophilin influences endocytic membrane fission is still unclear. Here, we show that dynamin-mediated membrane fission is potently inhibited in vitro when an excess of endophilin co-assembles with dynamin around membrane tubules. We further show by electron microscopy that endophilin intercalates between turns of the dynamin helix and impairs fission by preventing trans interactions between dynamin rungs that are thought to play critical roles in membrane constriction. In living cells, overexpression of endophilin delayed both fission and transferrin uptake. Together, our observations suggest that while endophilin helps shape endocytic tubules and recruit dynamin to endocytic sites, it can also block membrane fission when present in excess by inhibiting inter-dynamin interactions. The sequence of recruitment and the relative stoichiometry of the two proteins may be critical to regulated endocytic fission. PMID:28933693

Uptake of Sulfate but Not Phosphate by Mycobacterium tuberculosis Is Slower than That for Mycobacterium smegmatis

PubMed Central

Song, Houhui

2012-01-01

Knowledge of the metabolic pathways used by Mycobacterium tuberculosis during infection is important for understanding its nutrient requirements and host adaptation. However, uptake, the first step in the utilization of nutrients, is poorly understood for many essential nutrients, such as inorganic anions. Here, we show that M. tuberculosis utilizes nitrate as the sole nitrogen source, albeit at lower efficiency than asparagine, glutamate, and arginine. The growth of the porin triple mutant M. smegmatis ML16 in media with limiting amounts of nitrate and sulfate as sole nitrogen and sulfur sources, respectively, was delayed compared to that of the wild-type strain. The uptake of sulfate was 40-fold slower than that of the wild-type strain, indicating that the efficient uptake of these anions is dependent on porins. The uptake by M. tuberculosis of sulfate and phosphate was approximately 40- and 10-fold slower than that of M. smegmatis, respectively, which is consistent with the slower growth of M. tuberculosis. However, the uptake of these anions by M. tuberculosis is orders of magnitude faster than diffusion through lipid membranes, indicating that unknown outer membrane proteins are required to facilitate this process. PMID:22194452

Endotoxin-associated protein: a potent stimulus for human granulocytopoietic activity which may be accessory cell independent.

PubMed Central

Bjornson, B H; Agura, E; Harvey, J M; Johns, M; Andrews, R G; McCabe, W R

1988-01-01

Proteins coextracted with endotoxin, termed endotoxin-associated protein (EAP), have been shown to exert interleukin 1-like activities. The present studies demonstrate that EAP also exerts potent granulopoietic colony-stimulating activity (CSA) on human peripheral blood and bone marrow progenitor cells, comparable to that seen with various types of conditioned media. The CSA observed with EAP appeared to be heat (100 degrees C, 30 min) and trypsin resistant and partially pronase resistant. Similar resistance was observed with the porin proteins of the outer membrane of gram-negative bacteria, and similar CSA activity was observed with a purified porin preparation of Neisseria gonorrhoeae. The CSA of EAP could be demonstrated in human peripheral blood and bone marrow leukocytes rigorously depleted of monocytes, T lymphocytes, and B lymphocytes by treatment with specific monoclonal antibodies and complement. PMID:2836311

Water and solute transport across the peritoneal membrane.

PubMed

Morelle, Johann; Devuyst, Olivier

2015-09-01

We review the molecular mechanisms of peritoneal transport and discuss how a better understanding of these mechanisms is relevant for dialysis therapy. Peritoneal dialysis involves diffusion and osmosis through the highly vascularized peritoneal membrane. Computer simulations, expression studies and functional analyses in Aqp1 knockout mice demonstrated the critical role of the water channel aquaporin-1 (AQP1) in water removal during peritoneal dialysis. Pharmacologic regulation of AQP1, either through increased expression or gating, is associated with increased water transport in rodent models of peritoneal dialysis. Water transport is impaired during acute peritonitis, despite unchanged expression of AQP1, resulting from the increased microvascular area that dissipates the osmotic gradient across the membrane. In long-term peritoneal dialysis patients, the fibrotic interstitium also impairs water transport, resulting in ultrafiltration failure. Recent data suggest that stroke and drug intoxications might benefit from peritoneal dialysis and could represent novel applications of peritoneal transport in the future. A better understanding of the regulation of osmotic water transport across the peritoneum offers novel insights into the role of water channels in microvascular endothelia, the functional importance of structural changes in the peritoneal interstitium and the transport of water and solutes across biological membranes in general.

Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membranes

PubMed Central

Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

2009-01-01

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this work, we report the first proteomics-based characterization of nonenzymatically glycated proteins in human plasma and erythrocyte membranes from individuals with normal glucose tolerance, impaired glucose tolerance, and type 2 diabetes mellitus. Phenylboronate affinity chromatography was used to enrich glycated proteins and glycated tryptic peptides from both human plasma and erythrocyte membranes. The enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation-tandem mass spectrometry, resulting in the confident identification of 76 and 31 proteins from human plasma and erythrocyte membranes, respectively. Although most of the glycated proteins could be identified in samples from individuals with normal glucose tolerance, slightly higher numbers of glycated proteins and more glycation sites were identified in samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus. PMID:18396901

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

Liver mitochondrial membrane fluidity at early development of diabetes and its correlation with the respiration.

PubMed

Pérez-Hernández, Ismael H; Domínguez-Fuentes, Josué Misael; Palomar-Morales, Martín; Zazueta-Mendizabal, Ana Cecilia; Baiza-Gutman, Arturo; Mejía-Zepeda, Ricardo

2017-06-01

The biological membranes are important in cell function but, during development of diseases such as diabetes, they are impaired. Consequently, membrane-associated biological processes are impaired as well. The mitochondria are important organelles where oxidative phosphorylation takes place, a process closely related with the membranes. In general, it is accepted that the development process of diabetes decreases membrane fluidity. However, in some cases, it has been found to increase membrane fluidity of mitochondria but to decrease the Respiratory Control (RC) index. In this study we found an increase of membrane fluidity and an increase of the RC at an early phase of the development of a type 2 diabetes model. We measured the lipoperoxidation, analyzed the fatty acids composition by gas chromatography, and assessed membrane fluidity using three fluorescent monitors located at different depths inside the bilayer, dipyrenilpropane (DPyP), diphenylhexatriene (DPH), and trimethylammonium diphenylhexatriene (TMA-DPH). Our findings indicate that in the initial stage of diabetes development, when lipoperoxidation still is not significant, the membrane fluidity of liver mitochondria increases because of the increment in the unsaturated to saturated fatty acids ratio (U/S), thus producing an increase of the RC. The membrane fluidity is not the same at all depths in the bilayer. Contrary to the results obtained in mitochondria, the diabetes induced a decrease in the U/S fatty acids ratio of liver total lipids, indicating that the mitochondria might have an independent mechanism for regulating its fatty acids composition.

A novel osmosis membrane bioreactor-membrane distillation hybrid system for wastewater treatment and reuse.

PubMed

Nguyen, Nguyen Cong; Nguyen, Hau Thi; Chen, Shiao-Shing; Ngo, Huu Hao; Guo, Wenshan; Chan, Wen Hao; Ray, Saikat Sinha; Li, Chi-Wang; Hsu, Hung-Te

2016-06-01

A novel approach was designed to simultaneously enhance nutrient removal and reduce membrane fouling for wastewater treatment using an attached growth biofilm (AGB) integrated with an osmosis membrane bioreactor (OsMBR) system for the first time. In this study, a highly charged organic compound (HEDTA(3-)) was employed as a novel draw solution in the AGB-OsMBR system to obtain a low reverse salt flux, maintain a healthy environment for the microorganisms. The AGB-OsMBR system achieved a stable water flux of 3.62L/m(2)h, high nutrient removal of 99% and less fouling during a 60-day operation. Furthermore, the high salinity of diluted draw solution could be effectively recovered by membrane distillation (MD) process with salt rejection of 99.7%. The diluted draw solution was re-concentrated to its initial status (56.1mS/cm) at recovery of 9.8% after 6h. The work demonstrated that novel multi-barrier systems could produce high quality potable water from impaired streams. Copyright © 2016. Published by Elsevier Ltd.

Outer membrane proteins related to SusC and SusD are not required for Cytophaga hutchinsonii cellulose utilization.

PubMed

Zhu, Yongtao; Kwiatkowski, Kurt J; Yang, Tengteng; Kharade, Sampada S; Bahr, Constance M; Koropatkin, Nicole M; Liu, Weifeng; McBride, Mark J

2015-08-01

Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, employs a novel collection of cell-associated proteins to digest crystalline cellulose. Other Bacteroidetes rely on cell surface proteins related to the starch utilization system (Sus) proteins SusC and SusD to bind oligosaccharides and import them across the outer membrane for further digestion. These bacteria typically produce dozens of SusC-like porins and SusD-like oligosaccharide-binding proteins to facilitate utilization of diverse polysaccharides. C. hutchinsonii specializes in cellulose digestion and its genome has only two susC-like genes and two susD-like genes. Single and multiple gene deletions were constructed to determine if the susC-like and susD-like genes have roles in cellulose utilization. A mutant lacking all susC-like and all susD-like genes digested cellulose and grew on cellulose as well as wild-type cells. Further, recombinantly expressed SusD-like proteins CHU_0547 and CHU_0554 failed to bind cellulose or β-glucan hemicellulosic polysaccharides. The results suggest that the Bacteroidetes Sus paradigm for polysaccharide utilization may not apply to the cellulolytic bacterium C. hutchinsonii.

Impaired cortical mitochondrial function following TBI precedes behavioral changes

PubMed Central

Watson, William D.; Buonora, John E.; Yarnell, Angela M.; Lucky, Jessica J.; D’Acchille, Michaela I.; McMullen, David C.; Boston, Andrew G.; Kuczmarski, Andrew V.; Kean, William S.; Verma, Ajay; Grunberg, Neil E.; Cole, Jeffrey T.

2014-01-01

Traumatic brain injury (TBI) pathophysiology can be attributed to either the immediate, primary physical injury, or the delayed, secondary injury which begins minutes to hours after the initial injury and can persist for several months or longer. Because these secondary cascades are delayed and last for a significant time period post-TBI, they are primary research targets for new therapeutics. To investigate changes in mitochondrial function after a brain injury, both the cortical impact site and ipsilateral hippocampus of adult male rats 7 and 17 days after a controlled cortical impact (CCI) injury were examined. State 3, state 4, and uncoupler-stimulated rates of oxygen consumption, respiratory control ratios (RCRs) were measured and membrane potential quantified, and all were significantly decreased in 7 day post-TBI cortical mitochondria. By contrast, hippocampal mitochondria at 7 days showed only non-significant decreases in rates of oxygen consumption and membrane potential. NADH oxidase activities measured in disrupted mitochondria were normal in both injured cortex and hippocampus at 7 days post-CCI. Respiratory and phosphorylation capacities at 17 days post-CCI were comparable to naïve animals for both cortical and hippocampus mitochondria. However, unlike oxidative phosphorylation, membrane potential of mitochondria in the cortical lining of the impact site did not recover at 17 days, suggesting that while diminished cortical membrane potential at 17 days does not adversely affect mitochondrial capacity to synthesize ATP, it may negatively impact other membrane potential-sensitive mitochondrial functions. Memory status, as assessed by a passive avoidance paradigm, was not significantly impaired until 17 days after injury. These results indicate pronounced disturbances in cortical mitochondrial function 7 days after CCI which precede the behavioral impairment observed at 17 days. PMID:24550822

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

PubMed Central

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

2011-01-01

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

Relationship between red cell membrane fatty acids and adipokines in individuals with varying insulin sensitivity.

PubMed

Min, Y; Lowy, C; Islam, S; Khan, F S; Swaminathan, R

2011-06-01

Plasma leptin and adiponectin, and membrane phospholipid fatty acid composition are implicated into the mechanism of insulin resistance but no clear pattern has emerged. Hence, this study examined these variables in subjects presenting to the diabetic clinic for a diagnostic glucose tolerance test. Body composition, glucose, glycated hemoglobin, insulin, leptin, adiponectin, and red cell and plasma phospholipid fatty acids were assessed from 42 normal and 28 impaired glucose tolerant subjects. Insulin sensitivity was determined by homeostatic model assessment. The plasma phosphatidylcholine fatty acid composition of the impaired glucose tolerant subjects was similar to that of normal subjects. However, the impaired glucose tolerant subjects had significantly lower linoleic (P<0.05), eicosapentaenoic (P<0.05) and docosahexaenoic (P<0.01) acids in the red cell phosphatidylcholine and phosphatidylethanolamine compared with the normal subjects. Moreover, red cell phosphatidylcholine docosahexaenoic acid correlated positively with adiponectin (r=0.290, P<0.05) but negatively with leptin (r=-0.252, P<0.05), insulin (r=-0.335, P<0.01) and insulin resistance (r=-0.322, P<0.01). Plasma triglycerides, leptin and glucose combined predicted about 60% of variation in insulin level whereas insulin was the only component that predicted the membrane fatty acids. We postulate that membrane phospholipids fatty acids have an indirect role in determining insulin concentration but insulin has a major role in determining membrane fatty acid composition.

Early membrane events induced by salicylic acid in motor cells of the Mimosa pudica pulvinus.

PubMed

Saeedi, Saed; Rocher, Françoise; Bonmort, Janine; Fleurat-Lessard, Pierrette; Roblin, Gabriel

2013-04-01

Salicylic acid (o-hydroxy benzoic acid) (SA) induced a rapid dose-dependent membrane hyperpolarization (within seconds) and a modification of the proton secretion (within minutes) of Mimosa pudica pulvinar cells at concentrations higher than 0.1mM. Observations on plasma membrane vesicles isolated from pulvinar tissues showed that SA acted directly at the membrane level through a protonophore action as suggested by the inhibition of the proton gradient and the lack of effect on H(+)-ATPase catalytic activity. Comparative data obtained with protonophores (carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol) and inhibitors of ATPases (vanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol) corroborated this conclusion. Consequently, the collapse of the proton motive force led to an impairment in membrane functioning. This impairment is illustrated by the inhibition of the ion-driven turgor-mediated seismonastic reaction of the pulvinus following SA treatment. SA acted in a specific manner as its biosynthetic precursor benzoic acid induced much milder effects and the m- and p-OH benzoic acid derivatives did not trigger similar characteristic effects. Therefore, SA may be considered both a membrane signal molecule and a metabolic effector following its uptake in the cells.

In vivo administration of extracellular cGMP normalizes TNF-α and membrane expression of AMPA receptors in hippocampus and spatial reference memory but not IL-1β, NMDA receptors in membrane and working memory in hyperammonemic rats.

PubMed

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

2016-10-01

Patients with hepatic encephalopathy (HE) show working memory and visuo-spatial orientation deficits. Hyperammonemia is a main contributor to cognitive impairment in HE. Hyperammonemic rats show impaired spatial learning and learning ability in the Y maze. Intracerebral administration of extracellular cGMP restores learning in the Y-maze. The underlying mechanisms remain unknown. It also remains unknown whether extracellular cGMP improves neuroinflammation or restores spatial learning in hyperammonemic rats and if it affects differently reference and working memory. The aims of this work were: Spatial working and reference memory were assessed using the radial and Morris water mazes and neuroinflammation by immunohistochemistry and Western blot. Membrane expression of NMDA and AMPA receptor subunits was analyzed using the BS3 crosslinker. Extracellular cGMP was administered intracerebrally using osmotic minipumps. Chronic hyperammonemia induces neuroinflammation in hippocampus, with astrocytes activation and increased IL-1β, which are associated with increased NMDA receptors membrane expression and impaired working memory. This process is not affected by extracellular cGMP. Hyperammonemia also activates microglia and increases TNF-α, alters membrane expression of AMPA receptor subunits (increased GluA1 and reduced GluA2) and impairs reference memory. All these changes are reversed by extracellular cGMP. These results show that extracellular cGMP modulates spatial reference memory but not working memory. This would be mediated by modulation of TNF-α levels and of membrane expression of GluA1 and GluA2 subunits of AMPA receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

Inner membrane fusion mediates spatial distribution of axonal mitochondria

PubMed Central

Yu, Yiyi; Lee, Hao-Chih; Chen, Kuan-Chieh; Suhan, Joseph; Qiu, Minhua; Ba, Qinle; Yang, Ge

2016-01-01

In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, we developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. We found that stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distributions and morphology differentially. Knockdown of dOpa1 also impaired axonal transport of mitochondria. But the changed spatial distributions of mitochondria resulted primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, caused similar transport velocity impairment but different spatial distributions. Together, our data reveals that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution. PMID:26742817

Comparative proteomic analysis of outer membrane protein 43 (omp43)-deficient Bartonella henselae.

PubMed

Kang, Jun-Gu; Lee, Hee-Woo; Ko, Sungjin; Chae, Joon-Seok

2018-01-31

Outer membrane proteins (OMPs) of Gram-negative bacteria constitute the first line of defense protecting cells against environmental stresses including chemical, biophysical, and biological attacks. Although the 43-kDa OMP (OMP43) is major porin protein among Bartonella henselae -derived OMPs, its function remains unreported. In this study, OMP43-deficient mutant B. henselae (Δomp43) was generated to investigate OMP43 function. Interestingly, Δ omp 43 exhibited weaker proliferative ability than that of wild-type (WT) B. henselae . To study the differences in proteomic expression between WT and Δ omp 43, two-dimensional gel electrophoresis-based proteomic analysis was performed. Based on Clusters of Orthologus Groups functional assignments, 12 proteins were associated with metabolism, 7 proteins associated with information storage and processing, and 3 proteins associated with cellular processing and signaling. By semi-quantitative reverse transcriptase polymerase chain reaction, increases in tld D, efp, ntr X, pdh A, pur B, and ATPA mRNA expression and decreases in Rho and yfe A mRNA expression were confirmed in Δ omp 43. In conclusion, this is the first report showing that a loss of OMP43 expression in B. henselae leads to retarded proliferation. Furthermore, our proteomic data provide useful information for the further investigation of mechanisms related to the growth of B. henselae.

[Membrane and functional effects of vinpocetine and tocopherol in rats with experimental cerebral ischemia].

PubMed

Vishnevskiĭ, A A; Korotkevich, I G; Zhaparalieva, Ch O

2009-01-01

The membrane, antioxidant and functional effects of vinpocetine and a-tocopherol have been investigated under conditions of acute experimental cerebral ischemia in rats. Vinpocetine administration decreased accumulation of lysophospholipids in brain plasma membranes. Vinpocetine also blocked accumulation of conjugated dienes (CD). alpha-Tocopherol inhibited augmentation in CD content and did not reduce the level of lysophospholipids in brain plasma membranes. Functional consequences of membrane impairments were also detected in some behavioral tests and physical capabilities. Administration of both vinpocetine and alpha-tocopherol decreased manifestations of the altered parameters induced by cerebral ischemia and vinpocetine was more effective than alpha-tocopherol.

DJ-1 KNOCK-DOWN IMPAIRS ASTROCYTE MITOCHONDRIAL FUNCTION

PubMed Central

LARSEN, N. J.; AMBROSI, G.; MULLETT, S. J.; BERMAN, S. B.; HINKLE, D. A.

2012-01-01

Mitochondrial dysfunction has long been implicated in the pathogenesis of Parkinson’s disease (PD). PD brain tissues show evidence for mitochondrial respiratory chain Complex I deficiency. Pharmacological inhibitors of Complex I, such as rotenone, cause experimental parkinsonism. The cytoprotective protein DJ-1, whose deletion is sufficient to cause genetic PD, is also known to have mitochondria-stabilizing properties. We have previously shown that DJ-1 is over-expressed in PD astrocytes, and that DJ-1 deficiency impairs the capacity of astrocytes to protect co-cultured neurons against rotenone. Since DJ-1 modulated, astrocyte-mediated neuroprotection against rotenone may depend upon proper astrocytic mitochondrial functioning, we hypothesized that DJ-1 deficiency would impair astrocyte mitochondrial motility, fission/fusion dynamics, membrane potential maintenance, and respiration, both at baseline and as an enhancement of rotenone-induced mitochondrial dysfunction. In astrocyte-enriched cultures, we observed that DJ-1 knock-down reduced mitochondrial motility primarily in the cellular processes of both untreated and rotenone treated cells. In these same cultures, DJ-1 knock-down did not appreciably affect mitochondrial fission, fusion, or respiration, but did enhance rotenone-induced reductions in the mitochondrial membrane potential. In neuron–astrocyte co-cultures, astrocytic DJ-1 knock-down reduced astrocyte process mitochondrial motility in untreated cells, but this effect was not maintained in the presence of rotenone. In the same co-cultures, astrocytic DJ-1 knock-down significantly reduced mitochondrial fusion in the astrocyte cell bodies, but not the processes, under the same conditions of rotenone treatment in which DJ-1 deficiency is known to impair astrocyte-mediated neuroprotection. Our studies therefore demonstrated the following new findings: (i) DJ-1 deficiency can impair astrocyte mitochondrial physiology at multiple levels, (ii) astrocyte

Lysosomal impairment in Parkinson's disease.

PubMed

Dehay, Benjamin; Martinez-Vicente, Marta; Caldwell, Guy A; Caldwell, Kim A; Yue, Zhenyue; Cookson, Mark R; Klein, Christine; Vila, Miquel; Bezard, Erwan

2013-06-01

Impairment of autophagy-lysosomal pathways (ALPs) is increasingly regarded as a major pathogenic event in neurodegenerative diseases, including Parkinson's disease (PD). ALP alterations are observed in sporadic PD brains and in toxic and genetic rodent models of PD-related neurodegeneration. In addition, PD-linked mutations and post-translational modifications of α-synuclein impair its own lysosomal-mediated degradation, thereby contributing to its accumulation and aggregation. Furthermore, other PD-related genes, such as leucine-rich repeat kinase-2 (LRRK2), parkin, and phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), have been mechanistically linked to alterations in ALPs. Conversely, mutations in lysosomal-related genes, such as glucocerebrosidase (GBA) and lysosomal type 5 P-type ATPase (ATP13A2), have been linked to PD. New data offer mechanistic molecular evidence for such a connection, unraveling a causal link between lysosomal impairment, α-synuclein accumulation, and neurotoxicity. First, PD-related GBA deficiency/mutations initiate a positive feedback loop in which reduced lysosomal function leads to α-synuclein accumulation, which, in turn, further decreases lysosomal GBA activity by impairing the trafficking of GBA from the endoplasmic reticulum-Golgi to lysosomes, leading to neurodegeneration. Second, PD-related mutations/deficiency in the ATP13A2 gene lead to a general lysosomal impairment characterized by lysosomal membrane instability, impaired lysosomal acidification, decreased processing of lysosomal enzymes, reduced degradation of lysosomal substrates, and diminished clearance of autophagosomes, collectively contributing to α-synuclein accumulation and cell death. According to these new findings, primary lysosomal defects could potentially account for Lewy body formation and neurodegeneration in PD, laying the groundwork for the prospective development of new neuroprotective/disease-modifying therapeutic strategies

Noise analysis of antibiotic permeation through bacterial channels

NASA Astrophysics Data System (ADS)

Nestorovich, Ekaterina M.; Danelon, Christophe; Winterhalter, Mathias; Bezrukov, Sergey M.

2003-05-01

Statistical analysis of high-resolution current recordings from a single ion channel reconstituted into a planar lipid membrane allows us to study transport of antibiotics at the molecular detail. Working with the general bacterial porin, OmpF, we demonstrate that addition of zwitterionic β-lactam antibiotics to the membrane-bathing solution introduces transient interruptions in the small-ion current through the channel. Time-resolved measurements reveal that one antibiotic molecule blocks one of the monomers in the OmpF trimer for characteristic times from microseconds to hundreds of microseconds. Spectral noise analysis enables us to perform measurements over a wide range of changing parameters. In all cases studied, the residence time of an antibiotic molecule in the channel exceeds the estimated time for free diffusion by orders of magnitude. This demonstrates that, in analogy to substrate-specific channels that evolved to bind specific metabolite molecules, antibiotics have 'evolved' to be channel-specific. The charge distribution of an efficient antibiotic complements the charge distribution at the narrowest part of the bacterial porin. Interaction of these charges creates a zone of attraction inside the channel and compensates the penetrating molecule's entropy loss and desolvation energy. This facilitates antibiotic translocation through the narrowest part of the channel and accounts for higher antibiotic permeability rates.

Specific detection of membrane-toxic substances with a conductivity assay.

PubMed

Eich, J; Dürholt, H; Steger-Hartmann, T; Wagner, E

2000-03-01

A conductivity assay that represents a new biotest able to detect the effects of membrane-toxic compounds, e.g., detergents, organic solvents, and radical formers, on various organisms was previously described and developed. The conductivity assay measures ion leakage from cells, tissues, or whole plant and animal organisms whose membrane systems have been damaged by membrane-toxic compounds. In this study the specificity of the conductivity assay for membrane-toxic compounds was tested by comparing the electrolyte efflux from Elodea canadensis leaves during incubation with a well-known detergent (benzalkonium chloride) using different plant physiological and biochemical techniques (photochemical efficiency, plasmolysis capacity, NBT reduction, and electron microscopy of membranes of E. canadensis leaves). The comparison of the different methods proved that the electrolyte loss during benzalkonium chloride incubation determined in the conductivity assay is due to membrane impairment. The observed electrolyte loss correlated with a reduction of photochemical efficiency and a decrease in both plasmolysis and NBT reduction capacity. Furthermore, a disintegration of the plasmalemma could be seen in the electron micrographs. These results indicate that the measured electrolyte loss in the conductivity assay is a specific effect of membrane-toxic compounds. Copyright 2000 Academic Press.

Membrane properties involved in calcium-stimulated microparticle release from the plasma membranes of S49 lymphoma cells.

PubMed

Campbell, Lauryl E; Nelson, Jennifer; Gibbons, Elizabeth; Judd, Allan M; Bell, John D

2014-01-01

This study answered the question of whether biophysical mechanisms for microparticle shedding discovered in platelets and erythrocytes also apply to nucleated cells: cytoskeletal disruption, potassium efflux, transbilayer phospholipid migration, and membrane disordering. The calcium ionophore, ionomycin, disrupted the actin cytoskeleton of S49 lymphoma cells and produced rapid release of microparticles. This release was significantly inhibited by interventions that impaired calcium-activated potassium current. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated dismantling of the normal phospholipid transbilayer asymmetry. Rescue of the scrambling function at high ionophore concentration also resulted in enhanced particle shedding. The effect of membrane physical properties was addressed by varying the experimental temperature (32-42°C). A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammonium diphenylhexatriene and Patman revealed significant decrease in the level of apparent membrane order along that temperature range. These results demonstrated that biophysical mechanisms involved in microparticle release from platelets and erythrocytes apply also to lymphocytes.

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

Molecular insights into the m-AAA protease-mediated dislocation of transmembrane helices in the mitochondrial inner membrane.

PubMed

Lee, Seoeun; Lee, Hunsang; Yoo, Suji; Kim, Hyun

2017-12-08

Protein complexes involved in respiration, ATP synthesis, and protein import reside in the mitochondrial inner membrane; thus, proper regulation of these proteins is essential for cell viability. The m -AAA protease, a conserved hetero-hexameric AAA (ATPase associated with diverse cellular activities) protease, composed of the Yta10 and Yta12 proteins, regulates mitochondrial proteostasis by mediating protein maturation and degradation. It also recognizes and mediates the dislocation of membrane-embedded substrates, including foreign transmembrane (TM) segments, but the molecular mechanism involved in these processes remains elusive. This study investigated the role of the TM domains in the m -AAA protease by systematic replacement of one TM domain at a time in yeast. Our data indicated that replacement of the Yta10 TM2 domain abolishes membrane dislocation for only a subset of substrates, whereas replacement of the Yta12 TM2 domain impairs membrane dislocation for all tested substrates, suggesting different roles of the TM domains in each m -AAA protease subunit. Furthermore, m -AAA protease-mediated membrane dislocation was impaired in the presence of a large downstream hydrophilic moiety in a membrane substrate. This finding suggested that the m -AAA protease cannot dislocate large hydrophilic domains across the membrane, indicating that the membrane dislocation probably occurs in a lipid environment. In summary, this study highlights previously underappreciated biological roles of TM domains of the m -AAA proteases in mediating the recognition and dislocation of membrane-embedded substrates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Predicting mercury in mallard ducklings from mercury in chorioallantoic membranes

USGS Publications Warehouse

Heinz, G.H.; Hoffman, D.J.

2003-01-01

Methylmercury has been suspected as a cause of impaired reproduction in wild birds, but the confounding effects of other environmental stressors has made it difficult to determine how much mercury in the eggs of these wild species is harmful. Even when a sample egg can be collected from the nest of a wild bird and the mercury concentration in that egg compared to the laboratory-derived thresholds for reproductive impairment, additional information on the mercury levels in other eggs from that nest would be helpful in determining whether harmful levels of mercury were present in the clutch. The measurement of mercury levels in chorioallantoic membranes offers a possible way to estimate how much mercury was in a chick that hatched from an egg, and also in the whole fresh egg itself. While an embryo is developing, wastes are collected in a sac called the chorioallantoic membranes, which often remain inside the eggshell and can be collected for contaminant analysis. We fed methylmercury to captive mallards to generate a broad range of mercury levels in eggs, allowed the eggs to hatch normally, and then compared mercury concentrations in the hatchling versus the chorioallantoic membranes left behind in the eggshell. When the data from eggs laid by mercury- treated females were expressed as common logarithms, a linear equation was created by which the concentration of mercury in a duckling could be predicted from the concentration of mercury in the chorioallantoic membranes from the same egg. Therefore, if it were not possible to collect a sample egg from a clutch of wild bird eggs, the collection of the chorioallantoic membranes could be substituted, and the mercury predicted to be in the chick or whole egg could be compared to the thresholds of mercury that have been shown to cause harm in controlled feeding studies with pheasants, chickens, and mallards.

Composite polymer membranes for proton exchange membrane fuel cells operating at elevated temperatures and reduced humidities

NASA Astrophysics Data System (ADS)

Zhang, Tao

Proton Exchange Membrane Fuel Cells (PEMFCs) are the leading candidate in the fuel cell technology due to the high power density, solid electrolyte, and low operational temperature. However, PEMFCs operating in the normal temperature range (60-80°C) face problems including poor carbon monoxide tolerance and heat rejection. The poisoning effect can be significantly relieved by operating the fuel cell at elevated temperature, which also improves the heat rejection and electrochemical kinetics. Low relative humidity (RH) operation is also desirable to simplify the reactant humidification system. However, at elevated temperatures, reduced RH PEMFC performance is seriously impaired due to irreversible water loss from presently employed state-of-the-art polymer membrane, Nafion. This thesis focuses on developing polymer electrolyte membranes with high water retention ability for operation in elevated temperature (110-150°C), reduced humidity (˜50%RH) PEMFCs. One approach is to alter Nafion by adding inorganic particles such as TiO2, SiO2, Zr(HPO 4)2, etc. While the presence of these materials in Nafion has proven beneficial, a reduction or no improvement in the PEMFC performance of Nafion/TiO2 and Nafion/Zr(HPO4)2 membranes is observed with reduced particle sizes or increased particle loadings in Nafion. It is concluded that the PEMFC performance enhancement associated with addition of these inorganic particles was not due to the particle hydrophilicity. Rather, the particle, partially located in the hydrophobic region of the membrane, benefits the cell performance by altering the membrane structure. Water transport properties of some Nafion composite membranes were investigated by NMR methods including pulsed field gradient spin echo diffusion, spin-lattice relaxation, and spectral measurements. Compared to unmodified Nafion, composite membranes materials exhibit longer longitudinal relaxation time constant T1. In addition to the Nafion material, sulfonated styrene

Biaxial fatigue crack propagation behavior of perfluorosulfonic-acid membranes

NASA Astrophysics Data System (ADS)

Lin, Qiang; Shi, Shouwen; Wang, Lei; Chen, Xu; Chen, Gang

2018-04-01

Perfluorosulfonic-acid membranes have long been used as the typical electrolyte for polymer-electrolyte fuel cells, which not only transport proton and water but also serve as barriers to prevent reactants mixing. However, too often the structural integrity of perfluorosulfonic-acid membranes is impaired by membrane thinning or cracks/pinholes formation induced by mechanical and chemical degradations. Despite the increasing number of studies that report crack formation, such as crack size and shape, the underlying mechanism and driving forces have not been well explored. In this paper, the fatigue crack propagation behaviors of Nafion membranes subjected to biaxial loading conditions have been investigated. In particular, the fatigue crack growth rates of flat cracks in responses to different loading conditions are compared, and the impact of transverse stress on fatigue crack growth rate is clarified. In addition, the crack paths for slant cracks under both uniaxial and biaxial loading conditions are discussed, which are similar in geometry to those found after accelerated stress testing of fuel cells. The directions of initial crack propagation are calculated theoretically and compared with experimental observations, which are in good agreement. The findings reported here lays the foundation for understanding of mechanical failure of membranes.

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

Impaired activation of adenylyl cyclase in lung of the Basenji-greyhound model of airway hyperresponsiveness: decreased numbers of high affinity beta-adrenoceptors.

PubMed Central

Emala, C. W.; Aryana, A.; Hirshman, C. A.

1996-01-01

1. To evaluate mechanisms involved in the impaired beta-adrenoceptor stimulation of adenylyl cyclase in tissues from the Basenji-greyhound (BG) dog model of airway hyperresponsiveness, we compared agonist and antagonist binding affinity of beta-adrenoceptors, beta-adrenoceptor subtypes, percentage of beta-adrenoceptors sequestered, and coupling of the beta-adrenoceptor to Gs alpha in lung membranes from BG and control mongrel dogs. We found that lung membranes from the BG dog had higher total numbers of beta-adrenoceptors with a greater percentage of receptors of the beta 2 subtype as compared to mongrel lung membranes. 2. Agonist and antagonist binding affinity and the percentage of beta-adrenoceptors sequestered were not different in BG and mongrel dog lung membranes. However, the percentage of beta-adrenoceptors in the high affinity state for agonist was decreased in BG lung membranes suggesting an uncoupling of the receptor from Gs alpha. 3. Impaired coupling between the beta-adrenoceptor and G protein documented by the decreased numbers of beta-adrenoceptors in the high affinity state in BG lung membranes, is a plausible explanation for the reduced stimulation of adenylyl cyclase and the resultant reduction in airway smooth muscle relaxation in this model. PMID:8864536

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

PubMed

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

2014-01-01

Extracorporeal membrane oxygenation (ECMO) provides a rescue for children with severe cardiac failure. It has previously been shown that triiodothyronine (T3) improves cardiac function by modulating pyruvate oxidation during weaning. This study focused on fatty acid (FA) metabolism modulated by T3 for weaning from ECMO after cardiac injury. METHODS AND RESULTS: Nineteen immature piglets (9.1-15.3 kg) were separated into 3 groups with ECMO (6.5 h) and wean: normal circulation (Group-C); transient coronary occlusion (10 min) for ischemia-reperfusion (IR) followed by ECMO (Group-IR); and IR with T3 supplementation (Group-IR-T3). 13-Carbon ((13)C)-labeled lactate, medium-chain and long-chain FAs, was infused as oxidative substrates. Substrate fractional contribution (FC) to the citric acid cycle was analyzed by(13)C-nuclear magnetic resonance. ECMO depressed circulating T3 levels to 40% of the baseline at 4 h 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 [adenosine triphosphate]/[adenosine diphosphate] without either decreasing FC-FAs or elevating myocardial oxygen consumption over Group-C or -IR. T3 releases inhibition of lactate oxidation following IR 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.

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

Cognitive Impairment in Folate-Deficient Rats Corresponds to Depleted Brain Phosphatidylcholine and Is Prevented by Dietary Methionine without Lowering Plasma Homocysteine12

PubMed Central

Troen, Aron M.; Chao, Wei-Hsun; Crivello, Natalia A.; D'Anci, Kristen E.; Shukitt-Hale, Barbara; Smith, Don E.; Selhub, Jacob; Rosenberg, Irwin H.

2008-01-01

Poor folate status is associated with cognitive decline and dementia in older adults. Although impaired brain methylation activity and homocysteine toxicity are widely thought to account for this association, how folate deficiency impairs cognition is uncertain. To better define the role of folate deficiency in cognitive dysfunction, we fed rats folate-deficient diets (0 mg FA/kg diet) with or without supplemental L-methionine for 10 wk, followed by cognitive testing and tissue collection for hematological and biochemical analysis. Folate deficiency with normal methionine impaired spatial memory and learning; however, this impairment was prevented when the folate-deficient diet was supplemented with methionine. Under conditions of folate deficiency, brain membrane content of the methylated phospholipid phosphatidylcholine was significantly depleted, which was reversed with supplemental methionine. In contrast, neither elevated plasma homocysteine nor brain S-adenosylmethionine and S-adenosylhomocysteine concentrations predicted cognitive impairment and its prevention by methionine. The correspondence of cognitive outcomes to changes in brain membrane phosphatidylcholine content suggests that altered phosphatidylcholine and possibly choline metabolism might contribute to the manifestation of folate deficiency-related cognitive dysfunction. PMID:19022979

Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins.

PubMed

Sanjuán Szklarz, Luiza K; Kozjak-Pavlovic, Vera; Vögtle, F-Nora; Chacinska, Agnieszka; Milenkovic, Dusanka; Vogel, Sandra; Dürr, Mark; Westermann, Benedikt; Guiard, Bernard; Martinou, Jean-Claude; Borner, Christoph; Pfanner, Nikolaus; Meisinger, Chris

2007-04-20

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.

Impaired organic ion transport in proximal tubules of rats with Heymann nephritis.

PubMed

Park, E K; Hong, S K; Goldinger, J; Andres, G; Noble, B

1985-10-01

Organic ion transport across the basolateral membrane of proximal tubules was measured by means of the tissue slice technique in each of the four different stages of Heymann nephritis. Impairment of both organic anion and cation transport was detected early in Stage 2, and became more severe in Stage 3 of Heymann nephritis. The decreased transport function was associated with extensive damage to proximal tubule cells, including loss of brush border microvilli and basal infoldings. Despite these abnormalities of structure and function, oxygen consumption of proximal tubule cells remained essentially normal. Partial recovery of organic cation transport was noted late in Heymann nephritis (Stage 4). Recovery of the cation transport function was associated with a partial restoration of brush border microvilli and basal infoldings to proximal tubule cells. However, organic anion transport remained depressed throughout the entire course of disease. Impairment of organic ion transport in rats with Heymann nephritis appeared to result from damage to basolateral membrane transport elements rather than general deterioration of the metabolic machinery of proximal tubule cells. Decreased organic cation transport appeared to be the consequence of a reduction in the number of carrier sites, a phenomenon that could have resulted from decreased membrane surface area. However, the depression of organic anion transport was associated with decreased substrate affinity of the anion carrier, indicating that qualitative, rather than quantitative changes, were primarily responsible for that defect. Specific antibody-mediated damage to the anion transport elements in basolateral membranes of proximal tubules is postulated to occur in Heymann nephritis.

Triiodothyronine facilitates weaning from extracorporeal membrane oxygenation by improved mitochondrial substrate utilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Files, Matthew D.; Kajimoto, Masaki; Priddy, Colleen M.

2014-03-20

Extracorporeal membrane oxygenation (ECMO) provides a bridge to recovery after myocardial injury in infants and children, yet morbidity and mortality remain high. Weaning from the circuit requires adequate cardiac contractile function, which can be impaired by metabolic disturbances induced either by ischemia-reperfusion and / or by ECMO.

Desipramine induces disorder in cholesterol-rich membranes: implications for viral trafficking

NASA Astrophysics Data System (ADS)

Pakkanen, Kirsi; Salonen, Emppu; Mäkelä, Anna R.; Oker-Blom, Christian; Vattulainen, Ilpo; Vuento, Matti

2009-12-01

In this study, the effect of desipramine (DMI) on phospholipid bilayers and parvoviral entry was elucidated. In atomistic molecular dynamics simulations, DMI was found to introduce disorder in cholesterol-rich phospholipid bilayers. This was manifested by a decrease in the deuterium order parameter SCD as well as an increase in the membrane area. Disordering of the membrane suggested DMI to destabilize cholesterol-rich membrane domains (rafts) in cellular conditions. To relate the raft disrupting ability of DMI with novel biological relevance, we studied the intracellular effect of DMI using canine parvovirus (CPV), a virus known to interact with endosomal membranes and sphingomyelin, as an intracellular probe. DMI was found to cause retention of the virus in intracellular vesicular structures leading to the inhibition of viral proliferation. This implies that DMI has a deleterious effect on the viral traffic. As recycling endosomes and the internal vesicles of multivesicular bodies are known to contain raft components, the effect of desipramine beyond the plasma membrane step could be caused by raft disruption leading to impaired endosomal function and possibly have direct influence on the penetration of the virus through an endosomal membrane.

Electrospun cellulose acetate composites containing supported metal nanoparticles for antifungal membranes.

PubMed

Quirós, Jennifer; Gonzalo, Soledad; Jalvo, Blanca; Boltes, Karina; Perdigón-Melón, José Antonio; Rosal, Roberto

2016-09-01

Electrospun cellulose acetate composites containing silver and copper nanoparticles supported in sepiolite and mesoporous silica were prepared and tested as fungistatic membranes against the fungus Aspergillus niger. The nanoparticles were in the 3-50nm range for sepiolite supported materials and limited by the size of mesopores (5-8nm) in the case of mesoporous silica. Sepiolite and silica were well dispersed within the fibers, with larger aggregates in the micrometer range, and allowed a controlled release of metals to create a fungistatic environment. The effect was assessed using digital image analysis to evaluate fungal growth rate and fluorescence readings using a viability stain. The results showed that silver and copper nanomaterials significantly impaired the growth of fungi when the spores were incubated either in direct contact with particles or included in cellulose acetate composite membranes. The fungistatic effect took place on germinating spores before hyphae growth conidiophore formation. After 24h the cultures were separated from fungistatic materials and showed growth impairment only due to the prior exposure. Growth reduction was important for all the particles and membranes with respect to non-exposed controls. The effect of copper and silver loaded materials was not significantly different from each other with average reductions around 70% for bare particles and 50% for membranes. Copper on sepiolite was particularly efficient with a decrease of metabolic activity of up to 80% with respect to controls. Copper materials induced rapid maturation and conidiation with fungi splitting in sets of subcolonies. Metal-loaded nanomaterials acted as reservoirs for the controlled release of metals. The amount of silver or copper released daily by composite membranes represented roughly 1% of their total load of metals. Supported nanomaterials encapsulated in nanofibers allow formulating active membranes with high antifungal performance at the same time

Functional interplay between endothelial nitric oxide synthase and membrane type 1–matrix metalloproteinase in migrating endothelial cells

PubMed Central

Genís, Laura; Gonzalo, Pilar; Tutor, Antonio S.; Gálvez, Beatriz G.; Martínez-Ruiz, Antonio; Zaragoza, Carlos; Lamas, Santiago; Tryggvason, Karl; Apte, Suneel S.

2007-01-01

Nitric oxide (NO) is essential for vascular homeostasis and is also a critical modulator of angiogenesis; however, the molecular mechanisms of NO action during angiogenesis remain elusive. We have investigated the potential relationship between NO and membrane type 1–matrix metalloproteinase (MT1-MMP) during endothelial migration and capillary tube formation. Endothelial NO synthase (eNOS) colocalizes with MT1-MMP at motility-associated structures in migratory human endothelial cells (ECs); moreover, NO is produced at these structures and is released into the medium during EC migration. We have therefore addressed 2 questions: (1) the putative regulation of MT1-MMP by NO in migratory ECs; and (2) the requirement for MT1-MMP in NO-induced EC migration and tube formation. NO upregulates MT1-MMP membrane clustering on migratory human ECs, and this is accompanied by increased degradation of type I collagen substrate. MT1-MMP membrane expression and localization are impaired in lung ECs from eNOS-deficient mice, and these cells also show impaired migration and tube formation in vitro. Inhibition of MT1-MMP with a neutralizing antibody impairs NOinduced tube formation by human ECs, and NO-induced endothelial migration and tube formation are impaired in lung ECs from mice deficient in MT1-MMP. MT1-MMP thus appears to be a key molecular effector of NO during the EC migration and angiogenic processes, and is a potential therapeutic target for NO-associated vascular disorders. PMID:17606763

Study the Expression of ompf Gene in Esherichia coli Mutants.

PubMed

Jaktaji, R Pourahmad; Heidari, F

2013-09-01

The outer membrane porin proteins are the major factors in controlling the permeability of cell membrane. OmpF is an example of porin proteins in Esherichia coli. In normal growth condition a large amount of this protein is synthesised, but under stress condition, such as the presence of antibiotics in environment its expression is decreased inhibiting the entrance of antibiotics into cell. The expression of ompF is inhibited by antisense RNA transcribed from micF. In normal condition the expression of micF is low, but in the presence of antibiotics its expression is increased and causes multiple resistances to irrelevant antibiotics. The aims of this research were to study first, the intactness of micF and then quantify the expression of ompF in ciprofloxacin and tetracycline resistant mutants of E. coli. For this purpose the 5' end of micF was amplified and then sequenced. None of these mutants except one and its clone has a mutation in this gene. Then the relative expression of ompF in these mutants was quantified by real time PCR. There was no significant difference between ompF transcription of mutants and wild type strain. Based on this study and previous study it is concluded that low to intermediate levels of resistance to ciprofloxacin and tetracycline does not decrease ompF transcription.

Study the Expression of ompf Gene in Esherichia coli Mutants

PubMed Central

Jaktaji, R. Pourahmad; Heidari, F.

2013-01-01

The outer membrane porin proteins are the major factors in controlling the permeability of cell membrane. OmpF is an example of porin proteins in Esherichia coli. In normal growth condition a large amount of this protein is synthesised, but under stress condition, such as the presence of antibiotics in environment its expression is decreased inhibiting the entrance of antibiotics into cell. The expression of ompF is inhibited by antisense RNA transcribed from micF. In normal condition the expression of micF is low, but in the presence of antibiotics its expression is increased and causes multiple resistances to irrelevant antibiotics. The aims of this research were to study first, the intactness of micF and then quantify the expression of ompF in ciprofloxacin and tetracycline resistant mutants of E. coli. For this purpose the 5’ end of micF was amplified and then sequenced. None of these mutants except one and its clone has a mutation in this gene. Then the relative expression of ompF in these mutants was quantified by real time PCR. There was no significant difference between ompF transcription of mutants and wild type strain. Based on this study and previous study it is concluded that low to intermediate levels of resistance to ciprofloxacin and tetracycline does not decrease ompF transcription. PMID:24403654

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

Specific membrane lipid composition is important for plasmodesmata function in Arabidopsis.

PubMed

Grison, Magali S; Brocard, Lysiane; Fouillen, Laetitia; Nicolas, William; Wewer, Vera; Dörmann, Peter; Nacir, Houda; Benitez-Alfonso, Yoselin; Claverol, Stéphane; Germain, Véronique; Boutté, Yohann; Mongrand, Sébastien; Bayer, Emmanuelle M

2015-04-01

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of "native" PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes. © 2015 American Society of Plant Biologists. All rights reserved.

Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis

PubMed Central

Grison, Magali S.; Brocard, Lysiane; Fouillen, Laetitia; Nicolas, William; Wewer, Vera; Dörmann, Peter; Nacir, Houda; Benitez-Alfonso, Yoselin; Claverol, Stéphane; Germain, Véronique; Boutté, Yohann; Mongrand, Sébastien; Bayer, Emmanuelle M.

2015-01-01

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of “native” PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes. PMID:25818623

Dehydration rate determines the degree of membrane damage and desiccation tolerance in bryophytes.

PubMed

Cruz de Carvalho, Ricardo; Catalá, Myriam; Branquinho, Cristina; Marques da Silva, Jorge; Barreno, Eva

2017-03-01

Desiccation tolerant (DT) organisms are able to withstand an extended loss of body water and rapidly resume metabolism upon rehydration. This ability, however, is strongly dependent on a slow dehydration rate. Fast dehydration affects membrane integrity leading to intracellular solute leakage upon rehydration and thereby impairs metabolism recovery. We test the hypothesis that the increased cell membrane damage and membrane permeability observed under fast dehydration, compared with slow dehydration, is related to an increase in lipid peroxidation. Our results reject this hypothesis because following rehydration lipid peroxidation remains unaltered, a fact that could be due to the high increase of NO upon rehydration. However, in fast-dried samples we found a strong signal of red autofluorescence upon rehydration, which correlates with an increase in ROS production and with membrane leakage, particularly the case of phenolics. This could be used as a bioindicator of oxidative stress and membrane damage. © 2016 Scandinavian Plant Physiology Society.

Triiodothyronine Activates Lactate Oxidation Without Impairing Fatty Acid Oxidation and Improves Weaning From Extracorporeal Membrane Oxygenation

PubMed Central

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

2017-01-01

Background Extracorporeal membrane oxygenation (ECMO) provides a rescue for children with severe cardiac failure. It has previously been shown that triiodothyronine (T3) improves cardiac function by modulating pyruvate oxidation during weaning. This study focused on fatty acid (FA) metabolism modulated by T3 for weaning from ECMO after cardiac injury. Methods and Results Nineteen immature piglets (9.1–15.3 kg) were separated into 3 groups with ECMO (6.5 h) and wean: normal circulation (Group-C); transient coronary occlusion (10 min) for ischemia-reperfusion (IR) followed by ECMO (Group-IR); and IR with T3 supplementation (Group-IR-T3). 13-Carbon (13C)-labeled lactate, medium-chain and long-chain FAs, was infused as oxidative substrates. Substrate fractional contribution (FC) to the citric acid cycle was analyzed by 13C-nuclear magnetic resonance. ECMO depressed circulating T3 levels to 40% of the baseline at 4 h 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 [adenosine triphosphate]/[adenosine diphosphate] without either decreasing FC-FAs or elevating myocardial oxygen consumption over Group-C or -IR. Conclusions T3 releases inhibition of lactate oxidation following IR 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. PMID:25421230

N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic.

PubMed

Glozman, Rina; Okiyoneda, Tsukasa; Mulvihill, Cory M; Rini, James M; Barriere, Herve; Lukacs, Gergely L

2009-03-23

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

Escherichia coli mutants impaired in maltodextrin transport.

PubMed

Wandersman, C; Schwartz, M; Ferenci, T

1979-10-01

Wild-type Escherichia coli K-12 was found to grow equally well on maltose and on maltodextrins containing up to seven glucose residues. Three classes of mutants unable to grow on maltodextrins, but still able to grow on maltose, were investigated in detail. The first class, already known, was composed of phage lambda-resistant mutants, which lack the outer membrane protein coded by gene lamB. These mutants grow on maltose and maltotriose but not at all on maltotetraose and longer maltodextrins which cannot cross the outer membrane. A second class of mutants were affected in malE, the structural gene of the periplasmic maltose binding protein. The maltose binding proteins isolated from the new mutants were altered in their substrate binding properties, but not in a way that could account for the mutant phenotypes. Rather, the results of growth experiments and transport studies suggest that these malE mutants are impaired in their ability to transport maltodextrins across the outer membrane. This implies that the maltose binding protein (in wild-type strains) cooperates with the lambda receptor in permeation through the outer membrane. The last class of mutants described in this paper were affected in malG, or perhaps in an as yet undetected gene close to malG. They were defective in the transfer of maltodextrins from the periplasmic space to the cytoplasm but only slightly affected in the transport of maltose.

Red blood cell oxidative stress impairs oxygen delivery and induces red blood cell aging.

PubMed

Mohanty, Joy G; Nagababu, Enika; Rifkind, Joseph M

2014-01-01

Red Blood Cells (RBCs) need to deform and squeeze through narrow capillaries. Decreased deformability of RBCs is, therefore, one of the factors that can contribute to the elimination of aged or damaged RBCs from the circulation. This process can also cause impaired oxygen delivery, which contributes to the pathology of a number of diseases. Studies from our laboratory have shown that oxidative stress plays a significant role in damaging the RBC membrane and impairing its deformability. RBCs are continuously exposed to both endogenous and exogenous sources of reactive oxygen species (ROS) like superoxide and hydrogen peroxide (H2O2). The bulk of the ROS are neutralized by the RBC antioxidant system consisting of both non-enzymatic and enzymatic antioxidants including catalase, glutathione peroxidase and peroxiredoxin-2. However, the autoxidation of hemoglobin (Hb) bound to the membrane is relatively inaccessible to the predominantly cytosolic RBC antioxidant system. This inaccessibility becomes more pronounced under hypoxic conditions when Hb is partially oxygenated, resulting in an increased rate of autoxidation and increased affinity for the RBC membrane. We have shown that a fraction of peroxyredoxin-2 present on the RBC membrane may play a major role in neutralizing these ROS. H2O2 that is not neutralized by the RBC antioxidant system can react with the heme producing fluorescent heme degradation products (HDPs). We have used the level of these HDP as a measure of RBC oxidative Stress. Increased levels of HDP are detected during cellular aging and various diseases. The negative correlation (p < 0.0001) between the level of HDP and RBC deformability establishes a contribution of RBC oxidative stress to impaired deformability and cellular stiffness. While decreased deformability contributes to the removal of RBCs from the circulation, oxidative stress also contributes to the uptake of RBCs by macrophages, which plays a major role in the removal of RBCs from

Rapid Evolution of Culture-Impaired Bacteria During Adaptation to Biofilm Growth

PubMed Central

Penterman, Jon; Nguyen, Dao; Anderson, Erin; Staudinger, Benjamin J.; Greenberg, Everett P.; Lam, Joseph S.; Singh, Pradeep K.

2014-01-01

Summary Biofilm growth increases the fitness of bacteria in harsh conditions. However, bacteria from clinical and environmental biofilms can exhibit impaired growth in culture, even when the species involved are readily cultureable, and permissive conditions are used. Here we show that culture-impaired variants of Pseudomonas aeruginosa rapidly and abundantly evolve in laboratory biofilms. The culture-impaired phenotype is caused by mutations that alter the outer-membrane lipopolysaccharide structure. Within biofilms, the lipopolysaccharide mutations markedly increase bacterial fitness. However, outside the protected biofilm environment, the mutations sensitize the variants to killing by a self-produced antimicrobial agent. Thus, a biofilm-mediated adaptation produces a stark fitness trade off that compromises bacterial survival in culture. Trade offs like this could limit the ability of bacteria to transition between biofilm growth and the free-living state, and produce bacterial populations that escape detection by culture-based sampling. PMID:24412364

Sutureless Fixation of Amniotic Membrane for Therapy of Ocular Surface Disorders

PubMed Central

Kotomin, Ilya; Valtink, Monika; Hofmann, Kai; Frenzel, Annika; Morawietz, Henning; Werner, Carsten; Funk, Richard H. W.; Engelmann, Katrin

2015-01-01

Amniotic membrane is applied to the diseased ocular surface to stimulate wound healing and tissue repair, because it releases supportive growth factors and cytokines. These effects fade within about a week after application, necessitating repeated application. Generally, amniotic membrane is fixed with sutures to the ocular surface, but surgical intervention at the inflamed or diseased site can be detrimental. Therefore, we have developed a system for the mounting of amniotic membrane between two rings for application to a diseased ocular surface without surgical intervention (sutureless amniotic membrane transplantation). With this system, AmnioClip, amniotic membrane can be applied like a large contact lens. First prototypes were tested in an experiment on oneself for wearing comfort. The final system was tested on 7 patients in a pilot study. A possible influence of the ring system on the biological effects of amniotic membrane was analyzed by histochemistry and by analyzing the expression of vascular endothelial growth factor-A (VEGF-A), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF 2) and pigment epithelium-derived factor (PEDF) from amniotic membranes before and after therapeutic application. The final product, AmnioClip, showed good tolerance and did not impair the biological effects of amniotic membrane. VEGF-A and PEDF mRNA was expressed in amniotic membrane after storage and mounting before transplantation, but was undetectable after a 7-day application period. Consequently, transplantation of amniotic membranes with AmnioClip provides a sutureless and hence improved therapeutic strategy for corneal surface disorders. Trial Registration ClinicalTrials.gov NCT02168790 PMID:25955359

[Internal limiting membrane peeling as prophylaxis of epimacular membrane formation in eyes undergoing vitrectomy for rhegmatogenous retinal detachement].

PubMed

Hejsek, L; Dusová, J; Stepanov, A; Rozsíval, P

2014-06-01

Rhegmatogenous retinal detachment is a serious condition that can significantly impair visual function, even after a successful surgery. One of the complications that can significantly impair visual acuity in the postoperative period is a development of the epimacular membrane (ERM). The aim of this work is to monitor the effect of peeling of the internal limiting membrane (ILM) in the macula at the anatomical and functional results in the postoperative period, especially with regard to the development of ERM. Prospective study of 21 eyes, which underwent peeling of ILM during pars plana vitrectomy for rhegmatogenous retinal detachment (on detached macula). The ILM peeling was done without using decalin during this procedure. We tested best corrected visual acuity (BCVA) and followed fundus biomicroscopic findings. Proliferative vitreoretinopathy (PVR) was evaluated according to the recommendations of the Retina Society Terminology Committee. To exclude the development of ERM in the macula optical coherence tomography (OCT) was performed at the end of the 18-month follow-up period. In total, the results of 21 eyes of 21 patients who underwent PPV for rhegmatogenous retinal detachment were evaluated. In all of them was during PPV performed ILM peeling on detached macula, these are followed prospectively. ILM peeling without using decalin was sufficient in all eyes. All eyes with ILM peeling did not develop ERM at the end of the follow-up period. ILM peeling during PPV for rhegmatogenous retinal detachment reduces the risk of developing secondary ERM.

Exploration of a physiologically-inspired hearing-aid algorithm using a computer model mimicking impaired hearing.

PubMed

Jürgens, Tim; Clark, Nicholas R; Lecluyse, Wendy; Meddis, Ray

2016-01-01

To use a computer model of impaired hearing to explore the effects of a physiologically-inspired hearing-aid algorithm on a range of psychoacoustic measures. A computer model of a hypothetical impaired listener's hearing was constructed by adjusting parameters of a computer model of normal hearing. Absolute thresholds, estimates of compression, and frequency selectivity (summarized to a hearing profile) were assessed using this model with and without pre-processing the stimuli by a hearing-aid algorithm. The influence of different settings of the algorithm on the impaired profile was investigated. To validate the model predictions, the effect of the algorithm on hearing profiles of human impaired listeners was measured. A computer model simulating impaired hearing (total absence of basilar membrane compression) was used, and three hearing-impaired listeners participated. The hearing profiles of the model and the listeners showed substantial changes when the test stimuli were pre-processed by the hearing-aid algorithm. These changes consisted of lower absolute thresholds, steeper temporal masking curves, and sharper psychophysical tuning curves. The hearing-aid algorithm affected the impaired hearing profile of the model to approximate a normal hearing profile. Qualitatively similar results were found with the impaired listeners' hearing profiles.

Chronic ethanol administration inhibits calmodulin-dependent Ca++ uptake in synaptosomal membranes.

PubMed

Ross, D H

1986-06-01

Chronic ethanol administration inhibits ATP-dependent Ca++ uptake in a preparation of synaptic membranes prepared from mice following 1, 4 and 7 days of ethanol exposure in a liquid diet. Addition of calmodulin (2.5 micrograms) to membranes from mice receiving the control diet produced a slight stimulation of ATP dependent Ca++ uptake. Membranes from ETOH treated mice exhibited reduced capacity to take up Ca++ in ATP-dependent fashion. When calmodulin was added to membranes isolated from mice receiving ETOH on Days 1, 4 and 7 ATP-dependent Ca++ uptake was significantly stimulated (p less than 0.01) compared to (1) ETOH treated membranes in absence of calmodulin, and (2) control membranes. Behavioral tolerance as estimated by bar holding technique was found to be 25, 65 and 91 percent complete for Days 1, 4 and 7 respectively. These studies demonstrate that continued exposure of mice to ethanol via consumption of an ethanol containing liquid diet inhibits one of the mechanisms involving the cytosolic buffering of intracellular Ca++ in nerve terminals. This biochemical effect seen in parallel with the development of tolerance to ethanol impairment of bar holding suggests that increased cytosolic Ca++ may aid in central nervous system adaptation to ethanol.

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…

Ram locus is a key regulator to trigger multidrug resistance in Enterobacter aerogenes.

PubMed

Molitor, Alexander; James, Chloë E; Fanning, Séamus; Pagès, Jean-Marie; Davin-Regli, Anne

2018-02-01

Several genetic regulators belonging to AraC family are involved in the emergence of MDR isolates of E. aerogenes due to alterations in membrane permeability. Compared with the genetic regulator Mar, RamA may be more relevant towards the emergence of antibiotic resistance. Focusing on the global regulators, Mar and Ram, we compared the amino acid sequences of the Ram repressor in 59 clinical isolates and laboratory strains of E. aerogenes. Sequence types were associated with their corresponding multi-drug resistance phenotypes and membrane protein expression profiles using MIC and immunoblot assays. Quantitative gene expression analysis of the different regulators and their targets (porins and efflux pump components) were performed. In the majority of the MDR isolates tested, ramR and a region upstream of ramA were mutated but marR or marA were unchanged. Expression and cloning experiments highlighted the involvement of the ram locus in the modification of membrane permeability. Overexpression of RamA lead to decreased porin production and increased expression of efflux pump components, whereas overexpression of RamR had the opposite effects. Mutations or deletions in ramR, leading to the overexpression of RamA predominated in clinical MDR E. aerogenes isolates and were associated with a higher-level of expression of efflux pump components. It was hypothesised that mutations in ramR, and the self-regulating region proximal to ramA, probably altered the binding properties of the RamR repressor; thereby producing the MDR phenotype. Consequently, mutability of RamR may play a key role in predisposing E. aerogenes towards the emergence of a MDR phenotype.

Loss of ATP-dependent lysine uptake in the vacuolar membrane vesicles of Saccharomyces cerevisiae ypq1∆ mutant.

PubMed

Sekito, Takayuki; Nakamura, Kyosuke; Manabe, Kunio; Tone, Junichi; Sato, Yumika; Murao, Nami; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2014-01-01

Saccharomyces cerevisiae Ypq1p is a vacuolar membrane protein of the PQ-loop protein family. We found that ATP-dependent uptake activities of amino acids by vacuolar membrane vesicles were impaired by ypq1∆ mutation. Loss of lysine uptake was most remarkable, and the uptake was recovered by overproduction of Ypq1p. Ypq1p is thus involved in transport of amino acids into vacuoles.

Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

PubMed Central

Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

2014-01-01

The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein–protein and protein–lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid–protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status. PMID:24672530

Single-channel measurements of an N-acetylneuraminic acid-inducible outer membrane channel in Escherichia coli

PubMed Central

Giri, Janhavi; Tang, John M.; Wirth, Christophe; Peneff, Caroline M.

2012-01-01

NanC is an Escherichia coli outer membrane protein involved in sialic acid (Neu5Ac, i.e., N-acetylneuraminic acid) uptake. Expression of the NanC gene is induced and controlled by Neu5Ac. The transport mechanism of Neu5Ac is not known. The structure of NanC was recently solved (PDB code: 2WJQ) and includes a unique arrangement of positively charged (basic) side chains consistent with a role in acidic sugar transport. However, initial functional measurements of NanC failed to find its role in the transport of sialic acids, perhaps because of the ionic conditions used in the experiments. We show here that the ionic conditions generally preferred for measuring the function of outer-membrane porins are not appropriate for NanC. Single channels of NanC at pH 7.0 have: (1) conductance 100 pS to 800 pS in 100 mM KCl to 3 M KCl), (2) anion over cation selectivity (Vreversal = +16 mV in 250 mM KCl || 1 M KCl), and (3) two forms of voltage-dependent gating (channel closures above ±200 mV). Single-channel conductance decreases by 50% when HEPES concentration is increased from 100 μM to 100 mM in 250 mM KCl at pH 7.4, consistent with the two HEPES binding sites observed in the crystal structure. Studying alternative buffers, we find that phosphate interferes with the channel conductance. Single-channel conductance decreases by 19% when phosphate concentration is increased from 0 mM to 5 mM in 250 mM KCl at pH 8.0. Surprisingly, TRIS in the baths reacts with Ag|AgCl electrodes, producing artifacts even when the electrodes are on the far side of agar–KCl bridges. A suitable baseline solution for NanC is 250 mM KCl adjusted to pH 7.0 without buffer. PMID:22246445

DETRIMENTAL EFFECTS OF ACTIVE INTERNAL LIMITING MEMBRANE PEELING DURING EPIRETINAL MEMBRANE SURGERY: Microperimetric Analysis.

PubMed

Deltour, Jean-Baptiste; Grimbert, Pierre; Masse, Helene; Lebreton, Olivier; Weber, Michel

2017-03-01

The aim of the study was to assess the microperimetric consequences of active internal limiting membrane (ILM) peeling during idiopathic epimacular membrane (IEMM) surgery. This retrospective monocentric study included 32 eyes of 31 consecutive patients who underwent IEMM surgery. Internal limiting membrane integrity was assessed by ILM Blue staining after IEMM removal: peeling was spontaneous (Group S) or active (Group A). Preprocedure and postprocedure (1 and 6 months) examinations were performed using visual acuity determination, spectral domain optical coherence tomography and microperimetry. Twenty-two eyes had an "active ILM peeling" and 10 a "spontaneous ILM peeling." Both groups had comparable and significant improvements in visual acuity 6 months after surgery (+1.82 lines [+9 letters] [Group A] and +1.51 lines [+8 letters] [Group S], P < 0.01) associated with a significant reduction in optical coherence tomography central thickness (-99.9 μm [Group A], P < 0.01 and -62.2 μm [Group S], P = 0.05). Six months after surgery, the microperimetry showed more numerous and deeper microscotomas in the Group A than in the Group S (change in the number of microscotomas: 2.09 vs. -0.10, P = 0.06; change in deficit severity score: 13.18 dB vs. -2 dB, P < 0.01 for Group A and S, respectively). The number of microscotomas and also severity were increased in 63.6% of Group A patients and in only 20% of Group S patients. Microscotomas were most frequently located in IEMM and/or ILM areas. Internal limiting membrane peeling has progressively become generalized in IEMM surgery to reduce recurrences. This additional procedure does not change the postoperative visual acuity but increases the development of deeper microscotomas. The real impact on the quality of vision remains unclear. Active ILM peeling in IEMM surgery may be responsible for visual impairment related to its microtraumatic effects.

Omniphobic Membrane for Robust Membrane Distillation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, SH; Nejati, S; Boo, C

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 membranemore » 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.« less

Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes*

PubMed Central

Tietz, Stefanie; Puthiyaveetil, Sujith; Enlow, Heather M.; Yarbrough, Robert; Wood, Magnus; Semchonok, Dmitry A.; Lowry, Troy; Li, Zhirong; Jahns, Peter; Boekema, Egbert J.; Lenhert, Steven; Niyogi, Krishna K.; Kirchhoff, Helmut

2015-01-01

The structural organization of proteins in biological membranes can affect their function. Photosynthetic thylakoid membranes in chloroplasts have the remarkable ability to change their supramolecular organization between disordered and semicrystalline states. Although the change to the semicrystalline state is known to be triggered by abiotic factors, the functional significance of this protein organization has not yet been understood. Taking advantage of an Arabidopsis thaliana fatty acid desaturase mutant (fad5) that constitutively forms semicrystalline arrays, we systematically test the functional implications of protein crystals in photosynthetic membranes. Here, we show that the change into an ordered state facilitates molecular diffusion of photosynthetic components in crowded thylakoid membranes. The increased mobility of small lipophilic molecules like plastoquinone and xanthophylls has implications for diffusion-dependent electron transport and photoprotective energy-dependent quenching. The mobility of the large photosystem II supercomplexes, however, is impaired, leading to retarded repair of damaged proteins. Our results demonstrate that supramolecular changes into more ordered states have differing impacts on photosynthesis that favor either diffusion-dependent electron transport and photoprotection or protein repair processes, thus fine-tuning the photosynthetic energy conversion. PMID:25897076

Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers.

PubMed

Fusco, Giuliana; Chen, Serene W; Williamson, Philip T F; Cascella, Roberta; Perni, Michele; Jarvis, James A; Cecchi, Cristina; Vendruscolo, Michele; Chiti, Fabrizio; Cremades, Nunilo; Ying, Liming; Dobson, Christopher M; De Simone, Alfonso

2017-12-15

Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Biopores/membrane proteins in synthetic polymer membranes.

PubMed

Garni, Martina; Thamboo, Sagana; Schoenenberger, Cora-Ann; Palivan, Cornelia G

2017-04-01

Mimicking cell membranes by simple models based on the reconstitution of membrane proteins in lipid bilayers represents a straightforward approach to understand biological function of these proteins. This biomimetic strategy has been extended to synthetic membranes that have advantages in terms of chemical and mechanical stability, thus providing more robust hybrid membranes. We present here how membrane proteins and biopores have been inserted both in the membrane of nanosized and microsized compartments, and in planar membranes under various conditions. Such bio-hybrid membranes have new properties (as for example, permeability to ions/molecules), and functionality depending on the specificity of the inserted biomolecules. Interestingly, membrane proteins can be functionally inserted in synthetic membranes provided these have appropriate properties to overcome the high hydrophobic mismatch between the size of the biomolecule and the membrane thickness. Functional insertion of membrane proteins and biopores in synthetic membranes of compartments or in planar membranes is possible by an appropriate selection of the amphiphilic copolymers, and conditions of the self-assembly process. These hybrid membranes have new properties and functionality based on the specificity of the biomolecules and the nature of the synthetic membranes. Bio-hybrid membranes represent new solutions for the development of nanoreactors, artificial organelles or active surfaces/membranes that, by further gaining in complexity and functionality, will promote translational applications. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. Copyright © 2016. Published by Elsevier B.V.

Vitamin E-coated dialyzer membranes downregulate expression of monocyte adhesion and co-stimulatory molecules.

PubMed

Betjes, Michiel G H; Hoekstra, Franciska M E; Klepper, M; Postma, Saskia M; Vaessen, Leonard M B

2004-01-01

In patients on chronic hemodialysis leukocyte activation has been related to the impaired function of the immune system. In this study we investigated if the vitamin E-coated dialyzer membrane could reduce monocyte activation thereby improving cellular immunity. This hypothesis was tested in a prospective crossover trial in which 14 stable hemodialysis patients were switched from the baseline hemophane dialyzer to a vitamin E-coated and thereafter a polysulphone dialyzer membrane or vice versa. Monocyte MHC class I, CD54 and ICAM-1 expression was significantly downregulated when a vitamin E-coated or polysulphone dialyzer was used. The use of a vitamin E membrane specifically decreased monocyte CD40 and CD86 expression. Lectin induced T cell proliferation increased with the use of the vitamin E-coated membrane as compared to polysulphone and hemophane dialyzers. Vitamin E-coated dialyzers induced a less-activated phenotype of monocytes and may improve cellular immunity.

Outer membrane lipoprotein VacJ is required for the membrane integrity, serum resistance and biofilm formation of Actinobacillus pleuropneumoniae.

PubMed

Xie, Fang; Li, Gang; Zhang, Wanjiang; Zhang, Yanhe; Zhou, Long; Liu, Shuanghong; Liu, Siguo; Wang, Chunlai

2016-02-01

The outer membrane proteins of Actinobacillus pleuropneumoniae are mediators of infection, acting as targets for the host's defense system. The outer membrane lipoprotein VacJ is involved in serum resistance and intercellular spreading in several pathogenic bacteria. To investigate the role of VacJ in the pathogenicity of Actinobacillus pleuropneumoniae, the vacJ gene-deletion mutant MD12 ΔvacJ was constructed. The increased susceptibility to KCl, SDS plus EDTA, and several antibiotics in the MD12ΔvacJ mutant suggested that the stability of the outer membrane was impaired as a result of the mutation in the vacJ gene. The increased NPN fluorescence and significant cellular morphological variation in the MD12ΔvacJ mutant further demonstrated the crucial role of the VacJ lipoprotein in maintaining the outer membrane integrity of A. pleuropneumoniae. In addition, the MD12ΔvacJ mutant exhibited decreased survival from the serum and complement killing compared to the wild-type strain. Interestingly, the MD12ΔvacJ mutant showed reduced biofilm formation compared to the wild-type strain. To our knowledge, this is the first description of the VacJ lipoprotein contributing to bacterial biofilm formation. The data presented in this study illustrate the important role of the VacJ lipoprotein in the maintenance of cellular integrity, serum resistance, and biofilm formation in A. pleuropneumoniae. Copyright © 2015 Elsevier B.V. All rights reserved.

Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

PubMed

Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

2017-08-01

While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Enterobacter gergoviae membrane modifications are involved in the adaptive response to preservatives used in cosmetic industry.

PubMed

Périamé, Marina; Pagès, Jean-Marie; Davin-Regli, Anne

2015-01-01

The objective of this study was to understand the adaptive mechanisms in Enterobacter gergoviae which are involved in recurrent contaminations in cosmetic products that are incorporated with preservatives. Bacterial strains from two backgrounds were examined for a profound understanding of the mechanisms of adaptation against preservatives. It included a series of Ent. gergoviae strain-ATCC 33028 derivatives, isolated using increasing methylisothiazolinone-chloromethylisothiazolinone (MIT-CMIT) and triclosan concentrations. The other series was of Ent. gergoviae isolates from cosmetic products exhibiting MIT-CMIT and triclosan resistance. We evaluated the outer membrane protein modifications and efflux mechanisms activities responsible for the resistant trait via immunoblotting assays. Additionally, for understanding the efflux activity real-time efflux, experiments were performed. A cross-insusceptibility between preservatives and some disinfectants was observed in MIT-CMIT-resistant derivative isolates, but antibiotics susceptibility was not altered. Resistance to EDTA was significant in all preservatives insusceptible derivative strains, indicating modifications in the LPS layer. Furthermore, an array of real-time efflux assays indicated different activity levels while no variations were detected in porins and AcrAB-TolC pumps production. Overexpression of a specific flagellin-type protein was observed in one of the MIT-CMIT- and triclosan-resistant strains. Another candidate, a 25-kDa peroxiredoxin enzyme involved in oxidative detoxification, was identified to be overexpressed in MIT-CMIT derivative. A similar profile was also observed among strains isolated from cosmetic products. Our study highlights the existence of adaptive mechanisms such as overexpression of detoxifying enzymes, flagellin, modification of membrane structure/function in Ent. gergoviae. They might be involved in recurrent episodes of contaminations occurring in the cosmetic production

Creatine salts provide neuroprotection even after partial impairment of the creatine transporter.

PubMed

Adriano, E; Garbati, P; Salis, A; Damonte, G; Millo, E; Balestrino, M

2017-01-06

Creatine, a compound that is critical for energy metabolism of nervous cells, crosses the blood-brain barrier (BBB) and the neuronal plasma membrane with difficulty, and only using its specific transporter. In the hereditary condition where the creatine transporter is defective (creatine transporter deficiency) there is no creatine in the brain, and administration of creatine is useless lacking the transporter. The disease is severe and incurable. Creatine-derived molecules that could cross BBB and plasma membrane independently of the transporter might be useful to cure this condition. Moreover, such molecules could be useful also in stroke and other brain ischemic conditions. In this paper, we investigated three creatine salts, creatine ascorbate, creatine gluconate and creatine glucose. Of these, creatine glucose was ineffective after transporter block with guanidine acetic acid (GPA) administration. Creatine ascorbate was not superior to creatine in increasing tissue creatine and phosphocreatine content after transporter impairment, however even after such impairment it delayed synaptic failure during anoxia. Finally, creatine gluconate was superior to creatine in increasing tissue content of creatine after transporter block and slowed down PS disappearance during anoxia, an effect that creatine did not have. These findings suggest that coupling creatine to molecules having a specific transporter may be a useful strategy in creatine transporter deficiency. In particular, creatine ascorbate has effects comparable to those of creatine in normal conditions, while being superior to it under conditions of missing or impaired creatine transporter. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Arsenic impairs insulin signaling in differentiated neuroblastoma SH-SY5Y cells.

PubMed

Niyomchan, Apichaya; Visitnonthachai, Daranee; Suntararuks, Sumitra; Ngamsiri, Pronrumpa; Watcharasit, Piyajit; Satayavivad, Jutamaad

2018-05-01

A strong correlation between chronic arsenic exposure and neuropsychological disorders leads to a growing concern about a potential risk of arsenic related neurodegeneration. Evidently, brain insulin signaling contributes to physiological effects, including energy homeostasis, and learning and memory. Arsenic has been shown to impair insulin signaling in adipocytes and myocytes, however, this impairment has not yet been explored in neurons. Here we showed that NaAsO 2 caused significant reduction in basal levels of glucose, plasma membrane glucose transporter, GLUT 3 and Akt phosphorylation in differentiated human neuroblastoma SH-SY5Y cells. NaAsO 2 significantly decreased insulin-mediated glucose uptake, as well as GLUT1 and 3 membrane translocation. Furthermore, the ability of insulin to increase Akt phosphorylation, a well-recognized insulin signaling response, was significantly lessened by NaAsO 2 treatment. In addition, the classical tyrosine phosphorylation response of insulin was reduced by NaAsO 2 , as evidenced by reduction of insulin-induced tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1(IRS-1). Moreover, NaAsO 2 lowered the ratio of p110, a catalytic subunit to p85, a regulatory subunit of PI3K causing an imbalance between p110 and p85, the conditions reported to contribute to insulin sensitivity. Additionally, increment of IRS-1 interaction with GSK3β, and p85-PI3K were observed in NaAsO 2 treated cells. These molecular modulations may be mechanistically attributed to neuronal insulin signaling impairment by arsenic. Copyright © 2018 Elsevier B.V. All rights reserved.

Receptor clustering affects signal transduction at the membrane level in the reaction-limited regime

NASA Astrophysics Data System (ADS)

Caré, Bertrand R.; Soula, Hédi A.

2013-01-01

Many types of membrane receptors are found to be organized as clusters on the cell surface. We investigate the potential effect of such receptor clustering on the intracellular signal transduction stage. We consider a canonical pathway with a membrane receptor (R) activating a membrane-bound intracellular relay protein (G). We use Monte Carlo simulations to recreate biochemical reactions using different receptor spatial distributions and explore the dynamics of the signal transduction. Results show that activation of G by R is severely impaired by R clustering, leading to an apparent blunted biological effect compared to control. Paradoxically, this clustering decreases the half maximal effective dose (ED50) of the transduction stage, increasing the apparent affinity. We study an example of inter-receptor interaction in order to account for possible compensatory effects of clustering and observe the parameter range in which such interactions slightly counterbalance the loss of activation of G. The membrane receptors’ spatial distribution affects the internal stages of signal amplification, suggesting a functional role for membrane domains and receptor clustering independently of proximity-induced receptor-receptor interactions.

Membranes, methods of making membranes, and methods of separating gases using membranes

DOEpatents

Ho, W. S. Winston

2012-10-02

Membranes, methods of making membranes, and methods of separating gases using membranes are provided. The membranes can include at least one hydrophilic polymer, at least one cross-linking agent, at least one base, and at least one amino compound. The methods of separating gases using membranes can include contacting a gas stream containing at least one of CO.sub.2, H.sub.2S, and HCl with one side of a nonporous and at least one of CO.sub.2, H.sub.2S, and HCl selectively permeable membrane such that at least one of CO.sub.2, H.sub.2S, and HCl is selectively transported through the membrane.

WAVE2 forms a complex with PKA and is involved in PKA enhancement of membrane protrusions.

PubMed

Yamashita, Hiroshi; Ueda, Kazumitsu; Kioka, Noriyuki

2011-02-04

PKA contributes to many physiological processes, including glucose homeostasis and cell migration. The substrate specificity of PKA is low compared with other kinases; thus, complex formation with A-kinase-anchoring proteins is important for the localization of PKA in specific subcellular regions and the phosphorylation of specific substrates. Here, we show that PKA forms a complex with WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) in MDA-MB-231 breast cancer cells and mouse brain extracts. Two separate regions of WAVE2 are involved in WAVE2-PKA complex formation. This complex localizes to the leading edge of MDA-MB-231 cells. PKA activation results in enlargement of the membrane protrusion. WAVE2 depletion impairs PKA localization at membrane protrusions and the enlargement of membrane protrusion induced by PKA activation. Together, these results suggest that WAVE2 works as an A-kinase-anchoring protein that recruits PKA at membrane protrusions and plays a role in the enlargement of membrane protrusions induced by PKA activation.

Immunochemical and biological characterization of outer membrane proteins of Porphyromonas endodontalis.

PubMed Central

Ogawa, T; Kuribayashi, S; Shimauchi, H; Toda, T; Hamada, S

1992-01-01

Outer membrane proteins (OMP) of Porphyromonas endodontalis HG 370 (ATCC 35406) were prepared from the cell envelope fraction of the organisms. The cell envelope that had been obtained by sonication of the whole cells was extracted in 2% lithium dodecyl sulfate and then successively chromatographed with Sephacryl S-200 HR and DEAE-Sepharose Fast Flow. Two OMP fractions, OMP-I and OMP-II, were obtained, and their immunochemical properties and induction of specific antibodies were examined. The OMP-I preparation consisted of a major protein with an apparent molecular mass of 31 kDa and other moderate to minor proteins of 40.3, 51.4, 67, and 71.6 kDa, while the OMP-II preparation contained 14-, 15.5-, 27-, and 44-kDa proteins as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. OMP-I was found to form hydrophilic diffusion pores by incorporation into artificial liposomes composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that OMP-I exhibited significant porin activity. However, the liposomes containing heat-denatured OMP-I were scarcely active. Spontaneous and antigen-specific immunoglobulin M (IgM)-, IgG-, and IgA-secreting spot-forming cells (SFC) enzymatically dissociated into single-cell suspensions from chronically inflamed periapical tissues and were enumerated by enzyme-linked immunospot assay. In patients with radicular cysts or dental granulomas, the major isotype of spontaneous SFC was IgG. In radicular cysts, the OMP-II-specific IgG SFC represented 0.13% of the total IgG SFC, while the antigen-specific IgA or IgM SFC was not observed. It was also found that none of these mononuclear cells produced antibodies specific for OMP-I or lipopolysaccharide of P. endodontalis. Images PMID:1328059

[Effect of Membrane Wettability on Membrane Fouling and Chemical Durability of SPG Membranes].

PubMed

Zhang, Jing; Xiao, Tai-min; Zhang, Jing; Cao, Li-ya; Du, Ya-wei; Liu, Chun; Zhang, Lei

2015-05-01

Shirasu porous glass (SPG) membranes have been applied for microbubble aeration in aerobic wastewater treatment. In the present study, both hydrophilic and hydrophobic SPG membranes were used in a microbubble-aerated biofilm reactor with online chemical cleaning, and their membrane fouling and chemical durability were determined to be strongly dependent on the membrane wettability. The fouling layer formed on the surface of both membranes was confirmed to be mainly organic fouling, and the hydrophobic membrane showed a relatively stronger resistance to the organic fouling. The severe chemical corrosion of the hydrophilic membrane was observed due to exposure to the alkaline sodium hypochlorite solution used for chemical cleaning, which resulted in significant increases in the median pore diameter and the porosity. On the other hand, the pore structure of the hydrophobic membrane changed slightly when exposed to the alkaline sodium hypochlorite solution, suggesting its strong alkali-resistance due to the non-wetting surface. However, the surface hydrophobic groups of hydrophobic membrane could be oxidized by sodium hypochlorite solution, resulting in more wettable membrane surface. The hydrophobic membrane also showed better performance in the respects of oxygen transfer, contaminant removal and energy-saving. Therefore, the hydrophobic membrane seemed more appropriate to be applied for microbubble aeration in aerobic wastewater treatment process.

[Isoflavone genistein-8-c-glycoside prevents the oxidative damages in structure and function of rat liver microsomal membranes].

PubMed

Zavodnik, L B

2003-01-01

Bioflavonoids (polyhydroxyphenols) are ubiquitous components of plants, fruits and vegetables; these compounds are efficient scavengers of free oxygen radicals and peroxides. The aim of this study was to investigate the antioxidant and radioprotective effects of genistein-8-C-glicoside (G8CG), an isoflavone, isolated from the flowers of Lipinus luteusl L. G8CG prevents dose-dependently the destruction of the cytochrome P-450 and its conversion to an inactive form cytochrome P-420, inhibits membrane lipid peroxidation and membrane SH-group oxidation in isolated rat liver microsomal membranes under tert-butylhydroperoxide-induced oxidative stress. Single whole-body gamma-irradiation (1 Gy) of rats results in blood plasma and liver microsomal membrane lipid peroxidation, impairments of microsomal membrane structure and function. Rat treatment with G8CG (75 mg/kg) developed the clear protective effect, stabilized membrane structure and improved the parameters of the monooxygenase function. We can conclude that G8CG can be used as antioxidant and radioprotective agent.

Distinct membrane properties are differentially influenced by cardiolipin content and acyl chain composition in biomimetic membranes.

PubMed

Pennington, Edward Ross; Fix, Amy; Sullivan, E Madison; Brown, David A; Kennedy, Anthony; Shaikh, Saame Raza

2017-02-01

Cardiolipin (CL) has a critical role in maintaining mitochondrial inner membrane structure. In several conditions such as heart failure and aging, there is loss of CL content and remodeling of CL acyl chains, which are hypothesized to impair mitochondrial inner membrane biophysical organization. Therefore, this study discriminated how CL content and acyl chain composition influenced select properties of simple and complex mitochondrial mimicking model membranes. We focused on monolayer excess area/molecule (a measure of lipid miscibility), bilayer phase transitions, and microdomain organization. In monolayer compression studies, loss of tetralinoleoyl [(18:2) 4 ] CL content decreased the excess area/molecule. Replacement of (18:2) 4 CL acyl chains with tetraoleoyl [(18:1) 4 ] CL or tetradocosahexaenoyl [(22:6) 4 ] CL generally had little influence on monolayer excess area/molecule; in contrast, replacement of (18:2) 4 CL acyl chains with tetramyristoyl [(14:0) 4 ] CL increased monolayer excess area/molecule. In bilayers, calorimetric studies showed that substitution of (18:2) 4 CL with (18:1) 4 CL or (22:6) 4 CL lowered the phase transition temperature of phosphatidylcholine vesicles whereas (14:0) 4 CL had no effect. Finally, quantitative imaging of giant unilamellar vesicles revealed differential effects of CL content and acyl chain composition on microdomain organization, visualized with the fluorescent probe Texas Red DHPE. Notably, microdomain areas were decreased by differing magnitudes upon lowering of (18:2) 4 CL content and substitution of (18:2) 4 CL with (14:0) 4 CL or (22:6) 4 CL. Conversely, exchanging (18:2) 4 CL with (18:1) 4 CL increased microdomain area. Altogether, these data demonstrate that CL content and fatty acyl composition differentially target membrane physical properties, which has implications for understanding how CL regulates mitochondrial activity and the design of CL-specific therapeutics. Copyright © 2016 Elsevier B.V. All rights

Auditory processing efficiency deficits in children with developmental language impairments

NASA Astrophysics Data System (ADS)

Hartley, Douglas E. H.; Moore, David R.

2002-12-01

The ``temporal processing hypothesis'' suggests that individuals with specific language impairments (SLIs) and dyslexia have severe deficits in processing rapidly presented or brief sensory information, both within the auditory and visual domains. This hypothesis has been supported through evidence that language-impaired individuals have excess auditory backward masking. This paper presents an analysis of masking results from several studies in terms of a model of temporal resolution. Results from this modeling suggest that the masking results can be better explained by an ``auditory efficiency'' hypothesis. If impaired or immature listeners have a normal temporal window, but require a higher signal-to-noise level (poor processing efficiency), this hypothesis predicts the observed small deficits in the simultaneous masking task, and the much larger deficits in backward and forward masking tasks amongst those listeners. The difference in performance on these masking tasks is predictable from the compressive nonlinearity of the basilar membrane. The model also correctly predicts that backward masking (i) is more prone to training effects, (ii) has greater inter- and intrasubject variability, and (iii) increases less with masker level than do other masking tasks. These findings provide a new perspective on the mechanisms underlying communication disorders and auditory masking.

Science review: Mechanisms of impaired adrenal function in sepsis and molecular actions of glucocorticoids

PubMed Central

Prigent, Hélène; Maxime, Virginie; Annane, Djillali

2004-01-01

This review describes current knowledge on the mechanisms that underlie glucocorticoid insufficiency in sepsis and the molecular action of glucocorticoids. In patients with severe sepsis, numerous factors predispose to glucocorticoid insufficiency, including drugs, coagulation disorders and inflammatory mediators. These factors may compromise the hypothalamic–pituitary axis (i.e. secondary adrenal insufficiency) or the adrenal glands (i.e. primary adrenal failure), or may impair glucocorticoid access to target cells (i.e. peripheral tissue resistance). Irreversible anatomical damages to the hypothalamus, pituitary, or adrenal glands rarely occur. Conversely, transient functional impairment in hormone synthesis may be a common complication of severe sepsis. Glucocorticoids interact with a specific cytosolic glucocorticoid receptor, which undergoes conformational changes, sheds heat shock proteins and translocates to the nucleus. Glucocorticoids may also interact with membrane binding sites at the surface of the cells. The molecular action of glucocorticoids results in genomic and nongenomic effects. Direct and indirect transcriptional and post-transcriptional effects related to the cytosolic glucocorticoid receptor account for the genomic effects. Nongenomic effects are probably subsequent to cytosolic interaction between the glucocorticoid receptor and proteins, or to interaction between glucocorticoids and specific membrane binding sites. PMID:15312206

Influence of doxorubicin on model cell membrane properties: insights from in vitro and in silico studies.

PubMed

Alves, Ana Catarina; Magarkar, Aniket; Horta, Miguel; Lima, Jose L F C; Bunker, Alex; Nunes, Cláudia; Reis, Salette

2017-07-24

Despite doxorubicin being commonly used in chemotherapy there still remain significant holes in our knowledge regarding its delivery efficacy and an observed resistance mechanism that is postulated to involve the cell membrane. One possible mechanism is the efflux by protein P-gp, which is found predominantly in cholesterol enriched domains. Thereby, a hypothesis for the vulnerability of doxorubicin to efflux through P-gp is its enhanced affinity for the ordered cholesterol rich regions of the plasma membrane. Thus, we have studied doxorubicin's interaction with model membranes in a cholesterol rich, ordered environment and in liquid-disordered cholesterol poor environment. We have combined three separate experimental protocols: UV-Vis spectrophotometry, fluorescence quenching and steady-state anisotropy and computational molecular dynamics modeling. Our results show that the presence of cholesterol induces a change in membrane structure and doesn't impair doxorubicin's membrane partitioning, but reduces drug's influence on membrane fluidity without directly interacting with it. It is thus possible that the resistance mechanism that lowers the efficacy of doxorubicin, results from an increased density in membrane regions where the efflux proteins are present. This work represents a successful approach, combining experimental and computational studies of membrane based systems to unveil the behavior of drugs and candidate drug molecules.

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts.

PubMed

Kasahara, Kohji; Kaneda, Mizuho; Miki, Toshiaki; Iida, Kazuko; Sekino-Suzuki, Naoko; Kawashima, Ikuo; Suzuki, Hidenori; Shimonaka, Motoyuki; Arai, Morio; Ohno-Iwashita, Yoshiko; Kojima, Soichi; Abe, Mitsuhiro; Kobayashi, Toshihide; Okazaki, Toshiro; Souri, Masayoshi; Ichinose, Akitada; Yamamoto, Naomasa

2013-11-07

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

The effect of acute microgravity on mechanically-induced membrane damage and membrane-membrane fusion events

NASA Technical Reports Server (NTRS)

Clarke, M. S.; Vanderburg, C. R.; Feeback, D. L.; McIntire, L. V. (Principal Investigator)

2001-01-01

Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". Both membrane rupture and membrane resealing events mediated by membrane-membrane fusion characterize this response. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

The Effect of Acute Microgravity on Mechanically-Induced Membrane Damage and Membrane-Membrane Fusion Events

NASA Technical Reports Server (NTRS)

Clarke, Mark, S. F.; Vanderburg, Charles R.; Feedback, Daniel L.

2001-01-01

Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". This response is characterized by both membrane rupture and membrane resealing events mediated by membrane-membrane fusion. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

Disulfide Bond Formation and ToxR Activity in Vibrio cholerae

PubMed Central

Fengler, Vera H. I.; Boritsch, Eva C.; Tutz, Sarah; Seper, Andrea; Ebner, Hanna; Roier, Sandro; Schild, Stefan; Reidl, Joachim

2012-01-01

Virulence factor production in Vibrio cholerae is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation. PMID:23144706

Protective effects of physical exercise on MDMA-induced cognitive and mitochondrial impairment.

PubMed

Taghizadeh, Ghorban; Pourahmad, Jalal; Mehdizadeh, Hajar; Foroumadi, Alireza; Torkaman-Boutorabi, Anahita; Hassani, Shokoufeh; Naserzadeh, Parvaneh; Shariatmadari, Reyhaneh; Gholami, Mahdi; Rouini, Mohammad Reza; Sharifzadeh, Mohammad

2016-10-01

Debate continues about the effect of 3, 4-methylenedioxymethamphetamine (MDMA) on cognitive and mitochondrial function through the CNS. It has been shown that physical exercise has an important protective effect on cellular damage and death. Therefore, we investigated the effect of physical exercise on MDMA-induced impairments of spatial learning and memory as well as MDMA effects on brain mitochondrial function in rats. Male wistar rats underwent short-term (2 weeks) or long-term (4 weeks) treadmill exercise. After completion of exercise duration, acquisition and retention of spatial memory were evaluated by Morris water maze (MWM) test. Rats were intraperitoneally (I.P) injected with MDMA (5, 10, and 15mg/kg) 30min before the first training trial in 4 training days of MWM. Different parameters of brain mitochondrial function were measured including the level of ROS production, mitochondrial membrane potential (MMP), mitochondrial swelling, mitochondrial outermembrane damage, the amount of cytochrome c release from the mitochondria, and ADP/ATP ratio. MDMA damaged the spatial learning and memory in a dose-dependent manner. Brain mitochondria isolated from the rats treated with MDMA showed significant increase in ROS formation, collapse of MMP, mitochondrial swelling, and outer membrane damage, cytochrome c release from the mitochondria, and finally increased ADP/ATP ratio. This study also found that physical exercise significantly decreased the MDMA-induced impairments of spatial learning and memory and also mitochondrial dysfunction. The results indicated that MDMA-induced neurotoxicity leads to brain mitochondrial dysfunction and subsequent oxidative stress is followed by cognitive impairments. However, physical exercise could reduce these deleterious effects of MDMA through protective effects on brain mitochondrial function. Copyright © 2016 Elsevier Inc. All rights reserved.

Coexistence of Anti-Glomerular Basement Membrane Glomerulonephritis and Membranous Nephropathy in a Female Patient with Preserved Renal Function.

PubMed

Ogawara, Aoi; Harada, Makoto; Ichikawa, Tohru; Fujii, Kazuaki; Ehara, Takashi; Kobayashi, Mamoru

2017-12-01

Renal prognosis for anti-glomerular basement membrane (GBM) glomerulonephritis is poor. The greater the amount of anti-GBM antibody binding the antigen (type IV collagen of the glomerular basement membrane), the greater the number of crescents that develop in glomeruli, resulting in progression of renal impairment. Immunofluorescence staining reveals linear IgG depositions on glomerular capillary walls. Membranous nephropathy (MN) is one of the most common causes of nephrotic syndrome in middle-aged to elderly patients. Immune complex is deposited in the sub-epithelial space of the glomerulus resulting in the development of a membranous lesion. Immunofluorescence staining reveals granular IgG depositions on glomerular capillary walls. Coexisting anti-GBM glomerulonephritis and MN are rare and, here we report a case of coexisting anti-GBM glomerulonephritis and MN with preserved renal function. There are some cases of coexisting anti-GBM glomerulonephritis and MN do not show severely decreased renal function. A 76-year-old Japanese woman presented with nephrotic syndrome, microscopic hematuria, and was positive for anti-GBM antibody. Kidney biopsy revealed linear and granular IgG depositions in glomerular capillary walls, crescent formations, and electron-dense deposits in the sub-epithelial space. She was diagnosed with anti-GBM glomerulonephritis and MN. Steroid and cyclosporine therapy achieved complete remission, and kidney function was preserved. In conclusion, coexisting anti-GBM glomerulonephritis and MN can have preserved renal function. IgG subclass of deposited anti-GBM antibody may be associated with the severity of anti-GBM glomerulonephritis. In addition, in the case of nephrotic syndrome with hematuria, we should consider the possibility of coexisting anti-GBM glomerulonephritis and MN.

Parylene as a new membrane material for BioMEMS applications

NASA Astrophysics Data System (ADS)

Lu, Bo

The work in this thesis aims to use MEMS and microfabrication technologies to develop two types of parylene membrane devices for biomedical applications. The first device is the parylene membrane filter for cancer detection. The presence of circulating tumor cells (CTC) in patient blood is an important sign of cancer metastasis. However, currently there are two big challenges for CTC detection. First, CTCs are extremely rare, especially at the early stage of cancer metastasis. Secondly, CTCs are very fragile, and are very likely to be damaged during the capturing process. By using size-based membrane filtration through the specially designed parylene filters, together with a constant-pressure filtration system, we are able to capture the CTCs from patient blood with high capture efficiency, high viability, moderate enrichment, and high throughput. Both immunofluorescence enumeration and telomerase activity detection have been used to detect and differentiate the captured CTCs. The feasibility of further cell culture of the captured CTCs has also been demonstrated, which could be a useful way to increase the number of CTCs for future studies. Models of the time-dependent cell membrane damage are developed to predict and prevent CTC damage during this detection process. The results of clinical trials further demonstrate that the parylene membrane filter is a promising device for cancer detection. The second device is the parylene artificial Bruch's membrane for age-related macular degeneration (AMD). AMD is usually characterized by an impaired Bruch's membrane with much lowered permeability, which impedes the transportation of nutrients from choroid vessels to nourish the retinal pigment epithelial (RPE) cells and photoreceptors. Parylene is selected as a substitute material because of its good mechanical properties, transparency, biocompatibility, and machinability. More importantly, it is found that the permeability of submicron parylene is very similar to that of

Nanofiber Ion-Exchange Membranes for the Rapid Uptake and Recovery of Heavy Metals from Water

PubMed Central

Chitpong, Nithinart; Husson, Scott M.

2016-01-01

An evaluation of the performance of polyelectrolyte-modified nanofiber membranes was undertaken to determine their efficacy in the rapid uptake and recovery of heavy metals from impaired waters. The membranes were prepared by grafting poly(acrylic acid) (PAA) and poly(itaconic acid) (PIA) to cellulose nanofiber mats. Performance measurements quantified the dynamic ion-exchange capacity for cadmium (Cd), productivity, and recovery of Cd(II) from the membranes by regeneration. The dynamic binding capacities of Cd(II) on both types of nanofiber membrane were independent of the linear flow velocity, with a residence time of as low as 2 s. Analysis of breakthrough curves indicated that the mass flow rate increased rapidly at constant applied pressure after membranes approached equilibrium load capacity for Cd(II), apparently due to a collapse of the polymer chains on the membrane surface, leading to an increased porosity. This mechanism is supported by hydrodynamic radius (Rh) measurements for PAA and PIA obtained from dynamic light scattering, which show that Rh values decrease upon Cd(II) binding. Volumetric productivity was high for the nanofiber membranes, and reached 0.55 mg Cd/g/min. The use of ethylenediaminetetraacetic acid as regeneration reagent was effective in fully recovering Cd(II) from the membranes. Ion-exchange capacities were constant over five cycles of binding-regeneration. PMID:27999394

Nanofiber Ion-Exchange Membranes for the Rapid Uptake and Recovery of Heavy Metals from Water.

PubMed

Chitpong, Nithinart; Husson, Scott M

2016-12-20

An evaluation of the performance of polyelectrolyte-modified nanofiber membranes was undertaken to determine their efficacy in the rapid uptake and recovery of heavy metals from impaired waters. The membranes were prepared by grafting poly(acrylic acid) (PAA) and poly(itaconic acid) (PIA) to cellulose nanofiber mats. Performance measurements quantified the dynamic ion-exchange capacity for cadmium (Cd), productivity, and recovery of Cd(II) from the membranes by regeneration. The dynamic binding capacities of Cd(II) on both types of nanofiber membrane were independent of the linear flow velocity, with a residence time of as low as 2 s. Analysis of breakthrough curves indicated that the mass flow rate increased rapidly at constant applied pressure after membranes approached equilibrium load capacity for Cd(II), apparently due to a collapse of the polymer chains on the membrane surface, leading to an increased porosity. This mechanism is supported by hydrodynamic radius (R h ) measurements for PAA and PIA obtained from dynamic light scattering, which show that R h values decrease upon Cd(II) binding. Volumetric productivity was high for the nanofiber membranes, and reached 0.55 mg Cd/g/min. The use of ethylenediaminetetraacetic acid as regeneration reagent was effective in fully recovering Cd(II) from the membranes. Ion-exchange capacities were constant over five cycles of binding-regeneration.

Cubozoan Venom-Induced Cardiovascular Collapse Is Caused by Hyperkalemia and Prevented by Zinc Gluconate in Mice

PubMed Central

Yanagihara, Angel A.; Shohet, Ralph V.

2012-01-01

Chironex fleckeri (Australian box jellyfish) stings can cause acute cardiovascular collapse and death. We developed methods to recover venom with high specific activity, and evaluated the effects of both total venom and constituent porins at doses equivalent to lethal envenomation. Marked potassium release occurred within 5 min and hemolysis within 20 min in human red blood cells (RBC) exposed to venom or purified venom porin. Electron microscopy revealed abundant ∼12-nm transmembrane pores in RBC exposed to purified venom porins. C57BL/6 mice injected with venom showed rapid decline in ejection fraction with progression to electromechanical dissociation and electrocardiographic findings consistent with acute hyperkalemia. Recognizing that porin assembly can be inhibited by zinc, we found that zinc gluconate inhibited potassium efflux from RBC exposed to total venom or purified porin, and prolonged survival time in mice following venom injection. These findings suggest that hyperkalemia is the critical event following Chironex fleckeri envenomation and that rapid administration of zinc could be life saving in human sting victims. PMID:23251508

Neurologic Outcomes After Extracorporeal Membrane Oxygenation-A Systematic Review.

PubMed

Boyle, Katharine; Felling, Ryan; Yiu, Alvin; Battarjee, Wejdan; Schwartz, Jamie McElrath; Salorio, Cynthia; Bembea, Melania M

2018-06-11

The goal of this systematic review of the literature was to summarize neurologic outcomes following neonatal and pediatric extracorporeal membrane oxygenation. We conducted electronic searches of PubMed, Scopus, Web of Science, CINAHL, Cochrane, and EMBASE. Inclusion criteria included publication dates 2000-2016, patient ages 0-18 years, and use of standardized measures to evaluate outcomes after extracorporeal membrane oxygenation. We identified 3,497 unique citations; 60 full-text articles were included in the final review. Studies evaluated patients with congenital diaphragmatic hernia (7), cardiac disease (8), cardiac arrest (13), and mixed populations (32). Follow-up was conducted at hospital discharge in 10 studies (17%) and at a median of 26 months (interquartile range, 8-61 mo) after extracorporeal membrane oxygenation in 50 studies (83%). We found 55 outcome measures that assessed overall health and function (4), global cognitive ability (7), development (4), motor function (5), adaptive function (2), behavior/mood (6), hearing (2), quality of life (2), school achievement (5), speech and language (6), learning and memory (4), and attention and executive function (8). Overall, 10% to as many as 50% of children scored more than 2 SDS below the population mean on cognitive testing. Behavior problems were identified in 16-46% of children tested, and severe motor impairment was reported in 12% of children. Quality of life of former extracorporeal membrane oxygenation patients evaluated at school age or adolescence ranged from similar to healthy peers, to 31-53% having scores more than 1 SD below the population mean. This systematic review of the literature suggests that children who have undergone extracorporeal membrane oxygenation suffer from a wide range of disabilities. A meta-analysis was not feasible due to heterogeneity in pathologies, outcome measures, and age at follow-up, underscoring the importance of developing and employing a core set of outcomes

Sphingomyelin-induced inhibition of the plasma membrane calcium ATPase causes neurodegeneration in type A Niemann-Pick disease.

PubMed

Pérez-Cañamás, A; Benvegnù, S; Rueda, C B; Rábano, A; Satrústegui, J; Ledesma, M D

2017-05-01

Niemann-Pick disease type A (NPA) is a rare lysosomal storage disorder characterized by severe neurological alterations that leads to death in childhood. Loss-of-function mutations in the acid sphingomyelinase (ASM) gene cause NPA, and result in the accumulation of sphingomyelin (SM) in lysosomes and plasma membrane of neurons. Using ASM knockout (ASMko) mice as a NPA disease model, we investigated how high SM levels contribute to neural pathology in NPA. We found high levels of oxidative stress both in neurons from these mice and a NPA patient. Impaired activity of the plasma membrane calcium ATPase (PMCA) increases intracellular calcium. SM induces PMCA decreased activity, which causes oxidative stress. Incubating ASMko-cultured neurons in the histone deacetylase inhibitor, SAHA, restores PMCA activity and calcium homeostasis and, consequently, reduces the increased levels of oxidative stress. No recovery occurs when PMCA activity is pharmacologically impaired or genetically inhibited in vitro. Oral administration of SAHA prevents oxidative stress and neurodegeneration, and improves behavioral performance in ASMko mice. These results demonstrate a critical role for plasma membrane SM in neuronal calcium regulation. Thus, we identify changes in PMCA-triggered calcium homeostasis as an upstream mediator for NPA pathology. These findings can stimulate new approaches for pharmacological remediation in a disease with no current clinical treatments.

Energy transduction and signal averaging of fluctuating electric fields by a single protein ion channel.

PubMed

Verdia-Baguena, C; Gomez, V; Cervera, J; Ramirez, P; Mafe, S

2016-12-21

We demonstrate the electrical rectification and signal averaging of fluctuating signals using a biological nanostructure in aqueous solution: a single protein ion channel inserted in the lipid bilayer characteristic of cell membranes. The conversion of oscillating, zero time-average potentials into directional currents permits charging of a load capacitor to significant steady-state voltages within a few minutes in the case of the outer membrane porin F (OmpF) protein, a bacterial channel of Escherichia coli. The experiments and simulations show signal averaging effects at a more fundamental level than the traditional cell and tissue scales, which are characterized by ensembles of many ion channels operating simultaneously. The results also suggest signal transduction schemes with bio-electronic interfaces and ionic circuits where soft matter nanodiodes can be coupled to conventional electronic elements.

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.

Limb regeneration is impaired in an adult zebrafish model of diabetes mellitus.

PubMed

Olsen, Ansgar S; Sarras, Michael P; Intine, Robert V

2010-01-01

The zebrafish (Danio rerio) is an established model organism for the study of developmental processes, human disease, and tissue regeneration. We report that limb regeneration is severely impaired in our newly developed adult zebrafish model of type I diabetes mellitus. Intraperitoneal streptozocin injection of adult, wild-type zebrafish results in a sustained hyperglycemic state as determined by elevated fasting blood glucose values and increased glycation of serum protein. Serum insulin levels are also decreased and pancreas immunohistochemisty revealed a decreased amount of insulin signal in hyperglycemic fish. Additionally, the diabetic complications of retinal thinning and glomerular basement membrane thickening (early signs of retinopathy and nephropathy) resulting from the hyperglycemic state were evident in streptozocin-injected fish at 3 weeks. Most significantly, limb regeneration, following caudal fin amputation, is severely impaired in diabetic zebrafish and nonspecific toxic effects outside the pancreas were not found to contribute to impaired limb regeneration. This experimental system using adult zebrafish facilitates a broad spectrum of genetic and molecular approaches to study regeneration in the diabetic background. © 2010 by the Wound Healing Society.

Amphoteric Ion-Exchange Membranes with Significantly Improved Vanadium Barrier Properties for All-Vanadium Redox Flow Batteries.

PubMed

Nibel, Olga; Rojek, Tomasz; Schmidt, Thomas J; Gubler, Lorenz

2017-07-10

All-vanadium redox flow batteries (VRBs) have attracted considerable interest as promising energy-storage devices that can allow the efficient utilization of renewable energy sources. The membrane, which separates the porous electrodes in a redox flow cell, is one of the key components in VRBs. High rates of crossover of vanadium ions and water through the membrane impair the efficiency and capacity of a VRB. Thus, membranes with low permeation rate of vanadium species and water are required, also characterized by low resistance and stability in the VRB environment. Here, we present a new design concept for amphoteric ion-exchange membranes, based on radiation-induced grafting of vinylpyridine into an ethylene tetrafluoroethylene base film and a two-step functionalization to introduce cationic and anionic exchange sites, respectively. During long-term cycling, redox flow cells containing these membranes showed higher efficiency, less pronounced electrolyte imbalance, and significantly reduced capacity decay compared to the cells with the benchmark material Nafion 117. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

WAVE2 Forms a Complex with PKA and Is Involved in PKA Enhancement of Membrane Protrusions*

PubMed Central

Yamashita, Hiroshi; Ueda, Kazumitsu; Kioka, Noriyuki

2011-01-01

PKA contributes to many physiological processes, including glucose homeostasis and cell migration. The substrate specificity of PKA is low compared with other kinases; thus, complex formation with A-kinase-anchoring proteins is important for the localization of PKA in specific subcellular regions and the phosphorylation of specific substrates. Here, we show that PKA forms a complex with WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) in MDA-MB-231 breast cancer cells and mouse brain extracts. Two separate regions of WAVE2 are involved in WAVE2-PKA complex formation. This complex localizes to the leading edge of MDA-MB-231 cells. PKA activation results in enlargement of the membrane protrusion. WAVE2 depletion impairs PKA localization at membrane protrusions and the enlargement of membrane protrusion induced by PKA activation. Together, these results suggest that WAVE2 works as an A-kinase-anchoring protein that recruits PKA at membrane protrusions and plays a role in the enlargement of membrane protrusions induced by PKA activation. PMID:21119216

Age-Related Sensory Impairments and Risk of Cognitive Impairment

PubMed Central

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

2016-01-01

Background/Objectives To evaluate the associations of sensory impairments with the 10-year risk of cognitive impairment. Previous work has primarily focused on the relationship between a single sensory system and cognition. Design The Epidemiology of Hearing Loss Study (EHLS) is a longitudinal, population-based study of aging in the Beaver Dam, WI community. Baseline examinations were conducted in 1993 and follow-up exams have been conducted every 5 years. Setting General community Participants EHLS members without cognitive impairment at EHLS-2 (1998–2000). There were 1,884 participants (mean age = 66.7 years) with complete EHLS-2 sensory data and follow-up information. Measurements Cognitive impairment was 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 and 4 kHz) of > 25 decibel Hearing Level in either ear. Visual impairment was 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. Results Hearing, visual, and olfactory impairment were independently associated with cognitive impairment risk [Hearing: Hazard Ratio (HR) = 1.90, 95% Confidence Interval (C.I.) = 1.11, 3.26; Vision: HR = 2.05, 95% C.I. = 1.24, 3.38; Olfaction: HR = 3.92, 95% C.I. = 2.45, 6.26]. However, 85% with hearing impairment, 81% with visual impairment, and 76% with olfactory impairment did not develop cognitive impairment during follow-up. Conclusion The relationship between sensory impairment and cognitive impairment was not unique to one sensory system suggesting sensorineural health may be a marker of brain aging. The development of a combined sensorineurocognitive measure may be useful in uncovering mechanisms of healthy brain aging. PMID:27611845

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 = 2.45-6.26)). Nevertheless, 85% of participants with hearing impairment, 81% with visual impairment, and 76% with olfactory impairment did not develop cognitive impairment during follow-up. The relationship between sensory impairment and cognitive impairment was not unique to one sensory system, suggesting that sensorineural health may be a marker of brain aging. The development of a combined sensorineurocognitive measure may be useful in uncovering mechanisms of healthy brain aging. © 2016, Copyright the Authors Journal compilation © 2016, The American Geriatrics Society.

Recent Advances in Pd-Based Membranes for Membrane Reactors.

PubMed

Arratibel Plazaola, Alba; Pacheco Tanaka, David Alfredo; Van Sint Annaland, Martin; Gallucci, Fausto

2017-01-01

Palladium-based membranes for hydrogen separation have been studied by several research groups during the last 40 years. Much effort has been dedicated to improving the hydrogen flux of these membranes employing different alloys, supports, deposition/production techniques, etc. High flux and cheap membranes, yet stable at different operating conditions are required for their exploitation at industrial scale. The integration of membranes in multifunctional reactors (membrane reactors) poses additional demands on the membranes as interactions at different levels between the catalyst and the membrane surface can occur. Particularly, when employing the membranes in fluidized bed reactors, the selective layer should be resistant to or protected against erosion. In this review we will also describe a novel kind of membranes, the pore-filled type membranes prepared by Pacheco Tanaka and coworkers that represent a possible solution to integrate thin selective membranes into membrane reactors while protecting the selective layer. This work is focused on recent advances on metallic supports, materials used as an intermetallic diffusion layer when metallic supports are used and the most recent advances on Pd-based composite membranes. Particular attention is paid to improvements on sulfur resistance of Pd based membranes, resistance to hydrogen embrittlement and stability at high temperature.

Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

PubMed

Vu, Anh T; Wang, Xinying; Wickramasinghe, S Ranil; Yu, Bing; Yuan, Hua; Cong, Hailin; Luo, Yongli; Tang, Jianguo

2015-08-01

Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

PubMed Central

Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

1992-01-01

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the

Biomimetic membranes and methods of making biomimetic membranes

DOEpatents

Rempe, Susan; Brinker, Jeffrey C.; Rogers, David Michael; Jiang, Ying-Bing; Yang, Shaorong

2016-11-08

The present disclosure is directed to biomimetic membranes and methods of manufacturing such membranes that include structural features that mimic the structures of cellular membrane channels and produce membrane designs capable of high selectivity and high permeability or adsorptivity. The membrane structure, material and chemistry can be selected to perform liquid separations, gas separation and capture, ion transport and adsorption for a variety of applications.

Structure of a Type-1 Secretion System ABC Transporter.

PubMed

Morgan, Jacob L W; Acheson, Justin F; Zimmer, Jochen

2017-03-07

Type-1 secretion systems (T1SSs) represent a widespread mode of protein secretion across the cell envelope in Gram-negative bacteria. The T1SS is composed of an inner-membrane ABC transporter, a periplasmic membrane-fusion protein, and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing a conduit for the translocation of unfolded polypeptides. We show that ATP hydrolysis and assembly of the entire T1SS complex is necessary for protein secretion. Furthermore, we present a 3.15-Å crystal structure of AaPrtD, the ABC transporter found in the Aquifex aeolicus T1SS. The structure suggests a substrate entry window just above the transporter's nucleotide binding domains. In addition, highly kinked transmembrane helices, which frame a narrow channel not observed in canonical peptide transporters, are likely involved in substrate translocation. Overall, the AaPrtD structure supports a polypeptide transport mechanism distinct from alternating access. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Novel Membrane-Based Anti-Diabetic Action of Atorvastatin

PubMed Central

Horvath, Emily M.; Tackett, Lixuan; Elmendorf, Jeffrey S.

2008-01-01

We recently found that chromium picolinate (CrPic), a nutritional supplement thought to improve insulin sensitivity in individuals with impaired glucose tolerance, enhances insulin action by lowering plasma membrane (PM) cholesterol. Recent in vivo studies suggest that cholesterol-lowering statin drugs benefit insulin sensitivity in insulin-resistant patients, yet a mechanism is unknown. We report here that atorvastatin (ATV) diminished PM cholesterol by 22% (P<0.05) in 3T3-L1 adipocytes. As documented for CrPic, this small reduction in PM cholesterol enhanced insulin action. Replenishment of cholesterol mitigated the positive effects of ATV on insulin sensitivity. Co-treatment with CrPic and ATV did not amplify the extent of PM cholesterol loss or insulin sensitivity gain. In addition, analyses of insulin signal transduction suggest a non-signaling basis of both therapies. Our data reveal an unappreciated beneficial non-hepatic effect of statin action and highlight a novel mechanistic similarity between two recently recognized therapies of impaired glucose tolerance. PMID:18514061

Advanced Supported Liquid Membranes for Carbon Dioxide Control in Cabin Applications

NASA Technical Reports Server (NTRS)

Wickham, David T.; Gleason, Kevin J.; Engel, Jeffrey R.; Chullen, Cinda

2016-01-01

The development of new, robust, life support systems is critical to NASA's continued progress in space exploration. One vital function is maintaining the carbon dioxide (CO2) concentration in the cabin at levels that do not impair the health or performance of the crew. The carbon dioxide removal assembly (CDRA) is the current CO2 control technology on-board the International Space Station (ISS). Although the CDRA has met the needs of the ISS to date, the repeated cycling of the molecular sieve sorbent causes it to break down into small particles that clog filters or generate dust in the cabin. This reduces reliability and increases maintenance requirements. Another approach that has potential advantages over the current system is a membrane that separates CO2 from air. In this approach, cabin air contacts one side of the membrane while other side of the membrane is maintained at low pressure to create a driving force for CO2 transport across the membrane. In this application, the primary power requirement is for the pump that creates the low pressure and then pumps the CO2 to the oxygen recovery system. For such a membrane to be practical, it must have high CO2 permeation rate and excellent selectivity for CO2 over air. Unfortunately, conventional gas separation membranes do not have adequate CO2 permeability and selectivity to meet the needs of this application. However, the required performance could be obtained with a supported liquid membrane (SLM), which consists of a microporous material filled with a liquid that selectively reacts with CO2 over air. In a recently completed Phase II SBIR project, Reaction Systems, Inc. fabricated an SLM that is very close to meeting permeability and selectivity objectives for use in the Portable Life Support System (PLSS). This paper describes work carried out to evaluate its potential for use in the cabin.

How Membrane-Active Peptides Get into Lipid Membranes.

PubMed

Sani, Marc-Antoine; Separovic, Frances

2016-06-21

The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular

Membrane lipidomics in schizophrenia patients: a correlational study with clinical and cognitive manifestations.

PubMed

Tessier, C; Sweers, K; Frajerman, A; Bergaoui, H; Ferreri, F; Delva, C; Lapidus, N; Lamaziere, A; Roiser, J P; De Hert, M; Nuss, P

2016-10-04

Schizophrenia is a severe mental condition in which several lipid abnormalities-either structural or metabolic-have been described. We tested the hypothesis that an abnormality in membrane lipid composition may contribute to aberrant dopamine signaling, and thereby symptoms and cognitive impairment, in schizophrenia (SCZ) patients. Antipsychotic-medicated and clinically stable SCZ outpatients (n=74) were compared with matched healthy subjects (HC, n=40). A lipidomic analysis was performed in red blood cell (RBC) membranes examining the major phospholipid (PL) classes and their associated fatty acids (FAs). Clinical manifestations were examined using the positive and negative syndrome scale (PANSS). Cognitive function was assessed using the Continuous Performance Test, Salience Attribution Test and Wisconsin Card Sorting Test. Sphingomyelin (SM) percentage was the lipid abnormality most robustly associated with a schizophrenia diagnosis. Two groups of patients were defined. The first group (SCZ c/SM-) is characterized by a low SM membrane content. In this group, all other PL classes, plasmalogen and key polyunsaturated FAs known to be involved in brain function, were significantly modified, identifying a very specific membrane lipid cluster. The second patient group (SCZ c/SM+) was similar to HCs in terms of RBC membrane SM composition. Compared with SCZ c/SM+, SCZ c/SM- patients were characterized by significantly more severe PANSS total, positive, disorganized/cognitive and excited psychopathology. Cognitive performance was also significantly poorer in this subgroup. These data show that a specific RBC membrane lipid cluster is associated with clinical and cognitive manifestations of dopamine dysfunction in schizophrenia patients. We speculate that this membrane lipid abnormality influences presynaptic dopamine signaling.

Alternative energy efficient membrane bioreactor using reciprocating submerged membrane.

PubMed

Ho, J; Smith, S; Roh, H K

2014-01-01

A novel membrane bioreactor (MBR) pilot system, using membrane reciprocation instead of air scouring, was operated at constant high flux and daily fluctuating flux to demonstrate its application under peak and diurnal flow conditions. Low and stable transmembrane pressure was achieved at 40 l/m(2)/h (LMH) by use of repetitive membrane reciprocation. The results reveal that the inertial forces acting on the membrane fibers effectively propel foulants from the membrane surface. Reciprocation of the hollow fiber membrane is beneficial for the constant removal of solids that may build up on the membrane surface and inside the membrane bundle. The membrane reciprocation in the reciprocating MBR pilot consumed less energy than coarse air scouring used in conventional MBR systems. Specific energy consumption for the membrane reciprocation was 0.072 kWh/m(3) permeate produced at 40 LMH flux, which is 75% less than for a conventional air scouring system as reported in literature without consideration of energy consumption for biological aeration (0.29 kWh/m(3)). The daily fluctuating flux test confirmed that the membrane reciprocation is effective to handle fluctuating flux up to 50 LMH. The pilot-scale reciprocating MBR system successfully demonstrated that fouling can be controlled via 0.43 Hz membrane reciprocation with 44 mm or higher amplitude.

Hypocapnic but Not Metabolic Alkalosis Impairs Alveolar Fluid Reabsorption

PubMed Central

Myrianthefs, Pavlos M.; Briva, Arturo; Lecuona, Emilia; Dumasius, Vidas; Rutschman, David H.; Ridge, Karen M.; Baltopoulos, George J.; Sznajder, Jacob Iasha

2005-01-01

Acid-base disturbances, such as metabolic or respiratory alkalosis, are relatively common in critically ill patients. We examined the effects of alkalosis (hypocapnic or metabolic alkalosis) on alveolar fluid reabsorption in the isolated and continuously perfused rat lung model. We found that alveolar fluid reabsorption after 1 hour was impaired by low levels of CO2 partial pressure (PCO2; 10 and 20 mm Hg) independent of pH levels (7.7 or 7.4). In addition, PCO2 higher than 30 mm Hg or metabolic alkalosis did not have an effect on this process. The hypocapnia-mediated decrease of alveolar fluid reabsorption was associated with decreased Na,K-ATPase activity and protein abundance at the basolateral membranes of distal airspaces. The effect of low PCO2 on alveolar fluid reabsorption was reversible because clearance normalized after correcting the PCO2 back to normal levels. These data suggest that hypocapnic but not metabolic alkalosis impairs alveolar fluid reabsorption. Conceivably, correction of hypocapnic alkalosis in critically ill patients may contribute to the normalization of lung ability to clear edema. PMID:15764729

Hypocapnic but not metabolic alkalosis impairs alveolar fluid reabsorption.

PubMed

Myrianthefs, Pavlos M; Briva, Arturo; Lecuona, Emilia; Dumasius, Vidas; Rutschman, David H; Ridge, Karen M; Baltopoulos, George J; Sznajder, Jacob Iasha

2005-06-01

Acid-base disturbances, such as metabolic or respiratory alkalosis, are relatively common in critically ill patients. We examined the effects of alkalosis (hypocapnic or metabolic alkalosis) on alveolar fluid reabsorption in the isolated and continuously perfused rat lung model. We found that alveolar fluid reabsorption after 1 hour was impaired by low levels of CO2 partial pressure (PCO2; 10 and 20 mm Hg) independent of pH levels (7.7 or 7.4). In addition, PCO2 higher than 30 mm Hg or metabolic alkalosis did not have an effect on this process. The hypocapnia-mediated decrease of alveolar fluid reabsorption was associated with decreased Na,K-ATPase activity and protein abundance at the basolateral membranes of distal airspaces. The effect of low PCO2 on alveolar fluid reabsorption was reversible because clearance normalized after correcting the PCO2 back to normal levels. These data suggest that hypocapnic but not metabolic alkalosis impairs alveolar fluid reabsorption. Conceivably, correction of hypocapnic alkalosis in critically ill patients may contribute to the normalization of lung ability to clear edema.

Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts

PubMed Central

Schenkel, Laila C.; Singh, Ratnesh K.; Michel, Vera; Zeisel, Steven H.; da Costa, Kerry-Ann; Johnson, Amy R.; Mudd, Harvey S.; Bakovic, Marica

2015-01-01

Fibroblasts from a patient with postural orthostatic tachycardia syndrome (POTS), who presented with low plasma choline and betaine, were studied to determine the metabolic characteristics of the choline deficiency. Choline is required for the synthesis of the phospholipid phosphatidylcholine (PC) and for betaine, an important osmoregulator. Here, choline transport, lipid homeostasis, and mitochondria function were analyzed in skin fibroblasts from POTS and compared with control cells. The choline transporter-like protein 1/solute carrier 44A1 (CTL1/SLC44A1) and mRNA expression were 2–3 times lower in POTS fibroblasts, and choline uptake was reduced 60% (P < 0.05). Disturbances of membrane homeostasis were observed by reduced ratios between PC:phosphatidylethanolamine and sphingomyelin:cholesterol, as well as by modified phospholipid fatty acid composition. Choline deficiency also impaired mitochondria function, which was observed by a reduction in oxygen consumption, mitochondrial potential, and glycolytic activity. When POTS cells were treated with choline, transporter was up-regulated, and uptake of choline increased, offering an option for patient treatment. The characteristics of the POTS fibroblasts described here represent a first model of choline and CTL1/SLC44A1 deficiency, in which choline transport, membrane homeostasis, and mitochondrial function are impaired.—Schenkel, L. C., Singh, R. K., Michel, V., Zeisel, S. H., da Costa, K.-A., Johnson, A. R., Mudd, H. S., Bakovic, M. Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts. PMID:25466896

Membrane order in the plasma membrane and endocytic recycling compartment.

PubMed

Iaea, David B; Maxfield, Frederick R

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

The effect of activated carbon addition on membrane bioreactor processes for wastewater treatment and reclamation - A critical review.

PubMed

Skouteris, George; Saroj, Devendra; Melidis, Paraschos; Hai, Faisal I; Ouki, Sabèha

2015-06-01

This review concentrates on the effect of activated carbon (AC) addition to membrane bioreactors (MBRs) treating wastewaters. Use of AC-assisted MBRs combines adsorption, biodegradation and membrane filtration. This can lead to advanced removal of recalcitrant pollutants and mitigation of membrane fouling. The relative contribution of adsorption and biodegradation to overall removal achieved by an AC-assisted MBR process can vary, and "biological AC" may not fully develop due to competition of target pollutants with bulk organics in wastewater. Thus periodic replenishment of spent AC is necessary. Sludge retention time (SRT) governs the frequency of spent AC withdrawal and addition of fresh AC, and is an important parameter that significantly influences the performance of AC-assisted MBRs. Of utmost importance is AC dosage because AC overdose may aggravate membrane fouling, increase sludge viscosity, impair mass transfer and reduce sludge dewaterability. Copyright © 2015 Elsevier Ltd. All rights reserved.

Protective Effects of Myricetin on Acute Hypoxia-Induced Exercise Intolerance and Mitochondrial Impairments in Rats

PubMed Central

Zou, Dan; Liu, Peng; Chen, Ka; Xie, Qi; Liang, Xinyu; Bai, Qian; Zhou, Qicheng; Liu, Kai; Zhang, Ting; Zhu, Jundong; Mi, Mantian

2015-01-01

Purpose Exercise tolerance is impaired in hypoxia. The aim of this study was to evaluate the effects of myricetin, a dietary flavonoid compound widely found in fruits and vegetables, on acute hypoxia-induced exercise intolerance in vivo and in vitro. Methods Male rats were administered myricetin or vehicle for 7 days and subsequently spent 24 hours at a barometric pressure equivalent to 5000 m. Exercise capacity was then assessed through the run-to-fatigue procedure, and mitochondrial morphology in skeletal muscle cells was observed by transmission electron microscopy (TEM). The enzymatic activities of electron transfer complexes were analyzed using an enzyme-linked immuno-sorbent assay (ELISA). mtDNA was quantified by real-time-PCR. Mitochondrial membrane potential was measured by JC-1 staining. Protein expression was detected through western blotting, immunohistochemistry, and immunofluorescence. Results Myricetin supplementation significantly prevented the decline of run-to-fatigue time of rats in hypoxia, and attenuated acute hypoxia-induced mitochondrial impairment in skeletal muscle cells in vivo and in vitro by maintaining mitochondrial structure, mtDNA content, mitochondrial membrane potential, and activities of the respiratory chain complexes. Further studies showed that myricetin maintained mitochondrial biogenesis in skeletal muscle cells under hypoxic conditions by up-regulating the expressions of mitochondrial biogenesis-related regluators, in addition, AMP-activated protein kinase(AMPK) plays a crucial role in this process. Conclusions Myricetin may have important applications for improving physical performance under hypoxic environment, which may be attributed to the protective effect against mitochondrial impairment by maintaining mitochondrial biogenesis. PMID:25919288

Comprehensive Analysis of Transport Proteins Encoded Within the Genome of Bdellovibrio bacteriovorus

PubMed Central

Barabote, Ravi D.; Rendulic, Snjezana; Schuster, Stephan C.; Saier, Milton H.

2012-01-01

Bdellovibrio bacteriovorus is a bacterial parasite with an unusual lifestyle. It grows and reproduces in the periplasm of a host prey bacterium. The complete genome sequence of B. bacteriovorus has recently been reported. We have reanalyzed the transport proteins encoded within the B. bacteriovorus genome according to the current content of the transporter classification database (TCDB). A comprehensive analysis is given on the types and numbers of transport systems that B. bacteriovorus has. In this regard, the potential protein secretory capabilities of at least 4 types of inner membrane secretion systems and 5 types for outer membrane secretion are described. Surprisingly, B. bacteriovorus has a disproportionate percentage of cytoplasmic membrane channels and outer membrane porins. It has far more TonB/ExbBD-type systems and MotAB-type systems for energizing outer membrane transport and motility than does E. coli. Analysis of probable substrate specificities of its transporters provides clues to its metabolic preferences. Interesting examples of gene fusions and of potentially overlapping genes were also noted. Our analyses provide a comprehensive, detailed appreciation of the transport capabilities of B. bacteriovorus. They should serve as a guide for functional experimental analyses. PMID:17706914

Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors

PubMed Central

1994-01-01

The apparatus that permits protein translocation across the internal thylakoid membranes of chloroplasts is completely unknown, even though these membranes have been the subject of extensive biochemical analysis. We have used a genetic approach to characterize the translocation of Chlamydomonas cytochrome f, a chloroplast-encoded protein that spans the thylakoid once. Mutations in the hydrophobic core of the cytochrome f signal sequence inhibit the accumulation of cytochrome f, lead to an accumulation of precursor, and impair the ability of Chlamydomonas cells to grow photosynthetically. One hydrophobic core mutant also reduces the accumulation of other thylakoid membrane proteins, but not those that translocate completely across the membrane. These results suggest that the signal sequence of cytochrome f is required and is involved in one of multiple insertion pathways. Suppressors of two signal peptide mutations describe at least two nuclear genes whose products likely describe the translocation apparatus, and selected second-site chloroplast suppressors further define regions of the cytochrome f signal peptide. PMID:8034740

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.

Membrane order in the plasma membrane and endocytic recycling compartment

PubMed Central

Iaea, David B.; Maxfield, Frederick R.

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles. PMID:29125865

Outer membrane protein A of Acinetobacter baumannii induces differentiation of CD4+ T cells toward a Th1 polarizing phenotype through the activation of dendritic cells.

PubMed

Lee, Jun Sik; Lee, Je Chul; Lee, Chang-Min; Jung, In Duk; Jeong, Young-Il; Seong, Eun-Young; Chung, Hae-Young; Park, Yeong-Min

2007-06-30

Acinetobacter baumannii is an increasing hospital-acquired pathogen that causes a various type of infections, but little is known about the protective immune response to this microorganism. Outer membrane protein A of A. baumannii (AbOmpA) is a major porin protein and plays an important role in pathogenesis. We analyzed interaction between AbOmpA and dendritic cells (DCs) to characterize the role of this protein in promoting innate and adaptive immune responses. AbOmpA functionally activates bone marrow-derived DCs by augmenting expression of the surface markers, CD40, CD54, B7 family (CD80 and CD86) and major histocompatibility complex class I and II. AbOmpA induces production of Th1-promoting interleukin-12 from DCs and augments the syngeneic and allogeneic immunostimulatory capacity of DCs. AbOmpA stimulates production of interferon-gamma from T cells in mixed lymphocyte reactions, which suggesting Th1-polarizing capacity. CD4(+) T cells stimulated by AbOmpA-stimulated DCs show a Th1-polarizing cytokine profile. The expression of surface markers on DCs is mediated by both mitogen-activated protein kinases and NF-kappaB pathways. Our findings suggest that AbOmpA induces maturation of DCs and drives Th1 polarization, which are important properties for determining the nature of immune response against A. baumannii.

Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii.

PubMed

Rütgers, Mark; Muranaka, Ligia Segatto; Schulz-Raffelt, Miriam; Thoms, Sylvia; Schurig, Juliane; Willmund, Felix; Schroda, Michael

2017-12-01

A conserved reaction of all organisms exposed to heat stress is an increased expression of heat shock proteins (HSPs). Several studies have proposed that HSP expression in heat-stressed plant cells is triggered by an increased fluidity of the plasma membrane. Among the main lines of evidence in support of this model are as follows: (a) the degree of membrane lipid saturation was higher in cells grown at elevated temperatures and correlated with a lower amplitude of HSP expression upon a temperature upshift, (b) membrane fluidizers induce HSP expression at physiological temperatures, and (c) membrane rigidifier dimethylsulfoxide dampens heat-induced HSP expression. Here, we tested whether this holds also for Chlamydomonas reinhardtii. We show that heat-induced HSP expression in cells grown at elevated temperatures was reduced because they already contained elevated levels of cytosolic HSP70A/90A that apparently act as negative regulators of heat shock factor 1. We find that membrane rigidifier dimethylsulfoxide impaired translation under heat stress conditions and that membrane fluidizer benzyl alcohol not only induced HSP expression but also caused protein aggregation. These findings support the classical model for the cytosolic unfolded protein response, according to which HSP expression is induced by the accumulation of unfolded proteins. Hence, the membrane fluidity model should be reconsidered. © 2017 John Wiley & Sons Ltd.

PTK6 activation at the membrane regulates epithelial-mesenchymal transition in prostate cancer.

PubMed

Zheng, Yu; Wang, Zebin; Bie, Wenjun; Brauer, Patrick M; Perez White, Bethany E; Li, Jing; Nogueira, Veronique; Raychaudhuri, Pradip; Hay, Nissim; Tonetti, Debra A; Macias, Virgilia; Kajdacsy-Balla, André; Tyner, Angela L

2013-09-01

The intracellular tyrosine kinase protein tyrosine kinase 6 (PTK6) lacks a membrane-targeting SH4 domain and localizes to the nuclei of normal prostate epithelial cells. However, PTK6 translocates from the nucleus to the cytoplasm in human prostate tumor cells. Here, we show that while PTK6 is located primarily within the cytoplasm, the pool of active PTK6 in prostate cancer cells localizes to membranes. Ectopic expression of membrane-targeted active PTK6 promoted epithelial-mesenchymal transition in part by enhancing activation of AKT, thereby stimulating cancer cell migration and metastases in xenograft models of prostate cancer. Conversely, siRNA-mediated silencing of endogenous PTK6 promoted an epithelial phenotype and impaired tumor xenograft growth. In mice, PTEN deficiency caused endogenous active PTK6 to localize at membranes in association with decreased E-cadherin expression. Active PTK6 was detected at membranes in some high-grade human prostate tumors, and PTK6 and E-cadherin expression levels were inversely correlated in human prostate cancers. In addition, high levels of PTK6 expression predicted poor prognosis in patients with prostate cancer. Our findings reveal novel functions for PTK6 in the pathophysiology of prostate cancer, and they define this kinase as a candidate therapeutic target. Cancer Res; 73(17); 5426-37. ©2013 AACR.

Giant plasma membrane vesicles: models for understanding membrane organization.

PubMed

Levental, Kandice R; Levental, Ilya

2015-01-01

The organization of eukaryotic membranes into functional domains continues to fascinate and puzzle cell biologists and biophysicists. The lipid raft hypothesis proposes that collective lipid interactions compartmentalize the membrane into coexisting liquid domains that are central to membrane physiology. This hypothesis has proven controversial because such structures cannot be directly visualized in live cells by light microscopy. The recent observations of liquid-liquid phase separation in biological membranes are an important validation of the raft hypothesis and enable application of the experimental toolbox of membrane physics to a biologically complex phase-separated membrane. This review addresses the role of giant plasma membrane vesicles (GPMVs) in refining the raft hypothesis and expands on the application of GPMVs as an experimental model to answer some of key outstanding problems in membrane biology. Copyright © 2015 Elsevier Inc. All rights reserved.

Vaborbactam: Spectrum of Beta-Lactamase Inhibition and Impact of Resistance Mechanisms on Activity in Enterobacteriaceae

PubMed Central

Sun, Dongxu; Rubio-Aparicio, Debora; Nelson, Kirk; Tsivkovski, Ruslan; Griffith, David C.; Dudley, Michael N.

2017-01-01

ABSTRACT Vaborbactam (formerly RPX7009) is a new beta-lactamase inhibitor based on a cyclic boronic acid pharmacophore. The spectrum of beta-lactamase inhibition by vaborbactam and the impact of bacterial efflux and permeability on its activity were determined using a panel of strains with beta-lactamases cloned from various classes and a panel of Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing isogenic strains with various combinations of efflux and porin mutations. Vaborbactam is a potent inhibitor of class A carbapenemases, such as KPC, as well as an inhibitor of other class A (CTX-M, SHV, TEM) and class C (P99, MIR, FOX) beta-lactamases. Vaborbactam does not inhibit class D or class B carbapenemases. When combined with meropenem, vaborbactam had the highest potency compared to the potencies of vaborbactam in combination with other antibiotics against strains producing the KPC beta-lactamase. Consistent with broad-spectrum beta-lactamase inhibition, vaborbactam reduced the meropenem MICs for engineered isogenic strains of K. pneumoniae with increased meropenem MICs due to a combination of extended-spectrum beta-lactamase production, class C beta-lactamase production, and reduced permeability due to porin mutations. Vaborbactam crosses the outer membrane of K. pneumoniae using both OmpK35 and OmpK36, but OmpK36 is the preferred porin. Efflux by the multidrug resistance efflux pump AcrAB-TolC had a minimal impact on vaborbactam activity. Investigation of the vaborbactam concentration necessary for restoration of meropenem potency showed that vaborbactam at 8 μg/ml results in meropenem MICs of ≤2 μg/ml in the most resistant engineered strains containing multiple mutations. Vaborbactam is a highly active beta-lactamase inhibitor that restores the activity of meropenem and other beta-lactam antibiotics in beta-lactamase-producing bacteria, particularly KPC-producing carbapenem-resistant Enterobacteriaceae. PMID:28848018

Complexity of resistance mechanisms to imipenem in intensive care unit strains of Pseudomonas aeruginosa.

PubMed

Fournier, Damien; Richardot, Charlotte; Müller, Emeline; Robert-Nicoud, Marjorie; Llanes, Catherine; Plésiat, Patrick; Jeannot, Katy

2013-08-01

Pseudomonas aeruginosa can become resistant to carbapenems by both intrinsic (mutation-driven) and transferable (β-lactamase-based) mechanisms. Knowledge of the prevalence of these various mechanisms is important in intensive care units (ICUs) in order to define optimal prevention and therapeutic strategies. A total of 109 imipenem-non-susceptible (MIC >4 mg/L) strains of P. aeruginosa were collected in June 2010 from the ICUs of 26 French public hospitals. Their resistance mechanisms were characterized by phenotypic, enzymatic, western blotting and molecular methods. Single or associated imipenem resistance mechanisms were identified among the 109 strains. Seven isolates (6.4%) were found to produce a metallo-β-lactamase (one VIM-1, four VIM-2, one VIM-4 and one IMP-29). Porin OprD was lost in 94 (86.2%) strains as a result of mutations or gene disruption by various insertion sequences (ISPa1635, ISPa1328, IS911, ISPs1, IS51, IS222 and ISPa41). Thirteen other strains were shown to be regulatory mutants in which down-regulation of oprD was coupled with overexpressed efflux pumps CzcCBA (n = 1), MexXY (n = 9) and MexEF-OprN (n = 3). The lack of OprD was due to disruption of the oprD promoter by ISPsy2 in one strain and alteration of the porin signal sequence in another. Imipenem resistance in ICU P. aeruginosa strains may result from multiple mechanisms involving metallo-β-lactamase gene acquisition and genetic events (mutations and ISs) inactivating oprD, turning down its expression while increasing efflux activities or preventing insertion of porin OprD in the outer membrane. This diversity of mechanisms allows P. aeruginosa, more than any other nosocomial pathogen, to rapidly adapt to carbapenems in ICUs.

Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach.

PubMed

Alvarez, Angel H; Gutiérrez-Ortega, Abel; Hernández-Gutiérrez, Rodolfo

2015-10-01

Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD.

Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach

PubMed Central

Alvarez, Angel H.; Gutiérrez-Ortega, Abel; Hernández-Gutiérrez, Rodolfo

2015-01-01

Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD. PMID:26424916

An Effective Design of Electrically Conducting Thin-Film Composite (TFC) Membranes for Bio and Organic Fouling Control in Forward Osmosis (FO).

PubMed

Liu, Qing; Qiu, Guanglei; Zhou, Zhengzhong; Li, Jingguo; Amy, Gary Lee; Xie, Jianping; Lee, Jim Yang

2016-10-04

The organic foulants and bacteria in secondary wastewater treatment can seriously impair the membrane performance in a water treatment plant. The embedded electrode approach using an externally applied potential to repel organic foulants and inhibit bacterial adhesion can effectively reduce the frequency of membrane replacement. Electrode embedment in membranes is often carried out by dispensing a conductor (e.g., carbon nanotubes, or CNTs) in the membrane substrate, which gives rise to two problems: the leaching-out of the conductor and a percolation-limited membrane conductivity that results in an added energy cost. This study presents a facile method for the embedment of a continuous electrode in thin-film composite (TFC) forward osmosis (FO) membranes. Specifically, a conducting porous carbon paper is used as the understructure for the formation of a membrane substrate by the classical phase inversion process. The carbon paper and the membrane substrate polymer form an interpenetrating structure with good stability and low electrical resistance (only about 1Ω/□). The membrane-electrode assembly was deployed as the cathode of an electrochemical cell, and showed good resistance to organic and microbial fouling with the imposition of a 2.0 V DC voltage. The carbon paper-based FO TFC membranes also possess good mechanical stability for practical use.

Membrane fouling and wetting in membrane distillation and their mitigation by novel membranes with special wettability.

PubMed

Wang, Zhangxin; Lin, Shihong

2017-04-01

Membrane distillation (MD) has been identified as a promising technology to desalinate the hypersaline wastewaters from fracking and other industries. However, conventional hydrophobic MD membranes are highly susceptible to fouling and/or wetting by the hydrophobic and/or amphiphilic constituents in these wastewaters of complex compositions. This study systematically investigates the impact of the surface wetting properties on the membrane wetting and/or fouling behaviors in MD. Specifically, we compare the wetting and fouling resistance of three types of membranes of different wetting properties, including hydrophobic and omniphobic membranes as well as composite membranes with a hydrophobic substrate and a superhydrophilic top surface. We challenged the MD membranes with hypersaline feed solutions that contained a relatively high concentration of crude oil with and without added synthetic surfactants, Triton X-100. We found that the composite membranes with superhydrophilic top surface were robustly resistant to oil fouling in the absence of Triton X-100, but were subject to pore wetting in the presence of Triton X-100. On the other hand, the omniphobic membranes were easily fouled by oil-in-water emulsion without Triton X-100, but successfully sustained stable MD performance with Triton X-100 stabilized oil-in-water emulsion as the feed solution. In contrast, the conventional hydrophobic membranes failed readily regardless whether Triton X-100 was present, although via different mechanisms. These findings are corroborated by contact angle measures as well as oil-probe force spectroscopy. This study provides a holistic picture regarding how a hydrophobic membrane fails in MD and how we can leverage membranes with special wettability to prevent membrane failure in MD operations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cochlear compression: perceptual measures and implications for normal and impaired hearing.

PubMed

Oxenham, Andrew J; Bacon, Sid P

2003-10-01

This article provides a review of recent developments in our understanding of how cochlear nonlinearity affects sound perception and how a loss of the nonlinearity associated with cochlear hearing impairment changes the way sounds are perceived. The response of the healthy mammalian basilar membrane (BM) to sound is sharply tuned, highly nonlinear, and compressive. Damage to the outer hair cells (OHCs) results in changes to all three attributes: in the case of total OHC loss, the response of the BM becomes broadly tuned and linear. Many of the differences in auditory perception and performance between normal-hearing and hearing-impaired listeners can be explained in terms of these changes in BM response. Effects that can be accounted for in this way include poorer audiometric thresholds, loudness recruitment, reduced frequency selectivity, and changes in apparent temporal processing. All these effects can influence the ability of hearing-impaired listeners to perceive speech, especially in complex acoustic backgrounds. A number of behavioral methods have been proposed to estimate cochlear nonlinearity in individual listeners. By separating the effects of cochlear nonlinearity from other aspects of hearing impairment, such methods may contribute towards identifying the different physiological mechanisms responsible for hearing loss in individual patients. This in turn may lead to more accurate diagnoses and more effective hearing-aid fitting for individual patients. A remaining challenge is to devise a behavioral measure that is sufficiently accurate and efficient to be used in a clinical setting.

Electromagnetic fields (UHF) increase voltage sensitivity of membrane ion channels; possible indication of cell phone effect on living cells.

PubMed

Ketabi, N; Mobasheri, H; Faraji-Dana, R

2015-03-01

The effects of ultra high frequency (UHF) nonionizing electromagnetic fields (EMF) on the channel activities of nanopore forming protein, OmpF porin, were investigated. The voltage clamp technique was used to study the single channel activity of the pore in an artificial bilayer in the presence and absence of the electromagnetic fields at 910 to 990 MHz in real time. Channel activity patterns were used to address the effect of EMF on the dynamic, arrangement and dielectric properties of water molecules, as well as on the hydration state and arrangements of side chains lining the channel barrel. Based on the varied voltage sensitivity of the channel at different temperatures in the presence and absence of EMF, the amount of energy transferred to nano-environments of accessible groups was estimated to address the possible thermal effects of EMF. Our results show that the effects of EMF on channel activities are frequency dependent, with a maximum effect at 930 MHz. The frequency of channel gating and the voltage sensitivity is increased when the channel is exposed to EMF, while its conductance remains unchanged at all frequencies applied. We have not identified any changes in the capacitance and permeability of membrane in the presence of EMF. The effect of the EMF irradiated by cell phones is measured by Specific Absorption Rate (SAR) in artificial model of human head, Phantom. Thus, current approach applied to biological molecules and electrolytes might be considered as complement to evaluate safety of irradiating sources on biological matter at molecular level.

LAMP-2 absence interferes with plasma membrane repair and decreases T. cruzi host cell invasion.

PubMed

Couto, Natália Fernanda; Pedersane, Dina; Rezende, Luisa; Dias, Patrícia P; Corbani, Tayanne L; Bentini, Lívia C; Oliveira, Anny C S; Kelles, Ludmila F; Castro-Gomes, Thiago; Andrade, Luciana O

2017-06-01

Trypanosoma cruzi enters host cells by subverting the mechanism of cell membrane repair. In this process, the parasite induces small injuries in the host cell membrane leading to calcium entry and lysosomal exocytosis, which are followed by compensatory endocytosis events that drive parasites into host cells. We have previously shown that absence of both LAMP-1 and 2, major components of lysosomal membranes, decreases invasion of T. cruzi into host cells, but the mechanism by which they interfere with parasite invasion has not been described. Here we investigated the role of these proteins in parasitophorous vacuole morphology, host cell lysosomal exocytosis, and membrane repair ability. First, we showed that cells lacking only LAMP-2 present the same invasion phenotype as LAMP1/2-/- cells, indicating that LAMP-2 is an important player during T. cruzi invasion process. Second, neither vacuole morphology nor lysosomal exocytosis was altered in LAMP-2 lacking cells (LAMP2-/- and LAMP1/2-/- cells). We then investigated the ability of LAMP-2 deficient cells to perform compensatory endocytosis upon lysosomal secretion, the mechanism by which cells repair their membrane and T. cruzi ultimately enters cells. We observed that these cells perform less endocytosis upon injury when compared to WT cells. This was a consequence of impaired cholesterol traffic in cells lacking LAMP-2 and its influence in the distribution of caveolin-1 at the cell plasma membrane, which is crucial for plasma membrane repair. The results presented here show the major role of LAMP-2 in caveolin traffic and membrane repair and consequently in T. cruzi invasion.

LAMP-2 absence interferes with plasma membrane repair and decreases T. cruzi host cell invasion

PubMed Central

Rezende, Luisa; Bentini, Lívia C.; Oliveira, Anny C. S.

2017-01-01

Trypanosoma cruzi enters host cells by subverting the mechanism of cell membrane repair. In this process, the parasite induces small injuries in the host cell membrane leading to calcium entry and lysosomal exocytosis, which are followed by compensatory endocytosis events that drive parasites into host cells. We have previously shown that absence of both LAMP-1 and 2, major components of lysosomal membranes, decreases invasion of T. cruzi into host cells, but the mechanism by which they interfere with parasite invasion has not been described. Here we investigated the role of these proteins in parasitophorous vacuole morphology, host cell lysosomal exocytosis, and membrane repair ability. First, we showed that cells lacking only LAMP-2 present the same invasion phenotype as LAMP1/2-/- cells, indicating that LAMP-2 is an important player during T. cruzi invasion process. Second, neither vacuole morphology nor lysosomal exocytosis was altered in LAMP-2 lacking cells (LAMP2-/- and LAMP1/2-/- cells). We then investigated the ability of LAMP-2 deficient cells to perform compensatory endocytosis upon lysosomal secretion, the mechanism by which cells repair their membrane and T. cruzi ultimately enters cells. We observed that these cells perform less endocytosis upon injury when compared to WT cells. This was a consequence of impaired cholesterol traffic in cells lacking LAMP-2 and its influence in the distribution of caveolin-1 at the cell plasma membrane, which is crucial for plasma membrane repair. The results presented here show the major role of LAMP-2 in caveolin traffic and membrane repair and consequently in T. cruzi invasion. PMID:28586379

A framework for communication between visually impaired, hearing impaired and speech impaired using arduino

NASA Astrophysics Data System (ADS)

Sujatha, R.; Khandelwa, Prakhar; Gupta, Anusha; Anand, Nayan

2017-11-01

A long time ago our society accepted the notion of treating people with disabilities not as unviable and disabled but as differently-abled, recognizing their skills beyond their disabilities. The next step has to be taken by our scientific community, that is, to normalize lives of the people with disabilities and make it so as if they are no different to us. The primary step in this direction would be to normalize communication between people. People with an impaired speech or impaired vision or impaired hearing face difficulties while having a casual conversation with others. Any form of communication feels so strenuous that the impaired end up communicating just the important information and avoid a casual conversation. To normalize conversation between the impaired we need a simple and compact device which facilitates the conversation by providing the information in the desired form.

The interactions of peripheral membrane proteins with biological membranes

DOE PAGES

Johs, Alexander; Whited, A. M.

2015-07-29

The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

The C-terminal domain of TRPV4 is essential for plasma membrane localization.

PubMed

Becker, Daniel; Müller, Margarethe; Leuner, Kristina; Jendrach, Marina

2008-02-01

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.

Flow and fouling in membrane filters: Effects of membrane morphology

NASA Astrophysics Data System (ADS)

Sanaei, Pejman; Cummings, Linda J.

2015-11-01

Membrane filters are widely-used in microfiltration applications. Many types of filter membranes are produced commercially, for different filtration applications, but broadly speaking the requirements are to achieve fine control of separation, with low power consumption. The answer to this problem might seem obvious: select the membrane with the largest pore size and void fraction consistent with the separation requirements. However, membrane fouling (an inevitable consequence of successful filtration) is a complicated process, which depends on many parameters other than membrane pore size and void fraction; and which itself greatly affects the filtration process and membrane functionality. In this work we formulate mathematical models that can (i) account for the membrane internal morphology (internal structure, pore size & shape, etc.); (ii) fouling of membranes with specific morphology; and (iii) make some predictions as to what type of membrane morphology might offer optimum filtration performance.

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.

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

Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importancemore » of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.« less

Membrane Environment Exerts an Important Influence on Rac-Mediated Activation of Phospholipase Cγ2 ▿ †

PubMed Central

Everett, Katy L.; Buehler, Anja; Bunney, Tom D.; Margineanu, Anca; Baxendale, Rhona W.; Vatter, Petra; Retlich, Michael; Walliser, Claudia; Manning, Hugh B.; Neil, Mark A. A.; Dunsby, Christopher; French, Paul M. W.; Gierschik, Peter; Katan, Matilda

2011-01-01

We performed analyses of the molecular mechanisms involved in the regulation of phospholipase Cγ2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLCγ2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLCγ2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation. PMID:21245382

Second-Hand Cigarette Smoke Impairs Bacterial Phagocytosis in Macrophages by Modulating CFTR Dependent Lipid-Rafts

PubMed Central

Ni, Inzer; Ji, Changhoon; Vij, Neeraj

2015-01-01

Introduction First/Second-hand cigarette-smoke (FHS/SHS) exposure weakens immune defenses inducing chronic obstructive pulmonary disease (COPD) but the underlying mechanisms are not fully understood. Hence, we evaluated if SHS induced changes in membrane/lipid-raft (m-/r)-CFTR (cystic fibrosis transmembrane conductance regulator) expression/activity is a potential mechanism for impaired bacterial phagocytosis in COPD. Methods RAW264.7 murine macrophages were exposed to freshly prepared CS-extract (CSE) containing culture media and/or Pseudomonas-aeruginosa-PA01-GFP for phagocytosis (fluorescence-microscopy), bacterial survival (colony-forming-units-CFU), and immunoblotting assays. The CFTR-expression/activity and lipid-rafts were modulated by transient-transfection or inhibitors/inducers. Next, mice were exposed to acute/sub-chronic-SHS or room-air (5-days/3-weeks) and infected with PA01-GFP, followed by quantification of bacterial survival by CFU-assay. Results We investigated the effect of CSE treatment on RAW264.7 cells infected by PA01-GFP and observed that CSE treatment significantly (p<0.01) inhibits PA01-GFP phagocytosis as compared to the controls. We also verified this in murine model, exposed to acute/sub-chronic-SHS and found significant (p<0.05, p<0.02) increase in bacterial survival in the SHS-exposed lungs as compared to the room-air controls. Next, we examined the effect of impaired CFTR ion-channel-activity on PA01-GFP infection of RAW264.7 cells using CFTR172-inhibitor and found no significant change in phagocytosis. We also similarly evaluated the effect of a CFTR corrector-potentiator compound, VRT-532, and observed no significant rescue of CSE impaired PA01-GFP phagocytosis although it significantly (p<0.05) decreases CSE induced bacterial survival. Moreover, induction of CFTR expression in macrophages significantly (p<0.03) improves CSE impaired PA01-GFP phagocytosis as compared to the control. Next, we verified the link between m

Tannins Possessing Bacteriostatic Effect Impair Pseudomonas aeruginosa Adhesion and Biofilm Formation

PubMed Central

Trentin, Danielle S.; Silva, Denise B.; Amaral, Matheus W.; Zimmer, Karine R.; Silva, Márcia V.; Lopes, Norberto P.; Giordani, Raquel B.; Macedo, Alexandre J.

2013-01-01

Plants produce many compounds that are biologically active, either as part of their normal program of growth and development or in response to pathogen attack or stress. Traditionally, Anadenanthera colubrina, Commiphora leptophloeos and Myracrodruon urundeuva have been used by communities in the Brazilian Caatinga to treat several infectious diseases. The ability to impair bacterial adhesion represents an ideal strategy to combat bacterial pathogenesis, because of its importance in the early stages of the infectious process; thus, the search for anti-adherent compounds in plants is a very promising alternative. This study investigated the ability of stem-bark extracts from these three species to control the growth and prevent biofilm formation of Pseudomonas aeruginosa, an important opportunistic pathogen that adheres to surfaces and forms protective biofilms. A kinetic study (0–72 h) demonstrated that the growth of extract-treated bacteria was inhibited up to 9 h after incubation, suggesting a bacteriostatic activity. Transmission electron microscopy and fluorescence microscopy showed both viable and nonviable cells, indicating bacterial membrane damage; crystal violet assay and scanning electron microscopy demonstrated that treatment strongly inhibited biofilm formation during 6 and 24 h and that matrix production remained impaired even after growth was restored, at 24 and 48 h of incubation. Herein, we propose that the identified (condensed and hydrolyzable) tannins are able to inhibit biofilm formation via bacteriostatic properties, damaging the bacterial membrane and hindering matrix production. Our findings demonstrate the importance of this abundant class of Natural Products in higher plants against one of the most challenging issues in the hospital setting: biofilm resilience. PMID:23776646

[Principles of changes of structural organization of cell membranes and functional properties of erythrocytes in neurotic disorders].

PubMed

Riazantseva, N V; Novitskiĭ, V V

2003-02-01

Investigation into structural, metabolic, and functional conditions of red blood cells was performed in 24 patients with a neurosis (neurasthenia, disturbance of asaptation) with the aid of electrophoretic division of proteins of the erythrocyte membrane, thin-layer chromatography, fluorescent probing of membranes, evaluation of peroxidative oxidation process, scanning and transmission electron microscopy, laser diphractometry, photometry. The patients with neurotic disorders at the early period after the influence of psychogenic factors (up to 3 months) revealed disorganization of lipid and protein composition of the red cell membrane, increase in microviscosity of its lipid phase, impairment of surface architectonics and ultrastructure of red cells, decrease of a deformation ability and increase of aggregate properties of erythrocytes. The authors treat stability of erythrocytes' homeostasis under the long-term influence of psychogenic factors from a viewpoint of adaptive changes in organism under the influence of neurogenic factors.

Skin grafting impairs postsynaptic cutaneous vasodilator and sweating responses.

PubMed

Davis, Scott L; Shibasaki, Manabu; Low, David A; Cui, Jian; Keller, David M; Purdue, Gary F; Hunt, John L; Arnoldo, Brett D; Kowalske, Karen J; Crandall, Craig G

2007-01-01

This study tested the hypothesis that postsynaptic cutaneous vascular responses to endothelial-dependent and -independent vasodilators, as well as sweat gland function, are impaired in split-thickness grafted skin 5 to 9 months after surgery. Intradermal microdialysis membranes were placed in grafted and adjacent control skin, thereby allowing local delivery of the endothelial-dependent vasodilator, acetylcholine (ACh; 1 x 10(-7) to 1 x 10(-1) M at 10-fold increments) and the endothelial-independent nitric oxide donor, sodium nitroprusside (SNP; 5 x 10(-8) to 5 x 10(-2) M at 10-fold increments). Skin blood flow and sweat rate were simultaneously assessed over the semipermeable portion of the membrane. Cutaneous vascular conductance (CVC) was calculated from the ratio of laser Doppler-derived skin blood flow to mean arterial blood pressure. deltaCVC responses from baseline to these drugs were modeled via nonlinear regression curve fitting to identify the dose of ACh and SNP causing 50% of the maximal vasodilator response (EC50). A rightward shift in the CVC dose response curve for ACh was observed in grafted (EC50 = -2.61 +/- 0.44 log M) compared to adjacent control skin (EC50 = -3.34 +/- 0.46 log M; P = .003), whereas the mean EC50 for SNP was similar between grafted (EC50 = -4.21 +/- 0.94 log M) and adjacent control skin (EC50 = -3.87 +/- 0.65 log M; P = 0.332). Only minimal sweating to exogenous ACh was observed in grafted skin whereas normal sweating was observed in control skin. Increased EC50 and decreased maximal CVC responses to the exogenous administration of ACh suggest impairment of endothelial-dependent cutaneous vasodilator responses in grafted skin 5 to 9 months after surgery. Greatly attenuated sweating responses to ACh suggests either abnormal or an absence of functional sweat glands in the grafted skin.

CHARMM-GUI Membrane Builder toward realistic biological membrane simulations.

PubMed

Wu, Emilia L; Cheng, Xi; Jo, Sunhwan; Rui, Huan; Song, Kevin C; Dávila-Contreras, Eder M; Qi, Yifei; Lee, Jumin; Monje-Galvan, Viviana; Venable, Richard M; Klauda, Jeffery B; Im, Wonpil

2014-10-15

CHARMM-GUI Membrane Builder, http://www.charmm-gui.org/input/membrane, is a web-based user interface designed to interactively build all-atom protein/membrane or membrane-only systems for molecular dynamics simulations through an automated optimized process. In this work, we describe the new features and major improvements in Membrane Builder that allow users to robustly build realistic biological membrane systems, including (1) addition of new lipid types, such as phosphoinositides, cardiolipin (CL), sphingolipids, bacterial lipids, and ergosterol, yielding more than 180 lipid types, (2) enhanced building procedure for lipid packing around protein, (3) reliable algorithm to detect lipid tail penetration to ring structures and protein surface, (4) distance-based algorithm for faster initial ion displacement, (5) CHARMM inputs for P21 image transformation, and (6) NAMD equilibration and production inputs. The robustness of these new features is illustrated by building and simulating a membrane model of the polar and septal regions of E. coli membrane, which contains five lipid types: CL lipids with two types of acyl chains and phosphatidylethanolamine lipids with three types of acyl chains. It is our hope that CHARMM-GUI Membrane Builder becomes a useful tool for simulation studies to better understand the structure and dynamics of proteins and lipids in realistic biological membrane environments. Copyright © 2014 Wiley Periodicals, Inc.

FGFR3 mutation causes abnormal membranous ossification in achondroplasia.

PubMed

Di Rocco, Federico; Biosse Duplan, Martin; Heuzé, Yann; Kaci, Nabil; Komla-Ebri, Davide; Munnich, Arnold; Mugniery, Emilie; Benoist-Lasselin, Catherine; Legeai-Mallet, Laurence

2014-06-01

FGFR3 gain-of-function mutations lead to both chondrodysplasias and craniosynostoses. Achondroplasia (ACH), the most frequent dwarfism, is due to an FGFR3-activating mutation which results in impaired endochondral ossification. The effects of the mutation on membranous ossification are unknown. Fgfr3(Y367C/+) mice mimicking ACH and craniofacial analysis of patients with ACH and FGFR3-related craniosynostoses provide an opportunity to address this issue. Studying the calvaria and skull base, we observed abnormal cartilage and premature fusion of the synchondroses leading to modifications of foramen magnum shape and size in Fgfr3(Y367C/+) mice, ACH and FGFR3-related craniosynostoses patients. Partial premature fusion of the coronal sutures and non-ossified gaps in frontal bones were also present in Fgfr3(Y367C/+) mice and ACH patients. Our data provide strong support that not only endochondral ossification but also membranous ossification is severely affected in ACH. Demonstration of the impact of FGFR3 mutations on craniofacial development should initiate novel pharmacological and surgical therapeutic approaches.

The lipid composition modulates the influence of the bovine seminal plasma protein PDC-109 on membrane stability.

PubMed

Tannert, Astrid; Töpfer-Petersen, Edda; Herrmann, Andreas; Müller, Karin; Müller, Peter

2007-10-16

The bovine seminal plasma protein PDC-109 exerts an essential influence on the sperm cell plasma membrane during capacitation. However, by any mechanism, it has to be ensured that this function of the protein on sperm cells is not initiated too early, that is, upon ejaculation when PDC-109 and sperm cells come into first contact, but rather at later stages of sperm genesis in the female genital tract. To answer the question of whether changes of the bovine sperm lipid composition can modulate the effect of PDC-109 on sperm membranes, we have investigated the influence of PDC-109 on the integrity of (i) differently composed lipid vesicles and of (ii) membranes from human red blood cells and bovine spermatozoa. PDC-109 most effectively disturbed lipid membranes composed of choline-containing phospholipids and in the absence of cholesterol. The impact of the protein on lipid vesicles was attenuated in the presence of cholesterol or of noncholine-containing phospholipids, such as phosphatidylethanolamine or phosphatidylserine. An extraction of cholesterol from lipid or biological membranes using methyl-beta-cyclodextrin caused an increased membrane perturbation by PDC-109. Our results argue for a oppositional effect of PDC-109 during sperm cell genesis. We hypothesize that the lipid composition of ejaculated bull sperm cells allows a binding of PDC-109 without leading to an impairment of the plasma membrane. At later stages of sperm cell genesis upon release of cholesterol from sperm membranes, PDC-109 triggers a destabilization of the cells.

Membrane receptor-independent inhibitory effect of melatonin on androgen production in porcine theca cells.

PubMed

Wang, Heng; Pu, Yong; Luo, Lei; Li, Yunsheng; Zhang, Yunhai; Cao, Zubing

2018-06-01

Excessive secretion of androgens including androstenedione and testosterone in theca cells frequently causes female infertility in mammals. Melatonin is a potent inhibitor of androgen production in gonadal cells of several species in a membrane receptor-dependent manner. However, the function of melatonin in steroidogenesis of porcine theca cells remains unclear. Here we report that melatonin inhibits androgen biosynthesis independently of its membrane receptors in pigs. Using flow cytometry, immunofluorescence and RT-PCR we showed that the vast majority of cells isolated from the theca layer of antral follicles are indeed theca cells. Furthermore, we demonstrated that of the two of melatonin membrane receptors encoded in the porcine genome, theca cells exclusively express melatonin receptor 1B. Cell counting analysis indicated that different concentrations of melatonin did not alter the normal viability and proliferation of theca cells. Additionally, hormone radioimmunoassay and qPCR respectively showed that a high concentration of melatonin significantly repressed both androgen production and expression of steroidogenic genes involving StAR, CYP11A1, HSD3β and SET (P < 0.05), but did not impair progesterone production. Interestingly, these effects were not reversed by N-acetyl-2-benzyltryptamin, a melatonin membrane receptor antagonist. Overall, these results demonstrate that melatonin inhibits androgen production in porcine theca cells independently of its membrane receptor. Copyright © 2018 Elsevier Inc. All rights reserved.

Impaired Bioenergetics in Mutant Mitochondrial DNA Determines Cell Fate During Seizure-Like Activity.

PubMed

Kovac, Stjepana; Preza, Elisavet; Houlden, Henry; Walker, Matthew C; Abramov, Andrey Y

2018-04-27

Mutations in genes affecting mitochondrial proteins are increasingly recognised in patients with epilepsy, but the factors determining cell fate during seizure activity in these mutations remain unknown. Fluorescent dye imaging techniques were applied to fibroblast cell lines from patients suffering from common mitochondrial mutations and to age-matched controls. Using live cell imaging techniques in fibroblasts, we show that fibroblasts with mutations in the mitochondrial genome had reduced mitochondrial membrane potential and NADH pools and higher redox indices, indicative of respiratory chain dysfunction. Increasing concentrations of ferutinin, a Ca 2+ ionophore, led to oscillatory Ca 2+ signals in fibroblasts resembling dynamic Ca 2+ changes that occur during seizure-like activity. Co-monitoring of mitochondrial membrane potential (ΔΨ m ) changes induced by ferutinin showed accelerated membrane depolarisation and cell collapse in fibroblasts with mutations in the mitochondrial genome when compared to controls. Ca 2+ flash photolysis using caged Ca 2+ confirmed impaired Ca 2+ handling in fibroblasts with mitochondrial mutations. Findings indicate that intracellular Ca 2+ levels cannot be compensated during periods of hyperexcitability, leading to Ca 2+ overload and subsequent cell death in mitochondrial diseases.

Advanced Supported Liquid Membranes for Carbon Dioxide Control in Cabin Applications

NASA Technical Reports Server (NTRS)

Wickham, David T.; Gleason, Kevin J.; Engel, Jeffrey R.; Chullen, Cinda

2016-01-01

The development of new, robust, life support systems is critical to NASA's continued progress in space exploration. One vital function is maintaining the carbon dioxide (CO2) concentration in the cabin at levels that do not impair the health or performance of the crew. The CO2 removal assembly (CDRA) is the current CO2 control technology on-board the International Space Station (ISS). Although the CDRA has met the needs of the ISS to date, the repeated cycling of the molecular sieve sorbent causes it to break down into small particles that clog filters or generate dust in the cabin. This reduces reliability and increases maintenance requirements. Another approach that has potential advantages over the current system is a membrane that separates CO2 from air. In this approach, cabin air contacts one side of the membrane while other side of the membrane is maintained at low pressure to create a driving force for CO2 transport across the membrane. In this application, the primary power requirement is for the pump that creates the low pressure and then pumps the CO2 to the oxygen recovery system. For such a membrane to be practical, it must have high CO2 permeation rate and excellent selectivity for CO2 over air. Unfortunately, conventional gas separation membranes do not have adequate CO2 permeability and selectivity to meet the needs of this application. However, the required performance could be obtained with a supported liquid membrane (SLM), which consists of a microporous material filled with a liquid that selectively reacts with CO2 over air. In a recently completed Phase II SBIR project, Reaction Systems, Inc. fabricated an SLM that is very close to meeting permeability and selectivity objectives for use in the advanced space suit portable life support system. This paper describes work carried out to evaluate its potential for use in spacecraft cabin application.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation.

PubMed

Nasseau, M; Boublik, Y; Meier, W; Winterhalter, M; Fournier, D

2001-12-05

How can enzymes be protected against denaturation and proteolysis while keeping them in a fully functional state? One solution is to encapsulate the enzymes into liposomes, which enhances their stability against denaturation and proteases. However, the permeability barrier of the lipid membrane drastically reduces the activity of enzyme entrapped in the liposome by reducing the internal concentration of the substrate. To overcome this problem, we permeabilized the wall of the liposome by reconstitution of a porin from Escherichia coli. In this way, we recovered the full functionality of the enzyme while retaining the protection against denaturation and proteolytic enzymes. Copyright 2001 John Wiley & Sons, Inc.

Islet transplantation under the kidney capsule fully corrects the impaired skeletal muscle glucose transport system of streptozocin diabetic rats.

PubMed Central

Napoli, R; Davalli, A M; Hirshman, M F; Weitgasser, R; Weir, G C; Horton, E S

1996-01-01

Chronic insulin therapy improves but does not restore impaired insulin-mediated muscle glucose uptake in human diabetes or muscle glucose uptake, transport, and transporter translocation in streptozocin diabetic rats. To determine whether this inability is due to inadequate insulin replacement, we studied fasted streptozocin-induced diabetic Lewis rats either untreated or after islet transplantation under the kidney capsule. Plasma glucose was increased in untreated diabetics and normalized by the islet transplantation (110 +/- 5, 452 +/- 9, and 102 +/- 3 mg/dl in controls, untreated diabetics, and transplanted diabetics, respectively). Plasma membrane and intracellular microsomal membrane vesicles were prepared from hindlimb skeletal muscle of basal and maximally insulin-stimulated rats. Islet transplantation normalized plasma membrane carrier-mediated glucose transport Vmax, plasma membrane glucose transporter content, and insulin-induced transporter translocation. There were no differences in transporter intrinsic activity (Vmax/Ro) among the three groups. Microsomal membrane GLUT4 content was reduced by 30% in untreated diabetic rats and normal in transplanted diabetics, whereas the insulin-induced changes in microsomal membrane GLUT4 content were quantitatively similar in the three groups. There were no differences in plasma membrane GLUT1 among the groups and between basal and insulin stimulated states. Microsomal membrane GLUT1 content was increased 60% in untreated diabetics and normalized by the transplantation. In conclusion, an adequate insulin delivery in the peripheral circulation, obtained by islet transplantation, fully restores the muscle glucose transport system to normal in streptozocin diabetic rats. PMID:8617870

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

PubMed Central

van Geest, Marleen; Lolkema, Juke S.

2000-01-01

Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins. A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer. Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence. In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed. The latter studies search for signals in the polypeptide chain that direct the insertion process. Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies. PMID:10704472

Preparation and characterization of hydroxyapatite/gelatin composite membranes for immunoisolation

NASA Astrophysics Data System (ADS)

Chen, Jyh-Ping; Chang, Feng-Nian

2012-12-01

Composite membranes are fabricated from hydroxyapatite (HAP) and gelatin for immunoisolation of cells. The films were fabricated by crosslinking 5 wt%, 10 wt%, and 20 wt% gelatin with 1 wt% glutaraldehyde (GA) in the presence of HAP. Fourier transform infrared spectroscopy analysis confirms imide bond formation between GA and gelatin, while the crystal structure of HAP powder remains unchanged from X-ray diffraction analysis. The degree of crosslinking depends on crosslinking time and gelatin concentration. For 5% and 10% gelatin, the degree of crosslinking levels off at 90% within 48 h. From scanning electron microscopy micrographs, the microstructure of the composite membrane depends on the amount of gelatin used in the crosslinking reaction. The mechanical strength of the composite membrane could be enhanced by increasing the gelatin concentration. BET analysis indicates that pore size of the micropores on the surface HAP/gelatin agglomerates decreases with increasing gelatin concentration. However, the macropore, through which diffusion of molecules occurs, is larger at higher gelatin concentrations. The permeability coefficients of different molecules through a HAP/gelatin composite membrane increase with increasing gelatin concentration and is inversely correlated with the molecular weight of the molecule. For immunoisolation of cells, the diffusion of large molecules stimulated by the immune system can be rejected by a chamber constructed from the HAP/gelatin membrane. Insulinoma cells were encapsulated in alginate-poly-L-lysine-alginate microcapsules and enclosed in a HAP/gelatin chamber. The chamber did not impair the viability and function of insulinoma cells and cells can secrete insulin in response to glucose concentration change. The chamber is therefore useful for the physiologically controlled secretion of insulin in response to the blood glucose level. Intraperitoneal transplantation of the chamber into streptozotocin-induced diabetic SD rats could

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

PubMed

Katti, Sachin; Nyenhuis, Sarah B; Her, Bin; Srivastava, Atul K; Taylor, Alexander B; Hart, P John; Cafiso, David S; Igumenova, Tatyana I

2017-06-27

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca 2+ -dependent manner. In these cases, membrane association is triggered by Ca 2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd 2+ , in lieu of Ca 2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd 2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd 2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd 2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd 2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd 2+ -complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca 2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca 2+ ion binding to the C2 domain loop regions.

An Amphipathic Helix Directs Cellular Membrane Curvature Sensing and Function of the BAR Domain Protein PICK1.

PubMed

Herlo, Rasmus; Lund, Viktor K; Lycas, Matthew D; Jansen, Anna M; Khelashvili, George; Andersen, Rita C; Bhatia, Vikram; Pedersen, Thomas S; Albornoz, Pedro B C; Johner, Niklaus; Ammendrup-Johnsen, Ina; Christensen, Nikolaj R; Erlendsson, Simon; Stoklund, Mikkel; Larsen, Jannik B; Weinstein, Harel; Kjærulff, Ole; Stamou, Dimitrios; Gether, Ulrik; Madsen, Kenneth L

2018-05-15

BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila, disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Neuroacanthocytosis associated with a defect of the 4.1R membrane protein

PubMed Central

Orlacchio, Antonio; Calabresi, Paolo; Rum, Adriana; Tarzia, Anna; Salvati, Anna Maria; Kawarai, Toshitaka; Stefani, Alessandro; Pisani, Antonio; Bernardi, Giorgio; Cianciulli, Paolo; Caprari, Patrizia

2007-01-01

Background Neuroacanthocytosis (NA) denotes a heterogeneous group of diseases that are characterized by nervous system abnormalities in association with acanthocytosis in the patients' blood. The 4.1R protein of the erythrocyte membrane is critical for the membrane-associated cytoskeleton structure and in central neurons it regulates the stabilization of AMPA receptors on the neuronal surface at the postsynaptic density. We report clinical, biochemical, and genetic features in four patients from four unrelated families with NA in order to explain the cause of morphological abnormalities and the relationship with neurodegenerative processes. Case presentation All patients were characterised by atypical NA with a novel alteration of the erythrocyte membrane: a 4.1R protein deficiency. The 4.1R protein content was significantly lower in patients (3.40 ± 0.42) than in controls (4.41 ± 0.40, P < 0.0001), reflecting weakened interactions of the cytoskeleton with the membrane. In patients IV:1 (RM23), IV:3 (RM15), and IV:6 (RM16) the 4.1 deficiency seemed to affect the horizontal interactions of spectrin and an impairment of the dimer self-association into tetramers was detected. In patient IV:1 (RM16) the 4.1 deficiency seemed to affect the skeletal attachment to membrane and the protein band 3 was partially reduced. Conclusion A decreased expression pattern of the 4.1R protein was observed in the erythrocytes from patients with atypical NA, which might reflect the expression pattern in the central nervous system, especially basal ganglia, and might lead to dysfunction of AMPA-mediated glutamate transmission. PMID:17298666

Plant membrane proteomics.

PubMed

Ephritikhine, Geneviève; Ferro, Myriam; Rolland, Norbert

2004-12-01

Plant membrane proteins are involved in many different functions according to their location in the cell. For instance, the chloroplast has two membrane systems, thylakoids and envelope, with specialized membrane proteins for photosynthesis and metabolite and ion transporters, respectively. Although recent advances in sample preparation and analytical techniques have been achieved for the study of membrane proteins, the characterization of these proteins, especially the hydrophobic ones, is still challenging. The present review highlights recent advances in methodologies for identification of plant membrane proteins from purified subcellular structures. The interest of combining several complementary extraction procedures to take into account specific features of membrane proteins is discussed in the light of recent proteomics data, notably for chloroplast envelope, mitochondrial membranes and plasma membrane from Arabidopsis. These examples also illustrate how, on one hand, proteomics can feed bioinformatics for a better definition of prediction tools and, on the other hand, although prediction tools are not 100% reliable, they can give valuable information for biological investigations. In particular, membrane proteomics brings new insights over plant membrane systems, on both the membrane compartment where proteins are working and their putative cellular function.

Hearing Impairment, Mild Cognitive Impairment, and Dementia: A Meta-Analysis of Cohort Studies.

PubMed

Wei, Jingkai; Hu, Yirui; Zhang, Li; Hao, Qiang; Yang, Ruowei; Lu, Haidong; Zhang, Xuan; Chandrasekar, Eeshwar K

2017-01-01

To estimate a pooled association between hearing impairment and risk of mild cognitive impairment and dementia. PubMed, Embase, and Web of Science were searched for prospective cohort studies that examined the association between hearing impairment and risk of mild cognitive impairment and/or dementia. Random-effects models were fitted to estimate the summary risk ratios (RRs) and 95% confidence interval (CIs), which represents the pooled association between hearing impairment with risk of mild cognitive impairment and dementia, compared to subjects free of hearing impairment. Four studies on hearing impairment with mild cognitive impairment and 7 studies on hearing impairment with dementia were included in the meta-analysis. A total of 15,521 subjects were studied with follow-up periods between 2 and 16.8 years. Hearing impairment was associated with a greater risk of mild cognitive impairment (RR = 1.30, 95% CI: 1.12, 1.51) and dementia (RR = 2.39, 95% CI: 1.58, 3.61). The meta-analysis showed that hearing impairment is associated with a higher risk of mild cognitive impairment and dementia among older adults.

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

PubMed Central

Quon, Evan; Beh, Christopher T.

2015-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

Liver plasma membranes: an effective method to analyze membrane proteome.

PubMed

Cao, Rui; Liang, Songping

2012-01-01

Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

Impaired mitochondria and intracellular calcium transients in the salivary glands of obese rats.

PubMed

Ittichaicharoen, Jitjiroj; Apaijai, Nattayaporn; Tanajak, Pongpan; Sa-Nguanmoo, Piangkwan; Chattipakorn, Nipon; Chattipakorn, Siriporn C

2017-04-01

Long-term consumption of a high-fat diet (HFD) causes not only obese-insulin resistance, but is also associated with mitochondrial dysfunction in several organs. However, the effect of obese-insulin resistance on salivary glands has not been investigated. We hypothesized that obese-insulin resistance induced by HFD impaired salivary gland function by reducing salivation, increasing inflammation, and fibrosis, as well as impairing mitochondrial function and calcium transient signaling. Male Wistar rats (200-220 g) were fed either a ND or an HFD (n = 8/group) for 16 weeks. At the end of week 16, salivary flow rates, metabolic parameters, and plasma oxidative stress were determined. Rats were then sacrificed and submandibular glands were removed to determine inflammation, fibrosis, apoptosis, mitochondrial function and dynamics, and intracellular calcium transient signaling. Long-term consumption of an HFD caused obese-insulin resistance and increased oxidative stress, fibrosis, inflammation, and apoptosis in the salivary glands. In addition, impaired mitochondrial function, as indicated by increased mitochondrial reactive oxygen species, mitochondrial membrane depolarization, and mitochondrial swelling in salivary glands and impaired intracellular calcium regulation, as indicated by a reduced intracellular calcium transient rising rate, decay rates, and amplitude of salivary acinar cells, were observed in HFD-fed rats. However, salivary flow rate and level of aquaporin 5 protein were not different between both groups. Although HFD consumption did not affect salivation, it caused obese-insulin resistance, leading to pathophysiological alteration of salivary glands, including impaired intracellular calcium transients, increased oxidative stress and inflammation, and salivary mitochondrial dysfunction.

Epiretinal membrane: a treatable cause of visual disability in myotonic dystrophy type 1.

PubMed

Kersten, Hannah M; Roxburgh, Richard H; Child, Nicholas; Polkinghorne, Philip J; Frampton, Chris; Danesh-Meyer, Helen V

2014-01-01

A wide range of ocular abnormalities have been documented to occur in patients with myotonic dystrophy type 1. The objectives of this study were to investigate the macular and optic nerve morphology using optical coherence tomography in patients with myotonic dystrophy type 1. A total of 30 myotonic dystrophy type 1 patients and 28 controls were recruited for participation. All participants underwent a thorough ophthalmologic examination, including spectral-domain optical coherence tomography of the macula and retinal nerve fibre layer. Images were reviewed by a retinal specialist ophthalmologist, masked to the diagnosis of the participants. Average macular thickness was significantly greater in the myotonic dystrophy group compared to controls [327.3 μm vs. 308.5 μm (p < 0.001)]. Macular thickness was significantly greater (p < 0.005) in five of the nine macular regions. The increase in macular thickness was due to the increased prevalence of epiretinal membranes in the myotonic dystrophy patient group (p = 0.0002): 48.2 % of myotonic dystrophy patient eyes had evidence of epiretinal membrane, compared with 12.5 % of control eyes. Examination revealed that 56.7 % of myotonic dystrophy patients had an epiretinal membrane in at least one eye. Visual acuity was reduced due to the presence of epiretinal membrane in six patient eyes and none of the control eyes. The presence of an epiretinal membrane was significantly correlated with increasing age in the patient group. We report an increased prevalence of epiretinal membrane in the myotonic dystrophy type 1 group. This may be a previously under-recognised form of visual impairment in this group. Epiretinal membranes can be treated surgically. We suggest that, in addition to a comprehensive clinical examination, optical coherence tomography examination is implemented as part of an ophthalmological assessment for the myotonic dystrophy type 1 patient with reduced visual acuity.

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.

Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes.

PubMed

Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming

2014-01-01

Dimethyl sulfoxide (DMSO) is a polar organic solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacological and pathological effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species production, and subsequent cytochrome c release and caspase-3 activation. DMSO at concentrations of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO.

Dimethyl Sulfoxide Damages Mitochondrial Integrity and Membrane Potential in Cultured Astrocytes

PubMed Central

Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming

2014-01-01

Dimethyl sulfoxide (DMSO) is a polar organic solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacological and pathological effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species production, and subsequent cytochrome c release and caspase-3 activation. DMSO at concentrations of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO. PMID:25238609

Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts.

PubMed

Schenkel, Laila C; Singh, Ratnesh K; Michel, Vera; Zeisel, Steven H; da Costa, Kerry-Ann; Johnson, Amy R; Mudd, Harvey S; Bakovic, Marica

2015-05-01

Fibroblasts from a patient with postural orthostatic tachycardia syndrome (POTS), who presented with low plasma choline and betaine, were studied to determine the metabolic characteristics of the choline deficiency. Choline is required for the synthesis of the phospholipid phosphatidylcholine (PC) and for betaine, an important osmoregulator. Here, choline transport, lipid homeostasis, and mitochondria function were analyzed in skin fibroblasts from POTS and compared with control cells. The choline transporter-like protein 1/solute carrier 44A1 (CTL1/SLC44A1) and mRNA expression were 2-3 times lower in POTS fibroblasts, and choline uptake was reduced 60% (P < 0.05). Disturbances of membrane homeostasis were observed by reduced ratios between PC:phosphatidylethanolamine and sphingomyelin:cholesterol, as well as by modified phospholipid fatty acid composition. Choline deficiency also impaired mitochondria function, which was observed by a reduction in oxygen consumption, mitochondrial potential, and glycolytic activity. When POTS cells were treated with choline, transporter was up-regulated, and uptake of choline increased, offering an option for patient treatment. The characteristics of the POTS fibroblasts described here represent a first model of choline and CTL1/SLC44A1 deficiency, in which choline transport, membrane homeostasis, and mitochondrial function are impaired. © FASEB.

20 CFR 404.1598 - If you become disabled by another impairment(s).

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false If you become disabled by another impairment... Disability § 404.1598 If you become disabled by another impairment(s). If a new severe impairment(s) begins... are still disabled under § 404.1594. [50 FR 50136, Dec. 6, 1985] ...

20 CFR 404.1598 - If you become disabled by another impairment(s).

Code of Federal Regulations, 2013 CFR

2013-04-01

... 20 Employees' Benefits 2 2013-04-01 2013-04-01 false If you become disabled by another impairment... Disability § 404.1598 If you become disabled by another impairment(s). If a new severe impairment(s) begins... are still disabled under § 404.1594. [50 FR 50136, Dec. 6, 1985] ...

20 CFR 404.1598 - If you become disabled by another impairment(s).

Code of Federal Regulations, 2011 CFR

2011-04-01

... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false If you become disabled by another impairment... Disability § 404.1598 If you become disabled by another impairment(s). If a new severe impairment(s) begins... are still disabled under § 404.1594. [50 FR 50136, Dec. 6, 1985] ...

20 CFR 404.1598 - If you become disabled by another impairment(s).

Code of Federal Regulations, 2014 CFR

2014-04-01

... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false If you become disabled by another impairment... Disability § 404.1598 If you become disabled by another impairment(s). If a new severe impairment(s) begins... are still disabled under § 404.1594. [50 FR 50136, Dec. 6, 1985] ...

20 CFR 404.1598 - If you become disabled by another impairment(s).

Code of Federal Regulations, 2012 CFR

2012-04-01

... 20 Employees' Benefits 2 2012-04-01 2012-04-01 false If you become disabled by another impairment... Disability § 404.1598 If you become disabled by another impairment(s). If a new severe impairment(s) begins... are still disabled under § 404.1594. [50 FR 50136, Dec. 6, 1985] ...

Focus on membrane differentiation and membrane domains in the prokaryotic cell.

PubMed

Boekema, Egbert J; Scheffers, Dirk-Jan; van Bezouwen, Laura S; Bolhuis, Henk; Folea, I Mihaela

2013-01-01

A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different cellular processes. Typical membrane domains are found in bacteria where a specific membrane protein is abundantly expressed. Lipid rafts form another example. Despite the rareness of conventional organelles as found in eukaryotes, some bacteria are known to have an intricate internal cell membrane organization. Membrane proliferation can be divided into curvature and invaginations which can lead to internal compartmentalization. This study discusses some of the clearest examples of bacteria with such domains and internal membranes. The need for membrane specialization is highest among the heterogeneous group of bacteria which harvest light energy, such as photosynthetic bacteria and halophilic archaea. Most of the highly specialized membranes and domains, such as the purple membrane, chromatophore and chlorosome, are found in these autotrophic organisms. Otherwise the need for membrane differentiation is lower and variable, except for those structures involved in cell division. Microscopy techniques have given essential insight into bacterial membrane morphology. As microscopy will further contribute to the unraveling of membrane organization in the years to come, past and present technology in electron microscopy and light microscopy is discussed. Electron microscopy was the first to unravel bacterial morphology because it can directly visualize membranes with inserted proteins, which no other technique can do. Electron microscopy techniques developed in the 1950s and perfected in the following decades involve the thin sectioning and freeze fractioning of cells. Several studies from the golden age of these techniques show amazing examples of cell membrane morphology

G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

PubMed

Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

2017-09-01

G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion*

PubMed Central

Chong, Brandi M.; Russell, Tanya D.; Schaack, Jerome; Orlicky, David J.; Reigan, Philip; Ladinsky, Mark; McManaman, James L.

2011-01-01

Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion. PMID:21383012

Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes.

PubMed

Nallani, Madhavan; Andreasson-Ochsner, Mirjam; Tan, Cherng-Wen Darren; Sinner, Eva-Kathrin; Wisantoso, Yudi; Geifman-Shochat, Susana; Hunziker, Walter

2011-12-01

Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.

Use of hyperdry amniotic membrane in operations for cleft palate: a study in rats.

PubMed

Tsuno, Hiroaki; Noguchi, Makoto; Okabe, Motonori; Tomihara, Kei; Yoshida, Toshiko; Nikaido, Toshio

2015-04-01

The growth of maxillary bone and the development of dentition are often impaired in patients who have had pushback operations for repair of a cleft palate. There has been considerable discussion about the most suitable technique or material used in such repairs to resolve the problem. Hyperdry amniotic membrane, a new preservable material derived from human amnion, has recently been introduced in several procedures. We have evaluated its use during pushback surgery in animal studies to try to correct the inhibition of growth and development of the maxilla. Mucosal defects were created in 3-week-old rats, and then covered with hyperdry amniotic membrane or not. Healing was assessed by histological and morphological examination at 1 week and 7 weeks postoperatively. In the group treated with hyperdry amniotic membrane, submucosal tissue was reconstructed successfully during the early postoperative period. Lateral palatal growth was not inhibited as much, and medial inclination of the teeth was less, after a period of growth using this material. The results suggest that hyperdry amniotic membrane is a suitable new dressing material for use in the treatment of cleft palate. Copyright © 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Pretreatment and Membrane Hydrophilic Modification to Reduce Membrane Fouling

PubMed Central

Sun, Wen; Liu, Junxia; Chu, Huaqiang; Dong, Bingzhi

2013-01-01

The application of low pressure membranes (microfiltration/ultrafiltration) has undergone accelerated development for drinking water production. However, the major obstacle encountered in its popularization is membrane fouling caused by natural organic matter (NOM). This paper firstly summarizes the two factors causing the organic membrane fouling, including molecular weight (MW) and hydrophilicity/hydrophobicity of NOM, and then presents a brief introduction of the methods which can prevent membrane fouling such as pretreatment of the feed water (e.g., coagulation, adsorption, and pre-oxidation) and membrane hydrophilic modification (e.g., plasma modification, irradiation grafting modification, surface coating modification, blend modification, etc.). Perspectives of further research are also discussed. PMID:24956947

Recent developments on ion-exchange membranes and electro-membrane processes.

PubMed

Nagarale, R K; Gohil, G S; Shahi, Vinod K

2006-02-28

Rapid growth of chemical and biotechnology in diversified areas fuels the demand for the need of reliable green technologies for the down stream processes, which include separation, purification and isolation of the molecules. Ion-exchange membrane technologies are non-hazardous in nature and being widely used not only for separation and purification but their application also extended towards energy conversion devices, storage batteries and sensors etc. Now there is a quite demand for the ion-exchange membrane with better selectivities, less electrical resistance, high chemical, mechanical and thermal stability as well as good durability. A lot of work has been done for the development of these types of ion-exchange membranes during the past twenty-five years. Herein we have reviewed the preparation of various types of ion-exchange membranes, their characterization and applications for different electro-membrane processes. Primary attention has been given to the chemical route used for the membrane preparation. Several general reactions used for the preparation of ion-exchange membranes were described. Methodologies used for the characterization of these membranes and their applications were also reviewed for the benefit of readers, so that they can get all information about the ion-exchange membranes at one platform. Although there are large number of reports available regarding preparations and applications of ion-exchange membranes more emphasis were predicted for the usefulness of these membranes or processes for solving certain type of industrial or social problems. More efforts are needed to bring many products or processes to pilot scale and extent their applications.

Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells.

PubMed

Dufrêne, Y F

2001-02-01

The application of atomic force microscopy (AFM) to probe the ultrastructure and physical properties of microbial cell surfaces is reviewed. The unique capabilities of AFM can be summarized as follows: imaging surface topography with (sub)nanometer lateral resolution; examining biological specimens under physiological conditions; measuring local properties and interaction forces. AFM is being used increasingly for: (i) visualizing the surface ultrastructure of microbial cell surface layers, including bacterial S-layers, purple membranes, porin OmpF crystals and fungal rodlet layers; (ii) monitoring conformational changes of individual membrane proteins; (iii) examining the morphology of bacterial biofilms, (iv) revealing the nanoscale structure of living microbial cells, including fungi, yeasts and bacteria, (v) mapping interaction forces at microbial surfaces, such as van der Waals and electrostatic forces, solvation forces, and steric/bridging forces; and (vi) probing the local mechanical properties of cell surface layers and of single cells.

Nanothin Coculture Membranes with Tunable Pore Architecture and Thermoresponsive Functionality for Transfer-Printable Stem Cell-Derived Cardiac Sheets.

PubMed

Ryu, Seungmi; Yoo, Jin; Jang, Yeongseon; Han, Jin; Yu, Seung Jung; Park, Jooyeon; Jung, Seon Yeop; Ahn, Kyung Hyun; Im, Sung Gap; Char, Kookheon; Kim, Byung-Soo

2015-10-27

Coculturing stem cells with the desired cell type is an effective method to promote the differentiation of stem cells. The features of the membrane used for coculturing are crucial to achieving the best outcome. Not only should the membrane act as a physical barrier that prevents the mixing of the cocultured cell populations, but it should also allow effective interactions between the cells. Unfortunately, conventional membranes used for coculture do not sufficiently meet these requirements. In addition, cell harvesting using proteolytic enzymes following coculture impairs cell viability and the extracellular matrix (ECM) produced by the cultured cells. To overcome these limitations, we developed nanothin and highly porous (NTHP) membranes, which are ∼20-fold thinner and ∼25-fold more porous than the conventional coculture membranes. The tunable pore size of NTHP membranes at the nanoscale level was found crucial for the formation of direct gap junctions-mediated contacts between the cocultured cells. Differentiation of the cocultured stem cells was dramatically enhanced with the pore size-customized NTHP membrane system compared to conventional coculture methods. This was likely due to effective physical contacts between the cocultured cells and the fast diffusion of bioactive molecules across the membrane. Also, the thermoresponsive functionality of the NTHP membranes enabled the efficient generation of homogeneous, ECM-preserved, highly viable, and transfer-printable sheets of cardiomyogenically differentiated cells. The coculture platform developed in this study would be effective for producing various types of therapeutic multilayered cell sheets that can be differentiated from stem cells.

Membrane rupture generates single open membrane sheets during vaccinia virus assembly.

PubMed

Chlanda, Petr; Carbajal, Maria Alejandra; Cyrklaff, Marek; Griffiths, Gareth; Krijnse-Locker, Jacomine

2009-07-23

The biogenesis and dynamics of cellular membranes are governed by fusion and fission processes that ensure the maintenance of closed compartments. These principles also apply to viruses during acquisition of their envelope. Based on conventional electron microscopy (EM), however, it has been proposed that poxviruses assemble from membranes made de novo with "free" ends in the cytoplasm. Here, we analyze the origin and structure of poxvirus membranes in a close-to-native state and in three dimensions by using cryopreservation and electron tomography (ET). By cryo-EM, the precursor membrane of poxviruses appears as an open membrane sheet stabilized by a protein scaffold. ET shows that this membrane is derived from pre-existing cellular membranes that rupture to generate an open compartment, rather than being made de novo. Thus, poxvirus infection represents an excellent system to study how cytoplasmic membranes can form open sheets by a process distinct from well-defined mechanisms of membrane biogenesis.

Glucocorticoids in the prefrontal cortex enhance memory consolidation and impair working memory by a common neural mechanism

PubMed Central

Barsegyan, Areg; Mackenzie, Scott M.; Kurose, Brian D.; McGaugh, James L.; Roozendaal, Benno

2010-01-01

It is well established that acute administration of adrenocortical hormones enhances the consolidation of memories of emotional experiences and, concurrently, impairs working memory. These different glucocorticoid effects on these two memory functions have generally been considered to be independently regulated processes. Here we report that a glucocorticoid receptor agonist administered into the medial prefrontal cortex (mPFC) of male Sprague-Dawley rats both enhances memory consolidation and impairs working memory. Both memory effects are mediated by activation of a membrane-bound steroid receptor and depend on noradrenergic activity within the mPFC to increase levels of cAMP-dependent protein kinase. These findings provide direct evidence that glucocorticoid effects on both memory consolidation and working memory share a common neural influence within the mPFC. PMID:20810923

Water Membrane Evaporator

NASA Technical Reports Server (NTRS)

Ungar, Eugene K.; Almlie, Jay C.

2010-01-01

A water membrane evaporator (WME) has been conceived and tested as an alternative to the contamination-sensitive and corrosion-prone evaporators currently used for dissipating heat from space vehicles. The WME consists mainly of the following components: An outer stainless-steel screen that provides structural support for the components mentioned next; Inside and in contact with the stainless-steel screen, a hydrophobic membrane that is permeable to water vapor; Inside and in contact with the hydrophobic membrane, a hydrophilic membrane that transports the liquid feedwater to the inner surface of the hydrophobic membrane; Inside and in contact with the hydrophilic membrane, an annular array of tubes through which flows the spacecraft coolant carrying the heat to be dissipated; and An inner exclusion tube that limits the volume of feedwater in the WME. In operation, a pressurized feedwater reservoir is connected to the volume between the exclusion tube and the coolant tubes. Feedwater fills the volume, saturates the hydrophilic membrane, and is retained by the hydrophobic membrane. The outside of the WME is exposed to space vacuum. Heat from the spacecraft coolant is conducted through the tube walls and the water-saturated hydrophilic membrane to the liquid/vapor interface at the hydrophobic membrane, causing water to evaporate to space. Makeup water flows into the hydrophilic membrane through gaps between the coolant tubes.

Electrospun superhydrophobic membranes with unique structures for membrane distillation.

PubMed

Liao, Yuan; Loh, Chun-Heng; Wang, Rong; Fane, Anthony G

2014-09-24

With modest temperature demand, low operating pressure, and high solute rejection, membrane distillation (MD) is an attractive option for desalination, waste treatment, and food and pharmaceutical processing. However, large-scale practical applications of MD are still hindered by the absence of effective membranes with high hydrophobicity, high porosity, and adequate mechanical strength, which are important properties for MD permeation fluxes, stable long-term performance, and effective packing in modules without damage. This study describes novel design strategies for highly robust superhydrophobic dual-layer membranes for MD via electrospinning. One of the newly developed membranes comprises a durable and ultrathin 3-dimensional (3D) superhydrophobic skin and porous nanofibrous support whereas another was fabricated by electrospinning 3D superhydrophobic layers on a nonwoven support. These membranes exhibit superhydrophobicity toward distilled water, salty water, oil-in-water emulsion, and beverages, which enables them to be used not only for desalination but also for other processes. The superhydrophobic dual-layer membrane #3S-N with nanofibrous support has a competitive permeation flux of 24.6 ± 1.2 kg m(-2) h(-1) in MD (feed and permeate temperate were set as 333 and 293 K, respectively) due to the higher porosity of the nanofibrous scaffold. Meanwhile, the membranes with the nonwoven support exhibit greater mechanical strength due to this support combined with better long-term performance because of the thicker 3D superhydrophobic layers. The morphology, pore size, porosity, mechanical properties, and liquid enter pressure of water of these superhydrophobic composite membranes with two different structures are reported and compared with commercial polyvinylidene fluoride membranes.

Supported inorganic membranes

DOEpatents

Sehgal, Rakesh; Brinker, Charles Jeffrey

1998-01-01

Supported inorganic membranes capable of molecular sieving, and methods for their production, are provided. The subject membranes exhibit high flux and high selectivity. The subject membranes are substantially defect free and less than about 100 nm thick. The pores of the subject membranes have an average critical pore radius of less than about 5 .ANG., and have a narrow pore size distribution. The subject membranes are prepared by coating a porous substrate with a polymeric sol, preferably under conditions of low relative pressure of the liquid constituents of the sol. The coated substrate is dried and calcined to produce the subject supported membrane. Also provided are methods of derivatizing the surface of supported inorganic membranes with metal alkoxides. The subject membranes find use in a variety of applications, such as the separation of constituents of gaseous streams, as catalysts and catalyst supports, and the like.

Composite Membrane with Underwater-Oleophobic Surface for Anti-Oil-Fouling Membrane Distillation.

PubMed

Wang, Zhangxin; Hou, Deyin; Lin, Shihong

2016-04-05

In this study, we fabricated a composite membrane for membrane distillation (MD) by modifying a commercial hydrophobic polyvinylidene fluoride (PVDF) membrane with a nanocomposite coating comprising silica nanoparticles, chitosan hydrogel and fluoro-polymer. The composite membrane exhibits asymmetric wettability, with the modified surface being in-air hydrophilic and underwater oleophobic, and the unmodified surface remaining hydrophobic. By comparing the performance of the composite membrane and the pristine PVDF membrane in direct contact MD experiments using a saline emulsion with 1000 ppm crude oil (in water), we showed that the fabricated composite membrane was significantly more resistant to oil fouling compared to the pristine hydrophobic PVDF membrane. Force spectroscopy was conducted for the interaction between an oil droplet and the membrane surface using a force tensiometer. The difference between the composite membrane and the pristine PVDF membrane in their interaction with an oil droplet served to explain the difference in the fouling propensities between these two membranes observed in MD experiments. The results from this study suggest that underwater oleophobic coating can effectively mitigate oil fouling in MD operations, and that the fabricated composite membrane with asymmetric wettability can enable MD to desalinate hypersaline wastewater with high concentrations of hydrophobic contaminants.

Study of structural stability and damaging effect on membrane for four Aβ42 dimers

PubMed Central

Feng, Wei; Lei, Huimin; Si, Jiarui; Zhang, Tao

2017-01-01

Increasing evidence shows that Aβ oligomers are key pathogenic molecules in Alzheimer’s disease. Among Aβ oligomers, dimer is the smallest aggregate and toxic unit. Therefore, understanding its structural and dynamic properties is quite useful to prevent the formation and toxicity of the Aβ oligomers. In this study, we performed molecular dynamic simulations on four Aβ42 dimers, 2NCb, CNNC, NCNC and NCCN, within the hydrated DPPC membrane. Four Aβ42 dimers differ in the arrangements of two Aβ42 peptides. This study aims to investigate the impact of aggregation pattern of two Aβ peptides on the structural stability of the Aβ42 dimer and its disruption to the biological membrane. The MD results demonstrate that the NCCN, CNNC and NCNC have the larger structural fluctuation at the N-terminus of Aβ42 peptide, where the β-strand structure converts into the coil structure. The loss of the N-terminal β-strand further impairs the aggregate ability of Aβ42 dimer. In addition, inserting Aβ42 dimer into the membrane can considerably decrease the average APL of DPPC membrane. Moreover this decrease effect is largely dependent on the distance to the location of Aβ42 dimer and its secondary structure forms. Based on the results, the 2NCb is considered as a stable dimeric unit for aggregating the larger Aβ42 oligomer, and has a potent ability to disrupt the membrane. PMID:28594887

Relationship between the mobility of phosphocholine headgroups of liposomes and the hydrophobicity at the membrane interface: a characterization with spectrophotometric measurements.

PubMed

Shimanouchi, Toshinori; Sasaki, Masashi; Hiroiwa, Azusa; Yoshimoto, Noriko; Miyagawa, Kazuya; Umakoshi, Hiroshi; Kuboi, Ryoichi

2011-11-01

In this study, we investigated the dynamics of a membrane interface of liposomes prepared by eight zwitterionic phosphatidylcholines in terms of their headgroup mobility, with spectroscopic methods such as dielectric dispersion analysis (DDA), fluorescence spectroscopy. The DDA measurement is based on the response of the permanent dipole moment to a driving electric field and could give the information on the axial rotational Brownian motion of a headgroup with the permanent dipole moment. This motion depended on kinds of phospholipids, the diameter of the liposomes, and the temperature. The activation energy required to overcome the intermolecular force between headgroups of phospholipids depended on the strength of the interaction between headgroups such as hydrogen bonds and/or dipole-dipole interaction. Hydration at the phosphorous group of phospholipid and the molecular order of lipid membrane impaired the interaction between headgroups. Furthermore, the hydrophobicity of membrane surface increased parallel to the increase in headgroup mobility. It is, therefore, concluded that hydration of headgroup promoted its mobility to make the membrane surface hydrophobic. The lipid membrane in liquid crystalline phase or the lipid membrane with the larger curvature was more hydrophobic. Copyright © 2011 Elsevier B.V. All rights reserved.

Mild Cognitive Impairment

MedlinePlus

... your local chapter Join our online community Mild Cognitive Impairment Mild cognitive impairment (MCI) causes a slight ... About Symptoms Diagnosis Causes & risks Treatments About Mild Cognitive Impairment Prevalence of MCI Approximately 15 to 20 ...

Comparison and Analysis of Membrane Fouling between Flocculent Sludge Membrane Bioreactor and Granular Sludge Membrane Bioreactor

PubMed Central

Zhi-Qiang, Chen; Jun-Wen, Li; Yi-Hong, Zhang; Xuan, Wang; Bin, Zhang

2012-01-01

The goal of this study is to investigate the effect of inoculating granules on reducing membrane fouling. In order to evaluate the differences in performance between flocculent sludge and aerobic granular sludge in membrane reactors (MBRs), two reactors were run in parallel and various parameters related to membrane fouling were measured. The results indicated that specific resistance to the fouling layer was five times greater than that of mixed liquor sludge in the granular MBR. The floc sludge more easily formed a compact layer on the membrane surface, and increased membrane resistance. Specifically, the floc sludge had a higher moisture content, extracellular polymeric substances concentration, and negative surface charge. In contrast, aerobic granules could improve structural integrity and strength, which contributed to the preferable permeate performance. Therefore, inoculating aerobic granules in a MBR presents an effective method of reducing the membrane fouling associated with floc sludge the perspective of from the morphological characteristics of microbial aggregates. PMID:22859954

Autophagosomal membranes assemble at ER-plasma membrane contact sites.

PubMed

Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne

2017-01-01

The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

Membrane events in the acrosomal reaction of Limulus sperm. Membrane fusion, filament-membrane particle attachment, and the source and formation of new membrane surface

PubMed Central

1979-01-01

The membranes of Limulus (horseshoe crab) sperm were examined before and during the acrosomal reaction by using the technique of freeze- fracturing and thin sectioning. We focused on three areas. First, we examined stages in the fusion of the acrosomal vacuole with the cell surface. Fusion takes place in a particle-free zone which is surrounded by a circlet of particles on the P face of the plasma membrane and an underlying circlet of particles on the P face of the acrosomal vauole membrane. These circlets of particles are present before induction. Up to nine focal points of fusion occur within the particle-free zone. Second, we describe a system of fine filaments, each 30 A in diameter, which lies between the acrosomal vacuole and the plasma membrane. These filaments change their orientation as the vacuole opens, a process that takes place in less than 50 ms. Membrane particles seen on the P face of the acrosomal vacuole membrane change their orientation at the same time and in the same way as do the filaments, thus indicating that the membrane particles and filaments are probably connected. Third, we examined the source and the point of fusion of new membrane needed to cover the acrosomal process. This new membrane is almost certainly derived from the outer nuclear envelope and appears to insert into the plasma membrane in a particle-free area adjacent to an area rich in particles. The latter is the region where the particles are probably connected to the cytoplasmic filaments. The relevance of these observations in relation to the process of fertilization of this fantastic sperm is discussed. PMID:582596

Composite sensor membrane

DOEpatents

Majumdar, Arun [Orinda, CA; Satyanarayana, Srinath [Berkeley, CA; Yue, Min [Albany, CA

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.

The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes.

PubMed

Naito, Daisuke; Ogata, Takehiro; Hamaoka, Tetsuro; Nakanishi, Naohiko; Miyagawa, Kotaro; Maruyama, Naoki; Kasahara, Takeru; Taniguchi, Takuya; Nishi, Masahiro; Matoba, Satoaki; Ueyama, Tomomi

2015-12-15

Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function. Copyright © 2015 the American Physiological Society.

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.

Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

PubMed

Sun, Bingyun; Hood, Leroy

2014-06-06

The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

MTCL1 crosslinks and stabilizes non-centrosomal microtubules on the Golgi membrane.

PubMed

Sato, Yoshinori; Hayashi, Kenji; Amano, Yoshiko; Takahashi, Mikiko; Yonemura, Shigenobu; Hayashi, Ikuko; Hirose, Hiroko; Ohno, Shigeo; Suzuki, Atsushi

2014-11-04

Recent studies have revealed the presence of a microtubule subpopulation called Golgi-derived microtubules that support Golgi ribbon formation, which is required for maintaining polarized cell migration. CLASPs and AKAP450/CG-NAP are involved in their formation, but the underlying molecular mechanisms remain unclear. Here, we find that the microtubule-crosslinking protein, MTCL1, is recruited to the Golgi membranes through interactions with CLASPs and AKAP450/CG-NAP, and promotes microtubule growth from the Golgi membrane. Correspondingly, MTCL1 knockdown specifically impairs the formation of the stable perinuclear microtubule network to which the Golgi ribbon tethers and extends. Rescue experiments demonstrate that besides its crosslinking activity mediated by the N-terminal microtubule-binding region, the C-terminal microtubule-binding region plays essential roles in these MTCL1 functions through a novel microtubule-stabilizing activity. These results suggest that MTCL1 cooperates with CLASPs and AKAP450/CG-NAP in the formation of the Golgi-derived microtubules, and mediates their development into a stable microtubule network.

Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

PubMed

Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

2012-01-01

Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. Copyright © 2011 Elsevier B.V. All rights reserved.

Residues in the membrane-spanning domain core modulate conformation and fusogenicity of the HIV-1 envelope glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shang Liang; Hunter, Eric, E-mail: eric.hunter2@emory.ed

2010-09-01

The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant 'core' structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD 'core' do not influence specific virus-cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD 'core' delayed the kinetics and reduced the efficiency of cell-cell fusion mediated by Env. Although we observed no evidence that membrane fusionmore » mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process.« less

A novel high light-inducible carotenoid-binding protein complex in the thylakoid membranes of Synechocystis PCC 6803.

PubMed

Daddy, Soumana; Zhan, Jiao; Jantaro, Saowarath; He, Chenliu; He, Qingfang; Wang, Qiang

2015-03-30

Synechocystis sp. PCC 6803 is a model cyanobacterium extensively used to study photosynthesis. Here we reveal a novel high light-inducible carotenoid-binding protein complex (HLCC) in the thylakoid membranes of Synechocystis PCC 6803 cells exposed to high intensity light. Zeaxanthin and myxoxanthophyll accounted for 29.8% and 54.8%, respectively, of the carotenoids bound to the complex. Using Blue-Native PAGE followed by 2D SDS-PAGE and mass spectrometry, we showed that the HLCC consisted of Slr1128, IsiA, PsaD, and HliA/B. We confirmed these findings by SEAD fluorescence cross-linking and anti-PsaD immuno-coprecipitation analyses. The expression of genes encoding the protein components of the HLCC was enhanced by high light illumination and artificial oxidative stress. Deletion of these proteins resulted in impaired state transition and increased sensitivity to oxidative and/or high light stress, as indicated by increased membrane peroxidation. Therefore, the HLCC protects thylakoid membranes from extensive photooxidative damage, likely via a mechanism involving state transition.

Chemical degradation mechanisms of membranes for alkaline membrane fuel cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choe, Yoong-Kee; Henson, Neil J.; Kim, Yu Seung

2015-12-31

Chemical degradation mechanisms of membranes for alkaline membrane fuel cells have been investigated using density functional theory (DFT). We have elucidated that the aryl-ether moiety of membranes is one of the weakest site against attack of hydroxide ions. The results of DFT calculations for hydroxide initiated aryl-ether cleavage indicated that the aryl-ether cleavage occurred prior to degradation of cationic functional group. Such a weak nature of the aryl-ether group arises from the electron deficiency of the aryl group as well as the low bond dissociation energy. The DFT results suggests that removal of the aryl-ether group in the membrane shouldmore » enhance the stability of membranes under alkaline conditions. In fact, an ether fee poly(phenylene) membrane exhibits excellent stability against the attack from hydroxide ions.« less

Tannin-rich fraction from pomegranate rind damages membrane of Listeria monocytogenes.

PubMed

Li, Guanghui; Xu, Yunfeng; Wang, Xin; Zhang, Baigang; Shi, Chao; Zhang, Weisong; Xia, Xiaodong

2014-04-01

Pomegranate rind has been reported to inhibit several foodborne pathogens, and its antimicrobial activity has been attributed mainly to its tannin fraction. This study aimed to investigate the antimicrobial activity of the tannin-rich fraction from pomegranate rind (TFPR) against Listeria monocytogenes and its mechanism of action. The tannin-related components of TFPR were analyzed by high-performance liquid chromatography and liquid chromatography-mass spectrometry, and the minimum inhibitory concentration (MIC) of TFPR was determined using the agar dilution method. Extracellular potassium concentration, the release of cell constituents, intra- and extracellular ATP concentrations, membrane potential, and intracellular pH (pHin) were measured to elucidate a possible antibacterial mechanism. Punicalagin (64.2%, g/g) and ellagic acid (3.1%, g/g) were detected in TFPR, and the MICs of TFPR were determined to be 1.25-5.0 mg/mL for different L. monocytogenes strains. Treatment with TFPR induced a decrease of the intracellular ATP concentration, an increase of the extracellular concentrations of potassium and ATP, and the release of cell constituents. A reduction of pHin and cell membrane hyperpolarization were observed after treatment. Electron microscopic observations showed that the cell membrane structures of L. monocytogenes were apparently impaired by TFPR. It is concluded that TFPR could destroy the integrity of the cell membrane of L. monocytogenes, leading to a loss of cell homeostasis. These findings indicate that TFPR has the potential to be used as a food preservative in order to control L. monocytogenes contamination in food and reduce the risk of listeriosis.

Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

PubMed

Suetsugu, Shiro; Kurisu, Shusaku; Takenawa, Tadaomi

2014-10-01

All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes. Copyright © 2014 the

Protein Solvation in Membranes and at Water-Membrane Interfaces

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Chipot, Christophe; Wilson, Michael A.

2002-01-01

Different salvation properties of water and membranes mediate a host of biologically important processes, such as folding, insertion into a lipid bilayer, associations and functions of membrane proteins. These processes will be discussed in several examples involving synthetic and natural peptides. In particular, a mechanism by which a helical peptide becomes inserted into a model membrane will be described. Further, the molecular mechanism of recognition and association of protein helical segments in membranes will be discussed. These processes are crucial for proper functioning of a cell. A membrane-spanning domain of glycophorin A, which exists as a helical dimer, serves as the model system. For this system, the free energy of dissociation of the helices is being determined for both the wild type and a mutant, in which dimerization is disrupted.

Membrane processes

NASA Astrophysics Data System (ADS)

Staszak, Katarzyna

2017-11-01

The membrane processes have played important role in the industrial separation process. These technologies can be found in all industrial areas such as food, beverages, metallurgy, pulp and paper, textile, pharmaceutical, automotive, biotechnology and chemical industry, as well as in water treatment for domestic and industrial application. Although these processes are known since twentieth century, there are still many studies that focus on the testing of new membranes' materials and determining of conditions for optimal selectivity, i. e. the optimum transmembrane pressure (TMP) or permeate flux to minimize fouling. Moreover the researchers proposed some calculation methods to predict the membrane processes properties. In this article, the laboratory scale experiments of membrane separation techniques, as well their validation by calculation methods are presented. Because membrane is the "heart" of the process, experimental and computational methods for its characterization are also described.

Polymeric and Lipid Membranes-From Spheres to Flat Membranes and vice versa.

PubMed

Saveleva, Mariia S; Lengert, Ekaterina V; Gorin, Dmitry A; Parakhonskiy, Bogdan V; Skirtach, Andre G

2017-08-15

Membranes are important components in a number of systems, where separation and control of the flow of molecules is desirable. Controllable membranes represent an even more coveted and desirable entity and their development is considered to be the next step of development. Typically, membranes are considered on flat surfaces, but spherical capsules possess a perfect "infinite" or fully suspended membranes. Similarities and transitions between spherical and flat membranes are discussed, while applications of membranes are also emphasized.

Anion permselective membrane

NASA Technical Reports Server (NTRS)

Hodgdon, R. B.; Waite, W. A.

1982-01-01

The synthesis and fabrication of polymeric anion permselective membranes for redox systems are discussed. Variations of the prime candidate anion membrane formulation to achieve better resistance and/or lower permeability were explored. Processing parameters were evaluated to lower cost and fabricate larger sizes. The processing techniques to produce more membranes per batch were successfully integrated with the fabrication of larger membranes. Membranes of about 107 cm x 51 cm were made in excellent yield. Several measurements were made on the larger sample membranes. Among the data developed were water transport and transference numbers for these prime candidate membranes at 20 C. Other work done on this system included characterization of a number of specimens of candidate membranes which had been returned after service lives of up to sixteen months. Work with new polymer constituents, with new N.P.'s, catalysts and backing fabrics is discussed. Some work was also done to evaluate other proportions of the ingredients of the prime candidate system. The adoption of a flow selectivity test at elevated temperature was explored.

Evaluation method of membrane performance in membrane distillation process for seawater desalination.

PubMed

Chung, Seungjoon; Seo, Chang Duck; Choi, Jae-Hoon; Chung, Jinwook

2014-01-01

Membrane distillation (MD) is an emerging desalination technology as an energy-saving alternative to conventional distillation and reverse osmosis method. The selection of appropriate membrane is a prerequisite for the design of an optimized MD process. We proposed a simple approximation method to evaluate the performance of membranes for MD process. Three hollow fibre-type commercial membranes with different thicknesses and pore sizes were tested. Experimental results showed that one membrane was advantageous due to the highest flux, whereas another membrane was due to the lowest feed temperature drop. Regression analyses and multi-stage calculations were used to account for the trade-offeffects of flux and feed temperature drop. The most desirable membrane was selected from tested membranes in terms of the mean flux in a multi-stage process. This method would be useful for the selection of the membranes without complicated simulation techniques.

Electronic paramagnetic resonance investigation of the activity of Origanum vulgare L. essential oil on the Listeria monocytogenes membrane.

PubMed

Serio, A; Chiarini, M; Tettamanti, E; Paparella, A

2010-08-01

To evaluate the effect of oregano essential oil on Listeria monocytogenes cytoplasmic membrane. Nitroxide free-radical Electron Paramagnetic Resonance was applied on L. monocytogenes after 30 min exposure to oregano essential oil concentrations ranging from 0 to 1.25%. The impact of essential oil on the number of viable cells was evaluated by plate count. Growth dynamics of survivors in BHI and TSB were evaluated by turbidometry. After exposure to essential oil concentrations up to 0.50%, the membrane fluidity was changed and its order increased. When L. monocytogenes was exposed to higher concentrations, membrane order parameters slightly returned to the values of untreated cells. However, when the cells were exposed to EO in the presence of sodium azide, which impairs energy metabolism, the membrane fluidity was progressively enhanced, even at the lowest EO concentration (0.25%). Microbiological analyses confirmed a progressive reduction of viable count, at increasing essential oil concentrations. Both in BHI and TSB, the Lag phase length increased in treated cells with respect to controls, suggesting a cell damage recovery. The combined approach including microbiological and EPR analyses provided relevant information on membrane modification and cell response to essential oils. EPR approach was demonstrated to be an effective and helpful tool to comprehend the modifications exerted by essential oil on the bacterial membrane.

20 CFR 416.998 - If you become disabled by another impairment(s).

Code of Federal Regulations, 2012 CFR

2012-04-01

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20 CFR 416.998 - If you become disabled by another impairment(s).

Code of Federal Regulations, 2014 CFR

2014-04-01

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20 CFR 416.998 - If you become disabled by another impairment(s).

Code of Federal Regulations, 2013 CFR

2013-04-01

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20 CFR 416.998 - If you become disabled by another impairment(s).

Code of Federal Regulations, 2011 CFR

2011-04-01

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20 CFR 416.998 - If you become disabled by another impairment(s).

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false If you become disabled by another impairment... INCOME FOR THE AGED, BLIND, AND DISABLED Determining Disability and Blindness Continuing Or Stopping Disability Or Blindness § 416.998 If you become disabled by another impairment(s). If a new severe impairment...

Selective Permeability of PVA Membranes. I: Radiation-Crosslinked Membranes

NASA Technical Reports Server (NTRS)

Katz, Moshe G.; Wydeven, Theodore, Jr.

1981-01-01

The water and salt transport properties of ionizing radiation crosslinked poly(vinyl alcohol) (PVA) membranes were investigated. The studied membranes showed high permeabilities and low selectivities for both water and salt. The results were found to be in accord with a modified solution-diffusion model for transport across the membranes, in which pressure-dependent permeability coefficients are employed.

Selective permeability of PVA membranes. I - Radiation-crosslinked membranes

NASA Technical Reports Server (NTRS)

Katz, M. G.; Wydeven, T., Jr.

1981-01-01

The water and salt transport properties of ionizing radiation crosslinked poly(vinyl alcohol) (PVA) membranes were investigated. The studied membranes showed high permeabilities and low selectivities for both water and salt. The results were found to be in accord with a modified solution-diffusion model for transport across the membranes, in which pressure-dependent permeability coefficients are employed.

Palaeontological evidence of membrane relationship in step-by-step membrane fusion

PubMed Central

WANG, XIN; LIU, WENZHE; DU, KAIHE

2011-01-01

Studies on membrane fusion in living cells indicate that initiation of membrane fusion is a transient and hard to capture process. Despite previous research, membrane behaviour at this point is still poorly understood. Recent palaeobotanical research has revealed snapshots of membrane fusion in a 15-million-year-old fossil pinaceous cone. To reveal the membrane behaviour during the fusion, we conducted more observations on the same fossil material. Several discernible steps of membrane fusion have been fixed naturally and observed in the fossil material. This observation provides transmission electron microscope (TEM) images of the transient intermediate stage and clearly shows the relationship between membranes. Observing such a transient phenomenon in fossil material implies that the fixing was most likely accomplished quickly by a natural process. The mechanism behind this phenomenon is clearly worthy of further enquiry. PMID:21190428

Membrane Resonance Enables Stable and Robust Gamma Oscillations

PubMed Central

Moca, Vasile V.; Nikolić, Danko; Singer, Wolf; Mureşan, Raul C.

2014-01-01

Neuronal mechanisms underlying beta/gamma oscillations (20–80 Hz) are not completely understood. Here, we show that in vivo beta/gamma oscillations in the cat visual cortex sometimes exhibit remarkably stable frequency even when inputs fluctuate dramatically. Enhanced frequency stability is associated with stronger oscillations measured in individual units and larger power in the local field potential. Simulations of neuronal circuitry demonstrate that membrane properties of inhibitory interneurons strongly determine the characteristics of emergent oscillations. Exploration of networks containing either integrator or resonator inhibitory interneurons revealed that: (i) Resonance, as opposed to integration, promotes robust oscillations with large power and stable frequency via a mechanism called RING (Resonance INduced Gamma); resonance favors synchronization by reducing phase delays between interneurons and imposes bounds on oscillation cycle duration; (ii) Stability of frequency and robustness of the oscillation also depend on the relative timing of excitatory and inhibitory volleys within the oscillation cycle; (iii) RING can reproduce characteristics of both Pyramidal INterneuron Gamma (PING) and INterneuron Gamma (ING), transcending such classifications; (iv) In RING, robust gamma oscillations are promoted by slow but are impaired by fast inputs. Results suggest that interneuronal membrane resonance can be an important ingredient for generation of robust gamma oscillations having stable frequency. PMID:23042733

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)

Specific interaction of IM30/Vipp1 with cyanobacterial and chloroplast membranes results in membrane remodeling and eventually in membrane fusion.

PubMed

Heidrich, Jennifer; Thurotte, Adrien; Schneider, Dirk

2017-04-01

The photosynthetic light reaction takes place within the thylakoid membrane system in cyanobacteria and chloroplasts. Besides its global importance, the biogenesis, maintenance and dynamics of this membrane system are still a mystery. In the last two decades, strong evidence supported the idea that these processes involve IM30, the inner membrane-associated protein of 30kDa, a protein also known as the vesicle-inducing protein in plastids 1 (Vipp1). Even though we just only begin to understand the precise physiological function of this protein, it is clear that interaction of IM30 with membranes is crucial for biogenesis of thylakoid membranes. Here we summarize and discuss forces guiding IM30-membrane interactions, as the membrane properties as well as the oligomeric state of IM30 appear to affect proper interaction of IM30 with membrane surfaces. Interaction of IM30 with membranes results in an altered membrane structure and can finally trigger fusion of adjacent membranes, when Mg 2+ is present. Based on recent results, we finally present a model summarizing individual steps involved in IM30-mediated membrane fusion. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. Copyright © 2016 Elsevier B.V. All rights reserved.

Polymeric water filtration membranes

NASA Astrophysics Data System (ADS)

Paul, Mou

Nanofiltration (NF) membranes are used for separating salts and small neutral molecules. NF membranes show unique selectivity properties compared to reverse osmosis membranes as it can selectively pass monovalent salts and neutral molecules as a function of charge and molecular weight cut-off which are dependent on membrane characteristics and operating conditions. Dow Water and Process solutions has been a pioneer in the membrane based water purification field and Dow's role was instrumental in developing several NF membranes for different applications. However, the characterization of NF membranes and hence the development of structure-property relationship is challenging due to the nanoscale thin, crosslinked nature of the membrane. Recently significant efforts were employed to develop analytical capabilities to understand polymer structure and composition and it had been possible to achieve a structure-property relationship for NF membranes. This paper will highlight similar relationships and will also focus on the relationships of membrane structure with membrane transport properties and how this relationship influences products for different application areas such as in oil field, sweetener and minimum liquid discharge etc.

Ion-conducting membranes

DOEpatents

Masel, Richard I.; Sajjad, Syed Dawar; Gao, Yan; Liu, Zengcai; Chen, Qingmei

2017-12-26

An anion-conducting polymeric membrane comprises a terpolymer of styrene, vinylbenzyl-R.sub.s and vinylbenzyl-R.sub.x. R.sub.s is a positively charged cyclic amine group. R.sub.x is at least one constituent selected from the group consisting Cl, OH and a reaction product between an OH or Cl and a species other than a simple amine or a cyclic amine. The total weight of the vinylbenzyl-R.sub.x groups is greater than 0.3% of the total weight of the membrane. In a preferred embodiment, the membrane is a Helper Membrane that increases the faradaic efficiency of an electrochemical cell into which the membrane is incorporated, and also allows product formation at lower voltages than in cells without the Helper Membrane.

Glasslike Membrane Protein Diffusion in a Crowded Membrane.

PubMed

Munguira, Ignacio; Casuso, Ignacio; Takahashi, Hirohide; Rico, Felix; Miyagi, Atsushi; Chami, Mohamed; Scheuring, Simon

2016-02-23

Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion in vivo and in vitro using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities. Here, we use high-speed atomic force microscopy and a novel analysis methodology to analyze the pore forming protein lysenin in a highly crowded environment and document coexistence of several diffusion regimes within one membrane. We show the formation of local glassy phases, where proteins are trapped in neighbor-formed cages for time scales up to 10 s, which had not been previously experimentally reported for biological membranes. Furthermore, around solid-like patches and immobile molecules a slower glass phase is detected leading to protein trapping and creating a perimeter of decreased membrane diffusion.

Membrane preparation and solubilization.

PubMed

Roy, Ankita

2015-01-01

Membrane proteins play an essential role in several biological processes like ion transport, signal transduction, and electron transfer to name a few. For structural and functional studies of integral membrane proteins, it is critically important to isolate proteins from the membrane using biological detergents. Detergents disrupt the native lipid components of the native membrane and encase the membrane protein in an unnatural environment in aqueous solution. However, a particular membrane protein is best solubilized in a specific detergent; therefore, screening for the optimal detergent is essential. Apart from keeping the membrane protein monodispered in solution, the detergent has to be compatible with downstream processes to isolate and characterize a membrane protein. Over the past several years, a number of membrane proteins have been successfully isolated for structural and functional studies that allowed an outline of general strategies for isolating a novel membrane protein of interest. © 2015 Elsevier Inc. All rights reserved.

Examining the Role of Membrane Lipid Composition in Determining the Ethanol Tolerance of Saccharomyces cerevisiae

PubMed Central

Henderson, Clark M.

2014-01-01

Yeast (Saccharomyces cerevisiae) has an innate ability to withstand high levels of ethanol that would prove lethal to or severely impair the physiology of other organisms. Significant efforts have been undertaken to elucidate the biochemical and biophysical mechanisms of how ethanol interacts with lipid bilayers and cellular membranes. This research has implicated the yeast cellular membrane as the primary target of the toxic effects of ethanol. Analysis of model membrane systems exposed to ethanol has demonstrated ethanol's perturbing effect on lipid bilayers, and altering the lipid composition of these model bilayers can mitigate the effect of ethanol. In addition, cell membrane composition has been correlated with the ethanol tolerance of yeast cells. However, the physical phenomena behind this correlation are likely to be complex. Previous work based on often divergent experimental conditions and time-consuming low-resolution methodologies that limit large-scale analysis of yeast fermentations has fallen short of revealing shared mechanisms of alcohol tolerance in Saccharomyces cerevisiae. Lipidomics, a modern mass spectrometry-based approach to analyze the complex physiological regulation of lipid composition in yeast and other organisms, has helped to uncover potential mechanisms for alcohol tolerance in yeast. Recent experimental work utilizing lipidomics methodologies has provided a more detailed molecular picture of the relationship between lipid composition and ethanol tolerance. While it has become clear that the yeast cell membrane composition affects its ability to tolerate ethanol, the molecular mechanisms of yeast alcohol tolerance remain to be elucidated. PMID:24610851

Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

DOEpatents

Laible, Philip D; Hanson, Deborah K

2013-06-04

The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

A quinolinol-based small molecule with anti-MRSA activity that targets bacterial membrane and promotes fermentative metabolism.

PubMed

Nair, Dhanalakshmi R; Chen, Ji; Monteiro, João M; Josten, Michaele; Pinho, Mariana G; Sahl, Hans-Georg; Wu, Jimmy; Cheung, Ambrose

2017-10-01

In a loss-of-viability screen of small molecules against methicillin-resistant Staphylococcus aureus (MRSA) USA300, we found a small molecule, designated DNAC-2, which has an MIC of 8 μg ml -1 . DNAC-2 is a quinolinol derivative that is bactericidal at 2X MIC. Macromolecular synthesis assays at 2 × MIC of DNAC-2 revealed inhibition of DNA, cell wall, RNA and protein synthesis within fifteen to thirty minutes of treatment when compared to the untreated control. Transmission electron microscopy of DNAC-2-treated cells revealed a significantly thicker cell wall and impaired daughter cell separation. Exposure of USA300 cells to 1 × MIC of DNAC-2 resulted in mislocalization of PBP2 away from the septum in an FtsZ-independent manner. In addition, membrane localization with FM4-64, as well as depolarization study with DiOC 2 and lipophilic cation TPP+ displayed membrane irregularities and rapid membrane depolarization, respectively, in DNAC-2-treated cells vs -untreated control. However, DNAC-2 exhibited almost no toxicity toward eukaryotic membranes. Notably, DNAC-2 drives energy generation toward substrate level phosphorylation and the bacteria become more sensitive to DNAC-2 under anaerobic conditions. We propose that DNAC-2 affects USA300 by targeting the membrane, leading to partial membrane depolarization and subsequently affecting aerobic respiration and energy-dependent functional organization of macromolecular biosynthetic pathways. The multiple effects may have the desirable consequence of limiting the emergence of resistance to DNAC-2.

Epithelial Membrane Protein-2 in Human Proliferative Vitreoretinopathy and Epiretinal Membranes.

PubMed

Telander, David G; Yu, Alfred K; Forward, Krisztina I; Morales, Shawn A; Morse, Lawrence S; Park, Susanna S; Gordon, Lynn K

2016-06-01

To determine the level of epithelial membrane protein-2 (EMP2) expression in preretinal membranes from surgical patients with proliferative vitreoretinopathy (PVR) or epiretinal membranes (ERMs). EMP2, an integrin regulator, is expressed in the retinal pigment epithelium and understanding EMP2 expression in human retinal disease may help determine whether EMP2 is a potential therapeutic target. Preretinal membranes were collected during surgical vitrectomies after obtaining consents. The membranes were fixed, processed, sectioned, and protein expression of EMP2 was evaluated by immunohistochemistry. The staining intensity (SI) and percentage of positive cells (PP) in membranes were compared by masked observers. Membranes were categorized by their cause and type including inflammatory and traumatic. All of the membranes stained positive for EMP2. Proliferative vitreoretinopathy-induced membranes (all causes) showed greater expression of EMP2 than ERMs with higher SI (1.81 vs. 1.38; P = 0.07) and PP (2.08 vs. 1.54; P = 0.09). However all the PVR subgroups had similar levels of EMP2 expression without statistically significant differences by Kruskal-Wallis test. Inflammatory PVR had higher expression of EMP2 than ERMs (SI of 2.58 vs. 1.38); however, this was not statistically significant. No correlation was found between duration of PVR membrane and EMP2 expression. EMP2 was detected by RT-PCR in all samples (n = 6) tested. All studied ERMs and PVR membranes express EMP2. Levels of EMP2 trended higher in all PVR subgroups than in ERMs, especially in inflammatory and traumatic PVR. Future studies are needed to determine the role of EMP2 in the pathogenesis and treatment of various retinal conditions including PVR.

Membranous nephropathy

MedlinePlus

... glomerulonephritis; Membranous GN; Extramembranous glomerulonephritis; Glomerulonephritis - membranous; MGN Images Kidney anatomy References Appel GB, Radhakrishnan J. Glomerular disorders and nephrotic syndromes. In: Goldman L, ...

Impairment Rating Ambiguity in the United States: The Utah Impairment Guides for Calculating Workers' Compensation Impairments

PubMed Central

Hunter, Bradley; Bunkall, Larry D.; Holmes, Edward B.

2009-01-01

Since the implementation of workers' compensation, accurately and consistently rating impairment has been a concern for the employee and employer, as well as rating physicians. In an attempt to standardize and classify impairments, the American Medical Association (AMA) publishes the AMA Guides ("Guides"), and recently published its 6th edition of the AMA Guides. Common critiques of the AMA Guides 6th edition are that they are too complex, lacking in evidence-based methods, and rarely yield consistent ratings. Many states mandate use of some edition of the AMA Guides, but few states are adopting the current edition due to the increasing difficulty and frustration with their implementation. A clearer, simpler approach is needed. Some states have begun to develop their own supplemental guides to combat problems in complexity and validity. Likewise studies in Korea show that past methods for rating impairment are outdated and inconsistent, and call for measures to adapt current methods to Korea's specific needs. The Utah Supplemental Guides to the AMA Guides have been effective in increasing consistency in rating impairment. It is estimated that litigation of permanent impairment has fallen below 1% and Utah is now one of the least costly states for obtaining workers' compensation insurance, while maintaining a medical fee schedule above the national average. Utah's guides serve as a model for national or international impairment guides. PMID:19503678

Influences of acid-base property of membrane on interfacial interactions related with membrane fouling in a membrane bioreactor based on thermodynamic assessment.

PubMed

Zhao, Leihong; Qu, Xiaolu; Zhang, Meijia; Lin, Hongjun; Zhou, Xiaoling; Liao, Bao-Qiang; Mei, Rongwu; Hong, Huachang

2016-08-01

Failure of membrane hydrophobicity in predicting membrane fouling requires a more reliable indicator. In this study, influences of membrane acid base (AB) property on interfacial interactions in two different interaction scenarios in a submerged membrane bioreactor (MBR) were studied according to thermodynamic approaches. It was found that both the polyvinylidene fluoride (PVDF) membrane and foulant samples in the MBR had relatively high electron donor (γ(-)) component and low electron acceptor (γ(+)) component. For both of interaction scenarios, AB interaction was the major component of the total interaction. The results showed that, the total interaction monotonically decreased with membrane γ(-), while was marginally affected by membrane γ(+), suggesting that γ(-) could act as a reliable indicator for membrane fouling prediction. This study suggested that membrane modification for fouling mitigation should orient to improving membrane surface γ(-) component rather than hydrophilicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Separation membrane development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, M.W.

1998-08-01

A ceramic membrane has been developed to separate hydrogen from other gases. The method used is a sol-gel process. A thin layer of dense ceramic material is coated on a coarse ceramic filter substrate. The pore size distribution in the thin layer is controlled by a densification of the coating materials by heat treatment. The membrane has been tested by permeation measurement of the hydrogen and other gases. Selectivity of the membrane has been achieved to separate hydrogen from carbon monoxide. The permeation rate of hydrogen through the ceramic membrane was about 20 times larger than Pd-Ag membrane.

MitoQ regulates autophagy by inducing a pseudo-mitochondrial membrane potential

PubMed Central

Sun, Chao; Liu, Xiongxiong; Di, Cuixia; Wang, Zhenhua; Mi, Xiangquan; Liu, Yang; Zhao, Qiuyue; Mao, Aihong; Chen, Weiqiang; Gan, Lu; Zhang, Hong

2017-01-01

ABSTRACT During the process of oxidative phosphorylation, protons are pumped into the mitochondrial intermembrane space to establish a mitochondrial membrane potential (MMP). The electrochemical gradient generated allows protons to return to the matrix through the ATP synthase complex and generates ATP in the process. MitoQ is a lipophilic cationic drug that is adsorbed to the inner mitochondrial membrane; however, the cationic moiety of MitoQ remains in the intermembrane space. We found that the positive charges in MitoQ inhibited the activity of respiratory chain complexes I, III, and IV, reduced proton production, and decreased oxygen consumption. Therefore, a pseudo-MMP (PMMP) was formed via maintenance of exogenous positive charges. Proton backflow was severely impaired, leading to a decrease in ATP production and an increase in AMP production. Excess AMP activates AMP kinase, which inhibits the MTOR (mechanistic target of rapamycin) pathway and induces macroautophagy/autophagy. Therefore, we conclude that MitoQ increases PMMP via proton displacement with exogenous positive charges. In addition, PMMP triggered autophagy in hepatocellular carcinoma HepG2 cells via modification of mitochondrial bioenergetics pathways. PMID:28121478

MitoQ regulates autophagy by inducing a pseudo-mitochondrial membrane potential.

PubMed

Sun, Chao; Liu, Xiongxiong; Di, Cuixia; Wang, Zhenhua; Mi, Xiangquan; Liu, Yang; Zhao, Qiuyue; Mao, Aihong; Chen, Weiqiang; Gan, Lu; Zhang, Hong

2017-04-03

During the process of oxidative phosphorylation, protons are pumped into the mitochondrial intermembrane space to establish a mitochondrial membrane potential (MMP). The electrochemical gradient generated allows protons to return to the matrix through the ATP synthase complex and generates ATP in the process. MitoQ is a lipophilic cationic drug that is adsorbed to the inner mitochondrial membrane; however, the cationic moiety of MitoQ remains in the intermembrane space. We found that the positive charges in MitoQ inhibited the activity of respiratory chain complexes I, III, and IV, reduced proton production, and decreased oxygen consumption. Therefore, a pseudo-MMP (PMMP) was formed via maintenance of exogenous positive charges. Proton backflow was severely impaired, leading to a decrease in ATP production and an increase in AMP production. Excess AMP activates AMP kinase, which inhibits the MTOR (mechanistic target of rapamycin) pathway and induces macroautophagy/autophagy. Therefore, we conclude that MitoQ increases PMMP via proton displacement with exogenous positive charges. In addition, PMMP triggered autophagy in hepatocellular carcinoma HepG2 cells via modification of mitochondrial bioenergetics pathways.

Applicability of dynamic membrane technology in anaerobic membrane bioreactors.

PubMed

Ersahin, Mustafa Evren; Ozgun, Hale; Tao, Yu; van Lier, Jules B

2014-01-01

This study investigated the applicability of dynamic membrane technology in anaerobic membrane bioreactors for the treatment of high strength wastewaters. A monofilament woven fabric was used as support material for dynamic membrane formation. An anaerobic dynamic membrane bioreactor (AnDMBR) was operated under a variety of operational conditions, including different sludge retention times (SRTs) of 20 and 40 days in order to determine the effect of SRT on both biological performance and dynamic membrane filtration characteristics. High COD removal efficiencies exceeding 99% were achieved during the operation at both SRTs. Higher filtration resistances were measured during the operation at SRT of 40 days in comparison to SRT of 20 days, applying a stable flux of 2.6 L/m(2) h. The higher filtration resistances coincided with lower extracellular polymeric substances concentration in the bulk sludge at SRT of 40 days, likely resulting in a decreased particle flocculation. Results showed that dynamic membrane technology achieved a stable and high quality permeate and AnDMBRs can be used as a reliable and satisfactory technology for treatment of high strength wastewaters. Copyright © 2013 Elsevier Ltd. All rights reserved.

Novel magnetically induced membrane vibration (MMV) for fouling control in membrane bioreactors.

PubMed

Bilad, Muhammad R; Mezohegyi, Gergo; Declerck, Priscilla; Vankelecom, Ivo F J

2012-01-01

Conventional submerged membrane bioreactors (MBRs) rely on the coarse bubbles aeration to generate shear at the liquid-membrane interface to limit membrane fouling. Unfortunately, it is a very energy consuming method, still often resulting in a rapid decrease of membrane permeability and consequently in higher expenses. In this paper, the feasibility of a novel magnetically induced membrane vibration (MMV) system was studied in a lab-scale MBR treating synthetic wastewater. The effects on membrane fouling of applied electrical power of different operation strategies, of membrane flux and of the presence of multiple membranes on one vibrating engine on membrane fouling were investigated. The filtration performance was evaluated by determining the filtration resistance profiles and critical flux. The results showed clear advantages of the vibrating system over conventional MBR processes by ensuring higher fluxes at lower fouling rates. Intermittent vibration was found a promising strategy for both efficient fouling control and significant energy saving. The optimised MMV system is presumed to lead to significant energy and cost reduction in up-scaled MBR operations. Copyright © 2011 Elsevier Ltd. All rights reserved.

Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins.

PubMed Central

Gong, F C; Giddings, T H; Meehl, J B; Staehelin, L A; Galbraith, D W

1996-01-01

We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8700911

A forced-flow membrane reactor for transfructosylation using ceramic membrane.

PubMed

Nishizawa, K; Nakajima, M; Nabetani, H

2000-04-05

A forced-flow membrane reactor system for transfructosylation was investigated using several ceramic membranes having different pore sizes. beta-Fructofuranosidase from Aspergillus niger ATCC 20611 was immobilized chemically to the inner surface of a ceramic membrane activated by a silane-coupling reagent. Sucrose solution was forced through the ceramic membrane by crossflow filtration while transfructosylation took place. The saccharide composition of the product, which was a mixture of fructooligosaccharides (FOS), was a function of the permeate flux, which was easily controlled by pressure. Using 0.2 micrometer pore size of symmetric ceramic membrane, the volumetric productivity obtained was 3.87 kg m(-3) s(-1), which was 560 times higher than that in a reported batch system, with a short residence time of 11 s. The half-life of the immobilized enzyme in the membrane was estimated to be 35 days by a long-term operation. Copyright 2000 John Wiley & Sons, Inc.

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.

Membrane microdomains, rafts, and detergent-resistant membranes in plants and fungi.

PubMed

Malinsky, Jan; Opekarová, Miroslava; Grossmann, Guido; Tanner, Widmar

2013-01-01

The existence of specialized microdomains in plasma membranes, postulated for almost 25 years, has been popularized by the concept of lipid or membrane rafts. The idea that detergent-resistant membranes are equivalent to lipid rafts, which was generally abandoned after a decade of vigorous data accumulation, contributed to intense discussions about the validity of the raft concept. The existence of membrane microdomains, meanwhile, has been verified by unequivocal independent evidence. This review summarizes the current state of research in plants and fungi with respect to common aspects of both kingdoms. In these organisms, principally immobile microdomains large enough for microscopic detection have been visualized. These microdomains are found in the context of cell-cell interactions (plant symbionts and pathogens), membrane transport, stress, and polarized growth, and the data corroborate at least three mechanisms of formation. As documented in this review, modern methods of visualization of lateral membrane compartments are also able to uncover the functional relevance of membrane microdomains.

T Lymphocyte Activation Threshold and Membrane Reorganization Perturbations in Unique Culture Model

NASA Technical Reports Server (NTRS)

Adams, C. L.; Sams, C. F.

2000-01-01

Quantitative activation thresholds and cellular membrane reorganization are mechanisms by which resting T cells modulate their response to activating stimuli. Here we demonstrate perturbations of these cellular processes in a unique culture system that non-invasively inhibits T lymphocyte activation. During clinorotation, the T cell activation threshold is increased 5-fold. This increased threshold involves a mechanism independent of TCR triggering. Recruitment of lipid rafts to the activation site is impaired during clinorotation but does occur with increased stimulation. This study describes a situation in which an individual cell senses a change in its physical environment and alters its cell biological behavior.

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

Structure-function insights into direct lipid transfer between membranes by Mmm1-Mdm12 of ERMES.

PubMed

Kawano, Shin; Tamura, Yasushi; Kojima, Rieko; Bala, Siqin; Asai, Eri; Michel, Agnès H; Kornmann, Benoît; Riezman, Isabelle; Riezman, Howard; Sakae, Yoshitake; Okamoto, Yuko; Endo, Toshiya

2018-03-05

The endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES) physically links the membranes of the ER and mitochondria in yeast. Although the ER and mitochondria cooperate to synthesize glycerophospholipids, whether ERMES directly facilitates the lipid exchange between the two organelles remains controversial. Here, we compared the x-ray structures of an ERMES subunit Mdm12 from Kluyveromyces lactis with that of Mdm12 from Saccharomyces cerevisiae and found that both Mdm12 proteins possess a hydrophobic pocket for phospholipid binding. However in vitro lipid transfer assays showed that Mdm12 alone or an Mmm1 (another ERMES subunit) fusion protein exhibited only a weak lipid transfer activity between liposomes. In contrast, Mdm12 in a complex with Mmm1 mediated efficient lipid transfer between liposomes. Mutations in Mmm1 or Mdm12 impaired the lipid transfer activities of the Mdm12-Mmm1 complex and furthermore caused defective phosphatidylserine transport from the ER to mitochondrial membranes via ERMES in vitro. Therefore, the Mmm1-Mdm12 complex functions as a minimal unit that mediates lipid transfer between membranes. © 2018 Kawano et al.

Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag

PubMed Central

Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

2016-01-01

The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (l-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane. PMID:27120610

Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag.

PubMed

Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

2016-04-25

The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (L-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane.

Membrane lipid alterations in the metabolic syndrome and the role of dietary oils.

PubMed

Perona, Javier S

2017-09-01

The metabolic syndrome is a cluster of pathological conditions, including hypertension, hyperglycemia, hypertriglyceridemia, obesity and low HDL levels that is of great concern worldwide, as individuals with metabolic syndrome have an increased risk of type-2 diabetes and cardiovascular disease. Insulin resistance, the key feature of the metabolic syndrome, might be at the same time cause and consequence of impaired lipid composition in plasma membranes of insulin-sensitive tissues like liver, muscle and adipose tissue. Diet intervention has been proposed as a powerful tool to prevent the development of the metabolic syndrome, since healthy diets have been shown to have a protective role against the components of the metabolic syndrome. Particularly, dietary fatty acids are capable of modulating the deleterious effects of these conditions, among other mechanisms, by modifications of the lipid composition of the membranes in insulin-sensitive tissues. However, there is still scarce data based of high-level evidence on the effects of dietary oils on the effects of the metabolic syndrome and its components. This review summarizes the current knowledge on the effects of dietary oils on improving alterations of the components of the metabolic syndrome. It also examines their influence in the modulation of plasma membrane lipid composition and in the functionality of membrane proteins involved in insulin activity, like the insulin receptor, GLUT-4, CD36/FAT and ABCA-1, and their effect in the metabolism of glucose, fatty acids and cholesterol, and, in turn, the key features of the metabolic syndrome. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

Estrogens facilitate memory processing through membrane mediated mechanisms and alterations in spine density

PubMed Central

Luine, Victoria N.; Frankfurt, Maya

2012-01-01

Estrogens exert sustained, genomically mediated effects on memory throughout the female life cycle, but here we review new studies documenting rapid effects of estradiol on memory, which are exerted through membrane-mediated mechanisms. Use of recognition memory tasks in rats, shows that estrogens enhance memory consolidation within one hour. 17α-estradiol is more potent than 17β-estradiol, and the dose response relationship between estrogens and memory is an inverted U shape. Use of specific estrogen receptor (ER) agonists suggests mediation by an ERβ-like membrane receptor. Enhanced memory is associated with increased spine density and altered noradrenergic activity in the medial prefrontal cortex and hippocampus within 30 min. of administration. The environmental chemical, bisphenol-A, rapidly antagonizes enhancements in memory in both sexes possibly through actions on spines. Thus, estradiol and related compounds exert rapid alterations in cognition through non-genomic mechanisms, a finding which may provide a basis for better understanding and treating memory impairments. PMID:22981654

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin

DOE PAGES

Aksoyoglu, M. Alphan; Podgornik, Rudolf; Bezrukov, Sergey M.; ...

2016-07-27

Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different beta-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin alpha-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels aremore » consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. In conclusion, the obtained results demonstrate that the general concept of "polymers pushing polymers" is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores.« less

Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin

PubMed Central

Aksoyoglu, M. Alphan; Podgornik, Rudolf; Bezrukov, Sergey M.; Gurnev, Philip A.; Muthukumar, Murugappan; Parsegian, V. Adrian

2016-01-01

Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different β-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin α-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels are consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. The obtained results demonstrate that the general concept of “polymers pushing polymers” is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores. PMID:27466408

Characteristics of membrane fouling in submerged membrane bioreactor under sub-critical flux operation.

PubMed

Su, Y C; Huang, C P; Pan, Jill R; Lee, H C

2008-01-01

Recently, the membrane bioreactor (MBR) process has become one of the novel technologies to enhance the performance of biological treatment of wastewater. Membrane bioreactor process uses the membrane unit to replace a sediment tank, and this can greatly enhance treatment performance. However, membrane fouling in MBR restricts its widespread application because it leads to permeate flux decline, making more frequent membrane cleaning and replacement necessary, which then increases operating and maintenance costs. This study investigated the sludge characteristics in membrane fouling under sub-critical flux operation and also assessed the effect of shear stress on membrane fouling. Membrane fouling was slow under sub-critical flux operation. However, as filamentous microbes became dominant in the reactor, membrane fouling increased dramatically due to the increased viscosity and polysaccharides. A close link was found between membrane fouling and the amount of polysaccharides in soluble EPS. The predominant resistance was the cake resistance which could be minimized by increasing the shear stress. However, the resistance of colloids and solutes was not apparently reduced by increasing shear stress. Therefore, smaller particles such as macromolecules (e.g. polysaccharides) may play an important role in membrane fouling under sub-critical flux operation.

Tethered bilayer lipid membranes (tBLMs): interest and applications for biological membrane investigations.

PubMed

Rebaud, Samuel; Maniti, Ofelia; Girard-Egrot, Agnès P

2014-12-01

Biological membranes play a central role in the biology of the cell. They are not only the hydrophobic barrier allowing separation between two water soluble compartments but also a supra-molecular entity that has vital structural functions. Notably, they are involved in many exchange processes between the outside and inside cellular spaces. Accounting for the complexity of cell membranes, reliable models are needed to acquire current knowledge of the molecular processes occurring in membranes. To simplify the investigation of lipid/protein interactions, the use of biomimetic membranes is an approach that allows manipulation of the lipid composition of specific domains and/or the protein composition, and the evaluation of the reciprocal effects. Since the middle of the 80's, lipid bilayer membranes have been constantly developed as models of biological membranes with the ultimate goal to reincorporate membrane proteins for their functional investigation. In this review, after a brief description of the planar lipid bilayers as biomimetic membrane models, we will focus on the construction of the tethered Bilayer Lipid Membranes, the most promising model for efficient membrane protein reconstitution and investigation of molecular processes occurring in cell membranes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Membrane Bending by Protein Crowding

NASA Astrophysics Data System (ADS)

Stachowiak, Jeanne

2014-03-01

From endosomes and synaptic vesicles to the cristae of the mitochondria and the annulus of the nuclear pore, highly curved membranes are fundamental to the structure and physiology of living cells. The established view is that specific families of proteins are able to bend membranes by binding to them. For example, inherently curved proteins are thought to impose their structure on the membrane surface, while membrane-binding proteins with hydrophobic motifs are thought to insert into the membrane like wedges, driving curvature. However, computational models have recently revealed that these mechanisms would require specialized membrane-bending proteins to occupy nearly 100% of a curved membrane surface, an improbable physiological situation given the immense density and diversity of membrane-bound proteins, and the low expression levels of these specialized proteins within curved regions of the membrane. How then does curvature arise within the complex and crowded environment of cellular membranes? Our recent work using proteins involved in clathrin-mediated endocytosis, as well as engineered protein-lipid interactions, has suggested a new hypothesis - that lateral pressure generated by collisions between membrane-bound proteins can drive membrane bending. Specifically, by correlating membrane bending with quantitative optical measurements of protein density on synthetic membrane surfaces and simple physical models of collisions among membrane-bound proteins, we have demonstrated that protein-protein steric interactions can drive membrane curvature. These findings suggest that a simple imbalance in the concentration of membrane-bound proteins across a membrane surface can drive a membrane to bend, providing an efficient mechanism by which essentially any protein can contribute to shaping membranes.

Differential Expression of In Vivo and In Vitro Protein Profile of Outer Membrane of Acidovorax avenae Subsp. avenae

PubMed Central

Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Sun, Guochang

2012-01-01

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium. PMID:23166741

Membrane with supported internal passages

NASA Technical Reports Server (NTRS)

Gonzalez-Martin, Anuncia (Inventor); Salinas, Carlos E. (Inventor); Cisar, Alan J. (Inventor); Hitchens, G. Duncan (Inventor); Murphy, Oliver J. (Inventor)

2000-01-01

The invention provides an improved proton exchange membrane for use in electrochemical cells having internal passages parallel to the membrane surface comprising permanent tubes preferably placed at the ends of the fluid passages. The invention also provides an apparatus and process for making the membrane, membrane and electrode assemblies fabricated using the membrane, and the application of the membrane and electrode assemblies to a variety of devices, both electrochemical and otherwise. The passages in the membrane extend from one edge of the membrane to another and allow fluid flow through the membrane and give access directly to the membrane.

Visual Impairment

MedlinePlus

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Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.

PubMed

Li, Jingsong; Luo, Ruihua; Hooi, Shing Chuan; Ruga, Pilar; Zhang, Jiping; Meda, Paolo; Li, GuoDong

2005-03-22

Syncollin was first demonstrated to be a protein capable of affecting granule fusion in a cell-free system, but later studies revealed its luminal localization in zymogen granules. To determine its possible role in exocytosis in the intact cell, syncollin and a truncated form of the protein (lacking the N-terminal hydrophobic domain) were stably transfected in insulin-secreting INS-1 cells since these well-studied exocytotic cells appear not to express the protein per se. Studies by subcellular fractionation analysis, double immunofluorescence staining, and electron microscopy examination revealed that transfection of syncollin produced strong signals in the insulin secretory granules, whereas the product from transfecting the truncated syncollin was predominantly associated with the Golgi apparatus and to a lesser degree with the endoplasmic reticulum. The expressed products were associated with membranes and not the soluble fractions in either cytoplasm or the lumens of organelles. Importantly, insulin release stimulated by various secretagogues was severely impaired in cells expressing syncollin, but not affected by expressing truncated syncollin. Transfection of syncollin appeared not to impede insulin biosynthesis and processing, since cellular contents of proinsulin and insulin and the number of secretory granules were not altered. In addition, the early signals (membrane depolarization and Ca(2+) responses) for regulated insulin secretion were unaffected. These findings indicate that syncollin may be targeted to insulin secretory granules specifically and impair regulated secretion at a distal stage.

Proton exchange membranes prepared by grafting of styrene/divinylbenzene into crosslinked PTFE membranes

NASA Astrophysics Data System (ADS)

Li, Jingye; Ichizuri, Shogo; Asano, Saneto; Mutou, Fumihiro; Ikeda, Shigetoshi; Iida, Minoru; Miura, Takaharu; Oshima, Akihiro; Tabata, Yoneho; Washio, Masakazu

2005-07-01

Thin PTFE membranes were prepared by coating the PTFE dispersion onto the aluminum films. Thus the thin crosslinked PTFE (RX-PTFE) membranes were obtained by means of electron beam irradiation above the melting temperature of PTFE under oxygen-free atmosphere. The RX-PTFE membranes were pre-irradiated and grafted by styrene with or without divinylbenzene (DVB) in liquid phase. The existence of DVB accelerated the initial grafting rate. The styrene grafted RX-PTFE membranes are white colored, on the other hand, the styrene/DVB grafted RX-PTFE membranes are colorless. The proton exchange membranes (PEMs) were obtained by sulfonating the grafted membranes using chlorosulfonic acid. The ion exchange capacity (IEC) values of the PEMs ranging from 1.5 to 2.8 meq/g were obtained. The PEMs made from the styrene/DVB grafted membranes showed higher chemical stability than those of the styrene grafted membranes under oxidative circumstance.

Do Membranes Dream of Electric Tubes? Advanced Membranes Using Carbon Nanotube - Polymer Nanocomposites

NASA Astrophysics Data System (ADS)

de Lannoy, Charles-Francois Pedro Claude Karolek Ghislain

Membrane technologies represent an energy efficient, effective solution for treating municipal and commercial waters/wastewaters. Membranes are predominantly polymer-based and despite steady advances in polymeric materials, they continue to suffer from operational problems including biofouling and breakages. This work addresses these two disparate problems by developing novel CNT-polymer nanocomposite materials that contain variously functionalized carbon nanotubes (fCNTs) in low quantities (<0.5wt%). Several strategies have been employed to achieve highly functional CNT-polymer nanocomposite membranes including blend mixing, ionic charge association, and covalent cross-linking with monomer and oligomer constituents. These CNT-polymer nanocomposite membranes were compared to traditional polymer membranes across various properties including increased Young's Modulus, changes in surface hydrophilicity, fine control over molecular weight cut-off and flux, and surface electrical conductivity. Membranes with high surface electrical conductivity were further tested for their anti-biofouling properties. Finally, CNT stability and polymer compatibility were evaluated throughout membrane manufacture, use, and cleaning. The incorporation of CNTs mixed in bulk phase and linked through ionic associations in polymer matrices showed significant (50%) increases in Young's modulus for certain CNT functionalizations and derivatization percent. Membranes formed with high surface electrical conductivity demonstrated almost complete resistance to biofouling (> 95%) in long-term bacterially challenged experiments. CNTs and polymer mixtures that lacked covalent or ionic bonds were susceptible to significant (up to 10%) loss of CNTs during membrane non-solvent gelation and aggressive chemical cleaning treatment. Functionalized carbon nanotubes endow polymer membranes with their unique strength and electrically conductive properties. These added properties were demonstrated to greatly

Different Patterns of Theory of Mind Impairment in Mild Cognitive Impairment.

PubMed

Moreau, Noémie; Rauzy, Stéphane; Bonnefoi, Bernadette; Renié, Laurent; Martinez-Almoyna, Laurent; Viallet, François; Champagne-Lavau, Maud

2015-01-01

Theory of Mind refers to the ability to infer other’s mental states, their beliefs, intentions, or knowledge. To date, only two studies have reported the presence of Theory of Mind impairment in mild cognitive impairment (MCI). In the present study,we evaluated 20 MCI patients and compared them with 25 healthy control participants using two Theory of Mind tasks. The first task was a false belief paradigm as frequently used in the literature, and the second one was a referential communication task,assessing Theory of Mind in a real situation of interaction and which had never been used before in this population. The results showed that MCI patients presented difficulties inferring another person’s beliefs about reality and attributing knowledge to them in a situation of real-life interaction. Two different patterns of Theory of Mind emerged among the patients. In comparison with the control group, some MCI patients demonstrated impairment only in the interaction task and presented isolated episodicmemory impairment, while others were impaired in both Theory of Mind tasks and presented cognitive impairment impacting both episodic memory and executive functioning. Theory of Mind is thus altered in the very early stages of cognitive impairment even in real social interaction, which could impact precociously relationships in daily life.

Two-Dimensional-Material Membranes: A New Family of High-Performance Separation Membranes.

PubMed

Liu, Gongping; Jin, Wanqin; Xu, Nanping

2016-10-17

Two-dimensional (2D) materials of atomic thickness have emerged as nano-building blocks to develop high-performance separation membranes that feature unique nanopores and/or nanochannels. These 2D-material membranes exhibit extraordinary permeation properties, opening a new avenue to ultra-fast and highly selective membranes for water and gas separation. Summarized in this Minireview are the latest ground-breaking studies in 2D-material membranes as nanosheet and laminar membranes, with a focus on starting materials, nanostructures, and transport properties. Challenges and future directions of 2D-material membranes for wide implementation are discussed briefly. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Early developmental bisphenol-A exposure sex-independently impairs spatial memory by remodeling hippocampal dendritic architecture and synaptic transmission in rats

NASA Astrophysics Data System (ADS)

Liu, Zhi-Hua; Ding, Jin-Jun; Yang, Qian-Qian; Song, Hua-Zeng; Chen, Xiang-Tao; Xu, Yi; Xiao, Gui-Ran; Wang, Hui-Li

2016-08-01

Bisphenol-A (BPA, 4, 4‧-isopropylidene-2-diphenol), a synthetic xenoestrogen that widely used in the production of polycarbonate plastics, has been reported to impair hippocampal development and function. Our previous study has shown that BPA exposure impairs Sprague-Dawley (SD) male hippocampal dendritic spine outgrowth. In this study, the sex-effect of chronic BPA exposure on spatial memory in SD male and female rats and the related synaptic mechanism were further investigated. We found that chronic BPA exposure impaired spatial memory in both SD male and female rats, suggesting a dysfunction of hippocampus without gender-specific effect. Further investigation indicated that BPA exposure causes significant impairment of dendrite and spine structure, manifested as decreased dendritic complexity, dendritic spine density and percentage of mushroom shaped spines in hippocampal CA1 and dentate gyrus (DG) neurons. Furthermore, a significant reduction in Arc expression was detected upon BPA exposure. Strikingly, BPA exposure significantly increased the mIPSC amplitude without altering the mEPSC amplitude or frequency, accompanied by increased GABAARβ2/3 on postsynaptic membrane in cultured CA1 neurons. In summary, our study indicated that Arc, together with the increased surface GABAARβ2/3, contributed to BPA induced spatial memory deficits, providing a novel molecular basis for BPA achieved brain impairment.

Loss of Parkin Impairs Mitochondrial Function and Leads to Muscle Atrophy.

PubMed

Peker, Nesibe; Donipadi, Vinay; Sharma, Mridula; McFarlane, Craig; Kambadur, Ravi

2018-03-21

Parkinson's Disease is a neurodegenerative disease characterized by tremors, muscle stiffness and muscle weakness. Molecular genetic analysis confirmed that mutations in PARKIN and PINK1 genes, which play major roles in mitochondrial quality control and mitophagy, are frequently associated with Parkinson's Disease. PARKIN is an E3 ubiquitin ligase that translocates to mitochondria during loss of mitochondrial membrane potential to increase mitophagy. Although muscle dysfunction is noted in Parkinson's Disease, little is known about the involvement of PARKIN in the muscle phenotype of Parkinson's Disease. In this study, we report that the mitochondrial uncoupler CCCP promotes PINK1/PARKIN-mediated mitophagy in myogenic C2C12 cells. As a result of this excess mitophagy, we show that CCCP treatment of myotubes leads to the development of myotube atrophy in vitro. Surprisingly, we also found that siRNA-mediated knock down of Parkin results in accumulation of dysfunctional mitochondria, possibly due to impaired mitochondrial turnover. In addition, knock down of Parkin led to myotubular atrophy in vitro. Consistent with these in vitro results, Parkin knockout muscles showed impaired mitochondrial function and smaller myofiber area, suggesting that Parkin function is required for post-natal skeletal muscle growth and development.

Pilot demonstration of energy-efficient membrane bioreactor (MBR) using reciprocating submerged membrane.

PubMed

Ho, Jaeho; Smith, Shaleena; Patamasank, Jaren; Tontcheva, Petia; Kim, Gyu Dong; Roh, Hyung Keun

2015-03-01

Membrane bioreactor (MBR) is becoming popular for advanced wastewater treatment and water reuse. Air scouring to "shake" the membrane fibers is most suitable and applicable to maintain filtration without severe and rapidfouling. However, membrane fouling mitigating technologies are energy intensive. The goal of this research is to develop an alternative energy-saving MBR system to reduce energy consumption; a revolutionary system that will directly compete with air scouring technologies currently in the membrane water reuse market. The innovative MBR system, called reciprocation MBR (rMBR), prevents membrane fouling without the use of air scouring blowers. The mechanism featured is a mechanical reciprocating membrane frame that uses inertia to prevent fouling. Direct strong agitation of the fiber is also beneficial for the constant removal of solids built up on the membrane surface. The rMBR pilot consumes less energy than conventional coarse air scouring MBR systems. Specific energy consumption for membrane reciprocation for the pilot rMBR system was 0.072 kWh/m3 permeate produced at 40 LMH, which is 75% less than the conventional air scouring in an MBR system (0.29 kWh/m3). Reciprocation of the hollow-fiber membrane can overcome the hydrodynamic limitations of air scouring or cross-flow membrane systems with less energy consumption and/or higher energy efficiency.

SSTAR, a Stand-Alone Easy-To-Use Antimicrobial Resistance Gene Predictor.

PubMed

de Man, Tom J B; Limbago, Brandi M

2016-01-01

We present the easy-to-use Sequence Search Tool for Antimicrobial Resistance, SSTAR. It combines a locally executed BLASTN search against a customizable database with an intuitive graphical user interface for identifying antimicrobial resistance (AR) genes from genomic data. Although the database is initially populated from a public repository of acquired resistance determinants (i.e., ARG-ANNOT), it can be customized for particular pathogen groups and resistance mechanisms. For instance, outer membrane porin sequences associated with carbapenem resistance phenotypes can be added, and known intrinsic mechanisms can be included. Unique about this tool is the ability to easily detect putative new alleles and truncated versions of existing AR genes. Variants and potential new alleles are brought to the attention of the user for further investigation. For instance, SSTAR is able to identify modified or truncated versions of porins, which may be of great importance in carbapenemase-negative carbapenem-resistant Enterobacteriaceae. SSTAR is written in Java and is therefore platform independent and compatible with both Windows and Unix operating systems. SSTAR and its manual, which includes a simple installation guide, are freely available from https://github.com/tomdeman-bio/Sequence-Search-Tool-for-Antimicrobial-Resistance-SSTAR-. IMPORTANCE Whole-genome sequencing (WGS) is quickly becoming a routine method for identifying genes associated with antimicrobial resistance (AR). However, for many microbiologists, the use and analysis of WGS data present a substantial challenge. We developed SSTAR, software with a graphical user interface that enables the identification of known AR genes from WGS and has the unique capacity to easily detect new variants of known AR genes, including truncated protein variants. Current software solutions do not notify the user when genes are truncated and, therefore, likely nonfunctional, which makes phenotype predictions less accurate. SSTAR

Vitreoretinal interface abnormalities in middle-aged adults with visual impairment in the UK Biobank study: prevalence, impact on visual acuity and associations.

PubMed

McKibbin, Martin; Farragher, Tracey; Shickle, Darren

2017-01-01

The aim of this study was to determine the prevalence of vitreoretinal interface abnormalities (VRIA), the degree of visual impairment and associations with VRIA among adults, aged 40-69 years, in the UK Biobank study. Colour fundus photographs and spectral domain optical coherence tomography images were graded for 25% of the 8359 UK Biobank participants with mild visual impairment or worse (LogMAR >0.3 or Snellen <6/12) in at least one eye. The prevalence and contribution of VRIA to visual impairment was determined and multinomial logistic regression models were used to investigate association with known risk factors and other predetermined socioeconomic, biometric, lifestyle and medical variables for cases and matched controls. The minimum prevalence of any VRIA was 17.6% and 8.1% in the eyes with and without visual impairment, respectively. VRIA were identified as the primary cause of visual impairment in 3.6% of eyes. Although epiretinal membrane and vitreomacular traction were the most common VRIA, the degree of visual impairment was typically milder with these than with other VRIA. Visual impairment with a VRIA was positively associated with increasing age (relative risk ratio (RRR) 1.22 (95% CI 1.07 to 1.40)), female gender (RRR 1.28; 1.08 to 1.52) and Asian or Asian British ethnicity (RRR 1.60; 1.10 to 2.32). VRIA are common in middle-aged adults in the UK Biobank study, especially in eyes with visual impairment. VRIA were considered to be the primary cause of visual impairment in 3.6% of all eyes with visual impairment, although there was variation in the degree of visual impairment for each type of VRIA.

Anion permselective membrane

NASA Technical Reports Server (NTRS)

Alexander, S.; Hodgdon, R. B.

1977-01-01

The objective of NAS 3-20108 was the development and evaluation of improved anion selective membranes useful as efficient separators in a redox power storage cell system being constructed. The program was divided into three parts, (a) optimization of the selected candidate membrane systems, (b) investigation of alternative membrane/polymer systems, and (c) characterization of candidate membranes. The major synthesis effort was aimed at improving and optimizing as far as possible each candidate system with respect to three critical membrane properties essential for good redox cell performance. Substantial improvements were made in 5 candidate membrane systems. The critical synthesis variables of cross-link density, monomer ratio, and solvent composition were examined over a wide range. In addition, eight alternative polymer systems were investigated, two of which attained candidate status. Three other alternatives showed potential but required further research and development. Each candidate system was optimized for selectivity.

A vacuolar membrane protein Avt7p is involved in transport of amino acid and spore formation in Saccharomyces cerevisiae.

PubMed

Tone, Junichi; Yamanaka, Atsushi; Manabe, Kunio; Murao, Nami; Kawano-Kawada, Miyuki; Sekito, Takayuki; Kakinuma, Yoshimi

2015-01-01

Active transport systems for various amino acids operate in the vacuolar membrane of Saccharomyces cerevisiae. The gene families for vacuolar amino acid transporters were identified by reverse genetics experiments. In the AVT transporter family, Avt1p works for active uptake of amino acid into vacuole, and Avt3p, Avt4p, and Avt6p for active extrusion of amino acid from vacuole to cytosol. Here, we found green fluorescent protein-tagged Avt7p, an unidentified member of the AVT family, localized to the vacuolar membrane of S. cerevisiae. Disruption of the AVT7 gene enhanced both vacuolar contents of several amino acids and uptake activities of glutamine and proline by vacuolar membrane vesicles. Efficiency of spore formation was impaired by the disruption of the AVT7 gene, suggesting the physiological importance of Avt7p-dependent efflux of amino acid from vacuoles under nutrient-poor condition.

Solid-state membrane module

DOEpatents

Gordon, John Howard [Salt Lake City, UT; Taylor, Dale M [Murray, UT

2011-06-07

Solid-state membrane modules comprising at least one membrane unit, where the membrane unit has a dense mixed conducting oxide layer, and at least one conduit or manifold wherein the conduit or manifold comprises a dense layer and at least one of a porous layer and a slotted layer contiguous with the dense layer. The solid-state membrane modules may be used to carry out a variety of processes including the separating of any ionizable component from a feedstream wherein such ionizable component is capable of being transported through a dense mixed conducting oxide layer of the membrane units making up the membrane modules. For ease of construction, the membrane units may be planar.

Dialysis in rats with acute renal failure: evaluation of three different dialyzer membranes.

PubMed

Kränzlin, B; Gretz, N; Kirschfink, M; Mujais, S K

1996-11-01

Exposure to complement-activating cellulosic dialysis membranes has been claimed to adversely affect the course of acute renal failure (ARF). To test this hypothesis, male Sprague-Dawley rats were allocated to 2 groups: in Group 1, ARF was induced by bilateral renal artery clamping whereas in Group 2, animals underwent a sham procedure. In each group, rats were further allocated to undergo hemodialysis with either a Cuprophan, a Hemophan, or a polyacrylonitrile minidialyzer on Days 4 and 8 after surgery, or no dialysis. Renal function was measured by inulin clearance on the days after dialysis. Additionally, total complement activity (CH50) was estimated on Days 1, 2, 4, and 8, and complement factor C3 was detected immunohistochemically. The degree of renal failure and the rate of recovery of renal function were similar in all the ARF groups irrespective of whether they had undergone dialysis or not, or of the type of the dialysis membrane. Furthermore, there were no significant differences in the course of CH50 or in the amount and distribution of complement factor C3 in the kidney tissue between the rats of Groups 1 and 2. Our findings refute the hypothesis that in ischemic ARF exposure to complement-activating cellulosic dialysis membranes impairs the recovery of renal function in rats.

Glycine uptake by microvillous and basal plasma membrane vesicles from term human placentae.

PubMed

Dicke, J M; Verges, D; Kelley, L K; Smith, C H

1993-01-01

Like most amino acids, glycine is present in higher concentrations in the fetus than in the mother. Unlike most amino acids, animal studies suggest fetal concentrations of glycine are minimally in excess of those required for protein synthesis. Abnormal glycine utilization has also been demonstrated in small-for-gestational age human fetuses. The mechanism(s) of glycine uptake in the human placenta are unknown. In other mammalian cells glycine is a substrate for the A, ASC and Gly amino acid transport systems. In this study human placental glycine uptake was characterized using microvillous and basal plasma membrane vesicles each prepared from the same placenta. In both membranes glycine uptake was mediated predominantly by the sodium-dependent A system. Competitive inhibition studies suggest that in microvillous vesicles the small percentage of sodium-dependent glycine uptake not inhibited by methylaminoisobutyric acid (MeAIB) shares a transport system with glycine methyl ester and sarcosine, substrates of the Gly system in other tissues. In addition there are mediated sodium-independent and non-selective transport mechanisms in both plasma membranes. If fetal glycine availability is primarily contingent upon the common and highly regulated A system, glycine must compete with many other substrates potentially resulting in marginal fetal reserves, abnormal utilization and impaired growth.

Association between alcohol-impaired driving enforcement-related strategies and alcohol-impaired driving.

PubMed

Sanem, Julia R; Erickson, Darin J; Rutledge, Patricia C; Lenk, Kathleen M; Nelson, Toben F; Jones-Webb, Rhonda; Toomey, Traci L

2015-05-01

All states in the U.S. prohibit alcohol-impaired driving but active law enforcement is necessary for effectively reducing this behavior. Sobriety checkpoints, saturation patrols, open container laws, and media campaigns related to enforcement efforts are all enforcement-related strategies for reducing alcohol-impaired driving. We conducted surveys of all state patrol agencies and a representative sample of local law enforcement agencies to assess their use of alcohol-impaired driving enforcement-related strategies and to determine the relationship between these enforcement-related strategies and self-reported alcohol-impaired driving behavior obtained from the Behavioral Risk Factor Surveillance System. We found that sobriety checkpoints, saturation patrols, and enforcement of open container laws were associated with a lower prevalence of alcohol-impaired driving but, more importantly, a combination of enforcement-related strategies was associated with a greater decrease in alcohol-impaired driving than any individual enforcement-related activity. In addition, alcohol-impaired driving enforcement-related strategies were associated with decreased alcohol-impaired driving above and beyond their association with decreased binge drinking. Results suggest law enforcement agencies should give greater priority to using a combination of strategies rather than relying on any one individual enforcement activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Role of Ion Exchange Membranes in Membrane Capacitive Deionisation.

PubMed

Hassanvand, Armineh; Wei, Kajia; Talebi, Sahar; Chen, George Q; Kentish, Sandra E

2017-09-14

Ion-exchange membranes (IEMs) are unique in combining the electrochemical properties of ion exchange resins and the permeability of a membrane. They are being used widely to treat industrial effluents, and in seawater and brackish water desalination. Membrane Capacitive Deionisation (MCDI) is an emerging, energy efficient technology for brackish water desalination in which these ion-exchange membranes act as selective gates allowing the transport of counter-ions toward carbon electrodes. This article provides a summary of recent developments in the preparation, characterization, and performance of ion exchange membranes in the MCDI field. In some parts of this review, the most relevant literature in the area of electrodialysis (ED) is also discussed to better elucidate the role of the ion exchange membranes. We conclude that more work is required to better define the desalination performance of the proposed novel materials and cell designs for MCDI in treating a wide range of feed waters. The extent of fouling, the development of cleaning strategies, and further techno-economic studies, will add value to this emerging technique.

The Role of Ion Exchange Membranes in Membrane Capacitive Deionisation

PubMed Central

Hassanvand, Armineh; Wei, Kajia; Talebi, Sahar

2017-01-01

Ion-exchange membranes (IEMs) are unique in combining the electrochemical properties of ion exchange resins and the permeability of a membrane. They are being used widely to treat industrial effluents, and in seawater and brackish water desalination. Membrane Capacitive Deionisation (MCDI) is an emerging, energy efficient technology for brackish water desalination in which these ion-exchange membranes act as selective gates allowing the transport of counter-ions toward carbon electrodes. This article provides a summary of recent developments in the preparation, characterization, and performance of ion exchange membranes in the MCDI field. In some parts of this review, the most relevant literature in the area of electrodialysis (ED) is also discussed to better elucidate the role of the ion exchange membranes. We conclude that more work is required to better define the desalination performance of the proposed novel materials and cell designs for MCDI in treating a wide range of feed waters. The extent of fouling, the development of cleaning strategies, and further techno-economic studies, will add value to this emerging technique. PMID:28906442

Membrane Packing Problems: A short Review on computational Membrane Modeling Methods and Tools

PubMed Central

Sommer, Björn

2013-01-01

The use of model membranes is currently part of the daily workflow for many biochemical and biophysical disciplines. These membranes are used to analyze the behavior of small substances, to simulate transport processes, to study the structure of macromolecules or for illustrative purposes. But, how can these membrane structures be generated? This mini review discusses a number of ways to obtain these structures. First, the problem will be formulated as the Membrane Packing Problem. It will be shown that the theoretical problem of placing proteins and lipids onto a membrane area differ significantly. Thus, two sub-problems will be defined and discussed. Then, different – partly historical – membrane modeling methods will be introduced. And finally, membrane modeling tools will be evaluated which are able to semi-automatically generate these model membranes and thus, drastically accelerate and simplify the membrane generation process. The mini review concludes with advice about which tool is appropriate for which application case. PMID:24688707

Thermodynamic analysis of membrane fouling in a submerged membrane bioreactor and its implications.

PubMed

Hong, Huachang; Peng, Wei; Zhang, Meijia; Chen, Jianrong; He, Yiming; Wang, Fangyuan; Weng, Xuexiang; Yu, Haiying; Lin, Hongjun

2013-10-01

The thermodynamic interactions between membrane and sludge flocs in a submerged membrane bioreactor (MBR) were investigated. It was found that Lewis acid-base (AB) interaction predominated in the total interactions. The interaction energy composition of membrane-sludge flocs combination was quite similar to that of membrane-bovine serum albumin (BSA) combination, indicating the critical role of proteins in adhesion process. Detailed analysis revealed the existence of a repulsive energy barrier in membrane-foulants interaction. Calculation results demonstrated that small flocs possessed higher attractive interaction energy per unit mass, and therefore adhered to membrane surface more easily as compared to large flocs. Meanwhile, initial sludge adhesion would facilitate the following adhesion due to the reduced repulsive energy barrier. Membrane with high electron donor surface tension component was a favor option for membrane fouling abatement. These findings offered new insights into membrane fouling, and also provided significant implications for fouling control in MBRs. Copyright © 2013 Elsevier Ltd. All rights reserved.

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. © 2017 American Heart Association, Inc.

Solid-Supported Lipid Membranes: Formation, Stability and Applications

NASA Astrophysics Data System (ADS)

Goh, Haw Zan

insulating and structural properties. Preparation conditions are screened for those that are optimal for stBLM formation. Concentrations of lipid vesicles, hydrophobicity of SAMs, the presence of calcium and high concentrations of salt are identified as the key parameters. We show that stBLMs can be formed with vesicles of different compositions. Vesicle hemifusion opens up a new route in preserving the chemical compositions of stBLMs and facilitating membrane proteins incorporation. In Chapter 6, we visualize the hemifusion pathway of giant unilamellar vesicles (GUVs) with planar hydrophobic surfaces at the single vesicle level with fluorescence video microscopy. When a GUV hemifuses to a surface, its outer leaflet breaks apart and remains connected to the surface presumably through a hemifusion diaphragm. Lipids from the outer leaflet are transferred to the surface as a lipid monolayer that expands radially outward from the hemifusion diaphragm, thereby forming the loosely packed outer hemifusion zone. In Chapter 7, we develop an in vitro assay employing stBLMs and lipid vesicles to examine the functionality of GRASP in membrane tethering. Membrane-bound GRASP on opposing membranes dimerizes and tethers fluorescently-labeled vesicles to stBLMs. The fluorescence intensity of images taken at stBLM surfaces is used to quantify the tethering activity. Both wild type and mutant proteins were studied to shed light on the molecular mechanism of tethering. We show that the GRASP domain is sufficient and necessary for membrane tethering. In addition, the tethering capability of GRASP is impaired when the internal ligands and the binding pockets participating in dimerization are deleted and mutated. Membrane anchors, sizes of vesicles and membrane compositions are explored for their influence on the outcomes of the assay. Furthermore, preliminary analysis from neutron reflectivity measurements shows that both the internal ligands and binding pockets are exposed instead of buried

How the antimicrobial peptides destroy bacteria cell membrane: Translocations vs. membrane buckling

NASA Astrophysics Data System (ADS)

Golubovic, Leonardo; Gao, Lianghui; Chen, Licui; Fang, Weihai

2012-02-01

In this study, coarse grained Dissipative Particle Dynamics simulation with implementation of electrostatic interactions is developed in constant pressure and surface tension ensemble to elucidate how the antimicrobial peptide molecules affect bilayer cell membrane structure and kill bacteria. We find that peptides with different chemical-physical properties exhibit different membrane obstructing mechanisms. Peptide molecules can destroy vital functions of the affected bacteria by translocating across their membranes via worm-holes, or by associating with membrane lipids to form hydrophilic cores trapped inside the hydrophobic domain of the membranes. In the latter scenario, the affected membranes are strongly corrugated (buckled) in accord with very recent experimental observations [G. E. Fantner et al., Nat. Nanotech., 5 (2010), pp. 280-285].

Catalytic nanoporous membranes

DOEpatents

Pellin, Michael J; Hryn, John N; Elam, Jeffrey W

2013-08-27

A nanoporous catalytic membrane which displays several unique features Including pores which can go through the entire thickness of the membrane. The membrane has a higher catalytic and product selectivity than conventional catalysts. Anodic aluminum oxide (AAO) membranes serve as the catalyst substrate. This substrate is then subjected to Atomic Layer Deposition (ALD), which allows the controlled narrowing of the pores from 40 nm to 10 nm in the substrate by deposition of a preparatory material. Subsequent deposition of a catalytic layer on the inner surfaces of the pores reduces pore sizes to less than 10 nm and allows for a higher degree of reaction selectivity. The small pore sizes allow control over which molecules enter the pores, and the flow-through feature can allow for partial oxidation of reactant species as opposed to complete oxidation. A nanoporous separation membrane, produced by ALD is also provided for use in gaseous and liquid separations. The membrane has a high flow rate of material with 100% selectivity. Also provided is a method for producing a catalytic membrane having flow-through pores and discreet catalytic clusters adhering to the inside surfaces of the pores.

Resolution of Dialyzer Membrane-Associated Thrombocytopenia with Use of Cellulose Triacetate Membrane: A Case Report

PubMed Central

Olafiranye, Feyisayo; Kyaw, Win; Olafiranye, Oladipupo

2011-01-01

Blood and dialyzer membrane interaction can cause significant thrombocytopenia through the activation of complement system. The extent of this interaction determines the biocompatibility of the membrane. Although the newer synthetic membranes have been shown to have better biocompatibility profile than the cellulose-based membranes, little is known about the difference in biocompatibility between synthetic membrane and modified cellulose membrane. Herein, we report a case of a patient on hemodialysis who developed dialyzer-membrane-related thrombocytopenia with use of synthetic membrane (F200NR polysulfone). The diagnosis of dialyzer membrane-associated thrombocytopenia was suspected by the trend of platelet count before and after dialysis, and the absence of other possible causes of thrombocytopenia. We observed significant improvement in platelet count when the membrane was changed to modified cellulose membrane (cellulose triacetate). In patients at high risk for thrombocytopenia, the modified cellulose membrane could be a better alternative to the standard synthetic membranes during hemodialysis. PMID:21547252

Membrane Distillation of Meat Industry Effluent with Hydrophilic Polyurethane Coated Polytetrafluoroethylene Membranes

PubMed Central

Mostafa, M. G.; Zhu, Bo; Cran, Marlene; Dow, Noel; Milne, Nicholas; Desai, Dilip

2017-01-01

Meat rendering operations produce stick water waste which is rich in proteins, fats, and minerals. Membrane distillation (MD) may further recover water and valuable solids, but hydrophobic membranes are contaminated by the fats. Here, commercial hydrophobic polytetrafluorethylene (PTFE) membranes with a hydrophilic polyurethane surface layer (PU-PTFE) are used for the first time for direct contact MD (DCMD) on real poultry, fish, and bovine stick waters. Metal membrane microfiltration (MMF) was also used to capture fats prior to MD. Although the standard hydrophobic PTFE membranes failed rapidly, PU-PTFE membranes effectively processed all stick water samples to colourless permeate with sodium rejections >99%. Initial clean solution fluxes 5–6 L/m2/h declined to less than half during short 40% water recovery tests for all stick water samples. Fish stick water uniquely showed reduced fouling and up to 78% water recovery. Lost flux was easily restored by rinsing the membrane with clean water. MMF prior to MD removed 92% of fats, facilitating superior MD performance. Differences in fouling between stick waters were attributed to temperature polarisation from higher melt temperature fats and relative proportions to proteins. Hydrophilic coated MD membranes are applicable to stick water processing but further studies should consider membrane cleaning and longer-term stability. PMID:28961203

Catalytic nanoporous membranes

DOEpatents

Pellin, Michael J [Naperville, IL; Hryn, John N [Naperville, IL; Elam, Jeffrey W [Elmhurst, IL

2009-12-01

A nanoporous catalytic membrane which displays several unique features including pores which can go through the entire thickness of the membrane. The membrane has a higher catalytic and product selectivity than conventional catalysts. Anodic aluminum oxide (AAO) membranes serve as the catalyst substrate. This substrate is then subjected to Atomic Layer Deposition (ALD), which allows the controlled narrowing of the pores from 40 nm to 10 nm in the substrate by deposition of a preparatory material. Subsequent deposition of a catalytic layer on the inner surfaces of the pores reduces pore sizes to less than 10 nm and allows for a higher degree of reaction selectivity. The small pore sizes allow control over which molecules enter the pores, and the flow-through feature can allow for partial oxidation of reactant species as opposed to complete oxidation. A nanoporous separation membrane, produced by ALD is also provided for use in gaseous and liquid separations. The membrane has a high flow rate of material with 100% selectivity.

Actin filaments growing against an elastic membrane: Effect of membrane tension

NASA Astrophysics Data System (ADS)

Sadhu, Raj Kumar; Chatterjee, Sakuntala

2018-03-01

We study the force generation by a set of parallel actin filaments growing against an elastic membrane. The elastic membrane tries to stay flat and any deformation from this flat state, either caused by thermal fluctuations or due to protrusive polymerization force exerted by the filaments, costs energy. We study two lattice models to describe the membrane dynamics. In one case, the energy cost is assumed to be proportional to the absolute magnitude of the height gradient (gradient model) and in the other case it is proportional to the square of the height gradient (Gaussian model). For the gradient model we find that the membrane velocity is a nonmonotonic function of the elastic constant μ and reaches a peak at μ =μ* . For μ <μ* the system fails to reach a steady state and the membrane energy keeps increasing with time. For the Gaussian model, the system always reaches a steady state and the membrane velocity decreases monotonically with the elastic constant ν for all nonzero values of ν . Multiple filaments give rise to protrusions at different regions of the membrane and the elasticity of the membrane induces an effective attraction between the two protrusions in the Gaussian model which causes the protrusions to merge and a single wide protrusion is present in the system. In both the models, the relative time scale between the membrane and filament dynamics plays an important role in deciding whether the shape of elasticity-velocity curve is concave or convex. Our numerical simulations agree reasonably well with our analytical calculations.

Chapter 6: cubic membranes the missing dimension of cell membrane organization.

PubMed

Almsherqi, Zakaria A; Landh, Tomas; Kohlwein, Sepp D; Deng, Yuru

2009-01-01

Biological membranes are among the most fascinating assemblies of biomolecules: a bilayer less than 10 nm thick, composed of rather small lipid molecules that are held together simply by noncovalent forces, defines the cell and discriminates between "inside" and "outside", survival, and death. Intracellular compartmentalization-governed by biomembranes as well-is a characteristic feature of eukaryotic cells, which allows them to fulfill multiple and highly specialized anabolic and catabolic functions in strictly controlled environments. Although cellular membranes are generally visualized as flat sheets or closely folded isolated objects, multiple observations also demonstrate that membranes may fold into "unusual", highly organized structures with 2D or 3D periodicity. The obvious correlation of highly convoluted membrane organizations with pathological cellular states, for example, as a consequence of viral infection, deserves close consideration. However, knowledge about formation and function of these highly organized 3D periodic membrane structures is scarce, primarily due to the lack of appropriate techniques for their analysis in vivo. Currently, the only direct way to characterize cellular membrane architecture is by transmission electron microscopy (TEM). However, deciphering the spatial architecture solely based on two-dimensionally projected TEM images is a challenging task and prone to artifacts. In this review, we will provide an update on the current progress in identifying and analyzing 3D membrane architectures in biological systems, with a special focus on membranes with cubic symmetry, and their potential role in physiological and pathophysiological conditions. Proteomics and lipidomics approaches in defined experimental cell systems may prove instrumental to understand formation and function of 3D membrane morphologies.

Dystypia: isolated typing impairment without aphasia, apraxia or visuospatial impairment.

PubMed

Otsuki, Mika; Soma, Yoshiaki; Arihiro, Shoji; Watanabe, Yoshimasa; Moriwaki, Hiroshi; Naritomi, Hiroaki

2002-01-01

We report a 60-year-old right-handed Japanese man who showed an isolated persistent typing impairment without aphasia, agraphia, apraxia or any other neuropsychological deficit. We coined the term 'dystypia' for this peculiar neuropsychological manifestation. The symptom was caused by an infarction in the left frontal lobe involving the foot of the second frontal convolution and the frontal operculum. The patient's typing impairment was not attributable to a disturbance of the linguistic process, since he had no aphasia or agraphia. The impairment was not attributable to the impairment of the motor execution process either, since he had no apraxia. Thus, his typing impairment was deduced to be based on a disturbance of the intermediate process where the linguistic phonological information is converted into the corresponding performance. We hypothesized that there is a specific process for typing which branches from the motor programming process presented in neurolinguistic models. The foot of the left second frontal convolution and the operculum may play an important role in the manifestation of 'dystypia'. Copyright 2002 S. Karger AG, Basel

Hydrogen-enriched water restoration of impaired calcium propagation by arsenic in primary keratinocytes

NASA Astrophysics Data System (ADS)

Yu, Wei-Tai; Chiu, Yi-Ching; Lee, Chih-Hung; Yoshioka, Tohru; Yu, Hsin-Su

2013-11-01

Endemic contamination of artesian water for drinking by arsenic is known to cause several human cancers, including cancers of the skin, bladder, and lungs. In skin, multiple arsenic-induced Bowen's disease (As-BD) can develop into invasive cancers after decades of arsenic exposure. The characteristic histological features of As-BD include full-layer epidermal dysplasia, apoptosis, and abnormal proliferation. Calcium propagation is an essential cellular event contributing to keratinocyte differentiation, proliferation, and apoptosis, all of which occur in As-BD. This study investigated how arsenic interferes calcium propagation of skin keratinocytes through ROS production and whether hydrogen-enriched water would restore arsenic-impaired calcium propagation. Arsenic was found to induce oxidative stress and inhibit ATP- and thapsigaragin-induced calcium propagation. Pretreatment of arsenic-treated keratinocytes by hydrogen-enriched water or beta-mercaptoethanol with potent anti-oxidative effects partially restored the propagation of calcium by ATP and by thapsigaragin. It was concluded that arsenic may impair calcium propagation, likely through oxidative stress and interactions with thiol groups in membrane proteins.

Sulfated Titania-Silica Reinforced Nafion Nanocomposite Membranes for Proton Exchange Membrane Fuel Cells.

PubMed

Abu Sayeed, M D; Kim, Hee Jin; Gopalan, A I; Kim, Young Ho; Lee, Kwang-Pill; Choi, Sang-June

2015-09-01

Sulfated titania-silica (SO4(2-)-/TiO2-SiO2) composites were prepared by a sol-gel method with sulfate reaction and characterized by X-ray diffraction (XRD) and energy-dispersive X-ray spectroscopy (EDS). The nanometric diameter and geometry of the sulfated titania-silica (STS) was investigated by transmission electron microscopy (TEM). A small amount of the STS composite in the range of 0.5-3 wt% was then added as reinforcing into the Nafion membrane by water-assisted solution casting method to prepare STS reinforced Nafion nanocomposite membranes (STS-Nafion nanocomposite membranes). The additional functional groups, sulfate groups, of the nanocomposite membrane having more surface oxygenated groups enhanced the fuel cell membrane properties. The STS-Nafion nanocomposite membranes exhibited improved water uptake compared to that of neat Nafion membranes, whereas methanol uptake values were decreased dramatically improved thermal property of the prepared nanocomposite membranes were measured by thermogravimetric analysis (TGA). Furthermore, increased ion exchange capacity values were obtained by thermoacidic pretreatment of the nanocomposite membranes.

Asymmetric Membranes from Two Chemically Distinct Triblock Terpolymers Blended during Standard Membrane Fabrication.

PubMed

Li, Yuk Mun; Srinivasan, Divya; Vaidya, Parth; Gu, Yibei; Wiesner, Ulrich

2016-10-01

Deviating from the traditional formation of block copolymer derived isoporous membranes from one block copolymer chemistry, here asymmetric membranes with isoporous surface structure are derived from two chemically distinct block copolymers blended during standard membrane fabrication. As a first proof of principle, the fabrication of asymmetric membranes is reported, which are blended from two chemically distinct triblock terpolymers, poly(isoprene-b-styrene-b-(4-vinyl)pyridine) (ISV) and poly(isoprene-b-styrene-b-(dimethylamino)ethyl methacrylate) (ISA), differing in the pH-responsive hydrophilic segment. Using block copolymer self-assembly and nonsolvent induced phase separation process, pure and blended membranes are prepared by varying weight ratios of ISV to ISA. Pure and blended membranes exhibit a thin, selective layer of pores above a macroporous substructure. Observed permeabilities at varying pH values of blended membranes depend on relative triblock terpolymer composition. These results open a new direction for membrane fabrication through the use of mixtures of chemically distinct block copolymers enabling the tailoring of membrane surface chemistries and functionalities. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel high light-inducible carotenoid-binding protein complex in the thylakoid membranes of Synechocystis PCC 6803

PubMed Central

Daddy, Soumana; Zhan, Jiao; Jantaro, Saowarath; He, Chenliu; He, Qingfang; Wang, Qiang

2015-01-01

Synechocystis sp. PCC 6803 is a model cyanobacterium extensively used to study photosynthesis. Here we reveal a novel high light-inducible carotenoid-binding protein complex (HLCC) in the thylakoid membranes of Synechocystis PCC 6803 cells exposed to high intensity light. Zeaxanthin and myxoxanthophyll accounted for 29.8% and 54.8%, respectively, of the carotenoids bound to the complex. Using Blue-Native PAGE followed by 2D SDS-PAGE and mass spectrometry, we showed that the HLCC consisted of Slr1128, IsiA, PsaD, and HliA/B. We confirmed these findings by SEAD fluorescence cross-linking and anti-PsaD immuno-coprecipitation analyses. The expression of genes encoding the protein components of the HLCC was enhanced by high light illumination and artificial oxidative stress. Deletion of these proteins resulted in impaired state transition and increased sensitivity to oxidative and/or high light stress, as indicated by increased membrane peroxidation. Therefore, the HLCC protects thylakoid membranes from extensive photooxidative damage, likely via a mechanism involving state transition. PMID:25820628

Probing lipid membrane electrostatics

NASA Astrophysics Data System (ADS)

Yang, Yi

The electrostatic properties of lipid bilayer membranes play a significant role in many biological processes. Atomic force microscopy (AFM) is highly sensitive to membrane surface potential in electrolyte solutions. With fully characterized probe tips, AFM can perform quantitative electrostatic analysis of lipid membranes. Electrostatic interactions between Silicon nitride probes and supported zwitterionic dioleoylphosphatidylcholine (DOPC) bilayer with a variable fraction of anionic dioleoylphosphatidylserine (DOPS) were measured by AFM. Classical Gouy-Chapman theory was used to model the membrane electrostatics. The nonlinear Poisson-Boltzmann equation was numerically solved with finite element method to provide the potential distribution around the AFM tips. Theoretical tip-sample electrostatic interactions were calculated with the surface integral of both Maxwell and osmotic stress tensors on tip surface. The measured forces were interpreted with theoretical forces and the resulting surface charge densities of the membrane surfaces were in quantitative agreement with the Gouy-Chapman-Stern model of membrane charge regulation. It was demonstrated that the AFM can quantitatively detect membrane surface potential at a separation of several screening lengths, and that the AFM probe only perturbs the membrane surface potential by <2%. One important application of this technique is to estimate the dipole density of lipid membrane. Electrostatic analysis of DOPC lipid bilayers with the AFM reveals a repulsive force between the negatively charged probe tips and the zwitterionic lipid bilayers. This unexpected interaction has been analyzed quantitatively to reveal that the repulsion is due to a weak external field created by the internai membrane dipole moment. The analysis yields a dipole moment of 1.5 Debye per lipid with a dipole potential of +275 mV for supported DOPC membranes. This new ability to quantitatively measure the membrane dipole density in a noninvasive

Impaired Verb Fluency: A Sign of Mild Cognitive Impairment

ERIC Educational Resources Information Center

Ostberg, Per; Fernaeus, Sven-Erik; Hellstrom, Ake; Bogdanovic, Nenad; Wahlund, Lars Olof

2005-01-01

We assessed verb fluency vs. noun and letter-based fluency in 199 subjects referred for cognitive complaints including Subjective Cognitive Impairment, Mild Cognitive Impairment, and Alzheimer's disease. ANCOVAs and factor analyses identified verb, noun, and letter-based fluency as distinct tasks. Verb fluency performance in Mild Cognitive…