Building and Breaking the Cell Wall in Four Acts: A Kinesthetic and Tactile Role-Playing Exercise for Teaching Beta-Lactam Antibiotic Mechanism of Action and Resistance †

PubMed Central

Popovich, John; Stephens, Michelle; Celaya, Holly; Suwarno, Serena; Barclay, Shizuka; Yee, Emily; Dean, David A.; Farris, Megan; Haydel, Shelley E.

2018-01-01

“Building and breaking the cell wall” is designed to review the bacterial cell envelope, previously learned in lower-division biology classes, while introducing new topics such as antibiotics and bacterial antibiotic resistance mechanisms. We developed a kinesthetic and tactile modeling activity where students act as cellular components and construct the cell wall. In the first two acts, students model a portion of the gram-positive bacterial cell envelope and then demonstrate in detail how the peptidoglycan is formed. Act III involves student demonstration of the addition of β-lactam antibiotics to the environment and how they inhibit the formation of peptidoglycan, thereby preventing bacterial replication. Using Staphylococcus aureus as a model for gram-positive bacteria, students finish the activity (Act IV) by acting out how S. aureus often becomes resistant to β-lactam antibiotics. A high level of student engagement was observed, and the activity received positive feedback. In an assessment administered prior to and two months after the activity, significant improvements in scores were observed (p < 0.0001), demonstrating increased understanding and retention. This activity allows students to (i) visualize, role play, and kinesthetically “build” the cell envelope and form the peptidoglycan layer, (ii) understand the mechanism of action for β-lactam antibiotics, as well as how gene acquisition and protein changes result in resistance, and (iii) work cooperatively and actively to promote long-term retention of the subject material. PMID:29904519

Subpolar addition of new cell wall is directed by DivIVA in mycobacteria

PubMed Central

Meniche, Xavier; Otten, Renee; Siegrist, M. Sloan; Baer, Christina E.; Murphy, Kenan C.; Bertozzi, Carolyn R.; Sassetti, Christopher M.

2014-01-01

Mycobacteria are surrounded by a complex multilayered envelope and elongate at the poles. The principles that organize the coordinated addition of chemically diverse cell wall layers during polar extension remain unclear. We show that enzymes mediating the terminal cytosolic steps of peptidoglycan, arabinogalactan, and mycolic acid synthesis colocalize at sites of cell growth or division. The tropomyosin-like protein, DivIVA, is targeted to the negative curvature of the pole, is enriched at the growing end, and determines cell shape from this site. In contrast, cell wall synthetic complexes are concentrated at a distinct subpolar location. When viewed at subdiffraction resolution, new peptidoglycan is deposited at this subpolar site, and inert cell wall covers the DivIVA-marked tip. The differentiation between polar tip and cell wall synthetic complexes is also apparent at the biochemical level. Enzymes that generate mycolate precursors interact with DivIVA, but the final condensation of mycolic acids occurs in a distinct protein complex at the site of nascent cell wall addition. We propose an ultrastructural model of mycobacterial polar growth where new cell wall is added in an annular zone below the cell tip. This model may be broadly applicable to other bacterial and fungal organisms that grow via polar extension. PMID:25049412

The cell envelope stress response of Bacillus subtilis: from static signaling devices to dynamic regulatory network.

PubMed

Radeck, Jara; Fritz, Georg; Mascher, Thorsten

2017-02-01

The cell envelope stress response (CESR) encompasses all regulatory events that enable a cell to protect the integrity of its envelope, an essential structure of any bacterial cell. The underlying signaling network is particularly well understood in the Gram-positive model organism Bacillus subtilis. It consists of a number of two-component systems (2CS) and extracytoplasmic function σ factors that together regulate the production of both specific resistance determinants and general mechanisms to protect the envelope against antimicrobial peptides targeting the biogenesis of the cell wall. Here, we summarize the current picture of the B. subtilis CESR network, from the initial identification of the corresponding signaling devices to unraveling their interdependence and the underlying regulatory hierarchy within the network. In the course of detailed mechanistic studies, a number of novel signaling features could be described for the 2CSs involved in mediating CESR. This includes a novel class of so-called intramembrane-sensing histidine kinases (IM-HKs), which-instead of acting as stress sensors themselves-are activated via interprotein signal transfer. Some of these IM-HKs are involved in sensing the flux of antibiotic resistance transporters, a unique mechanism of responding to extracellular antibiotic challenge.

Electric field mediated loading of macromolecules in intact yeast cells is critically controlled at the wall level.

PubMed

Ganeva, V; Galutzov, B; Teissié, J

1995-12-13

The mechanism of electric field mediated macromolecule transfer inside an intact yeast cell was investigated by observing, under a microscope, the fluorescence associated to cells after pulsation in a buffer containing two different hydrophilic fluorescent dyes. In the case of a small probe such as propidium iodide, a long lived permeabilized state was induced by the field as classically observed on wall free systems. Penetration of a 70 kDa FITC dextran was obtained only by using drastic conditions and only a very limited number of yeast cells which took up macromolecules remained viable. Most dextrans were trapped in the wall. A dramatic improvement in transfer of dextrans was observed when the cells were treated by dithiothreitol before pulsation. A cytoplasmic protein leakage was detected after the electric treatment suggesting that an irreversible damage took place in the walls of many pulsed cells. Electroloading of macromolecules in intact yeast cells appears to be controlled by a field induced short lived alteration of the envelope organization.

Built-up outer wall and roofing sections for double walled envelope homes

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

Brodhead, B.

1980-01-01

A site built system that uses the inner envelope wall is described. Blocking and vertical nailers are attached to this wall and sheathed with foil faced drywall to create the envelope cavity. An outer layer of 3 1/2 in. of Expended Poly Styrene provides continuous solid insulation. The trusses are also sheathed in foil faced drywall and insulated with 5 1/2 in. of E.P.S. This effectively surrounds the building with a continuous vapor and infiltration barrier. Construction details as well as cost breakdowns are presented.

Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein

PubMed Central

Tsakalidou, E.; Anastasiou, R.; Vandenberghe, I.; van Beeumen, J.; Kalantzopoulos, G.

1999-01-01

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified. PMID:10223997

Modulating activity of vancomycin and daptomycin on the expression of autolysis cell-wall turnover and membrane charge genes in hVISA and VISA strains.

PubMed

Cafiso, Viviana; Bertuccio, Taschia; Spina, Daniela; Purrello, Simona; Campanile, Floriana; Di Pietro, Cinzia; Purrello, Michele; Stefani, Stefania

2012-01-01

Glycopeptides are still the gold standard to treat MRSA (Methicillin Resistant Staphylococcus aureus) infections, but their widespread use has led to vancomycin-reduced susceptibility [heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA) and Vancomycin-Intermediate-Staphylococcus aureus (VISA)], in which different genetic loci (regulatory, autolytic, cell-wall turnover and cell-envelope positive charge genes) are involved. In addition, reduced susceptibility to vancomycin can influence the development of resistance to daptomycin. Although the phenotypic and molecular changes of hVISA/VISA have been the focus of different papers, the molecular mechanisms responsible for these different phenotypes and for the vancomycin and daptomycin cross-resistance are not clearly understood. The aim of our study was to investigate, by real time RT-PCR, the relative quantitative expression of genes involved in autolysis (atl-lytM), cell-wall turnover (sceD), membrane charges (mprF-dltA) and regulatory mechanisms (agr-locus-graRS-walKR), in hVISA and VISA cultured with or without vancomycin and daptomycin, in order to better understand the molecular basis of vancomycin-reduced susceptibility and the modulating activity of vancomycin and daptomycin on the expression of genes implicated in their reduced susceptibility mechanisms. Our results show that hVISA and VISA present common features that distinguish them from Vancomycin-Susceptible Staphylococcus aureus (VSSA), responsible for the intermediate glycopeptide resistance i.e. an increased cell-wall turnover, an increased positive cell-wall charge responsible for a repulsion mechanism towards vancomycin and daptomycin, and reduced agr-functionality. Indeed, VISA emerges from hVISA when VISA acquires a reduced autolysis caused by a down-regulation of autolysin genes, atl/lytM, and a reduction of the net negative cell-envelope charge via dltA over-expression. Vancomycin and daptomycin, acting in a similar manner in hVISA and VISA, can influence their cross-resistance mechanisms promoting VISA behavior in hVISA and enhancing the cell-wall pathways responsible for the intermediate vancomycin resistance in VISA. Daptomycin can also induce a charge repulsion mechanism both in hVISA and VISA increasing the activity of the mprF.

Cell wall layers delimit cell groups derived from cell division in the foliose trebouxiophycean alga Prasiola japonica.

PubMed

Mine, Ichiro; Kinoshita, Urara; Kawashima, Shigetaka; Sekida, Satoko

2018-01-22

The cells in the foliose thallus of trebouxiophycean alga Prasiola japonica apparently develop into 2 × 2 cell groups composed of two two-celled groups, each of which is a pair of derivative cells of the latest cell division. In the present study, the structural features of cell walls of the alga P. japonica concerning the formation of the cell groups were investigated using histochemical methods. Thin cell layers stained by Calcofluor White appeared to envelope the two-celled and four-celled groups separately and, hence, separated them from neighboring cell groups, and the Calcofluor White-negative gaps between neighboring four-celled groups were specifically stained by lectins, such as soybean agglutinin, jacalin, and Vicia villosa lectin conjugated with fluorescein. These results indicated that the Calcofluor White-positive cell wall layer of parent cell that existed during two successive cell divisions structurally distinguished two-celled and four-celled groups from others in this alga. Moreover, the results suggested that the cell wall components of the Calcofluor White-negative gaps would possibly contribute to the formation of the planar thallus through lateral union of the cell groups.

Immunogenicity, biochemical and serological characterizations of ribosomal preparations from human oral strains of serotypes c and d of the bacterium Streptococcus mutans.

PubMed

Huis in 't Veld, J; Fischer, M

1984-01-01

Crude ribosomal preparations of Streptococcus mutans C67-1 (serotype c) and 50B4 (serotype d) contain protein RNA and carbohydrate. Sepharose CL-2B column chromatography of preparations yielded two distinct peaks. Cell-wall carbohydrates were predominantly present in peak I; the serological activity resided mainly in peak II. The preparations contained antigens which cross-reacted with several streptococcal Lancefield antisera. Antisera prepared against the preparations cross-reacted with cell-wall proteins (NaCl extracts) and Ag I/II, but not with cell-wall carbohydrate antigens (Rantz-Randall extracts). Thus, cell-envelope protein antigens in the preparations appear to be responsible for the serological activity. The unique properties of ribosomal preparations may, apart from serological cross-reactivity, be useful in the immunological protection against dental caries.

Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid.

PubMed

Zhang, Xinzheng; Xiang, Ye; Dunigan, David D; Klose, Thomas; Chipman, Paul R; Van Etten, James L; Rossmann, Michael G

2011-09-06

A cryoelectron microscopy 8.5 Å resolution map of the 1,900 Å diameter, icosahedral, internally enveloped Paramecium bursaria chlorella virus was used to interpret structures of the virus at initial stages of cell infection. A fivefold averaged map demonstrated that two minor capsid proteins involved in stabilizing the capsid are missing in the vicinity of the unique vertex. Reconstruction of the virus in the presence of host chlorella cell walls established that the spike at the unique vertex initiates binding to the cell wall, which results in the enveloped nucleocapsid moving closer to the cell. This process is concurrent with the release of the internal viral membrane that was linked to the capsid by many copies of a viral membrane protein in the mature infectous virus. Simultaneously, part of the trisymmetrons around the unique vertex disassemble, probably in part because two minor capsid proteins are absent, causing Paramecium bursaria chlorella virus and the cellular contents to merge, possibly as a result of enzyme(s) within the spike assembly. This may be one of only a few recordings of successive stages of a virus while infecting a eukaryotic host in pseudoatomic detail in three dimensions.

Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid

PubMed Central

Zhang, Xinzheng; Xiang, Ye; Dunigan, David D.; Klose, Thomas; Chipman, Paul R.; Van Etten, James L.; Rossmann, Michael G.

2011-01-01

A cryoelectron microscopy 8.5 Å resolution map of the 1,900 Å diameter, icosahedral, internally enveloped Paramecium bursaria chlorella virus was used to interpret structures of the virus at initial stages of cell infection. A fivefold averaged map demonstrated that two minor capsid proteins involved in stabilizing the capsid are missing in the vicinity of the unique vertex. Reconstruction of the virus in the presence of host chlorella cell walls established that the spike at the unique vertex initiates binding to the cell wall, which results in the enveloped nucleocapsid moving closer to the cell. This process is concurrent with the release of the internal viral membrane that was linked to the capsid by many copies of a viral membrane protein in the mature infectous virus. Simultaneously, part of the trisymmetrons around the unique vertex disassemble, probably in part because two minor capsid proteins are absent, causing Paramecium bursaria chlorella virus and the cellular contents to merge, possibly as a result of enzyme(s) within the spike assembly. This may be one of only a few recordings of successive stages of a virus while infecting a eukaryotic host in pseudoatomic detail in three dimensions. PMID:21873222

LytN, a Murein Hydrolase in the Cross-wall Compartment of Staphylococcus aureus, Is Involved in Proper Bacterial Growth and Envelope Assembly*

PubMed Central

Frankel, Matthew B.; Hendrickx, Antoni P. A.; Missiakas, Dominique M.; Schneewind, Olaf

2011-01-01

Cell cycle progression for the spherical microbe Staphylococcus aureus requires the coordinated synthesis and remodeling of peptidoglycan. The majority of these rearrangements takes place at the mid-cell, in a compartment designated the cross-wall. Secreted polypeptides endowed with a YSIRK-G/S signal peptide are directly delivered to the cross-wall compartment. One such YSIRK-containing protein is the murein hydrolase LytN. lytN mutations precipitate structural damage to the cross-wall and interfere with staphylococcal growth. Overexpression of lytN also affects growth and triggers rupture of the cross-wall. The lytN phenotype can be reversed by the controlled expression of lytN but not by adding purified LytN to staphylococcal cultures. LytN harbors LysM and CHAP domains, the latter of which functions as both an N-acetylmuramoyl-l-alanine amidase and d-alanyl-glycine endopeptidase. Thus, LytN secretion into the cross-wall promotes peptidoglycan separation and completion of the staphylococcal cell cycle. PMID:21784864

Cell Envelope of Corynebacteria: Structure and Influence on Pathogenicity

PubMed Central

Burkovski, Andreas

2013-01-01

To date the genus Corynebacterium comprises 88 species. More than half of these are connected to human and animal infections, with the most prominent member of the pathogenic species being Corynebacterium diphtheriae, which is also the type species of the genus. Corynebacterium species are characterized by a complex cell wall architecture: the plasma membrane of these bacteria is followed by a peptidoglycan layer, which itself is covalently linked to a polymer of arabinogalactan. Bound to this, an outer layer of mycolic acids is found which is functionally equivalent to the outer membrane of Gram-negative bacteria. As final layer, free polysaccharides, glycolipids, and proteins are found. The composition of the different substructures of the corynebacterial cell envelope and their influence on pathogenicity are discussed in this paper. PMID:23724339

Cell envelope of corynebacteria: structure and influence on pathogenicity.

PubMed

Burkovski, Andreas

2013-01-01

To date the genus Corynebacterium comprises 88 species. More than half of these are connected to human and animal infections, with the most prominent member of the pathogenic species being Corynebacterium diphtheriae, which is also the type species of the genus. Corynebacterium species are characterized by a complex cell wall architecture: the plasma membrane of these bacteria is followed by a peptidoglycan layer, which itself is covalently linked to a polymer of arabinogalactan. Bound to this, an outer layer of mycolic acids is found which is functionally equivalent to the outer membrane of Gram-negative bacteria. As final layer, free polysaccharides, glycolipids, and proteins are found. The composition of the different substructures of the corynebacterial cell envelope and their influence on pathogenicity are discussed in this paper.

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes*

PubMed Central

Chan, Yvonne Gar-Yun; Kim, Hwan Keun; Schneewind, Olaf; Missiakas, Dominique

2014-01-01

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan. PMID:24753256

Long clinostation influence on the localization of free and weakly bound calcium in cell walls of Funaria hygrometrica moss protonema cells

NASA Astrophysics Data System (ADS)

Nedukha, E. M.

The pyroantimonate method was used to study the localization of free and weakly bound calcium in cells of moss protonema of Funaria hygrometrica Hedw. cultivated on a clinostat (2 rev/min). Electroncytochemical study of control cells cultivated at 1 g revealed that granular precipitate marked chloroplasts, mitochondria, Golgi apparatus, lipid drops, nucleoplasma, nucleolus, nucleus membranes, cell walls and endoplasmic reticulum. In mitochondria the precipitate was revealed in stroma, in chloroplast it was found on thylakoids and envelope membranes. The cultivation of protonema on clinostat led to the intensification in cytochemical reaction product deposit. A considerable intensification of the reaction was noted in endomembranes, vacuoles, periplasmic space and cell walls. At the same time analysis of pectinase localization was made using the electroncytochemical method. A high reaction intensity in walls in comparison to that in control was found out to be a distinctive pecularity of the cells cultivated on clinostat. It testifies to the fact that increasing of freee calcium concentrations under conditions of clinostation is connected with pectinic substances hydrolysis and breaking of methoxy groups of pectins. Data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance of plant cells and the role of cell walls in gomeostasis of cell grown under conditions of simulated weighlessness.

Subcellular localization, interactions and dynamics of the phage-shock protein-like Lia response in Bacillus subtilis.

PubMed

Domínguez-Escobar, Julia; Wolf, Diana; Fritz, Georg; Höfler, Carolin; Wedlich-Söldner, Roland; Mascher, Thorsten

2014-05-01

The liaIH operon of Bacillus subtilis is the main target of the envelope stress-inducible two-component system LiaRS. Here, we studied the localization, interaction and cellular dynamics of Lia proteins to gain insights into the physiological role of the Lia response. We demonstrate that LiaI serves as the membrane anchor for the phage-shock protein A homologue LiaH. Under non-inducing conditions, LiaI locates in highly motile membrane-associated foci, while LiaH is dispersed throughout the cytoplasm. Under stress conditions, both proteins are strongly induced and colocalize in numerous distinct static spots at the cytoplasmic membrane. This behaviour is independent of MreB and does also not correlate with the stalling of the cell wall biosynthesis machinery upon antibiotic inhibition. It can be induced by antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis, while compounds that inhibit the cytoplasmic or extracytoplasmic steps do not trigger this response. Taken together, our data are consistent with a model in which the Lia system scans the cytoplasmic membrane for envelope perturbations. Upon their detection, LiaS activates the cognate response regulator LiaR, which in turn strongly induces the liaIH operon. Simultaneously, LiaI recruits LiaH to the membrane, presumably to protect the envelope and counteract the antibiotic-induced damage. © 2014 John Wiley & Sons Ltd.

Cell fusion and nuclear fusion in plants.

PubMed

Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

2016-12-01

Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall. Copyright © 2016 Elsevier Ltd. All rights reserved.

Binding of /sup 18/F by cell membranes and cell walls of Streptococcus mutans

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

Yotis, W.W.; Zeb, M.; McNulty, J.

1983-07-01

The binding of /sup 18/F to isolated cell membranes and cell walls of Streptococcus mutans GS-5 or other bacteria was assayed. The attachment of /sup 18/F to these cell envelopes proceeded slowly and reached equilibrium within 60 min. /sup 18/F binding was stimulated by Ca/sup 2 +/ (1 mM). The binding of /sup 18/F to cellular components was dependent upon the pH, as well as the amount of /sup 18/F and dose of the binder employed. The binding of /sup 18/F by cell walls prepared from fluoride-sensitive and fluoride-resistant cells of S. salivarius and S. mutans did not differ significantly.more » The pretreatment of cell walls or cell membranes for 60 min at 30 degrees C with 1 mg of RNase, DNase, or trypsin per ml did not influence the binding of /sup 18/F by the walls and membranes of S. mutans GS-5. However, prior exposure of cell membranes to sodium dodecyl sulfate caused a significant reduction in the number of /sup 18/F atoms bound by the membranes. In saturated assay systems, cell membranes of S. mutans GS-5 bound 10(15) to 10(16) atoms of /sup 18/F per mg (dry weight), whereas cell walls from S. mutans GS-5, FA-1, and HS-6 or Actinomyces viscosus T14V and T14AV bound 10(12) to 10(13) atoms of /sup 18/F per mg (dry weight). /sup 18/F in this quantity (10(12) to 10(13) atoms) cannot be detected with the fluoride electrode. The data provide, for the first time, a demonstration of /sup 18/F binding by cell membranes and walls of oral flora.« less

Physiological and Transcriptional Responses of Saccharomyces cerevisiae to d-Limonene Show Changes to the Cell Wall but Not to the Plasma Membrane

PubMed Central

Brennan, Timothy C. R.; Nielsen, Lars K.

2013-01-01

Monoterpenes can, upon hydrogenation, be used as light-fraction components of sustainable aviation fuels. Fermentative production of monoterpenes in engineered microorganisms, such as Saccharomyces cerevisiae, has gained attention as a potential route to deliver these next-generation fuels from renewable biomass. However, end product toxicity presents a formidable problem for microbial synthesis. Due to their hydrophobicity, monoterpene inhibition has long been attributed to membrane interference, but the molecular mechanism remains largely unsolved. In order to gain a better understanding of the mode of action, we analyzed the composition and structural integrity of the cell envelope as well as the transcriptional response of yeast cells treated with an inhibitory amount of d-limonene (107 mg/liter). We found no alterations in membrane fluidity, structural membrane integrity, or fatty acid composition after the solvent challenge. A 4-fold increase in the mean fluorescence intensity per cell (using calcofluor white stain) and increased sensitivity to cell wall-degrading enzymes demonstrated that limonene disrupts cell wall properties. Global transcript measurements confirmed the membrane integrity observations by showing no upregulation of ergosterol or fatty acid biosynthesis pathways, which are commonly overexpressed in yeast to reinforce membrane rigidity during ethanol exposure. Limonene shock did cause a compensatory response to cell wall damage through overexpression of several genes (ROM1, RLM1, PIR3, CTT1, YGP1, MLP1, PST1, and CWP1) involved with the cell wall integrity signaling pathway. This is the first report demonstrating that cell wall, rather than plasma membrane, deterioration is the main source of monoterpene inhibition. We show that limonene can alter the structure and function of the cell wall, which has a clear effect on cytokinesis. PMID:23542628

An Accessory Protein Required for Anchoring and Assembly of Amyloid Fibers in B. subtilis Biofilms

PubMed Central

Romero, Diego; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2011-01-01

Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibers, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibers. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibers to the cell wall and to assemble TasA into fibers. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibers can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibers. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibers that are not anchored to the cell. PMID:21477127

An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms.

PubMed

Romero, Diego; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2011-06-01

Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibres, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibres. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibres to the cell wall and to assemble TasA into fibres. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibres can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibres. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibres that are not anchored to the cell. © 2011 Blackwell Publishing Ltd.

The Baseplate of Lactobacillus delbrueckii Bacteriophage Ld17 Harbors a Glycerophosphodiesterase*

PubMed Central

Cornelissen, Anneleen; Sadovskaya, Irina; Vinogradov, Evgeny; Blangy, Stéphanie; Spinelli, Silvia; Casey, Eoghan; Mahony, Jennifer; Noben, Jean-Paul; Dal Bello, Fabio; Cambillau, Christian; van Sinderen, Douwe

2016-01-01

Glycerophosphodiester phosphodiesterases (GDPDs; EC 3.1.4.46) typically hydrolyze glycerophosphodiesters to sn-glycerol 3-phosphate (Gro3P) and their corresponding alcohol during patho/physiological processes in bacteria and eukaryotes. GDPD(-like) domains were identified in the structural particle of bacterial viruses (bacteriophages) specifically infecting Gram-positive bacteria. The GDPD of phage 17 (Ld17; GDPDLd17), representative of the group b Lactobacillus delbrueckii subsp. bulgaricus (Ldb)-infecting bacteriophages, was shown to hydrolyze, besides the simple glycerophosphodiester, two complex surface-associated carbohydrates of the Ldb17 cell envelope: the Gro3P decoration of the major surface polysaccharide d-galactan and the oligo(glycerol phosphate) backbone of the partially glycosylated cell wall teichoic acid, a minor Ldb17 cell envelope component. Degradation of cell wall teichoic acid occurs according to an exolytic mechanism, and Gro3P substitution is presumed to be inhibitory for GDPDLd17 activity. The presence of the GDPDLd17 homotrimer in the viral baseplate structure involved in phage-host interaction together with the dependence of native GDPD activity, adsorption, and efficiency of plating of Ca2+ ions supports a role for GDPDLd17 activity during phage adsorption and/or phage genome injection. In contrast to GDPDLd17, we could not identify any enzymatic activity for the GDPD-like domain in the neck passage structure of phage 340, a 936-type Lactococcus lactis subsp. lactis bacteriophage. PMID:27268053

2-aminoimidazoles potentiate ß-lactam antimicrobial activity against Mycobacterium tuberculosis by reducing ß-lactamase secretion and increasing cell envelope permeability

PubMed Central

Obregón-Henao, Andrés; Ackart, David F.; Podell, Brendan K.; Belardinelli, Juan M.; Jackson, Mary; Nguyen, Tuan V.; Blackledge, Meghan S.; Melander, Roberta J.; Melander, Christian; Johnson, Benjamin K.; Abramovitch, Robert B.

2017-01-01

There is an urgent need to develop new drug treatment strategies to control the global spread of drug-sensitive and multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis). The ß-lactam class of antibiotics is among the safest and most widely prescribed antibiotics, but they are not effective against M. tuberculosis due to intrinsic resistance. This study shows that 2-aminoimidazole (2-AI)-based small molecules potentiate ß-lactam antibiotics against M. tuberculosis. Active 2-AI compounds significantly reduced the minimal inhibitory and bactericidal concentrations of ß-lactams by increasing M. tuberculosis cell envelope permeability and decreasing protein secretion including ß-lactamase. Metabolic labeling and transcriptional profiling experiments revealed that 2-AI compounds impair mycolic acid biosynthesis, export and linkage to the mycobacterial envelope, counteracting an important defense mechanism reducing permeability to external agents. Additionally, other important constituents of the M. tuberculosis outer membrane including sulfolipid-1 and polyacyltrehalose were also less abundant in 2-AI treated bacilli. As a consequence of 2-AI treatment, M. tuberculosis displayed increased sensitivity to SDS, increased permeability to nucleic acid staining dyes, and rapid binding of cell wall targeting antibiotics. Transcriptional profiling analysis further confirmed that 2-AI induces transcriptional regulators associated with cell envelope stress. 2-AI based small molecules potentiate the antimicrobial activity of ß-lactams by a mechanism that is distinct from specific inhibitors of ß-lactamase activity and therefore may have value as an adjunctive anti-TB treatment. PMID:28749949

Thermal Insulating Concrete Wall Panel Design for Sustainable Built Environment

PubMed Central

Zhou, Ao; Wong, Kwun-Wah

2014-01-01

Air-conditioning system plays a significant role in providing users a thermally comfortable indoor environment, which is a necessity in modern buildings. In order to save the vast energy consumed by air-conditioning system, the building envelopes in envelope-load dominated buildings should be well designed such that the unwanted heat gain and loss with environment can be minimized. In this paper, a new design of concrete wall panel that enhances thermal insulation of buildings by adding a gypsum layer inside concrete is presented. Experiments have been conducted for monitoring the temperature variation in both proposed sandwich wall panel and conventional concrete wall panel under a heat radiation source. For further understanding the thermal effect of such sandwich wall panel design from building scale, two three-story building models adopting different wall panel designs are constructed for evaluating the temperature distribution of entire buildings using finite element method. Both the experimental and simulation results have shown that the gypsum layer improves the thermal insulation performance by retarding the heat transfer across the building envelopes. PMID:25177718

Thermal insulating concrete wall panel design for sustainable built environment.

PubMed

Zhou, Ao; Wong, Kwun-Wah; Lau, Denvid

2014-01-01

Air-conditioning system plays a significant role in providing users a thermally comfortable indoor environment, which is a necessity in modern buildings. In order to save the vast energy consumed by air-conditioning system, the building envelopes in envelope-load dominated buildings should be well designed such that the unwanted heat gain and loss with environment can be minimized. In this paper, a new design of concrete wall panel that enhances thermal insulation of buildings by adding a gypsum layer inside concrete is presented. Experiments have been conducted for monitoring the temperature variation in both proposed sandwich wall panel and conventional concrete wall panel under a heat radiation source. For further understanding the thermal effect of such sandwich wall panel design from building scale, two three-story building models adopting different wall panel designs are constructed for evaluating the temperature distribution of entire buildings using finite element method. Both the experimental and simulation results have shown that the gypsum layer improves the thermal insulation performance by retarding the heat transfer across the building envelopes.

Chemistry of Peptidoglycan in Mycobacterium tuberculosis Life Cycle: An off-the-wall Balance of Synthesis and Degradation.

PubMed

Squeglia, Flavia; Ruggiero, Alessia; Berisio, Rita

2018-02-21

The cell wall envelope of mycobacteria is structurally distinct from that of both Gram-positive and Gram-negative bacteria. In Mycobacterium tuberculosis, this cell wall has unique structural features and plays a crucial role in drug resistance and macrophage survival under stress conditions. Peptidoglycan is the major constituent of this cell wall, with an important structural role, giving structural strength, and counteracting the osmotic pressure of the cytoplasm. Synthesis of this complex polymer takes place in three stages that occur at three different locations in the cell, from the cytoplasm to the external side of the cell membrane, where polymerization occurs. A fine balance of peptidoglycan synthesis and degradation is responsible for a plethora of molecular mechanisms which are key to the pathogenicity of M. tuberculosis. Enlargement of mycobacterial cells can occur through the synthesis of new peptidoglycan, autolysis of old peptidoglycan, or a combination of both processes. Here, we discuss the chemical aspects of peptidoglycan synthesis and degradation, in relation to metabolic stages of M. tuberculosis. Going from inside the mycobacterial cytoplasm to outside its membrane, we describe the assembly line of peptidoglycan synthesis and polymerization, and continue with its depolymerization events and their consequences on mycobacterial life and resuscitation from dormancy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans.

PubMed

De, Arpan; Liao, Sumei; Bitoun, Jacob P; Roth, Randy; Beatty, Wandy L; Wu, Hui; Wen, Zezhang T

2017-09-01

Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG , encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, "squarish" dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation ( P < 0.01). Double and triple mutants with deficiency in brpA and/or psr , genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using the rgpG mutant. There were no major differences in growth rates between the wild-type strain and the rgpG brpA and rgpG psr double mutants, but the growth rate of the rgpG brpA psr triple mutant was reduced drastically ( P < 0.001). Under transmission electron microscopy, both double mutants resembled the rgpG mutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, the rgpG mutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and the brpA and psr single mutants. These results suggest that RgpG in S. mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope. IMPORTANCE Streptococcus mutans , a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans , indicative of a significant role of RGP in cell division and biofilm formation in S. mutans These results are novel not only in S. mutans , but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation in S. mutans This study reveals new potential targets to develop anticaries therapeutics. Copyright © 2017 American Society for Microbiology.

Performative building envelope design correlated to solar radiation and cooling energy consumption

NASA Astrophysics Data System (ADS)

Jacky, Thiodore; Santoni

2017-11-01

Climate change as an ongoing anthropogenic environmental challenge is predominantly caused by an amplification in the amount of greenhouse gases (GHGs), notably carbon dioxide (CO2) in building sector. Global CO2 emissions are emitted from HVAC (Heating, Ventilation, and Air Conditioning) occupation to provide thermal comfort in building. In fact, the amount of energy used for cooling or heating building is implication of building envelope design. Building envelope acts as interface layer of heat transfer between outdoor environment and the interior of a building. It appears as wall, window, roof and external shading device. This paper examines performance of various design strategy on building envelope to limit solar radiation and reduce cooling loads in tropical climate. The design strategies are considering orientation, window to wall ratio, material properties, and external shading device. This research applied simulation method using Autodesk Ecotect to investigate simultaneously between variations of wall and window ratio, shading device composition and the implication to the amount of solar radiation, cooling energy consumption. Comparative analysis on the data will determine logical variation between opening and shading device composition and cooling energy consumption. Optimizing the building envelope design is crucial strategy for reducing CO2 emissions and long-term energy reduction in building sector. Simulation technology as feedback loop will lead to better performative building envelope.

A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin.

PubMed

Kuznets, Galit; Vigonsky, Elena; Weissman, Ziva; Lalli, Daniela; Gildor, Tsvia; Kauffman, Sarah J; Turano, Paola; Becker, Jeffrey; Lewinson, Oded; Kornitzer, Daniel

2014-10-01

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7-/- mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.

Low-cost phase change material as an energy storage medium in building envelopes: Experimental and numerical analyses

DOE PAGES

Biswas, Kaushik; Abhari, Ramin

2014-10-03

A promising approach to increasing the energy efficiency of buildings is the implementation of a phase change material (PCM) in the building envelope. Numerous studies over the last two decades have reported the energy saving potential of PCMs in building envelopes, but their wide application has been inhibited, in part, by their high cost. This article describes a novel PCM made of naturally occurring fatty acids/glycerides trapped into high density polyethylene (HDPE) pellets and its performance in a building envelope application. The PCM-HDPE pellets were mixed with cellulose insulation and then added to an exterior wall of a test buildingmore » in a hot and humid climate, and tested over a period of several months, To demonstrate the efficacy of the PCM-enhanced cellulose insulation in reducing the building envelope heat gains and losses, side-by-side comparison was performed with another wall section filled with cellulose-only insulation. Further, numerical modeling of the test wall was performed to determine the actual impact of the PCM-HDPE pellets on wall-generated heating and cooling loads and the associated electricity consumption. The model was first validated using experimental data and then used for annual simulations using typical meteorological year (TMY3) weather data. Furthermore, this article presents the experimental data and numerical analyses showing the energy-saving potential of the new PCM.« less

Stud Walls With Continuous Exterior Insulation for Factory Built Housing: New York, New York (Fact Sheet), NREL (National Renewable Energy Laboratory)

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

None, None

The Advanced Envelope Research effort will provide factory homebuilders with high performance, cost-effective alternative envelope designs. In the near term, these technologies will play a central role in meeting stringent energy code requirements. For manufactured homes, the thermal requirements, last updated by statute in 1994, will move up to the more rigorous IECC 2012 levels in 2013, the requirements of which are consistent with site built and modular housing. This places added urgency on identifying envelope technologies that the industry can implement in the short timeframe. The primary goal of this research is to develop wall designs that meet themore » thermal requirements based on 2012 IECC standards. Given the affordable nature of manufactured homes, impact on first cost is a major consideration in developing the new envelope technologies. This work is part of a four-phase, multi-year effort. Phase 1 identified seven envelope technologies and provided a preliminary assessment of three selected methods for building high performance wall systems. Phase 2 focused on the development of viable product designs, manufacturing strategies, addressing code and structural issues, and cost analysis of the three selected options. An industry advisory committee helped critique and select the most viable solution to move further in the research - stud walls with continuous exterior insulation. Phase 3, the subject of the current report, focused on the design development of the selected wall concept and explored variations on the use of exterior foam insulation. The scope also included material selection, manufacturing and cost analysis, and prototyping and testing.« less

Technology Solutions Case Study: Stud Walls with Continuous Exterior Insulation for Factory Built Housing

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

None

The Advanced Envelope Research effort will provide factory homebuilders with high performance, cost-effective alternative envelope designs. In the near term, these technologies will play a central role in meeting stringent energy code requirements. For manufactured homes, the thermal requirements, last updated by statute in 1994, will move up to the more rigorous IECC 2012 levels in 2013, the requirements of which are consistent with site built and modular housing. This places added urgency on identifying envelope technologies that the industry can implement in the short timeframe. The primary goal of this research is to develop wall designs that meet themore » thermal requirements based on 2012 IECC standards. Given the affordable nature of manufactured homes, impact on first cost is a major consideration in developing the new envelope technologies. This work is part of a four-phase, multi-year effort. Phase 1 identified seven envelope technologies and provided a preliminary assessment of three selected methods for building high performance wall systems. Phase 2 focused on the development of viable product designs, manufacturing strategies, addressing code and structural issues, and cost analysis of the three selected options. An industry advisory committee helped critique and select the most viable solution to move further in the research — stud walls with continuous exterior insulation. Phase 3, the subject of the current report, focused on the design development of the selected wall concept and explored variations on the use of exterior foam insulation. The scope also included material selection, manufacturing and cost analysis, and prototyping and testing.« less

Advanced Envelope Research for Factory Built Housing, Phase 3. Design Development and Prototyping

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

Levy, E.; Kessler, B.; Mullens, M.

2014-01-01

The Advanced Envelope Research effort will provide factory homebuilders with high performance, cost-effective alternative envelope designs. In the near term, these technologies will play a central role in meeting stringent energy code requirements. For manufactured homes, the thermal requirements, last updated by statute in 1994, will move up to the more rigorous IECC 2012 levels in 2013, the requirements of which are consistent with site built and modular housing. This places added urgency on identifying envelope technologies that the industry can implement in the short timeframe. The primary goal of this research is to develop wall designs that meet themore » thermal requirements based on 2012 IECC standards. Given the affordable nature of manufactured homes, impact on first cost is a major consideration in developing the new envelope technologies. This work is part of a four-phase, multi-year effort. Phase 1 identified seven envelope technologies and provided a preliminary assessment of three selected methods for building high performance wall systems. Phase 2 focused on the development of viable product designs, manufacturing strategies, addressing code and structural issues, and cost analysis of the three selected options. An industry advisory committee helped critique and select the most viable solution to move further in the research -- stud walls with continuous exterior insulation. Phase 3, the subject of the current report, focused on the design development of the selected wall concept and explored variations on the use of exterior foam insulation. The scope also included material selection, manufacturing and cost analysis, and prototyping and testing.« less

Advanced Envelope Research for Factory Built Housing, Phase 3 -- Design Development and Prototyping

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

Levy, E.; Kessler, B.; Mullens, M.

2014-01-01

The Advanced Envelope Research effort will provide factory homebuilders with high performance, cost-effective alternative envelope designs. In the near term, these technologies will play a central role in meeting stringent energy code requirements. For manufactured homes, the thermal requirements, last updated by statute in 1994, will move up to the more rigorous IECC 2012 levels in 2013, the requirements of which are consistent with site built and modular housing. This places added urgency on identifying envelope technologies that the industry can implement in the short timeframe. The primary goal of this research is to develop wall designs that meet themore » thermal requirements based on 2012 IECC standards. Given the affordable nature of manufactured homes, impact on first cost is a major consideration in developing the new envelope technologies. This work is part of a four-phase, multi-year effort. Phase 1 identified seven envelope technologies and provided a preliminary assessment of three selected methods for building high performance wall systems. Phase 2 focused on the development of viable product designs, manufacturing strategies, addressing code and structural issues, and cost analysis of the three selected options. An industry advisory committee helped critique and select the most viable solution to move further in the research -- stud walls with continuous exterior insulation. Phase 3, the subject of the current report, focused on the design development of the selected wall concept and explored variations on the use of exterior foam insulation. The scope also included material selection, manufacturing and cost analysis, and prototyping and testing.« less

The effects of high-power microwaves on the ultrastructure of Bacillus subtilis.

PubMed

Kim, S-Y; Jo, E-K; Kim, H-J; Bai, K; Park, J-K

2008-07-01

To investigate the microbicidal mechanisms of high-power microwave (2.0 kW) irradiation on Bacillus subtilis and to determine the effect of this procedure on the ultrastructure of the cell wall. We performed viability test, examined cells using transmission electron microscopy (TEM), and measured the release of intracellular proteins and nucleic acids. The inactivation rate of B. subtilis by 2.0-kW microwave irradiation was higher than that of a domestic microwave (0.5 kW). Few proteins were released from either microwaved or boiled cells. However, the leakage of nucleic acids from 2.0-kW-microwaved cells was significantly higher than that of 0.5-kW-microwaved or boiled cells. Therefore, we examined ultrastructural alterations of microwaved or boiled cells to analyse the pattern of release of cytoplasmic contents. Although boiled cells did not show any ultrastructural changes on TEM, 2.0-kW-microwaved cells showed disruption of the cell wall. The microbicidal mechanisms of 2.0-kW microwave irradiation include damage to the microbial cell wall, breakage of the genomic DNA, and thermal coagulation of cytoplasmic proteins. TEM images showed that the cytoplasmic protein aggregation and cell envelope damage by microwave irradiation were different from the ultrastructural changes observed after boiling.

The Bacillus subtilis GntR family repressor YtrA responds to cell wall antibiotics.

PubMed

Salzberg, Letal I; Luo, Yun; Hachmann, Anna-Barbara; Mascher, Thorsten; Helmann, John D

2011-10-01

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism Bacillus subtilis. Ramoplanin strongly induces the LiaRS two-component regulatory system, while moenomycin almost exclusively induces genes that are part of the regulon of the extracytoplasmic function (ECF) σ factor σ(M). Ramoplanin additionally induces the ytrABCDEF and ywoBCD operons, which are not part of a previously characterized antibiotic-responsive regulon. Cluster analysis reveals that these two operons are selectively induced by a subset of cell wall antibiotics that inhibit lipid II function or recycling. Repression of both operons requires YtrA, which recognizes an inverted repeat in front of its own operon and in front of ywoB. These results suggest that YtrA is an additional regulator of cell envelope stress responses.

The Bacillus subtilis GntR Family Repressor YtrA Responds to Cell Wall Antibiotics▿§

PubMed Central

Salzberg, Letal I.; Luo, Yun; Hachmann, Anna-Barbara; Mascher, Thorsten; Helmann, John D.

2011-01-01

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism Bacillus subtilis. Ramoplanin strongly induces the LiaRS two-component regulatory system, while moenomycin almost exclusively induces genes that are part of the regulon of the extracytoplasmic function (ECF) σ factor σM. Ramoplanin additionally induces the ytrABCDEF and ywoBCD operons, which are not part of a previously characterized antibiotic-responsive regulon. Cluster analysis reveals that these two operons are selectively induced by a subset of cell wall antibiotics that inhibit lipid II function or recycling. Repression of both operons requires YtrA, which recognizes an inverted repeat in front of its own operon and in front of ywoB. These results suggest that YtrA is an additional regulator of cell envelope stress responses. PMID:21856850

Application of a Bacillus subtilis Whole-Cell Biosensor (PliaI-lux) for the Identification of Cell Wall Active Antibacterial Compounds.

PubMed

Kobras, Carolin Martina; Mascher, Thorsten; Gebhard, Susanne

2017-01-01

Whole-cell biosensors, based on the visualization of a reporter strain's response to a particular stimulus, are a robust and cost-effective means to monitor defined environmental conditions or the presence of chemical compounds. One specific field in which such biosensors are frequently applied is drug discovery, i.e., the screening of large numbers of bacterial or fungal strains for the production of antimicrobial compounds. We here describe the application of a luminescence-based Bacillus subtilis biosensor for the discovery of cell wall active substances. The system is based on the well-characterized promoter P liaI , which is induced in response to a wide range of conditions that cause cell envelope stress, particularly antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis. A simple "spot-on-lawn" assay, where colonies of potential producer strains are grown directly on a lawn of the reporter strain, allows for quantitative and time-resolved detection of antimicrobial compounds. Due to the very low technical demands of this procedure, we expect it to be easily applicable to a large variety of candidate producer strains and growth conditions.

Disruption of Brewers' yeast by hydrodynamic cavitation: Process variables and their influence on selective release.

PubMed

Balasundaram, B; Harrison, S T L

2006-06-05

Intracellular products, not secreted from the microbial cell, are released by breaking the cell envelope consisting of cytoplasmic membrane and an outer cell wall. Hydrodynamic cavitation has been reported to cause microbial cell disruption. By manipulating the operating variables involved, a wide range of intensity of cavitation can be achieved resulting in a varying extent of disruption. The effect of the process variables including cavitation number, initial cell concentration of the suspension and the number of passes across the cavitation zone on the release of enzymes from various locations of the Brewers' yeast was studied. The release profile of the enzymes studied include alpha-glucosidase (periplasmic), invertase (cell wall bound), alcohol dehydrogenase (ADH; cytoplasmic) and glucose-6-phosphate dehydrogenase (G6PDH; cytoplasmic). An optimum cavitation number Cv of 0.13 for maximum disruption was observed across the range Cv 0.09-0.99. The optimum cell concentration was found to be 0.5% (w/v, wet wt) when varying over the range 0.1%-5%. The sustained effect of cavitation on the yeast cell wall when re-circulating the suspension across the cavitation zone was found to release the cell wall bound enzyme invertase (86%) to a greater extent than the enzymes from other locations of the cell (e.g. periplasmic alpha-glucosidase at 17%). Localised damage to the cell wall could be observed using transmission electron microscopy (TEM) of cells subjected to less intense cavitation conditions. Absence of the release of cytoplasmic enzymes to a significant extent, absence of micronisation as observed by TEM and presence of a lower number of proteins bands in the culture supernatant on SDS-PAGE analysis following hydrodynamic cavitation compared to disruption by high-pressure homogenisation confirmed the selective release offered by hydrodynamic cavitation. Copyright 2006 Wiley Periodicals, Inc.

The Baseplate of Lactobacillus delbrueckii Bacteriophage Ld17 Harbors a Glycerophosphodiesterase.

PubMed

Cornelissen, Anneleen; Sadovskaya, Irina; Vinogradov, Evgeny; Blangy, Stéphanie; Spinelli, Silvia; Casey, Eoghan; Mahony, Jennifer; Noben, Jean-Paul; Dal Bello, Fabio; Cambillau, Christian; van Sinderen, Douwe

2016-08-05

Glycerophosphodiester phosphodiesterases (GDPDs; EC 3.1.4.46) typically hydrolyze glycerophosphodiesters to sn-glycerol 3-phosphate (Gro3P) and their corresponding alcohol during patho/physiological processes in bacteria and eukaryotes. GDPD(-like) domains were identified in the structural particle of bacterial viruses (bacteriophages) specifically infecting Gram-positive bacteria. The GDPD of phage 17 (Ld17; GDPDLd17), representative of the group b Lactobacillus delbrueckii subsp. bulgaricus (Ldb)-infecting bacteriophages, was shown to hydrolyze, besides the simple glycerophosphodiester, two complex surface-associated carbohydrates of the Ldb17 cell envelope: the Gro3P decoration of the major surface polysaccharide d-galactan and the oligo(glycerol phosphate) backbone of the partially glycosylated cell wall teichoic acid, a minor Ldb17 cell envelope component. Degradation of cell wall teichoic acid occurs according to an exolytic mechanism, and Gro3P substitution is presumed to be inhibitory for GDPDLd17 activity. The presence of the GDPDLd17 homotrimer in the viral baseplate structure involved in phage-host interaction together with the dependence of native GDPD activity, adsorption, and efficiency of plating of Ca(2+) ions supports a role for GDPDLd17 activity during phage adsorption and/or phage genome injection. In contrast to GDPDLd17, we could not identify any enzymatic activity for the GDPD-like domain in the neck passage structure of phage 340, a 936-type Lactococcus lactis subsp. lactis bacteriophage. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli.

PubMed

Lai, Ghee Chuan; Cho, Hongbaek; Bernhardt, Thomas G

2017-07-01

Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix.

Composition and immunoreactivity of the A60 complex and other cell fractions from Mycobacterium bovis BCG.

PubMed

Cocito, C; Vanlinden, F

1995-02-01

Surface static cultures of Mycobacterium bovis BCG contained cells embedded in an extracellular matrix, whose mechanical removal yielded free cells that were pressure disrupted and fractionated into cytoplasm and walls. Cell envelopes were either mechanically disrupted or extracted with detergents. Intracellular and extracellular fractions were analysed for proteins, polysaccharides, and antigen 6O (A60), a major complex immunodominant in tuberculosis. A60 was present in extracellular matrix, cytoplasm and walls: it represented a substantial portion of the proteins and polysaccharides of these fractions. While the protein/polysaccharide ratio varied according to the origin of A60 preparations, the electrophoretic patterns of A60 proteins (which accounted for the immunogenicity of the complex) remained unchanged. Western blots pointed to the proteins present within the 29-45 kDa range as the A60 components endowed with the highest immunogenicity level. Since the most heavily stained protein bands in SDS-PAGE patterns were located outside the region best recognized by antisera, a striking discordance was found between concentration and immunogenicity patterns of A60 proteins. The electrophoretic patterns of A60- and non-A60-proteins from cytoplasm were also different. A60 complexes in dot blots and some electrophoresed A60 proteins reacted with monoclonal antibodies directed against lipoarabinomannan (LAM), a highly immunogenic polymer of cell envelope. This contaminating compound was removed from A60 with organic solvents and detergents. SDS-PAGE and Western blot patterns of proteins from delipidated A60 were similar to those of native A60 proteins.

Genome-wide dynamics of a bacterial response to antibiotics that target the cell envelope

PubMed Central

2011-01-01

Background A decline in the discovery of new antibacterial drugs, coupled with a persistent rise in the occurrence of drug-resistant bacteria, has highlighted antibiotics as a diminishing resource. The future development of new drugs with novel antibacterial activities requires a detailed understanding of adaptive responses to existing compounds. This study uses Streptomyces coelicolor A3(2) as a model system to determine the genome-wide transcriptional response following exposure to three antibiotics (vancomycin, moenomycin A and bacitracin) that target distinct stages of cell wall biosynthesis. Results A generalised response to all three antibiotics was identified which involves activation of transcription of the cell envelope stress sigma factor σE, together with elements of the stringent response, and of the heat, osmotic and oxidative stress regulons. Attenuation of this system by deletion of genes encoding the osmotic stress sigma factor σB or the ppGpp synthetase RelA reduced resistance to both vancomycin and bacitracin. Many antibiotic-specific transcriptional changes were identified, representing cellular processes potentially important for tolerance to each antibiotic. Sensitivity studies using mutants constructed on the basis of the transcriptome profiling confirmed a role for several such genes in antibiotic resistance, validating the usefulness of the approach. Conclusions Antibiotic inhibition of bacterial cell wall biosynthesis induces both common and compound-specific transcriptional responses. Both can be exploited to increase antibiotic susceptibility. Regulatory networks known to govern responses to environmental and nutritional stresses are also at the core of the common antibiotic response, and likely help cells survive until any specific resistance mechanisms are fully functional. PMID:21569315

A Relay Network of Extracellular Heme-Binding Proteins Drives C. albicans Iron Acquisition from Hemoglobin

PubMed Central

Kuznets, Galit; Vigonsky, Elena; Weissman, Ziva; Lalli, Daniela; Gildor, Tsvia; Kauffman, Sarah J.; Turano, Paola; Becker, Jeffrey; Lewinson, Oded; Kornitzer, Daniel

2014-01-01

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7−/− mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope. PMID:25275454

Adamantylidene-substituted alkylphosphocholine TCAN26 is more active against Sporothrix schenckii than miltefosine.

PubMed

Borba-Santos, Luana Pereira; Ishida, Kelly; Calogeropoulou, Theodora; Souza, Wanderley de; Rozental, Sonia

2016-08-01

Sporotrichosis is the most frequent subcutaneous mycosis in the world and its increasing incidence has led to the search for new therapeutic options for its treatment. In this study, we demonstrated that three structural analogues of miltefosine (TCAN26, TC19, and TC70) showed inhibitory activity against Sporothrix schenckii sensu stricto and that TCAN26 was more active in vitro than miltefosine against several isolates. Scanning electron microscopy showed that S. schenckii exposure to TCAN26 resulted in cells that were slightly more elongated than untreated cells. Transmission electron microscopy showed that TCAN26 treatment induced loss of the regular cytoplasmic electron-density and altered the cell envelope (disruption of the cell membrane and cell wall, and increased cell wall thickness). Additionally, TCAN26 concentrations required to kill S. schenckii cells were lower than concentrations that were cytotoxic in mammalian cells, and TCAN26 was more selective than miltefosine. Thus, the adamantylidene-substituted alkylphosphocholine TCAN26 is a promising molecule for the development of novel antifungal compounds, although further investigations are required to elucidate the mode of action of TCAN26 in S. schenckii cells.

Mycobacterial glycolipids di-O-acylated trehalose and tri-O-acylated trehalose downregulate inducible nitric oxide synthase and nitric oxide production in macrophages.

PubMed

Espinosa-Cueto, Patricia; Escalera-Zamudio, Marina; Magallanes-Puebla, Alejandro; López-Marín, Luz María; Segura-Salinas, Erika; Mancilla, Raúl

2015-06-23

Tuberculosis (TB) remains a serious human health problem that affects millions of people in the world. Understanding the biology of Mycobacterium tuberculosis (Mtb) is essential for tackling this devastating disease. Mtb possesses a very complex cell envelope containing a variety of lipid components that participate in the establishment of the infection. We have previously demonstrated that di-O-acylated trehalose (DAT), a non-covalently linked cell wall glycolipid, inhibits the proliferation of T lymphocytes and the production of cytokines. In this work we show that DAT and the closely related tri-O-acylated trehalose (TAT) inhibits nitric oxide (NO) production and the inducible nitric oxide synthase (iNOS) expression in macrophages (MØ). These findings show that DAT and TAT are cell-wall located virulence factors that downregulate an important effector of the immune response against mycobacteria.

Integrated -Omics: A Powerful Approach to Understanding the Heterogeneous Lignification of Fibre Crops

PubMed Central

Gea, Guerriero; Kjell, Sergeant; Jean-François, Hausman

2013-01-01

Lignin and cellulose represent the two main components of plant secondary walls and the most abundant polymers on Earth. Quantitatively one of the principal products of the phenylpropanoid pathway, lignin confers high mechanical strength and hydrophobicity to plant walls, thus enabling erect growth and high-pressure water transport in the vessels. Lignin is characterized by a high natural heterogeneity in its composition and abundance in plant secondary cell walls, even in the different tissues of the same plant. A typical example is the stem of fibre crops, which shows a lignified core enveloped by a cellulosic, lignin-poor cortex. Despite the great value of fibre crops for humanity, however, still little is known on the mechanisms controlling their cell wall biogenesis, and particularly, what regulates their spatially-defined lignification pattern. Given the chemical complexity and the heterogeneous composition of fibre crops’ secondary walls, only the use of multidisciplinary approaches can convey an integrated picture and provide exhaustive information covering different levels of biological complexity. The present review highlights the importance of combining high throughput -omics approaches to get a complete understanding of the factors regulating the lignification heterogeneity typical of fibre crops. PMID:23708098

The laboratory investigation of surface envelope solitons: reflection from a vertical wall and collisions of solitons

NASA Astrophysics Data System (ADS)

Slunyaev, Alexey; Klein, Marco; Clauss, Günther F.

2016-04-01

Envelope soliton solutions are key elements governing the nonlinear wave dynamics within a simplified theory for unidirectional weakly modulated weakly nonlinear wave groups on the water surface. Within integrable models the solitons preserve their structure in collisions with other waves; they do not disperse and can carry energy infinitively long. Steep and short soliton-like wave groups have been shown to exist in laboratory tests [1] and, even earlier, in numerical simulations [2, 3]. Thus, long-living wave groups may play important role in the dynamics of intense sea waves and wave-structure interactions. The solitary wave groups may change the wave statistics and can be taken into account when developing approaches for the deterministic forecasting of dangerous waves, including so-called rogue waves. An experimental campaign has been conducted in the wave basin of the Technical University of Berlin on simulations of intense solitary wave groups. The first successful experimental observation of intense envelope solitons took place in this facility [1]. The new experiments aimed at following main goals: 1) to reproduce intense envelope solitons with different carrier wave lengths; 2) to estimate the rate of envelope soliton dissipation; 3) to consider the reflection of envelope solitons on a vertical wall; 4) to consider head-on collisions of envelope solitons, and 5) to consider overtaking interactions of envelope solitons. Up to 9 wave gauges were used in each experimental run, which enabled registration of the surface movement at different distances from the wavemaker, at different locations across the wave flume and near the wall. Besides surface displacements, the group envelope shapes were directly recorded, with use of phase shifts applied to the modulated waves generated by the wavemaker. [1] A. Slunyaev, G.F. Clauss, M. Klein, M. Onorato, Simulations and experiments of short intense envelope solitons of surface water waves. Phys. Fluids 25, 067105 (2013). [2] A.I. Dyachenko, V.E. Zakharov, On the formation of freak waves on the surface of deep water. JETP Lett. 88, 307 (2008). [3] A.V. Slunyaev, Numerical simulation of "limiting" envelope solitons of gravity waves on deep water. JETP 109, 676 (2009).

An ultrastructural study of sporidium formation during infection of a rhabditid nematode by large gun cells of Haptoglossa heteromorpha.

PubMed

Glockling, S L; Beakes, G W

2000-10-01

Recently fired gun cells of Haptoglossa heteromorpha, an aplanosporic nematode parasite, were examined ultrastructurally. The everted tubes of the fired cells had penetrated the cuticle of a nematode, and infective sporidia were developing inside the host body. The nematode cuticle was penetrated by the narrow, walled part of the tube below the needle chamber. The lower unwalled part of the tube tail formed the sporidium. The developing sporidium had a multilayered fibrous outer coating and the plasma membrane was separated from the wall in places. Sporidia contained biphasic membrane-bound vesicles that had been generated by the Golgi dictyosome during gun cell development. Immediately following gun cell firing, the nuclear envelope of the sporidium nucleus was not apparent, and the sporidium nucleus contained clusters of electron-dense particles concentrated in the nucleolar region. We compare the structures and organelles found in the mature gun cell with those in the fired cell and attempt to identify the membranous layers around the sporidium. Copyright 2000 Academic Press.

Membrane Organization and Cell Fusion During Mating in Fission Yeast Requires Multipass Membrane Protein Prm1

PubMed Central

Curto, M.-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M.-Henar

2014-01-01

The involvement of Schizosaccharomyces pombe prm1+ in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell–cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1+ and the dni+ genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell–cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell–cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome.

PubMed

Faheem, Muhammad; Martins-de-Sa, Diogo; Vidal, Julia F D; Álvares, Alice C M; Brandão-Neto, José; Bird, Louise E; Tully, Mark D; von Delft, Frank; Souto, Betulia M; Quirino, Betania F; Freitas, Sonia M; Barbosa, João Alexandre R G

2016-12-09

A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a K b of 1.8 × 10 5  M -1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space R g of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features.

Antimicrobial Peptides Targeting Gram-Positive Bacteria

PubMed Central

Malanovic, Nermina; Lohner, Karl

2016-01-01

Antimicrobial peptides (AMPs) have remarkably different structures as well as biological activity profiles, whereupon most of these peptides are supposed to kill bacteria via membrane damage. In order to understand their molecular mechanism and target cell specificity for Gram-positive bacteria, it is essential to consider the architecture of their cell envelopes. Before AMPs can interact with the cytoplasmic membrane of Gram-positive bacteria, they have to traverse the cell wall composed of wall- and lipoteichoic acids and peptidoglycan. While interaction of AMPs with peptidoglycan might rather facilitate penetration, interaction with anionic teichoic acids may act as either a trap for AMPs or a ladder for a route to the cytoplasmic membrane. Interaction with the cytoplasmic membrane frequently leads to lipid segregation affecting membrane domain organization, which affects membrane permeability, inhibits cell division processes or leads to delocalization of essential peripheral membrane proteins. Further, precursors of cell wall components, especially the highly conserved lipid II, are directly targeted by AMPs. Thereby, the peptides do not inhibit peptidoglycan synthesis via binding to proteins like common antibiotics, but form a complex with the precursor molecule, which in addition can promote pore formation and membrane disruption. Thus, the multifaceted mode of actions will make AMPs superior to antibiotics that act only on one specific target. PMID:27657092

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...

2015-09-15

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

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

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

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

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

The Experimental Research on Seismic Capacity of the Envelope Systems with Steel Frame

NASA Astrophysics Data System (ADS)

Li, Jiuyang; Wang, Bingbing; Li, Hengxu

2017-09-01

In this paper, according to the present application situation of the external envelope systems steel frame in the severe cold region, the stuffed composite wall panels are improved, the flexible connection with the steel frame is designed, the reduced scale specimens are made, the seismic capacity test is made and some indexes of the envelope systems such as bearing capacity, energy consumption and ductility, etc. are compared, which provide reference for the development and application of the steel frame envelope systems.

Adamantylidene-substituted alkylphosphocholine TCAN26 is more active against Sporothrix schenckii than miltefosine

PubMed Central

Borba-Santos, Luana Pereira; Ishida, Kelly; Calogeropoulou, Theodora; de Souza, Wanderley; Rozental, Sonia

2016-01-01

Sporotrichosis is the most frequent subcutaneous mycosis in the world and its increasing incidence has led to the search for new therapeutic options for its treatment. In this study, we demonstrated that three structural analogues of miltefosine (TCAN26, TC19, and TC70) showed inhibitory activity against Sporothrix schenckii sensu stricto and that TCAN26 was more active in vitro than miltefosine against several isolates. Scanning electron microscopy showed that S. schenckii exposure to TCAN26 resulted in cells that were slightly more elongated than untreated cells. Transmission electron microscopy showed that TCAN26 treatment induced loss of the regular cytoplasmic electron-density and altered the cell envelope (disruption of the cell membrane and cell wall, and increased cell wall thickness). Additionally, TCAN26 concentrations required to kill S. schenckii cells were lower than concentrations that were cytotoxic in mammalian cells, and TCAN26 was more selective than miltefosine. Thus, the adamantylidene-substituted alkylphosphocholine TCAN26 is a promising molecule for the development of novel antifungal compounds, although further investigations are required to elucidate the mode of action of TCAN26 in S. schenckii cells. PMID:27581121

Cell wall amidase AmiC1 is required for cellular communication and heterocyst development in the cyanobacterium Anabaena PCC 7120 but not for filament integrity.

PubMed

Berendt, Susanne; Lehner, Josef; Zhang, Yao Vincent; Rasse, Tobias M; Forchhammer, Karl; Maldener, Iris

2012-10-01

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.

A rule-based expert system applied to moisture durability of building envelopes

DOE PAGES

Boudreaux, Philip R.; Pallin, Simon B.; Accawi, Gina K.; ...

2018-01-09

The moisture durability of an envelope component such as a wall or roof is difficult to predict. Moisture durability depends on all the construction materials used, as well as the climate, orientation, air tightness, and indoor conditions. Modern building codes require more insulation and tighter construction but provide little guidance about how to ensure these energy-efficient assemblies remain moisture durable. Furthermore, as new products and materials are introduced, builders are increasingly uncertain about the long-term durability of their building envelope designs. Oak Ridge National Laboratory and the US Department of Energy’s Building America Program are applying a rule-based expert systemmore » methodology in a web tool to help designers determine whether a given wall design is likely to be moisture durable and provide expert guidance on moisture risk management specific to a wall design and climate. Finally, the expert system is populated with knowledge from both expert judgment and probabilistic hygrothermal simulation results.« less

A rule-based expert system applied to moisture durability of building envelopes

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

Boudreaux, Philip R.; Pallin, Simon B.; Accawi, Gina K.

The moisture durability of an envelope component such as a wall or roof is difficult to predict. Moisture durability depends on all the construction materials used, as well as the climate, orientation, air tightness, and indoor conditions. Modern building codes require more insulation and tighter construction but provide little guidance about how to ensure these energy-efficient assemblies remain moisture durable. Furthermore, as new products and materials are introduced, builders are increasingly uncertain about the long-term durability of their building envelope designs. Oak Ridge National Laboratory and the US Department of Energy’s Building America Program are applying a rule-based expert systemmore » methodology in a web tool to help designers determine whether a given wall design is likely to be moisture durable and provide expert guidance on moisture risk management specific to a wall design and climate. Finally, the expert system is populated with knowledge from both expert judgment and probabilistic hygrothermal simulation results.« less

Low-Cost Bio-Based Phase Change Materials as an Energy Storage Medium in Building Envelopes

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

Biswas, Kaushik; Abhari, Mr. Ramin; Shukla, Dr. Nitin

2015-01-01

A promising approach to increasing the energy efficiency of buildings is the implementation of phase change material (PCM) in building envelope systems. Several studies have reported the energy saving potential of PCM in building envelopes. However, wide application of PCMs in building applications has been inhibited, in part, by their high cost. This article describes a novel paraffin product made of naturally occurring fatty acids/glycerides trapped into high density polyethylene (HDPE) pellets and its performance in a building envelope application, with the ultimate goal of commercializing a low-cost PCM platform. The low-cost PCM pellets were mixed with cellulose insulation, installedmore » in external walls and field-tested under natural weatherization conditions for a period of several months. In addition, several PCM samples and PCM-cellulose samples were prepared under controlled conditions for laboratory-scale testing. The laboratory tests were performed to determine the phase change properties of PCM-enhanced cellulose insulation both at microscopic and macroscopic levels. This article presents the data and analysis from the exterior test wall and the laboratory-scale test data. PCM behavior is influenced by the weather and interior conditions, PCM phase change temperature and PCM distribution within the wall cavity, among other factors. Under optimal conditions, the field data showed up to 20% reduction in weekly heat transfer through an external wall due to the PCM compared to cellulose-only insulation.« less

Structural studies of the cell wall polysaccharide from Lactococcus lactis UC509.9.

PubMed

Vinogradov, Evgeny; Sadovskaya, Irina; Grard, Thierry; Murphy, James; Mahony, Jennifer; Chapot-Chartier, Marie-Pierre; van Sinderen, Douwe

2018-05-22

Lactococcus lactis is the most widely utilised starter bacterial species in dairy fermentations. The L. lactis cell envelope contains polysaccharides, which, among other known functions, serve as bacteriophage receptors. Our previous studies have highlighted the structural diversity of these so-called cell wall polysaccharides (CWPSs) among L. lactis strains that could account for the narrow host range of most lactococcal bacteriophages. In the present work, we studied the CWPS of L. lactis strain UC509.9, an Irish dairy starter strain that is host to the temperate and well-characterized P335-type phage Tuc2009. The UC509.9 CWPS structure was analyzed by methylation, deacetylation/deamination, Smith degradation and 2D NMR spectroscopy. The CWPS consists of a linear backbone composed of a tetrasaccharide repeat unit, partially substituted with a branched phosphorylated oligosaccharide having a common trisaccharide and three non-stoichiometric substitutions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mechanisms of Drug Resistance: Daptomycin Resistance

PubMed Central

Tran, Truc T.; Munita, Jose M.; Arias, Cesar A.

2016-01-01

Daptomycin (DAP) is a cyclic lipopeptide with in vitro activity against a variety of Gram-positive pathogens, including multidrug-resistant organisms. Since its introduction in clinical practice in 2003, DAP has become an important key front-line antibiotic for severe or deep-seated infections caused by Gram-positive organisms. Unfortunately, DAP-resistance (R) has been extensively documented in clinically important organisms such as Staphylococcus aureus, Enterococcus spp, and Streptococcus spp. Studies on the mechanisms of DAP-R in Bacillus subtilis and other Gram-positive bacteria indicate that the genetic pathways of DAP resistance are diverse and complex. However, a common phenomenon emerging from these mechanistic studies is that DAP-R is associated with important adaptive changes in cell wall and cell membrane homeostasis with critical changes in cell physiology. Findings related to these adaptive changes have offered novel insights into the genetics and molecular mechanisms of bacterial cell envelope stress response and the manner in which Gram-positive bacteria cope with the antimicrobial peptide attack and protect vital structures of the cell envelope such as the cell membrane. In this review, we will examine the most recent findings related to the molecular mechanisms of resistance to DAP in relevant Gram-positive pathogens and discuss the clinical implications for therapy against these important bacteria. PMID:26495887

Surface multiheme c-type cytochromes from Thermincola potens: Implications for dissimilatory metal reduction by Gram-positive bacteria

NASA Astrophysics Data System (ADS)

Carlson, H. K.; Iavarone, A. T.; Gorur, A.; Yeo, B. S.; Tran, R.; Melnyk, R. A.; Mathies, R. A.; Auer, M.; Coates, J. D.

2011-12-01

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they have been shown to be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or the humic substances analog, anthraquinone-2,6-disulfonate (AQDS). The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS and that several MHCs are localized to the cell wall or cell surface of T. potens. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results are the first direct evidence for cell-wall associated cytochromes and MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

Identification and characterization of smallest pore-forming protein in the cell wall of pathogenic Corynebacterium urealyticum DSM 7109.

PubMed

Abdali, Narges; Younas, Farhan; Mafakheri, Samaneh; Pothula, Karunakar R; Kleinekathöfer, Ulrich; Tauch, Andreas; Benz, Roland

2018-05-09

Corynebacterium urealyticum, a pathogenic, multidrug resistant member of the mycolata, is known as causative agent of urinary tract infections although it is a bacterium of the skin flora. This pathogenic bacterium shares with the mycolata the property of having an unusual cell envelope composition and architecture, typical for the genus Corynebacterium. The cell wall of members of the mycolata contains channel-forming proteins for the uptake of solutes. In this study, we provide novel information on the identification and characterization of a pore-forming protein in the cell wall of C. urealyticum DSM 7109. Detergent extracts of whole C. urealyticum cultures formed in lipid bilayer membranes slightly cation-selective pores with a single-channel conductance of 1.75 nS in 1 M KCl. Experiments with different salts and non-electrolytes suggested that the cell wall pore of C. urealyticum is wide and water-filled and has a diameter of about 1.8 nm. Molecular modelling and dynamics has been performed to obtain a model of the pore. For the search of the gene coding for the cell wall pore of C. urealyticum we looked in the known genome of C. urealyticum for a similar chromosomal localization of the porin gene to known porH and porA genes of other Corynebacterium strains. Three genes are located between the genes coding for GroEL2 and polyphosphate kinase (PKK2). Two of the genes (cur_1714 and cur_1715) were expressed in different constructs in C. glutamicum ΔporAΔporH and in porin-deficient BL21 DE3 Omp8 E. coli strains. The results suggested that the gene cur_1714 codes alone for the cell wall channel. The cell wall porin of C. urealyticum termed PorACur was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 4 kDa on tricine-containing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Biophysical characterization of the purified protein (PorACur) suggested indeed that cur_1714 is the gene coding for the pore-forming protein in C. urealyticum because the protein formed in lipid bilayer experiments the same pores as the detergent extract of whole cells. The study is the first report of a cell wall channel in the pathogenic C. urealyticum.

Identification of a novel gene cluster in the upstream region of the S-layer gene sbpA involved in cell wall metabolism of Lysinibacillus sphaericus CCM 2177 and characterization of the recombinantly produced autolysin and pyruvyl transferase.

PubMed

Pleschberger, Magdalena; Hildner, Florian; Rünzler, Dominik; Gelbmann, Nicola; Mayer, Harald F; Sleytr, Uwe B; Egelseer, Eva M

2013-05-01

The S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 assembles into a square (p4) lattice structure and recognizes a pyruvylated secondary cell wall polymer (SCWP) as the proper anchoring structure to the rigid cell wall layer. Sequencing of 8,004 bp in the 5'-upstream region of the S-layer gene sbpA led to five ORFs-encoding proteins involved in cell wall metabolism. After cloning and heterologous expression of ORF1 and ORF5 in Escherichia coli, the recombinant autolysin rAbpA and the recombinant pyruvyl transferase rCsaB were isolated, purified, and correct folding was confirmed by circular dichroism. Although rAbpA encoded by ORF1 showed amidase activity, it could attack whole cells of Ly. sphaericus CCM 2177 only after complete extraction of the S-layer lattice. Despite the presence of three S-layer-homology motifs on the N-terminal part, rAbpA did not show detectable affinity to peptidoglycan-containing sacculi, nor to isolated SCWP. As the molecular mass of the autolysin lies above the molecular exclusion limit of the S-layer, AbpA is obviously trapped within the rigid cell wall layer by the isoporous protein lattice. Immunogold-labeling of ultrathin-sectioned whole cells of Ly. sphaericus CCM 2177 with a polyclonal rabbit antiserum raised against rCsaB encoded by ORF5, and cell fractionation experiments demonstrated that the pyruvyl transferase was located in the cytoplasm, but not associated with cell envelope components including the plasma membrane. In enzymatic assays, rCsaB clearly showed pyruvyl transferase activity. By using RT-PCR, specific transcripts for each ORF could be detected. Cotranscription could be confirmed for ORF2 and ORF3.

How Listeria monocytogenes organizes its surface for virulence

PubMed Central

Carvalho, Filipe; Sousa, Sandra; Cabanes, Didier

2014-01-01

Listeria monocytogenes is a Gram-positive pathogen responsible for the manifestation of human listeriosis, an opportunistic foodborne disease with an associated high mortality rate. The key to the pathogenesis of listeriosis is the capacity of this bacterium to trigger its internalization by non-phagocytic cells and to survive and even replicate within phagocytes. The arsenal of virulence proteins deployed by L. monocytogenes to successfully promote the invasion and infection of host cells has been progressively unveiled over the past decades. A large majority of them is located at the cell envelope, which provides an interface for the establishment of close interactions between these bacterial factors and their host targets. Along the multistep pathways carrying these virulence proteins from the inner side of the cytoplasmic membrane to their cell envelope destination, a multiplicity of auxiliary proteins must act on the immature polypeptides to ensure that they not only maturate into fully functional effectors but also are placed or guided to their correct position in the bacterial surface. As the major scaffold for surface proteins, the cell wall and its metabolism are critical elements in listerial virulence. Conversely, the crucial physical support and protection provided by this structure make it an ideal target for the host immune system. Therefore, mechanisms involving fine modifications of cell envelope components are activated by L. monocytogenes to render it less recognizable by the innate immunity sensors or more resistant to the activity of antimicrobial effectors. This review provides a state-of-the-art compilation of the mechanisms used by L. monocytogenes to organize its surface for virulence, with special focus on those proteins that work “behind the frontline”, either supporting virulence effectors or ensuring the survival of the bacterium within its host. PMID:24809022

Time dependent wave envelope finite difference analysis of sound propagation

NASA Technical Reports Server (NTRS)

Baumeister, K. J.

1984-01-01

A transient finite difference wave envelope formulation is presented for sound propagation, without steady flow. Before the finite difference equations are formulated, the governing wave equation is first transformed to a form whose solution tends not to oscillate along the propagation direction. This transformation reduces the required number of grid points by an order of magnitude. Physically, the transformed pressure represents the amplitude of the conventional sound wave. The derivation for the wave envelope transient wave equation and appropriate boundary conditions are presented as well as the difference equations and stability requirements. To illustrate the method, example solutions are presented for sound propagation in a straight hard wall duct and in a two dimensional straight soft wall duct. The numerical results are in good agreement with exact analytical results.

Life Cycle Assessment of Wall Systems

NASA Astrophysics Data System (ADS)

Ramachandran, Sriranjani

Natural resource depletion and environmental degradation are the stark realities of the times we live in. As awareness about these issues increases globally, industries and businesses are becoming interested in understanding and minimizing the ecological footprints of their activities. Evaluating the environmental impacts of products and processes has become a key issue, and the first step towards addressing and eventually curbing climate change. Additionally, companies are finding it beneficial and are interested in going beyond compliance using pollution prevention strategies and environmental management systems to improve their environmental performance. Life-cycle Assessment (LCA) is an evaluative method to assess the environmental impacts associated with a products' life-cycle from cradle-to-grave (i.e. from raw material extraction through to material processing, manufacturing, distribution, use, repair and maintenance, and finally, disposal or recycling). This study focuses on evaluating building envelopes on the basis of their life-cycle analysis. In order to facilitate this analysis, a small-scale office building, the University Services Building (USB), with a built-up area of 148,101 ft2 situated on ASU campus in Tempe, Arizona was studied. The building's exterior envelope is the highlight of this study. The current exterior envelope is made of tilt-up concrete construction, a type of construction in which the concrete elements are constructed horizontally and tilted up, after they are cured, using cranes and are braced until other structural elements are secured. This building envelope is compared to five other building envelope systems (i.e. concrete block, insulated concrete form, cast-in-place concrete, steel studs and curtain wall constructions) evaluating them on the basis of least environmental impact. The research methodology involved developing energy models, simulating them and generating changes in energy consumption due to the above mentioned envelope types. Energy consumption data, along with various other details, such as building floor area, areas of walls, columns, beams etc. and their material types were imported into Life-Cycle Assessment software called ATHENA impact estimator for buildings. Using this four-stepped LCA methodology, the results showed that the Steel Stud envelope performed the best and less environmental impact compared to other envelope types. This research methodology can be applied to other building typologies.

Laboratory testing of a building envelope segment based on cellular concrete

NASA Astrophysics Data System (ADS)

Fořt, Jan; Pavlík, Zbyšek; Černý, Robert

2016-07-01

Hygrothermal performance of a building envelope based on cellular concrete blocks is studied in the paper. Simultaneously, the strain fields induced by the heat and moisture changes are monitored. The studied wall is exposed to the climatic load corresponding to the winter climatic conditions of the moderate year for Prague. The winter climatic exposure is chosen in order to simulate the critical conditions of the building structure from the point of view of material performance and temperature and humidity loading. The evaluation of hygrothermal performance of a researched wall is done on the basis of relative humidity and temperature profiles measured along the cross section of the cellular concrete blocks. Strain gauges are fixed on the wall surface in expected orientation of the blocks expansion. The obtained results show a good hygrothermal function of the analyzed cellular concrete wall and its insignificant strain.

Microstructural effects in drug release by solid and cellular polymeric dosage forms: A comparative study.

PubMed

Blaesi, Aron H; Saka, Nannaji

2017-11-01

In recent studies, we have introduced melt-processed polymeric cellular dosage forms to achieve both immediate drug release and predictable manufacture. Dosage forms ranging from minimally-porous solids to highly porous, open-cell and thin-walled structures were prepared, and the drug release characteristics investigated as the volume fraction of cells and the excipient molecular weight were varied. In the present study, both minimally-porous solid and cellular dosage forms consisting of various weight fractions of Acetaminophen drug and polyethylene glycol (PEG) excipient are prepared and analyzed. Microstructures of the solid forms and the cell walls range from single-phase solid solutions of the excipient and a small amount of drug molecules to two-phase composites of the excipient and tightly packed drug particles. Results of dissolution experiments show that the minimally-porous solid forms disintegrate and release drug by slow surface erosion. The erosion rate decreases as the drug weight fraction is increased. By contrast, the open-cell structures disintegrate rapidly by viscous exfoliation, and the disintegration time is independent of drug weight fraction. Drug release models suggest that the solid forms erode by convective mass transfer of the faster-eroding excipient if the drug volume fraction is small. At larger drug volume fractions, however, the slower-eroding drug particles hinder access of the free-flowing fluid to the excipient, thus slowing down erosion of the composite. Conversely, the disintegration rate of the cellular forms is limited by diffusion of the dissolution fluid into the excipient phase of the thin cell walls. Because the wall thickness is of the order of the drug particle size, and the particles are enveloped by the excipient during melt-processing, the drug particles cannot hinder diffusion through the excipient across the walls. Thus the disintegration time of the cellular forms is mostly unaffected by the volume fraction of drug in the walls. Copyright © 2017 Elsevier B.V. All rights reserved.

Remobilization of toxic heavy metals adsorbed to bacterial wall-clay composites.

PubMed Central

Flemming, C A; Ferris, F G; Beveridge, T J; Bailey, G W

1990-01-01

Significant quantities of Ag(I), Cu(II), and Cr(III) were bound to isolated Bacillus subtilis 168 walls, Escherichia coli K-12 envelopes, kaolinite and smectite clays, and the corresponding organic material-clay aggregates (1:1, wt/wt). These sorbed metals were leached with HNO3, Ca(NO3)2, EDTA, fulvic acid, and lysozyme at several concentrations over 48 h at room temperature. The remobilization of the sorbed metals depended on the physical properties of the organic and clay surfaces and on the character and concentration of the leaching agents. In general, the order of remobilization of metals was Cr much less than Ag less than Cu. Cr was very stable in the wall, clay, and composite systems; pH 3.0, 500 microM EDTA, 120-ppm [mg liter-1] fulvic acid, and 160-ppm Ca remobilized less than 32% (wt/wt) of sorbed Cr. Ag (45 to 87%) and Cu (up to 100%) were readily removed by these agents. Although each leaching agent was effective at mobilizing certain metals, elevated Ca or acidic pH produced the greatest overall mobility. The organic chelators were less effective. Lysozyme digestion of Bacillus walls remobilized Cu from walls and Cu-wall-kaolinite composites, but Ag, Cr, and smectite partially inhibited enzyme activity, and the metals remained insoluble. The extent of metal remobilization was not always dependent on increasing concentrations of leaching agents; for example, Ag mobility decreased with some clays and some composites treated with high fulvic acid, EDTA, and lysozyme concentrations. Sometimes the organic material-clay composites reacted in a manner distinctly different from that of their individual counterparts; e.g., 25% less Cu was remobilized from wall- and envelope-smectite composites than from walls, envelopes, or smectite individually in 500 microM EDTA. Alternatively, treatment with 160-ppm Ca removed 1.5 to 10 times more Ag from envelope-kaolinite composites than from the individual components. The particle size of the deposited metal may account for some of the stability changes; those metals that formed large, compact aggregates (Cr and Ag) as seen by transmission electron microscopy were less likely to be remobilized. In summary, it is apparent that remobilization of toxic heavy metals in sediments, soils, and the vadose zone is a complicated issue. Predictions based on single inorganic or organic component systems are too simplistic. Images PMID:2126702

Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria

PubMed Central

Carlson, Hans K.; Iavarone, Anthony T.; Gorur, Amita; Yeo, Boon Siang; Tran, Rosalie; Melnyk, Ryan A.; Mathies, Richard A.; Auer, Manfred; Coates, John D.

2012-01-01

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium. PMID:22307634

The lytic transglycosylase MltB connects membrane homeostasis and in vivo fitness of Acinetobacter baumannii.

PubMed

Crépin, Sébastien; Ottosen, Elizabeth N; Peters, Katharina; Smith, Sara N; Himpsl, Stephanie D; Vollmer, Waldemar; Mobley, Harry L T

2018-06-08

Acinetobacter baumannii has emerged as a leading nosocomial pathogen, infecting a wide range of anatomic sites including the respiratory tract and the bloodstream. In addition to being multi-drug resistant, little is known about the molecular basis of A. baumannii pathogenesis. To better understand A. baumannii virulence, a combination of a transposon-sequencing (TraDIS) screen and the neutropenic mouse model of bacteremia was used to identify the full set of fitness genes required during bloodstream infection. The lytic transglycosylase MltB was identified as a critical fitness factor. MltB cleaves the MurNAc-GlcNAc bond of peptidoglycan, which leads to cell wall remodeling. Here we show that MltB is part of a complex network connecting resistance to stresses, membrane homeostasis, biogenesis of pili and in vivo fitness. Indeed, inactivation of mltB not only impaired resistance to serum complement, cationic antimicrobial peptides and oxygen species, but also altered the cell envelope integrity, activated the envelope stress response, drastically reduced the number of pili at the cell surface and finally, significantly decreased colonization of both the bloodstream and the respiratory tract. This article is protected by copyright. All rights reserved. © 2018 John Wiley & Sons Ltd.

Envelope lipid-packing as a critical factor for the biological activity and stability of alphavirus particles isolated from mammalian and mosquito cells.

PubMed

Sousa, Ivanildo P; Carvalho, Carlos A M; Ferreira, Davis F; Weissmüller, Gilberto; Rocha, Gustavo M; Silva, Jerson L; Gomes, Andre M O

2011-01-21

Alphaviruses are enveloped arboviruses. The viral envelope is derived from the host cell and is positioned between two icosahedral protein shells (T = 4). Because the viral envelope contains glycoproteins involved in cell recognition and entry, the integrity of the envelope is critical for the success of the early events of infection. Differing levels of cholesterol in different hosts leads to the production of alphaviruses with distinct levels of this sterol loaded in the envelope. Using Mayaro virus, a New World alphavirus, we investigated the role of cholesterol on the envelope of alphavirus particles assembled in either mammalian or mosquito cells. Our results show that although quite different in their cholesterol content, Mayaro virus particles obtained from both cells share a similar high level of lateral organization in their envelopes. This organization, as well as viral stability and infectivity, is severely compromised when cholesterol is depleted from the envelope of virus particles isolated from mammalian cells, but virus particles isolated from mosquito cells are relatively unaffected by cholesterol depletion. We suggest that it is not cholesterol itself, but rather the organization of the viral envelope, that is critical for the biological activity of alphaviruses.

Pb-induced cellular defense system in the root meristematic cells of Allium sativum L.

PubMed

Jiang, Wusheng; Liu, Donghua

2010-03-02

Electron microscopy (EM) techniques enable identification of the main accumulations of lead (Pb) in cells and cellular organelles and observations of changes in cell ultrastructure. Although there is extensive literature relating to studies on the influence of heavy metals on plants, Pb tolerance strategies of plants have not yet been fully explained. Allium sativum L. is a potential plant for absorption and accumulation of heavy metals. In previous investigations the effects of different concentrations (10(-5) to 10(-3) M) of Pb were investigated in A. sativum, indicating a significant inhibitory effect on shoot and root growth at 10(-3) to 10(-4) M Pb. In the present study, we used EM and cytochemistry to investigate ultrastructural alterations, identify the synthesis and distribution of cysteine-rich proteins induced by Pb and explain the possible mechanisms of the Pb-induced cellular defense system in A. sativum. After 1 h of Pb treatment, dictyosomes were accompanied by numerous vesicles within cytoplasm. The endoplasm reticulum (ER) with swollen cisternae was arranged along the cell wall after 2 h. Some flattened cisternae were broken up into small closed vesicles and the nuclear envelope was generally more dilated after 4 h. During 24-36 h, phenomena appeared such as high vacuolization of cytoplasm and electron-dense granules in cell walls, vacuoles, cytoplasm and mitochondrial membranes. Other changes included mitochondrial swelling and loss of cristae, and vacuolization of ER and dictyosomes during 48-72 h. In the Pb-treatment groups, silver grains were observed in cell walls and in cytoplasm, suggesting the Gomori-Swift reaction can indirectly evaluate the Pb effects on plant cells. Cell walls can immobilize some Pb ions. Cysteine-rich proteins in cell walls were confirmed by the Gomori-Swift reaction. The morphological alterations in plasma membrane, dictyosomes and ER reflect the features of detoxification and tolerance under Pb stress. Vacuoles are ultimately one of main storage sites of Pb. Root meristematic cells of A. sativum exposed to lower Pb have a rapid and effective defense system, but with the increased level of Pb in the cytosol, cells were seriously injured.

Pb-induced cellular defense system in the root meristematic cells of Allium sativum L

PubMed Central

2010-01-01

Background Electron microscopy (EM) techniques enable identification of the main accumulations of lead (Pb) in cells and cellular organelles and observations of changes in cell ultrastructure. Although there is extensive literature relating to studies on the influence of heavy metals on plants, Pb tolerance strategies of plants have not yet been fully explained. Allium sativum L. is a potential plant for absorption and accumulation of heavy metals. In previous investigations the effects of different concentrations (10-5 to 10-3 M) of Pb were investigated in A. sativum, indicating a significant inhibitory effect on shoot and root growth at 10-3 to 10-4 M Pb. In the present study, we used EM and cytochemistry to investigate ultrastructural alterations, identify the synthesis and distribution of cysteine-rich proteins induced by Pb and explain the possible mechanisms of the Pb-induced cellular defense system in A. sativum. Results After 1 h of Pb treatment, dictyosomes were accompanied by numerous vesicles within cytoplasm. The endoplasm reticulum (ER) with swollen cisternae was arranged along the cell wall after 2 h. Some flattened cisternae were broken up into small closed vesicles and the nuclear envelope was generally more dilated after 4 h. During 24-36 h, phenomena appeared such as high vacuolization of cytoplasm and electron-dense granules in cell walls, vacuoles, cytoplasm and mitochondrial membranes. Other changes included mitochondrial swelling and loss of cristae, and vacuolization of ER and dictyosomes during 48-72 h. In the Pb-treatment groups, silver grains were observed in cell walls and in cytoplasm, suggesting the Gomori-Swift reaction can indirectly evaluate the Pb effects on plant cells. Conclusions Cell walls can immobilize some Pb ions. Cysteine-rich proteins in cell walls were confirmed by the Gomori-Swift reaction. The morphological alterations in plasma membrane, dictyosomes and ER reflect the features of detoxification and tolerance under Pb stress. Vacuoles are ultimately one of main storage sites of Pb. Root meristematic cells of A. sativum exposed to lower Pb have a rapid and effective defense system, but with the increased level of Pb in the cytosol, cells were seriously injured. PMID:20196842

A global regulatory system links virulence and antibiotic resistance to envelope homeostasis in Acinetobacter baumannii.

PubMed

Geisinger, Edward; Mortman, Nadav J; Vargas-Cuebas, Germán; Tai, Albert K; Isberg, Ralph R

2018-05-01

The nosocomial pathogen Acinetobacter baumannii is a significant threat due to its ability to cause infections refractory to a broad range of antibiotic treatments. We show here that a highly conserved sensory-transduction system, BfmRS, mediates the coordinate development of both enhanced virulence and resistance in this microorganism. Hyperactive alleles of BfmRS conferred increased protection from serum complement killing and allowed lethal systemic disease in mice. BfmRS also augmented resistance and tolerance against an expansive set of antibiotics, including dramatic protection from β-lactam toxicity. Through transcriptome profiling, we showed that BfmRS governs these phenotypes through global transcriptional regulation of a post-exponential-phase-like program of gene expression, a key feature of which is modulation of envelope biogenesis and defense pathways. BfmRS activity defended against cell-wall lesions through both β-lactamase-dependent and -independent mechanisms, with the latter being connected to control of lytic transglycosylase production and proper coordination of morphogenesis and division. In addition, hypersensitivity of bfmRS knockouts could be suppressed by unlinked mutations restoring a short, rod cell morphology, indicating that regulation of drug resistance, pathogenicity, and envelope morphogenesis are intimately linked by this central regulatory system in A. baumannii. This work demonstrates that BfmRS controls a global regulatory network coupling cellular physiology to the ability to cause invasive, drug-resistant infections.

School Tucked in an Envelope.

ERIC Educational Resources Information Center

AIA Journal, 1981

1981-01-01

At the Telluride (Colorado) school, the classroom wing was outfitted with a thick floor slab and a greenhouse space where heat is stored in waterfilled tubes. The gymnasium's southwestern wall was transformed into a Trombe wall by applying 2,000 square feet of glazing. (Author/MLF)

Ultrastructure of Bacterial Cells Infected with Bacteriophage PM2, a Lipid-containing Bacterial Virus

PubMed Central

Cota-Robles, Eugene; Espejo, Romilio Torres; Haywood, Patricia Williams

1968-01-01

The cytological pattern of infection of a host pseudomonad with PM2, a lipid-containing bacterial virus, was investigated by electron microscopy. Normal and infected cells frequently contain a myelin figure, which is found in the nucleoid region or at the periphery of the cell. The most striking finding in this investigation was that completed virions are found in the cell adjacent to or in association with the cytoplasmic membrane. This localization is precise; virions are not found elsewhere in infected cells. The completed virions occasionally appear to be attached to the cytoplasmic membrane. The virus contains a darkly staining core surrounded by a tripartite envelope of a thickness of approximately 70 A, which is identical to the thickness of the cytoplasmic membrane. Lysing cells appear to undergo extensive damage of the cytoplasmic membrane prior to rupture of the L layer of the cell wall. Images PMID:5742028

Superficial Macromolecular Arrays on the Cell Wall of Spirillum putridiconchylium

PubMed Central

Beveridge, T. J.; Murray, R. G. E.

1974-01-01

Electron microscopy of the cell envelope of Spirillum putridiconchylium, using negatively stained, thin-sectioned, and replicated freeze-etched preparations, showed two superficial wall layers forming a complex macromolecular pattern on the external surface. The outer structured layer was a linear array of particles overlying an inner tetragonal array of larger subunits. They were associated in a very regular fashion, and the complex was bonded to the outer, pitted surface of the lipopolysaccharide tripartite layer of the cell wall. The relationship of the components of the two structured layers was resolved with the aid of optical diffraction, combined with image filtering and reconstruction and linear and rotary integration techniques. The outer structural layer consisted of spherical 1.5-nm units set in double lines determined by the size and arrangement of 6- by 3-nm inner structural layer subunits, which bore one outer structural layer unit on each outer corner. The total effect of this arrangement was a double-ridged linear structure that was evident in surface replicas and negatively stained fragments of the whole wall. The packing of these units was not square but skewed by 2° off the perpendicular so that the “unit array” described by optical diffraction and linear integration appeared to be a deformed tetragon. The verity of the model was checked by using a photographically reduced image to produce an optical diffraction pattern for comparison with that of the actual layers. The correspondence was nearly perfect. Images PMID:4137219

The fast milk acidifying phenotype of Streptococcus thermophilus can be acquired by natural transformation of the genomic island encoding the cell-envelope proteinase PrtS.

PubMed

Dandoy, Damien; Fremaux, Christophe; de Frahan, Marie Henry; Horvath, Philippe; Boyaval, Patrick; Hols, Pascal; Fontaine, Laetitia

2011-08-30

In industrial fermentation processes, the rate of milk acidification by Streptococcus thermophilus is of major technological importance. The cell-envelope proteinase PrtS was previously shown to be a key determinant of the milk acidification activity in this species. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The presence or absence of PrtS divides the S. thermophilus strains into two phenotypic groups i.e. the slow and the fast acidifying strains. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and hence optimise the fermentation process of dairy products. In the present work, we developed for the first time a strategy based on natural transformation to confer the rapid acidification phenotype to slow acidifying starter strains of S. thermophilus. First, we established by gene disruption that (i) prtS, encoding the cell-envelope proteinase, is a key factor responsible for rapid milk acidification in fast acidifying strains, and that (ii) srtA, encoding sortase A, is not absolutely required to express the PrtS activity. Second, a 15-kb PCR product encompassing the prtS genomic island was transferred by natural transformation using the competence-inducing peptide in three distinct prtS-defective genetic backgrounds having or not a truncated sortase A gene. We showed that in all cases the milk acidification rate of transformants was significantly increased, reaching a level similar to that of wild-type fast acidifying strains. Furthermore, it appeared that the prtS-encoded activity does not depend on the prtS copy number or on its chromosomal integration locus. We have successfully used natural competence to transfer the prtS locus encoding the cell-envelope proteinase in three slow acidifying strains of S. thermophilus, allowing their conversion into fast acidifying derivatives. The efficient protocol developed in this article will provide the dairy industry with novel and optimised S. thermophilus starter strains.

The use of specific antibodies to mediate fusion between Sendai virus envelopes and living cells.

PubMed

Loyter, A; Tomasi, M; Gitman, A G; Etinger, L; Nussbaum, O

1984-01-01

Incubation of Sendai virus particles with non-ionic detergents such as Triton X-100 completely solubilizes the viral envelopes. Removal of the detergent from the supernatant (which contains the two main viral glycoproteins) leads to the formation of fusogenic, reconstituted viral envelopes. Soluble macromolecules such as DNA or proteins can be enclosed within the reconstituted vesicles, while membrane components can be inserted into the viral envelopes. Fusion of such loaded or 'hybrid' reconstituted envelopes with living cells in culture results in either microinjection or transfer of the viral components to the recipient cells. Thus such reconstituted envelopes can serve as efficient carriers for the introduction of macromolecules of biological interest into living cells in culture. A more specific vehicle has been constructed by chemically coupling anti-cell membrane antibodies (anti-human erythrocyte antibody) to the viral envelope. Such antibody-bearing intact virus particles or reconstituted envelopes bound to and fused with virus receptor-depleted cells. In addition, anti-Sendai virus antibodies were coupled to neuraminidase-treated human erythrocytes. Such antibodies mediated the binding and fusion of intact Sendai virus particles and their reconstituted envelopes to virus receptor-depleted cells.

Combined experimental and numerical evaluation of a prototype nano-PCM enhanced wallboard

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

Biswas, Kaushik; LuPh.D., Jue; Soroushian, Parviz

2014-01-01

In the United States, forty-eight (48) percent of the residential end-use energy consumption is spent on space heating and air conditioning. Reducing envelope-generated heating and cooling loads through application of phase change material (PCM)-enhanced building envelopes can facilitate maximizing the energy efficiency of buildings. Combined experimental testing and numerical modeling of PCM-enhanced envelope components are two important aspects of the evaluation of their energy benefits. An innovative phase change material (nano-PCM) was developed with PCM encapsulated with expanded graphite (interconnected) nanosheets, which is highly conductive for enhanced thermal storage and energy distribution, and is shape-stable for convenient incorporation into lightweightmore » building components. A wall with cellulose cavity insulation and prototype PCM-enhanced interior wallboards was built and tested in a natural exposure test (NET) facility in a hot-humid climate location. The test wall contained PCM wallboards and regular gypsum wallboard, for a side-by-side annual comparison study. Further, numerical modeling of the walls containing the nano-PCM wallboard was performed to determine its actual impact on wall-generated heating and cooling loads. The model was first validated using experimental data, and then used for annual simulations using Typical Meteorological Year (TMY3) weather data. This article presents the measured performance and numerical analysis evaluating the energy-saving potential of the nano-PCM-enhanced wallboard.« less

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

Levy, E.; Mullens, M.; Rath, P.

The Advanced Envelope Research effort will provide factory homebuilders with high performance, cost-effective envelope designs that can be effectively integrated into the plant production process while meeting the thermal requirements of the 2012 IECC standards. Given the affordable nature of manufactured homes, impact on first cost is a major consideration in developing new envelope technologies. This work is part of a multi-phase effort. Phase 1 identified seven envelope technologies and provided a preliminary assessment of three methods for building high performance walls. Phase 2 focused on developing viable product designs, manufacturing strategies, addressing code and structural issues, and cost analysismore » of the three selected options. An industry advisory committee helped narrow the research focus to perfecting a stud wall design with exterior continuous insulation (CI). Phase 3, completed in two stages, continued the design development effort, exploring and evaluating a range or methods for applying CI to factory built homes. The scope also included material selection, manufacturing and cost analysis, and prototyping and testing. During this phase, a home was built with CI, evaluated, and placed in service. The experience of building a mock up wall section with CI and then constructing on line a prototype home resolved important concerns about how to integrate the material into the production process. First steps were taken toward finding least expensive approaches for incorporating CI in standard factory building practices and a preliminary assessment suggested that even at this early stage the technology is attractive when viewed from a life cycle cost perspective.« less

Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

PubMed Central

Kim, Yoon-Sang; Wielgosz, Matthew M; Hargrove, Phillip; Kepes, Steven; Gray, John; Persons, Derek A; Nienhuis, Arthur W

2010-01-01

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34+ peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34+ cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34+ cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45+ cells in total bone marrow were comparable to that of the control, mock-transduced group (37–45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the γ-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the γ-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector. PMID:20372106

Antigen 85C Inhibition Restricts Mycobacterium tuberculosis Growth through Disruption of Cord Factor Biosynthesis

PubMed Central

Warrier, Thulasi; Tropis, Marielle; Werngren, Jim; Diehl, Anne; Gengenbacher, Martin; Schlegel, Brigitte; Schade, Markus; Oschkinat, Hartmut; Daffe, Mamadou; Hoffner, Sven; Eddine, Ali Nasser

2012-01-01

The antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital for Mycobacterium tuberculosis due to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential for M. tuberculosis virulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition of M. tuberculosis division in broth culture. I3-AG85 also inhibited M. tuberculosis survival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistant M. tuberculosis strains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of the M. tuberculosis envelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of the M. tuberculosis envelope. I3-AG85 inhibited the division of and reduced TDM synthesis in an M. tuberculosis strain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design. PMID:22290959

Phagocytosis of sperm by follicle cells of the carnivorous sponge Asbestopluma occidentalis (Porifera, Demospongiae).

PubMed

Riesgo, Ana

2010-06-01

During spermatogenesis of the carnivorous sponge Asbestopluma occidentalis, follicle cells that lined the spermatocysts phagocytosed unreleased mature sperm. Such follicle cells are part of the complex envelope that limits spermatocysts of A. occidentalis, which is also comprised of a collagen layer, a thick layer of intertwined cells, and spicules. Follicle cells showed vesicles containing single phagocytosed spermatozoa within their cytoplasm. Additionally, lipids and other inclusions were observed within the cytoplasm of follicle cells. It is likely that follicle cells recapture nutrients by phagocytosing spermatozoa and use them to form lipids and other inclusions. Such sperm phagocytosis is usually performed in higher invertebrates and vertebrates by Sertoli cells that are located in the testis wall. While Sertoli cells develop a wide range of functions such as creating a blood-testis barrier, providing crucial factors to ensure correct progression of spermatogenesis, and phagocytosis of aberrant, degenerating, and unreleased sperm cells, sponge follicle cells may only display phagocytotic activity on spermatogenic cells. Copyright 2010 Elsevier Ltd. All rights reserved.

Early evolution of large micro-organisms with cytological complexity revealed by microanalyses of 3.4 Ga organic-walled microfossils.

PubMed

Sugitani, K; Mimura, K; Takeuchi, M; Lepot, K; Ito, S; Javaux, E J

2015-11-01

The Strelley Pool Formation (SPF) is widely distributed in the East Pilbara Terrane (EPT) of the Pilbara Craton, Western Australia, and represents a Paleoarchean shallow-water to subaerial environment. It was deposited ~3.4 billion years ago and displays well-documented carbonate stromatolites. Diverse putative microfossils (SPF microfossils) were recently reported from several localities in the East Strelley, Panorama, Warralong, and Goldsworthy greenstone belts. Thus, the SPF provides unparalleled opportunities to gain insights into a shallow-water to subaerial ecosystem on the early Earth. Our new micro- to nanoscale ultrastructural and microchemical studies of the SPF microfossils show that large (20-70 μm) lenticular organic-walled flanged microfossils retain their structural integrity, morphology, and chain-like arrangements after acid (HF-HCl) extraction (palynology). Scanning and transmitted electron microscopy of extracted microfossils revealed that the central lenticular body is either alveolar or hollow, and the wall is continuous with the surrounding smooth to reticulated discoidal flange. These features demonstrate the evolution of large micro-organisms able to form an acid-resistant recalcitrant envelope or cell wall with complex morphology and to form colonial chains in the Paleoarchean era. This study provides evidence of the evolution of very early and remarkable biological innovations, well before the presumed late emergence of complex cells. © 2015 John Wiley & Sons Ltd.

Self-assembly of an electronically conductive network through microporous scaffolds.

PubMed

Sebastian, H Bri; Bryant, Steven L

2017-06-15

Electron transfer spanning significant distances through a microporous structure was established via the self-assembly of an electronically conductive iridium oxide nanowire matrix enveloping the pore walls. Microporous formations were simulated using two scaffold materials of varying physical and chemical properties; paraffin wax beads, and agar gel. Following infiltration into the micropores, iridium nanoparticles self-assembled at the pore wall/ethanol interface. Subsequently, cyclic voltammetry was employed to electrochemically crosslink the metal, erecting an interconnected, and electronically conductive metal oxide nanowire matrix. Electrochemical and spectral characterization techniques confirmed the formation of oxide nanowire matrices encompassing lengths of at least 1.6mm, 400× distances previously achieved using iridium nanoparticles. Nanowire matrices were engaged as biofuel cell anodes, where electrons were donated to the nanowires by a glucose oxidizing enzyme. Copyright © 2017 Elsevier Inc. All rights reserved.

Thermal element for maintaining minimum lamp wall temperature in fluorescent fixtures

DOEpatents

Siminovitch, Michael J.

1992-01-01

In a lighting fixture including a lamp and a housing, an improvement is disclosed for maintaining a lamp envelope area at a cooler, reduced temperature relative to the enclosed housing ambient. The improvement comprises a thermal element in thermal communication with the housing extending to and springably urging thermal communication with a predetermined area of the lamp envelope surface.

Ideal light concentrators with reflector gaps

DOEpatents

Winston, Roland

1980-01-01

A cylindrical or trough-like radiant energy concentration and collection device is provided. The device includes an energy absorber, a glazing enveloping the absorber and a reflective wall. The ideal contour of the reflective wall is determined with reference to a virtual absorber and not the actual absorber cross section.

In-Situ atomic force microscopic observation of ion beam bombarded plant cell envelopes

NASA Astrophysics Data System (ADS)

Sangyuenyongpipat, S.; Yu, L. D.; Brown, I. G.; Seprom, C.; Vilaithong, T.

2007-04-01

A program in ion beam bioengineering has been established at Chiang Mai University (CMU), Thailand, and ion beam induced transfer of plasmid DNA molecules into bacterial cells (Escherichia coli) has been demonstrated. However, a good understanding of the fundamental physical processes involved is lacking. In parallel work, onion skin cells have been bombarded with Ar+ ions at energy 25 keV and fluence1-2 × 1015 ions/cm2, revealing the formation of microcrater-like structures on the cell wall that could serve as channels for the transfer of large macromolecules into the cell interior. An in-situ atomic force microscope (AFM) system has been designed and installed in the CMU bio-implantation facility as a tool for the observation of these microcraters during ion beam bombardment. Here we describe some of the features of the in-situ AFM and outline some of the related work.

Molecular biology of mycoplasmas: from the minimum cell concept to the artificial cell.

PubMed

Cordova, Caio M M; Hoeltgebaum, Daniela L; Machado, Laís D P N; Santos, Larissa Dos

2016-01-01

Mycoplasmas are a large group of bacteria, sorted into different genera in the Mollicutes class, whose main characteristic in common, besides the small genome, is the absence of cell wall. They are considered cellular and molecular biology study models. We present an updated review of the molecular biology of these model microorganisms and the development of replicative vectors for the transformation of mycoplasmas. Synthetic biology studies inspired by these pioneering works became possible and won the attention of the mainstream media. For the first time, an artificial genome was synthesized (a minimal genome produced from consensus sequences obtained from mycoplasmas). For the first time, a functional artificial cell has been constructed by introducing a genome completely synthesized within a cell envelope of a mycoplasma obtained by transformation techniques. Therefore, this article offers an updated insight to the state of the art of these peculiar organisms' molecular biology.

N1N8-bis(gamma-glutamyl)spermidine cross-linking in epidermal-cell envelopes. Comparison of cross-link levels in normal and psoriatic cell envelopes.

PubMed Central

Martinet, N; Beninati, S; Nigra, T P; Folk, J E

1990-01-01

N1N8-Bis(gamma-glutamyl)spermidine was found in exhaustive proteolytic digests of isolated cell envelopes from human epidermis at levels comparable with those of epsilon-(gamma-glutamyl)lysine. Significantly higher than normal amounts of these compounds, particularly the bis(gamma-glutamyl)polyamine, were observed in envelopes from afflicted areas (scales) of psoriatic patients. These findings support the notions that N1N8-bis(gamma-glutamyl)spermidine, like epsilon-(gamma-glutamyl)lysine, functions in cell envelopes as an enzyme-generated protein cross-link and stabilizing force and that individuals with the chronic, recurrent skin disease, psoriasis, exhibit in involved epidermis abnormal cell-envelope-protein cross-linking. PMID:2241917

The diacylglycerol acyltransferase Rv3371 of Mycobacterium tuberculosis is required for growth arrest and involved in stress-induced cell wall alterations.

PubMed

Rastogi, Shivangi; Singh, Amit Kumar; Chandra, Gyan; Kushwaha, Pragati; Pant, Garima; Singh, Kavita; Mitra, Kalyan; Sashidhara, Koneni V; Krishnan, Manju Y

2017-05-01

Triacylglycerol (TAG) is important to mycobacteria both as cell envelope component and energy reservoir. Mycobacterium tuberculosis (Mtb) genome encodes at least 15 putative TAG synthase (tgs)s. We report that one of these genes, Rv3371, specific to pathogenic mycobacteria, when expressed in M. smegmatis leads to modifications in colony morphotype, bacterial architecture, cell surface properties and elevated TAG levels. Rv3371 was found to largely localize in the cell membrane. The Rv3371 promoter is minimally active during exponential growth in vitro, however, is up-regulated under stationary phase, hypoxia, nutrient starvation, nitrosative stress, low iron, in IFN-γ activated macrophages and infected mice. The low iron-induced expression of Rv3371 is likely due to the de-repression by Rv1404, which is probably activated by ideR. An Rv3371 deletion mutant of Mtb showed impaired non-replicating persistence in vitro and altered sensitivity to anti-mycobacterial drugs. In low iron medium, the Rv3371 deletion mutant showed reduced formation of TAG containing extracellular vesicles. Therefore Rv3371 is likely involved in Mtb growth arrest and cell wall alterations during persistence. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell envelope gene murG.

PubMed Central

Salmond, G P; Lutkenhaus, J F; Donachie, W D

1980-01-01

We report the identification, cloning, and mapping of a new cell envelope gene, murG. This lies in a group of five genes of similar phenotype (in the order murE murF murG murC ddl) all concerned with peptidoglycan biosynthesis. This group is in a larger cluster of at least 10 genes, all of which are involved in some way with cell envelope growth. Images PMID:6998962

Acute Modulation of Mycobacterial Cell Envelope Biogenesis by Front-Line Tuberculosis Drugs.

PubMed

Rodriguez-Rivera, Frances P; Zhou, Xiaoxue; Theriot, Julie A; Bertozzi, Carolyn R

2018-05-04

Front-line tuberculosis (TB) drugs have been characterized extensively in vitro and in vivo with respect to gene expression and cell viability. However, little work has been devoted to understanding their effects on the physiology of the cell envelope, one of the main targets of this clinical regimen. Herein, we use metabolic labeling methods to visualize the effects of TB drugs on cell envelope dynamics in mycobacterial species. We developed a new fluorophore-trehalose conjugate to visualize trehalose monomycolates of the mycomembrane using super-resolution microscopy. We also probed the relationship between mycomembrane and peptidoglycan dynamics using a dual metabolic labeling strategy. Finally, we found that metabolic labeling of both cell envelope structures reports on drug effects on cell physiology in two hours, far faster than a genetic sensor of cell envelope stress. Our work provides insight into acute drug effects on cell envelope biogenesis in live mycobacteria. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of five complete genome sequences for members of the class Peribacteria in the recently recognized Peregrinibacteria bacterial phylum

DOE PAGES

Anantharaman, Karthik; Brown, Christopher T.; Burstein, David; ...

2016-01-28

Five closely related populations of bacteria from the Candidate Phylum (CP) Peregrinibacteria, part of the bacterial Candidate Phyla Radiation (CPR), were sampled from filtered groundwater obtained from an aquifer adjacent to the Colorado River near the town of Rifle, CO, USA. Here, we present the first complete genome sequences for organisms from this phylum. These bacteria have small genomes and, unlike most organisms from other lineages in the CPR, have the capacity for nucleotide synthesis. They invest significantly in biosynthesis of cell wall and cell envelope components, including peptidoglycan, isoprenoids via the mevalonate pathway, and a variety of amino sugarsmore » including perosamine and rhamnose. The genomes encode an intriguing set of large extracellular proteins, some of which are very cysteine-rich and may function in attachment, possibly to other cells. Strain variation in these proteins is an important source of genotypic variety. Overall, the cell envelope features, combined with the lack of biosynthesis capacities for many required cofactors, fatty acids, and most amino acids point to a symbiotic lifestyle. Furthermore, phylogenetic analyses indicate that these bacteria likely represent a new class within the Peregrinibacteria phylum, although they ultimately may be recognized as members of a separate phylum. In conclusion, we propose the provisional taxonomic assignment as ‘ Candidatus Peribacter riflensis’, Genus Peribacter, Family Peribacteraceae, Order Peribacterales, Class Peribacteria in the phylum Peregrinibacteria.« less

Thermal element for maintaining minimum lamp wall temperature in fluorescent fixtures

DOEpatents

Siminovitch, M.J.

1992-11-10

In a lighting fixture including a lamp and a housing, an improvement is disclosed for maintaining a lamp envelope area at a cooler, reduced temperature relative to the enclosed housing ambient. The improvement comprises a thermal element in thermal communication with the housing extending to and springably urging thermal communication with a predetermined area of the lamp envelope surface. 12 figs.

Cell-autonomous defense, re-organization and trafficking of membranes in plant-microbe interactions.

PubMed

Dörmann, Peter; Kim, Hyeran; Ott, Thomas; Schulze-Lefert, Paul; Trujillo, Marco; Wewer, Vera; Hückelhoven, Ralph

2014-12-01

Plant cells dynamically change their architecture and molecular composition following encounters with beneficial or parasitic microbes, a process referred to as host cell reprogramming. Cell-autonomous defense reactions are typically polarized to the plant cell periphery underneath microbial contact sites, including de novo cell wall biosynthesis. Alternatively, host cell reprogramming converges in the biogenesis of membrane-enveloped compartments for accommodation of beneficial bacteria or invasive infection structures of filamentous microbes. Recent advances have revealed that, in response to microbial encounters, plasma membrane symmetry is broken, membrane tethering and SNARE complexes are recruited, lipid composition changes and plasma membrane-to-cytoskeleton signaling is activated, either for pre-invasive defense or for microbial entry. We provide a critical appraisal on recent studies with a focus on how plant cells re-structure membranes and the associated cytoskeleton in interactions with microbial pathogens, nitrogen-fixing rhizobia and mycorrhiza fungi. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

A Novel Family of Cell Wall-Related Proteins Regulated Differently during the Yeast Life Cycle

PubMed Central

Rodríguez-Peña, José Manuel; Cid, Víctor J.; Arroyo, Javier; Nombela, César

2000-01-01

The Saccharomyces cerevisiae Ygr189c, Yel040w, and Ylr213c gene products show significant homologies among themselves and with various bacterial β-glucanases and eukaryotic endotransglycosidases. Deletion of the corresponding genes, either individually or in combination, did not produce a lethal phenotype. However, the removal of YGR189c and YEL040w, but not YLR213c, caused additive sensitivity to compounds that interfere with cell wall construction, such as Congo red and Calcofluor White, and overexpression of YEL040w led to resistance to these compounds. These genes were renamed CRH1 and CRH2, respectively, for Congo red hypersensitive. By site-directed mutagenesis we found that the putative glycosidase domain of CRH1 was critical for its function in complementing hypersensitivity to the inhibitors. The involvement of CRH1 and CRH2 in the development of cell wall architecture was clearly shown, since the alkali-soluble glucan fraction in the crh1Δ crh2Δ strain was almost twice the level in the wild-type. Interestingly, the three genes were subject to different patterns of transcriptional regulation. CRH1 and YLR213c (renamed CRR1, for CRH related) were found to be cell cycle regulated and also expressed under sporulation conditions, whereas CRH2 expression did not vary during the mitotic cycle. Crh1 and Crh2 are localized at the cell surface, particularly in chitin-rich areas. Consistent with the observed expression patterns, Crh1–green fluorescent protein was found at the incipient bud site, around the septum area in later stages of budding, and in ascospore envelopes. Crh2 was found to localize mainly at the bud neck throughout the whole budding cycle, in mating projections and zygotes, but not in ascospores. These data suggest that the members of this family of putative glycosidases might exert a common role in cell wall organization at different stages of the yeast life cycle. PMID:10757808

Antibacterial effects of lactoferricin B.

PubMed

Vorland, L H; Ulvatne, H; Andersen, J; Haukland, H H; Rekdal, O; Svendsen, J S; Gutteberg, T J

1999-01-01

The antimicrobial peptide, lactoferricin, can be generated upon gastric pepsin cleavage of lactoferrin. We have examined the inhibitory efficacy of lactoferricin of bovine origin (Lf-cin B) on Escherichia coli, Proteus mirabilis and Staphylococcus aureus with or without a cell wall. We found that spheroplasts and protoplasts had a lower MIC than their counterparts with a cell wall. We also compared the efficacies of Lf-cin B (17-31) made of all L-amino acids and all D-amino acids. The peptide made of all D-amino acids was more active than the corresponding L-enantiomer. Furthermore, we examined the influence of Lf-cin B on the motility of E. coli and the influence of temperature on the susceptibility of bacteria exposed to Lf-cin B. Bacteria exposed to sub-MIC of Lf-cin B lost their motility. Bacteria exposed to Lf-cin B at 20 degrees C were more sensitive to Lf-cin B than when exposed at 37 degrees C. These findings indicate that the cell envelope is a limiting step for Lf-cin B to exert its antibiotic effect. We cannot rule out a receptor-mediated first step for Lf-cin B (17-31).

A transmission electron microscopy study of the diversity of Candida albicans cells induced by Euphorbia hirta L. leaf extract in vitro

PubMed Central

Basma, Abu Arra; Zuraini, Zakaria; Sasidharan, Sreenivasan

2011-01-01

Objective To determine the major changes in the microstructure of Candida albicans (C. albicans) after treatment with Euphorbia hirta (E. hirta) L. leaf extract. Methods Transmission electron microscopy was used to study the ultrastructural changes caused by E. hirta extract on C. albicans cells at various exposure time. Results It was found that the main abnormalities were the alterations in morphology, lysis and complete collapse of the yeast cells after 36 h of exposure to the extract. Whereas the control cultures showed a typical morphology of Candida with a uniform central density, typically structured nucleus, and a cytoplasm with several elements of endomembrane system and enveloped by a regular, intact cell wall. Conclusions The significant antifungal activity shown by this methanol extract of E. hirta L. suggests its potential against infections caused by C. albicans. The extract may be developed as an anticandidal agent. PMID:23569719

RanGAP1 is a continuous marker of the Arabidopsis cell division plane

PubMed Central

Xu, Xianfeng Morgan; Zhao, Qiao; Rodrigo-Peiris, Thushani; Brkljacic, Jelena; He, Chao Sylvia; Müller, Sabine; Meier, Iris

2008-01-01

In higher plants, the plane of cell division is faithfully predicted by the preprophase band (PPB). The PPB, a cortical ring of microtubules and F-actin, disassembles upon nuclear-envelope breakdown. During cytokinesis, the expanding cell plate fuses with the plasma membrane at the cortical division site, the site of the former PPB. The nature of the “molecular memory” that is left behind by the PPB and is proposed to guide the cell plate to the cortical division site is unknown. RanGAP is the GTPase activating protein of the small GTPase Ran, which provides spatial information for nucleocytoplasmic transport and various mitotic processes in animals. Here, we show that, in dividing root cells, Arabidopsis RanGAP1 concentrates at the PPB and remains associated with the cortical division site during mitosis and cytokinesis, requiring its N-terminal targeting domain. In a fass/ton2 mutant, which affects PPB formation, RanGAP1 recruitment to the PPB site is lost, while its PPB retention is microtubule-independent. RanGAP1 persistence at the cortical division site, but not its initial accumulation at the PPB requires the 2 cytokinesis-regulating kinesins POK1 and POK2. Depletion of RanGAP by inducible RNAi leads to oblique cell walls and cell-wall stubs in root cell files, consistent with cytokinesis defects. We propose that Arabidopsis RanGAP, a continuous positive protein marker of the plant division plane, has a role in spatial signaling during plant cell division. PMID:19011093

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.

PubMed

Song, Erwei; Zhu, Pengcheng; Lee, Sang-Kyung; Chowdhury, Dipanjan; Kussman, Steven; Dykxhoorn, Derek M; Feng, Yi; Palliser, Deborah; Weiner, David B; Shankar, Premlata; Marasco, Wayne A; Lieberman, Judy

2005-06-01

Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.

Moisture Performance of Energy-Efficient and Conventional Wood-Frame Wall Assemblies in a Mixed-Humid Climate

Treesearch

Samuel Glass; Vladimir Kochkin; S. Drumheller; Lance Barta

2015-01-01

Long-term moisture performance is a critical consideration for design and construction of building envelopes in energy-efficient buildings, yet field measurements of moisture characteristics for highly insulated wood-frame walls in mixed-humid climates are lacking. Temperature, relative humidity, and moisture content of wood framing and oriented strand board (OSB)...

Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells: fusion-mediated microinjection of ricin A and simian virus 40 DNA.

PubMed

Gitman, A G; Graessmann, A; Loyter, A

1985-11-01

Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA.

Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells: fusion-mediated microinjection of ricin A and simian virus 40 DNA.

PubMed Central

Gitman, A G; Graessmann, A; Loyter, A

1985-01-01

Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA. PMID:2997783

UNE FORME MICROTUBULAIRE ET PARACRISTALLINE DE RETICULUM ENDOPLASMIQUE DANS LES PHOTOCYTES DES ANNELIDES POLYNOINÆ

PubMed Central

Bassot, J. M.

1966-01-01

Luminous cells of polynoid worm elytra have been examined by methods of electron microscopy, with special attention focused on the fine structure of photogenic grains. These cells send apical prolongations into the mid-part of the elytra. The plasma membrane is very sinuous, and a special kind of desmosome links two portions of the same membrane. In addition to all the organelles which can be found in nonluminescent epithelial cells of the elytra, numerous photogenic grains are contained in their cytoplasm. These grains are composed of undulating microtubules measuring 200 A in diameter; their disposition in the grain is highly regular, and the grains appear as paracrystals. At the borders of the grains, the walls of the microtubules are often in continuity with those of the endoplasmic reticulum and with the external membrane of the nuclear envelope. Because of this fact, the microtubules of the grains may be considered a cytoplasmic organelle, representing a specialized form of the endoplasmic reticulum. The microtubules permit the repartition, inside and outside their walls, of two different products, one being forty-three times more abundant than the other; thus, the contact surface, in comparison to the volume, is greatly increased. The induction of the luminous reaction by change in the permeability of the microtubule walls, allowing contact between the two substances, is suggested as a working hypothesis. There is an evolution of the grains along the axis of the photocytes. The grains are often surrounded by progressively increasing amounts of glycogen. Their paracrystalline disposition is altered at the apex of the luminous cells. PMID:5971968

Cell envelopes of chemotaxis mutants of Escherichia coli rotate their flagella counterclockwise.

PubMed Central

Szupica, C J; Adler, J

1985-01-01

Flagella rotated exclusively counterclockwise in Escherichia coli cell envelopes prepared from wild-type cells, whose flagella rotated both clockwise and counterclockwise, from mutants rotating their flagella counterclockwise only, and even from mutants rotating their flagella primarily clockwise. Some factor needed for clockwise flagellar rotation appeared to be missing or defective in the cell envelopes. PMID:3884599

Expression of DNAJB12 or DNAJB14 Causes Coordinate Invasion of the Nucleus by Membranes Associated with a Novel Nuclear Pore Structure

PubMed Central

Goodwin, Edward C.; Motamedi, Nasim; Lipovsky, Alex; Fernández-Busnadiego, Rubén; DiMaio, Daniel

2014-01-01

DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for DNAJ-associated nuclear globular structures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity. PMID:24732912

The highly conserved MraZ protein is a transcriptional regulator in Escherichia coli

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

Eraso, Jesus M.; Markillie, Lye Meng; Mitchell, Hugh D.

2014-05-05

The mraZ and mraW genes are highly conserved in bacteria, both in sequence and location at the head of the division and cell wall (dcw) gene cluster. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin, and MraW is known to methylate ribosomal RNA, mraZ and mraW null mutants have no detectable growth phenotype in any species tested to date, hampering progress in understanding their physiological role. Here we show that overproduction of Escherichia coli MraZ perturbs cell division and the cell envelope, is more lethal at high levels or in minimal growth medium,more » and that MraW antagonizes these effects. MraZGFP localizes to the nucleoid, suggesting that it binds DNA. Indeed, purified MraZ directly binds a region upstream from its own promoter containing three direct repeats to regulate its own expression and that of downstream cell division and cell wall genes. MraZ-LacZ fusions are repressed by excess MraZ but not when DNA binding by MraZ is inhibited. RNAseq analysis indicates that MraZ is a global transcriptional regulator with numerous targets in addition to dcw genes. One of these targets, mioC, is directly bound by MraZ in a region with three direct repeats.« less

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

Levy, E.; Mullens, M.; Rath, P.

The Advanced Envelope Research effort will provide factory homebuilders with high performance, cost-effective envelope designs that can be effectively integrated into the plant production process while meeting the thermal requirements of the 2012 IECC standards. This work is part of a multiphase effort. Phase 1 identified seven envelope technologies and provided a preliminary assessment of three methods for building high performance walls. Phase 2 focused on developing viable product designs, manufacturing strategies, addressing code and structural issues, and cost analysis of the three selected options. An industry advisory committee helped narrow the research focus to perfecting a stud wall designmore » with exterior continuous insulation (CI). This report describes Phase 3, which was completed in two stages and continued the design development effort, exploring and evaluating a range or methods for applying CI to factory built homes. The scope also included material selection, manufacturing and cost analysis, and prototyping and testing. During this phase, a home was built with CI, evaluated, and placed in service. The experience of building a mock up wall section with CI and then constructing on line a prototype home resolved important concerns about how to integrate the material into the production process. First steps were taken toward finding least expensive approaches for incorporating CI in standard factory building practices and a preliminary assessment suggested that even at this early stage the technology is attractive when viewed from a life cycle cost perspective.« less

HIV envelope-mediated, CCR5/α4β7-dependent killing of CD4-negative γδ T cells which are lost during progression to AIDS.

PubMed

Li, Haishan; Pauza, C David

2011-11-24

HIV infects and replicates in CD4+ T cells but effects on host immunity and disease also involve depletion, hyper-activation, and modification of CD4-negative cell populations. In particular, the depletion of CD4-negative γδ T cells is common to all HIV+ individuals. We found that soluble or cell-associated envelope glycoproteins from CCR5-tropic strains of HIV could bind, activates the p38-caspase pathway, and induce the death of γδ cells. Envelope binding requires integrin α4β7 and chemokine receptor CCR5 which are at high levels and form a complex on the γδ T cell membrane. This receptor complex facilitated V3 loop binding to CCR5 in the absence of CD4-induced conformational changes. Cell death was increased by antigen stimulation after exposure to envelope glycoprotein. Direct signaling by envelope glycoprotein killed CD4-negative γδ T cells and reproduced a defect observed in all patients with HIV disease.

Spermatogenesis and sperm ultrastructure in the polychaete genus Ophryotrocha (Dorvilleidae)

NASA Astrophysics Data System (ADS)

Pfannenstiel, Hans-Dieter; Grünig, Charlotte

1990-06-01

The details of spermatogenesis and spermiogenesis are described for Ophryotrocha puerilis. The ultrastructure of mature sperm is shown for O. puerilis, O. hartmanni, O. gracilis, O. diadema, O. labronica, and O. notoglandulata. Clusters of sixteen cells each are proliferated by two stem cells in each setigerous segment of O. puerilis representing the very early stages of both oogenesis and spermatogenesis. In each spermatocyte-I cluster, the cells are interconnected by cytoplasmic bridges. Early, clusters are enveloped by peritoneal sheath cells. These transient gonad walls break down prior to meiosis. The meiotic processes may start in the clusters with the cells still interconnected, or during breakdown of the original cluster, giving rise to smaller subclusters of both spermatocytes I and spermatocytes II with various numbers of cells. Finally, spermatid tetrads are present. As spermiogenesis progresses, the tetrads disintegrate. Golgi vesicles in both spermatocytes and spermatids contain electron-dense material, presumably preacrosomal. The acrosome is formed by such vesicles. In the six species studied here, the acrosomes appear to be of a similar overall structure but are of different shape. Centrioles are usually located beneath the acrosome. The distal centriole forms the basal body of a flagellum-like cytoplasmic process. The microtubules of these flagellar equivalents do not show a normal ciliar arrangement. The flagellar equivalent appears to be non-motile. In O. hartmanni and in O. notoglandulata, a flagellar equivalent is missing. Microtubules originating from the proximal end of the distal centriole stretch to the nuclear envelope. This feature appears to be especially conspicuous in O. puerilis and in O. labronica. In O. labronica and in O. notoglandulata, bundles of microtubules paralleling the cell perimeter appear to stabilise the sperm. Various numbers of mitochondria are either randomly distributed around the nucleus or accumulate on one side, often directly under the acrosome.

Bacillus subtilis extracytoplasmic function (ECF) sigma factors and defense of the cell envelope

PubMed Central

Helmann, John D.

2016-01-01

Summary Bacillus subtilis provides a model for investigation of the bacterial cell envelope, the first line of defense against environmental threats. Extracytoplasmic function (ECF) sigma factors activate genes that confer resistance to agents that threaten the integrity of the envelope. Although their individual regulons overlap, σW is most closely associated with membrane-active agents, σX with cationic antimicrobial peptide resistance, and σV with resistance to lysozyme. Here, I highlight the role of the σM regulon, which is strongly induced by conditions that impair peptidoglycan synthesis and includes the core pathways of envelope synthesis and cell division, as well as stress-inducible alternative enzymes. Studies of these cell envelope stress responses provide insights into how bacteria acclimate to the presence of antibiotics. PMID:26901131

Susceptibility of Escherichia coli to Bactericidal Action of Lactoperoxidase, Peroxide, and Iodide or Thiocyanate

PubMed Central

Thomas, Edwin L.; Aune, Thomas M.

1978-01-01

The bactericidal action that results from lactoperoxidase-catalyzed oxidation of iodide or thiocyanate was studied, using Escherichia coli as the test organism. The susceptibility of intact cells to bactericidal action was compared with that of cells with altered cell envelopes. Exposure to ethylenediaminetetraacetic acid, to lysozyme and ethylenediaminetetraacetic acid, or to osmotic shock were used to alter the cell envelope. Bactericidal action was greatly increased when the cells were exposed to the lactoperoxidase-peroxide-iodide system at low temperatures, low cell density, or after alteration of the cell envelope. When thiocyanate was substituted for iodide, bactericidal activity was observed only at low cell density or after osmotic shock. Low temperature and low cell density lowered the rate of destruction of peroxide by the bacteria. Therefore, competition for peroxide between the bacteria and lactoperoxidase may influence the extent of bactericidal action. Alteration of the cell envelope had only a small effect on the rate of destruction of peroxide. Instead, the increased susceptibility of these altered cells suggested that bactericidal action required permeation of a reagent through the cell envelope. In addition to altering the cell envelope, these procedures partly depleted cells of oxidizable substrates and sulfhydryl components. Adding an oxidizable substrate did not decrease the susceptibility of the altered cells. On the other hand, mild reducing agents such as sulfhydryl compounds did partly reverse bactericidal action when added after exposure of cells to the peroxidase systems. These studies indicate that alteration of the metabolism, structure, or composition of bacterial cells can greatly increase their susceptibility to peroxidase bactericidal action. PMID:348097

Cation-induced transcriptional regulation of the dlt operon of Staphylococcus aureus.

PubMed

Koprivnjak, Tomaz; Mlakar, Vid; Swanson, Lindsey; Fournier, Benedicte; Peschel, Andreas; Weiss, Jerrold P

2006-05-01

Lipoteichoic and wall teichoic acids (TA) are highly anionic cell envelope-associated polymers containing repeating polyglycerol/ribitol phosphate moieties. Substitution of TA with D-alanine is important for modulation of many cell envelope-dependent processes, such as activity of autolytic enzymes, binding of divalent cations, and susceptibility to innate host defenses. D-Alanylation of TA is diminished when bacteria are grown in medium containing increased NaCl concentrations, but the effects of increased salt concentration on expression of the dlt operon encoding proteins mediating D-alanylation of TA are unknown. We demonstrate that Staphylococcus aureus transcriptionally represses dlt expression in response to high concentrations of Na(+) and moderate concentrations of Mg(2+) and Ca(2+) but not sucrose. Changes in dlt mRNA are induced within 15 min and sustained for several generations of growth. Mg(2+)-induced dlt repression depends on the ArlSR two-component system. Northern blotting, reverse transcription-PCR, and SMART-RACE analyses suggest that the dlt transcript begins 250 bp upstream of the dltA start codon and includes an open reading frame immediately upstream of dltA. Chloramphenicol transacetylase transcriptional fusions indicate that a region encompassing the 171 to 325 bp upstream of dltA is required for expression and Mg(2+)-induced repression of the dlt operon in S. aureus.

Cation-Induced Transcriptional Regulation of the dlt Operon of Staphylococcus aureus

PubMed Central

Koprivnjak, Tomaz; Mlakar, Vid; Swanson, Lindsey; Fournier, Benedicte; Peschel, Andreas; Weiss, Jerrold P.

2006-01-01

Lipoteichoic and wall teichoic acids (TA) are highly anionic cell envelope-associated polymers containing repeating polyglycerol/ribitol phosphate moieties. Substitution of TA with d-alanine is important for modulation of many cell envelope-dependent processes, such as activity of autolytic enzymes, binding of divalent cations, and susceptibility to innate host defenses. d-Alanylation of TA is diminished when bacteria are grown in medium containing increased NaCl concentrations, but the effects of increased salt concentration on expression of the dlt operon encoding proteins mediating d-alanylation of TA are unknown. We demonstrate that Staphylococcus aureus transcriptionally represses dlt expression in response to high concentrations of Na+ and moderate concentrations of Mg2+ and Ca2+ but not sucrose. Changes in dlt mRNA are induced within 15 min and sustained for several generations of growth. Mg2+-induced dlt repression depends on the ArlSR two-component system. Northern blotting, reverse transcription-PCR, and SMART-RACE analyses suggest that the dlt transcript begins 250 bp upstream of the dltA start codon and includes an open reading frame immediately upstream of dltA. Chloramphenicol transacetylase transcriptional fusions indicate that a region encompassing the 171 to 325 bp upstream of dltA is required for expression and Mg2+-induced repression of the dlt operon in S. aureus. PMID:16672616

Case Study of Envelope Sealing in Existing Multiunit Structures

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

Dentz, Jordan; Conlin, Francis; Podorson, David

2012-10-01

This report describes envelope air sealing that was included in the retrofit of a 244 unit low-rise multifamily housing complex in Durham, N.C. On average, total leakage was reduced by nearly half, from 19.7 ACH50 to 9.4 ACH50. Important air leakage locations identified included plumbing and electrical penetrations, dropped ceilings/soffits, windows, ducts and wall-to-floor intersections. Specifications and a pictorial guide were developed for contractors performing the work.

Nano-ranged low-energy ion-beam-induced DNA transfer in biological cells

NASA Astrophysics Data System (ADS)

Yu, L. D.; Wongkham, W.; Prakrajang, K.; Sangwijit, K.; Inthanon, K.; Thongkumkoon, P.; Wanichapichart, P.; Anuntalabhochai, S.

2013-06-01

Low-energy ion beams at a few tens of keV were demonstrated to be able to induce exogenous macromolecules to transfer into plant and bacterial cells. In the process, the ion beam with well controlled energy and fluence bombarded living cells to cause certain degree damage in the cell envelope in nanoscales to facilitate the macromolecules such as DNA to pass through the cell envelope and enter the cell. Consequently, the technique was applied for manipulating positive improvements in the biological species. This physical DNA transfer method was highly efficient and had less risk of side-effects compared with chemical and biological methods. For better understanding of mechanisms involved in the process, a systematic study on the mechanisms was carried out. Applications of the technique were also expanded from DNA transfer in plant and bacterial cells to DNA transfection in human cancer cells potentially for the stem cell therapy purpose. Low-energy nitrogen and argon ion beams that were applied in our experiments had ranges of 100 nm or less in the cell envelope membrane which was majorly composed of polymeric cellulose. The ion beam bombardment caused chain-scission dominant damage in the polymer and electrical property changes such as increase in the impedance in the envelope membrane. These nano-modifications of the cell envelope eventually enhanced the permeability of the envelope membrane to favor the DNA transfer. The paper reports details of our research in this direction.

Bacillus subtilis extracytoplasmic function (ECF) sigma factors and defense of the cell envelope.

PubMed

Helmann, John D

2016-04-01

Bacillus subtilis provides a model for investigation of the bacterial cell envelope, the first line of defense against environmental threats. Extracytoplasmic function (ECF) sigma factors activate genes that confer resistance to agents that threaten the integrity of the envelope. Although their individual regulons overlap, σ(W) is most closely associated with membrane-active agents, σ(X) with cationic antimicrobial peptide resistance, and σ(V) with resistance to lysozyme. Here, I highlight the role of the σ(M) regulon, which is strongly induced by conditions that impair peptidoglycan synthesis and includes the core pathways of envelope synthesis and cell division, as well as stress-inducible alternative enzymes. Studies of these cell envelope stress responses provide insights into how bacteria acclimate to the presence of antibiotics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Thermionic converter

DOEpatents

Fitzpatrick, G.O.

1987-05-19

A thermionic converter is set forth which includes an envelope having an electron collector structure attached adjacent to a wall. An electron emitter structure is positioned adjacent the collector structure and spaced apart from opposite wall. The emitter and collector structures are in a common chamber. The emitter structure is heated substantially only by thermal radiation. Very small interelectrode gaps can be maintained utilizing the thermionic converter whereby increased efficiency results. 10 figs.

Double Wall Framing Technique An Example of High Performance, Sustainable Building Envelope Technology

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

Kosny, Dr. Jan; Asiz, Andi; Shrestha, Som S

2015-01-01

Double wall technologies utilizing wood framing have been well-known and used in North American buildings for decades. Most of double wall designs use only natural materials such as wood products, gypsum, and cellulose fiber insulation, being one of few building envelope technologies achieving high thermal performance without use of plastic foams or fiberglass. Today, after several material and structural design modifications, these technologies are considered as highly thermally efficient, sustainable option for new constructions and sometimes, for retrofit projects. Following earlier analysis performed for U.S. Department of Energy by Fraunhofer CSE, this paper discusses different ways to build double wallsmore » and to optimize their thermal performance to minimize the space conditioning energy consumption. Description of structural configuration alternatives and thermal performance analysis are presented as well. Laboratory tests to evaluate thermal properties of used insulation and whole wall system thermal performance are also discussed in this paper. Finally, the thermal loads generated in field conditions by double walls are discussed utilizing results from a joined project performed by Zero Energy Building Research Alliance and Oak Ridge National Laboratory (ORNL), which made possible evaluation of the market viability of low-energy homes built in the Tennessee Valley. Experimental data recorded in two of the test houses built during this field study is presented in this work.« less

Operating envelope charts for the Langley 0.3-meter transonic cryogenic wind tunnel

NASA Technical Reports Server (NTRS)

Rallo, R. A.; Dress, D. A.; Siegle, H. J. A.

1986-01-01

To take full advantage of the unique Reynolds number capabilities of the 0.3-meter Transonic Cryogenic Tunnel (0.3-m TCT) at the NASA Langley Research Center, it was designed to accommodate test sections other than the original, octagonal, three-dimensional test section. A 20- by 60-cm two-dimensional test section was installed in 1976 and was extensively used, primarily for airfoil testing, through the fall of 1984. The tunnel was inactive during 1985 so that a new test section and improved high speed diffuser could be installed in the tunnel circuit. The new test section has solid adaptive top and bottom walls to reduce or eliminate wall interference for two-dimensional testing. The test section is 33- by 33-cm in cross section at the entrance and is 142 cm long. In the planning and running of past airfoil tests in the 0.3-m TCT, the use of operating envelope charts have proven very useful. These charts give the variation of total temperature and pressure with Mach number and Reynolds number. The operating total temperature range of the 0.3-m TCT is from about 78 K to 327 K with total pressures ranging from about 17.5 psia to 88 psia. This report presents the operating envelope charts for the 0.3-m TCT with the adaptive wall tes t section installed. They were all generated based on a 1-foot chord model. The Mach numbers vary from 0.1 to 0.95.

Peptidoglycan synthesis drives an FtsZ-treadmilling-independent step of cytokinesis.

PubMed

Monteiro, João M; Pereira, Ana R; Reichmann, Nathalie T; Saraiva, Bruno M; Fernandes, Pedro B; Veiga, Helena; Tavares, Andreia C; Santos, Margarida; Ferreira, Maria T; Macário, Vânia; VanNieuwenhze, Michael S; Filipe, Sérgio R; Pinho, Mariana G

2018-02-22

Peptidoglycan is the main component of the bacterial wall and protects cells from the mechanical stress that results from high intracellular turgor. Peptidoglycan biosynthesis is very similar in all bacteria; bacterial shapes are therefore mainly determined by the spatial and temporal regulation of peptidoglycan synthesis rather than by the chemical composition of peptidoglycan. The form of rod-shaped bacteria, such as Bacillus subtilis or Escherichia coli, is generated by the action of two peptidoglycan synthesis machineries that act at the septum and at the lateral wall in processes coordinated by the cytoskeletal proteins FtsZ and MreB, respectively. The tubulin homologue FtsZ is the first protein recruited to the division site, where it assembles in filaments-forming the Z ring-that undergo treadmilling and recruit later divisome proteins. The rate of treadmilling in B. subtilis controls the rates of both peptidoglycan synthesis and cell division. The actin homologue MreB forms discrete patches that move circumferentially around the cell in tracks perpendicular to the long axis of the cell, and organize the insertion of new cell wall during elongation. Cocci such as Staphylococcus aureus possess only one type of peptidoglycan synthesis machinery, which is diverted from the cell periphery to the septum in preparation for division. The molecular cue that coordinates this transition has remained elusive. Here we investigate the localization of S. aureus peptidoglycan biosynthesis proteins and show that the recruitment of the putative lipid II flippase MurJ to the septum, by the DivIB-DivIC-FtsL complex, drives peptidoglycan incorporation to the midcell. MurJ recruitment corresponds to a turning point in cytokinesis, which is slow and dependent on FtsZ treadmilling before MurJ arrival but becomes faster and independent of FtsZ treadmilling after peptidoglycan synthesis activity is directed to the septum, where it provides additional force for cell envelope constriction.

The Neomuran Revolution and Phagotrophic Origin of Eukaryotes and Cilia in the Light of Intracellular Coevolution and a Revised Tree of Life

PubMed Central

Cavalier-Smith, Thomas

2014-01-01

Three kinds of cells exist with increasingly complex membrane-protein targeting: Unibacteria (Archaebacteria, Posibacteria) with one cytoplasmic membrane (CM); Negibacteria with a two-membrane envelope (inner CM; outer membrane [OM]); eukaryotes with a plasma membrane and topologically distinct endomembranes and peroxisomes. I combine evidence from multigene trees, palaeontology, and cell biology to show that eukaryotes and archaebacteria are sisters, forming the clade neomura that evolved ∼1.2 Gy ago from a posibacterium, whose DNA segregation and cell division were destabilized by murein wall loss and rescued by the evolving novel neomuran endoskeleton, histones, cytokinesis, and glycoproteins. Phagotrophy then induced coevolving serial major changes making eukaryote cells, culminating in two dissimilar cilia via a novel gliding–fishing–swimming scenario. I transfer Chloroflexi to Posibacteria, root the universal tree between them and Heliobacteria, and argue that Negibacteria are a clade whose OM, evolving in a green posibacterium, was never lost. PMID:25183828

The neomuran revolution and phagotrophic origin of eukaryotes and cilia in the light of intracellular coevolution and a revised tree of life.

PubMed

Cavalier-Smith, Thomas

2014-09-02

Three kinds of cells exist with increasingly complex membrane-protein targeting: Unibacteria (Archaebacteria, Posibacteria) with one cytoplasmic membrane (CM); Negibacteria with a two-membrane envelope (inner CM; outer membrane [OM]); eukaryotes with a plasma membrane and topologically distinct endomembranes and peroxisomes. I combine evidence from multigene trees, palaeontology, and cell biology to show that eukaryotes and archaebacteria are sisters, forming the clade neomura that evolved ~1.2 Gy ago from a posibacterium, whose DNA segregation and cell division were destabilized by murein wall loss and rescued by the evolving novel neomuran endoskeleton, histones, cytokinesis, and glycoproteins. Phagotrophy then induced coevolving serial major changes making eukaryote cells, culminating in two dissimilar cilia via a novel gliding-fishing-swimming scenario. I transfer Chloroflexi to Posibacteria, root the universal tree between them and Heliobacteria, and argue that Negibacteria are a clade whose OM, evolving in a green posibacterium, was never lost. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Functional and proteomic analyses reveal that wxcB is involved in virulence, motility, detergent tolerance, and biofilm formation in Xanthomonas campestris pv. vesicatoria.

PubMed

Park, Hye-Jee; Jung, Ho Won; Han, Sang-Wook

2014-09-26

The bacterial envelope possesses diverse functions, including protection against environmental stress and virulence factors for host infection. Here, we report the function of wxcB in Xanthomonas campestris pv. vesicatoria (Xcv), a causal agent of bacterial leaf spot disease in tomato and pepper. To characterize roles of wxcB, we generated a knockout mutant (XcvΔwxcB) and found that the virulence of the mutant was weaker than that of the wild type in tomato plants. To predict the mechanism affected by wxcB, we compared protein expressions between the wild type and the mutant. Expression of 152 proteins showed a greater than 2-fold difference. Proteins involved in motility and cell wall/membrane were the most abundant. Through phenotypic assays, we further demonstrated that the mutant displayed reduced motility and tolerance to treatment, but it showed increased biofilm formation. Interestingly, the LPS profile was unchanged. These results lead to new insights into the functions of wxcB that is associated with cell wall/membrane functions, which contributes to pathogen virulence. Copyright © 2014 Elsevier Inc. All rights reserved.

Murine Sarcoma Virus Gene Expression: Transformants Which Express Viral Envelope Glycoprotein In The Absence Of The Major Internal Protein And Infectious Particles

PubMed Central

Bilello, John A.; Strand, Mette; August, J. T.

1974-01-01

Expression of the major internal protein and the envelope glycoprotein of murine C-type viruses in focus-derived lines of normal rat kidney cells infected with Kirsten murine sarcoma virus was measured by radioimmunoassay. Of the clones selected, which do not produce virus particles or the major viral structural protein, approximately half express the viral envelope glycoprotein at concentrations found in productively infected cells. Expression of the envelope glycoprotein did not appear to alter significantly the properties of the transformed cells in culture. PMID:4370209

Effect of alkali on the structure of cell envelopes of Chlamydia psittaci elementary bodies.

PubMed Central

Narita, T; Wyrick, P B; Manire, G P

1976-01-01

Suspensions of isolated cell envelopes of infectious elementary bodies (EB) of Chlamydia psittaci at alkaline pH showed a rapid, extensive decrease in absorbance, accompanied by the release of a cell envelope component in a sedimentable form. This phenomenon was observed both at 0 C and with envelopes which had been previously heated to 100 C. Monovalent and divalent cations effectively inhibited the turbidity loss, whereas ethylenediaminetetraacetate (EDTA) caused an accelerated decrease in turbidity. The turbidity loss observed after incubation of the envelopes at alkaline pH could be reversed to the level of the initial value by dialysis against distilled water containing Mg2+. Thin-section electron photomicrographs of purified EB exposed to alkaline buffer with EDTA revealed the loss of the internal contents of cells, but these cells still maintained their round shapes. The cell surface of treated EB appeared pitted in negatively stained preparations, whereas intact EB had a smooth surface. Electron microscopic studies on negatively stained preparations of the clear supernatant obtained after the treatment of the envelope with alkaline buffer containing EDTA demonstrated the presence of spherical particles, approximately 6 to 7 nm in diameter, and rodlike particles, which appeared to be made up of two or more spherical particles. Images PMID:1375

Termination unit

DOEpatents

Traeholt, Chresten; Willen, Dag; Roden, Mark; Tolbert, Jerry C.; Lindsay, David; Fisher, Paul W.; Nielsen, Carsten Thidemann

2016-05-03

Cable end section comprises end-parts of N electrical phases/neutral, and a thermally-insulation envelope comprising cooling fluid. The end-parts each comprises a conductor and are arranged with phase 1 innermost, N outermost surrounded by the neutral, electrical insulation being between phases and N and neutral. The end-parts comprise contacting surfaces located sequentially along the longitudinal extension of the end-section. A termination unit has an insulating envelope connected to a cryostat, special parts at both ends comprising an adapter piece at the cable interface and a closing end-piece terminating the envelope in the end-section. The special parts houses an inlet and/or outlet for cooling fluid. The space between an inner wall of the envelope and a central opening of the cable is filled with cooling fluid. The special part at the end connecting to the cryostat houses an inlet or outlet, splitting cooling flow into cable annular flow and termination annular flow.

Three-dimensional morphological and textural complexity of Archean putative microfossils from the Northeastern Pilbara Craton: indications of biogenicity of large (>15 microm) spheroidal and spindle-like structures.

PubMed

Sugitani, Kenichiro; Grey, Kathleen; Nagaoka, Tsutomu; Mimura, Koichi

2009-09-01

We recently reported a diverse assemblage of carbonaceous structures (thread-like, film-like, spheroidal, and spindle-like) from chert in the ca. 3.0 Ga Farrel Quartzite of the Gorge Creek Group in the Pilbara Craton, Western Australia. Results from a rigorous examination of occurrence, composition, morphological complexity, size distributions, and taphonomy provided presumptive evidence for biogenicity. In this study, we present new data of morphological and textural complexity of large (>15 microm) spheroidal and spindle-like structures, using an in-focus, 3-D image reconstruction system, which further raises the scale of credibility that these structures are microfossils. While many of the large spheroids are single-walled, and the wall is irregularly folded, a few specimens are partially blistered, double walled, or have a dimpled wall. The wall-surface texture varies from smooth and homogeneous (hyaline) to patchy, granular or reticulate. Such variation is best explained as resulting from taphonomic processes. Additionally, an inner solitary body, present in some large spheroids, is hollow and partially broken, which indicates a primary origin for this substructure. Spindle-like structures have two types of flange-like appendage; one is attached at the equatorial plane of the body, whereas the other appears to be attached peripherally. In both cases, the appendage tends to have a flat geometry, a tapering thickness, and constancy in shape, proportions, and dimensions. Spindle-wall surfaces are variously textured and heterogeneous. These morphological and textural complexities and heterogeneity refute potential abiogenic formation models for these structures, such as crystals coated with organic matter, fenestrae, and the diagenetic redistribution of carbonaceous matter. When coupled with other data from Raman spectroscopy, NanoSIMS analysis, and palynology, the evidence that these large carbonaceous structures are biogenic appears compelling, though it is still equivocal as to whether they are cells or outer envelopes of colonies of smaller cells.

Classification of Lactococcus lactis cell envelope proteinase based on gene sequencing, peptides formed after hydrolysis of milk, and computer modeling.

PubMed

Børsting, M W; Qvist, K B; Brockmann, E; Vindeløv, J; Pedersen, T L; Vogensen, F K; Ardö, Y

2015-01-01

Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from αS1-casein and β-casein. A model structure of Lc. lactis CEP based on the crystal structure of Streptococcus C5a CEP was used to investigate the AA positions in the substrate-binding region. New AA positions were suggested, which could be relevant for the cleavage specificity of CEP; however, these could only explain 2 out of 3 found subgroups. The third subgroup could be explained by 1 to 5 AA positions located opposite the substrate binding region. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The mechanistic basis of internal conductance: a theoretical analysis of mesophyll cell photosynthesis and CO2 diffusion.

PubMed

Tholen, Danny; Zhu, Xin-Guang

2011-05-01

Photosynthesis is limited by the conductance of carbon dioxide (CO(2)) from intercellular spaces to the sites of carboxylation. Although the concept of internal conductance (g(i)) has been known for over 50 years, shortcomings in the theoretical description of this process may have resulted in a limited understanding of the underlying mechanisms. To tackle this issue, we developed a three-dimensional reaction-diffusion model of photosynthesis in a typical C(3) mesophyll cell that includes all major components of the CO(2) diffusion pathway and associated reactions. Using this novel systems model, we systematically and quantitatively examined the mechanisms underlying g(i). Our results identify the resistances of the cell wall and chloroplast envelope as the most significant limitations to photosynthesis. In addition, the concentration of carbonic anhydrase in the stroma may also be limiting for the photosynthetic rate. Our analysis demonstrated that higher levels of photorespiration increase the apparent resistance to CO(2) diffusion, an effect that has thus far been ignored when determining g(i). Finally, we show that outward bicarbonate leakage through the chloroplast envelope could contribute to the observed decrease in g(i) under elevated CO(2). Our analysis suggests that physiological and anatomical features associated with g(i) have been evolutionarily fine-tuned to benefit CO(2) diffusion and photosynthesis. The model presented here provides a novel theoretical framework to further analyze the mechanisms underlying diffusion processes in the mesophyll.

The Mechanistic Basis of Internal Conductance: A Theoretical Analysis of Mesophyll Cell Photosynthesis and CO2 Diffusion1[W][OA

PubMed Central

Tholen, Danny; Zhu, Xin-Guang

2011-01-01

Photosynthesis is limited by the conductance of carbon dioxide (CO2) from intercellular spaces to the sites of carboxylation. Although the concept of internal conductance (gi) has been known for over 50 years, shortcomings in the theoretical description of this process may have resulted in a limited understanding of the underlying mechanisms. To tackle this issue, we developed a three-dimensional reaction-diffusion model of photosynthesis in a typical C3 mesophyll cell that includes all major components of the CO2 diffusion pathway and associated reactions. Using this novel systems model, we systematically and quantitatively examined the mechanisms underlying gi. Our results identify the resistances of the cell wall and chloroplast envelope as the most significant limitations to photosynthesis. In addition, the concentration of carbonic anhydrase in the stroma may also be limiting for the photosynthetic rate. Our analysis demonstrated that higher levels of photorespiration increase the apparent resistance to CO2 diffusion, an effect that has thus far been ignored when determining gi. Finally, we show that outward bicarbonate leakage through the chloroplast envelope could contribute to the observed decrease in gi under elevated CO2. Our analysis suggests that physiological and anatomical features associated with gi have been evolutionarily fine-tuned to benefit CO2 diffusion and photosynthesis. The model presented here provides a novel theoretical framework to further analyze the mechanisms underlying diffusion processes in the mesophyll. PMID:21441385

Time Dependence of Tip Morphology during Cellular/Dendritic Arrayed Growth

NASA Technical Reports Server (NTRS)

Song, H.; Tewari, S. N.

1996-01-01

Succinonitrile-1.9 wt pct acetone has been directionally solidified in 0.7 X 0.7-cm-square cross section pyrex ampoules in order to observe the cell/dendrite tip morphologies, not influenced by the 'wall effects', which are present during growth in the generally used thin (about 200 gm) crucibles. The tips do not maintain a steady-state shape, as is generally assumed. Instead, they fluctuate within a shape envelope. The extent of fluctuation increases with decreasing growth speed, as the micro structure changes from the dendritic to cellular. The influence of natural convection has been examined by comparing these morphologies with those grown, without convection, in the thin ampoules.

The structure of the perivascular compartment in the old canine brain: a case study.

PubMed

Criswell, Theodore P; Sharp, Matthew MacGregor; Dobson, Howard; Finucane, Ciara; Weller, Roy O; Verma, Ajay; Carare, Roxana O

2017-11-15

Dilatation of periarteriolar spaces in MRI of the ageing human brains occurs in white matter (WM), basal ganglia and midbrain but not in cerebral cortex. Perivenous collagenous occurs in periventricular but not in subcortical WM.Here we test the hypotheses that (a) the capacity for dilatation of periarteriolar spaces correlates with the anatomical distribution of leptomeningeal cells coating intracerebral arteries and (b) the regional development of perivenous collagenous in the WM correlates with the population of intramural cells in the walls of veins.The anatomical distribution of leptomeningeal and intramural cells related to cerebral blood vessels is best documented by electron microscopy, requiring perfusion-fixed tissue not available in human material. We therefore analysed perfusion-fixed brain from a 12-year-old Beagle dog as the canine brain represents the anatomical arrangement in the human brain. Results showed regional variation in the arrangement of leptomeningeal cells around blood vessels. Arterioles are enveloped by one complete layer of leptomeninges often with a second incomplete layer in the WM. Venules showed incomplete layers of leptomeningeal cells. Intramural cell expression was higher in the post-capillary venules of the subcortical WM when compared with periventricular WM, suggesting that periventricular collagenosis around venules may be due to a lower resistance in the venular walls. It appears that the regional variation in the capacity for dilatation of arteriolar perivascular spaces in the white WM may be related to the number of perivascular leptomeningeal cells surrounding vessels in different areas of the brain. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Technology Solutions Case Study: Cold Climate Foundation Wall Hygrothermal Research Facility, Cloquet, Minnesota

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

None

2014-09-01

This case study describes the University of Minnesota’s Cloquet Residential Research Facility (CRRF) in northern Minnesota, which features more than 2,500 ft2 of below-grade space for building systems foundation hygrothermal research. Here, the NorthernSTAR Building America Partnership team researches ways to improve the energy efficiency of the building envelope, including wall assemblies, basements, roofs, insulation, and air leakage.

Genetic control of T cell responsiveness to the Friend murine leukemia virus envelope antigen. Identification of class II loci of the H-2 as immune response genes

PubMed Central

1988-01-01

T cells primed specifically for the envelope glycoprotein of Friend murine leukemia helper virus (F-MuLV) were prepared by immunizing mice with a recombinant vaccinia virus that expressed the entire env gene of F-MuLV. Significant proliferative responses of F-MuLV envelope- specific, H-2a/b T cells were observed when the T cells were stimulated with antigen-pulsed peritoneal exudate cells (PEC) having the b allele at the K, A beta, A alpha, and E beta loci of the H-2. On the other hand, PEC having only the kappa allele at these loci did not induce the envelope-specific T cell proliferation, even when the PEC had the b allele at the E alpha, S, or D loci. F-MuLV envelope-specific proliferation of H-2a/b T cells under the stimulation of antigen- pulsed, H-2a/b PEC was specifically blocked with anti-I-Ab and anti-I- Ek mAbs but not with anti-Kb, anti-Kk, or anti-I-Ak mAbs. Moreover, (B10.MBR x A/WySn)F1 mice that have the b allele only at the K locus but not in I-A subregion were nonresponders to the envelope glycoprotein, and the bm12 mutation at the A beta locus completely abolished the T cell responsiveness to this antigen. These results indicate that proliferative T cells recognize a limited number of epitopes on F-MuLV envelope protein in the context of I-Ab, hybrid I- Ak/b, and/or hybrid I-Ek/b class II MHC molecules but fail to recognize the same envelope protein in the context of I-Ak or I-Ek molecules. This influence of the H-2I region on T cell recognition of the envelope glycoprotein appeared to control in vivo induction of protective immunity against Friend virus complex after immunization with the vaccinia-F-MuLV env vaccine. Thus, these results provide, for the first time, direct evidence for Ir gene-controlled responder/nonresponder phenotypes influencing the immune response to a pathogenic virus of mice. PMID:3141552

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype

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

Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4{sup +} T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependentmore » phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. - Highlights: • Jurkat T cells expressing the HIV-1 envelope fuse with THP-1 monocytes. • Heterokaryons display a dominant myeloid phenotype and monocyte function. • Heterokaryons exhibit activation features in the absence of activation agents. • Activation is not due to cell-cell interaction but requires cell-cell fusion. • The activated monocyte-like phenotype is mediated by TLR2/TLR4 signaling.« less

Cloning and characterization of human immunodeficiency virus type 1 variants diminished in the ability to induce syncytium-independent cytolysis.

PubMed Central

Stevenson, M; Haggerty, S; Lamonica, C; Mann, A M; Meier, C; Wasiak, A

1990-01-01

The phenomenon of interference was exploited to isolate low-abundance noncytopathic human immunodeficiency virus type 1 (HIV-1) variants from a primary HIV-1 isolate from an asymptomatic HIV-1-seropositive hemophiliac. Successive rounds of virus infection of a cytolysis-susceptible CD4+ cell line and isolation of surviving cells resulted in selective amplification of an HIV-1 variant reduced in the ability to induce cytolysis. The presence of a PvuII polymorphism facilitated subsequent amplification and cloning of cytopathic and noncytopathic HIV-1 variants from the primary isolate. Cloned virus stocks from cytopathic and noncytopathic variants exhibited similar replication kinetics, infectivity, and syncytium induction in susceptible host cells. The noncytopathic HIV-1 variant was unable, however, to induce single-cell killing in susceptible host cells. Construction of viral hybrids in which regions of cytopathic and noncytopathic variants were exchanged indicated that determinants for the noncytopathic phenotype map to the envelope glycoprotein. Sequence analysis of the envelope coding regions indicated the absence of two highly conserved N-linked glycosylation sites in the noncytopathic HIV-1 variant, which accompanied differences in processing of precursor gp160 envelope glycoprotein. These results demonstrate that determinants for syncytium-independent single-cell killing are located within the envelope glycoprotein and suggest that single-cell killing is profoundly influenced by alterations in envelope sequence which affect posttranslational processing of HIV-1 envelope glycoprotein within the infected cell. Images PMID:1695254

NKp44 receptor mediates interaction of the envelope glycoproteins from the West-Nile and dengue viruses with Natural Killer cells

PubMed Central

Hershkovitz, Oren; Rosental, Benyamin; Rosenberg, Lior Ann; Navarro-Sanchez, Martha Erika; Jivov, Sergey; Zilka, Alon; Gershoni-Yahalom, Orly; Brient-Litzler, Elodie; Bedouelle, Hugues; Ho, Joanna W.; Campbell, Kerry S.; Rager-Zisman, Bracha; Despres, Philippe; Porgador, Angel

2009-01-01

Dengue virus (DV) and West Nile virus (WNV) have become a global concern due to their widespread distribution and their ability to cause a variety of human diseases. Antiviral immune defenses involve natural killer (NK) cells. In the present study, we investigated the interaction between NK cells and these two flaviviruses. We show that the NK-activating receptor NKp44 is involved in virally-mediated NK activation through direct interaction with the flavivirus envelope protein. Recombinant NKp44 directly binds to purified DV and WNV envelope proteins and specifically to domain III of WNV envelope protein (EIII); it also binds to WNV virus-like particles (VLPs). These WNV-VLPs and WNV-EIII directly bind NK cells expressing high levels of NKp44. Functionally, interaction of NK cells with infective and inactivated WNV results in NKp44-mediated NK de-granulation. Finally, WNV infection of cells results in increased binding of recombinant NKp44 that is specifically inhibited by anti-WNV serum. WNV-infected target cells induce IFNγ secretion and augmented lysis by NKp44-expressing primary NK cells that are blocked by anti-NKp44 antibodies. Our findings show that triggering of NK cells by flavivirus is mediated by interaction of NKp44 with the flavivirus envelope protein. PMID:19635919

Envelope Structures of Gram-Positive Bacteria

PubMed Central

Rajagopal, Mithila; Walker, Suzanne

2016-01-01

Gram-positive organisms, including the pathogens Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecalis, have dynamic cell envelopes that mediate interactions with the environment and serve as the first line of defense against toxic molecules. Major components of the cell envelope include peptidoglycan, which is a well-established target for antibiotics, teichoic acids, capsular polysaccharides, surface proteins, and phospholipids. These components can undergo modification to promote pathogenesis, decrease susceptibility to antibiotics and host immune defenses, and enhance survival in hostile environments. This chapter will cover the structure, biosynthesis and important functions of major cell envelope components in Gram-positive bacteria. Possible targets for new antimicrobials will be noted. PMID:26919863

Study of an experimental methodology for thermal properties diagnostic of building envelop

NASA Astrophysics Data System (ADS)

Yang, Yingying; Sempy, Alain; Vogt Wu, Tingting; Sommier, Alain; Dumoulin, Jean; Batsale, Jean Christophe

2017-04-01

The building envelope plays a critical role in determining levels of comfort and building efficiency. Its real thermal properties characterization is of major interest to be able to diagnose energy efficiency performance of buildings (new construction and retrofitted existing old building). Research and development on a possible methodology for energy diagnostic of the building envelop is a hot topic and necessary trend. Many kinds of sensors and instruments are used for the studies. The application of infrared (IR) thermography in non-destructive evaluation has been widely employed for qualitative evaluations for building diagnostics; meanwhile, the IR thermography technology also has a large potentiality for the evaluation of the thermal characteristics of the building envelope. Some promising recent research studies have been carried out with such contactless measurement technique. Nevertheless, research efforts are still required for in situ measurements under natural environmental conditions. In order to develop new solutions for non-intrusive evaluation of local thermal performance, enabling quantitative assessment of thermal properties of buildings and materials, experiments were carried out on a multi-layer pratical scale wall fixed on a caisson placed in a climatic chamber. Six halogen lamps (1.5 kW for each lamp) placed in front of objective wall were used to emulate sunny conditions. The radiative heat flux emitted was monitored and modulated with time according to typical weather data set encountered in France. Both steady state and transient regime heat transfer were studied during these experiments. Contact sensors (thermocouples, heat flux meters, Peltier sensors) and non-contact sensors (thermal IR camera, pyranometer) were used to measure the temperatures and heat flux density evolution. It has to be noticed that the Peltier sensors have been tuned and used with a specific processing to set them compliant for heat flux density measurements. The measured data from different sensors were analysed and compared. The emissivity of wall surface and treated sensor surfaces were evaluated by using an IR camera with an adapted post-processing. Then, convective and radiative heat fluxes, at wall level, were estimated. Finally, the wall thermal properties can be calculated by using the measured temperatures and estimated heat fluxes using a dedicated thermal quadrupoles heat transfer model and an inverse method. This study aims at providing some guidelines for the choice of sensors, measurements protocol and adapted inverse model to be tested in real conditions on pilot situ scale. Aknowledgments : The Authors are very grateful to H2020 Built2Spec project for supporting this work.

SENSITIVITY OF THE HOUSE PRESSURE TEST FOR DUCT LEAKAGE TO VARIATIONS IN THE DISTRIBUTION OF AIR LEAKAGE IN THE HOUSE ENVELOPE

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

ANDREWS,J.W.

1998-12-01

The house pressure test for air leakage in ducts calculates the signed difference between the supply and return leakage from the response of the air pressure in the house to operation of the system fan. The currently accepted version of this calculation was based on particular assumptions about how the house envelope leakage is distributed between the walls, ceiling, and floor. This report generalizes the equation to account for an arbitrary distribution of envelope leakage. It concludes that the currently accepted equation is usually accurate to within {+-}5%, but in a small proportion of cases the results may diverge bymore » 50% or more.« less

Contribution of the Pmra Promoter to Expression of Genes in the Escherichia coli mra Cluster of Cell Envelope Biosynthesis and Cell Division Genes

PubMed Central

Mengin-Lecreulx, Dominique; Ayala, Juan; Bouhss, Ahmed; van Heijenoort, Jean; Parquet, Claudine; Hara, Hiroshi

1998-01-01

Recently, a promoter for the essential gene ftsI, which encodes penicillin-binding protein 3 of Escherichia coli, was precisely localized 1.9 kb upstream from this gene, at the beginning of the mra cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802–5811, 1997). Disruption of this promoter (Pmra) on the chromosome and its replacement by the lac promoter (Pmra::Plac) led to isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from Pmra to ftsW, the fifth gene downstream from ftsI, was provided in trans on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which Pmra::Plac promoter expression was repressed or fully induced. It was confirmed that the Pmra promoter is required for expression of the first nine genes of the mra cluster: mraZ (orfC), mraW (orfB), ftsL (mraR), ftsI, murE, murF, mraY, murD, and ftsW. Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced Pmra::Plac cells. This was correlated with an accumulation of the nucleotide precursors UDP–N-acetylglucosamine and UDP–N-acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP–N-acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of ftsZ, the penultimate gene from this cluster, was significantly reduced when Pmra expression was repressed. It was concluded that the transcription of the genes located downstream from ftsW in the mra cluster, from murG to ftsZ, is also mainly (but not exclusively) dependent on the Pmra promoter. PMID:9721276

Automated analysis of cell migration and nuclear envelope rupture in confined environments.

PubMed

Elacqua, Joshua J; McGregor, Alexandra L; Lammerding, Jan

2018-01-01

Recent in vitro and in vivo studies have highlighted the importance of the cell nucleus in governing migration through confined environments. Microfluidic devices that mimic the narrow interstitial spaces of tissues have emerged as important tools to study cellular dynamics during confined migration, including the consequences of nuclear deformation and nuclear envelope rupture. However, while image acquisition can be automated on motorized microscopes, the analysis of the corresponding time-lapse sequences for nuclear transit through the pores and events such as nuclear envelope rupture currently requires manual analysis. In addition to being highly time-consuming, such manual analysis is susceptible to person-to-person variability. Studies that compare large numbers of cell types and conditions therefore require automated image analysis to achieve sufficiently high throughput. Here, we present an automated image analysis program to register microfluidic constrictions and perform image segmentation to detect individual cell nuclei. The MATLAB program tracks nuclear migration over time and records constriction-transit events, transit times, transit success rates, and nuclear envelope rupture. Such automation reduces the time required to analyze migration experiments from weeks to hours, and removes the variability that arises from different human analysts. Comparison with manual analysis confirmed that both constriction transit and nuclear envelope rupture were detected correctly and reliably, and the automated analysis results closely matched a manual analysis gold standard. Applying the program to specific biological examples, we demonstrate its ability to detect differences in nuclear transit time between cells with different levels of the nuclear envelope proteins lamin A/C, which govern nuclear deformability, and to detect an increase in nuclear envelope rupture duration in cells in which CHMP7, a protein involved in nuclear envelope repair, had been depleted. The program thus presents a versatile tool for the study of confined migration and its effect on the cell nucleus.

A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity.

PubMed

York, Joanne; Nunberg, Jack H

2018-01-01

For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

Archaeal viruses at the cell envelope: entry and egress

PubMed Central

Quemin, Emmanuelle R. J.; Quax, Tessa E. F.

2015-01-01

The cell envelope represents the main line of host defense that viruses encounter on their way from one cell to another. The cytoplasmic membrane in general is a physical barrier that needs to be crossed both upon viral entry and exit. Therefore, viruses from the three domains of life employ a wide range of strategies for perforation of the cell membrane, each adapted to the cell surface environment of their host. Here, we review recent insights on entry and egress mechanisms of viruses infecting archaea. Due to the unique nature of the archaeal cell envelope, these particular viruses exhibit novel and unexpected mechanisms to traverse the cellular membrane. PMID:26097469

CD4+ T-cell recovery with suppressive ART-induced rapid sequence evolution in hepatitis C virus envelope but not NS3.

PubMed

Liu, Lin; Nardo, David; Li, Eric; Wang, Gary P

2016-03-13

CD4 T-cell depletion from HIV infection leads to a global decline in anti-hepatitis C virus (HCV) envelope neutralizing antibody (nAb) response, which may play a role in accelerating liver fibrosis. An increase in anti-HCV nAb titers has been reported during antiretroviral therapy (ART) but its impact on HCV remains poorly understood. The objective of this study is to determine the effects of ART on long-term HCV evolution. We examined HCV quasispecies structure and long-term evolution in HIV/HCV coinfected patients with ART-induced CD4 T-cell recovery, and compared with patients with CD4 T-cell depletion from delayed ART. We applied a single-variant sequencing (SVS) method to construct authentic viral quasispecies and compared sequence evolution in HCV envelope, the primary target for humoral immune responses, and NS3, a target for cellular immunity, between the two cohorts. The SVS method corrected biases known to skew the proportions of viral variants, revealing authentic HCV quasispeices structures. We observed higher rates of HCV envelope sequence evolution in patients with ART-induced CD4 T-cell recovery, compared with patients with CD4 T-cell depletion from delayed ART (P = 0.03). Evolutionary rates for NS3 were considerably lower than the rates for envelope (P < 0.01), with no significant difference observed between the two groups. ART-induced CD4 T-cell recovery results in rapid sequence evolution in HCV envelope, but not in NS3. These results suggest that suppressive ART disproportionally enhances HCV-specific humoral responses more than cellular responses, resulting in rapid sequence evolution in HCV envelope but not NS3.

A baculovirus polyhedron envelope protein: immunogold localization in infected cells and mature polyhedra.

PubMed

Russell, R L; Rohrmann, G F

1990-01-01

A polyclonal antiserum against a trpE fusion protein containing the complete open reading frame of the polyhedron envelope (PE) protein from the nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was used for immunogold staining and electron microscopic examination of polyhedra, isolated polyhedron envelopes, and infected insect cells at selected times postinfection. The antiserum specifically stained the peripheral envelope of mature polyhedra and also stained the envelope structure which remained after polyhedra were dissolved in dilute alkaline solutions. In OpMNPV-infected Lymantria dispar cells, the PE protein was detected by 48 hr postinfection (hr p.i.) but specific localization and staining of developing polyhedra were not evident. However, by 72 hr p.i. substantial and preferential staining of the periphery of developing polyhedra was evident even though a distinct polyhedron envelope was not yet observed. In addition, the periphery of fibrillar structures was stained by the PE antiserum. By 96 hr p.i., mature envelopes surrounded polyhedra and these polyhedron envelopes were stained with the PE antibody. The progression of PE protein staining during polyhedron morphogenesis indicates that the PE protein accumulates and becomes associated with developing polyhedra in the nucleus between 48 and 72 hr p.i. Very late in infection the mature polyhedron envelope forms on the polyhedron surface. The apparent affinity of the PE protein for the surface of maturing polyhedra suggests that it may be a major component of the polyhedron envelope or may form the matrix for the deposition of other components which contribute to the mature envelope. Immunogold staining and protease digestion experiments indicate that protein is an essential component of the polyhedron envelope.

The cell envelope proteome of Aggregatibacter actinomycetemcomitans

PubMed Central

Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

2014-01-01

Summary The cell envelope of Gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 28% of the predicted ORFs in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, while others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity. PMID:25055881

The complex chemistry of outflow cavity walls exposed: the case of low-mass protostars

NASA Astrophysics Data System (ADS)

Drozdovskaya, Maria N.; Walsh, Catherine; Visser, Ruud; Harsono, Daniel; van Dishoeck, Ewine F.

2015-08-01

Complex organic molecules are ubiquitous companions of young low-mass protostars. Recent observations suggest that their emission stems, not only from the traditional hot corino, but also from offset positions. In this work, 2D physicochemical modelling of an envelope-cavity system is carried out. Wavelength-dependent radiative transfer calculations are performed and a comprehensive gas-grain chemical network is used to simulate the physical and chemical structure. The morphology of the system delineates three distinct regions: the cavity wall layer with time-dependent and species-variant enhancements; a torus rich in complex organic ices, but not reflected in gas-phase abundances and the remaining outer envelope abundant in simpler solid and gaseous molecules. Strongly irradiated regions, such as the cavity wall layer, are subject to frequent photodissociation in the solid phase. Subsequent recombination of the photoproducts leads to frequent reactive desorption, causing gas-phase enhancements of several orders of magnitude. This mechanism remains to be quantified with laboratory experiments. Direct photodesorption is found to be relatively inefficient. If radicals are not produced directly in the icy mantle, the formation of complex organics is impeded. For efficiency, a sufficient number of FUV photons needs to penetrate the envelope, and elevated cool dust temperatures need to enable grain-surface radical mobility. As a result, a high stellar luminosity and a sufficiently wide cavity favour chemical complexity. Furthermore within this paradigm, complex organics are demonstrated to have unique lifetimes and be grouped into early (formaldehyde, ketene, methanol, formic acid, methyl formate, acetic acid and glycolaldehyde) and late (acetaldehyde, dimethyl ether and ethanol) species.

Study of potential nonconformities of a new recreation center building's envelope

NASA Astrophysics Data System (ADS)

Stanescu, M.; Kajl, S.; Lamarche, L.

2016-09-01

This article presents a building envelope's analysis in order to verify the compliance with mandatory provisions of the Model National Energy Code for Buildings in Canada (MNECB 1997). Because some of the requirements are «not met», investigations were carried out to provide justifications in order to prove that the building can be considered as an exception to the mandatory provisions of MNECB. Therefore, we evaluate the impact of three (3) potential nonconformities of the building's walls on the building energy performance. In regards to article 3.1.1.1.4 of MNECB, there is an exception if it can be proved that permanent process (like heat recovery of refrigeration compressors) can produce at all times enough heat that no other heating source is required. First of all, by using simulation, we were able to indicate that almost all building's heating will be provided by energy recovery from ice rinks refrigeration systems (99.2%). Secondly, by using an energy analysis carried out with HEAT2 software, we can show that the increase of heating energy demand caused by the 3 studied walls is very low. This represents an increase of the heating energy demand of only 0.2%, and this, regardless of the heat recovery process. Because the nonconforming wall sections are small (0.97% of the envelope area), this mainly explains the minor impact in terms of building performance. In conclusion, according to the results obtained, we were able to recommend the building for consideration as an exception to the mandatory provisions of MNECB.

A novel intermediate in processing of murine leukemia virus envelope glycoproteins. Proteolytic cleavage in the late Golgi region.

PubMed

Bedgood, R M; Stallcup, M R

1992-04-05

The intracellular processing of the murine leukemia virus envelope glycoprotein precursor Pr85 to the mature products gp70 and p15e was analyzed in the mouse T-lymphoma cell line W7MG1. Kinetic (pulse-chase) analysis of synthesis and processing, coupled with endoglycosidase (endo H) and neuraminidase digestions revealed the existence of a novel high molecular weight processing intermediate, gp95, containing endo H-resistant terminally glycosylated oligosaccharide chains. In contrast to previously published conclusions, our data indicate that proteolytic cleavage of the envelope precursor occurs after the acquisition of endo H-resistant chains and terminal glycosylation and thus after the mannosidase II step. In the same W7MG1 cell line, the type and order of murine leukemia virus envelope protein processing events was identical to that for the mouse mammary tumor virus envelope protein. Interestingly, complete mouse mammary tumor virus envelope protein processing requires the addition of glucocorticoid hormone, whereas murine leukemia virus envelope protein processing occurs constitutively in these W7MG1 cells. We propose that all retroviral envelope proteins share a common processing pathway in which proteolytic processing is a late event that follows acquisition of endo H resistance and terminal glycosylation.

Mutations altering the gammaretrovirus endoproteolytic motif affect glycosylation of the envelope glycoprotein and early events of the virus life cycle

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

Argaw, Takele; Wilson, Carolyn A., E-mail: carolyn.wilson@fda.hhs.gov

Previously, we found that mutation of glutamine to proline in the endoproteolytic cleavage signal of the PERV-C envelope (RQKK to RPKK) resulted in non-infectious vectors. Here, we show that RPKK results in a non-infectious vector when placed in not only a PERV envelope, but also the envelope of a related gammaretrovirus, FeLV-B. The amino acid substitutions do not prevent envelope precursor cleavage, viral core and genome assembly, or receptor binding. Rather, the mutations result in the formation of hyperglycosylated glycoprotein and a reduction in the reverse transcribed minus strand synthesis and undetectable 2-LTR circular DNA in cells exposed to vectorsmore » with these mutated envelopes. Our findings suggest novel functions associated with the cleavage signal sequence that may affect trafficking through the glycosylation machinery of the cell. Further, the glycosylation status of the envelope appears to impact post-binding events of the viral life cycle, either membrane fusion, internalization, or reverse transcription. - Highlights: • Env cleavage signal impacts infectivity of gammaretroviruses. • Non-infectious mutants have hyper-glycosylated envelope that bind target cells. • Non-infectious mutants have defects in the formation of the double-stranded DNA. • Env cleavage motif has functions beyond cleavage of the env precursor.« less

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype.

PubMed

Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique; Licona-Limón, Ileana; Huerta, Leonor

2017-03-01

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4 + T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation of Envelope-Modified Baculoviruses for Gene Delivery into Mammalian Cells.

PubMed

Hofmann, Christian

2016-01-01

Genetically modified baculoviruses can efficiently deliver and express genes in mammalian cells. The major prerequisite for the expression of a gene transferred by baculovirus is its control by a promoter that is active in mammalian cells. This chapter describes methods for producing second generation baculovirus vectors through modification of their envelope. Envelope modified baculoviruses offer additional new applications of the system, such as their use in in vivo gene delivery, targeting, and vaccination. Methods of generating a recombinant baculovirus vector with a modified envelope and its amplification and purification, including technical scale production, are discussed. A variety of notes give clues regarding specific technical procedures. Finally, methods to analyze the virus and transduction procedures are presented.

Fractal avalanche ruptures in biological membranes

NASA Astrophysics Data System (ADS)

Gözen, Irep; Dommersnes, Paul; Czolkos, Ilja; Jesorka, Aldo; Lobovkina, Tatsiana; Orwar, Owe

2010-11-01

Bilayer membranes envelope cells as well as organelles, and constitute the most ubiquitous biological material found in all branches of the phylogenetic tree. Cell membrane rupture is an important biological process, and substantial rupture rates are found in skeletal and cardiac muscle cells under a mechanical load. Rupture can also be induced by processes such as cell death, and active cell membrane repair mechanisms are essential to preserve cell integrity. Pore formation in cell membranes is also at the heart of many biomedical applications such as in drug, gene and short interfering RNA delivery. Membrane rupture dynamics has been studied in bilayer vesicles under tensile stress, which consistently produce circular pores. We observed very different rupture mechanics in bilayer membranes spreading on solid supports: in one instance fingering instabilities were seen resulting in floral-like pores and in another, the rupture proceeded in a series of rapid avalanches causing fractal membrane fragmentation. The intermittent character of rupture evolution and the broad distribution in avalanche sizes is consistent with crackling-noise dynamics. Such noisy dynamics appear in fracture of solid disordered materials, in dislocation avalanches in plastic deformations and domain wall magnetization avalanches. We also observed similar fractal rupture mechanics in spreading cell membranes.

State transitions of actin cortices in vitro and in vivo

NASA Astrophysics Data System (ADS)

Tan, Tzer Han; Keren, Kinneret; Mackintosh, Fred; Schmidt, Christoph; Fakhri, Nikta

Most animal cells are enveloped by a thin layer of actin cortex which governs the cell mechanics. A functional cortex must be rigid to provide mechanical support while being flexible to allow for rapid restructuring events such as cell division. To satisfy these requirements, the actin cortex is highly dynamic with fast actin turnover and myosin-driven contractility. The regulatory mechanism responsible for the transition between a mechanically stable state and a restructuring state is not well understood. Here, we develop a technique to map the dynamics of reconstituted actin cortices in emulsion droplets using IR fluorescent single-walled carbon nanotubes (SWNTs). By increasing crosslinker concentration, we find that a homogeneous cortex transitions to an intermediate state with broken rotational symmetry and a globally contractile state which further breaks translational symmetry. We apply this new dynamic mapping technique to cortices of live starfish oocytes in various developmental stages. To identify the regulatory mechanism for steady state transitions, we subject the oocytes to actin and myosin disrupting drugs.

Mycolic acids: deciphering and targeting the Achilles' heel of the tubercle bacillus

PubMed Central

Nataraj, Vijayashankar; Varela, Cristian; Javid, Asma; Singh, Albel; Besra, Gurdyal S.

2015-01-01

Summary Mycolic acids are unique long chain fatty acids found in the lipid‐rich cell walls of mycobacteria including the tubercle bacillus M ycobacterium tuberculosis. Essential for viability and virulence, enzymes involved in the biosynthesis of mycolic acids represent novel targets for drug development. This is particularly relevant to the impact on global health given the rise of multidrug resistant and extensively drug resistant strains of M . tuberculosis. In this review, we discuss recent advances in our understanding of how mycolic acid are synthesised, especially the potential role of specialised fatty acid synthase complexes. Also, we examine the role of a recently reported mycolic acid transporter MmpL3 with reference to several reports of the targeting of this transporter by diverse compounds with anti‐M . tuberculosis activity. Additionally, we consider recent findings that place mycolic acid biosynthesis in the context of the cell biology of the bacterium, viz its localisation and co‐ordination with the bacterial cytoskeleton, and its role beyond maintaining cell envelope integrity. PMID:26135034

Genetic Interaction Maps in Escherichia coli Reveal Functional Crosstalk among Cell Envelope Biogenesis Pathways

PubMed Central

Vlasblom, James; Gagarinova, Alla; Phanse, Sadhna; Graham, Chris; Yousif, Fouad; Ding, Huiming; Xiong, Xuejian; Nazarians-Armavil, Anaies; Alamgir, Md; Ali, Mehrab; Pogoutse, Oxana; Pe'er, Asaf; Arnold, Roland; Michaut, Magali; Parkinson, John; Golshani, Ashkan; Whitfield, Chris; Wodak, Shoshana J.; Moreno-Hagelsieb, Gabriel; Greenblatt, Jack F.; Emili, Andrew

2011-01-01

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among >235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target. PMID:22125496

Genetic interaction maps in Escherichia coli reveal functional crosstalk among cell envelope biogenesis pathways.

PubMed

Babu, Mohan; Díaz-Mejía, J Javier; Vlasblom, James; Gagarinova, Alla; Phanse, Sadhna; Graham, Chris; Yousif, Fouad; Ding, Huiming; Xiong, Xuejian; Nazarians-Armavil, Anaies; Alamgir, Md; Ali, Mehrab; Pogoutse, Oxana; Pe'er, Asaf; Arnold, Roland; Michaut, Magali; Parkinson, John; Golshani, Ashkan; Whitfield, Chris; Wodak, Shoshana J; Moreno-Hagelsieb, Gabriel; Greenblatt, Jack F; Emili, Andrew

2011-11-01

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target.

Role of the nuclear envelope in the pathogenesis of age-related bone loss and osteoporosis

PubMed Central

Vidal, Christopher; Bermeo, Sandra; Fatkin, Diane; Duque, Gustavo

2012-01-01

The nuclear envelope is the most important border in the eukaryotic cell. The role of the nuclear envelope in cell differentiation and function is determined by a constant interaction between the elements of the nuclear envelope and the transcriptional regulators involved in signal transcription pathways. Among those components of the nuclear envelope, there is a growing evidence that changes in the expression of A-type lamins, which are essential components of the nuclear lamina, are associated with age-related changes in bone affecting the capacity of differentiation of mesenchymal stem cells into osteoblasts, favoring adipogenesis and affecting the function and survival of the osteocytes. Overall, as A-type lamins are considered as the 'guardians of the soma', these proteins are also essential for the integrity and quality of the bone and pivotal for the longevity of the musculoskeletal system. PMID:23951459

Geometric and Kinematic Structure of the Outflow/Envelope System of L1527 Revealed by Subarcsecond-resolution Observation of CS

NASA Astrophysics Data System (ADS)

Oya, Yoko; Sakai, Nami; Lefloch, Bertrand; López-Sepulcre, Ana; Watanabe, Yoshimasa; Ceccarelli, Cecilia; Yamamoto, Satoshi

2015-10-01

Subarcsecond-resolution images of the rotational line emissions of CS and c-C3H2 obtained toward the low-mass protostar IRAS 04368+2557 in L1527 with the Atacama Large Millimeter/submillimeter Array are investigated to constrain the orientation of the outflow/envelope system. The distribution of CS consists of an envelope component extending from north to south and a faint butterfly shaped outflow component. The kinematic structure of the envelope is well reproduced by a simple ballistic model of an infalling rotating envelope. Although the envelope has a nearly edge-on configuration, we find that the western side of the envelope faces the observer. This configuration is opposite to the direction of the large-scale (˜104 AU) outflow suggested previously from the 12CO (J = 3-2) observation, and to the morphology of infrared reflection near the protostar (˜200 AU). The latter discrepancy could originate from high extinction by the outflow cavity of the western side, or may indicate that the outflow axis is not parallel to the rotation axis of the envelope. Position-velocity diagrams show the accelerated outflow cavity wall, and its kinematic structure in the 2000 AU scale is explained by a standard parabolic model with the inclination angle derived from the analysis of the envelope. The different orientation of the outflow between the small and large scale implies a possibility of precession of the outflow axis. The shape and the velocity of the outflow in the vicinity of the protostar are compared with those of other protostars.

[Cell entry mechanisms of coronaviruses].

PubMed

Taguchi, Fumihiro; Matsuyama, Shutoku

2009-12-01

Enveloped viruses enter into cells via fusion of their envelope and cellular membrane. Spike (S) protein of coronavirus (CoV) is responsible for entry events. We studied the cell entry mechanisms of two different CoVs, murine coronavirus mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV). MHV-JHM that induces syncytia in infected cells entered directly from cell surface, i.e., fusion of envelope and plasma membrane, whereas SARS-CoV and MHV-2 that fail to induce syncytia entered via endosome in a protease-dependent fashion, i.e., fusion of envelope and endosomal membrane. The latter viruses entered directly from cell surface, when receptor-bound viruses were treated with proteases that activate fusion activity of their S proteins. The entry pathway of SARS-CoV could influence the severity of the disease. It was also reveled that a highly neurovirulent JHM spread in a receptor-independent fashion, which could result in a high neuropathogenicity of the virus.

Detecting cell death with optical coherence tomography and envelope statistics

NASA Astrophysics Data System (ADS)

Farhat, Golnaz; Yang, Victor X. D.; Czarnota, Gregory J.; Kolios, Michael C.

2011-02-01

Currently no standard clinical or preclinical noninvasive method exists to monitor cell death based on morphological changes at the cellular level. In our past work we have demonstrated that quantitative high frequency ultrasound imaging can detect cell death in vitro and in vivo. In this study we apply quantitative methods previously used with high frequency ultrasound to optical coherence tomography (OCT) to detect cell death. The ultimate goal of this work is to use these methods for optically-based clinical and preclinical cancer treatment monitoring. Optical coherence tomography data were acquired from acute myeloid leukemia cells undergoing three modes of cell death. Significant increases in integrated backscatter were observed for cells undergoing apoptosis and mitotic arrest, while necrotic cells induced a decrease. These changes appear to be linked to structural changes observed in histology obtained from the cell samples. Signal envelope statistics were analyzed from fittings of the generalized gamma distribution to histograms of envelope intensities. The parameters from this distribution demonstrated sensitivities to morphological changes in the cell samples. These results indicate that OCT integrated backscatter and first order envelope statistics can be used to detect and potentially differentiate between modes of cell death in vitro.

Thermionic converter

DOEpatents

Fitzpatrick, Gary O.

1987-05-19

A thermionic converter (10) is set forth which includes an envelope (12) having an electron collector structure (22) attached adjacent to a wall (16). An electron emitter structure (24) is positioned adjacent the collector structure (22) and spaced apart from opposite wall (14). The emitter (24) and collector (22) structures are in a common chamber (20). The emitter structure (24) is heated substantially only by thermal radiation. Very small interelectrode gaps (28) can be maintained utilizing the thermionic converter (10) whereby increased efficiency results.

Dissecting Escherichia coli Outer Membrane Biogenesis Using Differential Proteomics

PubMed Central

Martorana, Alessandra M.; Motta, Sara; Di Silvestre, Dario; Falchi, Federica; Dehò, Gianni; Mauri, Pierluigi; Sperandeo, Paola; Polissi, Alessandra

2014-01-01

The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality. PMID:24967819

Synthesis and assembly of Hepatitis B virus envelope protein-derived particles in Escherichia coli.

PubMed

Li, Hao; Onbe, Keisuke; Liu, Qiushi; Iijima, Masumi; Tatematsu, Kenji; Seno, Masaharu; Tada, Hiroko; Kuroda, Shun' Ichi

2017-08-19

Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nanocarrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The ΔNC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ΔNC mutant was optimized by the N-end rule for E. coli expression, the modified ΔNC mutant (mΔNC) was efficiently expressed as particles in E. coli. The molecular mass of mΔNC particle was approx. 670 kDa, and the diameter was 28.5 ± 6.2 nm (mean ± SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mΔNC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Imaging mycobacterial growth and division with a fluorogenic probe.

PubMed

Hodges, Heather L; Brown, Robert A; Crooks, John A; Weibel, Douglas B; Kiessling, Laura L

2018-05-15

Control and manipulation of bacterial populations requires an understanding of the factors that govern growth, division, and antibiotic action. Fluorescent and chemically reactive small molecule probes of cell envelope components can visualize these processes and advance our knowledge of cell envelope biosynthesis (e.g., peptidoglycan production). Still, fundamental gaps remain in our understanding of the spatial and temporal dynamics of cell envelope assembly. Previously described reporters require steps that limit their use to static imaging. Probes that can be used for real-time imaging would advance our understanding of cell envelope construction. To this end, we synthesized a fluorogenic probe that enables continuous live cell imaging in mycobacteria and related genera. This probe reports on the mycolyltransferases that assemble the mycolic acid membrane. This peptidoglycan-anchored bilayer-like assembly functions to protect these cells from antibiotics and host defenses. Our probe, quencher-trehalose-fluorophore (QTF), is an analog of the natural mycolyltransferase substrate. Mycolyltransferases process QTF by diverting their normal transesterification activity to hydrolysis, a process that unleashes fluorescence. QTF enables high contrast continuous imaging and the visualization of mycolyltransferase activity in cells. QTF revealed that mycolyltransferase activity is augmented before cell division and localized to the septa and cell poles, especially at the old pole. This observed localization suggests that mycolyltransferases are components of extracellular cell envelope assemblies, in analogy to the intracellular divisomes and polar elongation complexes. We anticipate QTF can be exploited to detect and monitor mycobacteria in physiologically relevant environments.

Systematic approach to in-depth understanding of photoelectrocatalytic bacterial inactivation mechanisms by tracking the decomposed building blocks.

PubMed

Sun, Hongwei; Li, Guiying; Nie, Xin; Shi, Huixian; Wong, Po-Keung; Zhao, Huijun; An, Taicheng

2014-08-19

A systematic approach was developed to understand, in-depth, the mechanisms involved during the inactivation of bacterial cells using photoelectrocatalytic (PEC) processes with Escherichia coli K-12 as the model microorganism. The bacterial cells were found to be inactivated and decomposed primarily due to attack from photogenerated H2O2. Extracellular reactive oxygen species (ROSs), such as H2O2, may penetrate into the bacterial cell and cause dramatically elevated intracellular ROSs levels, which would overwhelm the antioxidative capacity of bacterial protective enzymes such as superoxide dismutase and catalase. The activities of these two enzymes were found to decrease due to the ROSs attacks during PEC inactivation. Bacterial cell wall damage was then observed, including loss of cell membrane integrity and increased permeability, followed by the decomposition of cell envelope (demonstrated by scanning electronic microscope images). One of the bacterial building blocks, protein, was found to be oxidatively damaged due to the ROSs attacks, as well. Leakage of cytoplasm and biomolecules (bacterial building blocks such as proteins and nucleic acids) were evident during prolonged PEC inactivation process. The leaked cytoplasmic substances and cell debris could be further degraded and, ultimately, mineralized with prolonged PEC treatment.

Ultrastructure and Pathology of Microsporidium phytoseiuli n. sp. Infecting the Predatory Mite, Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae)

PubMed

Bjørnson; Steiner; Keddie

1996-11-01

Ultrastructure and pathology of Microsporidium phytoseiuli n. sp. infecting the predatory mite Phytoseiulus persimilis Athias-Henriot is described using light and transmission electron microscopy. Infected mites showed no gross, external symptoms. All observed stages of the parasite had unpaired nuclei. Schizonts were commonly observed within nuclei of digestive cells of the ventriculus and within the cytoplasm of cells lining the cecal wall and in muscle tissue underlying it. Sporoblasts and spores occurred in the nuclei and cytoplasm of digestive cells within the ventriculus, in cortical regions of the sub- and supraesophageal ganglia, within the cecal wall and muscle tissue, and in parenchyma cells underlying the cuticle. Mature spores were also observed in developing eggs within gravid females. These were broad- to elongate-ovoid, measured 4.33 ± 0.35 x 1.27 ± 0.15 &mgr;m (electron micrographs), 5.37 ± 0.46 x 2.22 ± 0.17 &mgr;m (fixed and stained), and 5.88 ± 0.34 x 2.22 ± 0.19 &mgr;m (fresh) and had an isolfilar polar filament coiled 12 to 15 times within the posterior two-thirds. Within cells, individual spores appeared to be in direct contact with host cytoplasm, while groups of spores were infrequently observed within interfacial envelopes. Groups of 4, 8, to more than 16 spores were observed by light microscopy, while 8 was the maximum observed by electron microscopy. No spores were observed in Tetranychus urticae, a mite used as food during this study.

Gravitational waves from vacuum first-order phase transitions: From the envelope to the lattice

NASA Astrophysics Data System (ADS)

Cutting, Daniel; Hindmarsh, Mark; Weir, David J.

2018-06-01

We conduct large scale numerical simulations of gravitational wave production at a first-order vacuum phase transition. We find a power law for the gravitational wave power spectrum at high wave number which falls off as k-1.5 rather than the k-1 produced by the envelope approximation. The peak of the power spectrum is shifted to slightly lower wave numbers from that of the envelope approximation. The envelope approximation reproduces our results for the peak power less well, agreeing only to within an order of magnitude. After the bubbles finish colliding, the scalar field oscillates around the true vacuum. An additional feature is produced in the UV of the gravitational wave power spectrum, and this continues to grow linearly until the end of our simulation. The additional feature peaks at a length scale close to the bubble wall thickness and is shown to have a negligible contribution to the energy in gravitational waves, providing the scalar field mass is much smaller than the Planck mass.

Changes in the position and volume of inactive X chromosomes during the G0/G1 transition.

PubMed

Lyu, Guoliang; Tan, Tan; Guan, Yiting; Sun, Lei; Liang, Qianjin; Tao, Wei

2018-04-21

In female mammals, each cell silences one X chromosome by converting it into transcriptionally inert heterochromatin. The inactivation is concomitant with epigenetic changes including methylation of specific histone residues and incorporation of macroH2A. Such epigenetic changes may exert influence on the positioning of the inactive X chromosome (Xi) within the nucleus beyond the level of chromatin structure. However, the dynamic positioning of the inactive X chromosome during cell cycle remains unclear. Here, we show that H3K27me3 is a cell-cycle-independent marker for the inactivated X chromosomes in WI38 cells. By utilizing this marker, three types of Xi locations in the nuclei are classified, which are envelope position (associated with envelope), mid-position (between the envelope and nucleolus), and nucleolus position (associated with the nucleolus). Moreover, serial-section analysis revealed that the inactive X chromosomes in the mid-position appear to be sparser and less condensed than those associated with the nuclear envelope or nucleolus. During the transition from G0 to G1 phase, the inactive X chromosomes tend to move from the envelope position to the nucleolus position in WI38 cells. Our results imply a role of chromosome positioning in maintaining the organization of the inactive X chromosomes in different cell phases.

Single-round selection yields a unique retroviral envelope utilizing GPR172A as its host receptor.

PubMed

Mazari, Peter M; Linder-Basso, Daniela; Sarangi, Anindita; Chang, Yehchung; Roth, Monica J

2009-04-07

The recognition by a viral envelope of its cognate host-cell receptor is the initial critical step in defining the viral host-range and tissue specificity. This study combines a single-round of selection of a random envelope library with a parallel cDNA screen for receptor function to identify a distinct retroviral envelope/receptor pair. The 11-aa targeting domain of the modified feline leukemia virus envelope consists of a constrained peptide. Critical to the binding of the constrained peptide envelope to its cellular receptor are a pair of internal cysteines and an essential Trp required for maintenance of titers >10(5) lacZ staining units per milliliter. The receptor used for viral entry is the human GPR172A protein, a G-protein-coupled receptor isolated from osteosarcoma cells. The ability to generate unique envelopes capable of using tissue- or disease-specific receptors marks an advance in the development of efficient gene-therapy vectors.

Role of phosphatidylserine receptors in enveloped virus infection.

PubMed

Morizono, Kouki; Chen, Irvin S Y

2014-04-01

We recently demonstrated that a soluble protein, Gas6, can facilitate viral entry by bridging viral envelope phosphatidylserine to Axl, a receptor tyrosine kinase expressed on target cells. The interaction between phosphatidylserine, Gas6, and Axl was originally shown to be a molecular mechanism through which phagocytes recognize phosphatidylserine exposed on dead cells. Since our initial report, several groups have confirmed that Axl/Gas6, as well as other phosphatidylserine receptors, facilitate entry of dengue, West Nile, and Ebola viruses. Virus binding by viral envelope phosphatidylserine is now a viral entry mechanism generalized to many families of viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most of the known human phosphatidylserine-recognizing molecules, including MFG-E8, TIM-1, -3, and -4, CD300a, BAI1, and stabilin-1 and -2, for their abilities to facilitate virus binding and infection. Using pseudotyped lentiviral vectors, we found that a soluble phosphatidylserine-binding protein, MFG-E8, enhances transduction. Cell surface receptors TIM-1 and -4 also enhance virus binding/transduction. The extent of enhancement by these molecules varies, depending on the type of pseudotyping envelope proteins. Mutated MFG-E8, which binds viral envelope phosphatidylserine without bridging virus to cells, but, surprisingly, not annexin V, which has been used to block phagocytosis of dead cells by concealing phosphatidylserine, efficiently blocks these phosphatidylserine-dependent viral entry mechanisms. These results provide insight into understanding the role of viral envelope phosphatidylserine in viral infection. Envelope phosphatidylserine has previously been shown to be important for replication of various envelope viruses, but details of this mechanism(s) were unclear. We were the first to report that a bifunctional serum protein, Gas6, bridges envelope phosphatidylserine to a cell surface receptor, Axl. Recent studies demonstrated that many envelope viruses, including vaccinia, dengue, West Nile, and Ebola viruses, utilize Axl/Gas6 to facilitate their entry, suggesting that the phosphatidylserine-mediated viral entry mechanism can be shared by various enveloped viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most human phosphatidylserine-recognizing molecules for their abilities to facilitate viral infection. The results provide insights into the role(s) of envelope phosphatidylserine in viral infection, which can be applicable to the development of novel antiviral reagents that block phosphatidylserine-mediated viral entry.

Identification of an Envelope Protein from the FRD Family of Human Endogenous Retroviruses (HERV-FRD) Conferring Infectivity and Functional Conservation among Simians

PubMed Central

Blaise, Sandra; Ruggieri, Alessia; Dewannieux, Marie; Cosset, François-Loic; Heidmann, Thierry

2004-01-01

A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians—from New World monkeys to humans—are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation—located in the TM subunit—was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution. PMID:14694139

Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians.

PubMed

Blaise, Sandra; Ruggieri, Alessia; Dewannieux, Marie; Cosset, François-Loic; Heidmann, Thierry

2004-01-01

A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians-from New World monkeys to humans-are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation-located in the TM subunit-was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution.

Theoretical prediction of gold vein location in deposits originated by a wall magma intrusion

NASA Astrophysics Data System (ADS)

Martin, Pablo; Maass-Artigas, Fernando; Cortés-Vega, Luis

2016-05-01

The isotherm time-evolution resulting from the intrusion of a hot dike in a cold rock is analized considering the general case of nonvertical walls. This is applied to the theoretical prediction of the gold veins location due to isothermal evolution. As in previous treatments earth surface effects are considered and the gold veins are determined by the envelope of the isotherms. The locations of the gold veins in the Callao mines of Venezuela are now well predicted. The new treatment is now more elaborated and complex that in the case of vertical walls, performed in previous papers, but it is more adequated to the real cases as the one in El Callao, where the wall is not vertical.

Electromagnetic fields backscattered from an s-shaped inlet cavity with an absorber coating on its inner walls

NASA Technical Reports Server (NTRS)

Burkholder, R. J.; Chuang, C. W.; Pathak, P. H.

1987-01-01

The EM backscatter from a two-dimensional S-shaped inlet cavity is analyzed using three different techniques, namely a hybrid combination of asymptotic high frequency and modal methods, an integral equation method, and the geometrical optics ray method, respectively. This inlet has a thin absorber coating on its perfectly conducting inner walls and the planar interior termination is made perfectly conducting. The effect of the absorber on the inner wall is treated via a perturbation scheme in the hybrid approach where it is assumed that the loss is sufficiently small for the method to be valid. The results are compared with the backscatter from a straight inlet cavity to evaluate the effect of offsetting the termination in the S-bend configuration such that it is not visible from the open end of the inlet. The envelope of the backscatter pattern for the straight inlet is always seen to peak around the forward axis due to the large return from the directly visible termination, and the pattern envelope tapers off away from the forward axis. Offsetting the termination causes the envelope of the backscatter pattern to flatten out, thereby reducing the return near the forward axis by several dB. The absorber coating reduces the pattern level of the straight inlet in directions away from the forward axis but has little effect on the peak near the axis; furthermore, the absorber coating is seen to consistently reduce the backscatter from the S-bend inlet for almost all incidence angles. The hybrid method gives excellent agreement with experimental data and with the integral equation solution, whereas, the geometrical optics ray tracing method is able to generally predict the average of the bachscatter pattern but not the pattern details.

Optimization for energy efficiency of underground building envelope thermal performance in different climate zones of China

NASA Astrophysics Data System (ADS)

Shi, Luyang; Liu, Jing; Zhang, Huibo

2017-11-01

The object of this article is to investigate the influence of thermal performance of envelopes in shallow-buried buildings on energy consumption for different climate zones of China. For the purpose of this study, an effective building energy simulation tool (DeST) developed by Tsinghua University was chosen to model the heat transfer in underground buildings. Based on the simulative results, energy consumption for heating and cooling for the whole year was obtained. The results showed that the relationship between energy consumption and U-value of envelopes for underground buildings is different compared with above-ground buildings: improving thermal performance of exterior walls cannot reduce energy consumption, on the contrary, may result in more energy cost. Besides, it is can be derived that optimized U-values of underground building envelopes vary with climate zones of China in this study. For severe cold climate zone, the optimized U-value of underground building envelopes is 0.8W/(m2·K); for cold climate zone, the optimized U-value is 1.5W/(m2·K); for warm climate zone, the U-value is 2.0W/(m2·K).

Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

PubMed

Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

2016-03-10

In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy.

PubMed

Tsai, Y-S; Shiau, A-L; Chen, Y-F; Tsai, H-T; Tzai, T-S; Wu, C-L

2010-01-01

The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells.

PubMed

Humbert, Olivier; Gisch, Don W; Wohlfahrt, Martin E; Adams, Amie B; Greenberg, Phil D; Schmitt, Tom M; Trobridge, Grant D; Kiem, Hans-Peter

2016-08-01

Lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) have demonstrated great promise in gene therapy trials employing hematopoietic stem cell and T-cells. The VSV-G envelope confers broad tropism and stability to the vector but is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. We previously showed that cocal pseudotyped LVs offer an excellent alternative to VSV-G vectors because of their broad tropism and resistance to human serum inactivation. In this study, we demonstrate that cocal LVs transduce CD34(+) and CD4(+) T-cells more efficiently than VSV-G LVs and share the same receptor(s) for cell entry. 293T-cells stably expressing the cocal envelope produced significantly higher LV titers than VSV-G expressing cells. We developed cocal pseudotyped, third-generation, self-inactivating LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T-cell receptor, which achieved concentrated titers above 10(8) IU/ml and were successfully adapted for growth in suspension, serum-free culture. The resulting LVs were at least as effective as standard LVs in transducing CD34(+) and CD4(+) T-cells. Our stable cocal LV producer cell lines should facilitate the production of large-scale, high titer clinical grade vectors.

Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium.

PubMed Central

Pace, J; Chai, T J

1989-01-01

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water. Images PMID:2782869

A common pathway for p10 and calyx proteins in progressive stages of polyhedron envelope assembly in AcMNPV-infected Spodoptera frugiperda larvae.

PubMed

Lee, S Y; Poloumienko, A; Belfry, S; Qu, X; Chen, W; MacAfee, N; Morin, B; Lucarotti, C; Krause, M

1996-01-01

The assembly of the polyhedron envelope in baculovirus-infected cells has been the subject of several studies, yet it is still poorly understood. We have used immunogold-labelled antibodies to two baculovirus proteins, p10 and calyx (also referred to as polyhedron envelope protein or PEP), to follow envelope assembly in AcMNPV-infected tissues of Spodoptera frugiperda larvae. We show that, in wild type virus, both proteins colocalize in fibrillar structures and associated electron-dense spacers which progress to encircle the polyhedra, as well as in completed polyhedron envelopes. In cells infected with polyhedrin-negative (PH-) viruses, an unusual proliferation of these spacers was observed suggesting a deregulatory event in the envelope assembly process. Results of Northern and Western blot analysis revealed that synthesis of P10 and calyx mRNA and proteins in PH- AcMNPV is unaffected as compared to wild type virus. Taken together, the observed physical and compositional connection between fibrillar structures, spacers and polyhedron envelopes, as well as the abnormal appearance of the spacers in PH- mutants, provide further evidence in support of a cooperative role of these structures in the assembly of the polyhedron envelope.

The nuclear envelope from basic biology to therapy.

PubMed

Worman, Howard J; Foisner, Roland

2010-02-01

The nuclear envelope has long been a focus of basic research for a highly specialized group of cell biologists. More recently, an expanding group of scientists and physicians have developed a keen interest in the nuclear envelope since mutations in the genes encoding lamins and associated proteins have been shown to cause a diverse range of human diseases often called laminopathies or nuclear envelopathies. Most of these diseases have tissue-selective phenotypes, suggesting that the nuclear envelope must function in cell-type- and developmental-stage-specific processes such as chromatin organization, regulation of gene expression, controlled nucleocytoplasmic transport and response to stress in metazoans. On 22-23 April 2009, Professor Christopher Hutchison organized the 4th British Nuclear Envelope Disease and Chromatin Organization meeting at the College of St Hild and St Bede at Durham University, sponsored by the Biochemical Society. In attendance were investigators with one common interest, the nuclear envelope, but with diverse expertise and training in animal and plant cell biology, genetics, developmental biology and medicine. We were each honoured to be keynote speakers. This issue of Biochemical Society Transactions contains papers written by some of the presenters at this scientifically exciting meeting, held in a bucolic setting where the food was tasty and the wine flowed freely. Perhaps at the end of this excellent meeting more questions were raised than answered, which will stimulate future research. However, what became clear is that the nuclear envelope is a cellular structure with critical functions in addition to its traditional role as a barrier separating the nuclear and cytoplasmic compartments in interphase eukaryotic cells.

Bacillus megaterium sporal peptidoglycan synthesis studied by high-resolution autoradiography.

PubMed

Frehel, C; Ryter, A

1980-11-01

Cells of a Dap- Lys- mutant strain of Bacillus megaterium were pulse labeled with [3H]diaminopimelic acid at different times of growth and sporulation. They were processed for radioactivity measurements and high-resolution autoradiography either just after the pulse or after a chase in a nonradioactive medium until refractile forespores started to appear at time (t)4,5. In the pulse-labeled cells, autoradiographs and radioactivity measurements showed that the radioactivity incorporated during a pulse decreased abruptly after t0 and stayed at a low level until t5, although the forespore wall and cortex were formed between t4 and t5. In the pulse-chased bacteria, the acid-insoluble radioactivity, as well as the number of silver grains on autoradiographs, increased during the chase in cells labeled at t1 to t2, whereas it decreased in those labeled before t0. Furthermore, analysis of silver grain distribution showed that, in stage IV bacteria, grains were distributed at the outside of the forespore, mostly on the sporangium cell wall, when pulse-labeling occurred before or at t0; they were located along the cortex and in the forespore cytoplasm when labeling was made at t1 or t2. These facts show that [3H]diaminopimelic acid necessary for spore envelope synthesis was incorporated before their morphological appearance. Free or small diaminopimelic acid precursors entered the sporangium between t1 and t2. The appearance of silver grains in the forespore cytoplasm suggests that the forespore is implicated in sporal peptidoglycan synthesis.

Suberin: the biopolyester at the frontier of plants

NASA Astrophysics Data System (ADS)

Graça, José

2015-10-01

Suberin is a lipophilic macromolecule found in specialized plant cell walls, wherever insulation or protection towards the surroundings is needed. Suberized cells form the periderm, the tissue that envelops secondary stems as part of the bark, and develop as the sealing tissue after wounding or leaf abscission. Suberin is a complex polyester built from poly-functional long-chain fatty acids (suberin acids) and glycerol. The suberin acids composition of a number of plant tissues and species is now established, but how the polyester macromolecule is assembled within the suberized cell walls is not known. In the last years contributions from several areas have however significantly enriched our understanding of suberin. The primary structure of the polyester, i.e. how the suberin acids and glycerol are sequentially linked was revealed, together with the stereochemistry of the mid-chain functional groups some suberin acids have; solid-state NMR studies showed the presence of methylene chains spatially separated and with different molecular mobility; biophysical studies showed the membrane behaviour of suberin acids derivatives, allowing new insights on structure-properties relationships; and a number of candidate genes were conclusively related to suberin biosynthesis. The comprehension of suberin as a macromolecule will be essential to understand its vital protective roles in plants and how they will deal with eventual environmental changes. Suberin is also expected to be a source for high-performing bio-based chemicals, taking advantage of the structural uniqueness of their constituent suberin acids.

Streptococcus pyogenes Sortase Mutants Are Highly Susceptible to Killing by Host Factors Due to Aberrant Envelope Physiology

PubMed Central

Raz, Assaf; Tanasescu, Ana-Maria; Zhao, Anna M.; Serrano, Anna; Alston, Tricia; Sol, Asaf; Bachrach, Gilad; Fischetti, Vincent A.

2015-01-01

Cell wall anchored virulence factors are critical for infection and colonization of the host by Gram-positive bacteria. Such proteins have an N-terminal leader sequence and a C-terminal sorting signal, composed of an LPXTG motif, a hydrophobic stretch, and a few positively charged amino acids. The sorting signal halts translocation across the membrane, allowing sortase to cleave the LPXTG motif, leading to surface anchoring. Deletion of sortase prevents the anchoring of virulence factors to the wall; the effects on bacterial physiology however, have not been thoroughly characterized. Here we show that deletion of Streptococcus pyogenes sortase A leads to accumulation of sorting intermediates, particularly at the septum, altering cellular morphology and physiology, and compromising membrane integrity. Such cells are highly sensitive to cathelicidin, and are rapidly killed in blood and plasma. These phenomena are not a loss-of-function effect caused by the absence of anchored surface proteins, but specifically result from the accumulation of sorting intermediates. Reduction in the level of sorting intermediates leads to a return of the sortase mutant to normal morphology, while expression of M protein with an altered LPXTG motif in wild type cells leads to toxicity in the host environment, similar to that observed in the sortase mutant. These unanticipated effects suggest that inhibition of sortase by small-molecule inhibitors could similarly lead to the rapid elimination of pathogens from an infected host, making such inhibitors much better anti-bacterial agents than previously believed. PMID:26484774

Novel role of the LPS core glycosyltransferase WapH for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis.

PubMed

Benforte, Florencia C; Colonnella, Maria A; Ricardi, Martiniano M; Solar Venero, Esmeralda C; Lizarraga, Leonardo; López, Nancy I; Tribelli, Paula M

2018-01-01

Psychrotroph microorganisms have developed cellular mechanisms to cope with cold stress. Cell envelopes are key components for bacterial survival. Outer membrane is a constituent of Gram negative bacterial envelopes, consisting of several components, such as lipopolysaccharides (LPS). In this work we investigated the relevance of envelope characteristics for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis by analyzing a mini Tn5 wapH mutant strain, encoding a core LPS glycosyltransferase. Our results showed that wapH strain is impaired to grow under low temperature but not for cold survival. The mutation in wapH, provoked a strong aggregative phenotype and modifications of envelope nanomechanical properties such as lower flexibility and higher turgor pressure, cell permeability and surface area to volume ratio (S/V). Changes in these characteristics were also observed in the wild type strain grown at different temperatures, showing higher cell flexibility but lower turgor pressure under cold conditions. Cold shock experiments indicated that an acclimation period in the wild type is necessary for cell flexibility and S/V ratio adjustments. Alteration in cell-cell interaction capabilities was observed in wapH strain. Mixed cells of wild type and wapH strains, as well as those of the wild type strain grown at different temperatures, showed a mosaic pattern of aggregation. These results indicate that wapH mutation provoked marked envelope alterations showing that LPS core conservation appears as a novel essential feature for active growth under cold conditions.

Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

PubMed Central

Poon, Betty; Chen, Irvin S. Y.

1998-01-01

In vitro infection by human T-cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2) can result in syncytium formation, facilitating viral entry. Using cell lines that were susceptible to HTLV-2-mediated syncytium formation but were nonfusogenic with HTLV-1, we constructed chimeric envelopes between HTLV-1 and -2 and assayed for the ability to induce syncytia in BJAB cells and HeLa cells. We have identified a fusion domain composed of the first 64 amino acids at the amino terminus of the HTLV-2 transmembrane protein, p21, the retention of which was required for syncytium induction. Construction of replication-competent HTLV genomic clones allowed us to correlate the ability of HTLV-2 to induce syncytia with the ability to replicate in BJAB cells. Differences in the ability to induce syncytia were not due to differences in the levels of total or cell membrane-associated envelope or in the formation of multimers. Therefore, we have localized a fusion domain within the amino terminus of the transmembrane protein of HTLV-2 envelope that is necessary for syncytium induction and viral replication. PMID:9499049

Influence of PCMs in thermal insulation on thermal behaviour of building envelopes

NASA Astrophysics Data System (ADS)

Dydek, K.; Furmański, P.; Łapka, P.

2016-09-01

A model of heat transfer through a wall consisting of a layer of concrete and PCM enhanced thermal insulation is considered. The model accounts for heat conduction in both layers, thermal radiation and heat absorption/release due to phase change in the insulation as well as time variation in the ambient temperature and insolation. Local thermal equilibrium between encapsulated PCM and light-weight thermal insulation was assumed. Radiation emission, absorption and scattering were also accounted for in the model. Comparison of different cases of heat flow through the building envelope was carried out. These cases included presence or absence of PCM and thermal radiation in the insulation, effect of emissivity of the PCM microcapsules as well as an effect of solar radiation or its lack on the ambient side of the envelope. Two ways of the PCM distribution in thermal insulation were also considered. The results of simulations were presented for conditions corresponding to the mean summer and winter seasons in Warsaw. It was found that thermal radiation plays an important role in heat transfer through thermal insulation layer of the wall while the presence of the PCM in it significantly contributes to damping of temperature fluctuations and a decrease in heat fluxes flowing into or lost by the interior of the building. The similar effect was observed for a decrease in emissivity of the microcapsules containing PCM.

Whole cell quenched flow analysis.

PubMed

Chiang, Ya-Yu; Haeri, Sina; Gizewski, Carsten; Stewart, Joanna D; Ehrhard, Peter; Shrimpton, John; Janasek, Dirk; West, Jonathan

2013-12-03

This paper describes a microfluidic quenched flow platform for the investigation of ligand-mediated cell surface processes with unprecedented temporal resolution. A roll-slip behavior caused by cell-wall-fluid coupling was documented and acts to minimize the compression and shear stresses experienced by the cell. This feature enables high-velocity (100-400 mm/s) operation without impacting the integrity of the cell membrane. In addition, rotation generates localized convection paths. This cell-driven micromixing effect causes the cell to become rapidly enveloped with ligands to saturate the surface receptors. High-speed imaging of the transport of a Janus particle and fictitious domain numerical simulations were used to predict millisecond-scale biochemical switching times. Dispersion in the incubation channel was characterized by microparticle image velocimetry and minimized by using a horizontal Hele-Shaw velocity profile in combination with vertical hydrodynamic focusing to achieve highly reproducible incubation times (CV = 3.6%). Microfluidic quenched flow was used to investigate the pY1131 autophosphorylation transition in the type I insulin-like growth factor receptor (IGF-1R). This predimerized receptor undergoes autophosphorylation within 100 ms of stimulation. Beyond this demonstration, the extreme temporal resolution can be used to gain new insights into the mechanisms underpinning a tremendous variety of important cell surface events.

A nuptially transmitted ichthyosporean symbiont of Tenebrio molitor (Coleoptera: Tenebrionidae).

PubMed

Lord, Jeffrey C; Hartzer, Kris L; Kambhampati, Srinivas

2012-01-01

The yellow mealworm, Tenebrio molitor, harbors a symbiont that has spores with a thick, laminated wall and infects the fat body and ventral nerve chord of adult and larval beetles. In adult males, there is heavy infection of the epithelial cells of the testes and between testes lobes with occasional penetration of the lobes. Spores are enveloped in the spermatophores when they are formed at the time of mating and transferred to the female's bursa copulatrix. Infection has not been found in the ovaries. The sequence of the nuclear small subunit rDNA indicates that the symbiont is a member of the Ichthyosporea, a class of protists near the animal-fungi divergence. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

Technical - Economic Research for Passive Buildings

NASA Astrophysics Data System (ADS)

Miniotaite, Ruta

2017-10-01

A newly constructed passive house must save 80 % of heat resources; otherwise it is not a passive house. The heating energy demand of a passive building is less than 15 kWh/m2 per year. However, a passive house is something more than just an energy-saving house. This concept involves sustainable, high-quality, valuable, healthy and durable construction. Features of a passive house: high insulation of envelope components, high-quality windows, good tightness of the building, regenerative ventilation system and elimination of thermal bridges. The Energy Performance of Buildings Directive (EPBD) 61 requires all new public buildings to become near-zero energy buildings by 2019 and will be extended to all new buildings by 2021. This concept involves sustainable, high-quality, valuable, healthy and durable construction. Foundation, walls and roofs are the most essential elements of a house. The type of foundation for a private house is selected considering many factors. The article examines technological and structural solutions for passive buildings foundation, walls and roofs. The technical-economic comparison of the main structures of a passive house revealed that it is cheaper to install an adequately designed concrete slab foundation than to build strip or pile foundation and the floor separately. Timber stud walls are the cheapest wall option for a passive house and 45-51% cheaper compared to other options. The comparison of roofs and ceilings showed that insulation of the ceiling is 25% more efficient than insulation of the roof. The comparison of the main envelope elements efficiency by multiple-criteria evaluation methods showed that it is economically feasible to install concrete slab on ground foundation, stud walls with sheet cladding and a pitched roof with insulated ceiling.

Behavior of sandwich panels in a fire

NASA Astrophysics Data System (ADS)

Chelekova, Eugenia

2018-03-01

For the last decades there emerged a vast number of buildings and structures erected with the use of sandwich panels. The field of application for this construction material is manifold, especially in the construction of fire and explosion hazardous buildings. In advanced evacu-ation time calculation methods the coefficient of heat losses is defined with dire regard to fire load features, but without account to thermal and physical characteristics of building envelopes, or, to be exact, it is defined for brick and concrete walls with gross heat capacity. That is why the application of the heat loss coefficient expression obtained for buildings of sandwich panels is impossible because of different heat capacity of these panels from the heat capacities of brick and concrete building envelopes. The article conducts an analysis and calculation of the heal loss coefficient for buildings and structures of three layer sandwich panels as building envelopes.

[Fine structure of glial cells in the central nervous system of the tapeworm Grillotia erinaceus (Cestoda: Trypanorhyncha)].

PubMed

Biserova, N M

2008-01-01

The problem of glial cells existing in parasitic and free living flatworms is correlated with organization of parenchyma in platyhelmintes. In the contrary to the widespread opinion that myelin-like envelopes and glial cells do not exist in the nervous system of parasitic flatworms, it has been shown by ultrastructural researches that Amphilina foliacea (Cestoda, Amphilinidea) has well developed glial cells and myelin-like envelopes in the ganglia and main cords, which include both glial cells and intercellular components. The aim of our research was to reveal and investigate in details structural components corresponding to the concept of the glial cell in the CNS of Grillotia erinaceus (Cestoda: Trypanorhyncha). Three types of glial cells have been found. The first type is the fibroblast-like glial cells; cells locate in the cerebral ganglion, contain in cytoplasm and extract out fibrillar matrix, form desmosomes and have supporting function. The glial cells of the second type form myeline-like envelope of the giant axons and bulbar nerves in scolex and have laminar cytoplasm. These cells are numerous and exceed in number the neurons bodies into the nerve. The glial cells of the third type form multilayer envelopes in the main nerve cords; extra cellular fibers and gap-junctions take place between the layers. There are contacts between the glial cells of the third type and excretory epithelium but specialized contacts with neurons have been not found. The existing of glial cells in free living and parasitic flatworms is discussed.

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

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

Armstrong, S.K.

1986-01-01

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

Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae*

PubMed Central

Wesener, Darryl A.; Levengood, Matthew R.

2017-01-01

The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis. In each species, the galactan is constructed from uridine 5′-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope. PMID:28039359

Antineoplastic And Antiviral Properties Of Merocyanine 540

NASA Astrophysics Data System (ADS)

Sieber, Fritz

1989-03-01

Simultaneous exposure to the lipophilic photosensitizer, merocyanine 540, and light in the presence of serum (or certain serum components) and oxygen kills leukemia cells, lymphoma cells, neuroblastoma cells, cell-free enveloped viruses, cell-associated enveloped viruses, and virus-infected cells. The same treatment spares pluripotent hematopoietic stem cells, mature erythrocytes, factor VIII, von Willebrand factor, and probably other blood components. Merocyanine 540-mediated photosensitization is now being evaluated clinically as a means to eliminate residual tumor cells from autologous remission bone marrow grafts and preclinically as a means to inactivate pathogenic viruses in blood products.

Electron microscopic examination of uncultured soil-dwelling bacteria.

PubMed

Amako, Kazunobu; Takade, Akemi; Taniai, Hiroaki; Yoshida, Shin-ichi

2008-05-01

Bacteria living in soil collected from a rice paddy in Fukuoka, Japan, were examined by electron microscopy using a freeze-substitution fixation method. Most of the observed bacteria could be categorized, based on the structure of the cell envelope and overall morphology, into one of five groups: (i) bacterial spore; (ii) Gram-positive type; (iii) Gram-negative type; (iv) Mycobacterium like; and (v) Archaea like. However, a few of the bacteria could not be readily categorized into one of these groups because they had unique cell wall structures, basically resembling those of Gram-negative bacteria, but with the layer corresponding to the peptidoglycan layer in Gram-negative bacteria being extremely thick, like that of the cortex of a bacterial spore. The characteristic morphological features found in many of these uncultured, soil-dwelling cells were the nucleoid being in a condensed state and the cytoplasm being shrunken. We were able to produce similar morphologies in vitro using a Salmonella sp. by culturing under low-temperature, low-nutrient conditions, similar to those found in some natural environments. These unusual morphologies are therefore hypothesized to be characteristic of bacteria in resting or dormant stages.

Type III secretion system effector proteins: double agents in bacterial disease and plant defense.

PubMed

Alfano, James R; Collmer, Alan

2004-01-01

Many phytopathogenic bacteria inject virulence effector proteins into plant cells via a Hrp type III secretion system (TTSS). Without the TTSS, these pathogens cannot defeat basal defenses, grow in plants, produce disease lesions in hosts, or elicit the hypersensitive response (HR) in nonhosts. Pathogen genome projects employing bioinformatic methods to identify TTSS Hrp regulon promoters and TTSS pathway targeting signals suggest that phytopathogenic Pseudomonas, Xanthomonas, and Ralstonia spp. harbor large arsenals of effectors. The Hrp TTSS employs customized cytoplasmic chaperones, conserved export components in the bacterial envelope (also used by the TTSS of animal pathogens), and a more specialized set of TTSS-secreted proteins to deliver effectors across the plant cell wall and plasma membrane. Many effectors can act as molecular double agents that betray the pathogen to plant defenses in some interactions and suppress host defenses in others. Investigations of the functions of effectors within plant cells have demonstrated the plasma membrane and nucleus as subcellular sites for several effectors, revealed some effectors to possess cysteine protease or protein tyrosine phosphatase activity, and provided new clues to the coevolution of bacterium-plant interactions.

Nuclear envelope: positioning nuclei and organizing synapses

PubMed Central

Razafsky, David; Hodzic, Didier

2015-01-01

The nuclear envelope plays an essential role in nuclear positioning within cells and tissues. This review highlights advances in understanding the mechanisms of nuclear positioning during skeletal muscle and central nervous system development. New findings, particularly about Atype lamins and Nesprin1, may link nuclear envelope integrity to synaptic integrity. Thus synaptic defects, rather than nuclear mispositioning, may underlie human pathologies associated with mutations of nuclear envelope proteins. PMID:26079712

Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models

PubMed Central

Yokoi, Fumiaki; Dang, Mai T.; Yang, Guang; Li, JinDong; Doroodchi, Atbin; Zhou, Tong; Li, Yuqing

2011-01-01

Myoclonus-dystonia (M-D) is a movement disorder characterized by myoclonic jerks with dystonia. DYT11 M-D is caused by mutations in SGCE which codes for ε-sarcoglycan. SGCE is maternally imprinted and paternally expressed. Abnormal nuclear envelope has been reported in mouse models of DYT1 generalized torsion dystonia. However, it is not known whether similar alterations occur in DYT11 M-D. We developed a mouse model of DYT11 M-D using paternally-inherited Sgce heterozygous knockout (Sgce KO) mice and reported that they had myoclonus and motor coordination and learning deficits in the beam-walking test. However, the specific brain regions that contribute to these phenotypes have not been identified. Since ε-sarcoglycan is highly expressed in the cerebellar Purkinje cells, here we examined the nuclear envelope in these cells using a transmission electron microscope and found that they are abnormal in Sgce KO mice. Our results put DYT11 M-D in a growing family of nuclear envelopathies. To analyze the effect of loss of ε-sarcoglycan function in the cerebellar Purkinje cells, we produced paternally-inherited cerebellar Purkinje cell-specific Sgce conditional knockout (Sgce pKO) mice. Sgce pKO mice showed motor learning deficits, while they did not show abnormal nuclear envelope in the cerebellar Purkinje cells, robust motor deficits, or myoclonus. The results suggest that ε-sarcoglycan in the cerebellar Purkinje cells contributes to the motor learning, while loss of ε-sarcoglycan in other brain regions may contribute to nuclear envelope abnormality, myoclonus and motor coordination deficits. PMID:22040906

Identification of the heart wall and chamber based on temporal change of ultrasonic scatterer distribution

NASA Astrophysics Data System (ADS)

Takahashi, Kohei; Taki, Hirofumi; Kanai, Hiroshi

2017-07-01

In most current methods for evaluating the cardiac function by ultrasound, the heart wall area is identified manually by an examiner. To eliminate examiner dependence and to improve usability, an automatic heart wall identification method is desirable. Identification based on only echogenicity often fails because of low echogenicity of some areas of the heart wall. In the present study, to determine more essential features, we focused on the relative temporal change of ultrasonic scatterer distribution and proposed three features for identification of the heart wall and the chamber: cross-correlation of RF signals, that of envelopes, and spatial dispersion of movement vectors in small regions. In an in vivo experiment, using echogenicity and the three features, we identified the heart wall and the chamber in the left ventricular long-axis view, resulting in criteria of separability J of 1.69, 1.40, and 3.02 using these features compared with the result of 0.979 using echogenicity.

Modeling the Effect of Olivocochlear Efferents on the Subcortical Envelope Following Response in Humans

DTIC Science & Technology

2016-11-28

olivocochlear reflex (MOCR), a feedback mechanism that controls gain of the outer hair cells, is thought to provide protection and enhancement for a listener in...effectively reduce the outer hair cell gain, depending on the stimulus frequency, level, and timing. Human Envelope Following Responses (EFRs

Bacillus subtilis MreB paralogues have different filament architectures and lead to shape remodelling of a heterologous cell system.

PubMed

Soufo, Hervé Joël Defeu; Graumann, Peter L

2010-12-01

Like many bacteria, Bacillus subtilis cells contain three actin-like MreB proteins. We show that the three paralogues, MreB, Mbl and MreBH, have different filament architectures in a heterologous cell system, and form straight filaments, helices or ring structures, different from the regular helical arrangement in B. subtilis cells. However, when coexpressed, they colocalize into a single filamentous helical structure, showing that the paralogues influence each other's filament architecture. Ring-like MreBH structures can be converted into MreB-like helical filaments by a single point mutation affecting subunit contacts, showing that MreB paralogues feature flexible filament arrangements. Time-lapse and FRAP experiments show that filaments can extend as well as shrink at both ends, and also show internal rearrangement, suggesting that filaments consist of overlapping bundles of shorter filaments that continuously turn over. Upon induction in Escherichia coli cells, B. subtilis MreB (BsMreB) filaments push the cells into strikingly altered cell morphology, showing that MreB filaments can change cell shape. E. coli cells with a weakened cell wall were ruptured upon induction of BsMreB filaments, suggesting that the bacterial actin orthologue may exert force against the cell membrane and envelope, and thus possibly plays an additional mechanical role in bacteria. © 2010 Blackwell Publishing Ltd.

Transgene vaccination using Ulex europaeus agglutinin I (UEA-1) for targeted mucosal immunization against HIV-1 envelope.

PubMed

Wang, Xinhai; Kochetkova, Irina; Haddad, Asmahan; Hoyt, Teri; Hone, David M; Pascual, David W

2005-05-31

Receptor-mediated gene transfer using an M cell ligand has been shown to be an efficient method for mucosal DNA immunization. To investigate further into alternative M cell ligands, the plant lectin, Ulex europaeus agglutinin I (UEA-1), was tested. UEA-1 binds to human intestinal Caco-2 cells, and these cells can be transfected with poly-l-lysine (PL)-conjugated UEA-1 for expression of reporter cDNAs. When tested in vivo, mice nasally immunized with UEA-1-PL complexed to plasmid encoding HIV-1 envelope showed elevated systemic and mucosal antibody responses, and these were supported by tissue antibody-forming cells. Likewise, elevated envelope-specific CTLs were induced. Thus, UEA-1 mediated DNA delivery represents an alternative mucosal formulation for inducing humoral and cellular immunity against HIV-1.

HIV envelope glycoprotein imaged at high resolution | Center for Cancer Research

Cancer.gov

The outer surface of the human immunodeficiency virus (HIV) is surrounded by an envelope studded with spike-shaped glycoproteins called Env that help the deadly virus identify, bind, and infect cells. When unbound, Env exists in a “closed” conformational state. Upon binding with target cells, such as CD4+ T cells, the protein transitions to an “open” configuration. Given that

Photodynamic therapy using hemagglutinating virus of Japan envelope (HVJ-E): a novel therapeutic approach for the treatment of hormone antagonistic prostate cancer

NASA Astrophysics Data System (ADS)

Inai, Mizuho; Yamauchi, Masaya; Honda, Norihiro; Hazama, Hisanao; Tachikawa, Shoji; Nakamura, Hiroyuki; Nishida, Tomoki; Yasuda, Hidehiro; Kaneda, Yasufumi; Awazu, Kunio

2015-03-01

Traditional treatment options for prostate cancer are insufficient to cure advanced drug-resistant prostate cancer. Thus, as an alternative form of cancer therapy, photodynamic therapy (PDT) has become the main subject of intense investigation as a possible treatment modality. In this study, ultraviolet-inactivated viral vector, called hemagglutinating virus of Japan envelope (HVJ-E) was utilized to establish an effective delivery system for photosensitizer. Lipidated protoporphyrin IX (PpIX lipid) was inserted in HVJ-E by centrifugation to create a new drug delivering system that allows selective accumulation of photosensitizers in cancer cells. To study in vitro drug release mechanism of porphyrus envelope, the ultra-high voltage electron microscope tomography was applied. Next, to evaluate the photodynamic efficiency of porphyrus envelope for hormone antagonistic prostate cancer cells (PC-3), uptake of porphyrus envelope derived PpIX lipid and PpIX induced from exogenously administered precursor of 5-aminolevulinic acid hydrochloride (5-ALA) were compared by measuring fluorescence intensity of PpIX. Finally, to evaluate the efficacy of porphyrus envelope-PDT, laser light at a wavelength of 405 nm was irradiated to PC-3 cells. As a result, incorporation of porphyrus envelope-derived PpIX lipid occurred via membrane fusion, giving the highest fluorescence intensity when compared to 5-ALA-induced PpIX. Also, results from PDT experiment revealed the 28.6 × 103-fold and 206-fold increase in therapeutic efficacy when compared to those of PDT using 5-ALA induced PpIX and PpIX lipid, respectively. Our findings suggest how porphyrus envelope can induce efficient accumulation of PpIX lipid, which can enhance the therapeutic efficacy of PDT against hormone antagonistic prostate cancer.

Mechanism of Dissolution of Envelopes of the Extreme Halophile Halobacterium cutirubrum1

PubMed Central

Onishi, H.; Kushner, D. J.

1966-01-01

Onishi, H. (National Research Council, Ottawa, Ontario, Canada), and D. J. Kushner. Mechanism of dissolution of envelopes of the extreme halophile Halobacterium cutirubrum. J. Bacteriol. 91:646–652. 1966.—Envelopes of Halobacterium cutirubrum dissolved rapidly in media of low ionic strength. Heating partially inhibited breakdown, probably because of nonspecific protein coagulation rather than inactivation of a lytic enzyme(s). Dissolution of envelopes in water did not involve splitting of peptide bonds or protein-lipid bonds, or any extensive breakdown of carbohydrate polymers. Dissolution was increased by alcohols and urea, even at high salt concentrations, but was not affected by metabolic inhibitors. Thus, no evidence was found for a dilution-activated lytic enzyme that contributes to envelope breakdown. Cells of H. cutirubrum were stable in 2 m NaCl, but lysis occurred in 2 m KCl or NH4Cl. This lysis did not involve an extensive breakdown of the envelope. No evidence for different sites of Na+, K+, and NH4+ action was obtained from the pattern of release of envelope constituents in different concentrations of these salts. Ultracentrifugation studies showed that adding salts to envelopes that had been dissolved in water led to a nonspecific reaggregation of envelope material. No difference was seen between the effects of KCl and NaCl, except at 3 to 4 m concentrations where KCl caused more aggregation. The preferential effect of Na+ on intact cells is probably due to its ability specifically to prevent leakage rather than to an overall effect on envelope integrity. Images PMID:5883109

A laboratory facility for research on wind-driven rain intrusion in building envelope assemblies

Treesearch

Samuel V. Glass

2010-01-01

Moisture management is critical for durable, energy-efficient buildings. To address the need for research on wind-driven rain intrusion in wall assemblies, the U.S. Forest Products Laboratory is developing a new facility. This paper describes the underlying principle of this facility and its capabilities.

Seamless metal-clad fiber-reinforced organic matrix composite structures and process for their manufacture

NASA Technical Reports Server (NTRS)

Bluck, Raymond M. (Inventor); Bush, Harold G. (Inventor); Johnson, Robert R. (Inventor)

1990-01-01

A metallic outer sleeve is provided which is capable of enveloping a hollow metallic inner member having continuous reinforcing fibers attached to the distal end thereof. The inner member is then introduced into outer sleeve until inner member is completely enveloped by outer sleeve. A liquid matrix member is then injected into space between inner member and outer sleeve. A pressurized heat transfer medium is flowed through the inside of inner member, thereby forming a fiber reinforced matrix composite material. The wall thicknesses of both inner member and outer sleeve are then reduced to the appropriate size by chemical etching, to adjust the thermal expansion coefficient of the metal-clad composite structure to the desired value. thereby forming a fiber reinforced matrix composite material. The wall thicknesses of both inner member and outer sleeve are then reduced to the appropriate size by chemical etching, to adjust the thermal expansion coefficient of the metal-clad composite structure to the desired value. The novelty of this invention resides in the development of a efficient method of producing seamless metal clad fiber reinforced organic matrix composite structures.

Assessment of energy and economic performance of office building models: a case study

NASA Astrophysics Data System (ADS)

Song, X. Y.; Ye, C. T.; Li, H. S.; Wang, X. L.; Ma, W. B.

2016-08-01

Energy consumption of building accounts for more than 37.3% of total energy consumption while the proportion of energy-saving buildings is just 5% in China. In this paper, in order to save potential energy, an office building in Southern China was selected as a test example for energy consumption characteristics. The base building model was developed by TRNSYS software and validated against the recorded data from the field work in six days out of August-September in 2013. Sensitivity analysis was conducted for energy performance of building envelope retrofitting; five envelope parameters were analyzed for assessing the thermal responses. Results indicated that the key sensitivity factors were obtained for the heat-transfer coefficient of exterior walls (U-wall), infiltration rate and shading coefficient (SC), of which the sum sensitivity factor was about 89.32%. In addition, the results were evaluated in terms of energy and economic analysis. The analysis of sensitivity validated against some important results of previous studies. On the other hand, the cost-effective method improved the efficiency of investment management in building energy.

Insect Gut Symbiont Susceptibility to Host Antimicrobial Peptides Caused by Alteration of the Bacterial Cell Envelope*

PubMed Central

Kim, Jiyeun Kate; Son, Dae Woo; Kim, Chan-Hee; Cho, Jae Hyun; Marchetti, Roberta; Silipo, Alba; Sturiale, Luisa; Park, Ha Young; Huh, Ye Rang; Nakayama, Hiroshi; Fukatsu, Takema; Molinaro, Antonio; Lee, Bok Luel

2015-01-01

The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system because of its cultivatable Burkholderia symbionts. This system is greatly advantageous because it allows the acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it is possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis. PMID:26116716

Regulation of Flagellum Biosynthesis in Response to Cell Envelope Stress in Salmonella enterica Serovar Typhimurium

PubMed Central

2018-01-01

ABSTRACT Flagellum-driven motility of Salmonella enterica serovar Typhimurium facilitates host colonization. However, the large extracellular flagellum is also a prime target for the immune system. As consequence, expression of flagella is bistable within a population of Salmonella, resulting in flagellated and nonflagellated subpopulations. This allows the bacteria to maximize fitness in hostile environments. The degenerate EAL domain protein RflP (formerly YdiV) is responsible for the bistable expression of flagella by directing the flagellar master regulatory complex FlhD4C2 with respect to proteolytic degradation. Information concerning the environmental cues controlling expression of rflP and thus about the bistable flagellar biosynthesis remains ambiguous. Here, we demonstrated that RflP responds to cell envelope stress and alterations of outer membrane integrity. Lipopolysaccharide (LPS) truncation mutants of Salmonella Typhimurium exhibited increasing motility defects due to downregulation of flagellar gene expression. Transposon mutagenesis and genetic profiling revealed that σ24 (RpoE) and Rcs phosphorelay-dependent cell envelope stress response systems sense modifications of the lipopolysaccaride, low pH, and activity of the complement system. This subsequently results in activation of RflP expression and degradation of FlhD4C2 via ClpXP. We speculate that the presence of diverse hostile environments inside the host might result in cell envelope damage and would thus trigger the repression of resource-costly and immunogenic flagellum biosynthesis via activation of the cell envelope stress response. PMID:29717015

Immunomodulatory effects of exosomes produced by virus-infected cells.

PubMed

Petrik, Juraj

2016-08-01

Viruses have developed a spectrum of ways to modify cellular pathways to hijack the cell machinery for the synthesis of their nucleic acid and proteins. Similarly, they use intracellular vesicular mechanisms of trafficking for their assembly and eventual release, with a number of viruses acquiring their envelope from internal or plasma cell membranes. There is an increasing number of reports on viral exploitation of cell secretome pathways to avoid recognition and stimulation of the immune response. Extracellular vesicles (EV) containing viral particles have been shown to shield viruses after exiting the host cell, in some cases challenging the boundaries between viral groups traditionally characterised as enveloped and non-enveloped. Apart from viral particles, EV can spread the virus also carrying viral genome and can modify the target cells through their cargo of virus-coded miRNAs and proteins as well as selectively packaged cellular mRNAs, miRNAs, proteins and lipids, differing in composition and quantities from the cell of origin. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Genetics Home Reference: mandibuloacral dysplasia

MedlinePlus

... proteins act as scaffolding (supporting) components of the nuclear envelope, which is the membrane that surrounds the nucleus in cells. The nuclear envelope regulates the movement of molecules into and ...

Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models.

PubMed

Yokoi, Fumiaki; Dang, Mai T; Yang, Guang; Li, Jindong; Doroodchi, Atbin; Zhou, Tong; Li, Yuqing

2012-02-01

Myoclonus-dystonia (M-D) is a movement disorder characterized by myoclonic jerks with dystonia. DYT11 M-D is caused by mutations in SGCE which codes for ɛ-sarcoglycan. SGCE is maternally imprinted and paternally expressed. Abnormal nuclear envelope has been reported in mouse models of DYT1 generalized torsion dystonia. However, it is not known whether similar alterations occur in DYT11 M-D. We developed a mouse model of DYT11 M-D using paternally inherited Sgce heterozygous knockout (Sgce KO) mice and reported that they had myoclonus and motor coordination and learning deficits in the beam-walking test. However, the specific brain regions that contribute to these phenotypes have not been identified. Since ɛ-sarcoglycan is highly expressed in the cerebellar Purkinje cells, here we examined the nuclear envelope in these cells using a transmission electron microscope and found that they are abnormal in Sgce KO mice. Our results put DYT11 M-D in a growing family of nuclear envelopathies. To analyze the effect of loss of ɛ-sarcoglycan function in the cerebellar Purkinje cells, we produced paternally inherited cerebellar Purkinje cell-specific Sgce conditional knockout (Sgce pKO) mice. Sgce pKO mice showed motor learning deficits, while they did not show abnormal nuclear envelope in the cerebellar Purkinje cells, robust motor deficits, or myoclonus. The results suggest that ɛ-sarcoglycan in the cerebellar Purkinje cells contributes to the motor learning, while loss of ɛ-sarcoglycan in other brain regions may contribute to nuclear envelope abnormality, myoclonus and motor coordination deficits. Copyright © 2011 Elsevier B.V. All rights reserved.

Selective induction of cell-mediated immunity and protection of rhesus macaques from chronic SHIV{sub KU2} infection by prophylactic vaccination with a conserved HIV-1 envelope peptide-cocktail

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

Nehete, Pramod N.; Nehete, Bharti P.; Hill, Lori

2008-01-05

Infection of Indian-origin rhesus macaques by the simian human immunodeficiency virus (SHIV) is considered to be a suitable preclinical model for directly testing efficacy of vaccine candidates based on the HIV-1 envelope. We used this model for prophylactic vaccination with a peptide-cocktail comprised of highly conserved HIV-1 envelope sequences immunogenic/antigenic in macaques and humans. Separate groups of macaques were immunized with the peptide-cocktail by intravenous and subcutaneous routes using autologous dendritic cells (DC) and Freund's adjuvant, respectively. The vaccine elicited antigen specific IFN-{gamma}-producing cells and T-cell proliferation, but not HIV-neutralizing antibodies. The vaccinated animals also exhibited efficient cross-clade cytolytic activitymore » against target cells expressing envelope proteins corresponding to HIV-1 strains representative of multiple clades that increased after intravenous challenge with pathogenic SHIV{sub KU2}. Virus-neutralizing antibodies were either undetectable or present only transiently at low levels in the control as well as vaccinated monkeys after infection. Significant control of plasma viremia leading to undetectable levels was achieved in majority of vaccinated monkeys compared to mock-vaccinated controls. Monkeys vaccinated with the peptide-cocktail using autologous DC, compared to Freund's adjuvant, and the mock-vaccinated animals, showed significantly higher IFN-{gamma} production, higher levels of vaccine-specific IFN-{gamma} producing CD4{sup +} cells and significant control of plasma viremia. These results support DC-based vaccine delivery and the utility of the conserved HIV-1 envelope peptide-cocktail, capable of priming strong cell-mediated immunity, for potential inclusion in HIV vaccination strategies.« less

Comprehensive analysis of mycolic acid subclass and molecular species composition of Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105).

PubMed

Uenishi, Yuko; Fujita, Yukiko; Kusunose, Naoto; Yano, Ikuya; Sunagawa, Makoto

2008-02-01

The mycobacterial cell envelope consists of a characteristic cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. Since mycolic acids have recently been reported to play crucial roles in host immune response, detailed molecular characterization of mycolic acid subclasses and sub-subclasses of CWS from Mycobacterium bovis BCG Tokyo 172 (SMP-105) was performed. Mycolic acids were liberated by alkali hydrolysis from SMP-105, and their methyl esters were separated by silica gel TLC into three subclasses: alpha-, methoxy-, and keto-mycolates. Each mycolate subclass was further separated by silver nitrate (AgNO(3))-coated silica gel TLC into sub-subclasses. Molecular weights of individual mycolic acid were determined by MALDI-TOF mass spectrometry. alpha-Mycolates were sub-grouped into cis, cis-dicyclopropanoic (alpha1), and cis-monocyclopropanoic-cis-monoenoic (alpha2) series; methoxy-mycolates were sub-grouped into cis-monocyclopropanoic (m1), trans-monocyclopropanoic (m2), trans-monoenoic (m3), cis-monocyclopropanoic-trans-monoenoic (m4), cis-monoenoic (m5), and cis-monocyclopropanoic-cis-monoenoic (m6) series; and keto-mycolates were sub-grouped into cis-monocyclopropanoic (k1), trans-monocyclopropanoic (k2), trans-monoenoic (k3), cis-monoenoic (k4), and cis-monocyclopropanoic-cis-monoenoic (k5) series. The position of each functional group, including cyclopropane rings and methoxy and keto groups, was determined by analysis of the meromycolates with fast atom bombardment (FAB) mass spectrometry and FAB mass-mass spectrometry, and the cis/trans ratio of cyclopropane rings and double bonds were determined by NMR analysis of methyl mycolates. Mycolic acid subclass and molecular species composition of SMP-105 showed characteristic features including newly-identified cis-monocyclopropanoic-trans-monoenoic mycolic acid (m4).

DltX of Bacillus thuringiensis Is Essential for D-Alanylation of Teichoic Acids and Resistance to Antimicrobial Response in Insects

PubMed Central

Kamar, Rita; Réjasse, Agnès; Jéhanno, Isabelle; Attieh, Zaynoun; Courtin, Pascal; Chapot-Chartier, Marie-Pierre; Nielsen-Leroux, Christina; Lereclus, Didier; el Chamy, Laure; Kallassy, Mireille; Sanchis-Borja, Vincent

2017-01-01

The dlt operon of Gram-positive bacteria is required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TAs). Addition of D-alanine to TAs reduces the negative charge of the cell envelope thereby preventing cationic antimicrobial peptides (CAMPs) from reaching their target of action on the bacterial surface. In most gram-positive bacteria, this operon consists of five genes dltXABCD but the involvement of the first ORF (dltX) encoding a small protein of unknown function, has never been investigated. The aim of this study was to establish whether this protein is involved in the D-alanylation process in Bacillus thuringiensis. We, therefore constructed an in frame deletion mutant of dltX, without affecting the expression of the other genes of the operon. The growth characteristics of the dltX mutant and those of the wild type strain were similar under standard in vitro conditions. However, disruption of dltX drastically impaired the resistance of B. thuringiensis to CAMPs and significantly attenuated its virulence in two insect species. Moreover, high-performance liquid chromatography studies showed that the dltX mutant was devoid of D-alanine, and electrophoretic mobility measurements indicated that the cells carried a higher negative surface charge. Scanning electron microscopy experiments showed morphological alterations of these mutant bacteria, suggesting that depletion of D-alanine from TAs affects cell wall structure. Our findings suggest that DltX is essential for the incorporation of D-alanyl esters into TAs. Therefore, DltX plays a direct role in the resistance to CAMPs, thus contributing to the survival of B. thuringiensis in insects. To our knowledge, this work is the first report examining the involvement of dltX in the D-alanylation of TAs. PMID:28824570

Isolated receptor binding domains of HTLV-1 and HTLV-2 envelopes bind Glut-1 on activated CD4+ and CD8+ T cells

PubMed Central

Kinet, Sandrina; Swainson, Louise; Lavanya, Madakasira; Mongellaz, Cedric; Montel-Hagen, Amélie; Craveiro, Marco; Manel, Nicolas; Battini, Jean-Luc; Sitbon, Marc; Taylor, Naomi

2007-01-01

Background We previously identified the glucose transporter Glut-1, a member of the multimembrane-spanning facilitative nutrient transporter family, as a receptor for both HTLV-1 and HTLV-2. However, a recent report concluded that Glut-1 cannot serve as a receptor for HTLV-1 on CD4 T cells: This was based mainly on their inability to detect Glut-1 on this lymphocyte subset using the commercial antibody mAb1418. It was therefore of significant interest to thoroughly assess Glut-1 expression on CD4 and CD8 T cells, and its association with HTLV-1 and -2 envelope binding. Results As previously reported, ectopic expression of Glut-1 but not Glut-3 resulted in significantly augmented binding of tagged proteins harboring the receptor binding domains of either HTLV-1 or HTLV-2 envelope glycoproteins (H1RBD or H2RBD). Using antibodies raised against the carboxy-terminal peptide of Glut-1, we found that Glut-1 expression was significantly increased in both CD4 and CD8 cells following TCR stimulation. Corresponding increases in the binding of H1RBD as well as H2RBD, not detected on quiescent T cells, were observed following TCR engagement. Furthermore, increased Glut-1 expression was accompanied by a massive augmentation in glucose uptake in TCR-stimulated CD4 and CD8 lymphocytes. Finally, we determined that the apparent contradictory results obtained by Takenouchi et al were due to their monitoring of Glut-1 with a mAb that does not bind cells expressing endogenous Glut-1, including human erythrocytes that harbor 300,000 copies per cell. Conclusion Transfection of Glut-1 directly correlates with the capacities of HTLV-1 and HTLV-2 envelope-derived ligands to bind cells. Moreover, Glut-1 is induced by TCR engagement, resulting in massive increases in glucose uptake and binding of HTLV-1 and -2 envelopes to both CD4 and CD8 T lymphocytes. Therefore, Glut-1 is a primary binding receptor for HTLV-1 and HTLV-2 envelopes on activated CD4 as well as CD8 lymphocytes. PMID:17504522

On the application of a new thermal diagnostic model: the passive elements equivalent in term of ventilation inside a room

NASA Astrophysics Data System (ADS)

El Khattabi, El Mehdi; Mharzi, Mohamed; Raefat, Saad; Meghari, Zouhair

2018-05-01

In this paper, the thermal equivalence of the passive elements of a room in a building located in Fez-Morocco has been studied. The possibility of replacing them with a semi-passive element such as ventilation has been appraised. For this aim a Software in Fortran taking into account the meteorological external conditions along with different parameters of the building envelope has been performed. A new computational approach is adapted to determinate the temperature distribution throughout the building multilayer walls. A novel equation gathering the internal temperature with the external conditions, and the building envelope has been deduced in transient state.

Canine distemper virus matrix protein influences particle infectivity, particle composition, and envelope distribution in polarized epithelial cells and modulates virulence.

PubMed

Dietzel, Erik; Anderson, Danielle E; Castan, Alexandre; von Messling, Veronika; Maisner, Andrea

2011-07-01

In paramyxoviruses, the matrix (M) protein mediates the interaction between the envelope and internal proteins during particle assembly and egress. In measles virus (MeV), M mutations, such as those found in subacute sclerosing panencephalitis (SSPE) strains, and differences in vaccine and wild-type M proteins can affect the strength of interaction with the envelope glycoproteins, assembly efficiency, and spread. However, the contribution of the M protein to the replication and pathogenesis of the closely related canine distemper virus (CDV) has not been characterized. To this end this, we generated a recombinant wild-type CDV carrying a vaccine strain M protein. The recombinant virus retained the parental growth phenotype in VerodogSLAMtag cells, but displayed an increased particle-to-infectivity ratio very similar to that of the vaccine strain, likely due to inefficient H protein incorporation. Even though infectious virus was released only from the apical surface, consistent with the release polarity of the wild-type CDV strain, envelope protein distribution in polarized epithelial cells reproduced the bipolar pattern seen in vaccine strain-infected cells. Most notably, the chimeric virus was completely attenuated in ferrets and caused only a mild and transient leukopenia, indicating that the differences in particle infectivity and envelope protein sorting mediated by the vaccine M protein contribute importantly to vaccine strain attenuation.

Gene Targeting of Envoplakin, a Cytoskeletal Linker Protein and Precursor of the Epidermal Cornified Envelope

PubMed Central

Määttä, Arto; DiColandrea, Teresa; Groot, Karen; Watt, Fiona M.

2001-01-01

Envoplakin, a member of the plakin family of cytoskeletal linker proteins, is localized in desmosomes of stratified epithelial cells and is a component of the epidermal cornified envelope. Gene targeting in mouse embryonic stem cells was used to generate a null allele of envoplakin. No envoplakin transcripts from the targeted allele could be detected in the skin of newborn mice. Mice homozygous for the targeted allele were born in the normal Mendelian ratio and were fertile. They did not develop any discernible pathological phenotype up to the age of 1 year. The ultrastructural appearance of cornified envelopes from adult epidermis was indistinguishable between wild-type and knockout mice, and there was no evidence that the absence of envoplakin affected the subcellular distribution of periplakin or desmoplakin, two other plakins found in desmosomes. The proportion of immature cornified envelopes in the epidermis of newborn mice was greater in envoplakin-null animals than in heterozygous littermates or wild-type mice, and the envelopes had a larger surface area. This correlated with a slight delay in barrier acquisition during embryonic development. We conclude that although envoplakin is part of the scaffolding on which the cornified envelope is assembled, it is not essential for envelope formation or epidermal barrier function. PMID:11564887

Estimating envelope thermal characteristics from single point in time thermal images

NASA Astrophysics Data System (ADS)

Alshatshati, Salahaldin Faraj

Energy efficiency programs implemented nationally in the U.S. by utilities have rendered savings which have cost on average 0.03/kWh. This cost is still well below generation costs. However, as the lowest cost energy efficiency measures are adopted, this the cost effectiveness of further investment declines. Thus there is a need to more effectively find the most opportunities for savings regionally and nationally, so that the greatest cost effectiveness in implementing energy efficiency can be achieved. Integral to this process. are at scale energy audits. However, on-site building energy audits process are expensive, in the range of US1.29/m2-$5.37/m2 and there are an insufficient number of professionals to perform the audits. Energy audits that can be conducted at-scale and at low cost are needed. Research is presented that addresses at community-wide scales characterization of building envelope thermal characteristics via drive-by and fly-over GPS linked thermal imaging. A central question drives this research: Can single point-in-time thermal images be used to infer U-values and thermal capacitances of walls and roofs? Previous efforts to use thermal images to estimate U-values have been limited to rare steady exterior weather conditions. The approaches posed here are based upon the development two models first is a dynamic model of a building envelope component with unknown U-value and thermal capacitance. The weather conditions prior to the thermal image are used as inputs to the model. The model is solved to determine the exterior surface temperature, ultimately predicted the temperature at the thermal measurement time. The model U-value and thermal capacitance are tuned in order to force the error between the predicted surface temperature and the measured surface temperature from thermal imaging to be near zero. This model is developed simply to show that such a model cannot be relied upon to accurately estimate the U-value. The second is a data-based methodology. This approach integrates the exterior surface temperature measurements, historical utility data, and easily accessible or potentially easily accessible housing data. A Random Forest model is developed from a training subset of residences for which the envelope U-value is known. This model is used to predict the envelope U-value for a validation set of houses with unknown U-value. Demonstrated is an ability to estimate the wall/roof U-value with an R-squared value in the range of 0.97 and 0.96 respectively, using as few as 9 and 24 training houses for respectively wall and ceiling U-value estimation. The implication of this research is significant, offering the possibility of auditing residences remotely at-scale via aerial and drive-by thermal imaging.

Virus-mimetic nanovesicles as a versatile antigen-delivery system

PubMed Central

Zhang, Pengfei; Chen, Yixin; Zeng, Yun; Shen, Chenguang; Li, Rui; Guo, Zhide; Li, Shaowei; Zheng, Qingbing; Chu, Chengchao; Wang, Zhantong; Zheng, Zizheng; Tian, Rui; Ge, Shengxiang; Zhang, Xianzhong; Xia, Ning-Shao; Liu, Gang; Chen, Xiaoyuan

2015-01-01

It is a critically important challenge to rapidly design effective vaccines to reduce the morbidity and mortality of unexpected pandemics. Inspired from the way that most enveloped viruses hijack a host cell membrane and subsequently release by a budding process that requires cell membrane scission, we genetically engineered viral antigen to harbor into cell membrane, then form uniform spherical virus-mimetic nanovesicles (VMVs) that resemble natural virus in size, shape, and specific immunogenicity with the help of surfactants. Incubation of major cell membrane vesicles with surfactants generates a large amount of nano-sized uniform VMVs displaying the native conformational epitopes. With the diverse display of epitopes and viral envelope glycoproteins that can be functionally anchored onto VMVs, we demonstrate VMVs to be straightforward, robust and tunable nanobiotechnology platforms for fabricating antigen delivery systems against a wide range of enveloped viruses. PMID:26504197

Site-specific volumetric analysis of lung tumour motion

NASA Astrophysics Data System (ADS)

Pepin, Eric W.; Wu, Huanmei; Sandison, George A.; Langer, Mark; Shirato, Hiroki

2010-06-01

The treatment of lung cancer with radiation therapy is hindered by respiratory motion. Real-time adjustments to compensate for this motion are hampered by mechanical system latencies and imaging-rate restrictions. To better understand tumour motion behaviour for adaptive image-guided radiation therapy of lung cancer, the volume of a tumour's motion space was investigated. Motion data were collected by tracking an implanted fiducial using fluoroscopy at 30 Hz during treatment sessions. A total of 637 treatment fractions from 31 tumours were used in this study. For each fraction, data points collected from three consecutive breathing cycles were used to identify instantaneous tumour location. A convex hull was created over these data points, defining the tumour motion envelope. The study sought a correlation between the tumour location in the lung and the convex hull's volume and shape. It was found that tumours located in the upper apex had smaller motion envelopes (<50 mm3), whereas tumours located near the chest wall or diaphragm had larger envelopes (>70 mm3). Tumours attached to fixed anatomical structures had small motion spaces. Three general shapes described the tumour motion envelopes: 50% of motion envelopes enclosed largely 1D oscillation, 38% enclosed an ellipsoid path, 6% enclosed an arced path and 6% were of hybrid shape. This location-space correlation suggests it may be useful in developing a predictive model, but more work needs to be done to verify it.

Mutation of a Broadly Conserved Operon (RL3499-RL3502) from Rhizobium leguminosarum Biovar viciae Causes Defects in Cell Morphology and Envelope Integrity▿†

PubMed Central

Vanderlinde, Elizabeth M.; Magnus, Samantha A.; Tambalo, Dinah D.; Koval, Susan F.; Yost, Christopher K.

2011-01-01

The bacterial cell envelope is of critical importance to the function and survival of the cell; it acts as a barrier against harmful toxins while allowing the flow of nutrients into the cell. It also serves as a point of physical contact between a bacterial cell and its host. Hence, the cell envelope of Rhizobium leguminosarum is critical to cell survival under both free-living and symbiotic conditions. Transposon mutagenesis of R. leguminosarum strain 3841 followed by a screen to isolate mutants with defective cell envelopes led to the identification of a novel conserved operon (RL3499-RL3502) consisting of a putative moxR-like AAA+ ATPase, a hypothetical protein with a domain of unknown function (designated domain of unknown function 58), and two hypothetical transmembrane proteins. Mutation of genes within this operon resulted in increased sensitivity to membrane-disruptive agents such as detergents, hydrophobic antibiotics, and alkaline pH. On minimal media, the mutants retain their rod shape but are roughly 3 times larger than the wild type. On media containing glycine or peptides such as yeast extract, the mutants form large, distorted spheres and are incapable of sustained growth under these culture conditions. Expression of the operon is maximal during the stationary phase of growth and is reduced in a chvG mutant, indicating a role for this sensor kinase in regulation of the operon. Our findings provide the first functional insight into these genes of unknown function, suggesting a possible role in cell envelope development in Rhizobium leguminosarum. Given the broad conservation of these genes among the Alphaproteobacteria, the results of this study may also provide insight into the physiological role of these genes in other Alphaproteobacteria, including the animal pathogen Brucella. PMID:21357485

Identification of Cell Wall Synthesis Regulatory Genes Controlling Biomass Characteristics and Yield in Rice (Oryza Sativa)

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

Peng, Zhaohua PEng; Ronald, Palmela; Wang, Guo-Liang

This project aims to identify the regulatory genes of rice cell wall synthesis pathways using a cell wall removal and regeneration system. We completed the gene expression profiling studies following the time course from cell wall removal to cell wall regeneration in rice suspension cells. We also completed, total proteome, nuclear subproteome and histone modification studies following the course from cell wall removal and cell wall regeneration process. A large number of differentially expressed regulatory genes and proteins were identified. Meanwhile, we generated RNAi and over-expression transgenic rice for 45 genes with at least 10 independent transgenic lines for eachmore » gene. In addition, we ordered T-DNA and transposon insertion mutants for 60 genes from Korea, Japan, and France and characterized the mutants. Overall, we have mutants and transgenic lines for over 90 genes, exceeded our proposed goal of generating mutants for 50 genes. Interesting Discoveries a) Cell wall re-synthesis in protoplasts may involve a novel cell wall synthesis mechanism. The synthesis of the primary cell wall is initiated in late cytokinesis with further modification during cell expansion. Phragmoplast plays an essential role in cell wall synthesis. It services as a scaffold for building the cell plate and formation of a new cell wall. Only one phragmoplast and one new cell wall is produced for each dividing cell. When the cell wall was removed enzymatically, we found that cell wall re-synthesis started from multiple locations simultaneously, suggesting that a novel mechanism is involved in cell wall re-synthesis. This observation raised many interesting questions, such as how the starting sites of cell wall synthesis are determined, whether phragmoplast and cell plate like structures are involved in cell wall re-synthesis, and more importantly whether the same set of enzymes and apparatus are used in cell wall re-synthesis as during cytokinesis. Given that many known cell wall synthesis pathway genes are induced by removal of cell wall, some cell wall synthesis apparatus must be shared in both cases. The cell wall re-synthesis mechanism may have broad application because our preliminary assay indicates that the cell wall characteristics are highly different from those produced during cytokinesis. A thorough understanding on the regulation of cell wall re-synthesis may lead to improvement of cell wall characteristics. b) Removal of cell wall results in chromatin decondensation Another interesting observation was that removal of cell wall was associated with substantial chromatin change. Our DNA DAPI stain, chromatin MNase digestion, histone modification proteomics, protein differential expression analysis, and DNA oligo array studies all supported that substantial chromatin change was associated with removal of cell wall treatment. It is still under investigation if the chromatin change is associated with activation of cell wall synthesis genes, in which chromatin remodeling is required. Another possibility is that the cell wall is required for stabilizing the chromatin structure in plant cells. Given that spindle fiber is directly connected with both chromatin structure and cell wall synthesis, it is possible that there is an intrinsic connection between cell wall and chromatin.« less

A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

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

Mourez, Thomas; APHP, GH Saint-Louis-Lariboisiere, Laboratoire de Bacteriologie-Virologie, F-75010 Paris; Universite Paris 7 Denis Diderot, F-75010 Paris

2011-10-25

We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimericmore » particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.« less

Identification of a major polypeptide of the nuclear pore complex

PubMed Central

1982-01-01

The nuclear pore complex is a prominent structural component of the nuclear envelope that appears to regulate nucleoplasmic molecular movement. Up to now, none of its polypeptides have been defined. To identify possible pore complex proteins, we fractionated rat liver nuclear envelopes and microsomal membranes with strong protein perturbants into peripheral and intrinsic membrane proteins, and compared these fractions on SDS gels. From this analysis, we identified a prominent 190-kilodalton intrinsic membrane polypeptide that occurs specifically in nuclear envelopes. Lectin binding studies indicate that this polypeptide (gp 190) is the major nuclear envelope glycoprotein. Upon treatment of nuclear envelopes with Triton X-100, gp 190 remains associated with a protein substructure of the nuclear envelope consisting of pore complexes and nuclear lamina. We prepared monospecific antibodies to gp 190 for immunocytochemical localization. Immunofluorescence staining of tissue culture cells suggests that gp 190 occurs exclusively in the nucleus during interphase. This polypeptide becomes dispersed throughout the cell in mitotic prophase when the nuclear envelope is disassembled, and subsequently returns to the nuclear surfaces during telophase when the nuclear envelope is reconstructed. Immunoferritin labeling of Triton-treated rat liver nuclei demonstrates that gp 190 occurs exclusively in the nuclear pore complex, in the regions of the cytoplasmic (and possibly nucleoplasmic) pore complex annuli. A polypeptide that cross-reacts with gp 190 is present in diverse vertebrate species, as shown by antibody labeling of nitrocellulose SDS gel transfers. On the basis of its biochemical characteristics, we suggest that gp 190 may be involved in anchoring the pore complex to nuclear envelope membranes. PMID:7153248

Direction of flagellar rotation in bacterial cell envelopes.

PubMed Central

Ravid, S; Eisenbach, M

1984-01-01

Cell envelopes with functional flagella, isolated from wild-type strains of Escherichia coli and Salmonella typhimurium by formation of spheroplasts with penicillin and subsequent osmotic lysis, demonstrate counterclockwise (CCW)-biased rotation when energized with an electron donor for respiration, DL-lactate. Since the direction of flagellar rotation in bacteria is central to the expression of chemotaxis, we studied the cause of this bias. Our main observations were: (i) spheroplasts acquired a clockwise (CW) bias if instead of being lysed they were further incubated with penicillin; (ii) repellents temporarily caused CW rotation of tethered bacteria and spheroplasts but not of their derived cell envelopes; (iii) deenergizing CW-rotating cheV bacteria by KCN or arsenate treatment caused CCW bias; (iv) cell envelopes isolated from CW-rotating cheC and cheV mutants retained the CW bias, unlike envelopes isolated from cheB and cheZ mutants, which upon cytoplasmic release lost this bias and acquired CCW bias; and (v) an inwardly directed, artificially induced proton current rotated tethered envelopes in CCW direction, but an outwardly directed current was unable to rotate the envelopes. It is concluded that (i) a cytoplasmic constituent is required for the expression of CW rotation (or repression of CCW rotation) in strains which are not defective in the switch; (ii) in the absence of this cytoplasmic constituent, the motor is not reversible in such strains, and it probably is mechanically constricted so as to permit CCW sense of rotation only; (iii) the requirement of CW rotation for ATP is not at the level of the motor or the switch but at one of the preceding functional steps of the chemotaxis machinery; (iv) the cheC and cheV gene products are associated with the cytoplasmic membrane; and (v) direct interaction between the switch-motor system and the repellent sensors is improbable. Images PMID:6370958

A targeted mutation within the feline leukemia virus (FeLV) envelope protein immunosuppressive domain to improve a canarypox virus-vectored FeLV vaccine.

PubMed

Schlecht-Louf, Géraldine; Mangeney, Marianne; El-Garch, Hanane; Lacombe, Valérie; Poulet, Hervé; Heidmann, Thierry

2014-01-01

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the "mechanical" function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be "switched off" by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.

CASI Work Plan: Calendar Year 2013

DTIC Science & Technology

2013-02-15

reducing energy consumption in existing Army barracks using innovative heating, ventilating , and air-conditioning (HVAC) and building envelope...to enhance the effective “R-value” of insulation, thus reducing energy transfer through walls while maintaining comfortable temperatures for... consumption below ASHRAE’s goal for new facilities. This project will determine how effective the daylighting strategies are in providing adequate

Simulated Aging and Characterization of Phase Change Materials for Thermal Management of Building Envelopes

DTIC Science & Technology

2015-09-01

materials of a PCM wall or ceiling panel. BioPCMat™ absorbs heat in the daytime and releases that heat during the night. The dimension of the typical...micrographs of Energain PCM samples showed evidence of melting and re- ERDC/CERL TR-15-23 32 crystallization ; however, there was no significant

Cell envelope stress in mycobacteria is regulated by the novel signal transduction ATPase IniR in response to trehalose

PubMed Central

van Winden, Vincent J. C.; Sparrius, Marion; van de Weerd, Robert; Speer, Alexander; Ummels, Roy; Sherman, David R.

2017-01-01

The cell envelope of mycobacteria is a highly unique and complex structure that is functionally equivalent to that of Gram-negative bacteria to protect the bacterial cell. Defects in the integrity or assembly of this cell envelope must be sensed to allow the induction of stress response systems. The promoter that is specifically and most strongly induced upon exposure to ethambutol and isoniazid, first line drugs that affect cell envelope biogenesis, is the iniBAC promoter. In this study, we set out to identify the regulator of the iniBAC operon in Mycobacterium marinum using an unbiased transposon mutagenesis screen in a constitutively iniBAC-expressing mutant background. We obtained multiple mutants in the mce1 locus as well as mutants in an uncharacterized putative transcriptional regulator (MMAR_0612). This latter gene was shown to function as the iniBAC regulator, as overexpression resulted in constitutive iniBAC induction, whereas a knockout mutant was unable to respond to the presence of ethambutol and isoniazid. Experiments with the M. tuberculosis homologue (Rv0339c) showed identical results. RNAseq experiments showed that this regulatory gene was exclusively involved in the regulation of the iniBAC operon. We therefore propose to name this dedicated regulator iniBAC Regulator (IniR). IniR belongs to the family of signal transduction ATPases with numerous domains, including a putative sugar-binding domain. Upon testing different sugars, we identified trehalose as an activator and metabolic cue for iniBAC activation, which could also explain the effect of the mce1 mutations. In conclusion, cell envelope stress in mycobacteria is regulated by IniR in a cascade that includes trehalose. PMID:29281637

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

PubMed Central

Millet, Jean Kaoru; Whittaker, Gary R.

2016-01-01

Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay. PMID:28018942

The formation of zona radiata in Pseudosciaena crocea revealed by light and transmission electron microscopy.

PubMed

Ma, Xiao-Xin; Zhu, Jun-Quan; Zhou, Hong; Yang, Wan-Xi

2012-02-01

The egg envelope is an essential structure occurring during oogenesis. It plays an important role during the process of fertilization in the large yellow croaker Pseudosciaena crocea. Elucidation of egg envelope formation helps us to understand fertilization mechanisms in teleosts. In the present work, we studied the formation of egg envelope in P. crocea by light microscopy, as well as by transmission and scanning electron microscopy. Four layers exist outside the oocyte plasmalemma, i.e., theca cell layer, basal membrane, granulosa cell layer and zona radiata. According to our observation, zona radiata is a multilaminar structure just like the same structure reported in teleosts, but the origin of this structure is a little different. Before it is formed, a peripheral space filled with different density of vesicles is the place where zona radiata is formed. Zona radiata (Z1) is secreted only by oocyte itself, it belongs to the primary envelope; zona radiata 2 (Z2) and zona radiata 3 (Z3) belong to the secondary envelope, because the two layers are formed after granulosa cells appear, and microvilli participate this process. It is very interesting that Z2 and Z3 are situated between Z1 and the granulosa cell first, but they translocate to the other side of Z1. This microanatomy difference may due to the participation of microvilli. The new finding about egg envelope formation in P. crocea will help us to do further investigation on fertilization mechanisms and will make artificial breeding possible which may contribute to the resource recovery of this species. Copyright © 2011 Elsevier Ltd. All rights reserved.

Numerical simulation of dark envelope soliton in plasma

NASA Astrophysics Data System (ADS)

Wang, Fang-Ping; Han, Juan-fang; Zhang, Jie; Gao, Dong-Ning; Li, Zhong-Zheng; Duan, Wen-Shan; Zhang, Heng

2018-03-01

One-dimensional (1-D) particle-in-cell simulation is used to study the propagation of dark envelop solitons described by the nonlinear Schrödinger equation (NLSE) in electron-ion plasmas. The rational solution of the NLSE is presented, which is proposed as an effective tool for studying the dark envelope soliton in plasma. It is demonstrated by our numerical simulation that there is dark envelope soliton in electron-ion plasmas. The numerical results are in good agreements with the analytical ones from the NLSE which is obtained from the reductive perturbation method. The limitation of the amplitude of dark envelop solitons in plasma is noticed.

Architecture of dermatophyte cell Walls: Electron microscopic and biochemical analysis

NASA Technical Reports Server (NTRS)

Nozawa, Y.; Kitajima, Y.

1984-01-01

A review with 83 references on the cell wall structure of dermatophytes is presented. Topics discussed include separation and preparation of cell walls; microstructure of cell walls by electron microscopy; chemical composition of cell walls; structural model of cell walls; and morphological structure of cell walls.

Antibody-mediated targeting of replication-competent retroviral vectors.

PubMed

Tai, Chien-Kuo; Logg, Christopher R; Park, Jinha M; Anderson, W French; Press, Michael F; Kasahara, Noriyuki

2003-05-20

Replication-competent murine leukemia virus (MLV) vectors can be engineered to achieve high efficiency gene transfer to solid tumors in vivo and tumor-restricted replication, however their safety can be further enhanced by redirecting tropism of the virus envelope. We have therefore tested the targeting capability and replicative stability of ecotropic and amphotropic replication-competent retrovirus (RCR) vectors containing two tandem repeats from the immunoglobulin G-binding domain of Staphylococcal protein A inserted into the proline-rich "hinge" region of the envelope, which enables modular use of antibodies of various specificities for vector targeting. The modified envelopes were efficiently expressed and incorporated into virions, were capable of capturing monoclonal anti-HER2 antibodies, and mediated efficient binding of the virus-antibody complex to HER2-positive target cells. While infectivity was markedly reduced by pseudotyping with targeted envelopes alone, coexpression of wild-type envelope rescued efficient cellular entry. Both ecotropic and amphotropic RCR vector/anti-HER2 antibody complexes achieved significant enhancement of transduction on murine target cells overexpressing HER2, which could be competed by preincubation with excess free antibodies. Interestingly, HER2-expressing human breast cancer cells did not show enhancement of transduction despite efficient antibody-mediated cell surface binding, suggesting that target cell-specific parameters markedly affect the efficiency of post-binding entry processes. Serial replication of targeted vectors resulted in selection of Z domain deletion variants, but reduction of the overall size of the vector genome enhanced its stability. Application of antibody-mediated targeting to the initial localization of replication-competent virus vectors to tumor sites will thus require optimized target selection and vector design.

Arabidopsis Regenerating Protoplast: A Powerful Model System for Combining the Proteomics of Cell Wall Proteins and the Visualization of Cell Wall Dynamics

PubMed Central

Yokoyama, Ryusuke; Kuki, Hiroaki; Kuroha, Takeshi; Nishitani, Kazuhiko

2016-01-01

The development of a range of sub-proteomic approaches to the plant cell wall has identified many of the cell wall proteins. However, it remains difficult to elucidate the precise biological role of each protein and the cell wall dynamics driven by their actions. The plant protoplast provides an excellent means not only for characterizing cell wall proteins, but also for visualizing the dynamics of cell wall regeneration, during which cell wall proteins are secreted. It therefore offers a unique opportunity to investigate the de novo construction process of the cell wall. This review deals with sub-proteomic approaches to the plant cell wall through the use of protoplasts, a methodology that will provide the basis for further exploration of cell wall proteins and cell wall dynamics. PMID:28248244

Genetics Home Reference: triple A syndrome

MedlinePlus

... understood. Within cells, ALADIN is found in the nuclear envelope, the structure that surrounds the nucleus and ... protein from reaching its proper location in the nuclear envelope. The absence of ALADIN in the nuclear ...

Role of Cysteines in Stabilizing the Randomized Receptor Binding Domains within Feline Leukemia Virus Envelope Proteins.

PubMed

Valdivieso-Torres, Leonardo; Sarangi, Anindita; Whidby, Jillian; Marcotrigiano, Joseph; Roth, Monica J

2015-12-30

Retargeting of gammaretroviral envelope proteins has shown promising results in the isolation of novel isolates with therapeutic potential. However, the optimal conditions required to obtain high-affinity retargeted envelope proteins with narrow tropism are not understood. This study highlights the advantage of constrained peptides within receptor binding domains and validates the random library screening technique of obtaining novel retargeted Env proteins. Using a modified vector backbone to screen the envelope libraries on 143B osteosarcoma cells, three novel and unique retargeted envelopes were isolated. The use of complex disulfide bonds within variable regions required for receptor binding is found within natural gammaretroviral envelope isolates. Interestingly, two of the isolates, named AII and BV2, have a pair of cysteines located within the randomized region of 11 amino acids similar to that identified within the CP Env, an isolate identified in a previous Env library screen on the human renal carcinoma Caki-1 cell line. The amino acids within the randomized region of AII and BV2 envelopes that are essential for viral infection have been identified in this study and include these cysteine residues. Through mutagenesis studies, the putative disulfide bond pairs including and beyond the randomized region were examined. In parallel, the disulfide bonds of CP Env were identified using mass spectrometry. The results indicate that this pair of cysteines creates the structural context to position key hydrophobic (F and W) and basic (K and H) residues critical for viral titer and suggest that AII, BV2, and CP internal cysteines bond together in distinct ways. Retargeted gammaretroviral particles have broad applications for therapeutic use. Although great advances have been achieved in identifying new Env-host cell receptor pairs, the rules for designing optimal Env libraries are still unclear. We have found that isolates with an additional pair of cysteines within the randomized region have the highest transduction efficiencies. This emphasizes the importance of considering cysteine pairs in the design of new libraries. Furthermore, our data clearly indicate that these cysteines are essential for viral infectivity by presenting essential residues to the host cell receptor. These studies facilitate the screening of Env libraries for functional entry into target cells, allowing the identification of novel gammaretroviral Envs targeting alternative host cell receptors for gene and protein delivery. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A Role for Caenorhabditis elegans Importin IMA-2 in Germ Line and Embryonic Mitosis

PubMed Central

Geles, Kenneth G.; Johnson, Jeffrey J.; Jong, Sena; Adam, Stephen A.

2002-01-01

The importin α family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin α proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin α proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype. ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis. PMID:12221121

Role of the Phosphatidylserine Receptor TIM-1 in Enveloped-Virus Entry

PubMed Central

Moller-Tank, Sven; Kondratowicz, Andrew S.; Davey, Robert A.; Rennert, Paul D.

2013-01-01

The cell surface receptor T cell immunoglobulin mucin domain 1 (TIM-1) dramatically enhances filovirus infection of epithelial cells. Here, we showed that key phosphatidylserine (PtdSer) binding residues of the TIM-1 IgV domain are critical for Ebola virus (EBOV) entry through direct interaction with PtdSer on the viral envelope. PtdSer liposomes but not phosphatidylcholine liposomes competed with TIM-1 for EBOV pseudovirion binding and transduction. Further, annexin V (AnxV) substituted for the TIM-1 IgV domain, supporting a PtdSer-dependent mechanism. Our findings suggest that TIM-1-dependent uptake of EBOV occurs by apoptotic mimicry. Additionally, TIM-1 enhanced infection of a wide range of enveloped viruses, including alphaviruses and a baculovirus. As further evidence of the critical role of enveloped-virion-associated PtdSer in TIM-1-mediated uptake, TIM-1 enhanced internalization of pseudovirions and virus-like proteins (VLPs) lacking a glycoprotein, providing evidence that TIM-1 and PtdSer-binding receptors can mediate virus uptake independent of a glycoprotein. These results provide evidence for a broad role of TIM-1 as a PtdSer-binding receptor that mediates enveloped-virus uptake. Utilization of PtdSer-binding receptors may explain the wide tropism of many of these viruses and provide new avenues for controlling their virulence. PMID:23698310

Death of mitochondria during programmed cell death of leaf mesophyll cells.

PubMed

Selga, Tūrs; Selga, Maija; Pāvila, Vineta

2005-12-01

The role of plant mitochondria in the programmed cell death (PCD) is widely discussed. However, spectrum and sequence of mitochondrial structural changes during different types of PCD in leaves are poorly described. Pea, cucumber and rye plants were grown under controlled growing conditions. A part of them were sprinkled with ethylene releaser to accelerate cell death. During yellowing the palisade parenchyma mitochondria were attracted to nuclear envelope. Mitochondrial matrix became electron translucent. Mitochondria entered vacuole by invagination of tonoplast and formed multivesicular bodies. Ethephon treatment increased the frequency of sticking of mitochondria to the nuclear envelope or chloroplasts and peroxisomes. Mitochondria divided by different mechanisms and became enclosed in Golgi and ER derived authopagic vacuoles or in the central vacuole. Several fold increase of the diameter of cristae became typical. In all cases mitochondria were attached to nuclear envelope. It can be considered as structural mechanism of promoting of PCD.

Data on the association of the nuclear envelope protein Sun1 with nucleoli.

PubMed

Moujaber, Ossama; Omran, Nawal; Kodiha, Mohamed; Pié, Brigitte; Cooper, Ellis; Presley, John F; Stochaj, Ursula

2017-08-01

SUN proteins participate in diverse cellular activities, many of which are connected to the nuclear envelope. Recently, the family member SUN1 has been linked to novel biological activities. These include the regulation of nucleoli, intranuclear compartments that assemble ribosomal subunits. We show that SUN1 associates with nucleoli in several mammalian epithelial cell lines. This nucleolar localization is not shared by all cell types, as SUN1 concentrates at the nuclear envelope in ganglionic neurons and non-neuronal satellite cells. Database analyses and Western blotting emphasize the complexity of SUN1 protein profiles in different mammalian cells. We constructed a STRING network which identifies SUN1-related proteins as part of a larger network that includes several nucleolar proteins. Taken together, the current data highlight the diversity of SUN1 proteins and emphasize the possible links between SUN1 and nucleoli.

Noninfectious virus-like particles produced by Moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents

PubMed Central

Sharma, Sanjai; Murai, Fukashi; Miyanohara, Atsushi; Friedmann, Theodore

1997-01-01

Retrovirus packaging cell lines expressing the Moloney murine leukemia virus gag and pol genes but lacking virus envelope genes produce virus-like particles constitutively, whether or not they express a transcript from an integrated retroviral provirus. In the absence of a proviral transcript, the assembled particles contain processed gag and reverse transcriptase, and particles made by cells expressing an integrated lacZ provirus also contain viral RNA. The virus-like particles from both cell types are enveloped and are secreted/budded into the extracellular space but are noninfectious. Their physicochemical properties are similar to those of mature retroviral particles. The noninfectious gag pol RNA particles can readily be made infectious by the addition of lipofection reagents to produce preparations with titers of up to 105 colony-forming units per ml. PMID:9380714

Thermal envelope field measurements in an energy-efficient office and dormitory

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

Christian, J.E.

1983-04-01

A 345-m/sup 2/ earth-covered structure located at Oak Ridge National Laboratory is the focus of a DOE-sponsored building envelope research project. Based on field-measured data, heating the office and dormitory building over the 1981-1982 heating season cost $1.70/m/sup 2/ ($0.16/ft/sup 2/), assuming the cost of electricity to be $0.057/kWh. The building's thermal integrity factor is 0.016 kWh/m/sup 2/ /sup 0/C (2.8 Btu/ft/sup 2/ /sup 0/F). A preliminary DOE-2 model estimates the monthly electric energy needs for heating to be within 5% of our field data-derived estimates. DOE-2 building simulations suggest that this earth-covered, passively solar heated office dormitory saves 30%more » of the space heating and 26% of the cooling costs of an energy-efficient above grade structure. A preliminary winter energy balance has been generated from data collected in February and March and provides a fractional breakdown of thermal losses and gains. Performances have been isolated for several of the energy-conserving components: the earth-covered roof, the bermed wall, and the nonvented Trombe wall. The earth-covered roof system showed an overall thermal transmittance of 0.18 W/m/sup 2/ /sup 0/C (R = 31 h ft/sup 2/ /sup 0/F Btu/sup -1/). The thermocouple wells in the earth surrounding the building indicate that burying a wall is more energy efficient than berming. During one week in February, the Trombe wall produced a 50% greater net thermal gain to the building than an equivalent area of south-facing windows.« less

Characterization of the Sclerotinia sclerotiorum cell wall proteome.

PubMed

Liu, Longzhou; Free, Stephen J

2016-08-01

We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)-anchored cell wall proteins and 30 non-GPI-anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes. © 2015 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.

Proteins required for lipopolysaccharide assembly in Escherichia coli form a trans-envelope complex†

PubMed Central

Chng, Shu-Sin; Gronenberg, Luisa S.; Kahne, Daniel

2010-01-01

The viability of Gram-negative organisms is dependent on the proper placement of lipopolysaccharide (LPS) in the outer leaflet of its outer membrane. LPS is synthesized inside the cell and transported to the surface by seven essential Lpt proteins. How these proteins cooperate to transport LPS is unknown. We show that these Lpt proteins can be found in a membrane fraction that contains inner and outer membranes, and that they co-purify. This constitutes the first evidence that the Lpt proteins form a trans-envelope complex. We suggest that this protein bridge provides a route for LPS transport across the cell envelope. PMID:20446753

Human Cytomegalovirus nuclear egress and secondary envelopment are negatively affected in the absence of cellular p53

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

Kuan, Man I; O’Dowd, John M.; Chughtai, Kamila

2016-10-15

Human Cytomegalovirus (HCMV) infection is compromised in cells lacking p53, a transcription factor that mediates cellular stress responses. In this study we have investigated compromised functional virion production in cells with p53 knocked out (p53KOs). Infectious center assays found most p53KOs released functional virions. Analysis of electron micrographs revealed modestly decreased capsid production in infected p53KOs compared to wt. Substantially fewer p53KOs displayed HCMV-induced infoldings of the inner nuclear membrane (IINMs). In p53KOs, fewer capsids were found in IINMs and in the cytoplasm. The deficit in virus-induced membrane remodeling within the nucleus of p53KOs was mirrored in the cytoplasm, withmore » a disproportionately smaller number of capsids re-enveloped. Reintroduction of p53 substantially recovered these deficits. Overall, the absence of p53 contributed to inhibition of the formation and function of IINMs and re-envelopment of the reduced number of capsids able to reach the cytoplasm. -- Highlights: •The majority of p53KO cells release fewer functional virions than wt cells. •Nucleocapsids do not efficiently exit the nucleus in p53KO cells. •Infoldings of the inner nuclear membrane are not efficiently formed in p53KO cells. •Cytoplasmic capsids are not efficiently re-enveloped in p53KO cells. •Reintroduction of p53 largely ameliorates these phenotypes.« less

The Tubular Sheaths Encasing Methanosaeta thermophila Filaments Are Functional Amyloids*

PubMed Central

Dueholm, Morten S.; Larsen, Poul; Finster, Kai; Stenvang, Marcel R.; Christiansen, Gunna; Vad, Brian S.; Bøggild, Andreas; Otzen, Daniel E.; Nielsen, Per Halkjær

2015-01-01

Archaea are renowned for their ability to thrive in extreme environments, although they can be found in virtually all habitats. Their adaptive success is linked to their unique cell envelopes that are extremely resistant to chemical and thermal denaturation and that resist proteolysis by common proteases. Here we employ amyloid-specific conformation antibodies and biophysical techniques to show that the extracellular cell wall sheaths encasing the methanogenic archaea Methanosaeta thermophila PT are functional amyloids. Depolymerization of sheaths and subsequent MS/MS analyses revealed that the sheaths are composed of a single major sheath protein (MspA). The amyloidogenic nature of MspA was confirmed by in vitro amyloid formation of recombinant MspA under a wide range of environmental conditions. This is the first report of a functional amyloid from the archaeal domain of life. The amyloid nature explains the extreme resistance of the sheath, the elastic properties that allow diffusible substrates to penetrate through expandable hoop boundaries, and how the sheaths are able to split and elongate outside the cell. The archaeal sheath amyloids do not share homology with any of the currently known functional amyloids and clearly represent a new function of the amyloid protein fold. PMID:26109065

The molecular mechanisms on glomangiopericytoma invasion

PubMed Central

2013-01-01

Purpose To observed the imaging and pathological features of the glomangiopericytoma. Experimental design In this paper we report a typical case of glomangiopericytoma arising in the skull base area and summarize the clinical manifestations, imaging and pathological features of such diseases. Results Immunohistochemical staining confirmed the tumor cells were strongly positive to Vim, SMA, MSA and negative to CD31, CD34. Partial cells were positive to FVIII. The imaging can’t confirm the diagnosis but indicate the the tumor has intact envelope.The cells in the tumor envelope is positive to Vim and negative SMA and FVIII. These findings were compatible with glomangiopericytoma and the cells in the tumor envelope is not glomangiopericytoma cells. Conclusion In view of the clinical and pathological features of the glomangiopericytoma, we believe that the surgery is the best treatment so far and the tumor can be resected completely. The above results can be preliminary reason to explain the low recurrence of such diseases. PMID:24074285

The Role of Auxin in Cell Wall Expansion

PubMed Central

2018-01-01

Plant cells are surrounded by cell walls, which are dynamic structures displaying a strictly regulated balance between rigidity and flexibility. Walls are fairly rigid to provide support and protection, but also extensible, to allow cell growth, which is triggered by a high intracellular turgor pressure. Wall properties regulate the differential growth of the cell, resulting in a diversity of cell sizes and shapes. The plant hormone auxin is well known to stimulate cell elongation via increasing wall extensibility. Auxin participates in the regulation of cell wall properties by inducing wall loosening. Here, we review what is known on cell wall property regulation by auxin. We focus particularly on the auxin role during cell expansion linked directly to cell wall modifications. We also analyze downstream targets of transcriptional auxin signaling, which are related to the cell wall and could be linked to acid growth and the action of wall-loosening proteins. All together, this update elucidates the connection between hormonal signaling and cell wall synthesis and deposition. PMID:29565829

The Role of Auxin in Cell Wall Expansion.

PubMed

Majda, Mateusz; Robert, Stéphanie

2018-03-22

Plant cells are surrounded by cell walls, which are dynamic structures displaying a strictly regulated balance between rigidity and flexibility. Walls are fairly rigid to provide support and protection, but also extensible, to allow cell growth, which is triggered by a high intracellular turgor pressure. Wall properties regulate the differential growth of the cell, resulting in a diversity of cell sizes and shapes. The plant hormone auxin is well known to stimulate cell elongation via increasing wall extensibility. Auxin participates in the regulation of cell wall properties by inducing wall loosening. Here, we review what is known on cell wall property regulation by auxin. We focus particularly on the auxin role during cell expansion linked directly to cell wall modifications. We also analyze downstream targets of transcriptional auxin signaling, which are related to the cell wall and could be linked to acid growth and the action of wall-loosening proteins. All together, this update elucidates the connection between hormonal signaling and cell wall synthesis and deposition.

Electron-Beam Dynamics for an Advanced Flash-Radiography Accelerator

DOE PAGES

Ekdahl, Carl

2015-11-17

Beam dynamics issues were assessed for a new linear induction electron accelerator being designed for multipulse flash radiography of large explosively driven hydrodynamic experiments. Special attention was paid to equilibrium beam transport, possible emittance growth, and beam stability. Especially problematic would be high-frequency beam instabilities that could blur individual radiographic source spots, low-frequency beam motion that could cause pulse-to-pulse spot displacement, and emittance growth that could enlarge the source spots. Furthermore, beam physics issues were examined through theoretical analysis and computer simulations, including particle-in-cell codes. Beam instabilities investigated included beam breakup, image displacement, diocotron, parametric envelope, ion hose, and themore » resistive wall instability. The beam corkscrew motion and emittance growth from beam mismatch were also studied. It was concluded that a beam with radiographic quality equivalent to the present accelerators at Los Alamos National Laboratory will result if the same engineering standards and construction details are upheld.« less

Electron-beam dynamics for an advanced flash-radiography accelerator

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

Ekdahl, Carl August Jr.

2015-06-22

Beam dynamics issues were assessed for a new linear induction electron accelerator. Special attention was paid to equilibrium beam transport, possible emittance growth, and beam stability. Especially problematic would be high-frequency beam instabilities that could blur individual radiographic source spots, low-frequency beam motion that could cause pulse-to-pulse spot displacement, and emittance growth that could enlarge the source spots. Beam physics issues were examined through theoretical analysis and computer simulations, including particle-in cell (PIC) codes. Beam instabilities investigated included beam breakup (BBU), image displacement, diocotron, parametric envelope, ion hose, and the resistive wall instability. Beam corkscrew motion and emittance growth frommore » beam mismatch were also studied. It was concluded that a beam with radiographic quality equivalent to the present accelerators at Los Alamos will result if the same engineering standards and construction details are upheld.« less

Fusion between perinuclear virions and the outer nuclear membrane requires the fusogenic activity of herpes simplex virus gB.

PubMed

Wright, Catherine C; Wisner, Todd W; Hannah, Brian P; Eisenberg, Roselyn J; Cohen, Gary H; Johnson, David C

2009-11-01

Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic "fusion loops." These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.

Russian Apartment Building Thermal Response Models for Retrofit Selection and Verification

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

Armstrong, Peter R.; Dirks, James A.; Reilly, Raymond W.

2000-08-21

The Enterprise Housing Divestiture Project (EHDP) aims to identify cost-effective energy efficiency and conservation measures for Russian apartment buildings and to implement these measures in the entire stock of buildings undergoing divestiture in six cities. Short-term measurements of infiltration and exterior wall heat-loss coefficient were made in the cities of Cheropovets, Orenburg, Petrozavodsk, Ryazan, and Vladimir. Long-term monitoring equipment was installed in six or more buildings in the aforementioned and in the city of Volxhov. The results of these measurements will be used to calibrate models used to select optimal retrofit packages and to verify energy savings. The retrofit categoriesmore » representing the largest technical potential in these buildings are envelope, heat recovery, and heating/hot water system improvements. This paper describes efforts to establish a useful thermal model calibration process. The model structures and analytical methods for obtaining building parameters from time series weather, energy use, and thermal response data are developed. Our experience applying these methods to two, nominally identical 5-story apartment buildings in the city of Ryazan is presented. Building envelope UA?s inferred from measured whole-building thermal response data are compared with UA?s based on window and wall U-values (the latter obtained by ASTM in-situ measurements of 20 wall sections in various Ryazan panel buildings) as well. The UA's obtained by these completely independent measurements differ by less than 10%.« less

Thermal-envelope field measurements in an energy-efficient office/dormitory

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

Christian, J.E.

1982-01-01

A 345 m/sup 2/ earth-covered structure located at the Oak Ridge National Laboratory is the focus of a DOE sponsored building-envelope research project. To heat the office/dormitory building over the 1981-1982 heating season would cost $1.70/m/sup 2/ ($0.16/ft/sup 2/), assuming $0.07/kWh. The thermal-integrity factor is 0.016 kWh/m/sup 2/ /sup 0/C (2.8 Btu/ft/sup 2/ /sup 0/F). A preliminary DOE-II model estimates the monthly electric energy needs for heating within 5% of field data derived estimates. DOE-II building simulations suggest that this earth-covered/passively heated office dormitory saves 30% for space heating and 26% for cooling compared to an energy efficient above grademore » structure. A preliminary winter energy balance has been generated from data collected in February and March providing a fractional breakdown of thermal losses and gains. A number of the energy-conserving component performances have been isolated; earth-covered roof, bermed wall, and nonvented trombe wall. The earth-covered roof system showed an overall thermal transmittance of 0.18 W/m/sup 2///sup 0/C (R=31 hr ft/sup 2/ /sup 0/F/Btu). The thermocouple wells located in the earth surrounding the building indicate the additional energy savings of burying over berming. For one week in February the trombe wall produced a 50% greater net thermal gain to the building then south facing windows per equivalent unit area.« less

Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance

PubMed Central

Ene, Iuliana V.; Walker, Louise A.; Schiavone, Marion; Lee, Keunsook K.; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A. R.; Munro, Carol A.

2015-01-01

ABSTRACT The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. PMID:26220968

Mouthparts in Leptotrombidium larvae (Acariformes: Trombiculidae).

PubMed

Shatrov, Andrey B; Takahashi, Mamoru; Misumi, Hitoko; Takahashi, Yumi

2016-04-01

Mouthparts of Leptotrombidium larvae (Acariformes: Trombiculidae), potential vectors of tsutsugamushi disease agents, were studied in detail using light microscopy, scanning electron microscopy, and transmission electron microscopy. The mouthparts incorporated within the pseudotagma gnathosoma are composed of the infracapitulum ventrally and the chelicerae dorsally. The ventral wall of the infracapitulum is formed by a wide mentum posteriorly and a narrowed malapophysis anteriorly. The malapophysis firmly envelops the distal cheliceral portions by its lateral walls. The lateral lips of the malapophysis are flexible structures hiding the cheliceral blades in inactive condition and turning back forming a type of temporary sucker closely applied to the host skin during feeding. The roof of the infracapitulum is formed by a weakly sclerotized labrum anteriorly and a cervix with the capitular apodemes extending posteriorly. The labral muscles are lacking. The capitular apodemes serve as origin for pharyngeal dilators running to the dorsal wall of the pharynx fused with the bottom of the infracapitulum. The basal cheliceral segments are separated from each other besides the very posterior portions where they are movably joined by the inner walls. The sigmoid pieces serve for insertion of the cheliceral elevators originating at the posterior portions of the basal segments. The movable digits reveal the solid basal sclerite and the cheliceral blade curved upward with a tricuspid cap on its tip. Dendrites of nerve cells run along the digits to their tips. The ganglia are placed within the basal segments just behind the movable digits. The chelicerae also reveal well developed flexible fixed digits overhanging the basal portions of the blades. The gnathosoma possesses several sets of extrinsic muscles originating at the scutum and at the soft cuticle behind it. Laterally, the gnathosoma bears five-segmented palps with a trifurcate palpal claw. © 2016 Wiley Periodicals, Inc.

Antagonistic Effect of Monovalent Cations in Maintenance of Cellular Integrity of a Marine Bacterium1

PubMed Central

De Voe, Irving W.; Oginsky, Evelyn L.

1969-01-01

The susceptibility of a marine bacterium, designated isolate c-A1, to lysis in distilled water and in salt solutions has been found to be a function of Na+ concentration. Optical densities of cells pre-exposed to 0.05 m MgCl2 were maintained in 1.0 m KCl, whereas those of cells pre-exposed to 1.0 m NaCl were not maintained at any KCl concentration tested. Cells transferred from MgCl2 to low concentrations of NaCl underwent more extensive lysis than did those transferred to distilled water. The degree of disruption of cells transferred to distilled water from mixtures of 0.05 m MgCl2 and NaCl (0 to 1.0 m) was dependent on the concentration of NaCl; similar results were obtained with LiCl, but not with KCl. In electron micrographs of thin sections, c-A1 cell envelopes consisted of two double-track layers which fractured and peeled apart on lysis after pre-exposure to NaCl-MgCl2 mixtures. Envelope eruptions or “hernias” occurred only in lysed cells pre-exposed to NaCl alone. No evidence for a functional lytic enzyme was found. Comparative studies on a terrestrial pseudomonad with a multilayered envelope indicated that preexposure to NaCl did not enhance the susceptibility of this cell to lysis in distilled water. The lytic susceptibility of the marine bacterium is considered to be the consequence of competition between specific monovalent cations and Mg++ for electrostatic interactions with components of the cell envelope of this organism. Images PMID:5788707

Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance.

PubMed

Ene, Iuliana V; Walker, Louise A; Schiavone, Marion; Lee, Keunsook K; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A R; Munro, Carol A; Brown, Alistair J P

2015-07-28

The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by a network of cell wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how cell wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical cell wall remodeling enzymes control cell wall flexibility, and its regulation is strongly dependent on host nutritional inputs. We also demonstrate an entirely new level of cell wall dynamism, where significant architectural changes and structural realignment occur within seconds of an osmotic shock. Copyright © 2015 Ene et al.

Archaeal S-Layers: Overview and Current State of the Art.

PubMed

Rodrigues-Oliveira, Thiago; Belmok, Aline; Vasconcellos, Deborah; Schuster, Bernhard; Kyaw, Cynthia M

2017-01-01

In contrast to bacteria, all archaea possess cell walls lacking peptidoglycan and a number of different cell envelope components have also been described. A paracrystalline protein surface layer, commonly referred to as S-layer, is present in nearly all archaea described to date. S-layers are composed of only one or two proteins and form different lattice structures. In this review, we summarize current understanding of archaeal S-layer proteins, discussing topics such as structure, lattice type distribution among archaeal phyla and glycosylation. The hexagonal lattice type is dominant within the phylum Euryarchaeota, while in the Crenarchaeota this feature is mainly associated with specific orders. S-layers exclusive to the Crenarchaeota have also been described, which are composed of two proteins. Information regarding S-layers in the remaining archaeal phyla is limited, mainly due to organism description through only culture-independent methods. Despite the numerous applied studies using bacterial S-layers, few reports have employed archaea as a study model. As such, archaeal S-layers represent an area for exploration in both basic and applied research.

Archaeal S-Layers: Overview and Current State of the Art

PubMed Central

Rodrigues-Oliveira, Thiago; Belmok, Aline; Vasconcellos, Deborah; Schuster, Bernhard; Kyaw, Cynthia M.

2017-01-01

In contrast to bacteria, all archaea possess cell walls lacking peptidoglycan and a number of different cell envelope components have also been described. A paracrystalline protein surface layer, commonly referred to as S-layer, is present in nearly all archaea described to date. S-layers are composed of only one or two proteins and form different lattice structures. In this review, we summarize current understanding of archaeal S-layer proteins, discussing topics such as structure, lattice type distribution among archaeal phyla and glycosylation. The hexagonal lattice type is dominant within the phylum Euryarchaeota, while in the Crenarchaeota this feature is mainly associated with specific orders. S-layers exclusive to the Crenarchaeota have also been described, which are composed of two proteins. Information regarding S-layers in the remaining archaeal phyla is limited, mainly due to organism description through only culture-independent methods. Despite the numerous applied studies using bacterial S-layers, few reports have employed archaea as a study model. As such, archaeal S-layers represent an area for exploration in both basic and applied research. PMID:29312266

Morphological and biochemical features of Borrelia burgdorferi pleomorphic forms

PubMed Central

Herranen, Anni; Schwarzbach, Armin; Gilbert, Leona

2015-01-01

The spirochaete bacterium Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, the most common tick-borne infection in the northern hemisphere. There is a long-standing debate regarding the role of pleomorphic forms in Lyme disease pathogenesis, while very little is known about the characteristics of these morphological variants. Here, we present a comprehensive analysis of B. burgdorferi pleomorphic formation in different culturing conditions at physiological temperature. Interestingly, human serum induced the bacterium to change its morphology to round bodies (RBs). In addition, biofilm-like colonies in suspension were found to be part of B. burgdorferi’s normal in vitro growth. Further studies provided evidence that spherical RBs had an intact and flexible cell envelope, demonstrating that they are not cell wall deficient, or degenerative as previously implied. However, the RBs displayed lower metabolic activity compared with spirochaetes. Furthermore, our results indicated that the different pleomorphic variants were distinguishable by having unique biochemical signatures. Consequently, pleomorphic B. burgdorferi should be taken into consideration as being clinically relevant and influence the development of novel diagnostics and treatment protocols. PMID:25564498

Importance of MAP Kinases during Protoperithecial Morphogenesis in Neurospora crassa

PubMed Central

Jeffree, Chris E.; Oborny, Radek; Boonyarungsrit, Patid; Read, Nick D.

2012-01-01

In order to produce multicellular structures filamentous fungi combine various morphogenetic programs that are fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; confirmed that all three MAP kinase cascades are required for sexual development; and showed that the three different cascades each have distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion. Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal adhesion, and envelopment during the construction of fertilizable protoperithecia. PMID:22900028

Isolation, Chemical Composition, and Ultrastructural Features of the Cell Membrane of the Mycoplasma-Like Organism Spiroplasma citri

PubMed Central

Razin, Shmuel; Hasin, Miriam; Ne'eman, Zvi; Rottem, Shlomo

1973-01-01

Thin sections of Spiroplasma citri, a mycoplasma-like organism isolated from citrus infected with “Stubborn” disease, showed the organisms to be limited by a single trilaminar plasma membrane. An additional outer layer could, however, be frequently seen in freeze-etched preparations of unwashed cells. The organisms were found to be extremely sensitive to lysis by osmotic shock. The cell membrane of S. citri isolated in this way resembled that of mycoplasmas in ultrastructure and gross chemical composition. The isolated membranes showed the characteristic trilaminar shape in section and the typical particle-studded fracture faces in freeze-etched preparations. Protein and lipid formed over 80% of the total dry weight of the membrane, which had a density of ~1.180 g/cm3. Cholesterol constituted over 20% of the total membrane lipid. Phosphatidyl-glycerol, synthesized by the organisms, was the major phospholipid. Significant amounts of hexosamine (15 to 35 μg/mg of membrane protein) could be found in the membrane preparations. Our results support the thesis that S. citri does not possess a cell wall, either of the gram-positive or the gram-negative type, though it may be coated by some other type of an envelope or by a slime layer, at least temporarily. Images PMID:4127633

Cdk1 Activates Pre-mitotic Nuclear Envelope Dynein Recruitment and Apical Nuclear Migration in Neural Stem Cells.

PubMed

Baffet, Alexandre D; Hu, Daniel J; Vallee, Richard B

2015-06-22

Dynein recruitment to the nuclear envelope is required for pre-mitotic nucleus-centrosome interactions in nonneuronal cells and for apical nuclear migration in neural stem cells. In each case, dynein is recruited to the nuclear envelope (NE) specifically during G2 via two nuclear pore-mediated mechanisms involving RanBP2-BicD2 and Nup133-CENP-F. The mechanisms responsible for cell-cycle control of this behavior are unknown. We now find that Cdk1 serves as a direct master controller for NE dynein recruitment in neural stem cells and HeLa cells. Cdk1 phosphorylates conserved sites within RanBP2 and activates BicD2 binding and early dynein recruitment. Late recruitment is triggered by a Cdk1-induced export of CENP-F from the nucleus. Forced NE targeting of BicD2 overrides Cdk1 inhibition, fully rescuing dynein recruitment and nuclear migration in neural stem cells. These results reveal how NE dynein recruitment is cell-cycle regulated and identify the trigger mechanism for apical nuclear migration in the brain. Copyright © 2015 Elsevier Inc. All rights reserved.

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

PubMed

Kirschman, Junghwa; Qi, Mingli; Ding, Lingmei; Hammonds, Jason; Dienger-Stambaugh, Krista; Wang, Jaang-Jiun; Lapierre, Lynne A; Goldenring, James R; Spearman, Paul

2018-03-01

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C 560-649 ) and examined the consequences on Env trafficking and incorporation into particles. FIP1C 560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW 795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane. IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes trapped inside the cell within the endosomal recycling compartment. Intracellular trapping resulted in a loss of envelope protein on released particles and a corresponding loss of infectivity. Mutations of specific trafficking motifs in the envelope protein tail prevented its trapping in the recycling compartment. These results establish that trafficking to the endosomal recycling compartment is an essential step in HIV envelope protein particle incorporation. Copyright © 2018 American Society for Microbiology.

Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria.

PubMed

Thomas, Pious; Sekhar, Aparna Chandra

2014-01-01

It is generally believed that endophytic microorganisms are intercellular inhabitants present in either cultivable or non-cultivable form primarily as root colonizers. The objective of this study was to determine whether the actively mobile micro-particles observed in the intracellular matrix of fresh tissue sections of banana included endophytic bacteria. Tissue sections (50-100 µm) from apical leaf sheaths of surface-disinfected suckers (cv. Grand Naine) displayed 'Brownian motion'-reminiscent abundant motile micro-particles under bright-field and phase-contrast (×1000), which appeared similar in size and motility to the pure cultures of endophytes previously isolated from banana. Observations on callus, embryonic cells and protoplasts with intact cell wall/plasma membrane confirmed their cytoplasmic nature. The motility of these entities reduced or ceased upon tissue fixation or staining with safranin/crystal violet (0.5 % w/v), but continued uninterrupted following treatment with actin-disrupting drugs, ruling out the possibility of micro-organelles like peroxisomes. Staining with 2,3,5-triphenyl tetrazolium chloride (TTC) confirmed them to be live bacteria with similar observations after dilute safranin (0.005 %) treatment. Tissue staining with SYTO-9 coupled with epi-fluorescence or confocal laser scanning microscopy showed bacterial colonization along the peri-space between cell wall and plasma membrane initially. SYTO-9 counterstaining on TTC- or safranin-treated tissue and those subjected to enzymatic permeabilization revealed the cytoplasmic bacteria. These included organisms moving freely in the cytoplasm and those adhering to the nuclear envelope or vacuoles and the intravacuolar colonizers. The observations appeared ubiquitous to different genomes and genotypes of banana. Plating the tissue homogenate on nutrient media seldom yielded colony growth. This study, supported largely by live cell video-imaging, demonstrates enormous intracellular colonization in bananas by normally non-cultivable endophytic bacteria in two niches, namely cytoplasmic and periplasmic, designated as 'Cytobacts' and 'Peribacts', respectively. The integral intracellular association with their clonal perpetuation suggests a mutualistic relationship between endophytes and the host.

Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria

PubMed Central

Thomas, Pious; Sekhar, Aparna Chandra

2014-01-01

It is generally believed that endophytic microorganisms are intercellular inhabitants present in either cultivable or non-cultivable form primarily as root colonizers. The objective of this study was to determine whether the actively mobile micro-particles observed in the intracellular matrix of fresh tissue sections of banana included endophytic bacteria. Tissue sections (50–100 µm) from apical leaf sheaths of surface-disinfected suckers (cv. Grand Naine) displayed ‘Brownian motion’-reminiscent abundant motile micro-particles under bright-field and phase-contrast (×1000), which appeared similar in size and motility to the pure cultures of endophytes previously isolated from banana. Observations on callus, embryonic cells and protoplasts with intact cell wall/plasma membrane confirmed their cytoplasmic nature. The motility of these entities reduced or ceased upon tissue fixation or staining with safranin/crystal violet (0.5 % w/v), but continued uninterrupted following treatment with actin-disrupting drugs, ruling out the possibility of micro-organelles like peroxisomes. Staining with 2,3,5-triphenyl tetrazolium chloride (TTC) confirmed them to be live bacteria with similar observations after dilute safranin (0.005 %) treatment. Tissue staining with SYTO-9 coupled with epi-fluorescence or confocal laser scanning microscopy showed bacterial colonization along the peri-space between cell wall and plasma membrane initially. SYTO-9 counterstaining on TTC- or safranin-treated tissue and those subjected to enzymatic permeabilization revealed the cytoplasmic bacteria. These included organisms moving freely in the cytoplasm and those adhering to the nuclear envelope or vacuoles and the intravacuolar colonizers. The observations appeared ubiquitous to different genomes and genotypes of banana. Plating the tissue homogenate on nutrient media seldom yielded colony growth. This study, supported largely by live cell video-imaging, demonstrates enormous intracellular colonization in bananas by normally non-cultivable endophytic bacteria in two niches, namely cytoplasmic and periplasmic, designated as ‘Cytobacts’ and ‘Peribacts’, respectively. The integral intracellular association with their clonal perpetuation suggests a mutualistic relationship between endophytes and the host. PMID:24790123

A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis

PubMed Central

Faria-Blanc, Nuno; Mortimer, Jenny C.; Dupree, Paul

2018-01-01

Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous system has been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants. PMID:29636762

A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis.

PubMed

Faria-Blanc, Nuno; Mortimer, Jenny C; Dupree, Paul

2018-01-01

Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous system has been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants.

Differential histone modification and protein expression associated with cell wall removal and regeneration in rice (Oryza sativa).

PubMed

Tan, Feng; Zhang, Kangling; Mujahid, Hana; Verma, Desh Pal S; Peng, Zhaohua

2011-02-04

The cell wall is a critical extracellular structure that provides protection and structural support in plant cells. To study the biological function of the cell wall and the regulation of cell wall resynthesis, we examined cellular responses to enzymatic removal of the cell wall in rice (Oryza sativa) suspension cells using proteomic approaches. We find that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Nucleus DAPI stain and MNase digestion further show that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses reveal that substantial histone modification changes, particularly H3K18(AC) and H3K23(AC), are associated with the removal and regeneration of the cell wall. Label-free quantitative proteome analyses further reveal that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, along with cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.

Plant cell wall proteomics: the leadership of Arabidopsis thaliana

PubMed Central

Albenne, Cécile; Canut, Hervé; Jamet, Elisabeth

2013-01-01

Plant cell wall proteins (CWPs) progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses, and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose, and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cell walls need to be modified and customized during plant development and in response to environmental cues, thus contributing to plant adaptation. CWPs play essential roles in all these physiological processes and particularly in the dynamics of cell walls, which requires organization and rearrangements of polysaccharides as well as cell-to-cell communication. In the last 10 years, plant cell wall proteomics has greatly contributed to a wider knowledge of CWPs. This update will deal with (i) a survey of plant cell wall proteomics studies with a focus on Arabidopsis thaliana; (ii) the main protein families identified and the still missing peptides; (iii) the persistent issue of the non-canonical CWPs; (iv) the present challenges to overcome technological bottlenecks; and (v) the perspectives beyond cell wall proteomics to understand CWP functions. PMID:23641247

Influence of the bud neck on nuclear envelope fission in Saccharomyces cerevisiae.

PubMed

Melloy, Patricia G; Rose, Mark D

2017-09-15

Studies have shown that nuclear envelope fission (karyokinesis) in budding yeast depends on cytokinesis, but not distinguished whether this was a direct requirement, indirect, because of cell cycle arrest, or due to bud neck-localized proteins impacting both processes. To determine the requirements for karyokinesis, we examined mutants conditionally defective for bud emergence and/or nuclear migration. The common mutant phenotype was completion of the nuclear division cycle within the mother cell, but karyokinesis did not occur. In the cdc24 swe1 mutant, at the non-permissive temperature, multiple nuclei accumulated within the unbudded cell, with connected nuclear envelopes. Upon return to the permissive temperature, the cdc24 swe1 mutant initiated bud emergence, but only the nucleus spanning the neck underwent fission suggesting that the bud neck region is important for fission initiation. The neck may be critical for either mechanical reasons, as the contractile ring might facilitate fission, or for regulatory reasons, as the site of a protein network regulating nuclear envelope fission, mitotic exit, and cytokinesis. We also found that 77-85% of pairs of septin mutant nuclei completed nuclear envelope fission. In addition, 27% of myo1Δ mutant nuclei completed karyokinesis. These data suggested that fission is not dependent on mechanical contraction at the bud neck, but was instead controlled by regulatory proteins there. Copyright © 2017 Elsevier Inc. All rights reserved.

Cell Wall Structure of Coccoid Green Algae as an Important Trade-Off Between Biotic Interference Mechanisms and Multidimensional Cell Growth.

PubMed

Dunker, Susanne; Wilhelm, Christian

2018-01-01

Coccoid green algae can be divided in two groups based on their cell wall structure. One group has a highly chemical resistant cell wall (HR-cell wall) containing algaenan. The other group is more susceptible to chemicals (LR-cell wall - Low resistant cell wall). Algaenan is considered as important molecule to explain cell wall resistance. Interestingly, cell wall types (LR- and HR-cell wall) are not in accordance with the taxonomic classes Chlorophyceae and Trebouxiophyceae, which makes it even more interesting to consider the ecological function. It was already shown that algaenan helps to protect against virus, bacterial and fungal attack, but in this study we show for the first time that green algae with different cell wall properties show different sensitivity against interference competition with the cyanobacterium Microcystis aeruginosa . Based on previous work with co-cultures of M. aeruginosa and two green algae ( Acutodesmus obliquus and Oocystis marssonii ) differing in their cell wall structure, it was shown that M. aeruginosa could impair only the growth of the green algae if they belong to the LR-cell wall type. In this study it was shown that the sensitivity to biotic interference mechanism shows a more general pattern within coccoid green algae species depending on cell wall structure.

Involvement of bacterial TonB-dependent signaling in the generation of an oligogalacturonide damage-associated molecular pattern from plant cell walls exposed to Xanthomonas campestris pv. campestris pectate lyases

PubMed Central

2012-01-01

Background Efficient perception of attacking pathogens is essential for plants. Plant defense is evoked by molecules termed elicitors. Endogenous elicitors or damage-associated molecular patterns (DAMPs) originate from plant materials upon injury or pathogen activity. While there are comparably well-characterized examples for DAMPs, often oligogalacturonides (OGAs), generated by the activity of fungal pathogens, endogenous elicitors evoked by bacterial pathogens have been rarely described. In particular, the signal perception and transduction processes involved in DAMP generation are poorly characterized. Results A mutant strain of the phytopathogenic bacterium Xanthomonas campestris pv. campestris deficient in exbD2, which encodes a component of its unusual elaborate TonB system, had impaired pectate lyase activity and caused no visible symptoms for defense on the non-host plant pepper (Capsicum annuum). A co-incubation of X. campestris pv. campestris with isolated cell wall material from C. annuum led to the release of compounds which induced an oxidative burst in cell suspension cultures of the non-host plant. Lipopolysaccharides and proteins were ruled out as elicitors by polymyxin B and heat treatment, respectively. After hydrolysis with trifluoroacetic acid and subsequent HPAE chromatography, the elicitor preparation contained galacturonic acid, the monosaccharide constituent of pectate. OGAs were isolated from this crude elicitor preparation by HPAEC and tested for their biological activity. While small OGAs were unable to induce an oxidative burst, the elicitor activity in cell suspension cultures of the non-host plants tobacco and pepper increased with the degree of polymerization (DP). Maximal elicitor activity was observed for DPs exceeding 8. In contrast to the X. campestris pv. campestris wild type B100, the exbD2 mutant was unable to generate elicitor activity from plant cell wall material or from pectin. Conclusions To our knowledge, this is the second report on a DAMP generated by bacterial features. The generation of the OGA elicitor is embedded in a complex exchange of signals within the framework of the plant-microbe interaction of C. annuum and X. campestris pv. campestris. The bacterial TonB-system is essential for the substrate-induced generation of extracellular pectate lyase activity. This is the first demonstration that a TonB-system is involved in bacterial trans-envelope signaling in the context of a pathogenic interaction with a plant. PMID:23082751

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

PubMed Central

Ao, Jie; Chinnici, Jennifer L.; Maddi, Abhiram

2015-01-01

A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix. PMID:26048011

Caenorhabditis elegans polo-like kinase PLK-1 is required for merging parental genomes into a single nucleus.

PubMed

Rahman, Mohammad M; Munzig, Mandy; Kaneshiro, Kiyomi; Lee, Brandon; Strome, Susan; Müller-Reichert, Thomas; Cohen-Fix, Orna

2015-12-15

Before the first zygotic division, the nuclear envelopes of the maternal and paternal pronuclei disassemble, allowing both sets of chromosomes to be incorporated into a single nucleus in daughter cells after mitosis. We found that in Caenorhabditis elegans, partial inactivation of the polo-like kinase PLK-1 causes the formation of two nuclei, containing either the maternal or paternal chromosomes, in each daughter cell. These two nuclei gave rise to paired nuclei in all subsequent cell divisions. The paired-nuclei phenotype was caused by a defect in forming a gap in the nuclear envelopes at the interface between the two pronuclei during the first mitotic division. This was accompanied by defects in chromosome congression and alignment of the maternal and paternal metaphase plates relative to each other. Perturbing chromosome congression by other means also resulted in failure to disassemble the nuclear envelope between the two pronuclei. Our data further show that PLK-1 is needed for nuclear envelope breakdown during early embryogenesis. We propose that during the first zygotic division, PLK-1-dependent chromosome congression and metaphase plate alignment are necessary for the disassembly of the nuclear envelope between the two pronuclei, ultimately allowing intermingling of the maternal and paternal chromosomes. © 2015 Rahman et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells

PubMed Central

Gu, Mingyu; LaJoie, Dollie; Chen, Opal S.; von Appen, Alexander; Ladinsky, Mark S.; Redd, Michael J.; Nikolova, Linda; Bjorkman, Pamela J.; Sundquist, Wesley I.; Ullman, Katharine S.; Frost, Adam

2017-01-01

Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In Schizosaccharomyces pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope. PMID:28242692

The type 2 dengue virus envelope protein interacts with small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9).

PubMed

Chiu, Mei-Wui; Shih, Hsiu-Ming; Yang, Tsung-Han; Yang, Yun-Liang

2007-05-01

Dengue viruses are mosquito-borne flaviviruses and may cause the life-threatening dengue hemorrhagic fever and dengue shock syndrome. Its envelope protein is responsible mainly for the virus attachment and entry to host cells. To identify the human cellular proteins interacting with the envelope protein of dengue virus serotype 2 inside host cells, we have performed a screening with the yeast-two-hybrid-based "Functional Yeast Array". Interestingly, the small ubiquitin-like modifier-1 conjugating enzyme 9 protein, modulating cellular processes such as those regulating signal transduction and cell growth, was one of the candidates interacting with the dengue virus envelope protein. With co-precipitation assay, we have demonstrated that it indeed could interact directly with the Ubc9 protein. Site-directed mutagenesis has demonstrated that Ubc9 might interact with the E protein via amino acid residues K51 and K241. Furthermore, immunofluorescence microscopy has shown that the DV2E-EGFP proteins tended to progress toward the nuclear membrane and co-localized with Flag-Ubc9 proteins around the nuclear membrane in the cytoplasmic side, and DV2E-EGFP also shifted the distribution of Flag-Ubc9 from evenly in the nucleus toward concentrating around the nuclear membrane in the nucleic side. In addition, over-expression of Ubc9 could reduce the plaque formation of the dengue virus in mammalian cells. This is the first report that DV envelope proteins can interact with the protein of sumoylation system and Ubc9 may involve in the host defense system to prevent virus propagation.

Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

PubMed Central

Levin, David E.

2011-01-01

The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed. PMID:22174182

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

Hun, Diana E.; Atchley, Jerald Allen; Childs, Phillip W.

Estimates for 2010 indicate that infiltration in residential buildings was responsible for 2.85 quads of energy (DOE 2014), which is about 3% of the total energy consumed in the US. One of the mechanisms being implemented to reduce this energy penalty is the use of air barriers as part of the building envelope. These technologies decrease airflow through major leakage sites such as oriented strand board (OSB) joints, and gaps around penetrations (e.g., windows, doors, pipes, electrical outlets) as indicated by Hun et al. (2014). However, most air barrier materials do not properly address leakage spots such as wall-to-roof jointsmore » and wall-to-foundation joints because these are difficult to seal, and because air barrier manufacturers usually do not provide adequate instructions for these locations. The present study focuses on characterizing typical air leakage sites in wall assemblies with air barrier materials.« less

Comparison of SPHC Hydrocode Results with Penetration Equations and Results of Other Codes

NASA Technical Reports Server (NTRS)

Evans, Steven W.; Stallworth, Roderick; Stellingwerf, Robert F.

2004-01-01

The SPHC hydrodynamic code was used to simulate impacts of spherical aluminum projectiles on a single-wall aluminum plate and on a generic Whipple shield. Simulations were carried out in two and three dimensions. Projectile speeds ranged from 2 kilometers per second to 10 kilometers per second for the single-wall runs, and from 3 kilometers per second to 40 kilometers per second for the Whipple shield runs. Spallation limit results of the single-wall simulations are compared with predictions from five standard penetration equations, and are shown to fall comfortably within the envelope of these analytical relations. Ballistic limit results of the Whipple shield simulations are compared with results from the AUTODYN-2D and PAM-SHOCK-3D codes presented in a paper at the Hypervelocity Impact Symposium 2000 and the Christiansen formulation of 2003.

Antibody to the gp120 V1/V2 loops and CD4+and CD8+ T-cell responses in protection from SIVmac251 vaginal acquisition and persistent viremia

PubMed Central

Gordon, Shari N.; Doster, Melvin N; Kines, Rhonda C.; Keele, Brandon F; Cofano, Egidio Brocca; Guan, Yongjun; Pegu, Poonam; Liyanage, Namal P.M.; Vaccari, Monica; Cuburu, Nicolas; Buck, Christopher B.; Ferrari, Guido; Montefiori, David; Piatak, Mike; Lifson, Jeffrey D; Xenophontos, Anastasia M.; Venzon, David; Robert-Guroff, Marjorie; Graham, Barney S.; Lowy, Douglas R.; Schiller, John T.; Franchini, Genoveffa

2015-01-01

The human papilloma virus pseudovirions (HPV-PsVs) approach is an effective gene-delivery system that can prime or boost an immune response in the vaginal tract of non human primates and mice. Intra-vaginal vaccination with HPV-PsVs expressing SIV genes, combined with an intra-muscular gp120 protein injection, induced humoral and cellular SIV-specific responses in macaques. Priming systemic immune responses with intramuscular immunization with ALVAC-SIV vaccines, followed by intra-vaginal HPV-PsV-SIV/gp120 boosting, expanded and/or recruited T-cells in the female genital tract. Using a stringent repeated low dose intra-vaginal challenge with the highly pathogenic SIVmac251, we show that while these regimens did not demonstrate significant protection from virus acquisition, they provided control of viremia in a number of animals. High avidity antibody responses to the envelope gp120 V1/V2 region correlated with delayed SIVmac251 acquisition, while virus levels in mucosal tissues were inversely correlated with anti-envelope CD4+T-cell responses. CD8+T-cell depletion in animals with controlled viremia caused an increase in tissue virus load in some animals, suggesting a role for CD8+T-cells in virus control. This study highlights the importance of CD8+ cells and anti-envelope CD4+ T-cell in curtailing virus replication and anti-envelope V1/V2 antibodies in preventing SIVmac251 acquisition. PMID:25398324

Advanced Insulation for High Performance Cost-Effective Wall, Roof, and Foundation Systems Final Report

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

Costeux, Stephane; Bunker, Shanon

The objective of this project was to explore and potentially develop high performing insulation with increased R/inch and low impact on climate change that would help design highly insulating building envelope systems with more durable performance and lower overall system cost than envelopes with equivalent performance made with materials available today. The proposed technical approach relied on insulation foams with nanoscale pores (about 100 nm in size) in which heat transfer will be decreased. Through the development of new foaming methods, of new polymer formulations and new analytical techniques, and by advancing the understanding of how cells nucleate, expand andmore » stabilize at the nanoscale, Dow successfully invented and developed methods to produce foams with 100 nm cells and 80% porosity by batch foaming at the laboratory scale. Measurements of the gas conductivity on small nanofoam specimen confirmed quantitatively the benefit of nanoscale cells (Knudsen effect) to increase insulation value, which was the key technical hypotheses of the program. In order to bring this technology closer to a viable semi-continuous/continuous process, the project team modified an existing continuous extrusion foaming process as well as designed and built a custom system to produce 6" x 6" foam panels. Dow demonstrated for the first time that nanofoams can be produced in a both processes. However, due to technical delays, foam characteristics achieved so far fall short of the 100 nm target set for optimal insulation foams. In parallel with the technology development, effort was directed to the determination of most promising applications for nanocellular insulation foam. Voice of Customer (VOC) exercise confirmed that demand for high-R value product will rise due to building code increased requirements in the near future, but that acceptance for novel products by building industry may be slow. Partnerships with green builders, initial launches in smaller markets (e.g. EIFS), and efforts to drive cost down will help acceptance in residential and commercial retrofit and new construction.« less

A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

PubMed

Huberman, Lori B; Murray, Andrew W

2014-01-01

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

A Model for Cell Wall Dissolution in Mating Yeast Cells: Polarized Secretion and Restricted Diffusion of Cell Wall Remodeling Enzymes Induces Local Dissolution

PubMed Central

Huberman, Lori B.; Murray, Andrew W.

2014-01-01

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559

Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability.

PubMed

Takaki, Tohru; Montagner, Marco; Serres, Murielle P; Le Berre, Maël; Russell, Matt; Collinson, Lucy; Szuhai, Karoly; Howell, Michael; Boulton, Simon J; Sahai, Erik; Petronczki, Mark

2017-07-24

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability.

Envelope Protein Dynamics in Paramyxovirus Entry

PubMed Central

Plattet, Philippe; Plemper, Richard K.

2013-01-01

ABSTRACT Paramyxoviruses include major pathogens with significant global health and economic impact. This large family of enveloped RNA viruses infects cells by employing two surface glycoproteins that tightly cooperate to fuse their lipid envelopes with the target cell plasma membrane, an attachment and a fusion (F) protein. Membrane fusion is believed to depend on receptor-induced conformational changes within the attachment protein that lead to the activation and subsequent refolding of F. While structural and mechanistic studies have considerably advanced our insight into paramyxovirus cell adhesion and the structural basis of F refolding, how precisely the attachment protein links receptor engagement to F triggering remained poorly understood. Recent reports based on work with several paramyxovirus family members have transformed our understanding of the triggering mechanism of the membrane fusion machinery. Here, we review these recent findings, which (i) offer a broader mechanistic understanding of the paramyxovirus cell entry system, (ii) illuminate key similarities and differences between entry strategies of different paramyxovirus family members, and (iii) suggest new strategies for the development of novel therapeutics. PMID:23820396

Envelope protein dynamics in paramyxovirus entry.

PubMed

Plattet, Philippe; Plemper, Richard K

2013-07-02

Paramyxoviruses include major pathogens with significant global health and economic impact. This large family of enveloped RNA viruses infects cells by employing two surface glycoproteins that tightly cooperate to fuse their lipid envelopes with the target cell plasma membrane, an attachment and a fusion (F) protein. Membrane fusion is believed to depend on receptor-induced conformational changes within the attachment protein that lead to the activation and subsequent refolding of F. While structural and mechanistic studies have considerably advanced our insight into paramyxovirus cell adhesion and the structural basis of F refolding, how precisely the attachment protein links receptor engagement to F triggering remained poorly understood. Recent reports based on work with several paramyxovirus family members have transformed our understanding of the triggering mechanism of the membrane fusion machinery. Here, we review these recent findings, which (i) offer a broader mechanistic understanding of the paramyxovirus cell entry system, (ii) illuminate key similarities and differences between entry strategies of different paramyxovirus family members, and (iii) suggest new strategies for the development of novel therapeutics.

Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability

PubMed Central

Takaki, Tohru; Montagner, Marco; Serres, Murielle P.; Le Berre, Maël; Russell, Matt; Collinson, Lucy; Szuhai, Karoly; Howell, Michael; Boulton, Simon J.; Sahai, Erik; Petronczki, Mark

2017-01-01

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability. PMID:28737169

A study of the native cell wall structures of the marine alga Ventricaria ventricosa (Siphonocladales, Chlorophyceae) using atomic force microscopy.

PubMed

Eslick, Enid M; Beilby, Mary J; Moon, Anthony R

2014-04-01

A substantial proportion of the architecture of the plant cell wall remains unknown with a few cell wall models being proposed. Moreover, even less is known about the green algal cell wall. Techniques that allow direct visualization of the cell wall in as near to its native state are of importance in unravelling the spatial arrangement of cell wall structures and hence in the development of cell wall models. Atomic force microscopy (AFM) was used to image the native cell wall of living cells of Ventricaria ventricosa (V. ventricosa) at high resolution under physiological conditions. The cell wall polymers were identified mainly qualitatively via their structural appearance. The cellulose microfibrils (CMFs) were easily recognizable and the imaging results indicate that the V. ventricosa cell wall has a cross-fibrillar structure throughout. We found the native wall to be abundant in matrix polysaccharides existing in different curing states. The soft phase matrix polysaccharides susceptible by the AFM scanning tip existed as a glutinous fibrillar meshwork, possibly incorporating both the pectic- and hemicellulosic-type substances. The hard phase matrix producing clearer images, revealed coiled fibrillar structures associated with CMFs, sometimes being resolved as globular structures by the AFM tip. The coiling fibrillar structures were also seen in the images of isolated cell wall fragments. The mucilaginous component of the wall was discernible from the gelatinous cell wall matrix as it formed microstructural domains over the surface. AFM has been successful in imaging the native cell wall and revealing novel findings such as the 'coiling fibrillar structures' and cell wall components which have previously not been seen, that is, the gelatinous matrix phase.

Wall Boundary Layer Measurements for the NASA Langley Transonic Dynamics Tunnel

NASA Technical Reports Server (NTRS)

Wieseman, Carol D.; Bennett, Robert M.

2007-01-01

Measurements of the boundary layer parameters in the NASA Langley Transonic Dynamics tunnel were conducted during extensive calibration activities following the facility conversion from a Freon-12 heavy-gas test medium to R-134a. Boundary-layer rakes were mounted on the wind-tunnel walls, ceiling, and floor. Measurements were made over the range of tunnel operation envelope in both heavy gas and air and without a model in the test section at three tunnel stations. Configuration variables included open and closed east sidewall wall slots, for air and R134a test media, reentry flap settings, and stagnation pressures over the full range of tunnel operation. The boundary layer thickness varied considerably for the six rakes. The thickness for the east wall was considerably larger that the other rakes and was also larger than previously reported. There generally was some reduction in thickness at supersonic Mach numbers, but the effect of stagnation pressure, and test medium were not extensive.

HIV envelope glycoprotein imaged at high resolution | Center for Cancer Research

Cancer.gov

The outer surface of the human immunodeficiency virus (HIV) is surrounded by an envelope studded with spike-shaped glycoproteins called Env that help the deadly virus identify, bind, and infect cells. When unbound, Env exists in a “closed” conformational state. Upon binding with target cells, such as CD4+ T cells, the protein transitions to an “open” configuration. Given that Env is the only viral protein expressed on HIV’s surface, knowing its detailed structure—especially in the unbound state—may be critical for designing antibodies and vaccines against HIV.

Altered Cell Wall Plasticity Can Restrict Plant Growth under Ammonium Nutrition.

PubMed

Podgórska, Anna; Burian, Maria; Gieczewska, Katarzyna; Ostaszewska-Bugajska, Monika; Zebrowski, Jacek; Solecka, Danuta; Szal, Bożena

2017-01-01

Plants mainly utilize inorganic forms of nitrogen (N), such as nitrate (NO 3 - ) and ammonium (NH 4 + ). However, the composition of the N source is important, because excess of NH 4 + promotes morphological disorders. Plants cultured on NH 4 + as the sole N source exhibit serious growth inhibition, commonly referred to as "ammonium toxicity syndrome." NH 4 + -mediated suppression of growth may be attributable to both repression of cell elongation and reduction of cell division. The precondition for cell enlargement is the expansion of the cell wall, which requires the loosening of the cell wall polymers. Therefore, to understand how NH 4 + nutrition may trigger growth retardation in plants, properties of their cell walls were analyzed. We found that Arabidopsis thaliana using NH 4 + as the sole N source has smaller cells with relatively thicker cell walls. Moreover, cellulose, which is the main load-bearing polysaccharide revealed a denser assembly of microfibrils. Consequently, the leaf blade tissue showed elevated tensile strength and indicated higher cell wall stiffness. These changes might be related to changes in polysaccharide and ion content of cell walls. Further, NH 4 + toxicity was associated with altered activities of cell wall modifying proteins. The lower activity and/or expression of pectin hydrolyzing enzymes and expansins might limit cell wall expansion. Additionally, the higher activity of cell wall peroxidases can lead to higher cross-linking of cell wall polymers. Overall, the NH 4 + -mediated inhibition of growth is related to a more rigid cell wall structure, which limits expansion of cells. The changes in cell wall composition were also indicated by decreased expression of Feronia , a receptor-like kinase involved in the control of cell wall extension.

Altered Cell Wall Plasticity Can Restrict Plant Growth under Ammonium Nutrition

PubMed Central

Podgórska, Anna; Burian, Maria; Gieczewska, Katarzyna; Ostaszewska-Bugajska, Monika; Zebrowski, Jacek; Solecka, Danuta; Szal, Bożena

2017-01-01

Plants mainly utilize inorganic forms of nitrogen (N), such as nitrate (NO3–) and ammonium (NH4+). However, the composition of the N source is important, because excess of NH4+ promotes morphological disorders. Plants cultured on NH4+ as the sole N source exhibit serious growth inhibition, commonly referred to as “ammonium toxicity syndrome.” NH4+-mediated suppression of growth may be attributable to both repression of cell elongation and reduction of cell division. The precondition for cell enlargement is the expansion of the cell wall, which requires the loosening of the cell wall polymers. Therefore, to understand how NH4+ nutrition may trigger growth retardation in plants, properties of their cell walls were analyzed. We found that Arabidopsis thaliana using NH4+ as the sole N source has smaller cells with relatively thicker cell walls. Moreover, cellulose, which is the main load-bearing polysaccharide revealed a denser assembly of microfibrils. Consequently, the leaf blade tissue showed elevated tensile strength and indicated higher cell wall stiffness. These changes might be related to changes in polysaccharide and ion content of cell walls. Further, NH4+ toxicity was associated with altered activities of cell wall modifying proteins. The lower activity and/or expression of pectin hydrolyzing enzymes and expansins might limit cell wall expansion. Additionally, the higher activity of cell wall peroxidases can lead to higher cross-linking of cell wall polymers. Overall, the NH4+-mediated inhibition of growth is related to a more rigid cell wall structure, which limits expansion of cells. The changes in cell wall composition were also indicated by decreased expression of Feronia, a receptor-like kinase involved in the control of cell wall extension. PMID:28848567

Removal of either N-glycan site from the envelope receptor binding domain of Moloney and Friend but not AKV mouse ecotropic gammaretroviruses alters receptor usage

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

Knoper, Ryan C.; Ferrarone, John; Yan Yuhe

2009-09-01

Three N-linked glycosylation sites were removed from the envelope glycoproteins of Friend, Moloney, and AKV mouse ecotropic gammaretroviruses: gs1 and gs2, in the receptor binding domain; and gs8, in a region implicated in post-binding cell fusion. Mutants were tested for their ability to infect rodent cells expressing 4 CAT-1 receptor variants. Three mutants (Mo-gs1, Mo-gs2, and Fr-gs1) infect NIH 3T3 and rat XC cells, but are severely restricted in Mus dunni cells and Lec8, a Chinese hamster cell line susceptible to ecotropic virus. This restriction is reproduced in ferret cells expressing M. dunni dCAT-1, but not in cells expressing NIHmore » 3T3 mCAT-1. Virus binding assays, pseudotype assays, and the use of glycosylation inhibitors further suggest that restriction is primarily due to receptor polymorphism and, in M. dunni cells, to glycosylation of cellular proteins. Virus envelope glycan size or type does not affect infectivity. Thus, host range variation due to N-glycan deletion is receptor variant-specific, cell-specific, virus type-specific, and glycan site-specific.« less

An additional simple denitrification bioreactor using packed gel envelopes applicable to industrial wastewater treatment.

PubMed

Morita, Masahiko; Uemoto, Hiroaki; Watanabe, Atsushi

2007-08-15

A simple denitrification bioreactor for nitrate-containing wastewater without organic compounds was developed. This bioreactor consisted of packed gel envelopes in a single tank. Each envelope comprised two plates of gels containing Paracoccus denitrificans cells with an internal space between the plates. As an electron donor for denitrification, ethanol was injected into the internal space and not directly into the wastewater. P. denitrificans cells in the gel reduced nitrate to nitrogen gas by using the injected ethanol. Nitrate-containing desulfurization wastewater derived from a coal-fired thermal power plant was continuously treated with 20 packed gel envelopes (size, 1,000 x 900 x 12 mm; surface area, 1.44 m(2)) in a reactor tank (volume 1.5 m(3)). When the total nitrogen concentration in the inflow was around 150 mg-N x L(-1), the envelopes removed approximately 60-80% of the total nitrogen, and the maximum nitrogen removal rate was 5.0 g-N x day(-1) per square meter of the gel surface. This value corresponded to the volumetric nitrogen removal performance of 0.109 kg-N x m(-3) x day(-1). In each envelope, a high utilization efficiency of the electron donor was attained, although more than the double amount of the electron donor was empirically injected in the present activated sludge system to achieve denitrification when compared with the theoretical value. The bioreactor using the envelopes would be extremely effective as an additional denitrification system because these envelopes can be easily installed in the vacant spaces of preinstalled water treatment systems, without requiring additional facilities for removing surplus ethanol and sludge. (c) 2007 Wiley Periodicals, Inc.

Endomembrane Cation Transporters and Membrane Trafficking

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

Sze, Heven

Multicellular, as well as unicellular, organisms have evolved mechanisms to regulate ion and pH homeostasis in response to developmental cues and to a changing environment. The working hypothesis is that the balance of fluxes mediated by diverse transporters at the plasma membrane and in subcellular organelles determines ionic cellular distribution, which is critical for maintenance of membrane potential, pH control, osmolality, transport of nutrients, and protein activity. An emerging theme in plant cell biology is that cells respond and adapt to diverse cues through changes of the dynamic endomembrane system. Yet we know very little about the transporters that mightmore » influence the operation of the secretory system in plants. Here we focus on transporters that influence alkali cation and pH homeostasis, mainly in the endomembrane/ secretory system. The endomembrane system of eukaryote cells serves several major functions: i) sort cargo (e.g. enzymes, transporters or receptors) to specific destinations, ii) modulate the protein and lipid composition of membrane domains through remodeling, and iii) determine and alter the properties of the cell wall through synthesis and remodeling. We had uncovered a novel family of predicted cation/H + exchangers (CHX) and K + efflux antiporters (KEA) that are prevalent in higher plants, but rare in metazoans. We combined phylogenetic and transcriptomic analyses with molecular genetic, cell biological and biochemical studies, and have published the first reports on functions of plant CHXs and KEAs. CHX studied to date act at the endomembrane system where their actions are distinct from the better-studied NHX (Na/K-H + exchangers). Arabidopsis thaliana CHX20 in guard cells modulate stomatal opening, and thus is significant for vegetative survival. Other CHXs ensure reproductive success on dry land, as they participate in organizing pollen walls, targeting of pollen tubes to the ovule or promoting fertilization. Based on localization and mutant analyses, we conclude that CHXs modulate the ion balance, pH or both in micro-regions of endoplasmic reticulum, endosomes and prevacuolar compartment (PVC), and so influence membrane trafficking and signaling resulting in proper osmoregulation in guard cells and seed formation. We also demonstrated for the first time that AtKEA2 associates with chloroplasts, especially at the two poles of developing plastids. These results show that AtKEA1 and AtKEA2 transporters in specific microdomains of the inner envelope link local osmotic, ionic, and pH homeostasis to plastid division and thylakoid membrane formation. The first 3-D structure model of AtCHX was generated, and architecture-directed mutagenesis identified critical residues of the transport core giving insights to the transport mode of this family. Thus we have revealed for the first time crucial roles of an unknown K +/H + transport family on plant growth (KEA), gas exchange, pollen cell wall, and different phases of reproduction (CHXs). The dynamic endomembrane of plant cells is integral to cytokinesis, cell expansion, defense, and cell wall formation, thus these studies are directly relevant to the mission of the Department of Energy and to a better understanding of determinants for enhancing plant biomass and plant tolerance to abiotic stress.« less

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

PubMed

Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

2011-12-01

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

The alpha-TIF (VP16) homologue (ETIF) of equine herpesvirus 1 is essential for secondary envelopment and virus egress.

PubMed

von Einem, Jens; Schumacher, Daniel; O'Callaghan, Dennis J; Osterrieder, Nikolaus

2006-03-01

The equine herpesvirus 1 (EHV-1) alpha-trans-inducing factor homologue (ETIF; VP16-E) is a 60-kDa virion component encoded by gene 12 (ORF12) that transactivates the immediate-early gene promoter. Here we report on the function of EHV-1 ETIF in the context of viral infection. An ETIF-null mutant from EHV-1 strain RacL11 (vL11deltaETIF) was constructed and analyzed. After transfection of vL11deltaETIF DNA into RK13 cells, no infectious virus could be reconstituted, and only single infected cells or small foci containing up to eight infected cells were detected. In contrast, after transfection of vL11deltaETIF DNA into a complementing cell line, infectious virus could be recovered, indicating the requirement of ETIF for productive virus infection. The growth defect of vL11deltaETIF could largely be restored by propagation on the complementing cell line, and growth on the complementing cell line resulted in incorporation of ETIF in mature and secreted virions. Low- and high-multiplicity infections of RK13 cells with phenotypically complemented vL11deltaETIF virus resulted in titers of virus progeny similar to those used for infection, suggesting that input ETIF from infection was recycled. Ultrastructural studies of vL11deltaETIF-infected cells demonstrated a marked defect in secondary envelopment at cytoplasmic membranes, resulting in very few enveloped virions in transport vesicles or extracellular space. Taken together, our results demonstrate that ETIF has an essential function in the replication cycle of EHV-1, and its main role appears to be in secondary envelopment.

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

PubMed Central

Hirai-Yuki, Asuka; Hensley, Lucinda; Whitmire, Jason K.

2016-01-01

ABSTRACT Hepatitis A virus (HAV) is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV) are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replication in vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood) sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99%) progeny virions were released apically from Caco-2 cells, whereas basolateral (64%) versus apical (36%) release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (<0.02%/h) in HepG2-N6 cells, arguing against this as a mechanism for differences in membrane envelopment of serum versus fecal virus. High concentrations of human bile acids converted eHAV to nonenveloped virions, whereas virus present in bile from HAV-infected Ifnar1−/− Ifngr1−/− and Mavs−/− mice banded over a range of densities extending from that of eHAV to that of nonenveloped virions. We conclude that nonenveloped virions shed in feces are derived from eHAV released across the canalicular membrane and stripped of membranes by the detergent action of bile acids within the proximal biliary canaliculus. PMID:27923925

TIM1 (HAVCR1) Is Not Essential for Cellular Entry of Either Quasi-enveloped or Naked Hepatitis A Virions

PubMed Central

Das, Anshuman; Hirai-Yuki, Asuka; González-López, Olga; Rhein, Bethany; Moller-Tank, Sven; Brouillette, Rachel; Hensley, Lucinda; Misumi, Ichiro; Lovell, William; Cullen, John M.; Whitmire, Jason K.; Maury, Wendy

2017-01-01

ABSTRACT Receptor molecules play key roles in the cellular entry of picornaviruses, and TIM1 (HAVCR1) is widely accepted to be the receptor for hepatitis A virus (HAV), an unusual, hepatotropic human picornavirus. However, its identification as the hepatovirus receptor predated the discovery that hepatoviruses undergo nonlytic release from infected cells as membrane-cloaked, quasi-enveloped HAV (eHAV) virions that enter cells via a pathway distinct from naked, nonenveloped virions. We thus revisited the role of TIM1 in hepatovirus entry, examining both adherence and infection/replication in cells with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-engineered TIM1 knockout. Cell culture-derived, gradient-purified eHAV bound Huh-7.5 human hepatoma cells less efficiently than naked HAV at 4°C, but eliminating TIM1 expression caused no difference in adherence of either form of HAV, nor any impact on infection and replication in these cells. In contrast, TIM1-deficient Vero cells showed a modest reduction in quasi-enveloped eHAV (but not naked HAV) attachment and replication. Thus, TIM1 facilitates quasi-enveloped eHAV entry in Vero cells, most likely by binding phosphatidylserine (PtdSer) residues on the eHAV membrane. Both Tim1−/− Ifnar1−/− and Tim4−/− Ifnar1−/− double-knockout mice were susceptible to infection upon intravenous challenge with infected liver homogenate, with fecal HAV shedding and serum alanine aminotransferase (ALT) elevations similar to those in Ifnar1−/− mice. However, intrahepatic HAV RNA and ALT elevations were modestly reduced in Tim1−/−Ifnar1−/− mice compared to Ifnar1−/− mice challenged with a lower titer of gradient-purified HAV or eHAV. We conclude that TIM1 is not an essential hepatovirus entry factor, although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver injury in mice. PMID:28874468

Effect of heat and moisture transport and storage properties of building stones on the hygrothermal performance of historical building envelopes

NASA Astrophysics Data System (ADS)

KoÅáková, Dana; Kočí, Václav; Žumár, Jaromír; Keppert, Martin; Holčapek, Ondřej; Vejmelková, Eva; Černý, Robert

2016-12-01

The heat and moisture transport and storage parameters of three different natural stones used on the Czech territory since medieval times are determined experimentally, together with the basic physical properties and mechanical parameters. The measured data are applied as input parameters in the computational modeling of hygrothermal performance of building envelopes made of the analyzed stones. Test reference year climatic data of three different locations within the Czech Republic are used as boundary conditions on the exterior side. Using the simulated hygric and thermal performance of particular stone walls, their applicability is assessed in a relation to the geographical and climatic conditions. The obtained results indicate that all three investigated stones are highly resistant to weather conditions, freeze/thaw cycles in particular.

Signal Processing for Determining Water Height in Steam Pipes with Dynamic Surface Conditions

NASA Technical Reports Server (NTRS)

Lih, Shyh-Shiuh; Lee, Hyeong Jae; Bar-Cohen, Yoseph

2015-01-01

An enhanced signal processing method based on the filtered Hilbert envelope of the auto-correlation function of the wave signal has been developed to monitor the height of condensed water through the steel wall of steam pipes with dynamic surface conditions. The developed signal processing algorithm can also be used to estimate the thickness of the pipe to determine the cut-off frequency for the low pass filter frequency of the Hilbert Envelope. Testing and analysis results by using the developed technique for dynamic surface conditions are presented. A multiple array of transducers setup and methodology are proposed for both the pulse-echo and pitch-catch signals to monitor the fluctuation of the water height due to disturbance, water flow, and other anomaly conditions.

Tetrad pollen formation in Annona (Annonaceae): proexine formation andbinding mechanism.

PubMed

Tsou, Chih-Hua; Fu, Yu-Lan

2002-05-01

Meiotic tetrads of Annona glabra and A. montana build up a well-developed proexine (protectum, probaculum, and pronexine) at the proximal side but only a thin pronexine at the distal side during the tetrad stage. The callosic envelope is only partially digested by the end of tetrad stage. The remaining, undigested part is composed of the intersporal mass and thin peripheral layers, and the latter is conjunct with the distal pronexine of the microspore. In this remaining callosic structure celluloses are also present. Later on, due to the continuous slow decomposition of this callose-cellulose structure and microspore expansion, microspores break up the callose-cellulose envelope. Because all the four microspores are bound together by the callose-cellulose structure, they move out of the chamber in rotation. Eventually the thin pronexine is pulled toward the center of the tetrad and the well-developed proexine becomes the distal wall. These descriptions of the partial digestion of callosic envelope, the transformation from a callose-cellulose structure to the binding system of tetrad pollen, and microspore rotation in Annona are unusual in the angiosperms.

Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.

PubMed

Rappa, Germana; Santos, Mark F; Green, Toni M; Karbanová, Jana; Hassler, Justin; Bai, Yongsheng; Barsky, Sanford H; Corbeil, Denis; Lorico, Aurelio

2017-02-28

Extracellular membrane vesicles (EVs) function as vehicles of intercellular communication, but how the biomaterials they carry reach the target site in recipient cells is an open question. We report that subdomains of Rab7+ late endosomes and nuclear envelope invaginations come together to create a sub-nuclear compartment, where biomaterials associated with CD9+ EVs are delivered. EV-derived biomaterials were also found in the nuclei of host cells. The inhibition of nuclear import and export pathways abrogated the nuclear localization of EV-derived biomaterials or led to their accumulation therein, respectively, suggesting that their translocation is dependent on nuclear pores. Nuclear envelope invagination-associated late endosomes were observed in ex vivo biopsies in both breast carcinoma and associated stromal cells. The transcriptome of stromal cells exposed to cancer cell-derived CD9+ EVs revealed that the regulation of eleven genes, notably those involved in inflammation, relies on the nuclear translocation of EV-derived biomaterials. Our findings uncover a new cellular pathway used by EVs to reach nuclear compartment.

Role of HIV-2 envelope in Lv2-mediated restriction

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

Reuter, Sandra; Kaumanns, Patrick; Buschhorn, Sabine B.

2005-02-05

We have characterized envelope protein pseudotyped HIV-2 particles derived from two HIV-2 isolates termed prCBL23 and CBL23 in order to define the role of the envelope protein for the Lv2-mediated restriction to infection. Previously, it has been described that the primary isolate prCBL23 is restricted to infection of several human cell types, whereas the T cell line adapted isolate CBL23 is not restricted in these cell types. Molecular cloning of the two isolates revealed that the env and the gag gene are responsible for the observed phenotype and that this restriction is mediated by Lv2, which is distinct from Ref1/Lv1more » (Schmitz, C., Marchant, D., Neil, S.J., Aubin, K., Reuter, S., Dittmar, M.T., McKnight, A., Kizhatil, K., Albritton, L.M., 2004. Lv2, a novel postentry restriction, is mediated by both capsid and envelope. J. Virol. 78 (4), 2006-2016). We generated pseudotyped viruses consisting of HIV-2 (ROD-A{delta}env-GFP, ROD-A{delta}env-RFP, or ROD-A{delta}env-REN) and the prCBL23 or CBL23 envelope proteins as well as chimeric proteins between these envelopes. We demonstrate that a single amino acid exchange at position 74 in the surface unit of CBL23-Env confers restriction to infection. This single point mutation causes tighter CD4 binding, resulting in a less efficient fusion into the cytosol of the restricted cell line. Prevention of endosome formation and prevention of endosome acidification enhance infectivity of the restricted particles for GHOST/X4 cells indicating a degradative lysosomal pathway as a cause for the reduced cytosolic entry. The described restriction to infection of the primary isolate prCBL23 is therefore largely caused by an entry defect. A remaining restriction to infection (19-fold) is preserved when endosomal acidification is prevented. This restriction to infection is also dependent on the presence of the point mutation at position 74 (G74E)« less

NUCLEAR ENVELOPE-ASSOCIATED RESUMPTION OF RNA SYNTHESIS IN LATE MITOSIS OF HELA CELLS

PubMed Central

Simmons, T.; Heywood, P.; Hodge, L.

1973-01-01

The restitution of RNA synthesis in cultures progressing from metaphase into interphase (G1) has been investigated in synchronized HeLa S3 cells by using inhibitors of macro-molecular synthesis and the technique of electron microscope autoradiography. The rate of incorporation of radioactive uridine into RNA approached interphase levels in the absence of renewed protein synthesis. In contrast, maintenance of this rate in G1 was dependent upon renewed protein synthesis. Restoration of synthesis of heterogeneous nuclear RNA occurred under conditions that inhibited production of ribosomal precursor RNA. In autoradiographs of individual cells exposed to radioactive uridine, silver grains were first detected after nuclear envelope reformation at the periphery of the chromosome mass but before chromosomal decondensation. These data are consistent with the following interpretation. Multiple RNA polymerase activities persist through mitosis and are involved in the initiation of RNA synthesis in early telophase at sites on the nuclear envelope. PMID:4752403

The Cell Wall of the Human Fungal Pathogen Aspergillus fumigatus: Biosynthesis, Organization, Immune Response, and Virulence.

PubMed

Latgé, Jean-Paul; Beauvais, Anne; Chamilos, Georgios

2017-09-08

More than 90% of the cell wall of the filamentous fungus Aspergillus fumigatus comprises polysaccharides. Biosynthesis of the cell wall polysaccharides is under the control of three types of enzymes: transmembrane synthases, which are anchored to the plasma membrane and use nucleotide sugars as substrates, and cell wall-associated transglycosidases and glycosyl hydrolases, which are responsible for remodeling the de novo synthesized polysaccharides and establishing the three-dimensional structure of the cell wall. For years, the cell wall was considered an inert exoskeleton of the fungal cell. The cell wall is now recognized as a living organelle, since the composition and cellular localization of the different constitutive cell wall components (especially of the outer layers) vary when the fungus senses changes in the external environment. The cell wall plays a major role during infection. The recognition of the fungal cell wall by the host is essential in the initiation of the immune response. The interactions between the different pattern-recognition receptors (PRRs) and cell wall pathogen-associated molecular patterns (PAMPs) orientate the host response toward either fungal death or growth, which would then lead to disease development. Understanding the molecular determinants of the interplay between the cell wall and host immunity is fundamental to combatting Aspergillus diseases.

Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development

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

Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra

Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18more » cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture.« less

Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

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

Ponec, M.; Weerheim, A.; Havekes, L.

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis resulting in changes in the plasma membrane composition. Hydrocortisonemore » stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.« less

Structural and mechanistic studies of measles virus illuminate paramyxovirus entry.

PubMed

Plemper, Richard K; Brindley, Melinda A; Iorio, Ronald M

2011-06-01

Measles virus (MeV), a member of the paramyxovirus family of enveloped RNA viruses and one of the most infectious viral pathogens identified, accounts for major pediatric morbidity and mortality worldwide although coordinated efforts to achieve global measles control are in place. Target cell entry is mediated by two viral envelope glycoproteins, the attachment (H) and fusion (F) proteins, which form a complex that achieves merger of the envelope with target cell membranes. Despite continually expanding knowledge of the entry strategies employed by enveloped viruses, our molecular insight into the organization of functional paramyxovirus fusion complexes and the mechanisms by which the receptor binding by the attachment protein triggers the required conformational rearrangements of the fusion protein remain incomplete. Recently reported crystal structures of the MeV attachment protein in complex with its cellular receptors CD46 or SLAM and newly developed functional assays have now illuminated some of the fundamental principles that govern cell entry by this archetype member of the paramyxovirus family. Here, we review these advances in our molecular understanding of MeV entry in the context of diverse entry strategies employed by other members of the paramyxovirus family.

The Role of the Nuclear Envelope Protein MAN1 in Mesenchymal Stem Cell Differentiation.

PubMed

Bermeo, Sandra; Al-Saedi, Ahmed; Kassem, Moustapha; Vidal, Christopher; Duque, Gustavo

2017-12-01

Mutations in MAN1, a protein of the nuclear envelope, cause bone phenotypes characterized by hyperostosis. The mechanism of this pro-osteogenic phenotype remains unknown. We increased and decreased MAN1 expression in mesenchymal stem cells (MSC) upon which standard osteogenic and adipogenic differentiation were performed. MAN1 knockdown increased osteogenesis and mineralization. In contrast, osteogenesis remained stable upon MAN1 overexpression. Regarding a mechanism, we found that low levels of MAN1 facilitated the nuclear accumulation of regulatory smads and smads-related complexes, with a concurrently high expression of nuclear β-Catenin. In addition, we found adipogenesis to be decreased in both conditions, although predominantly affected by MAN1 overexpression. Finally, lamin A, a protein of the nuclear envelope that regulates MSC differentiation, was unaffected by changes in MAN1. In conclusion, our studies demonstrated that lower levels of MAN1 in differentiating MSC are associated with higher osteogenesis and lower adipogenesis. High levels of MAN1 only affected adipogenesis. These effects could have an important role in the understanding of the role of the proteins of the nuclear envelope in bone formation. J. Cell. Biochem. 118: 4425-4435, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains.

PubMed

Han, Mee-Jung; Lee, Sang Yup; Hong, Soon Ho

2012-04-01

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane beta-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

The Impact of Microfibril Orientations on the Biomechanics of Plant Cell Walls and Tissues.

PubMed

Ptashnyk, Mariya; Seguin, Brian

2016-11-01

The microscopic structure and anisotropy of plant cell walls greatly influence the mechanical properties, morphogenesis, and growth of plant cells and tissues. The microscopic structure and properties of cell walls are determined by the orientation and mechanical properties of the cellulose microfibrils and the mechanical properties of the cell wall matrix. Viewing the shape of a plant cell as a square prism with the axis aligning with the primary direction of expansion and growth, the orientation of the microfibrils within the side walls, i.e. the parts of the cell walls on the sides of the cells, is known. However, not much is known about their orientation at the upper and lower ends of the cell. Here we investigate the impact of the orientation of cellulose microfibrils within the upper and lower parts of the plant cell walls by solving the equations of linear elasticity numerically. Three different scenarios for the orientation of the microfibrils are considered. We also distinguish between the microstructure in the side walls given by microfibrils perpendicular to the main direction of the expansion and the situation where the microfibrils are rotated through the wall thickness. The macroscopic elastic properties of the cell wall are obtained using homogenization theory from the microscopic description of the elastic properties of the cell wall microfibrils and wall matrix. It is found that the orientation of the microfibrils in the upper and lower parts of the cell walls affects the expansion of the cell in the lateral directions and is particularly important in the case of forces acting on plant cell walls and tissues.

High temperature induced disruption of the cell wall integrity and structure in Pleurotus ostreatus mycelia.

PubMed

Qiu, Zhiheng; Wu, Xiangli; Gao, Wei; Zhang, Jinxia; Huang, Chenyang

2018-05-30

Fungal cells are surrounded by a tight cell wall to protect them from harmful environmental conditions and to resist lysis. The synthesis and assembly determine the shape, structure, and integrity of the cell wall during the process of mycelial growth and development. High temperature is an important abiotic stress, which affects the synthesis and assembly of cell walls. In the present study, the chitin and β-1,3-glucan concentrations in the cell wall of Pleurotus ostreatus mycelia were changed after high-temperature treatment. Significantly higher chitin and β-1,3-glucan concentrations were detected at 36 °C than those incubated at 28 °C. With the increased temperature, many aberrant chitin deposition patches occurred, and the distribution of chitin in the cell wall was uneven. Moreover, high temperature disrupts the cell wall integrity, and P. ostreatus mycelia became hypersensitive to cell wall-perturbing agents at 36 °C. The cell wall structure tended to shrink or distorted after high temperature. The cell walls were observed to be thicker and looser by using transmission electron microscopy. High temperature can decrease the mannose content in the cell wall and increase the relative cell wall porosity. According to infrared absorption spectrum, high temperature broke or decreased the glycosidic linkages. Finally, P. ostreatus mycelial cell wall was easily degraded by lysing enzymes after high-temperature treatment. In other words, the cell wall destruction caused by high temperature may be a breakthrough for P. ostreatus to be easily infected by Trichoderma.

Tegument Assembly and Secondary Envelopment of Alphaherpesviruses

PubMed Central

Owen, Danielle J.; Crump, Colin M.; Graham, Stephen C.

2015-01-01

Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called “tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei. PMID:26393641

Endoplasmic reticulum-derived reactive oxygen species (ROS) is involved in toxicity of cell wall stress to Candida albicans.

PubMed

Yu, Qilin; Zhang, Bing; Li, Jianrong; Zhang, Biao; Wang, Honggang; Li, Mingchun

2016-10-01

The cell wall is an important cell structure in both fungi and bacteria, and hence becomes a common antimicrobial target. The cell wall-perturbing agents disrupt synthesis and function of cell wall components, leading to cell wall stress and consequent cell death. However, little is known about the detailed mechanisms by which cell wall stress renders fungal cell death. In this study, we found that ROS scavengers drastically attenuated the antifungal effect of cell wall-perturbing agents to the model fungal pathogen Candida albicans, and these agents caused remarkable ROS accumulation and activation of oxidative stress response (OSR) in this fungus. Interestingly, cell wall stress did not cause mitochondrial dysfunction and elevation of mitochondrial superoxide levels. Furthermore, the iron chelator 2,2'-bipyridyl (BIP) and the hydroxyl radical scavengers could not attenuate cell wall stress-caused growth inhibition and ROS accumulation. However, cell wall stress up-regulated expression of unfold protein response (UPR) genes, enhanced protein secretion and promoted protein folding-related oxidation of Ero1, an important source of ROS production. These results indicated that oxidation of Ero1 in the endoplasmic reticulum (ER), rather than mitochondrial electron transport and Fenton reaction, contributed to cell wall stress-related ROS accumulation and consequent growth inhibition. Our findings uncover a novel link between cell wall integrity (CWI), ER function and ROS production in fungal cells, and shed novel light on development of strategies promoting the antifungal efficacy of cell wall-perturbing agents against fungal infections. Copyright © 2016 Elsevier Inc. All rights reserved.

Regulation of cell wall biosynthesis.

PubMed

Zhong, Ruiqin; Ye, Zheng-Hua

2007-12-01

Plant cell walls differ in their amount and composition among various cell types and even in different microdomains of the wall of a given cell. Plants must have evolved regulatory mechanisms controlling biosynthesis, targeted secretion, and assembly of wall components to achieve the heterogeneity in cell walls. A number of factors, including hormones, the cytoskeleton, glycosylphosphatidylinositol-anchored proteins, phosphoinositides, and sugar nucleotide supply, have been implicated in the regulation of cell wall biosynthesis or deposition. In the past two years, there have been important discoveries in transcriptional regulation of secondary wall biosynthesis. Several transcription factors in the NAC and MYB families have been shown to be the key switches for activation of secondary wall biosynthesis. These studies suggest a transcriptional network comprised of a hierarchy of transcription factors is involved in regulating secondary wall biosynthesis. Further investigation and integration of the regulatory players participating in the making of cell walls will certainly lead to our understanding of how wall amounts and composition are controlled in a given cell type. This may eventually allow custom design of plant cell walls on the basis of our needs.

Substitution of specific cysteine residues in E1 glycoprotein of classical swine fever virus strain Brescia affects formation of E1-E2 heterodimers and alters virulence in swine

USDA-ARS?s Scientific Manuscript database

E1, along with E^rns and E2, is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini and E^rns loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1,...

Relating Nanoscale Accessibility within Plant Cell Walls to Improved Enzyme Hydrolysis Yields in Corn Stover Subjected to Diverse Pretreatments.

PubMed

Crowe, Jacob D; Zarger, Rachael A; Hodge, David B

2017-10-04

Simultaneous chemical modification and physical reorganization of plant cell walls via alkaline hydrogen peroxide or liquid hot water pretreatment can alter cell wall structural properties impacting nanoscale porosity. Nanoscale porosity was characterized using solute exclusion to assess accessible pore volumes, water retention value as a proxy for accessible water-cell walls surface area, and solute-induced cell wall swelling to measure cell wall rigidity. Key findings concluded that delignification by alkaline hydrogen peroxide pretreatment decreased cell wall rigidity and that the subsequent cell wall swelling resulted increased nanoscale porosity and improved enzyme binding and hydrolysis compared to limited swelling and increased accessible surface areas observed in liquid hot water pretreated biomass. The volume accessible to a 90 Å dextran probe within the cell wall was found to be correlated to both enzyme binding and glucose hydrolysis yields, indicating cell wall porosity is a key contributor to effective hydrolysis yields.

Following the compositional changes of fresh grape skin cell walls during the fermentation process in the presence and absence of maceration enzymes.

PubMed

Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Trygg, Johan; Vivier, Melané A

2015-03-18

Cell wall profiling technologies were used to follow compositional changes that occurred in the skins of grape berries (from two different ripeness levels) during fermentation and enzyme maceration. Multivariate data analysis showed that the fermentation process yielded cell walls enriched in hemicellulose components because pectin was solubilized (and removed) with a reduction as well as exposure of cell wall proteins usually embedded within the cell wall structure. The addition of enzymes caused even more depectination, and the enzymes unravelled the cell walls enabling better access to, and extraction of, all cell wall polymers. Overripe grapes had cell walls that were extensively hydrolyzed and depolymerized, probably by natural grape-tissue-ripening enzymes, and this enhanced the impact that the maceration enzymes had on the cell wall monosaccharide profile. The combination of the techniques that were used is an effective direct measurement of the hydrolysis actions of maceration enzymes on the cell walls of grape berry skin.

Regulation of nuclear envelope dynamics via APC/C is necessary for the progression of semi-open mitosis in Schizosaccharomyces japonicus.

PubMed

Aoki, Keita; Shiwa, Yuh; Takada, Hiraku; Yoshikawa, Hirofumi; Niki, Hironori

2013-09-01

Three types of mitosis, which are open, closed or semi-open mitosis, function in eukaryotic cells, respectively. The open mitosis involves breakage of the nuclear envelope before nuclear division, whereas the closed mitosis proceeds with an intact nuclear envelope. To understand the mechanism and significance of three types of mitotic division in eukaryotes, we investigated the process of semi-open mitosis, in which the nuclear envelope is only partially broken, in the fission yeast Schizosaccharomyces japonicus. In anaphase-promoting complex/cyclosome (APC/C) mutants of Sz. japonicus, the nuclear envelope remained relatively intact during anaphase, resulting in impaired semi-open mitosis. As a suppressor of apc2 mutant, a mutation of Oar2, which was a 3-oxoacyl-[acyl carrier protein] reductase, was obtained. The level of the Oar2, which had two destruction-box motifs recognized by APC/C, was increased in APC/C mutants. Furthermore, the defective semi-open mitosis observed in an apc2 mutant was restored by mutated oar2+. Based on these findings, we propose that APC/C regulates the dynamics of the nuclear envelope through degradation of Oar2 dependent on APC/C during the metaphase-to-anaphase transition of semi-open mitosis in Sz. japonicus. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Employing proteomic analysis to compare Paracoccidioides lutzii yeast and mycelium cell wall proteins.

PubMed

Araújo, Danielle Silva; de Sousa Lima, Patrícia; Baeza, Lilian Cristiane; Parente, Ana Flávia Alves; Melo Bailão, Alexandre; Borges, Clayton Luiz; de Almeida Soares, Célia Maria

2017-11-01

Paracoccidioidomycosis is an important systemic mycosis caused by thermodimorphic fungi of the Paracoccidioides genus. During the infective process, the cell wall acts at the interface between the fungus and the host. In this way, the cell wall has a key role in growth, environment sensing and interaction, as well as morphogenesis of the fungus. Since the cell wall is absent in mammals, it may present molecules that are described as target sites for new antifungal drugs. Despite its importance, up to now few studies have been conducted employing proteomics in for the identification of cell wall proteins in Paracoccidioides spp. Here, a detailed proteomic approach, including cell wall-fractionation coupled to NanoUPLC-MS E , was used to study and compare the cell wall fractions from Paracoccidioides lutzii mycelia and yeast cells. The analyzed samples consisted of cell wall proteins extracted by hot SDS followed by extraction by mild alkali. In summary, 512 proteins constituting different cell wall fractions were identified, including 7 predicted GPI-dependent cell wall proteins that are potentially involved in cell wall metabolism. Adhesins previously described in Paracoccidioides spp. such as enolase, glyceraldehyde-3-phosphate dehydrogenase were identified. Comparing the proteins in mycelium and yeast cells, we detected some that are common to both fungal phases, such as Ecm33, and some specific proteins, as glucanase Crf1. All of those proteins were described in the metabolism of cell wall. Our study provides an important elucidation of cell wall composition of fractions in Paracoccidioides, opening a way to understand the fungus cell wall architecture. Copyright © 2017 Elsevier B.V. All rights reserved.

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus.

PubMed

Hirai-Yuki, Asuka; Hensley, Lucinda; Whitmire, Jason K; Lemon, Stanley M

2016-12-06

Hepatitis A virus (HAV) is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV) are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replication in vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood) sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99%) progeny virions were released apically from Caco-2 cells, whereas basolateral (64%) versus apical (36%) release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (<0.02%/h) in HepG2-N6 cells, arguing against this as a mechanism for differences in membrane envelopment of serum versus fecal virus. High concentrations of human bile acids converted eHAV to nonenveloped virions, whereas virus present in bile from HAV-infected Ifnar1 -/- Ifngr1 -/- and Mavs -/- mice banded over a range of densities extending from that of eHAV to that of nonenveloped virions. We conclude that nonenveloped virions shed in feces are derived from eHAV released across the canalicular membrane and stripped of membranes by the detergent action of bile acids within the proximal biliary canaliculus. HAV is a hepatotropic, fecally/orally transmitted picornavirus that can cause severe hepatitis in humans. Recent work reveals that it has an unusual life cycle. Virus is found in cell culture supernatant fluids in two mature, infectious forms: one wrapped in membranes (quasi-enveloped) and another that is nonenveloped. Membrane-wrapped virions circulate in blood during acute infection and are resistant to neutralizing antibodies, likely facilitating HAV dissemination within the liver. On the other hand, virus shed in feces is nonenveloped and highly stable, facilitating epidemic spread and transmission to naive hosts. Factors controlling the biogenesis of these two distinct forms of the virus in infected humans are not understood. Here we characterize vectorial release of quasi-enveloped virions from polarized epithelial cell cultures and provide evidence that bile acids strip membranes from eHAV following its secretion into the biliary tract. These results enhance our understanding of the life cycle of this unusual picornavirus. Copyright © 2016 Hirai-Yuki et al.

Aberrant localization of lamin B receptor (LBR) in cellular senescence in human cells

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

Arai, Rumi; En, Atsuki; Ukekawa, Ryo

2016-05-13

5-Bromodeoxyuridine (BrdU), a thymidine analogue, induces cellular senescence in mammalian cells. BrdU induces cellular senescence probably through the regulation of chromatin because BrdU destabilizes or disrupts nucleosome positioning and decondenses heterochromatin. Since heterochromatin is tethered to the nuclear periphery through the interaction with the nuclear envelope proteins, we examined the localization of the several nuclear envelope proteins such as lamins, lamin-interacting proteins, nuclear pore complex proteins, and nuclear transport proteins in senescent cells. We have shown here that lamin B receptor (LBR) showed a change in localization in both BrdU-induced and replicative senescent cells.

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.

Engineering cell wall synthesis mechanism for enhanced PHB accumulation in E. coli.

PubMed

Zhang, Xing-Chen; Guo, Yingying; Liu, Xu; Chen, Xin-Guang; Wu, Qiong; Chen, Guo-Qiang

2018-01-01

The rigidity of bacterial cell walls synthesized by a complicated pathway limit the cell shapes as coccus, bar or ellipse or even fibers. A less rigid bacterium could be beneficial for intracellular accumulation of poly-3-hydroxybutyrate (PHB) as granular inclusion bodies. To understand how cell rigidity affects PHB accumulation, E. coli cell wall synthesis pathway was reinforced and weakened, respectively. Cell rigidity was achieved by thickening the cell walls via insertion of a constitutive gltA (encoding citrate synthase) promoter in front of a series of cell wall synthesis genes on the chromosome of several E. coli derivatives, resulting in 1.32-1.60 folds increase of Young's modulus in mechanical strength for longer E. coli cells over-expressing fission ring FtsZ protein inhibiting gene sulA. Cell rigidity was weakened by down regulating expressions of ten genes in the cell wall synthesis pathway using CRISPRi, leading to elastic cells with more spaces for PHB accumulation. The regulation on cell wall synthesis changes the cell rigidity: E. coli with thickened cell walls accumulated only 25% PHB while cell wall weakened E. coli produced 93% PHB. Manipulation on cell wall synthesis mechanism adds another possibility to morphology engineering of microorganisms. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA.

PubMed

Gao, Qiuqiang; Liou, Liang-Chun; Ren, Qun; Bao, Xiaoming; Zhang, Zhaojie

2014-03-03

The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ 0 ). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ 0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ 0 cells to SDS after salt treatment, while overexpression of SCW11 results in higher sensitivity. In addition, salt stress in ρ 0 cells induces high levels of reactive oxygen species (ROS), which further damages the cell wall, causing cells to become more sensitive towards the cell wall-perturbing agent.

TRACING INFALL AND ROTATION ALONG THE OUTFLOW CAVITY WALLS OF THE L483 PROTOSTELLAR ENVELOPE

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

Leung, Gigi Y.C.; Lim, Jeremy; Takakuwa, Shigehisa

2016-12-10

Single-dish observations in CS(7–6) reveal emission extending out to thousands of au along the outflow axis of low-mass protostars and having a velocity gradient in the opposite direction to that of their outflows. This emission has been attributed to dense and warm gas flowing outward along the walls of bipolar outflow cavities. Here, we present combined single-dish and interferometric CS(7–6) maps for the low-mass protostar L483, revealing a newly discovered compact central component (radius ≲800 au) and previously unknown features in its extended component (visible out to ∼4000 au). The velocity gradient and skewed (toward the redshifted side) brightness distributionmore » of the extended component are detectable out to a radius of ∼2000 au, but not beyond. The compact central component exhibits a velocity gradient in the same direction as, but which is steeper than that of, the extended component. Furthermore, both components exhibit a velocity gradient with an approximately constant magnitude across the outflow axis, apparent in the extended component not just through but also away from the center out to 2000 au. We point out contradictions between our results and model predictions for outflowing gas and propose a new model in which all of the aforementioned emission can be qualitatively explained by gas inflowing along the outflow cavity walls of a rigidly rotating envelope. Our model also can explain the extended CS(7–6) emission observed around other low-mass protostars.« less

Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls.

PubMed

Marcus, Susan E; Verhertbruggen, Yves; Hervé, Cécile; Ordaz-Ortiz, José J; Farkas, Vladimir; Pedersen, Henriette L; Willats, William G T; Knox, J Paul

2008-05-22

Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure. Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types. These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re-considered in relation to the potential masking of cell wall epitopes by other cell wall components.

The Interplay between Cell Wall Mechanical Properties and the Cell Cycle in Staphylococcus aureus

PubMed Central

Bailey, Richard G.; Turner, Robert D.; Mullin, Nic; Clarke, Nigel; Foster, Simon J.; Hobbs, Jamie K.

2014-01-01

The nanoscale mechanical properties of live Staphylococcus aureus cells during different phases of growth were studied by atomic force microscopy. Indentation to different depths provided access to both local cell wall mechanical properties and whole-cell properties, including a component related to cell turgor pressure. Local cell wall properties were found to change in a characteristic manner throughout the division cycle. Splitting of the cell into two daughter cells followed a local softening of the cell wall along the division circumference, with the cell wall on either side of the division circumference becoming stiffer. Once exposed, the newly formed septum was found to be stiffer than the surrounding, older cell wall. Deeper indentations, which were affected by cell turgor pressure, did not show a change in stiffness throughout the division cycle, implying that enzymatic cell wall remodeling and local variations in wall properties are responsible for the evolution of cell shape through division. PMID:25468333

KRE5 Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in Candida glabrata

PubMed Central

Sasaki, Masato; Ito, Fumie; Aoyama, Toshio; Sato-Okamoto, Michiyo; Takahashi-Nakaguchi, Azusa; Chibana, Hiroji; Shibata, Nobuyuki

2016-01-01

The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in Candida glabrata, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of KRE5, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall β-1,6-glucan content. KRE5 repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of β-1,6-glucan content. These results indicate that KRE5 is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in C. glabrata. PMID:27548283

Imaging the Dynamics of Cell Wall Polymer Deposition in the Unicellular Model Plant, Penium margaritaceum.

PubMed

Domozych, David; Lietz, Anna; Patten, Molly; Singer, Emily; Tinaz, Berke; Raimundo, Sandra C

2017-01-01

The unicellular green alga, Penium margaritaceum, represents a novel and valuable model organism for elucidating cell wall dynamics in plants. This organism's cell wall contains several polymers that are highly similar to those found in the primary cell walls of land plants. Penium is easily grown in laboratory culture and is effectively manipulated in various experimental protocols including microplate assays and correlative microscopy. Most importantly, Penium can be live labeled with cell wall-specific antibodies or other probes and returned to culture where specific cell wall developmental events can be monitored. Additionally, live cells can be rapidly cryo-fixed and cell wall surface microarchitecture can be observed with variable pressure scanning electron microscopy. Here, we describe the methodology for maintaining Penium for experimental cell wall enzyme studies.

Cell walls of the dimorphic fungal pathogens Sporothrix schenckii and Sporothrix brasiliensis exhibit bilaminate structures and sloughing of extensive and intact layers

PubMed Central

Walker, Louise A.; Niño-Vega, Gustavo; Mora-Montes, Héctor M.; Neves, Gabriela W. P.; Villalobos-Duno, Hector; Barreto, Laura; Garcia, Karina; Franco, Bernardo; Martínez-Álvarez, José A.; Munro, Carol A.; Gow, Neil A. R.

2018-01-01

Sporotrichosis is a subcutaneous mycosis caused by pathogenic species of the Sporothrix genus. A new emerging species, Sporothrix brasiliensis, is related to cat-transmitted sporotrichosis and has severe clinical manifestations. The cell wall of pathogenic fungi is a unique structure and impacts directly on the host immune response. We reveal and compare the cell wall structures of Sporothrix schenckii and S. brasiliensis using high-pressure freezing electron microscopy to study the cell wall organization of both species. To analyze the components of the cell wall, we also used infrared and 13C and 1H NMR spectroscopy and the sugar composition was determined by quantitative high-performance anion-exchange chromatography. Our ultrastructural data revealed a bi-layered cell wall structure for both species, including an external microfibrillar layer and an inner electron-dense layer. The inner and outer layers of the S. brasiliensis cell wall were thicker than those of S. schenckii, correlating with an increase in the chitin and rhamnose contents. Moreover, the outer microfibrillar layer of the S. brasiliensis cell wall had longer microfibrils interconnecting yeast cells. Distinct from those of other dimorphic fungi, the cell wall of Sporothrix spp. lacked α-glucan component. Interestingly, glycogen α-particles were identified in the cytoplasm close to the cell wall and the plasma membrane. The cell wall structure as well as the presence of glycogen α-particles varied over time during cell culture. The structural differences observed in the cell wall of these Sporothrix species seemed to impact its uptake by monocyte-derived human macrophages. The data presented here show a unique cell wall structure of S. brasiliensis and S. schenckii during the yeast parasitic phase. A new cell wall model for Sporothrix spp. is therefore proposed that suggests that these fungi molt sheets of intact cell wall layers. This observation may have significant effects on localized and disseminated immunopathology. PMID:29522522

Requirement of cholesterol in the viral envelope for dengue virus infection.

PubMed

Carro, Ana C; Damonte, Elsa B

2013-06-01

The role of cholesterol in the virus envelope or in the cellular membranes for dengue virus (DENV) infection was examined by depletion with methyl-beta-cyclodextrin (MCD) or nystatin. Pretreatment of virions with MCD or nystatin significantly reduced virus infectivity in a dose-dependent manner. By contrast, pre-treatment of diverse human cell lines with MCD or nystatin did not affect DENV infection. The four DENV serotypes were similarly inactivated by cholesterol-extracting drugs and infectivity was partially rescued when virion suspensions were treated with MCD in the presence of bovine serum. The addition of serum or exogenous water-soluble cholesterol after MCD treatment did not produce a reversion of MCD inactivating effect. Furthermore, virion treatment with extra cholesterol exerted also a virucidal effect. Binding and uptake of cholesterol-deficient DENV into the host cell were not impaired, whereas the next step of fusion between virion envelope and endosome membrane leading to virion uncoating and release of nucleocapsids to the cytoplasm appeared to be prevented, as determined by the retention of capsid protein in cells infected with MCD inactivated-DENV virions. Thereafter, the infection was almost completely inhibited, given the failure of viral RNA synthesis and viral protein expression in cells infected with MCD-treated virions. These data suggest that envelope cholesterol is a critical factor in the fusion process for DENV entry. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

PubMed

Santos, Clelton A; Janissen, Richard; Toledo, Marcelo A S; Beloti, Lilian L; Azzoni, Adriano R; Cotta, Monica A; Souza, Anete P

2015-10-01

The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development. Copyright © 2015 Elsevier B.V. All rights reserved.

The receptor-like kinase AtVRLK1 regulates secondary cell wall thickening.

PubMed

Huang, Cheng; Zhang, Rui; Gui, Jinshan; Zhong, Yu; Li, Laigeng

2018-04-20

During the growth and development of land plants, some specialized cells, such as tracheary elements, undergo secondary cell wall thickening. Secondary cell walls contain additional lignin, compared with primary cell walls, thus providing mechanical strength and potentially improving defenses against pathogens. However, the molecular mechanisms that initiate wall thickening are unknown. In this study, we identified an Arabidopsis thaliana leucine-rich repeat receptor-like kinase, encoded by AtVRLK1 (Vascular-Related RLK 1), that is specifically expressed in cells undergoing secondary cell wall thickening. Suppression of AtVRLK1expression resulted in a range of phenotypes that included retarded early elongation of the inflorescence stem, shorter fibers, slower root growth, and shorter flower filaments. In contrast, upregulation of AtVRLK1 led to longer fiber cells, reduced secondary cell wall thickening in fiber and vessel cells, and defects in anther dehiscence. Molecular and cellular analyses showed that downregulation of AtVRLK1 promoted secondary cell wall thickening and upregulation of AtVRLK1 enhanced cell elongation and inhibited secondary cell wall thickening. We propose that AtVRLK1 functions as a signaling component in coordinating cell elongation and cell wall thickening during growth and development. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Delineating CD4 dependency of HIV-1: Adaptation to infect low level CD4 expressing target cells widens cellular tropism but severely impacts on envelope functionality

PubMed Central

Beauparlant, David; Rusert, Peter; Magnus, Carsten; Weber, Jacqueline; Uhr, Therese; Clapham, Paul R.; Metzner, Karin J.

2017-01-01

A hallmark of HIV-1 infection is the continuously declining number of the virus’ predominant target cells, activated CD4+ T cells. With diminishing CD4+ T cell levels, the capacity to utilize alternate cell types and receptors, including cells that express low CD4 receptor levels such as macrophages, thus becomes crucial. To explore evolutionary paths that allow HIV-1 to acquire a wider host cell range by infecting cells with lower CD4 levels, we dissected the evolution of the envelope-CD4 interaction under in vitro culture conditions that mimicked the decline of CD4high target cells, using a prototypic subtype B, R5-tropic strain. Adaptation to CD4low targets proved to severely alter envelope functions including trimer opening as indicated by a higher affinity to CD4 and loss in shielding against neutralizing antibodies. We observed a strikingly decreased infectivity on CD4high target cells, but sustained infectivity on CD4low targets, including macrophages. Intriguingly, the adaptation to CD4low targets altered the kinetic of the entry process, leading to rapid CD4 engagement and an extended transition time between CD4 and CCR5 binding during entry. This phenotype was also observed for certain central nervous system (CNS) derived macrophage-tropic viruses, highlighting that the functional perturbation we defined upon in vitro adaptation to CD4low targets occurs in vivo. Collectively, our findings suggest that CD4low adapted envelopes may exhibit severe deficiencies in entry fitness and shielding early in their evolution. Considering this, adaptation to CD4low targets may preferentially occur in a sheltered and immune-privileged environment such as the CNS to allow fitness restoring compensatory mutations to occur. PMID:28264054

Spatio-temporal diversification of the cell wall matrix materials in the developing stomatal complexes of Zea mays.

PubMed

Giannoutsou, E; Apostolakos, P; Galatis, B

2016-11-01

The matrix cell wall materials, in developing Zea mays stomatal complexes are asymmetrically distributed, a phenomenon appearing related to the local cell wall expansion and deformation, the establishment of cell polarity, and determination of the cell division plane. In cells of developing Zea mays stomatal complexes, definite cell wall regions expand determinately and become locally deformed. This differential cell wall behavior is obvious in the guard cell mother cells (GMCs) and the subsidiary cell mother cells (SMCs) that locally protrude towards the adjacent GMCs. The latter, emitting a morphogenetic stimulus, induce polarization/asymmetrical division in SMCs. Examination of immunolabeled specimens revealed that homogalacturonans (HGAs) with a high degree of de-esterification (2F4- and JIM5-HGA epitopes) and arabinogalactan proteins are selectively distributed in the extending and deformed cell wall regions, while their margins are enriched with rhamnogalacturonans (RGAs) containing highly branched arabinans (LM6-RGA epitope). In SMCs, the local cell wall matrix differentiation constitutes the first structural event, indicating the establishment of cell polarity. Moreover, in the premitotic GMCs and SMCs, non-esterified HGAs (2F4-HGA epitope) are preferentially localized in the cell wall areas outlining the cytoplasm where the preprophase band is formed. In these areas, the forthcoming cell plate fuses with the parent cell walls. These data suggest that the described heterogeneity in matrix cell wall materials is probably involved in: (a) local cell wall expansion and deformation, (b) the transduction of the inductive GMC stimulus, and (c) the determination of the division plane in GMCs and SMCs.

Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

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

Maric, Martina; Haugo, Alison C.; Dauer, William

2014-07-15

Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but ismore » enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.« less

Immunogold scanning electron microscopy can reveal the polysaccharide architecture of xylem cell walls

PubMed Central

Sun, Yuliang; Juzenas, Kevin

2017-01-01

Abstract Immunofluorescence microscopy (IFM) and immunogold transmission electron microscopy (TEM) are the two main techniques commonly used to detect polysaccharides in plant cell walls. Both are important in localizing cell wall polysaccharides, but both have major limitations, such as low resolution in IFM and restricted sample size for immunogold TEM. In this study, we have developed a robust technique that combines immunocytochemistry with scanning electron microscopy (SEM) to study cell wall polysaccharide architecture in xylem cells at high resolution over large areas of sample. Using multiple cell wall monoclonal antibodies (mAbs), this immunogold SEM technique reliably localized groups of hemicellulosic and pectic polysaccharides in the cell walls of five different xylem structures (vessel elements, fibers, axial and ray parenchyma cells, and tyloses). This demonstrates its important advantages over the other two methods for studying cell wall polysaccharide composition and distribution in these structures. In addition, it can show the three-dimensional distribution of a polysaccharide group in the vessel lateral wall and the polysaccharide components in the cell wall of developing tyloses. This technique, therefore, should be valuable for understanding the cell wall polysaccharide composition, architecture and functions of diverse cell types. PMID:28398585

Building a plant cell wall at a glance.

PubMed

Lampugnani, Edwin R; Khan, Ghazanfar Abbas; Somssich, Marc; Persson, Staffan

2018-01-29

Plant cells are surrounded by a strong polysaccharide-rich cell wall that aids in determining the overall form, growth and development of the plant body. Indeed, the unique shapes of the 40-odd cell types in plants are determined by their walls, as removal of the cell wall results in spherical protoplasts that are amorphic. Hence, assembly and remodeling of the wall is essential in plant development. Most plant cell walls are composed of a framework of cellulose microfibrils that are cross-linked to each other by heteropolysaccharides. The cell walls are highly dynamic and adapt to the changing requirements of the plant during growth. However, despite the importance of plant cell walls for plant growth and for applications that we use in our daily life such as food, feed and fuel, comparatively little is known about how they are synthesized and modified. In this Cell Science at a Glance article and accompanying poster, we aim to illustrate the underpinning cell biology of the synthesis of wall carbohydrates, and their incorporation into the wall, in the model plant Arabidopsis . © 2018. Published by The Company of Biologists Ltd.

Cell wall evolution and diversity

PubMed Central

Fangel, Jonatan U.; Ulvskov, Peter; Knox, J. P.; Mikkelsen, Maria D.; Harholt, Jesper; Popper, Zoë A.; Willats, William G.T.

2012-01-01

Plant cell walls display a considerable degree of diversity in their compositions and molecular architectures. In some cases the functional significance of a particular cell wall type appears to be easy to discern: secondary cells walls are often reinforced with lignin that provides durability; the thin cell walls of pollen tubes have particular compositions that enable their tip growth; lupin seed cell walls are characteristically thickened with galactan used as a storage polysaccharide. However, more frequently the evolutionary mechanisms and selection pressures that underpin cell wall diversity and evolution are unclear. For diverse green plants (chlorophytes and streptophytes) the rapidly increasing availability of transcriptome and genome data sets, the development of methods for cell wall analyses which require less material for analysis, and expansion of molecular probe sets, are providing new insights into the diversity and occurrence of cell wall polysaccharides and associated biosynthetic genes. Such research is important for refining our understanding of some of the fundamental processes that enabled plants to colonize land and to subsequently radiate so comprehensively. The study of cell wall structural diversity is also an important aspect of the industrial utilization of global polysaccharide bio-resources. PMID:22783271

Enhanced Thermal Performance of Mosques in Qatar

NASA Astrophysics Data System (ADS)

Touma, A. Al; Ouahrani, D.

2017-12-01

Qatar has an abundance of mosques that significantly contribute to the increasing energy consumption in the country. Little attention has been given to providing mitigation methods that limit the energy demands of mosques without violating the worshippers’ thermal comfort. Most of these researches dealt with enhancing the mosque envelope through the addition of insulation layers. Since most mosque walls in Qatar are mostly already insulated, this study proposes the installation of shading on the mosque roof that is anticipated to yield similar energy savings in comparison with insulated roofs. An actual mosque in Qatar, which is a combination of six different spaces consisting of men and women’s prayer rooms, ablutions and toilets, was simulated and yielded a total annual energy demand of 619.55 kWh/m2. The mosque, whose walls are already insulated, yielded 9.1% energy savings when an insulation layer was added to its roof whereas it produced 6.2% energy savings when a shading layer was added above this roof. As the reconstruction of the roof envelope is practically unrealistic in existing mosques, the addition of shading to the roof was found to produce comparable energy savings. Lastly, it was found that new mosques with thin-roof insulation and shading tend to be more energy-efficient than those with thick-roof insulation.

Destructive effects of butyrate on the cell envelope of Helicobacter pylori.

PubMed

Yonezawa, Hideo; Osaki, Takako; Hanawa, Tomoko; Kurata, Satoshi; Zaman, Cynthia; Woo, Timothy Derk Hoong; Takahashi, Motomichi; Matsubara, Sachie; Kawakami, Hayato; Ochiai, Kuniyasu; Kamiya, Shigeru

2012-04-01

Helicobacter pylori can be found in the oral cavity and is mostly detected by the use of PCR techniques. Growth of H. pylori is influenced by various factors in the mouth, such as the oral microflora, saliva and other antimicrobial substances, all of which make colonization of the oral cavity by H. pylori difficult. In the present study, we analysed the effect of the cell supernatant of a representative periodontal bacterium Porphyromonas gingivalis on H. pylori and found that the cell supernatant destroyed the H. pylori cell envelope. As P. gingivalis produces butyric acid, we focused our research on the effects of butyrate and found that it significantly inhibited the growth of H. pylori. H. pylori cytoplasmic proteins and DNA were detected in the extracellular environment after treatment with butyrate, suggesting that the integrity of the cell envelope was compromised and indicating that butyrate has a bactericidal effect on H. pylori. In addition, levels of extracellular H. pylori DNA increased following treatment with the cell supernatant of butyric acid-producing bacteria, indicating that the cell supernatant also has a bactericidal effect and that this may be due to its butyric acid content. In conclusion, butyric acid-producing bacteria may play a role in affecting H. pylori colonization of the oral cavity.

Distribution of alginate and cellulose and regulatory role of calcium in the cell wall of the brown alga Ectocarpus siliculosus (Ectocarpales, Phaeophyceae).

PubMed

Terauchi, Makoto; Nagasato, Chikako; Inoue, Akira; Ito, Toshiaki; Motomura, Taizo

2016-08-01

This work investigated a correlation between the three-dimensional architecture and compound-components of the brown algal cell wall. Calcium greatly contributes to the cell wall integrity. Brown algae have a unique cell wall consisting of alginate, cellulose, and sulfated polysaccharides. However, the relationship between the architecture and the composition of the cell wall is poorly understood. Here, we investigated the architecture of the cell wall and the effect of extracellular calcium in the sporophyte and gametophyte of the model brown alga, Ectocarpus siliculosus (Dillwyn) Lyngbye, using transmission electron microscopy, histochemical, and immunohistochemical studies. The lateral cell wall of vegetative cells of the sporophyte thalli had multilayered architecture containing electron-dense and negatively stained fibrils. Electron tomographic analysis showed that the amount of the electron-dense fibrils and the junctions was different between inner and outer layers, and between the perpendicular and tangential directions of the cell wall. By immersing the gametophyte thalli in the low-calcium (one-eighth of the normal concentration) artificial seawater medium, the fibrous layers of the lateral cell wall of vegetative cells became swollen. Destruction of cell wall integrity was also induced by the addition of sorbitol. The results demonstrated that electron-dense fibrils were composed of alginate-calcium fibrous gels, and electron negatively stained fibrils were crystalline cellulose microfibrils. It was concluded that the spatial arrangement of electron-dense fibrils was different between the layers and between the directions of the cell wall, and calcium was necessary for maintaining the fibrous layers in the cell wall. This study provides insights into the design principle of the brown algal cell wall.

Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development.

PubMed

Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra; Peck, Matthew L; Vega-Sánchez, Miguel E; Williams, Brian; Chiniquy, Dawn M; Saha, Prasenjit; Pattathil, Sivakumar; Conlin, Brian; Zhu, Lan; Hahn, Michael G; Willats, William G T; Scheller, Henrik V; Ronald, Pamela C; Bartley, Laura E

2016-10-01

Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Screening and characterization of plant cell walls using carbohydrate microarrays.

PubMed

Sørensen, Iben; Willats, William G T

2011-01-01

Plant cells are surrounded by cell walls built largely from complex carbohydrates. The primary walls of growing plant cells consist of interdependent networks of three polysaccharide classes: cellulose, cross-linking glycans (also known as hemicelluloses), and pectins. Cellulose microfibrils are tethered together by cross-linking glycans, and this assembly forms the major load-bearing component of primary walls, which is infiltrated with pectic polymers. In the secondary walls of woody tissues, pectins are much reduced and walls are reinforced with the phenolic polymer lignin. Plant cell walls are essential for plant life and also have numerous industrial applications, ranging from wood to nutraceuticals. Enhancing our knowledge of cell wall biology and the effective use of cell wall materials is dependent to a large extent on being able to analyse their fine structures. We have developed a suite of techniques based on microarrays probed with monoclonal antibodies with specificity for cell wall components, and here we present practical protocols for this type of analysis.

Characteristic thickened cell walls of the bracts of the 'eternal flower' Helichrysum bracteatum.

PubMed

Nishikawa, Kuniko; Ito, Hiroaki; Awano, Tatsuya; Hosokawa, Munetaka; Yazawa, Susumu

2008-07-01

Helichrysum bracteatum is called an 'eternal flower' and has large, coloured, scarious bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum. DAPI (4'6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized light microscopy were used for observation of cells, including cell wall morphology. Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not observed in the petal and bract tissues of Chrysanthemum morifolium. Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves. The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they may be a new cell type.

Peptidoglycan turnover and recycling in Gram-positive bacteria.

PubMed

Reith, Jan; Mayer, Christoph

2011-10-01

Bacterial cells are protected by an exoskeleton, the stabilizing and shape-maintaining cell wall, consisting of the complex macromolecule peptidoglycan. In view of its function, it could be assumed that the cell wall is a static structure. In truth, however, it is steadily broken down by peptidoglycan-cleaving enzymes during cell growth. In this process, named cell wall turnover, in one generation up to half of the preexisting peptidoglycan of a bacterial cell is released from the wall. This would result in a massive loss of cell material, if turnover products were not be taken up and recovered. Indeed, in the Gram-negative model organism Escherichia coli, peptidoglycan recovery has been recognized as a complex pathway, named cell wall recycling. It involves about a dozen dedicated recycling enzymes that convey cell wall turnover products to peptidoglycan synthesis or energy pathways. Whether Gram-positive bacteria also recover their cell wall is currently questioned. Given the much larger portion of peptidoglycan in the cell wall of Gram-positive bacteria, however, recovery of the wall material would provide an even greater benefit in these organisms compared to Gram-negatives. Consistently, in many Gram-positives, orthologs of recycling enzymes were identified, indicating that the cell wall may also be recycled in these organisms. This mini-review provides a compilation of information about cell wall turnover and recycling in Gram-positive bacteria during cell growth and division, including recent findings relating to muropeptide recovery in Bacillus subtilis and Clostridium acetobutylicum from our group. Furthermore, the impact of cell wall turnover and recycling on biotechnological processes is discussed.

RADIOAUTOGRAPHIC STUDY OF CELL WALL DEPOSITION IN GROWING PLANT CELLS

PubMed Central

Ray, Peter M.

1967-01-01

Segments cut from growing oat coleoptiles and pea stems were fed glucose-3H in presence and absence of the growth hormone indoleacetic acid (IAA). By means of electron microscope radioautography it was demonstrated that new cell wall material is deposited both at the wall surface (apposition) and within the preexisting wall structure (internally). Quantitative profiles for the distribution of incorporation with position through the thickness of the wall were obtained for the thick outer wall of epidermal cells. With both oat coleoptile and pea stem epidermal outer walls, it was found that a larger proportion of the newly synthesized wall material appeared to become incorporated within the wall in the presence of IAA. Extraction experiments on coleoptile tissue showed that activity that had been incorporated into the cell wall interior represented noncellulosic constituents, mainly hemicelluloses, whereas cellulose was deposited largely or entirely by apposition. It seems possible that internal incorporation of hemicelluloses plays a role in the cell wall expansion process that is involved in cell growth. PMID:6064369

The Tubular Sheaths Encasing Methanosaeta thermophila Filaments Are Functional Amyloids.

PubMed

Dueholm, Morten S; Larsen, Poul; Finster, Kai; Stenvang, Marcel R; Christiansen, Gunna; Vad, Brian S; Bøggild, Andreas; Otzen, Daniel E; Nielsen, Per Halkjær

2015-08-14

Archaea are renowned for their ability to thrive in extreme environments, although they can be found in virtually all habitats. Their adaptive success is linked to their unique cell envelopes that are extremely resistant to chemical and thermal denaturation and that resist proteolysis by common proteases. Here we employ amyloid-specific conformation antibodies and biophysical techniques to show that the extracellular cell wall sheaths encasing the methanogenic archaea Methanosaeta thermophila PT are functional amyloids. Depolymerization of sheaths and subsequent MS/MS analyses revealed that the sheaths are composed of a single major sheath protein (MspA). The amyloidogenic nature of MspA was confirmed by in vitro amyloid formation of recombinant MspA under a wide range of environmental conditions. This is the first report of a functional amyloid from the archaeal domain of life. The amyloid nature explains the extreme resistance of the sheath, the elastic properties that allow diffusible substrates to penetrate through expandable hoop boundaries, and how the sheaths are able to split and elongate outside the cell. The archaeal sheath amyloids do not share homology with any of the currently known functional amyloids and clearly represent a new function of the amyloid protein fold. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Lactobacillus casei BL23 Produces Microvesicles Carrying Proteins That Have Been Associated with Its Probiotic Effect

PubMed Central

Domínguez Rubio, A. Paula; Martínez, Jimena H.; Martínez Casillas, Diana C.; Coluccio Leskow, Federico; Piuri, Mariana; Pérez, Oscar E.

2017-01-01

Archaea, bacteria, and eukarya secrete membrane microvesicles (MVs) as a mechanism for intercellular communication. We report the isolation and characterization of MVs from the probiotic strain Lactobacillus casei BL23. MVs were characterized using analytical high performance techniques, DLS, AFM and TEM. Similar to what has been described for other Gram-positive bacteria, MVs were on the nanometric size range (30–50 nm). MVs carried cytoplasmic components such as DNA, RNA and proteins. Using a proteomic approach (LC-MS), we identified a total of 103 proteins; 13 exclusively present in the MVs. The MVs content included cell envelope associated and secretory proteins, heat and cold shock proteins, several metabolic enzymes, proteases, structural components of the ribosome, membrane transporters, cell wall-associated hydrolases and phage related proteins. In particular, we identified proteins described as mediators of Lactobacillus’ probiotic effects such as p40, p75 and the product of LCABL_31160, annotated as an adhesion protein. The presence of these proteins suggests a role for the MVs in the bacteria-gastrointestinal cells interface. The expression and further encapsulation of proteins into MVs of GRAS (Generally Recognized as Safe) bacteria could represent a scientific novelty, with applications in food, nutraceuticals and clinical therapies. PMID:28979244

Antibiotic resistance in Staphylococcus aureus. Current status and future prospects.

PubMed

Foster, Timothy J

2017-05-01

The major targets for antibiotics in staphylococci are (i) the cell envelope, (ii) the ribosome and (iii) nucleic acids. Several novel targets emerged from recent targeted drug discovery programmes including the ClpP protease and FtsZ from the cell division machinery. Resistance can either develop by horizontal transfer of resistance determinants encoded by mobile genetic elements viz plasmids, transposons and the staphylococcal cassette chromosome or by mutations in chromosomal genes. Horizontally acquired resistance can occur by one of the following mechanisms: (i) enzymatic drug modification and inactivation, (ii) enzymatic modification of the drug binding site, (iii) drug efflux, (iv) bypass mechanisms involving acquisition of a novel drug-resistant target, (v) displacement of the drug to protect the target. Acquisition of resistance by mutation can result from (i) alteration of the drug target that prevents the inhibitor from binding, (ii) derepression of chromosomally encoded multidrug resistance efflux pumps and (iii) multiple stepwise mutations that alter the structure and composition of the cell wall and/or membrane to reduce drug access to its target. This review focuses on development of resistance to currently used antibiotics and examines future prospects for new antibiotics and informed use of drug combinations. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Interactions of Condensed Tannins with Saccharomyces cerevisiae Yeast Cells and Cell Walls: Tannin Location by Microscopy.

PubMed

Mekoue Nguela, Julie; Vernhet, Aude; Sieczkowski, Nathalie; Brillouet, Jean-Marc

2015-09-02

Interactions between grape tannins/red wine polyphenols and yeast cells/cell walls was previously studied within the framework of red wine aging and the use of yeast-derived products as an alternative to aging on lees. Results evidenced a quite different behavior between whole cells (biomass grown to elaborate yeast-derived products, inactivated yeast, and yeast inactivated after autolysis) and yeast cell walls (obtained from mechanical disruption of the biomass). Briefly, whole cells exhibited a high capacity to irreversibly adsorb grape and wine tannins, whereas only weak interactions were observed for cell walls. This last point was quite unexpected considering the literature and called into question the real role of cell walls in yeasts' ability to fix tannins. In the present work, tannin location after interactions between grape and wine tannins and yeast cells and cell walls was studied by means of transmission electron microscopy, light epifluorescence, and confocal microscopy. Microscopy observations evidenced that if tannins interact with cell walls, and especially cell wall mannoproteins, they also diffuse freely through the walls of dead cells to interact with their plasma membrane and cytoplasmic components.

Deltabaculoviruses encode a functional type I budded virus envelope fusion protein

USDA-ARS?s Scientific Manuscript database

Envelope fusion proteins (F proteins) are major constituents of budded viruses (BVs) of alpha- and betabaculoviruses (Baculoviridae) and are essential for the systemic infection of insect larvae and insect cells in culture. An F protein homolog gene was absent in gammabaculoviruses. Here we show tha...

At the border: the plasma membrane-cell wall continuum.

PubMed

Liu, Zengyu; Persson, Staffan; Sánchez-Rodríguez, Clara

2015-03-01

Plant cells rely on their cell walls for directed growth and environmental adaptation. Synthesis and remodelling of the cell walls are membrane-related processes. During cell growth and exposure to external stimuli, there is a constant exchange of lipids, proteins, and other cell wall components between the cytosol and the plasma membrane/apoplast. This exchange of material and the localization of cell wall proteins at certain spots in the plasma membrane seem to rely on a particular membrane composition. In addition, sensors at the plasma membrane detect changes in the cell wall architecture, and activate cytoplasmic signalling schemes and ultimately cell wall remodelling. The apoplastic polysaccharide matrix is, on the other hand, crucial for preventing proteins diffusing uncontrollably in the membrane. Therefore, the cell wall-plasma membrane link is essential for plant development and responses to external stimuli. This review focuses on the relationship between the cell wall and plasma membrane, and its importance for plant tissue organization. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Plant and algal cell walls: diversity and functionality

PubMed Central

Popper, Zoë A.; Ralet, Marie-Christine; Domozych, David S.

2014-01-01

Background Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore, wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes (plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. Scope The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every aspect of plant and algal physiology that will present many of the major challenges in future cell wall research. PMID:25453142

Plant and algal cell walls: diversity and functionality.

PubMed

Popper, Zoë A; Ralet, Marie-Christine; Domozych, David S

2014-10-01

Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore,wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes ( plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every aspect of plant and algal physiology that will present many of the major challenges in future cell wall research.

DBIO Best Thesis Award: Mechanics, Dynamics, and Organization of the Bacterial Cytoskeleton and Cell Wall

NASA Astrophysics Data System (ADS)

Wang, Siyuan

2012-02-01

Bacteria come in a variety of shapes. While the peptidoglycan (PG) cell wall serves as an exoskeleton that defines the static cell shape, the internal bacterial cytoskeleton mediates cell shape by recruiting PG synthesis machinery and thus defining the pattern of cell-wall synthesis. While much is known about the chemistry and biology of the cytoskeleton and cell wall, much of their biophysics, including essential aspects of the functionality, dynamics, and organization, remain unknown. This dissertation aims to elucidate the detailed biophysical mechanisms of cytoskeleton guided wall synthesis. First, I find that the bacterial cytoskeleton MreB contributes nearly as much to the rigidity of an Escherichia coli cell as the cell wall. This conclusion implies that the cytoskeletal polymer MreB applies meaningful force to the cell wall, an idea favored by theoretical modeling of wall growth, and suggests an evolutionary origin of cytoskeleton-governed cell rigidity. Second, I observe that MreB rotates around the long axis of E. coli, and the motion depends on wall synthesis. This is the first discovery of a cell-wall assembly driven molecular motor in bacteria. Third, I prove that both cell-wall synthesis and the PG network have chiral ordering, which is established by the spatial pattern of MreB. This work links the molecular structure of the cytoskeleton and of the cell wall with organismal-scale behavior. Finally, I develop a mathematical model of cytoskeleton-cell membrane interactions, which explains the preferential orientation of different cytoskeleton components in bacteria.

Wall effects in continuous microfluidic magneto-affinity cell separation.

PubMed

Wu, Liqun; Zhang, Yong; Palaniapan, Moorthi; Roy, Partha

2010-05-01

Continuous microfluidic magneto-affinity cell separator combines unique microscale flow phenomenon with advantageous nanobead properties, to isolate cells with high specificity. Owing to the comparable size of the cell-bead complexes and the microchannels, the walls of the microchannel exert a strong influence on the separation of cells by this method. We present a theoretical and experimental study that provides a quantitative description of hydrodynamic wall interactions and wall rolling velocity of cells. A transient convection model describes the transport of cells in two-phase microfluidic flow under the influence of an external magnetic field. Transport of cells along the microchannel walls is also considered via an additional equation. Results show the variation of cell flux in the fluid phases and the wall as a function of a dimensionless parameter arising in the equations. Our results suggest that conditions may be optimized to maximize cell separation while minimizing contact with the wall surfaces. Experimentally measured cell rolling velocities on the wall indicate the presence of other near-wall forces in addition to fluid shear forces. Separation of a human colon carcinoma cell line from a mixture of red blood cells, with folic acid conjugated 1 microm and 200 nm beads, is reported.

Arsenate arrests flagellar rotation in cytoplasm-free envelopes of bacteria.

PubMed Central

Margolin, Y; Barak, R; Eisenbach, M

1994-01-01

The effect of arsenate on flagellar rotation in cytoplasm-free flagellated envelopes of Escherichia coli and Salmonella typhimurium was investigated. Flagellar rotation ceased as soon as the envelopes were exposed to arsenate. Inclusion of phosphate intracellularly (but not extracellular) prevented the inhibition by arsenate. In a parallel experiment, the rotation was not affected by inclusion of an ATP trap (hexokinase and glucose) within the envelopes. It is concluded that arsenate affects the motor in a way other than reversible deenergization. This may be an irreversible damage to the cell or direct inhibition of the motor by arsenate. The latter possibility suggests that a process of phosphorylation or phosphate binding is involved in the motor function. PMID:8071237

The Cell Nucleus Serves as a Mechanotransducer of Tissue Damage-Induced Inflammation.

PubMed

Enyedi, Balázs; Jelcic, Mark; Niethammer, Philipp

2016-05-19

Tissue damage activates cytosolic phospholipase A2 (cPLA2), releasing arachidonic acid (AA), which is oxidized to proinflammatory eicosanoids by 5-lipoxygenase (5-LOX) on the nuclear envelope. How tissue damage is sensed to activate cPLA2 is unknown. We investigated this by live imaging in wounded zebrafish larvae, where damage of the fin tissue causes osmotic cell swelling at the wound margin and the generation of a chemotactic eicosanoid signal. Osmotic swelling of cells and their nuclei activates cPla2 by translocating it from the nucleoplasm to the nuclear envelope. Elevated cytosolic Ca(2+) was necessary but not sufficient for cPla2 translocation, and nuclear swelling was required in parallel. cPla2 translocation upon nuclear swelling was reconstituted in isolated nuclei and appears to be a simple physical process mediated by tension in the nuclear envelope. Our data suggest that the nucleus plays a mechanosensory role in inflammation by transducing cell swelling and lysis into proinflammatory eicosanoid signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Origin of the cell nucleus, mitosis and sex: roles of intracellular coevolution.

PubMed

Cavalier-Smith, Thomas

2010-02-04

The transition from prokaryotes to eukaryotes was the most radical change in cell organisation since life began, with the largest ever burst of gene duplication and novelty. According to the coevolutionary theory of eukaryote origins, the fundamental innovations were the concerted origins of the endomembrane system and cytoskeleton, subsequently recruited to form the cell nucleus and coevolving mitotic apparatus, with numerous genetic eukaryotic novelties inevitable consequences of this compartmentation and novel DNA segregation mechanism. Physical and mutational mechanisms of origin of the nucleus are seldom considered beyond the long-standing assumption that it involved wrapping pre-existing endomembranes around chromatin. Discussions on the origin of sex typically overlook its association with protozoan entry into dormant walled cysts and the likely simultaneous coevolutionary, not sequential, origin of mitosis and meiosis. I elucidate nuclear and mitotic coevolution, explaining the origins of dicer and small centromeric RNAs for positionally controlling centromeric heterochromatin, and how 27 major features of the cell nucleus evolved in four logical stages, making both mechanisms and selective advantages explicit: two initial stages (origin of 30 nm chromatin fibres, enabling DNA compaction; and firmer attachment of endomembranes to heterochromatin) protected DNA and nascent RNA from shearing by novel molecular motors mediating vesicle transport, division, and cytoplasmic motility. Then octagonal nuclear pore complexes (NPCs) arguably evolved from COPII coated vesicle proteins trapped in clumps by Ran GTPase-mediated cisternal fusion that generated the fenestrated nuclear envelope, preventing lethal complete cisternal fusion, and allowing passive protein and RNA exchange. Finally, plugging NPC lumens by an FG-nucleoporin meshwork and adopting karyopherins for nucleocytoplasmic exchange conferred compartmentation advantages. These successive changes took place in naked growing cells, probably as indirect consequences of the origin of phagotrophy. The first eukaryote had 1-2 cilia and also walled resting cysts; I outline how encystation may have promoted the origin of meiotic sex. I also explain why many alternative ideas are inadequate. Nuclear pore complexes are evolutionary chimaeras of endomembrane- and mitosis-related chromatin-associated proteins. The keys to understanding eukaryogenesis are a proper phylogenetic context and understanding organelle coevolution: how innovations in one cell component caused repercussions on others.

Pulmonary Langerhans cell histiocytosis with cervical lymph node involvement, and coexistence with pulmonary tuberculosis and right pneumothorax: a case report and review of literature.

PubMed

Gao, Limin; Li, Huifang; Li, Gandi; Liu, Weiping; Li, Jinnan; Zhang, Wenyan

2015-01-01

We report an uncommon 22-year-old male Pulmonary Langerhans Cell Histiocytosis (PLCH) case which co-existed with pulmonary tuberculosis (TB). Unlike the common PLCH cases, this PLCH case has cervical lymph node involvement and right pneumothorax. The diagnosis was established by the imaging of lung and the biopsies of the lung and left neck lymph node. Imaging of the chest showed characteristic small nodules and thin-walled cysts and right pneumothorax. The LCH cells in the lung and left neck lymph node were characterized by large convoluted nuclei with cerebriform indentations of the nuclear envelope and longitudinal grooves. The nuclei contained small eosinophilic nucleoli and moderate amount cytoplasm. Immunohistochemically, the histiocytoid cells were positive for Langerin, CD1a and S-100. Acid-fast bacilli were found in sputum and lung biopsy tissue. To the best of our knowledge, this is the first case of PLCH with cervical lymph node involvement, and coexisted with pulmonary tuberculosis, right pneumothorax. A contribution of this case and review three of the five cases of PLCH with extrapulmonary involvement to lymph nodes resolved spontaneously after smoking cessation constitute a novel addition that it is inappropriate to regard pulmonary/nodal LCH as multi-organ or disseminated disease, and the treatment methods are the same whether the PLCH patient with lymph node involvement or not.

C. elegans Nuclear Envelope Proteins Emerin, MAN1, Lamin, and Nucleoporins Reveal Unique Timing of Nuclear Envelope Breakdown during Mitosis

PubMed Central

Lee, Kenneth K.; Gruenbaum, Yosef; Spann, Perah; Liu, Jun; Wilson, Katherine L.

2000-01-01

Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes. PMID:10982402

Physics of Cellular Movements

NASA Astrophysics Data System (ADS)

Sackmann, Erich; Keber, Felix; Heinrich, Doris

2010-04-01

The survival of cells depends on perpetual active motions, including (a) bending excitations of the soft cell envelopes, (b) the bidirectional transport of materials and organelles between the cell center and the periphery, and (c) the ongoing restructuring of the intracellular macromolecular scaffolds mediating global cell changes associated with cell adhesion locomotion and phagocytosis. Central questions addressed are the following: How can this bustling motion of extremely complex soft structures be characterized and measured? What are the major driving forces? Further topics include (a) the active dynamic control of global shape changes by the interactive coupling of the aster-like soft scaffold of microtubules and the network of actin filaments associated with the cell envelope (the actin cortex) and (b) the generation of propulsion forces by solitary actin gelation waves propagating within the actin cortex.

In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses

PubMed Central

Tani, Hideki; Limn, Chang Kwang; Yap, Chan Choo; Onishi, Masayoshi; Nozaki, Masami; Nishimune, Yoshitake; Okahashi, Nobuo; Kitagawa, Yoshinori; Watanabe, Rie; Mochizuki, Rika; Moriishi, Kohji; Matsuura, Yoshiharu

2003-01-01

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy. PMID:12941888

Fission yeast Lem2 and Man1 perform fundamental functions of the animal cell nuclear lamina.

PubMed

Gonzalez, Yanira; Saito, Akira; Sazer, Shelley

2012-01-01

In animal cells the nuclear lamina, which consists of lamins and lamin-associated proteins, serves several functions: it provides a structural scaffold for the nuclear envelope and tethers proteins and heterochromatin to the nuclear periphery. In yeast, proteins and large heterochromatic domains including telomeres are also peripherally localized, but there is no evidence that yeast have lamins or a fibrous nuclear envelope scaffold. Nonetheless, we found that the Lem2 and Man1 proteins of the fission yeast Schizosaccharomyces pombe, evolutionarily distant relatives of the Lap2/Emerin/Man1 (LEM) sub-family of animal cell lamin-associated proteins, perform fundamental functions of the animal cell lamina. These integral inner nuclear membrane localized proteins, with nuclear localized DNA binding Helix-Extension-Helix (HEH) domains, impact nuclear envelope structure and integrity, are essential for the enrichment of telomeres at the nuclear periphery and by means of their HEH domains anchor chromatin, most likely transcriptionally repressed heterochromatin, to the nuclear periphery. These data indicate that the core functions of the nuclear lamina are conserved between fungi and animal cells and can be performed in fission yeast, without lamins or other intermediate filament proteins.

Characterization of a 3rd Generation Lentiviral Vector Pseudotyped With Nipah Virus Envelope Proteins For Endothelial Cell Transduction

PubMed Central

Witting, Scott R.; Vallanda, Priya; Gamble, Aisha L.

2013-01-01

Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, 3rd generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared to VSV pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in 3rd generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells. PMID:23698741

Characterization of a third generation lentiviral vector pseudotyped with Nipah virus envelope proteins for endothelial cell transduction.

PubMed

Witting, S R; Vallanda, P; Gamble, A L

2013-10-01

Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, third generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared with vesicular stomatitis virus pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in third generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells.

Fast kinetic studies of plasmid DNA transfer in intact yeast cells mediated by electropulsation.

PubMed

Ganeva, V; Galutzov, B; Teissie, J

1995-09-25

Intact yeast cell Electrotransformation process was investigated. It is a two step process. The plasmid must be pre-mixed and present in contact with the cells during the pulse. During the millisecond field pulse, plasmid DNA is associated to the envelope. It therefore crosses the membrane by a process which lasts several seconds as shown by its sensitivity to a post pulse addition of DNase. Electrotransformation is not supported by an electrophoretic transfer due to the external field nor by a free diffusion across the electropermeabilized envelope. DNA is first bound during the field pulse and then is transferred by a still unknown active process due to cell metabolism.

Direct observation of nanoparticle-cancer cell nucleus interactions.

PubMed

Dam, Duncan Hieu M; Lee, Jung Heon; Sisco, Patrick N; Co, Dick T; Zhang, Ming; Wasielewski, Michael R; Odom, Teri W

2012-04-24

We report the direct visualization of interactions between drug-loaded nanoparticles and the cancer cell nucleus. Nanoconstructs composed of nucleolin-specific aptamers and gold nanostars were actively transported to the nucleus and induced major changes to the nuclear phenotype via nuclear envelope invaginations near the site of the construct. The number of local deformations could be increased by ultrafast, light-triggered release of the aptamers from the surface of the gold nanostars. Cancer cells with more nuclear envelope folding showed increased caspase 3 and 7 activity (apoptosis) as well as decreased cell viability. This newly revealed correlation between drug-induced changes in nuclear phenotype and increased therapeutic efficacy could provide new insight for nuclear-targeted cancer therapy.

Structure, function, and biosynthesis of plant cell walls: proceedings of the seventh annual symposium in botany

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

Dugger, W.M.; Bartnicki-Garcia, S.

Papers in the following areas were included in these symposium proceedings: (1) cell wall chemistry and biosynthesis; (2) cell wall hydrolysis and associated physiology; (3) cellular events associated with cell wall biosynthesis; and (4) interactions of plant cell walls with pathogens and related responses. Papers have been individually abstracted for the data base. (ACR)

Construction of fusogenic vesicles bearing specific antibodies. Targeting of reconstituted Sendai virus envelopes towards neuraminidase-treated human erythrocytes.

PubMed

Gitman, A G; Loyter, A

1984-08-10

The cross-linking reagents succinimidyl-4-(p-maleimidophenyl)-butyrate and N-succinimidyl-3-(2-pyridyldithio)-propionate were used to covalently attach antibodies against human erythrocytes to the thiol-containing paraffin, dodecanethiol. The complex formed, dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody was inserted into the membranes of reconstituted Sendai virus envelopes. This was achieved by addition of the dodecanethiol-maleimidophenylbutyrate-antibody to a detergent solution (Triton X-100) containing the viral envelope phospholipids and glycoproteins. Removal of the detergent led to the formation of vesicles containing the viral glycoprotein and the dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody complexes within the same membrane. Reconstituted Sendai virus envelope-bearing antibodies against human erythrocytes were able to fuse with human erythrocytes (as was reflected by reconstituted Sendai virus envelope-induced hemolysis) from which the natural virus receptors were removed by treatment with neuraminidase. Thus, it appears that anti-human erythrocyte antibodies could substitute for the viral binding protein (hemagglutinin/neuraminidase glycoprotein) in mediating functional binding of the virus particles to the cell plasma membranes. Furthermore, from the results of the present work, it may be inferred that in addition to being the viral-binding protein, hemagglutinin/neuraminidase glycoprotein actively participates in the process of virus-cell fusion.

Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes.

PubMed

Petrovsky, Roman; Krohne, Georg; Großhans, Jörg

2018-03-01

The nuclear envelope has a stereotypic morphology consisting of a flat double layer of the inner and outer nuclear membrane, with interspersed nuclear pores. Underlying and tightly linked to the inner nuclear membrane is the nuclear lamina, a proteinous layer of intermediate filament proteins and associated proteins. Physiological, experimental or pathological alterations in the constitution of the lamina lead to changes in nuclear morphology, such as blebs and lobulations. It has so far remained unclear whether the morphological changes depend on the differentiation state and the specific lamina protein. Here we analysed the ultrastructural morphology of the nuclear envelope in intestinal stem cells and differentiated enterocytes in adult Drosophila flies, in which the proteins Lam, Kugelkern or a farnesylated variant of LamC were overexpressed. Surprisingly, we detected distinct morphological features specific for the respective protein. Lam induced envelopes with multiple layers of membrane and lamina, surrounding the whole nucleus whereas farnesylated LamC induced the formation of a thick fibrillary lamina. In contrast, Kugelkern induced single-layered and double-layered intranuclear membrane structures, which are likely be derived from infoldings of the inner nuclear membrane or of the double layer of the envelope. Copyright © 2018 Elsevier GmbH. All rights reserved.

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

PubMed Central

Orlean, Peter

2012-01-01

The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking vary during growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells, covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphosphatidylinositol membrane anchors, β1,3- and β1,6-linked glucans, and chitin; the reactions that cross-link wall components; and the possible functions of enzymatic and nonenzymatic cell wall proteins. PMID:23135325

Retention of Proanthocyanidin in Wine-like Solution Is Conferred by a Dynamic Interaction between Soluble and Insoluble Grape Cell Wall Components.

PubMed

Bindon, Keren A; Li, Sijing; Kassara, Stella; Smith, Paul A

2016-11-09

For better understanding of the factors that impact proanthocyanidin (PA) adsorption by insoluble cell walls or interaction with soluble cell wall-derived components, application of a commercial polygalacturonase enzyme preparation was investigated to modify grape cell wall structure. Soluble and insoluble cell wall material was isolated from the skin and mesocarp components of Vitis vinifera Shiraz grapes. It was observed that significant depolymerization of the insoluble grape cell wall occurred following enzyme application to both grape cell wall fractions, with increased solubilization of rhamnogalacturonan-enriched, low molecular weight polysaccharides. However, in the case of grape mesocarp, the solubilization of protein from cell walls (in buffer) was significant and increased only slightly by the enzyme treatment. Enzyme treatment significantly reduced the adsorption of PA by insoluble cell walls, but this effect was observed only when material solubilized from grape cell walls had been removed. The loss of PA through interaction with the soluble cell wall fraction was observed to be greater for mesocarp than skin cell walls. Subsequent experiments on the soluble mesocarp cell wall fraction confirmed a role for protein in the precipitation of PA. This identified a potential mechanism by which extracted grape PA may be lost from wine during vinification, as a precipitate with solubilized grape mesocarp proteins. Although protein was a minor component in terms of total concentration, losses of PA via precipitation with proteins were in the order of 50% of available PA. PA-induced precipitation could proceed until all protein was removed from solution and may account for the very low levels of residual protein observed in red wines. The results point to a dynamic interaction of grape insoluble and soluble components in modulating PA retention in wine.

The transcription factor Rap1p is required for tolerance to cell-wall perturbing agents and for cell-wall maintenance in Saccharomyces cerevisiae.

PubMed

Azad, Gajendra Kumar; Singh, Vikash; Baranwal, Shivani; Thakare, Mayur Jankiram; Tomar, Raghuvir S

2015-01-02

Yeast repressor activator protein (Rap1p) is involved in genomic stability and transcriptional regulation. We explored the function of Rap1p in yeast physiology using Rap1p truncation mutants. Our results revealed that the N-terminal truncation of Rap1p (Rap1ΔN) leads to hypersensitivity towards elevated temperature and cell-wall perturbing agents. Cell wall analysis showed an increase in the chitin and glucan content in Rap1ΔN cells as compared with wild type cells. Accordingly, mutant cells had a twofold thicker cell wall, as observed by electron microscopy. Furthermore, Rap1ΔN cells had increased levels of phosphorylated Slt2p, a MAP kinase of the cell wall integrity pathway. Mutant cells also had elevated levels of cell wall integrity response transcripts. Taken together, our findings suggest a connection between Rap1p and cell wall homeostasis. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Bacterial cell-wall recycling

PubMed Central

Johnson, Jarrod W.; Fisher, Jed F.; Mobashery, Shahriar

2012-01-01

Many Gram-negative and Gram-positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many—and quite possibly all—bacteria, the peptidoglycan fragments are recovered and recycled. While cell-wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell-wall–targeting antibiotics and to regulate resistance mechanisms. In several Gram-negative pathogens, anhydro-MurNAc-peptide cell-wall fragments regulate AmpC β-lactamase induction. In some Gram-positive organisms, short peptides derived from the cell wall regulate the induction of both β-lactamase and β-lactam-resistant penicillin-binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell-wall-targeted antibiotics. Indeed, such inhibitors improve the potency of β-lactams in vitro against inducible AmpC β-lactamase-producing bacteria. We describe the key steps of cell-wall remodeling and recycling, the regulation of resistance mechanisms by cell-wall recycling, and recent advances toward the discovery of cell-wall recycling inhibitors. PMID:23163477

Characteristic Thickened Cell Walls of the Bracts of the ‘Eternal Flower’ Helichrysum bracteatum

PubMed Central

Nishikawa, Kuniko; Ito, Hiroaki; Awano, Tatsuya; Hosokawa, Munetaka; Yazawa, Susumu

2008-01-01

Background and Aims Helichrysum bracteatum is called an ‘eternal flower’ and has large, coloured, scarious bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum. Methods DAPI (4'6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized light microscopy were used for observation of cells, including cell wall morphology. Key Results Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not observed in the petal and bract tissues of Chrysanthemum morifolium. Conclusions Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves. The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they may be a new cell type. PMID:18436550

Mechanical feedback coordinates cell wall expansion and assembly in yeast mating morphogenesis

PubMed Central

2018-01-01

The shaping of individual cells requires a tight coordination of cell mechanics and growth. However, it is unclear how information about the mechanical state of the wall is relayed to the molecular processes building it, thereby enabling the coordination of cell wall expansion and assembly during morphogenesis. Combining theoretical and experimental approaches, we show that a mechanical feedback coordinating cell wall assembly and expansion is essential to sustain mating projection growth in budding yeast (Saccharomyces cerevisiae). Our theoretical results indicate that the mechanical feedback provided by the Cell Wall Integrity pathway, with cell wall stress sensors Wsc1 and Mid2 increasingly activating membrane-localized cell wall synthases Fks1/2 upon faster cell wall expansion, stabilizes mating projection growth without affecting cell shape. Experimental perturbation of the osmotic pressure and cell wall mechanics, as well as compromising the mechanical feedback through genetic deletion of the stress sensors, leads to cellular phenotypes that support the theoretical predictions. Our results indicate that while the existence of mechanical feedback is essential to stabilize mating projection growth, the shape and size of the cell are insensitive to the feedback. PMID:29346368

Wall relaxation and the driving forces for cell expansive growth

NASA Technical Reports Server (NTRS)

Cosgrove, D. J.

1987-01-01

When water uptake by growing cells is prevented, the turgor pressure and the tensile stress in the cell wall are reduced by continued wall loosening. This process, termed in vivo stress relaxation, provides a new way to study the dynamics of wall loosening and to measure the wall yield threshold and the physiological wall extensibility. Stress relaxation experiments indicate that wall stress supplies the mechanical driving force for wall yielding. Cell expansion also requires water absorption. The driving force for water uptake during growth is created by wall relaxation, which lowers the water potential of the expanding cells. New techniques for measuring this driving force show that it is smaller than believed previously; in elongating stems it is only 0.3 to 0.5 bar. This means that the hydraulic resistance of the water transport pathway is small and that rate of cell expansion is controlled primarily by wall loosening and yielding.

Two endogenous proteins that induce cell wall extension in plants

NASA Technical Reports Server (NTRS)

McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.

1992-01-01

Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

The roles of thermal insulation and heat storage in the energy performance of the wall materials: a simulation study.

PubMed

Long, Linshuang; Ye, Hong

2016-04-07

A high-performance envelope is the prerequisite and foundation to a zero energy building. The thermal conductivity and volumetric heat capacity of a wall are two thermophysical properties that strongly influence the energy performance. Although many case studies have been performed, the results failed to give a big picture of the roles of these properties in the energy performance of an active building. In this work, a traversal study on the energy performance of a standard room with all potential wall materials was performed for the first time. It was revealed that both heat storage materials and insulation materials are suitable for external walls. However, the importances of those materials are distinct in different situations: the heat storage plays a primary role when the thermal conductivity of the material is relatively high, but the effect of the thermal insulation is dominant when the conductivity is relatively low. Regarding internal walls, they are less significant to the energy performance than the external ones, and they need exclusively the heat storage materials with a high thermal conductivity. These requirements for materials are consistent under various climate conditions. This study may provide a roadmap for the material scientists interested in developing high-performance wall materials.

The roles of thermal insulation and heat storage in the energy performance of the wall materials: a simulation study

PubMed Central

Long, Linshuang; Ye, Hong

2016-01-01

A high-performance envelope is the prerequisite and foundation to a zero energy building. The thermal conductivity and volumetric heat capacity of a wall are two thermophysical properties that strongly influence the energy performance. Although many case studies have been performed, the results failed to give a big picture of the roles of these properties in the energy performance of an active building. In this work, a traversal study on the energy performance of a standard room with all potential wall materials was performed for the first time. It was revealed that both heat storage materials and insulation materials are suitable for external walls. However, the importances of those materials are distinct in different situations: the heat storage plays a primary role when the thermal conductivity of the material is relatively high, but the effect of the thermal insulation is dominant when the conductivity is relatively low. Regarding internal walls, they are less significant to the energy performance than the external ones, and they need exclusively the heat storage materials with a high thermal conductivity. These requirements for materials are consistent under various climate conditions. This study may provide a roadmap for the material scientists interested in developing high-performance wall materials. PMID:27052186

Retrofitted green roofs and walls and improvements in thermal comfort

NASA Astrophysics Data System (ADS)

Feitosa, Renato Castiglia; Wilkinson, Sara

2017-06-01

Increased urbanization has led to a worsening in the quality of life for many people living in large cities in respect of the urban heat island effect and increases of indoor temperatures in housing and other buildings. A solution may be to retrofit existing environments to their former conditions, with a combination of green infrastructures applied to existing walls and rooftops. Retrofitted green roofs may attenuate housing temperature. However, with tall buildings, facade areas are much larger compared to rooftop areas, the role of green walls in mitigating extreme temperatures is more pronounced. Thus, the combination of green roofs and green walls is expected to promote a better thermal performance in the building envelope. For this purpose, a modular vegetated system is adopted for covering both walls and rooftops. Rather than temperature itself, the heat index, which comprises the combined effect of temperature and relative humidity is used in the evaluation of thermal comfort in small scale experiments performed in Sydney - Australia, where identical timber framed structures prototypes (vegetated and non-vegetated) are compared. The results have shown a different understanding of thermal comfort improvement regarding heat index rather than temperature itself. The combination of green roof and walls has a valid role to play in heat index attenuation.

Pectinous cell wall thickenings formation - A common defense strategy of plants to cope with Pb.

PubMed

Krzesłowska, Magdalena; Rabęda, Irena; Basińska, Aneta; Lewandowski, Michał; Mellerowicz, Ewa J; Napieralska, Anna; Samardakiewicz, Sławomir; Woźny, Adam

2016-07-01

Lead, one of the most abundant and hazardous trace metals affecting living organisms, has been commonly detected in plant cell walls including some tolerant plants, mining ecotypes and hyperaccumulators. We have previously shown that in tip growing Funaria sp. protonemata cell wall is remodeled in response to lead by formation of thickenings rich in low-methylesterified pectins (pectin epitope JIM5 - JIM5-P) able to bind metal ions, which accumulate large amounts of Pb. Hence, it leads to the increase of cell wall capacity for Pb compartmentalization. Here we show that diverse plant species belonging to different phyla (Arabidopsis, hybrid aspen, star duckweed), form similar cell wall thickenings in response to Pb. These thickenings are formed in tip growing cells such as the root hairs, and in diffuse growing cells such as meristematic and root cap columella cells of root apices in hybrid aspen and Arabidopsis and in mesophyll cells in star duckweed fronds. Notably, all analyzed cell wall thickenings were abundant in JIM5-P and accumulated high amounts of Pb. In addition, the co-localization of JIM5-P and Pb commonly occurred in these cells. Hence, cell wall thickenings formed the extra compartment for Pb accumulation. In this way plant cells increased cell wall capacity for compartmentalization of this toxic metal, protecting protoplast from its toxicity. As cell wall thickenings occurred in diverse plant species and cell types differing in the type of growth we may conclude that pectinous cell wall thickenings formation is a widespread defense strategy of plants to cope with Pb. Moreover, detection of natural defense strategy, increasing plant cell walls capacity for metal accumulation, reveals a promising direction for enhancing plant efficiency in phytoremediation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genetic and biochemical characterization of the GH72 family of cell wall transglycosylases in Neurospora crassa.

PubMed

Ao, Jie; Free, Stephen J

2017-04-01

The Neurospora crassa genome encodes five GH72 family transglycosylases, and four of these enzymes (GEL-1, GEL-2, GEL-3 and GEL-5) have been found to be present in the cell wall proteome. We carried out an extensive genetic analysis on the role of these four transglycosylases in cell wall biogenesis and demonstrated that the transglycosylases are required for the formation of a normal cell wall. As suggested by the proteomic analysis, we found that multiple transglycosylases were being expressed in N. crassa cells and that different combinations of the enzymes are required in different cell types. The combination of GEL-1, GEL-2 and GEL-5 is required for the growth of vegetative hyphae, while the GEL-1, GEL-2, GEL-3 combination is needed for the production of aerial hyphae and conidia. Our data demonstrates that the enzymes are redundant with partially overlapping enzymatic activities, which provides the fungus with a robust cell wall biosynthetic system. Characterization of the transglycosylase-deficient mutants demonstrated that the incorporation of cell wall proteins was severely compromised. Interestingly, we found that the transglycosylase-deficient mutant cell walls contained more β-1,3-glucan than the wild type cell wall. Our results demonstrate that the GH72 transglycosylases are not needed for the incorporation of β-1,3-glucan into the cell wall, but they are required for the incorporation of cell wall glycoprotein into the cell wall. Copyright © 2017 Elsevier Inc. All rights reserved.

A Cell Wall Proteome and Targeted Cell Wall Analyses Provide Novel Information on Hemicellulose Metabolism in Flax.

PubMed

Chabi, Malika; Goulas, Estelle; Leclercq, Celine C; de Waele, Isabelle; Rihouey, Christophe; Cenci, Ugo; Day, Arnaud; Blervacq, Anne-Sophie; Neutelings, Godfrey; Duponchel, Ludovic; Lerouge, Patrice; Hausman, Jean-François; Renaut, Jenny; Hawkins, Simon

2017-09-01

Experimentally-generated (nanoLC-MS/MS) proteomic analyses of four different flax organs/tissues (inner-stem, outer-stem, leaves and roots) enriched in proteins from 3 different sub-compartments (soluble-, membrane-, and cell wall-proteins) was combined with publically available data on flax seed and whole-stem proteins to generate a flax protein database containing 2996 nonredundant total proteins. Subsequent multiple analyses (MapMan, CAZy, WallProtDB and expert curation) of this database were then used to identify a flax cell wall proteome consisting of 456 nonredundant proteins localized in the cell wall and/or associated with cell wall biosynthesis, remodeling and other cell wall related processes. Examination of the proteins present in different flax organs/tissues provided a detailed overview of cell wall metabolism and highlighted the importance of hemicellulose and pectin remodeling in stem tissues. Phylogenetic analyses of proteins in the cell wall proteome revealed an important paralogy in the class IIIA xyloglucan endo-transglycosylase/hydrolase (XTH) family associated with xyloglucan endo-hydrolase activity.Immunolocalisation, FT-IR microspectroscopy, and enzymatic fingerprinting indicated that flax fiber primary/S1 cell walls contained xyloglucans with typical substituted side chains as well as glucuronoxylans in much lower quantities. These results suggest a likely central role of xyloglucans and endotransglucosylase/hydrolase activity in flax fiber formation and cell wall remodeling processes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots.

PubMed Central

Freshour, G.; Clay, R. P.; Fuller, M. S.; Albersheim, P.; Darvill, A. G.; Hahn, M. G.

1996-01-01

The plant cell wall is a dynamic structure that plays important roles in growth and development and in the interactions of plants with their environment and other organisms. We have used monoclonal antibodies that recognize different carbohydrate epitopes present in plant cell-wall polysaccharides to locate these epitopes in roots of developing Arabidopsis thaliana seedlings. An epitope in the pectic polysaccharide rhamnogalacturonan I is observed in the walls of epidermal and cortical cells in mature parts of the root. This epitope is inserted into the walls in a developmentally regulated manner. Initially, the epitope is observed in atrichoblasts and later appears in trichoblasts and simultaneously in cortical cells. A terminal [alpha]-fucosyl-containing epitope is present in almost all of the cell walls in the root. An arabinosylated (1->6)-[beta]-galactan epitope is also found in all of the cell walls of the root with the exception of lateral root-cap cell walls. It is striking that these three polysaccharide epitopes are not uniformly distributed (or accessible) within the walls of a given cell, nor are these epitopes distributed equally across the two walls laid down by adjacent cells. Our results further suggest that the biosynthesis and differentiation of primary cell walls in plants are precisely regulated in a temporal, spatial, and developmental manner. PMID:12226270

Atomic force microscopy stiffness tomography on living Arabidopsis thaliana cells reveals the mechanical properties of surface and deep cell-wall layers during growth.

PubMed

Radotić, Ksenija; Roduit, Charles; Simonović, Jasna; Hornitschek, Patricia; Fankhauser, Christian; Mutavdžić, Dragosav; Steinbach, Gabor; Dietler, Giovanni; Kasas, Sandor

2012-08-08

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A computational approach for inferring the cell wall properties that govern guard cell dynamics.

PubMed

Woolfenden, Hugh C; Bourdais, Gildas; Kopischke, Michaela; Miedes, Eva; Molina, Antonio; Robatzek, Silke; Morris, Richard J

2017-10-01

Guard cells dynamically adjust their shape in order to regulate photosynthetic gas exchange, respiration rates and defend against pathogen entry. Cell shape changes are determined by the interplay of cell wall material properties and turgor pressure. To investigate this relationship between turgor pressure, cell wall properties and cell shape, we focused on kidney-shaped stomata and developed a biomechanical model of a guard cell pair. Treating the cell wall as a composite of the pectin-rich cell wall matrix embedded with cellulose microfibrils, we show that strong, circumferentially oriented fibres are critical for opening. We find that the opening dynamics are dictated by the mechanical stress response of the cell wall matrix, and as the turgor rises, the pectinaceous matrix stiffens. We validate these predictions with stomatal opening experiments in selected Arabidopsis cell wall mutants. Thus, using a computational framework that combines a 3D biomechanical model with parameter optimization, we demonstrate how to exploit subtle shape changes to infer cell wall material properties. Our findings reveal that proper stomatal dynamics are built on two key properties of the cell wall, namely anisotropy in the form of hoop reinforcement and strain stiffening. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd and Society for Experimental Biology.

Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

PubMed

Lacour, William; Adjili, Salim; Blaising, Julie; Favier, Arnaud; Monier, Karine; Mezhoud, Sarra; Ladavière, Catherine; Place, Christophe; Pécheur, Eve-Isabelle; Charreyre, Marie-Thérèse

2016-08-01

Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Virus-induced gene silencing offers a functional genomics platform for studying plant cell wall formation.

PubMed

Zhu, Xiaohong; Pattathil, Sivakumar; Mazumder, Koushik; Brehm, Amanda; Hahn, Michael G; Dinesh-Kumar, S P; Joshi, Chandrashekhar P

2010-09-01

Virus-induced gene silencing (VIGS) is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus (TRV)-based VIGS system to study functions of genes involved in either primary or secondary cell wall formation in Nicotiana benthamiana plants. A 3-week post-VIGS time frame is sufficient to observe phenotypic alterations in the anatomical structure of stems and chemical composition of the primary and secondary cell walls. We used cell wall glycan-directed monoclonal antibodies to demonstrate that alteration of cell wall polymer synthesis during the secondary growth phase of VIGS plants has profound effects on the extractability of components from woody stem cell walls. Therefore, TRV-based VIGS together with cell wall component profiling methods provide a high-throughput gene discovery platform for studying plant cell wall formation from a bioenergy perspective.

Plant cell wall signalling and receptor-like kinases.

PubMed

Wolf, Sebastian

2017-02-15

Communication between the extracellular matrix and the cell interior is essential for all organisms as intrinsic and extrinsic cues have to be integrated to co-ordinate development, growth, and behaviour. This applies in particular to plants, the growth and shape of which is governed by deposition and remodelling of the cell wall, a rigid, yet dynamic, extracellular network. It is thus generally assumed that cell wall surveillance pathways exist to monitor the state of the wall and, if needed, elicit compensatory responses such as altered expression of cell wall remodelling and biosynthesis genes. Here, I highlight recent advances in the field of cell wall signalling in plants, with emphasis on the role of plasma membrane receptor-like kinase complexes. In addition, possible roles for cell wall-mediated signalling beyond the maintenance of cell wall integrity are discussed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Nipah virion entry kinetics, composition, and conformational changes determined by enzymatic virus-like particles and new flow virometry tools.

PubMed

Landowski, Matthew; Dabundo, Jeffrey; Liu, Qian; Nicola, Anthony V; Aguilar, Hector C

2014-12-01

Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. Drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are notably cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. NiV's attachment (G) and fusion (F) envelope glycoproteins mediate viral binding to the ephrinB2/ephrinB3 cell receptors and virus-cell membrane fusion, respectively. The NiV matrix protein (M) can autonomously induce NiV assembly and budding. Using a β-lactamase (βLa) reporter/NiV-M chimeric protein, we produced NiVLPs expressing NiV-G and wild-type or mutant NiV-F on their surfaces. By preloading target cells with the βLa fluorescent substrate CCF2-AM, we obtained viral entry kinetic curves that correlated with the NiV-F fusogenic phenotypes, validating NiVLPs as suitable viral entry kinetic tools and suggesting overall relatively slower viral entry than cell-cell fusion kinetics. Additionally, the proportions of F and G on individual NiVLPs and the extent of receptor-induced conformational changes in NiV-G were measured via flow virometry, allowing the proper interpretation of the viral entry kinetic phenotypes. The significance of these findings in the viral entry field extends beyond NiV to other paramyxoviruses and enveloped viruses. Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. For example, drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are especially cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. Our new tools allowed us the high-throughput measurement of viral entry kinetics, glycoprotein proportions on individual viral particles, and receptor-induced conformational changes in viral glycoproteins on viral surfaces. The significance of these findings extends beyond NiV to other paramyxoviruses and enveloped viruses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

PubMed Central

Porwal, Manvi; Cohen, Sarah; Snoussi, Kenza; Popa-Wagner, Ruth; Anderson, Fenja; Dugot-Senant, Nathalie; Wodrich, Harald; Dinsart, Christiane; Kleinschmidt, Jürgen A.; Panté, Nelly; Kann, Michael

2013-01-01

Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca++ efflux from the lumen between inner and outer nuclear membrane we found that Ca++ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis. PMID:24204256

The Specific Nature of Plant Cell Wall Polysaccharides 1

PubMed Central

Nevins, Donald J.; English, Patricia D.; Albersheim, Peter

1967-01-01

Polysaccharide compositions of cell walls were assessed by quantitative analyses of the component sugars. Cell walls were hydrolyzed in 2 n trifluoroacetic acid and the liberated sugars reduced to their respective alditols. The alditols were acetylated and the resulting alditol acetates separated by gas chromatography. Quantitative assay of the alditol acetates was accomplished by electronically integrating the detector output of the gas chromatograph. Myo-inositol, introduced into the sample prior to hydrolysis, served as an internal standard. The cell wall polysaccharide compositions of plant varieties within a given species are essentially identical. However, differences in the sugar composition were observed in cell walls prepared from different species of the same as well as of different genera. The fact that the wall compositions of different varieties of the same species are the same indicates that the biosynthesis of cell wall polysaccharides is genetically regulated. The cell walls of various morphological parts (roots, hypocotyls, first internodes and primary leaves) of bean plants were each found to have a characteristic sugar composition. It was found that the cell wall sugar composition of suspension-cultured sycamore cells could be altered by growing the cells on different carbon sources. This demonstrates that the biosynthesis of cell wall polysaccharides can be manipulated without fatal consequences. PMID:16656594

Dynamic changes in transcriptome and cell wall composition underlying brassinosteroid-mediated lignification of switchgrass suspension cells.

PubMed

Rao, Xiaolan; Shen, Hui; Pattathil, Sivakumar; Hahn, Michael G; Gelineo-Albersheim, Ivana; Mohnen, Debra; Pu, Yunqiao; Ragauskas, Arthur J; Chen, Xin; Chen, Fang; Dixon, Richard A

2017-01-01

Plant cell walls contribute the majority of plant biomass that can be used to produce transportation fuels. However, the complexity and variability in composition and structure of cell walls, particularly the presence of lignin, negatively impacts their deconstruction for bioenergy. Metabolic and genetic changes associated with secondary wall development in the biofuel crop switchgrass ( Panicum virgatum ) have yet to be reported. Our previous studies have established a cell suspension system for switchgrass, in which cell wall lignification can be induced by application of brassinolide (BL). We have now collected cell wall composition and microarray-based transcriptome profiles for BL-induced and non-induced suspension cultures to provide an overview of the dynamic changes in transcriptional reprogramming during BL-induced cell wall modification. From this analysis, we have identified changes in candidate genes involved in cell wall precursor synthesis, cellulose, hemicellulose, and pectin formation and ester-linkage generation. We have also identified a large number of transcription factors with expression correlated with lignin biosynthesis genes, among which are candidates for control of syringyl (S) lignin accumulation. Together, this work provides an overview of the dynamic compositional changes during brassinosteroid-induced cell wall remodeling, and identifies candidate genes for future plant genetic engineering to overcome cell wall recalcitrance.

Clindamycin Affects Group A Streptococcus Virulence Factors and Improves Clinical Outcome.

PubMed

Andreoni, Federica; Zürcher, Claudia; Tarnutzer, Andrea; Schilcher, Katrin; Neff, Andrina; Keller, Nadia; Marques Maggio, Ewerton; Poyart, Claire; Schuepbach, Reto A; Zinkernagel, Annelies S

2017-01-15

Group A Streptococcus (GAS) has acquired an arsenal of virulence factors, promoting life-threatening invasive infections such as necrotizing fasciitis. Current therapeutic regimens for necrotizing fasciitis include surgical debridement and treatment with cell wall-active antibiotics. Addition of clindamycin (CLI) is recommended, although clinical evidence is lacking. Reflecting the current clinical dilemma, an observational study showed that only 63% of the patients with severe invasive GAS infection received CLI. This work thus aimed to address whether CLI improves necrotizing fasciitis outcome by modulating virulence factors of CLI-susceptible and CLI-resistant GAS in vitro and in vivo. Treatment with CLI reduced extracellular DNase Sda1 and streptolysin O (SLO) activity in vivo, whereas subinhibitory CLI concentrations induced expression and activity of SLO, DNase, and Streptococcus pyogenes cell envelope protease in vitro. Our in vivo results suggest that CLI should be administered as soon as possible to patients with necrotizing fasciitis, while our in vitro studies emphasize that a high dosage of CLI is essential. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

TIM1 (HAVCR1) Is Not Essential for Cellular Entry of Either Quasi-enveloped or Naked Hepatitis A Virions.

PubMed

Das, Anshuman; Hirai-Yuki, Asuka; González-López, Olga; Rhein, Bethany; Moller-Tank, Sven; Brouillette, Rachel; Hensley, Lucinda; Misumi, Ichiro; Lovell, William; Cullen, John M; Whitmire, Jason K; Maury, Wendy; Lemon, Stanley M

2017-09-05

Receptor molecules play key roles in the cellular entry of picornaviruses, and TIM1 (HAVCR1) is widely accepted to be the receptor for hepatitis A virus (HAV), an unusual, hepatotropic human picornavirus. However, its identification as the hepatovirus receptor predated the discovery that hepatoviruses undergo nonlytic release from infected cells as membrane-cloaked, quasi-enveloped HAV (eHAV) virions that enter cells via a pathway distinct from naked, nonenveloped virions. We thus revisited the role of TIM1 in hepatovirus entry, examining both adherence and infection/replication in cells with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-engineered TIM1 knockout. Cell culture-derived, gradient-purified eHAV bound Huh-7.5 human hepatoma cells less efficiently than naked HAV at 4°C, but eliminating TIM1 expression caused no difference in adherence of either form of HAV, nor any impact on infection and replication in these cells. In contrast, TIM1-deficient Vero cells showed a modest reduction in quasi-enveloped eHAV (but not naked HAV) attachment and replication. Thus, TIM1 facilitates quasi-enveloped eHAV entry in Vero cells, most likely by binding phosphatidylserine (PtdSer) residues on the eHAV membrane. Both Tim1 -/- Ifnar1 -/- and Tim4 -/- Ifnar1 -/- double-knockout mice were susceptible to infection upon intravenous challenge with infected liver homogenate, with fecal HAV shedding and serum alanine aminotransferase (ALT) elevations similar to those in Ifnar1 -/- mice. However, intrahepatic HAV RNA and ALT elevations were modestly reduced in Tim1 -/- Ifnar1 -/- mice compared to Ifnar1 -/- mice challenged with a lower titer of gradient-purified HAV or eHAV. We conclude that TIM1 is not an essential hepatovirus entry factor, although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver injury in mice. IMPORTANCE T cell immunoglobulin and mucin-containing domain protein 1 (TIM1) was reported more than 2 decades ago to be an essential cellular receptor for hepatitis A virus (HAV), a picornavirus in the Hepatovirus genus, resulting in its designation as "hepatitis A virus cellular receptor 1" (HAVCR1) by the Human Genome Organization Gene Nomenclature Committee. However, recent studies have shown that HAV exists in nature as both naked, nonenveloped (HAV) virions and membrane-cloaked, quasi-enveloped infectious virus (eHAV), prompting us to revisit the role of TIM1 in viral entry. We show here that TIM1 (HAVCR1) is not an essential cellular receptor for HAV entry into cultured cells or required for viral replication and pathogenesis in permissive strains of mice, although it may facilitate early stages of infection by binding phosphatidylserine on the eHAV surface. This work thus corrects the published record and sets the stage for future efforts to identify specific hepatovirus entry factors. Copyright © 2017 Das et al.

Remarkable proanthocyanidin adsorption properties of monastrell pomace cell wall material highlight its potential use as an alternative fining agent in red wine production.

PubMed

Bautista-Ortín, Ana Belén; Ruiz-García, Yolanda; Marín, Fátima; Molero, Noelia; Apolinar-Valiente, Rafael; Gómez-Plaza, Encarna

2015-01-21

The existence of interactions between the polysaccharides of vegetal cell walls and proanthocyanins makes this cell wall material an interesting option for its use as a fining agent to reduce the level of proanthocyanins in wines. Pomace wastes from the winery are widely available and a source of cell wall material, and the identification of varieties whose pomace cell walls present high proanthocyanin binding capacity and of processing methods that could enhance their adsorption properties could be of great interest. This study compared the proanthocyanin adsorption properties of pomace cell wall material from three different grape varieties (Monastrell, Cabernet Sauvignon, and Syrah), and the results were compared with those obtained using fresh grape cell walls. Also, the effect of the vinification method has been studied. Analysis of the proanthocyanidins in the solution after reaction with the cell wall material, using phloroglucinolysis and size exclusion chromatography, provided quantitative and qualitative information on the adsorbed and nonadsorbed compounds. A highlight of this study was the observation that Monastrell pomace cell wall material showed a strong affinity for proanthocyanidins, with values similar to that obtained for fresh grapes cell walls, and a preferential binding of high molecular mass proanthocyanidins, so these pomace cell walls could be used in wines to reduce astringency. The use of maceration enzymes during vinification had little effect on the retention capacity of the pomace cell walls obtained from this vinification, although an increase in the retention of low molecular mass proanthocyanidins was observed, and this might have implications for wine sensory properties.

Visualizing chemical functionality in plant cell walls

DOE PAGES

Zeng, Yining; Himmel, Michael E.; Ding, Shi-You

2017-11-30

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructivelymore » and in real-time during conversion processes. Microscopic characterization has revealed heterogeneity in the distribution of chemical features, which would otherwise be hidden in bulk analysis. Key microscopic features include cell wall type, wall layering, and wall composition - especially cellulose and lignin distributions. Microscopic tools, such as atomic force microscopy, stimulated Raman scattering microscopy, and fluorescence microscopy, have been applied to investigations of cell wall structure and chemistry from the native wall to wall treated by thermal chemical pretreatment and enzymatic hydrolysis. While advancing our current understanding of plant cell wall recalcitrance and deconstruction, microscopic tools with improved spatial resolution will steadily enhance our fundamental understanding of cell wall function.« less

Visualizing chemical functionality in plant cell walls

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

Zeng, Yining; Himmel, Michael E.; Ding, Shi-You

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructivelymore » and in real-time during conversion processes. Microscopic characterization has revealed heterogeneity in the distribution of chemical features, which would otherwise be hidden in bulk analysis. Key microscopic features include cell wall type, wall layering, and wall composition - especially cellulose and lignin distributions. Microscopic tools, such as atomic force microscopy, stimulated Raman scattering microscopy, and fluorescence microscopy, have been applied to investigations of cell wall structure and chemistry from the native wall to wall treated by thermal chemical pretreatment and enzymatic hydrolysis. While advancing our current understanding of plant cell wall recalcitrance and deconstruction, microscopic tools with improved spatial resolution will steadily enhance our fundamental understanding of cell wall function.« less

Visualizing chemical functionality in plant cell walls.

PubMed

Zeng, Yining; Himmel, Michael E; Ding, Shi-You

2017-01-01

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructively and in real-time during conversion processes. Microscopic characterization has revealed heterogeneity in the distribution of chemical features, which would otherwise be hidden in bulk analysis. Key microscopic features include cell wall type, wall layering, and wall composition-especially cellulose and lignin distributions. Microscopic tools, such as atomic force microscopy, stimulated Raman scattering microscopy, and fluorescence microscopy, have been applied to investigations of cell wall structure and chemistry from the native wall to wall treated by thermal chemical pretreatment and enzymatic hydrolysis. While advancing our current understanding of plant cell wall recalcitrance and deconstruction, microscopic tools with improved spatial resolution will steadily enhance our fundamental understanding of cell wall function.

B cell clonal lineage alterations upon recombinant HIV-1 envelope immunization of Rhesus macaques

USDA-ARS?s Scientific Manuscript database

Broadly neutralizing HIV-1 antibodies (bNAbs) isolated from infected subjects display protective potential in animal models. Their elicitation by immunization is thus highly desirable. The HIV-1 envelope glycoprotein (Env) is the sole viral target of bnAbs, but is also targeted by binding, non-neutr...

Formation of wood secondary cell wall may involve two type cellulose synthase complexes in Populus.

PubMed

Xi, Wang; Song, Dongliang; Sun, Jiayan; Shen, Junhui; Li, Laigeng

2017-03-01

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.

Intracellular self-assembly based multi-labeling of key viral components: Envelope, capsid and nucleic acids.

PubMed

Wen, Li; Lin, Yi; Zhang, Zhi-Ling; Lu, Wen; Lv, Cheng; Chen, Zhi-Liang; Wang, Han-Zhong; Pang, Dai-Wen

2016-08-01

Envelope, capsid and nucleic acids are key viral components that are all involved in crucial events during virus infection. Thus simultaneous labeling of these key components is an indispensable prerequisite for monitoring comprehensive virus infection process and dissecting virus infection mechanism. Baculovirus was genetically tagged with biotin on its envelope protein GP64 and enhanced green fluorescent protein (EGFP) on its capsid protein VP39. Spodoptera frugiperda 9 (Sf9) cells were infected by the recombinant baculovirus and subsequently fed with streptavidin-conjugated quantum dots (SA-QDs) and cell-permeable nucleic acids dye SYTO 82. Just by genetic engineering and virus propagation, multi-labeling of envelope, capsid and nucleic acids was spontaneously accomplished during virus inherent self-assembly process, significantly simplifying the labeling process while maintaining virus infectivity. Intracellular dissociation and transportation of all the key viral components, which was barely reported previously, was real-time monitored based on the multi-labeling approach, offering opportunities for deeply understanding virus infection and developing anti-virus treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Incorporation of Hepatitis C Virus E1 and E2 Glycoproteins: The Keystones on a Peculiar Virion

PubMed Central

Vieyres, Gabrielle; Dubuisson, Jean; Pietschmann, Thomas

2014-01-01

Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2. Their structure and mode of fusion remain unknown, and so does the virion architecture. The organization of the HCV envelope shell in particular is subject to discussion as it incorporates or associates with host-derived lipoproteins, to an extent that the biophysical properties of the virion resemble more very-low-density lipoproteins than of any virus known so far. The recent development of novel cell culture systems for HCV has provided new insights on the assembly of this atypical viral particle. Hence, the extensive E1E2 characterization accomplished for the last two decades in heterologous expression systems can now be brought into the context of a productive HCV infection. This review describes the biogenesis and maturation of HCV envelope glycoproteins, as well as the interplay between viral and host factors required for their incorporation in the viral envelope, in a way that allows efficient entry into target cells and evasion of the host immune response. PMID:24618856

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

PubMed Central

Adu-Gyamfi, Emmanuel; Johnson, Kristen A.; Fraser, Mark E.; Scott, Jordan L.; Soni, Smita P.; Jones, Keaton R.; Digman, Michelle A.; Gratton, Enrico; Tessier, Charles R.

2015-01-01

ABSTRACT Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. IMPORTANCE The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. PMID:26136573

Beam-dynamics codes used at DARHT

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

Ekdahl, Jr., Carl August

Several beam simulation codes are used to help gain a better understanding of beam dynamics in the DARHT LIAs. The most notable of these fall into the following categories: for beam production – Tricomp Trak orbit tracking code, LSP Particle in cell (PIC) code, for beam transport and acceleration – XTR static envelope and centroid code, LAMDA time-resolved envelope and centroid code, LSP-Slice PIC code, for coasting-beam transport to target – LAMDA time-resolved envelope code, LSP-Slice PIC code. These codes are also being used to inform the design of Scorpius.

Mechanosensation Dynamically Coordinates Polar Growth and Cell Wall Assembly to Promote Cell Survival.

PubMed

Davì, Valeria; Tanimoto, Hirokazu; Ershov, Dmitry; Haupt, Armin; De Belly, Henry; Le Borgne, Rémi; Couturier, Etienne; Boudaoud, Arezki; Minc, Nicolas

2018-04-23

How growing cells cope with size expansion while ensuring mechanical integrity is not known. In walled cells, such as those of microbes and plants, growth and viability are both supported by a thin and rigid encasing cell wall (CW). We deciphered the dynamic mechanisms controlling wall surface assembly during cell growth, using a sub-resolution microscopy approach to monitor CW thickness in live rod-shaped fission yeast cells. We found that polar cell growth yielded wall thinning and that thickness negatively influenced growth. Thickness at growing tips exhibited a fluctuating behavior with thickening phases followed by thinning phases, indicative of a delayed feedback promoting thickness homeostasis. This feedback was mediated by mechanosensing through the CW integrity pathway, which probes strain in the wall to adjust synthase localization and activity to surface growth. Mutants defective in thickness homeostasis lysed by rupturing the wall, demonstrating its pivotal role for walled cell survival. Copyright © 2018 Elsevier Inc. All rights reserved.

The Cell Wall-Associated Proteins in the Dimorphic Pathogenic Species of Paracoccidioides.

PubMed

Puccia, Rosana; Vallejo, Milene C; Longo, Larissa V G

2017-01-01

Paracoccidioides brasiliensis and P. lutzii cause human paracoccidioidomycosis (PCM). They are dimorphic ascomycetes that grow as filaments at mild temperatures up to 28oC and as multibudding pathogenic yeast cells at 37oC. Components of the fungal cell wall have an important role in the interaction with the host because they compose the cell outermost layer. The Paracoccidioides cell wall is composed mainly of polysaccharides, but it also contains proportionally smaller rates of proteins, lipids, and melanin. The polysaccharide cell wall composition and structure of Paracoccidioides yeast cells, filamentous and transition phases were studied in detail in the past. Other cell wall components have been better analyzed in the last decades. The present work gives to the readers a detailed updated view of cell wall-associated proteins. Proteins that have been localized at the cell wall compartment using antibodies are individually addressed. We also make an overview about PCM, the Paracoccidioides cell wall structure, secretion mechanisms, and fungal extracellular vesicles. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Unexpected features of exponentially growing Tobacco Bright Yellow-2 cell suspension culture in relation to excreted extracellular polysaccharides and cell wall composition.

PubMed

Issawi, Mohammad; Muhieddine, Mohammad; Girard, Celine; Sol, Vincent; Riou, Catherine

2017-10-01

This article presents a new insight about TBY-2 cells; from extracellular polysaccharides secretion to cell wall composition during cell suspension culture. In the medium of cells taken 2 days after dilution (end of lag phase), a two unit pH decrease from 5.38 to 3.45 was observed and linked to a high uronic acid (UA) amount secretion (47.8%) while, in 4 and 7 day-old spent media, pH increased and UA amounts decreased 35.6 and 42.3% UA, respectively. To attain deeper knowledge of the putative link between extracellular polysaccharide excretion and cell wall composition, we determined cell wall UA and neutral sugar composition of cells from D2 to D12 cultures. While cell walls from D2 and D3 cells contained a large amount of uronic acid (twice as much as the other analysed cell walls), similar amounts of neutral sugar were detected in cells from lag to end of exponential phase cells suggesting an enriched pectin network in young cultures. Indeed, monosaccharide composition analysis leads to an estimated percentage of pectins of 56% for D3 cell wall against 45% D7 cell walls indicating that the cells at the mid-exponential growth phase re-organized their cell wall linked to a decrease in secreted UA that finally led to a stabilization of the spent medium pH to 5.4. In conclusion, TBY-2 cell suspension from lag to stationary phase showed cell wall remodeling that could be of interest in drug interaction and internalization study.

Investigation of Plant Cell Wall Properties: A Study of Contributions from the Nanoscale to the Macroscale Impacting Cell Wall Recalcitrance

NASA Astrophysics Data System (ADS)

Crowe, Jacob Dillon

Biochemical conversion of lignocellulosic biomass to fuel ethanol is one of a few challenging, yet opportune technologies that can reduce the consumption of petroleum-derived transportation fuels, while providing parallel reductions in greenhouse gas emissions. Biomass recalcitrance, or resistance to deconstruction, is a major technical challenge that limits effective conversion of biomass to fermentable sugars, often requiring a costly thermochemical pretreatment step to improve biomass deconstruction. Biomass recalcitrance is imparted largely by the secondary cell wall, a complex polymeric matrix of cell wall polysaccharides and aromatic heteropolymers, that provides structural stability to cells and enables plant upright growth. Polymers within the cell wall can vary both compositionally and structurally depending upon plant species and anatomical fraction, and have varied responses to thermochemical pretreatments. Cell wall properties impacting recalcitrance are still not well understood, and as a result, the goal of this dissertation is to investigate structural features of the cell wall contributing to recalcitrance (1) in diverse anatomical fractions of a single species, (2) in response to diverse pretreatments, and (3) resulting from genetic modification. In the first study, feedstock cell wall heterogeneity was investigated in anatomical (stem, leaf sheaths, and leaf blades) and internode fractions of switchgrass at varying tissue maturities. Lignin content was observed as the key contributor to recalcitrance in maturing stem tissues only, with non-cellulosic substituted glucuronoarabinoxylans and pectic polysaccharides contributing to cell wall recalcitrance in leaf sheath and leaf blades. Hydroxycinnamate (i.e., saponifiable p-coumarate and ferulate) content along with xylan and pectin extractability decreased with tissue maturity, suggesting lignification is only one component imparting maturity specific cell wall recalcitrance. In the second study, alkaline hydrogen peroxide and liquid hot water pretreatments were shown to alter structural properties impacting nanoscale porosity in corn stover. Delignification by alkaline hydrogen peroxide pretreatment decreased cell wall rigidity, with subsequent cell wall swelling resulting in increased nanoscale porosity and improved enzymatic hydrolysis compared to limited swelling and increased accessible surface areas observed in liquid hot water pretreated biomass. The volume accessible to a 90 A dextran probe within the cell wall was found to be positively correlated to both enzyme binding and glucose hydrolysis yields, indicating cell wall porosity is a key contributor to effective hydrolysis yields. In the third study, the effect of altered xylan content and structure was investigated in irregular xylem (irx) Arabidopsis thaliana mutants to understand the role xylan plays in secondary cell wall development and organization. Higher xylan extractability and lower cellulose crystallinity observed in irx9 and irx15 irx15-L mutants compared to wild type indicated altered xylan integration into the secondary cell wall. Nanoscale cell wall organization observed using multiple microscopy techniques was impacted to some extent in all irx mutants, with disorganized cellulose microfibril layers in sclerenchyma secondary cell walls likely resulting from irregular xylan structure and content. Irregular secondary cell wall microfibril layers showed heterogeneous nanomechanical properties compared to wild type, which translated to mechanical deficiencies observed in stem tensile tests. These results suggest nanoscale defects in cell wall strength can correspond to macroscale phenotypes.

[Au]/[Ag]-catalysed expedient synthesis of branched heneicosafuranosyl arabinogalactan motif of Mycobacterium tuberculosis cell wall

NASA Astrophysics Data System (ADS)

Thadke, Shivaji A.; Mishra, Bijoyananda; Islam, Maidul; Pasari, Sandip; Manmode, Sujit; Rao, Boddu Venkateswara; Neralkar, Mahesh; Shinde, Ganesh P.; Walke, Gulab; Hotha, Srinivas

2017-01-01

Emergence of multidrug-resistant and extreme-drug-resistant strains of Mycobacterium tuberculosis (MTb) can cause serious socioeconomic burdens. Arabinogalactan present on the cellular envelope of MTb is unique and is required for its survival; access to arabinogalactan is essential for understanding the biosynthetic machinery that assembles it. Isolation from Nature is a herculean task and, as a result, chemical synthesis is the most sought after technique. Here we report a convergent synthesis of branched heneicosafuranosyl arabinogalactan (HAG) of MTb. Key furanosylations are performed using [Au]/[Ag] catalysts. The synthesis of HAG is achieved by the repetitive use of three reactions namely 1,2-trans furanoside synthesis by propargyl 1,2-orthoester donors, unmasking of silyl ether, and conversion of n-pentenyl furanosides into 1,2-orthoesters. Synthesis of HAG is achieved in 47 steps (with an overall yield of 0.09%) of which 21 are installation of furanosidic linkages in a stereoselective manner.

Crystal structure of Bacillus anthracis transpeptidase enzyme CapD.

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

Wu, R.; Richter, S.; Zhang, R.

2009-09-04

Bacillus anthracis elaborates a poly-{gamma}-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the {gamma}-glutamyltranspeptidase CapD with and without {alpha}-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-{gamma}-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-{gamma}-glutamatemore » binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro{sup 427}, Gly{sup 428}, and Gly{sup 429} activate the catalytic residue of the enzyme, Thr{sup 352}, and stabilize an oxyanion hole via main chain amide hydrogen bonds.« less

Insights into teichoic acid biosynthesis by Bifidobacterium bifidum PRL2010.

PubMed

Colagiorgi, Angelo; Turroni, Francesca; Mancabelli, Leonardo; Serafini, Fausta; Secchi, Andrea; van Sinderen, Douwe; Ventura, Marco

2015-09-01

Bifidobacteria are colonizers of the human gut, where they are interacting with their host as well as with other members of the intestinal microbiota. Teichoic acids (TAs) have previously been shown to play an important role in modulating microbe-host interactions in the human gut. However, so far, there is a paucity of information regarding the presence of TAs in the cell envelope of bifidobacteria. In silico analyses targeting the chromosomes of all 48 (sub)species that currently represent the genus Bifidobacterium revealed the presence of genes responsible for TA biosynthesis, suggesting that bifidobacteria contain both wall TAs and lipoteichoic acids. Transcriptome analyses of the infant gut commensal Bifidobacterium bifidum PRL2010 highlighted that the transcription of the presumptive TA biosynthetic loci is modulated in response to environmental conditions reflecting those of the human gut. Furthermore, chemical characterization of TAs produced by PRL2010 indicates the presence of lipoteichoic acids. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The bacterial envelope as a target for novel anti-MRSA antibiotics.

PubMed

Van Bambeke, Françoise; Mingeot-Leclercq, Marie-Paule; Struelens, Marc J; Tulkens, Paul M

2008-03-01

Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate S. aureus (VISA) are spreading worldwide, making the search for antibiotics directed against new targets a high priority. Drugs that anchor in the bacterial membrane (e.g. ceragenins and lipopeptides) or that target the bacterial membrane and proteic (lipoglycopeptides) or lipidic (glycodepsipeptides) cell wall precursors seem to have the most potential because they show a fast and extensive bactericidal effect and are probably less prone to select for resistance owing to the difficulty in modifying their targets in a way that is compatible with bacterial survival. The efficacy of lipopeptides and lipoglycopeptides has been demonstrated in the treatment of skin and skin structure infections, and bacteremia caused by resistant S. aureus. Ceragenins and glycodepsipeptides are restricted to topical applications because of their unsatisfactory safety profile. The mode of action, pharmacological and microbiological properties and target indications of these anti-MRSA agents, which function by disturbing membrane integrity, are reviewed in this article.

Breaching the nuclear envelope in development and disease

PubMed Central

Hatch, Emily

2014-01-01

In eukaryotic cells the nuclear genome is enclosed by the nuclear envelope (NE). In metazoans, the NE breaks down in mitosis and it has been assumed that the physical barrier separating nucleoplasm and cytoplasm remains intact during the rest of the cell cycle and cell differentiation. However, recent studies suggest that nonmitotic NE remodeling plays a critical role in development, virus infection, laminopathies, and cancer. Although the mechanisms underlying these NE restructuring events are currently being defined, one common theme is activation of protein kinase C family members in the interphase nucleus to disrupt the nuclear lamina, demonstrating the importance of the lamina in maintaining nuclear integrity. PMID:24751535

Case Study of Envelope Sealing in Existing Multiunit Structures

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

Dentz, J.; Conlin, F.; Podorson, D.

2012-10-01

Envelope air sealing was included in the retrofit of a 244 unit low-rise multifamily housing complex in Durham, N.C. Pre- and post-retrofit enclosure leakage tests were conducted on 51 units and detailed diagnostics were performed on 16. On average, total leakage was reduced by nearly half, from 19.7 ACH50 to 9.4 ACH50. Costs for air sealing were $0.31 per square foot of conditioned floor area, lower than estimates found in the National Residential Efficiency Measures Database (NREMD) and other sources, perhaps due in part to the large-scale production nature of the project. Modeling with BEopt software -- using an estimatemore » of 85% of the envelope air leakage going to the outside (based on guarded tests performed at the site) -- calculated a space conditioning energy cost savings of 15% to 21% due to the air sealing retrofit. Important air leakage locations identified included plumbing and electrical penetrations, dropped ceilings/soffits, windows, ducts and wall-to-floor intersections. Previous repair activity had created significant leakage locations as well. Specifications and a pictorial guide were developed for contractors performing the work.« less

The Acid Growth Theory of auxin-induced cell elongation is alive and well

NASA Technical Reports Server (NTRS)

Rayle, D. L.; Cleland, R. E.

1992-01-01

Plant cells elongate irreversibly only when load-bearing bonds in the walls are cleaved. Auxin causes the elongation of stem and coleoptile cells by promoting wall loosening via cleavage of these bonds. This process may be coupled with the intercalation of new cell wall polymers. Because the primary site of auxin action appears to be the plasma membrane or some intracellular site, and wall loosening is extracellular, there must be communication between the protoplast and the wall. Some "wall-loosening factor" must be exported from auxin-impacted cells, which sets into motion the wall loosening events. About 20 years ago, it was suggested that the wall-loosening factor is hydrogen ions. This idea and subsequent supporting data gave rise to the Acid Growth Theory, which states that when exposed to auxin, susceptible cells excrete protons into the wall (apoplast) at an enhanced rate, resulting in a decrease in apoplastic pH. The lowered wall pH then activates wall-loosening processes, the precise nature of which is unknown. Because exogenous acid causes a transient (1-4 h) increase in growth rate, auxin must also mediate events in addition to wall acidification for growth to continue for an extended period of time. These events may include osmoregulation, cell wall synthesis, and maintenance of the capacity of walls to undergo acid-induced wall loosening. At present, we do not know if these phenomena are tightly coupled to wall acidification or if they are the products of multiple independent signal transduction pathways.

The composition of West Nile virus lipid envelope unveils a role of sphingolipid metabolism in flavivirus biogenesis.

PubMed

Martín-Acebes, Miguel A; Merino-Ramos, Teresa; Blázquez, Ana-Belén; Casas, Josefina; Escribano-Romero, Estela; Sobrino, Francisco; Saiz, Juan-Carlos

2014-10-01

West Nile virus (WNV) is an emerging zoonotic mosquito-borne flavivirus responsible for outbreaks of febrile illness and meningoencephalitis. The replication of WNV takes place on virus-modified membranes from the endoplasmic reticulum of the host cell, and virions acquire their envelope by budding into this organelle. Consistent with this view, the cellular biology of this pathogen is intimately linked to modifications of the intracellular membranes, and the requirement for specific lipids, such as cholesterol and fatty acids, has been documented. In this study, we evaluated the impact of WNV infection on two important components of cellular membranes, glycerophospholipids and sphingolipids, by mass spectrometry of infected cells. A significant increase in the content of several glycerophospholipids (phosphatidylcholine, plasmalogens, and lysophospholipids) and sphingolipids (ceramide, dihydroceramide, and sphingomyelin) was noticed in WNV-infected cells, suggesting that these lipids have functional roles during WNV infection. Furthermore, the analysis of the lipid envelope of WNV virions and recombinant virus-like particles revealed that their envelopes had a unique composition. The envelopes were enriched in sphingolipids (sphingomyelin) and showed reduced levels of phosphatidylcholine, similar to sphingolipid-enriched lipid microdomains. Inhibition of neutral sphingomyelinase (which catalyzes the hydrolysis of sphingomyelin into ceramide) by either pharmacological approaches or small interfering RNA-mediated silencing reduced the release of flavivirus virions as well as virus-like particles, suggesting a role of sphingomyelin-to-ceramide conversion in flavivirus budding and confirming the importance of sphingolipids in the biogenesis of WNV. Importance: West Nile virus (WNV) is a neurotropic flavivirus spread by mosquitoes that can infect multiple vertebrate hosts, including humans. There is no specific vaccine or therapy against this pathogen licensed for human use. Since the multiplication of this virus is associated with rearrangements of host cell membranes, we analyzed the effect of WNV infection on different cellular lipids that constitute important membrane components. The levels of multiple lipid species were increased in infected cells, pointing to the induction of major alterations of cellular lipid metabolism by WNV infection. Interestingly, certain sphingolipids, which were increased in infected cells, were also enriched in the lipid envelope of the virus, thus suggesting a potential role during virus assembly. We further verified the role of sphingolipids in the production of WNV by means of functional analyses. This study provides new insight into the formation of flavivirus infectious particles and the involvement of sphingolipids in the WNV life cycle. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Composition of West Nile Virus Lipid Envelope Unveils a Role of Sphingolipid Metabolism in Flavivirus Biogenesis

PubMed Central

Martín-Acebes, Miguel A.; Merino-Ramos, Teresa; Blázquez, Ana-Belén; Casas, Josefina; Escribano-Romero, Estela

2014-01-01

ABSTRACT West Nile virus (WNV) is an emerging zoonotic mosquito-borne flavivirus responsible for outbreaks of febrile illness and meningoencephalitis. The replication of WNV takes place on virus-modified membranes from the endoplasmic reticulum of the host cell, and virions acquire their envelope by budding into this organelle. Consistent with this view, the cellular biology of this pathogen is intimately linked to modifications of the intracellular membranes, and the requirement for specific lipids, such as cholesterol and fatty acids, has been documented. In this study, we evaluated the impact of WNV infection on two important components of cellular membranes, glycerophospholipids and sphingolipids, by mass spectrometry of infected cells. A significant increase in the content of several glycerophospholipids (phosphatidylcholine, plasmalogens, and lysophospholipids) and sphingolipids (ceramide, dihydroceramide, and sphingomyelin) was noticed in WNV-infected cells, suggesting that these lipids have functional roles during WNV infection. Furthermore, the analysis of the lipid envelope of WNV virions and recombinant virus-like particles revealed that their envelopes had a unique composition. The envelopes were enriched in sphingolipids (sphingomyelin) and showed reduced levels of phosphatidylcholine, similar to sphingolipid-enriched lipid microdomains. Inhibition of neutral sphingomyelinase (which catalyzes the hydrolysis of sphingomyelin into ceramide) by either pharmacological approaches or small interfering RNA-mediated silencing reduced the release of flavivirus virions as well as virus-like particles, suggesting a role of sphingomyelin-to-ceramide conversion in flavivirus budding and confirming the importance of sphingolipids in the biogenesis of WNV. IMPORTANCE West Nile virus (WNV) is a neurotropic flavivirus spread by mosquitoes that can infect multiple vertebrate hosts, including humans. There is no specific vaccine or therapy against this pathogen licensed for human use. Since the multiplication of this virus is associated with rearrangements of host cell membranes, we analyzed the effect of WNV infection on different cellular lipids that constitute important membrane components. The levels of multiple lipid species were increased in infected cells, pointing to the induction of major alterations of cellular lipid metabolism by WNV infection. Interestingly, certain sphingolipids, which were increased in infected cells, were also enriched in the lipid envelope of the virus, thus suggesting a potential role during virus assembly. We further verified the role of sphingolipids in the production of WNV by means of functional analyses. This study provides new insight into the formation of flavivirus infectious particles and the involvement of sphingolipids in the WNV life cycle. PMID:25122799

Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability

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

Pattathil, Sivakumar; Ingwers, Miles W.; Victoriano, Olivia L.

The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and themore » overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. In conclusion, to our knowledge, this is the first direct evidence, delineated by glycomic analyses, that abiotic stress affects cell wall ultrastructure. This study is also unique in that glycome profiling of pine needles has never before been reported.« less

Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability.

PubMed

Pattathil, Sivakumar; Ingwers, Miles W; Victoriano, Olivia L; Kandemkavil, Sindhu; McGuire, Mary Anne; Teskey, Robert O; Aubrey, Doug P

2016-01-01

The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and the overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin-associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. To our knowledge, this is the first direct evidence, delineated by glycomic analyses, that abiotic stress affects cell wall ultrastructure. This study is also unique in that glycome profiling of pine needles has never before been reported.

Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability

DOE PAGES

Pattathil, Sivakumar; Ingwers, Miles W.; Victoriano, Olivia L.; ...

2016-06-24

The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and themore » overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. In conclusion, to our knowledge, this is the first direct evidence, delineated by glycomic analyses, that abiotic stress affects cell wall ultrastructure. This study is also unique in that glycome profiling of pine needles has never before been reported.« less

Vesicles between plasma membrane and cell wall prior to visible senescence of Iris and Dendrobium flowers.

PubMed

Kamdee, Channatika; Kirasak, Kanjana; Ketsa, Saichol; van Doorn, Wouter G

2015-09-01

Cut Iris flowers (Iris x hollandica, cv. Blue Magic) show visible senescence about two days after full opening. Epidermal cells of the outer tepals collapse due to programmed cell death (PCD). Transmission electron microscopy (TEM) showed irregular swelling of the cell walls, starting prior to cell collapse. Compared to cells in flowers that had just opened, wall thickness increased up to tenfold prior to cell death. Fibrils were visible in the swollen walls. After cell death very little of the cell wall remained. Prior to and during visible wall swelling, vesicles (paramural bodies) were observed between the plasma membrane and the cell walls. The vesicles were also found in groups and were accompanied by amorphous substance. They usually showed a single membrane, and had a variety of diameters and electron densities. Cut Dendrobium hybrid cv. Lucky Duan flowers exhibited visible senescence about 14 days after full flower opening. Paramural bodies were also found in Dendrobium tepal epidermis and mesophyll cells, related to wall swelling and degradation. Although alternative explanations are well possible, it is hypothesized that paramural bodies carry enzymes involved in cell wall breakdown. The literature has not yet reported such bodies in association with senescence/PCD. Copyright © 2015 Elsevier GmbH. All rights reserved.

Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos.

PubMed

Zhang, Qisen; Zhang, Xiaoqi; Pettolino, Filomena; Zhou, Gaofeng; Li, Chengdao

2016-02-01

Barley (Hordeum vulgare L.) seed germination initiates many important biological processes such as DNA, membrane and mitochondrial repairs. However, little is known on cell wall modifications in germinating embryos. We have investigated cell wall polysaccharide composition change, gene transcription and alternative splicing events in four barley varieties at 24h and 48 h germination. Cell wall components in germinating barley embryos changed rapidly, with increases in cellulose and (1,3)(1,4)-β-D-glucan (20-100%) within 24h, but decreases in heteroxylan and arabinan (3-50%). There were also significant changes in the levels of type I arabinogalactans and heteromannans. Alternative splicing played very important roles in cell wall modifications. At least 22 cell wall transcripts were detected to undergo either alternative 3' splicing, alternative 5' splicing or intron retention type of alternative splicing. These genes coded enzymes catalyzing synthesis and degradation of cellulose, heteroxylan, (1,3)(1,4)-β-D-glucan and other cell wall polymers. Furthermore, transcriptional regulation also played very important roles in cell wall modifications. Transcript levels of primary wall cellulase synthase, heteroxylan synthesizing and nucleotide sugar inter-conversion genes were very high in germinating embryos. At least 50 cell wall genes changed transcript levels significantly. Expression patterns of many cell wall genes coincided with changes in polysaccharide composition. Our data showed that cell wall polysaccharide metabolism was very active in germinating barley embryos, which was regulated at both transcriptional and post-transcriptional levels. Copyright © 2015 Elsevier GmbH. All rights reserved.

Dynamic Assembly of Brambleberry Mediates Nuclear Envelope Fusion during Early Development

PubMed Central

Abrams, Elliott W.; Zhang, Hong; Marlow, Florence L.; Kapp, Lee; Lu, Sumei; Mullins, Mary C.

2012-01-01

Summary To accommodate the large cells following zygote formation, early blastomeres employ modified cell divisions. Karyomeres are one such modification, a mitotic intermediate wherein individual chromatin masses are surrounded by nuclear envelope, which then fuse to form a single mononucleus. We identified brambleberry, a maternal-effect zebrafish mutant that disrupts karyomere fusion resulting in formation of multiple micronuclei. brambleberry is a previously unannotated gene homologous to Kar5p, which participates in nuclear fusion in yeast. We demonstrate that Brambleberry is required for pronuclear fusion following fertilization in zebrafish. As karyomeres form, Brambleberry localizes to the nuclear envelope with prominent puncta evident near karyomere-karyomere interfaces corresponding to membrane fusion sites. Our studies identify the first factor acting in karyomere fusion and suggest that specialized proteins are necessary for proper nuclear division in large dividing blastomeres. PMID:22863006

The Antibacterial Activity of Coriolus versicolor Methanol Extract and Its Effect on Ultrastructural Changes of Staphylococcus aureus and Salmonella Enteritidis.

PubMed

Matijašević, Danka; Pantić, Milena; Rašković, Božidar; Pavlović, Vladimir; Duvnjak, Dunja; Sknepnek, Aleksandra; Nikšić, Miomir

2016-01-01

The antibacterial activity of methanol extract obtained from fruiting body of industrially grown basidiomycete Coriolus versicolor was examined. The Minimum Inhibitory Concentration (MIC) values against various bacteria ranged from 0.625 to 20 mg mL(-1). C. versicolor expressed bactericidal activity against both Gram-positive and Gram-negative bacteria. The growth curves of Staphylococcus aureus and Salmonella enterica serovar Enteritidis, measured at 630 nm, and confirmed with macrodilution method showed that the obtained extract could inhibit the growth of tested bacteria. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the loss of 260-nm-absorbing material were used to examine the ultrastructural changes in bacteria induced by the extract. When S. aureus was exposed to the MIC of C. versicolor, elongated and malformed cells were observed by SEM, while S. Enteritidis treated cells appeared shorter and aggregated with ruptured cell walls. TEM revealed the formation of non-membrane-enclosed bodies and depleted inner content of S. aureus. Larger and irregular periplasmic space and deformed and scattered components of the cell envelope were observed in treated S. Enteritidis. The loss of 260-nm-absorbing material indicated that the disruptive action of the extract on cytoplasmic membrane was more pronounced in S. aureus than in S. Enteritidis treated cells. The UV and FTIR spectrophotometric analyses revealed diverse composition of C. versicolor extract and high content of total phenolics. Altogether, mushroom extracts could be used to develop nutraceuticals or drugs effective against pathogenic microorganisms.

Two-Level Chebyshev Filter Based Complementary Subspace Method: Pushing the Envelope of Large-Scale Electronic Structure Calculations.

PubMed

Banerjee, Amartya S; Lin, Lin; Suryanarayana, Phanish; Yang, Chao; Pask, John E

2018-06-12

We describe a novel iterative strategy for Kohn-Sham density functional theory calculations aimed at large systems (>1,000 electrons), applicable to metals and insulators alike. In lieu of explicit diagonalization of the Kohn-Sham Hamiltonian on every self-consistent field (SCF) iteration, we employ a two-level Chebyshev polynomial filter based complementary subspace strategy to (1) compute a set of vectors that span the occupied subspace of the Hamiltonian; (2) reduce subspace diagonalization to just partially occupied states; and (3) obtain those states in an efficient, scalable manner via an inner Chebyshev filter iteration. By reducing the necessary computation to just partially occupied states and obtaining these through an inner Chebyshev iteration, our approach reduces the cost of large metallic calculations significantly, while eliminating subspace diagonalization for insulating systems altogether. We describe the implementation of the method within the framework of the discontinuous Galerkin (DG) electronic structure method and show that this results in a computational scheme that can effectively tackle bulk and nano systems containing tens of thousands of electrons, with chemical accuracy, within a few minutes or less of wall clock time per SCF iteration on large-scale computing platforms. We anticipate that our method will be instrumental in pushing the envelope of large-scale ab initio molecular dynamics. As a demonstration of this, we simulate a bulk silicon system containing 8,000 atoms at finite temperature, and obtain an average SCF step wall time of 51 s on 34,560 processors; thus allowing us to carry out 1.0 ps of ab initio molecular dynamics in approximately 28 h (of wall time).

Modelling cell wall growth using a fibre-reinforced hyperelastic-viscoplastic constitutive law

NASA Astrophysics Data System (ADS)

Huang, R.; Becker, A. A.; Jones, I. A.

2012-04-01

A fibre-reinforced hyperelastic-viscoplastic model using a finite strain Finite Element (FE) analysis is presented to study the expansive growth of cell walls. Based on the connections between biological concepts and plasticity theory, e.g. wall-loosening and plastic yield, wall-stiffening and plastic hardening, the modelling of cell wall growth is established within a framework of anisotropic viscoplasticity aiming to represent the corresponding biology-controlled behaviour of a cell wall. In order to model in vivo growth, special attention is paid to the differences between a living cell and an isolated wall. The proposed hyperelastic-viscoplastic theory provides a unique framework to clarify the interplay between cellulose microfibrils and cell wall matrix and how this interplay regulates sustainable growth in a particular direction while maintaining the mechanical strength of the cell walls by new material deposition. Moreover, the effect of temperature is taken into account. A numerical scheme is suggested and FE case studies are presented and compared with experimental data.

Potent virucidal effect of pheophorbide a and pyropheophorbide a on enveloped viruses.

PubMed

Bouslama, Lamjed; Hayashi, Kyoko; Lee, Jung-Bum; Ghorbel, Abdelwahed; Hayashi, Toshimitsu

2011-01-01

In this study, we evaluated the inhibitory effect of ethanol and aqueous extracts from a stem of Opuntia ficus indica on replication of three kinds of viruses: two enveloped viruses [herpes simplex virus type 2 (HSV-2), influenza A virus (IFV-A)], and one non-enveloped virus [poliovirus type 1 (PV-1)]. Only ethanol extract from the cactus stem showed significant antiviral activity in vitro. Two chlorophyll derivatives, pheophorbide a and pyropheophorbide a, were isolated as active substances exhibiting potent virucidal effects on HSV-2 and IFV-A, but no activity against PV-1 was observed. These findings suggest that these active compounds might recognize specific glycoproteins of enveloped viruses, precluding their binding to host cell receptors and inhibiting viral infections.

(Hydroxyproline-rich glycoproteins of the plant cell wall)

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

Varner, J.E.

1990-01-01

We are studying the chemistry and architecture of plant cells walls, the extracellular matrices that taken together shape the plant and provide mechanical support for the plant. Cell walls are dynamic structures that regulate, or are the site of, many physiological processes, in addition to being the cells' first line of defense against invading pathogens. In the past year we have examined the role of the cell wall enzyme ascorbic acid oxidase as related to the structure of the wall and its possible interactions with hydroxyproline-rich glycoproteins of the wall.

A Structurally Specialized Uniform Wall Layer is Essential for Constructing Wall Ingrowth Papillae in Transfer Cells

PubMed Central

Xia, Xue; Zhang, Hui-Ming; Offler, Christina E.; Patrick, John W.

2017-01-01

Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans-differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta. Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated. PMID:29259611

A Structurally Specialized Uniform Wall Layer is Essential for Constructing Wall Ingrowth Papillae in Transfer Cells.

PubMed

Xia, Xue; Zhang, Hui-Ming; Offler, Christina E; Patrick, John W

2017-01-01

Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans -differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta . Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated.

Isolation of a novel cell wall architecture mutant of rice with defective Arabidopsis COBL4 ortholog BC1 required for regulated deposition of secondary cell wall components.

PubMed

Sato, Kanna; Suzuki, Ryu; Nishikubo, Nobuyuki; Takenouchi, Sachi; Ito, Sachiko; Nakano, Yoshimi; Nakaba, Satoshi; Sano, Yuzou; Funada, Ryo; Kajita, Shinya; Kitano, Hidemi; Katayama, Yoshihiro

2010-06-01

The plant secondary cell wall is a highly ordered structure composed of various polysaccharides, phenolic components and proteins. Its coordinated regulation of a number of complex metabolic pathways and assembly has not been resolved. To understand the molecular mechanisms that regulate secondary cell wall synthesis, we isolated a novel rice mutant, cell wall architecture1 (cwa1), that exhibits an irregular thickening pattern in the secondary cell wall of sclerenchyma, as well as culm brittleness and reduced cellulose content in mature internodes. Light and transmission electron microscopy revealed that the cwa1 mutant plant has regions of local aggregation in the secondary cell walls of the cortical fibers in its internodes, showing uneven thickness. Ultraviolet microscopic observation indicated that localization of cell wall phenolic components was perturbed and that these components abundantly deposited at the aggregated cell wall regions in sclerenchyma. Therefore, regulation of deposition and assembly of secondary cell wall materials, i.e. phenolic components, appear to be disturbed by mutation of the cwa1 gene. Genetic analysis showed that cwa1 is allelic to brittle culm1 (bc1), which encodes the glycosylphosphatidylinositol-anchored COBRA-like protein specifically in plants. BC1 is known as a regulator that controls the culm mechanical strength and cellulose content in the secondary cell walls of sclerenchyma, but the precise function of BC1 has not been resolved. Our results suggest that CWA1/BC1 has an essential role in assembling cell wall constituents at their appropriate sites, thereby enabling synthesis of solid and flexible internodes in rice.

Cell Wall Remodeling by a Synthetic Analog Reveals Metabolic Adaptation in Vancomycin Resistant Enterococci.

PubMed

Pidgeon, Sean E; Pires, Marcos M

2017-07-21

Drug-resistant bacterial infections threaten to overburden our healthcare system and disrupt modern medicine. A large class of potent antibiotics, including vancomycin, operate by interfering with bacterial cell wall biosynthesis. Vancomycin-resistant enterococci (VRE) evade the blockage of cell wall biosynthesis by altering cell wall precursors, rendering them drug insensitive. Herein, we reveal the phenotypic plasticity and cell wall remodeling of VRE in response to vancomycin in live bacterial cells via a metabolic probe. A synthetic cell wall analog was designed and constructed to monitor cell wall structural alterations. Our results demonstrate that the biosynthetic pathway for vancomycin-resistant precursors can be hijacked by synthetic analogs to track the kinetics of phenotype induction. In addition, we leveraged this probe to interrogate the response of VRE cells to vancomycin analogs and a series of cell wall-targeted antibiotics. Finally, we describe a proof-of-principle strategy to visually inspect drug resistance induction. Based on our findings, we anticipate that our metabolic probe will play an important role in further elucidating the interplay among the enzymes involved in the VRE biosynthetic rewiring.

Do plant cell walls have a code?

PubMed

Tavares, Eveline Q P; Buckeridge, Marcos S

2015-12-01

A code is a set of rules that establish correspondence between two worlds, signs (consisting of encrypted information) and meaning (of the decrypted message). A third element, the adaptor, connects both worlds, assigning meaning to a code. We propose that a Glycomic Code exists in plant cell walls where signs are represented by monosaccharides and phenylpropanoids and meaning is cell wall architecture with its highly complex association of polymers. Cell wall biosynthetic mechanisms, structure, architecture and properties are addressed according to Code Biology perspective, focusing on how they oppose to cell wall deconstruction. Cell wall hydrolysis is mainly focused as a mechanism of decryption of the Glycomic Code. Evidence for encoded information in cell wall polymers fine structure is highlighted and the implications of the existence of the Glycomic Code are discussed. Aspects related to fine structure are responsible for polysaccharide packing and polymer-polymer interactions, affecting the final cell wall architecture. The question whether polymers assembly within a wall display similar properties as other biological macromolecules (i.e. proteins, DNA, histones) is addressed, i.e. do they display a code? Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria

PubMed Central

Mistou, Michel-Yves; Sutcliffe, Iain C.; van Sorge, Nina M.

2016-01-01

The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. PMID:26975195

Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria.

PubMed

Mistou, Michel-Yves; Sutcliffe, Iain C; van Sorge, Nina M

2016-07-01

The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. © FEMS 2016.

Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared Microspectroscopy

PubMed Central

Devaux, Marie-Françoise; Jamme, Frédéric; André, William; Bouchet, Brigitte; Alvarado, Camille; Durand, Sylvie; Robert, Paul; Saulnier, Luc; Bonnin, Estelle; Guillon, Fabienne

2018-01-01

Tracking enzyme localization and following the local biochemical modification of the substrate should help explain the recalcitrance of lignocellulosic plant cell walls to enzymatic degradation. Time-lapse studies using conventional imaging require enzyme labeling and following the biochemical modifications of biopolymers found in plant cell walls, which cannot be easily achieved. In the present work, synchrotron facilities have been used to image the enzymatic degradation of lignocellulosic biomass without labeling the enzyme or the cell walls. Multichannel autofluorescence imaging of the protein and phenolic compounds after excitation at 275 nm highlighted the presence or absence of enzymes on cell walls and made it possible to track them during the reaction. Image analysis was used to quantify the fluorescence intensity variations. Consistent variations in the enzyme concentration were found locally for cell cavities and their surrounding cell walls. Microfluidic FT-IR microspectroscopy allowed for time-lapse tracking of local changes in the polysaccharides in cell walls during degradation. Hemicellulose degradation was found to occur prior to cellulose degradation using a Celluclast® preparation. Combining the fluorescence and FT-IR information yielded the conclusion that enzymes did not bind to lignified cell walls, which were consequently not degraded. Fluorescence multiscale imaging and FT-IR microspectroscopy showed an unexpected variability both in the initial biochemical composition and the degradation pattern, highlighting micro-domains in the cell wall of a given cell. Fluorescence intensity quantification showed that the enzymes were not evenly distributed, and their amount increased progressively on degradable cell walls. During degradation, adjacent cells were separated and the cell wall fragmented until complete degradation. PMID:29515611

Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

PubMed

Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

2015-01-01

Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

A unified wall function for compressible turbulence modelling

NASA Astrophysics Data System (ADS)

Ong, K. C.; Chan, A.

2018-05-01

Turbulence modelling near the wall often requires a high mesh density clustered around the wall and the first cells adjacent to the wall to be placed in the viscous sublayer. As a result, the numerical stability is constrained by the smallest cell size and hence requires high computational overhead. In the present study, a unified wall function is developed which is valid for viscous sublayer, buffer sublayer and inertial sublayer, as well as including effects of compressibility, heat transfer and pressure gradient. The resulting wall function applies to compressible turbulence modelling for both isothermal and adiabatic wall boundary conditions with the non-zero pressure gradient. Two simple wall function algorithms are implemented for practical computation of isothermal and adiabatic wall boundary conditions. The numerical results show that the wall function evaluates the wall shear stress and turbulent quantities of wall adjacent cells at wide range of non-dimensional wall distance and alleviate the number and size of cells required.

Growth and cell wall changes in stem organs under microgravity and hypergravity conditions

NASA Astrophysics Data System (ADS)

Hoson, Takayuki; Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro

Gravity strongly influences plant growth and development, which is fundamentally brought about by modifications to the properties of the cell wall. We have examined the changes in growth and cell wall properties in seedling organs under hypergravity conditions produced by centrifugation and under microgravity conditions in space. Hypergravity stimuli have been shown to decrease the growth rate of various seedling organs. When hypergravity suppressed elongation growth, a decrease in cell wall extensibility (an increase in cell wall rigidity) was induced. Hypergravity has also been shown to increase cell wall thickness in various mate-rials. In addition, a polymerization of certain matrix polysaccharides was brought about by hypergravity: in dicotyledons hypergravity increased the molecular size of xyloglucans, whereas hypergravity increased that of 1,3,1,4-β-glucans in monocotyledonous Gramineae. These mod-ifications to cell wall metabolism may be responsible for a decrease in cell wall extensibility, leading to growth suppression under hypergravity conditions. How then does microgravity in-fluence growth and cell wall properties? Here, there was a possibility that microgravity might induce changes similar to those by hypergravity, because plants have evolved and adapted to 1 g condition for more than 400 million years. However, the changes observed under microgravity conditions in space were just opposite to those induced by hypergravity: stimulation of elonga-tion growth, an increase in cell wall extensibility, and a decrease in cell wall thickness as well as depolymerization of cell wall polysaccharides were brought about in space. Furthermore, growth and cell wall properties varied in proportion to the logarithm of the magnitude of grav-ity in the range from microgravity to hypergravity, as shown in the dose-response relation in light and hormonal responses. Thus, microgravity may be a `stress-less' environment for plant seedlings to grow and develop. Preliminary results obtained by recent Space Seed experiment in the Kibo Module on the International Space Station (PI: S. Kamisaka) suggest that this hypothesis is also applicable to mature Arabidopsis plants.

Profiling the Hydrolysis of Isolated Grape Berry Skin Cell Walls by Purified Enzymes.

PubMed

Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Vivier, Melané A

2015-09-23

The unraveling of crushed grapes by maceration enzymes during winemaking is difficult to study because of the complex and rather undefined nature of both the substrate and the enzyme preparations. In this study we simplified both the substrate, by using isolated grape skin cell walls, and the enzyme preparations, by using purified enzymes in buffered conditions, to carefully follow the impact of the individual and combined enzymes on the grape skin cell walls. By using cell wall profiling techniques we could monitor the compositional changes in the grape cell wall polymers due to enzyme activity. Extensive enzymatic hydrolysis, achieved with a preparation of pectinases or pectinases combined with cellulase or hemicellulase enzymes, completely removed or drastically reduced levels of pectin polymers, whereas less extensive hydrolysis only opened up the cell wall structure and allowed extraction of polymers from within the cell wall layers. Synergistic enzyme activity was detectable as well as indications of specific cell wall polymer associations.

A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance

PubMed Central

Watterson, Daniel; Robinson, Jodie; Chappell, Keith J.; Butler, Mark S.; Edwards, David J.; Fry, Scott R.; Bermingham, Imogen M.; Cooper, Matthew A.; Young, Paul R.

2016-01-01

Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus. PMID:26976324

Direct Observation of Nanoparticle-Cancer Cell Nucleus Interactions

PubMed Central

Dam, Duncan Hieu M.; Lee, Jung Heon; Sisco, Patrick N.; Co, Dick T.; Zhang, Ming; Wasielewski, Michael R.; Odom, Teri W.

2012-01-01

We report the direct visualization of interactions between drug-loaded nanoparticles and the cancer cell nucleus. Nanoconstructs composed of nucleolin-specific aptamers and gold nanostars were actively transported to the nucleus and induced major changes to the nuclear phenotype via nuclear envelope invaginations near the site of the construct. The number of local deformations could be increased by ultra-fast, light-triggered release of the aptamers from the surface of the gold nanostars. Cancer cells with more nuclear envelope folding showed increased caspase 3 and 7 activity (apoptosis) as well as decreased cell viability. This newly revealed correlation between drug-induced changes in nuclear phenotype and increased therapeutic efficacy could provide new insight for nuclear-targeted cancer therapy. PMID:22424173

Outside-in control -Does plant cell wall integrity regulate cell cycle progression?

PubMed

Gigli-Bisceglia, Nora; Hamann, Thorsten

2018-04-13

During recent years it has become accepted that plant cell walls are not inert objects surrounding all plant cells but are instead highly dynamic, plastic structures. They are involved in a large number of cell biological processes and contribute actively to plant growth, development and interaction with environment. Therefore, it is not surprising that cellular processes can control plant cell wall integrity while, simultaneously, cell wall integrity can influence cellular processes. In yeast and animal cells such a bi-directional relationship also exists between the yeast/animal extra-cellular matrices and the cell cycle. In yeast, the cell wall integrity maintenance mechanism and a dedicated plasmamembrane integrity checkpoint are mediating this relationship. Recent research has yielded insights into the mechanism controlling plant cell wall metabolism during cytokinesis. However, knowledge regarding putative regulatory pathways controlling adaptive modifications in plant cell cycle activity in response to changes in the state of the plant cell wall are not yet identified. In this review, we summarize similarities and differences in regulatory mechanisms coordinating extra cellular matrices and cell cycle activity in animal and yeast cells, discuss the available evidence supporting the existence of such a mechanism in plants and suggest that the plant cell wall integrity maintenance mechanism might also control cell cycle activity in plant cells. This article is protected by copyright. All rights reserved.

Phenotype-Based Screening of Small Molecules to Modify Plant Cell Walls Using BY-2 Cells.

PubMed

Okubo-Kurihara, Emiko; Matsui, Minami

2018-01-01

The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.

Genetics of Capsular Polysaccharides and Cell Envelope (Glyco)lipids

PubMed Central

Daffé, Mamadou; Crick, Dean C.; Jackson, Mary

2014-01-01

This chapter summarizes what is currently known of the structures, physiological roles, involvement in pathogenicity and biogenesis of a variety of non-covalently bound cell envelope lipids and glycoconjugates of Mycobacterium tuberculosis and other Mycobacterium species. Topics addressed in this chapter include phospholipids; phosphatidylinositol mannosides; triglycerides; isoprenoids and related compounds (polyprenyl phosphate, menaquinones, carotenoids, non-carotenoid cyclic isoprenoids); acyltrehaloses (lipooligosaccharides, trehalose mono- and di-mycolates, sulfolipids, di- and poly-acyltrehaloses); mannosyl-beta-1-phosphomycoketides; glycopeptidolipids; phthiocerol dimycocerosates, para-hydroxybenzoic acids and phenolic glycolipids; mycobactins; mycolactones; and capsular polysaccharides. PMID:25485178

The plant cell wall in the feeding sites of cyst nematodes.

PubMed

Bohlmann, Holger; Sobczak, Miroslaw

2014-01-01

Plant parasitic cyst nematodes (genera Heterodera and Globodera) are serious pests for many crops. They enter the host roots as migratory second stage juveniles (J2) and migrate intracellularly toward the vascular cylinder using their stylet and a set of cell wall degrading enzymes produced in the pharyngeal glands. They select an initial syncytial cell (ISC) within the vascular cylinder or inner cortex layers to induce the formation of a multicellular feeding site called a syncytium, which is the only source of nutrients for the parasite during its entire life. A syncytium can consist of more than hundred cells whose protoplasts are fused together through local cell wall dissolutions. While the nematode produces a cocktail of cell wall degrading and modifying enzymes during migration through the root, the cell wall degradations occurring during syncytium development are due to the plants own cell wall modifying and degrading proteins. The outer syncytial cell wall thickens to withstand the increasing osmotic pressure inside the syncytium. Furthermore, pronounced cell wall ingrowths can be formed on the outer syncytial wall at the interface with xylem vessels. They increase the surface of the symplast-apoplast interface, thus enhancing nutrient uptake into the syncytium. Processes of cell wall degradation, synthesis and modification in the syncytium are facilitated by a variety of plant proteins and enzymes including expansins, glucanases, pectate lyases and cellulose synthases, which are produced inside the syncytium or in cells surrounding the syncytium.

Cell wall of pathogenic yeasts and implications for antimycotic therapy.

PubMed

Cassone, A

1986-01-01

Yeast cell wall is a complex, multilayered structure where amorphous, granular and fibrillar components interact with each other to confer both the specific cell shape and osmotic protection against lysis. Thus it is widely recognized that as is the case with bacteria, yeast cell wall is a major potential target for selective chemotherapeutic drugs. Despite intensive research, very few such drugs have been discovered and none has found substantial application in human diseases to date. Among the different cell wall components, beta-glucan and chitin are the fibrillar materials playing a fundamental role in the overall rigidity and resistance of the wall. Inhibition of the metabolism of these polymers, therefore, should promptly lead to lysis. This indeed occurs and aculeacin, echinocandin and polyoxins are examples of agents producing such an action. Particular attention should be focused on chitin synthesis. Although quantitatively a minor cell wall component, chitin is important in the mechanism of dimorphic transition, especially in Candida albicans, a major human opportunistic pathogen. This transition is associated with increased invasiveness and general virulence of the fungus. Yeast cell wall may also limit the effect of antifungals which owe their action to disturbance of the cytoplasmic membrane or of cell metabolism. Indeed, the cell wall may hinder access to the cell interior both under growing conditions and, particularly, during cell ageing in the stationary phase, when important structural changes occur in the cell wall due to unbalanced wall growth (phenotypic drug resistance).

Biochemical Requirements of Virus Wrapping by the Endoplasmic Reticulum: Involvement of ATP and Endoplasmic Reticulum Calcium Store during Envelopment of African Swine Fever Virus

PubMed Central

Cobbold, Christian; Brookes, Sharon M.; Wileman, Thomas

2000-01-01

Enwrapment by membrane cisternae has emerged recently as a mechanism of envelopment for large enveloped DNA viruses, such as herpesviruses, poxviruses, and African swine fever (ASF) virus. For both ASF virus and the poxviruses, wrapping is a multistage process initiated by the recruitment of capsid proteins onto membrane cisternae of the endoplasmic reticulum (ER) or associated ER-Golgi intermediate membrane compartments. Capsid assembly induces progressive bending of membrane cisternae into the characteristic shape of viral particles, and envelopment provides virions with two membranes in one step. We have used biochemical assays for ASF virus capsid recruitment, assembly, and envelopment to define the cellular processes important for the enwrapment of viruses by membrane cisternae. Capsid assembly on the ER membrane, and envelopment by ER cisternae, were inhibited when cells were depleted of ATP or depleted of calcium by incubation with A23187 and EDTA or the ER calcium ATPase inhibitor, thapsigargin. Electron microscopy analysis showed that cells depleted of calcium were unable to assemble icosahedral particles. Instead, assembly sites contained crescent-shaped and bulbous structures and, in rare cases, empty closed five-sided particles. Interestingly, recruitment of the capsid protein from the cytosol onto the ER membrane did not require ATP or an intact ER calcium store. The results show that following recruitment of the virus capsid protein onto the ER membrane, subsequent stages of capsid assembly and enwrapment are dependent on ATP and are regulated by the calcium gradients present across the ER membrane cisternae. PMID:10666244

Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes

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

Cosgrove, Daniel J.

The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the ‘Young's modulus’ of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potentialmore » pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics.« less

My body is a cage: mechanisms and modulation of plant cell growth.

PubMed

Braidwood, Luke; Breuer, Christian; Sugimoto, Keiko

2014-01-01

388 I. 388 II. 389 III. 389 IV. 390 V. 391 VI. 393 VII. 394 VIII. 398 399 References 399 SUMMARY: The wall surrounding plant cells provides protection from abiotic and biotic stresses, and support through the action of turgor pressure. However, the presence of this strong elastic wall also prevents cell movement and resists cell growth. This growth can be likened to extending a house from the inside, using extremely high pressures to push out the walls. Plants must increase cell volume in order to explore their environment, acquire nutrients and reproduce. Cell wall material must stretch and flow in a controlled manner and, concomitantly, new cell wall material must be deposited at the correct rate and site to prevent wall and cell rupture. In this review, we examine biomechanics, cell wall structure and growth regulatory networks to provide a 'big picture' of plant cell growth. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

The connection of cytoskeletal network with plasma membrane and the cell wall

PubMed Central

Liu, Zengyu; Persson, Staffan; Zhang, Yi

2015-01-01

The cell wall provides external support of the plant cells, while the cytoskeletons including the microtubules and the actin filaments constitute an internal framework. The cytoskeletons contribute to the cell wall biosynthesis by spatially and temporarily regulating the transportation and deposition of cell wall components. This tight control is achieved by the dynamic behavior of the cytoskeletons, but also through the tethering of these structures to the plasma membrane. This tethering may also extend beyond the plasma membrane and impact on the cell wall, possibly in the form of a feedback loop. In this review, we discuss the linking components between the cytoskeletons and the plasma membrane, and/or the cell wall. We also discuss the prospective roles of these components in cell wall biosynthesis and modifications, and aim to provide a platform for further studies in this field. PMID:25693826

Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation.

PubMed

Watanabe, Yoichiro; Schneider, Rene; Barkwill, Sarah; Gonzales-Vigil, Eliana; Hill, Joseph L; Samuels, A Lacey; Persson, Staffan; Mansfield, Shawn D

2018-06-05

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs. Copyright © 2018 the Author(s). Published by PNAS.

Identification of potential cell wall component that allows Taka-amylase A adsorption in submerged cultures of Aspergillus oryzae.

PubMed

Sato, Hiroki; Toyoshima, Yoshiyuki; Shintani, Takahiro; Gomi, Katsuya

2011-12-01

We observed that α-amylase (Taka-amylase A; TAA) activity in the culture broth disappeared in the later stage of submerged cultivation of Aspergillus oryzae. This disappearance was caused by adsorption of TAA onto the cell wall of A. oryzae and not due to protein degradation by extracellular proteolytic enzymes. To determine the cell wall component(s) that allows TAA adsorption efficiently, the cell wall was fractionated by stepwise alkali treatment and enzymatic digestion. Consequently, alkali-insoluble cell wall fractions exhibited high levels of TAA adsorption. In addition, this adsorption capacity was significantly enhanced by treatment of the alkali-insoluble fraction with β-glucanase, which resulted in the concomitant increase in the amount of chitin in the resulting fraction. In contrast, the adsorption capacity was diminished by treating the cell wall fraction with chitinase. These results suggest that the major component that allows TAA adsorption is chitin. However, both the mycelium and the cell wall demonstrated the inability to allow TAA adsorption in the early stage of cultivation, despite chitin content in the cell wall being identical in both early and late stages of cultivation. These results suggest the existence of unidentified factor(s) that could prevent the adsorption of TAA onto the cell wall. Such factor(s) is most likely removed or diminished from the cell wall following longer cultivation periods.

A model of cell wall expansion based on thermodynamics of polymer networks

NASA Technical Reports Server (NTRS)

Veytsman, B. A.; Cosgrove, D. J.

1998-01-01

A theory of cell wall extension is proposed. It is shown that macroscopic properties of cell walls can be explained through the microscopic properties of interpenetrating networks of cellulose and hemicellulose. The qualitative conclusions of the theory agree with the existing experimental data. The dependence of the cell wall yield threshold on the secretion of the wall components is discussed.

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

PubMed Central

Romaniuk, Joseph A. H.; Cegelski, Lynette

2015-01-01

The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms. PMID:26370936

Cellular immune responses in patients with hepatitis B surface antigen seroclearance induced by antiviral therapy

PubMed Central

2011-01-01

Background The mechanisms by which chronic hepatitis B is completely resolved through antiviral therapy are unknown, and the contribution of acquired T cell immunity to hepatitis B surface antigen (HBsAg) seroclearance has not been investigated. Therefore, we measured the T-cell responses to core and envelope antigens in patients with HBsAg seroclearance. Methods Fourteen subjects with HBsAg seroclearance following antiviral treatment for chronic hepatitis B, 7 HBeAg-positive immunotolerant HBV carriers and 9 HBeAg-negative inactive HBsAg carriers were recruited. HBV-specific T-cell responses to recombinant HBV core (rHBcAg) and envelope (rHBsAg) proteins and pools of core and envelope peptides were measured using an ELISPOT assay detecting interferon-gamma and intracellular cytokine staining (ICS) assays detecting interferon-gamma or interleukin 2. Results Interferon-gamma ELISPOT assays showed a low frequency of weak responses to the rHBsAg and S peptide pool in the HBsAg seroclearance group, and the response frequency to the rHBcAg and the C peptide pool was higher than to the rHBsAg (P < 0.001) and S peptide pool (P = 0.001) respectively. A higher response frequency to C than S peptide pools was confirmed in the interferon-gamma ICS assays for both CD4+ (P = 0.033) and CD8+ (P = 0.040) T cells in the HBsAg seroclearance group. The responses to C and S antigens in the inactive carriers were similar. Conclusions There was a low frequency of CD4+ and CD8+ T cell immune responses to envelope antigens in Chinese subjects with HBsAg seroclearance following antiviral therapy. It is unlikely that these immune responses are responsible for HBsAg seroclearance in these subjects. PMID:21320337

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