Identification and Deletion of Tft1, a Predicted Glycosyltransferase Necessary for Cell Wall β-1,3;1,4-Glucan Synthesis in Aspergillus fumigatus

PubMed Central

Samar, Danial; Kieler, Joshua B.; Klutts, J. Stacey

2015-01-01

Aspergillus fumigatus is an environmental mold that causes severe, often fatal invasive infections in immunocompromised patients. The search for new antifungal drug targets is critical, and the synthesis of the cell wall represents a potential area to find such a target. Embedded within the main β-1,3-glucan core of the A. fumigatus cell wall is a mixed linkage, β-D-(1,3;1,4)-glucan. The role of this molecule or how it is synthesized is unknown, though it comprises 10% of the glucans within the wall. While this is not a well-studied molecule in fungi, it has been studied in plants. Using the sequences of two plant mixed linkage glucan synthases, a single ortholog was identified in A. fumigatus (Tft1). A strain lacking this enzyme (tft1Δ) was generated along with revertant strains containing the native gene under the control of either the native or a strongly expressing promoter. Immunofluorescence staining with an antibody against β-(1,3;1,4)-glucan and biochemical quantification of this polysaccharide in the tft1Δ strain demonstrated complete loss of this molecule. Reintroduction of the gene into the knockout strain yielded reappearance in amounts that correlated with expected expression of the gene. The loss of Tft1 and mixed linkage glucan yielded no in vitro growth phenotype. However, there was a modest increase in virulence for the tft1Δ strain in a wax worm model. While the precise roles for β-(1,3;1,4)-glucan within A. fumigatus cell wall are still uncertain, it is clear that Tft1 plays a pivotal role in the biosynthesis of this cell wall polysaccharide. PMID:25723175

Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans

PubMed Central

2018-01-01

Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized. No differences were seen in its Ywp1, but it exhibited greater adhesiveness, sensitivity to wall perturbing agents, and exposure of its underlying β-1,3-glucan layer to external antibodies. The molecular basis for this greater epitope accessibility has not been determined, but has facilitated exploration of how these properties change as a function of cell growth and morphology. In addition, previously engineered strains with reduced quantities of Ywp1 in their cell walls were also found to have greater β-1,3-glucan exposure, indicating that Ywp1 itself contributes to the masking of wall epitopes, which may be important for understanding the anti-adhesive effect of Ywp1. Ectopic production of Ywp1 by hyphae, which reduces the adhesivity of these filamentous forms of C. albicans, was similarly found to reduce exposure of the β-1,3-glucan in their walls. To monitor Ywp1 in the cell wall irrespective of its accessibility, green fluorescent protein (Gfp) was genetically inserted into wall-anchored Ywp1 using a bifunctional cassette that also allowed production from a single transfection of a soluble, anchor-free version. The wall-anchored Ywp1-Gfp-Ywp1 accumulated in the wall of the yeast forms but not hyphae, and appeared to have properties similar to native Ywp1, including its adhesion-inhibiting effect. Some pseudohyphal walls also detectably accumulated this probe. Strains of C. albicans with tandem hemagglutinin (HA) epitopes inserted into wall-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and β-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system. PMID:29329339

Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans.

PubMed

Granger, Bruce L

2018-01-01

Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized. No differences were seen in its Ywp1, but it exhibited greater adhesiveness, sensitivity to wall perturbing agents, and exposure of its underlying β-1,3-glucan layer to external antibodies. The molecular basis for this greater epitope accessibility has not been determined, but has facilitated exploration of how these properties change as a function of cell growth and morphology. In addition, previously engineered strains with reduced quantities of Ywp1 in their cell walls were also found to have greater β-1,3-glucan exposure, indicating that Ywp1 itself contributes to the masking of wall epitopes, which may be important for understanding the anti-adhesive effect of Ywp1. Ectopic production of Ywp1 by hyphae, which reduces the adhesivity of these filamentous forms of C. albicans, was similarly found to reduce exposure of the β-1,3-glucan in their walls. To monitor Ywp1 in the cell wall irrespective of its accessibility, green fluorescent protein (Gfp) was genetically inserted into wall-anchored Ywp1 using a bifunctional cassette that also allowed production from a single transfection of a soluble, anchor-free version. The wall-anchored Ywp1-Gfp-Ywp1 accumulated in the wall of the yeast forms but not hyphae, and appeared to have properties similar to native Ywp1, including its adhesion-inhibiting effect. Some pseudohyphal walls also detectably accumulated this probe. Strains of C. albicans with tandem hemagglutinin (HA) epitopes inserted into wall-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and β-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system.

Single-molecule electrocatalysis by single-walled carbon nanotubes.

PubMed

Xu, Weilin; Shen, Hao; Kim, Yoon Ji; Zhou, Xiaochun; Liu, Guokun; Park, Jiwoong; Chen, Peng

2009-12-01

We report a single-molecule fluorescence study of electrocatalysis by single-walled carbon nanotubes (SWNTs) at single-reaction resolution. Applying super-resolution optical imaging, we find that the electrocatalysis occurs at discrete, nanometer-dimension sites on SWNTs. Single-molecule kinetic analysis leads to an electrocatalytic mechanism, allowing quantification of the reactivity and heterogeneity of individual reactive sites. Combined with conductivity measurements, this approach will be powerful to interrogate how the electronic structure of SWNTs affects the electrocatalytic interfacial charge transfer, a process fundamental to photoelectrochemical cells.

Nanomechanics of Yeast Surfaces Revealed by AFM

NASA Astrophysics Data System (ADS)

Dague, Etienne; Beaussart, Audrey; Alsteens, David

Despite the large and well-documented characterization of the microbial cell wall in terms of chemical composition, the determination of the mechanical properties of surface molecules in relation to their function remains a key challenge in cell biology.The emergence of powerful tools allowing molecular manipulations has already revolutionized our understanding of the surface properties of fungal cells. At the frontier between nanophysics and molecular biology, atomic force microscopy (AFM), and more specifically single-molecule force spectroscopy (SMFS), has strongly contributed to our current knowledge of the cell wall organization and nanomechanical properties. However, due to the complexity of the technique, measurements on live cells are still at their infancy.In this chapter, we describe the cell wall composition and recapitulate the principles of AFM as well as the main current methodologies used to perform AFM measurements on live cells, including sample immobilization and tip functionalization.The current status of the progress in probing nanomechanics of the yeast surface is illustrated through three recent breakthrough studies. Determination of the cell wall nanostructure and elasticity is presented through two examples: the mechanical response of mannoproteins from brewing yeasts and elasticity measurements on lacking polysaccharide mutant strains. Additionally, an elegant study on force-induced unfolding and clustering of adhesion proteins located at the cell surface is also presented.

Interaction of Vascular Smooth Muscle Cells Under Low Shear Stress

NASA Technical Reports Server (NTRS)

Seidel, Charles L.

1998-01-01

The blood vessel wall consists of three cellular layers, an outer adventitial, a middle medial and an inner intimal layer. When the blood vessel forms in the embryo it begins as a tube composed of a single cell type called endothelial cells. Over time, other cells are recruited from the surrounding tissue to form additional layers on the outer surface of the endothelial tube. The cells that are recruited are called mesenchymal cells. Mesenchymal cells are responsible for the production of connective tissue that holds the blood vessel together and for developing into vascular smooth muscle cells that are responsible for regulating the diameter of the vessel (1) and therefore, blood flow. In a fully developed blood vessel, the endothelial cells make- up the majority of cells in the intimal layer while the mesenchymal cells make-up the majority of cells in the medial and adventitial layers. Within the medial layer of a mature vessel, cells are organized into multiple circular layers of alternating bands of connective tissue and cells. The cell layer is composed of a mixture of mesenchymal cells that have not developed into smooth muscle cells and fully developed smooth muscle cells (2). The assembly and organization of complex tissues is directed in part by a signaling system composed of proteins on the cell surface called adhesion molecules. Adhesion molecules enable cells to recognize each other as well as the composition of the connective tissue in which they reside (3). It was hypothesized that the different cell types that compose the vascular wall possess different adhesion molecules that enable them to recognize each other and through this recognition system, form the complex layered organization of the vascular wall. In other words, the layered organization is an intrinsic property of the cells. If this hypothesis is correct then the different cells that make up the vessel wall, when mixed together, should organize themselves into a layered structure resembling an intact blood vessel. Experiments described below were designed to test this hypothesis.

A glycosylphosphatidylinositol anchor is required for membrane localization but dispensable for cell wall association of chitin deacetylase 2 in Cryptococcus neoformans.

PubMed

Gilbert, Nicole M; Baker, Lorina G; Specht, Charles A; Lodge, Jennifer K

2012-01-01

Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. The surface of a pathogenic microbe is a major interface with its host. In fungi, the outer surface consists of a complex matrix known as the cell wall, which includes polysaccharides, proteins, and other molecules. The mammalian host recognizes many of these surface molecules and mounts appropriate responses to combat the microbial infection. Cryptococcus neoformans is a serious fungal pathogen that kills over 600,000 people annually. It converts most of its chitin, a cell wall polysaccharide, to chitosan, which is necessary for virulence. Chitin deacetylase enzymes have been identified in the cell wall, and our studies were undertaken to understand how the deacetylase is linked to the wall and where it has activity. Our results have implications for the current model of chitosan biosynthesis and further challenge the paradigm of covalent linkages between cell wall proteins and polysaccharides through a lipid modification of the protein.

Quantification of plant cell coupling with live-cell microscopy.

PubMed

Liesche, Johannes; Schulz, Alexander

2015-01-01

Movement of nutrients and signaling compounds from cell to cell is an essential process for plant growth and development. To understand processes such as carbon allocation, cell communication, and reaction to pathogen attack it is important to know a specific molecule's capacity to pass a specific cell wall interface. Transport through plasmodesmata, the cell wall channels that directly connect plant cells, is regulated not only by a fixed size exclusion limit, but also by physiological and pathological adaptation. The noninvasive approach described here offers the possibility of precisely determining the plasmodesmata-mediated cell wall permeability for small molecules in living cells.The method is based on photoactivation of the fluorescent tracer caged fluorescein. Non-fluorescent caged fluorescein is applied to a target tissue, where it is taken up passively into all cells. Imaged by confocal microscopy, loaded tracer is activated by UV illumination in a target cell and its spread to neighboring cells monitored. When combined with high-speed acquisition by resonant scanning or spinning disc confocal microscopy, the high signal-to-noise ratio of photoactivation allows collection of three-dimensional (3D) time series. These contain all necessary functional and anatomical data to measure cell coupling in complex tissues noninvasively.

Bioinspired Layer-by-Layer Microcapsules Based on Cellulose Nanofibers with Switchable Permeability.

PubMed

Paulraj, Thomas; Riazanova, Anastasia V; Yao, Kun; Andersson, Richard L; Müllertz, Anette; Svagan, Anna J

2017-04-10

Green, all-polysaccharide based microcapsules with mechanically robust capsule walls and fast, stimuli-triggered, and switchable permeability behavior show great promise in applications based on selective and timed permeability. Taking a cue from nature, the build-up and composition of plant primary cell walls inspired the capsule wall assembly, because the primary cell walls in plants exhibit high mechanical properties despite being in a highly hydrated state, primarily owing to cellulose microfibrils. The microcapsules (16 ± 4 μm in diameter) were fabricated using the layer-by-layer technique on sacrificial CaCO 3 templates, using plant polysaccharides (pectin, cellulose nanofibers, and xyloglucan) only. In water, the capsule wall was permeable to labeled dextrans with a hydrodynamic diameter of ∼6.6 nm. Upon exposure to NaCl, the porosity of the capsule wall quickly changed allowing larger molecules (∼12 nm) to permeate. However, the porosity could be restored to its original state by removal of NaCl, by which permeants became trapped inside the capsule's core. The high integrity of cell wall was due to the CNF and the ON/OFF alteration of the permeability properties, and subsequent loading/unloading of molecules, could be repeated several times with the same capsule demonstrating a robust microcontainer with controllable permeability properties.

Single-molecule analysis of the major glycopolymers of pathogenic and non-pathogenic yeast cells

NASA Astrophysics Data System (ADS)

El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Alsteens, David; Sarazin, Aurore; Jouault, Thierry; Dufrêne, Yves F.

2013-05-01

Most microbes are coated with carbohydrates that show remarkable structural variability and play a crucial role in mediating microbial-host interactions. Understanding the functions of cell wall glycoconjugates requires detailed knowledge of their molecular organization, diversity and heterogeneity. Here we use atomic force microscopy (AFM) with tips bearing specific probes (lectins, antibodies) to analyze the major glycopolymers of pathogenic and non-pathogenic yeast cells at molecular resolution. We show that non-ubiquitous β-1,2-mannans are largely exposed on the surface of native cells from pathogenic Candida albicans and C. glabrata, the former species displaying the highest glycopolymer density and extensions. We also find that chitin, a major component of the inner layer of the yeast cell wall, is much more abundant in C. albicans. These differences in molecular properties, further supported by flow cytometry measurements, may play an important role in strengthening cell wall mechanics and immune interactions. This study demonstrates that single-molecule AFM, combined with immunological and fluorescence methods, is a powerful platform in fungal glycobiology for probing the density, distribution and extension of specific cell wall glycoconjugates. In nanomedicine, we anticipate that this new form of AFM-based nanoglycobiology will contribute to the development of sugar-based drugs, immunotherapeutics, vaccines and diagnostics.

Understanding decay resistance, dimensional stability and strength changes in heat treated and acetylated wood

Treesearch

Roger M. Rowell; Rebecca E. Ibach; James McSweeny; Thomas Nilsson

2009-01-01

Reductions in hygroscopicity, increased dimensional stability and decay resistance of heat-treated wood depend on decomposition of a large portion of the hemicelluloses in the wood cell wall. In theory, these hemicelluloses are converted to small organic molecules, water and volatile furan-type intermediates that can polymerize in the cell wall. Reductions in...

Revealing of Saccharomyces cerevisiae yeast cell wall proteins capable of binding thioflavin T, a fluorescent dye specifically interacting with amyloid fibrils.

PubMed

Gorkovskii, A A; Bezsonov, E E; Plotnikova, T A; Kalebina, T S; Kulaev, I S

2009-11-01

Proteins binding thioflavin T leading to its specific fluorescence were discovered in a fraction of noncovalently bound Saccharomyces cerevisiae yeast cell wall mannoproteins. Thioflavin-binding proteins display high resistance to trypsin digestion in solution. These data are the first experimental evidence for the presence of proteins whose properties are characteristic of amyloids in yeast cell wall, except for data on glucanotransferase Bgl2p that has amyloid properties. Our data suggest the anchoring of these proteins in the cell wall by a trypsin-sensitive part of the protein molecule. Experiments with a mutant strain devoid of the BGL2 gene suggest the compensation of absent amyloid-like protein Bgl2p by increase in contents of thioflavin-binding proteins in the cell wall.

Discovery of Novel Cell Wall-Active Compounds Using PywaC, a Sensitive Reporter of Cell Wall Stress, in the Model Gram-Positive Bacterium Bacillus subtilis

PubMed Central

Czarny, T. L.; Perri, A. L.; French, S.

2014-01-01

The emergence of antibiotic resistance in recent years has radically reduced the clinical efficacy of many antibacterial treatments and now poses a significant threat to public health. One of the earliest studied well-validated targets for antimicrobial discovery is the bacterial cell wall. The essential nature of this pathway, its conservation among bacterial pathogens, and its absence in human biology have made cell wall synthesis an attractive pathway for new antibiotic drug discovery. Herein, we describe a highly sensitive screening methodology for identifying chemical agents that perturb cell wall synthesis, using the model of the Gram-positive bacterium Bacillus subtilis. We report on a cell-based pilot screen of 26,000 small molecules to look for cell wall-active chemicals in real time using an autonomous luminescence gene cluster driven by the promoter of ywaC, which encodes a guanosine tetra(penta)phosphate synthetase that is expressed under cell wall stress. The promoter-reporter system was generally much more sensitive than growth inhibition testing and responded almost exclusively to cell wall-active antibiotics. Follow-up testing of the compounds from the pilot screen with secondary assays to verify the mechanism of action led to the discovery of 9 novel cell wall-active compounds. PMID:24687489

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.

Two ATP Binding Cassette G Transporters, Rice ATP Binding Cassette G26 and ATP Binding Cassette G15, Collaboratively Regulate Rice Male Reproduction1[OPEN

PubMed Central

Zhao, Guochao; Shi, Jianxin; Liang, Wanqi; Xue, Feiyang; Luo, Qian; Zhu, Lu; Qu, Guorun; Chen, Mingjiao; Schreiber, Lukas; Zhang, Dabing

2015-01-01

Male reproduction in higher plants requires the support of various metabolites, including lipid molecules produced in the innermost anther wall layer (the tapetum), but how the molecules are allocated among different anther tissues remains largely unknown. Previously, rice (Oryza sativa) ATP binding cassette G15 (ABCG15) and its Arabidopsis (Arabidopsis thaliana) ortholog were shown to be required for pollen exine formation. Here, we report the significant role of OsABCG26 in regulating the development of anther cuticle and pollen exine together with OsABCG15 in rice. Cytological and chemical analyses indicate that osabcg26 shows reduced transport of lipidic molecules from tapetal cells for anther cuticle development. Supportively, the localization of OsABCG26 is on the plasma membrane of the anther wall layers. By contrast, OsABCG15 is polarly localized in tapetal plasma membrane facing anther locules. osabcg26 osabcg15 double mutant displays an almost complete absence of anther cuticle and pollen exine, similar to that of osabcg15 single mutant. Taken together, we propose that OsABCG26 and OsABCG15 collaboratively regulate rice male reproduction: OsABCG26 is mainly responsible for the transport of lipidic molecules from tapetal cells to anther wall layers, whereas OsABCG15 mainly is responsible for the export of lipidic molecules from the tapetal cells to anther locules for pollen exine development. PMID:26392263

Cell shape acquisition and maintenance in rodlike bacteria

NASA Astrophysics Data System (ADS)

van Teeffelen, Sven; Wingreen, Ned; Gitai, Zemer

2010-03-01

The shape of rodlike bacteria such as Escherichia coli is mainly governed by the expansion and reorganization of the peptidoglycan cell wall. The cell wall is a huge, mostly single-layered molecule of stiff glycan strands that typically run perpendicular to the long axis and are crosslinked by short peptides. The wall resists the excess pressure from inside the cell. Although much is known about the enzymes that synthesize the wall, the mechanisms by which the cell maintains a constant rod diameter and uniform glycan strand orientation during growth remain unknown. Here we present quantitative results on the structure and dynamics of two essential proteins, which are believed to play an important role in cell wall synthesis. In particular, we have focused on the filament-forming protein MreB, an actin homolog that forms a long helical bundle along the inner membrane of the cell, and penicillin-binding protein 2, an essential protein for peptide bond formation in the periplasm. Based on their interplay we discuss the possibility of MreB serving as a guide and ruler for cell wall synthesis.

Surface enhanced Raman scattering analyses of individual silver nanoaggregates on living single yeast cell wall

NASA Astrophysics Data System (ADS)

Sujith, Athiyanathil; Itoh, Tamitake; Abe, Hiroko; Anas, Abdul Aziz; Yoshida, Kenichi; Biju, Vasudevanpillai; Ishikawa, Mitsuru

2008-03-01

We labeled the living yeast cell surface (Saccharomyces cerevisiae strain W303-1A) by silver nanoparticles which can form nanoaggregates and found to show surface enhanced Raman scattering (SERS) activity. Blinking of SERS and its polarization dependence reveal that SERS signals are from amplified electromagnetic field at nanometric Ag nanoparticles gaps with single or a few molecules sensitivity. We tentatively assigned SERS spectra from a yeast cell wall to mannoproteins. Nanoaggregate-by-nanoaggregate variations and temporal fluctuations of SERS spectra are discussed in terms of inhomogeneous mannoprotein distribution on a cell wall and possible ways of Ag nanoaggregate adsorption, respectively.

Solution NMR analyses of the anticodon arms of proteinogenic and non-proteinogenic tRNAGly

PubMed Central

Chang, Andrew T.; Nikonowicz, Edward P.

2012-01-01

Although the fate of most tRNA molecules in the cell is aminoacylation and delivery to the ribosome, some tRNAs are destined to fulfill other functional roles. In addition to their central role in translation, tRNA molecules participate in processes such as regulation of gene expression, bacterial cell wall biosynthesis, viral replication, antibiotic biosynthesis, and suppression of alternative splicing. In bacteria, glycyl-tRNA molecules with anticodon sequences GCC and UCC exhibit multiple extra-translational functions including transcriptional regulation and cell wall biosynthesis. We have determined the high-resolution structures of three glycyl-tRNA anticodon arms with anticodon sequences GCC and UCC. Two of the tRNA molecules are proteinogenic (tRNAGly,GCC and tRNAGly,UCC) and the third is non-proteinogenic (np-tRNAGly,UCC) and participates in cell wall biosynthesis. The UV-monitored thermal melting curves show that the anticodon arm of tRNAGly,UCC with a loop-closing C-A+ base pair melts at a 10 °C lower temperature than those of tRNAGly,GCC or np-tRNAGly,UCC. U-A and C-G pairs close the loops of the later two molecules and enhance stem stability. Mg2+ stabilizes the tRNAGly,UCC anticodon arm and lessens the Tm differential. The structures of the three tRNAGly anticodon arms exhibit small differences between one another, but none of them form the classical U-turn motif. The anticodon loop of tRNAGly,GCC becomes more dynamic and disordered in the presence of multivalent cations, whereas metal ion coordination in the anticodon loops of tRNAGly,UCC and np-tRNAGly,UCC establishes conformational homogeneity. The conformational similarity of the molecules is greater than their functional differences might suggest. Because aminoacylation of the full-length tRNA molecules is accomplished by one tRNA synthetase, the similar structural context of the loop may facilitate efficient recognition of each of the anticodon sequences. PMID:22468768

Discovery of novel cell wall-active compounds using P ywaC, a sensitive reporter of cell wall stress, in the model gram-positive bacterium Bacillus subtilis.

PubMed

Czarny, T L; Perri, A L; French, S; Brown, E D

2014-06-01

The emergence of antibiotic resistance in recent years has radically reduced the clinical efficacy of many antibacterial treatments and now poses a significant threat to public health. One of the earliest studied well-validated targets for antimicrobial discovery is the bacterial cell wall. The essential nature of this pathway, its conservation among bacterial pathogens, and its absence in human biology have made cell wall synthesis an attractive pathway for new antibiotic drug discovery. Herein, we describe a highly sensitive screening methodology for identifying chemical agents that perturb cell wall synthesis, using the model of the Gram-positive bacterium Bacillus subtilis. We report on a cell-based pilot screen of 26,000 small molecules to look for cell wall-active chemicals in real time using an autonomous luminescence gene cluster driven by the promoter of ywaC, which encodes a guanosine tetra(penta)phosphate synthetase that is expressed under cell wall stress. The promoter-reporter system was generally much more sensitive than growth inhibition testing and responded almost exclusively to cell wall-active antibiotics. Follow-up testing of the compounds from the pilot screen with secondary assays to verify the mechanism of action led to the discovery of 9 novel cell wall-active compounds. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Draft Genome of the Invasive Walking Stick, Medauroidea extradendata, Reveals Extensive Lineage-Specific Gene Family Expansions of Cell Wall Degrading Enzymes in Phasmatodea

PubMed Central

Brand, Philipp; Lin, Wei; Johnson, Brian R.

2018-01-01

Plant cell wall components are the most abundant macromolecules on Earth. The study of the breakdown of these molecules is thus a central question in biology. Surprisingly, plant cell wall breakdown by herbivores is relatively poorly understood, as nearly all early work focused on the mechanisms used by symbiotic microbes to breakdown plant cell walls in insects such as termites. Recently, however, it has been shown that many organisms make endogenous cellulases. Insects, and other arthropods, in particular have been shown to express a variety of plant cell wall degrading enzymes in many gene families with the ability to break down all the major components of the plant cell wall. Here we report the genome of a walking stick, Medauroidea extradentata, an obligate herbivore that makes uses of endogenously produced plant cell wall degrading enzymes. We present a draft of the 3.3Gbp genome along with an official gene set that contains a diversity of plant cell wall degrading enzymes. We show that at least one of the major families of plant cell wall degrading enzymes, the pectinases, have undergone a striking lineage-specific gene family expansion in the Phasmatodea. This genome will be a useful resource for comparative evolutionary studies with herbivores in many other clades and will help elucidate the mechanisms by which metazoans breakdown plant cell wall components. PMID:29588379

Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Jin, Hong; Heller, Daniel A.; Kalbacova, Marie; Kim, Jong-Ho; Zhang, Jingqing; Boghossian, Ardemis A.; Maheshri, Narendra; Strano, Michael S.

2010-04-01

An emerging concept in cell signalling is the natural role of reactive oxygen species such as hydrogen peroxide (H2O2) as beneficial messengers in redox signalling pathways. The nature of H2O2 signalling is confounded, however, by difficulties in tracking it in living systems, both spatially and temporally, at low concentrations. Here, we develop an array of fluorescent single-walled carbon nanotubes that can selectively record, in real time, the discrete, stochastic quenching events that occur as H2O2 molecules are emitted from individual human epidermal carcinoma cells stimulated by epidermal growth factor. We show mathematically that such arrays can distinguish between molecules originating locally on the cell membrane from other contributions. We find that epidermal growth factor induces 2 nmol H2O2 locally over a period of 50 min. This platform promises a new approach to understanding the signalling of reactive oxygen species at the cellular level.

A Versatile Click-Compatible Monolignol Probe to Study Lignin Deposition in Plant Cell Walls

PubMed Central

Pandey, Jyotsna L.; Wang, Bo; Diehl, Brett G.; Richard, Tom L.; Chen, Gong; Anderson, Charles T.

2015-01-01

Lignin plays important structural and functional roles in plants by forming a hydrophobic matrix in secondary cell walls that enhances mechanical strength and resists microbial decay. While the importance of the lignin matrix is well documented and the biosynthetic pathways for monolignols are known, the process by which lignin precursors or monolignols are transported and polymerized to form this matrix remains a subject of considerable debate. In this study, we have synthesized and tested an analog of coniferyl alcohol that has been modified to contain an ethynyl group at the C-3 position. This modification enables fluorescent tagging and imaging of this molecule after its incorporation into plant tissue by click chemistry-assisted covalent labeling with a fluorescent azide dye, and confers a distinct Raman signature that could be used for Raman imaging. We found that this monolignol analog is incorporated into in vitro-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings. Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana plants and labeling with an Alexa-594 azide dye revealed the precise locations of new lignin polymerization. Results from this study indicate that this molecule can provide high-resolution localization of lignification during plant cell wall maturation and lignin matrix assembly. PMID:25884205

Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM

NASA Astrophysics Data System (ADS)

Alsteens, David; Dupres, Vincent; McEvoy, Kevin; Wildling, Linda; Gruber, Hermann J.; Dufrêne, Yves F.

2008-09-01

Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls.

Methods of expressing and detecting activity of expansin in plant cells

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

Hood, Elizabeth E.; Yoon, Sangwoong

A method of expressing heterologous expansin in a plant cell is provided where a nucleic acid molecule encoding expansin is introduced into the plant cell and in an embodiment is operably linked to a promoter preferentially expressing in the seed tissue of the plant, and in another embodiment is linked to a promoter preferentially expressing in the embryo tissue of the seed. An embodiment provides the nucleic acid molecule is operably linked to a second nucleic acid molecule that directs expression to the endoplasmic reticulum, vacuole or cell wall. Plants and plant parts expressing expansin are provided. An assay formore » detection of expansin activity is also provided.« less

Reciprocal Interactions between Cadmium-Induced Cell Wall Responses and Oxidative Stress in Plants

PubMed Central

Loix, Christophe; Huybrechts, Michiel; Vangronsveld, Jaco; Gielen, Marijke; Keunen, Els; Cuypers, Ann

2017-01-01

Cadmium (Cd) pollution renders many soils across the world unsuited or unsafe for food- or feed-orientated agriculture. The main mechanism of Cd phytotoxicity is the induction of oxidative stress, amongst others through the depletion of glutathione. Oxidative stress can damage lipids, proteins, and nucleic acids, leading to growth inhibition or even cell death. The plant cell has a variety of tools to defend itself against Cd stress. First and foremost, cell walls might prevent Cd from entering and damaging the protoplast. Both the primary and secondary cell wall have an array of defensive mechanisms that can be adapted to cope with Cd. Pectin, which contains most of the negative charges within the primary cell wall, can sequester Cd very effectively. In the secondary cell wall, lignification can serve to immobilize Cd and create a tougher barrier for entry. Changes in cell wall composition are, however, dependent on nutrients and conversely might affect their uptake. Additionally, the role of ascorbate (AsA) as most important apoplastic antioxidant is of considerable interest, due to the fact that oxidative stress is a major mechanism underlying Cd toxicity, and that AsA biosynthesis shares several links with cell wall construction. In this review, modifications of the plant cell wall in response to Cd exposure are discussed. Focus lies on pectin in the primary cell wall, lignification in the secondary cell wall and the importance of AsA in the apoplast. Regarding lignification, we attempt to answer the question whether increased lignification is merely a consequence of Cd toxicity, or rather an elicited defense response. We propose a model for lignification as defense response, with a central role for hydrogen peroxide as substrate and signaling molecule. PMID:29163592

Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins.

PubMed

Peaucelle, Alexis; Louvet, Romain; Johansen, Jorunn N; Höfte, Herman; Laufs, Patrick; Pelloux, Jérome; Mouille, Grégory

2008-12-23

Plant organs are produced from meristems in a characteristic pattern. This pattern, referred to as phyllotaxis, is thought to be generated by local gradients of an information molecule, auxin. Some studies propose a key role for the mechanical properties of the cell walls in the control of organ outgrowth. A major cell-wall component is the linear alpha-1-4-linked D-GalAp pectic polysaccharide homogalacturonan (HG), which plays a key role in cell-to-cell cohesion. HG is deposited in the cell wall in a highly (70%-80%) methyl-esterified form and is subsequently de-methyl-esterified by pectin methyl-esterases (PME, EC 3.1.1.11). PME activity is itself regulated by endogenous PME inhibitor (PMEI) proteins. PME action modulates cell-wall-matrix properties and plays a role in the control of cell growth. Here, we show that the formation of flower primordia in the Arabidopsis shoot apical meristem is accompanied by the de-methyl-esterification of pectic polysaccharides in the cell walls. In addition, experimental perturbation of the methyl-esterification status of pectins within the meristem dramatically alters the phyllotactic pattern. These results demonstrate that regulated de-methyl-esterification of pectins is a key event in the outgrowth of primordia and possibly also in phyllotactic patterning.

Solution nuclear magnetic resonance analyses of the anticodon arms of proteinogenic and nonproteinogenic tRNA(Gly).

PubMed

Chang, Andrew T; Nikonowicz, Edward P

2012-05-01

Although the fate of most tRNA molecules in the cell is aminoacylation and delivery to the ribosome, some tRNAs are destined to fulfill other functional roles. In addition to their central role in translation, tRNA molecules participate in processes such as regulation of gene expression, bacterial cell wall biosynthesis, viral replication, antibiotic biosynthesis, and suppression of alternative splicing. In bacteria, glycyl-tRNA molecules with anticodon sequences GCC and UCC exhibit multiple extratranslational functions, including transcriptional regulation and cell wall biosynthesis. We have determined the high-resolution structures of three glycyl-tRNA anticodon arms with anticodon sequences GCC and UCC. Two of the tRNA molecules are proteinogenic (tRNA(Gly,GCC) and tRNA(Gly,UCC)), and the third is nonproteinogenic (np-tRNA(Gly,UCC)) and participates in cell wall biosynthesis. The UV-monitored thermal melting curves show that the anticodon arm of tRNA(Gly,UCC) with a loop-closing C-A(+) base pair melts at a temperature 10 °C lower than those of tRNA(Gly,GCC) and np-tRNA(Gly,UCC). U-A and C-G pairs close the loops of the latter two molecules and enhance stem stability. Mg(2+) stabilizes the tRNA(Gly,UCC) anticodon arm and reduces the T(m) differential. The structures of the three tRNA(Gly) anticodon arms exhibit small differences among one another, but none of them form the classical U-turn motif. The anticodon loop of tRNA(Gly,GCC) becomes more dynamic and disordered in the presence of multivalent cations, whereas metal ion coordination in the anticodon loops of tRNA(Gly,UCC) and np-tRNA(Gly,UCC) establishes conformational homogeneity. The conformational similarity of the molecules is greater than their functional differences might suggest. Because aminoacylation of full-length tRNA molecules is accomplished by one tRNA synthetase, the similar structural context of the loop may facilitate efficient recognition of each of the anticodon sequences.

ATP-binding cassette transporter 1 participates in LDL oxidation by artery wall cells.

PubMed

Reddy, Srinivasa T; Hama, Susan; Ng, Carey; Grijalva, Victor; Navab, Mohamad; Fogelman, Alan M

2002-11-01

We have previously reported that products of the lipoxygenase pathway, hydroperoxyoctadecadienoic acid and hydroperoxyeicosatetraenoic acid, as well as cholesterol linoleate hydroperoxides, collectively termed seeding molecules, are removed by apolipoprotein A-I (apoA-I) from the artery wall cells and render low density lipoprotein (LDL) resistant to oxidation by human artery wall cells. The mechanisms by which oxidized lipids are transported and/or transferred to lipoproteins and the pathways by which apoA-I facilitates their removal remain unclear. ATP-binding cassette transporter 1 (ABCA1) is known to facilitate the release of cellular phospholipids and cholesterol from the plasma membrane to apoA-I and high density lipoprotein. Therefore, we evaluated whether ABCA1 participates in LDL oxidation. In this report, we show that (1) chemical inhibitors of ABCA1 function, glyburide and DIDS, block artery wall cell-mediated oxidative modification of LDL, (2) inhibition of ABCA1 with the use of antisense (but not sense) oligonucleotides prevents LDL-induced lipid hydroperoxide formation and LDL-induced monocyte chemotactic activity by the artery wall cells, and (3) oxysterols that induce ABCA1 expression, such as 22(R)hydroxycholesterol, enhance cell-mediated LDL oxidation. Furthermore, we also show that 22(R)hydroxycholesterol induces the production of reactive oxygen species in the artery wall cells, which can be removed by incubating the artery wall cells with apoA-I. Our data suggest that ABCA1 plays an important role in artery wall cell-mediated modification/oxidation of LDL by modulating the release of reactive oxygen species from artery wall cells that are necessary for LDL oxidation.

Scanning Electron Microscope Examination of Cotton Linters and Wood Pulp Fibers before and after Nitration and Gun Propellant Manufacture

DTIC Science & Technology

1983-03-01

both types of cellulose , the cell walls are still intact with the microfibrils showing little damage. The cellulose microfibrils consist of long chains...25 2. Model of Cellulose Microfibril ... S............. .............. .26 3. Model of Plant Cell Wall Bonding of Microfibril Bundles...molecules protrude above and below the plane of the cellulose ribbon.J’ (See Figures 2 and 3,) Bundles of these cellulose ribbons are called microfibrils

Metabolic labelling of the carbohydrate core in bacterial peptidoglycan and its applications

PubMed Central

Liang, Hai; DeMeester, Kristen E.; Hou, Ching-Wen; Parent, Michelle A.; Caplan, Jeffrey L.; Grimes, Catherine L.

2017-01-01

Bacterial cells are surrounded by a polymer known as peptidoglycan (PG), which protects the cell from changes in osmotic pressure and small molecule insults. A component of this material, N-acetyl-muramic acid (NAM), serves as a core structural element for innate immune recognition of PG fragments. We report the synthesis of modifiable NAM carbohydrate derivatives and the installation of these building blocks into the backbone of Gram-positive and Gram-negative bacterial PG utilizing metabolic cell wall recycling and biosynthetic machineries. Whole cells are labelled via click chemistry and visualized using super-resolution microscopy, revealing higher resolution PG structural details and allowing the cell wall biosynthesis, as well as its destruction in immune cells, to be tracked. This study will assist in the future identification of mechanisms that the immune system uses to recognize bacteria, glean information about fundamental cell wall architecture and aid in the design of novel antibiotics. PMID:28425464

Roles of Cells from the Arterial Vessel Wall in Atherosclerosis.

PubMed

Wang, Di; Wang, Zhiyan; Zhang, Lili; Wang, Yi

2017-01-01

Atherosclerosis has been identified as a chronic inflammatory disease of the arterial vessel wall. Accumulating evidence indicates that different cells from the tunica intima, media, adventitia, and perivascular adipose tissue not only comprise the intact and normal arterial vessel wall but also participate all in the inflammatory response of atherosclerosis via multiple intricate pathways. For instance, endothelial dysfunction has historically been considered to be the initiator of the development of atherosclerosis. The migration and proliferation of smooth muscle cells also play a pivotal role in the progression of atherosclerosis. Additionally, the fibroblasts from the adventitia and adipocytes from perivascular adipose tissue have received considerable attention given their special functions that contribute to atherosclerosis. In addition, numerous types of cytokines produced by different cells from the arterial vessel wall, including endothelium-derived relaxing factors, endothelium-derived contracting factors, tumor necrosis factors, interleukin, adhesion molecules, interferon, and adventitium-derived relaxing factors, have been implicated in atherosclerosis. Herein, we summarize the possible roles of different cells from the entire arterial vessel wall in the pathogenesis of atherosclerosis.

The role of endoxyloglucan transferase in the organization of plant cell walls.

PubMed

Nishitani, K

1997-01-01

The plant cell wall plays a central role in morphogenesis as well as responsiveness to environmental signals. Xyloglucans are the principal component of the plant cell wall matrix and serve as cross-links between cellulose microfibrils to form the cellulose-xyloglucan framework. Endoxyloglucan transferase (EXGT), which was isolated and characterized in 1992, is an enzyme that mediates molecular grafting reaction between xyloglucan molecules. Structural studies on cDNAs encoding EXGT and its related proteins have disclosed the ubiquitous presence in the plant kingdom of a large multigene family of xyloglucan-related proteins (XRPs). Each XRP functions as either hydrolase or transferase acting on xyloglucans and is considered to be responsible for rearrangement of the cellulose-xyloglucan framework, the processes essential for the construction, modification, and degradation of plant cell walls. Different XRP genes exhibit potentially different expression profiles with respect to tissue specificity and responsiveness to hormonal and mechanical signals. The molecular approach to individual XRP genes will open a new path for exploring the controlling mechanisms by which the plant cell wall is constructed and reformed during plant growth and development.

Biochemical and Immunocytological Characterizations of Arabidopsis Pollen Tube Cell Wall1[C][W][OA

PubMed Central

Dardelle, Flavien; Lehner, Arnaud; Ramdani, Yasmina; Bardor, Muriel; Lerouge, Patrice; Driouich, Azeddine; Mollet, Jean-Claude

2010-01-01

During plant sexual reproduction, pollen germination and tube growth require development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis (Arabidopsis thaliana) pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified homogalacturonan and arabinogalactan protein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1→5)-α-l-arabinan, the side chains of rhamnogalacturonan I. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono O-acetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that the Arabidopsis pollen tube wall has its own characteristics compared with other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics. PMID:20547702

Biochemical and immunocytological characterizations of Arabidopsis pollen tube cell wall.

PubMed

Dardelle, Flavien; Lehner, Arnaud; Ramdani, Yasmina; Bardor, Muriel; Lerouge, Patrice; Driouich, Azeddine; Mollet, Jean-Claude

2010-08-01

During plant sexual reproduction, pollen germination and tube growth require development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis (Arabidopsis thaliana) pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified homogalacturonan and arabinogalactan protein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1-->5)-alpha-L-arabinan, the side chains of rhamnogalacturonan I. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono O-acetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that the Arabidopsis pollen tube wall has its own characteristics compared with other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics.

Chemical Organization of the Cell Wall Polysaccharide Core of Malassezia restricta

PubMed Central

Stalhberger, Thomas; Simenel, Catherine; Clavaud, Cécile; Eijsink, Vincent G. H.; Jourdain, Roland; Delepierre, Muriel; Latgé, Jean-Paul; Breton, Lionel; Fontaine, Thierry

2014-01-01

Malassezia species are ubiquitous residents of human skin and are associated with several diseases such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. Malassezia restricta is one of the most predominant yeasts of the healthy human skin, its cell wall has been investigated in this paper. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharide core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% β-(1,3)-glucan, and 70% β-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant, and β-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is β-(1,6)-glucans, which are large molecules composed of a linear β-(1,6)-glucan chains with β-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both β-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here show that M. restricta has a polysaccharide organization very different of all fungal species analyzed to date. PMID:24627479

Chemical organization of the cell wall polysaccharide core of Malassezia restricta.

PubMed

Stalhberger, Thomas; Simenel, Catherine; Clavaud, Cécile; Eijsink, Vincent G H; Jourdain, Roland; Delepierre, Muriel; Latgé, Jean-Paul; Breton, Lionel; Fontaine, Thierry

2014-05-02

Malassezia species are ubiquitous residents of human skin and are associated with several diseases such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. Malassezia restricta is one of the most predominant yeasts of the healthy human skin, its cell wall has been investigated in this paper. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharide core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% β-(1,3)-glucan, and 70% β-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant, and β-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is β-(1,6)-glucans, which are large molecules composed of a linear β-(1,6)-glucan chains with β-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both β-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here show that M. restricta has a polysaccharide organization very different of all fungal species analyzed to date.

N-Acyl-Homoserine Lactone Primes Plants for Cell Wall Reinforcement and Induces Resistance to Bacterial Pathogens via the Salicylic Acid/Oxylipin Pathway[C][W][OPEN

PubMed Central

Schenk, Sebastian T.; Hernández-Reyes, Casandra; Samans, Birgit; Stein, Elke; Neumann, Christina; Schikora, Marek; Reichelt, Michael; Mithöfer, Axel; Becker, Annette; Kogel, Karl-Heinz; Schikora, Adam

2014-01-01

The ability of plants to monitor their surroundings, for instance the perception of bacteria, is of crucial importance. The perception of microorganism-derived molecules and their effector proteins is the best understood of these monitoring processes. In addition, plants perceive bacterial quorum sensing (QS) molecules used for cell-to-cell communication between bacteria. Here, we propose a mechanism for how N-acyl-homoserine lactones (AHLs), a group of QS molecules, influence host defense and fortify resistance in Arabidopsis thaliana against bacterial pathogens. N-3-oxo-tetradecanoyl-l-homoserine lactone (oxo-C14-HSL) primed plants for enhanced callose deposition, accumulation of phenolic compounds, and lignification of cell walls. Moreover, increased levels of oxylipins and salicylic acid favored closure of stomata in response to Pseudomonas syringae infection. The AHL-induced resistance seems to differ from the systemic acquired and the induced systemic resistances, providing new insight into inter-kingdom communication. Consistent with the observation that short-chain AHLs, unlike oxo-C14-HSL, promote plant growth, treatments with C6-HSL, oxo-C10-HSL, or oxo-C14-HSL resulted in different transcriptional profiles in Arabidopsis. Understanding the priming induced by bacterial QS molecules augments our knowledge of plant reactions to bacteria and suggests strategies for using beneficial bacteria in plant protection. PMID:24963057

Notch signalling coordinates tissue growth and wing fate specification in Drosophila.

PubMed

Rafel, Neus; Milán, Marco

2008-12-01

During the development of a given organ, tissue growth and fate specification are simultaneously controlled by the activity of a discrete number of signalling molecules. Here, we report that these two processes are extraordinarily coordinated in the Drosophila wing primordium, which extensively proliferates during larval development to give rise to the dorsal thoracic body wall and the adult wing. The developmental decision between wing and body wall is defined by the opposing activities of two secreted signalling molecules, Wingless and the EGF receptor ligand Vein. Notch signalling is involved in the determination of a variety of cell fates, including growth and cell survival. We present evidence that growth of the wing primordium mediated by the activity of Notch is required for wing fate specification. Our data indicate that tissue size modulates the activity range of the signalling molecules Wingless and Vein. These results highlight a crucial role of Notch in linking proliferation and fate specification in the developing wing primordium.

Decreasing methylation of pectin caused by nitric oxide leads to higher aluminium binding in cell walls and greater aluminium sensitivity of wheat roots

PubMed Central

Sun, Chengliang; Lu, Lingli; Yu, Yan; Liu, Lijuan; Hu, Yan; Ye, Yiquan; Jin, Chongwei; Lin, Xianyong

2016-01-01

Nitric oxide (NO) is an important bioactive molecule involved in cell wall metabolism, which has been recognized as a major target of aluminium (Al) toxicity. We have investigated the effects of Al-induced NO production on cell wall composition and the subsequent Al-binding capacity in roots of an Al-sensitive cultivar of wheat (Triticum aestivum L. cv. Yang-5). We found that Al exposure induced NO accumulation in the root tips. Eliminating NO production with an NO scavenger (cPTIO) significantly alleviated the Al-induced inhibition of root growth and thus reduced Al accumulation. Elimination of NO, however, did not significantly affect malate efflux or rhizosphere pH changes under Al exposure. Levels of cell wall polysaccharides (pectin, hemicelluloses 1, and hemicelluloses 2) and pectin methylesterase activity, as well as pectin demethylation in the root apex, significantly increased under Al treatment. Exogenous cPTIO application significantly decreased pectin methylesterase activity and increased the degree of methylation of pectin in the root cell wall, thus decreasing the Al-binding capacity of pectin. These results suggest that the Al-induced enhanced production of NO decreases cell wall pectin methylation, thus increasing the Al-binding capacity of pectin and negatively regulating Al tolerance in wheat. PMID:26663393

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins

PubMed Central

Ghamrawi, Sarah; Gastebois, Amandine; Zykwinska, Agata; Vandeputte, Patrick; Marot, Agnès; Mabilleau, Guillaume; Cuenot, Stéphane; Bouchara, Jean-Philippe

2015-01-01

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence. PMID:26038837

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins.

PubMed

Ghamrawi, Sarah; Gastebois, Amandine; Zykwinska, Agata; Vandeputte, Patrick; Marot, Agnès; Mabilleau, Guillaume; Cuenot, Stéphane; Bouchara, Jean-Philippe

2015-01-01

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.

Uncovering plant-pathogen crosstalk through apoplastic proteomic studies.

PubMed

Delaunois, Bertrand; Jeandet, Philippe; Clément, Christophe; Baillieul, Fabienne; Dorey, Stéphan; Cordelier, Sylvain

2014-01-01

Plant pathogens have evolved by developing different strategies to infect their host, which in turn have elaborated immune responses to counter the pathogen invasion. The apoplast, including the cell wall and extracellular space outside the plasma membrane, is one of the first compartments where pathogen-host interaction occurs. The plant cell wall is composed of a complex network of polysaccharides polymers and glycoproteins and serves as a natural physical barrier against pathogen invasion. The apoplastic fluid, circulating through the cell wall and intercellular spaces, provides a means for delivering molecules and facilitating intercellular communications. Some plant-pathogen interactions lead to plant cell wall degradation allowing pathogens to penetrate into the cells. In turn, the plant immune system recognizes microbial- or damage-associated molecular patterns (MAMPs or DAMPs) and initiates a set of basal immune responses, including the strengthening of the plant cell wall. The establishment of defense requires the regulation of a wide variety of proteins that are involved at different levels, from receptor perception of the pathogen via signaling mechanisms to the strengthening of the cell wall or degradation of the pathogen itself. A fine regulation of apoplastic proteins is therefore essential for rapid and effective pathogen perception and for maintaining cell wall integrity. This review aims to provide insight into analyses using proteomic approaches of the apoplast to highlight the modulation of the apoplastic protein patterns during pathogen infection and to unravel the key players involved in plant-pathogen interaction.

From microgravity to osmotic conditions: mechanical integration of plant cells in response to stress

NASA Astrophysics Data System (ADS)

Wojtaszek, Przemyslaw; Kasprowicz, Anna; Michalak, Michal; Janczara, Renata; Volkmann, Dieter; Baluska, Frantisek

Chemical reactions and interactions between molecules are commonly thought of as being at the basis of Life. Research of recent years, however, is more and more evidently indicating that physical forces are profoundly affecting the functioning of life at all levels of its organiza-tion. To detect and to respond to such forces, plant cells need to be integrated mechanically. Cell walls are the outermost functional zone of plant cells. They surround the individual cells, and also form a part of the apoplast. In cell suspensions, cell walls are embedded in the cul-ture medium which can be considered as a superapoplast. Through physical and chemical interactions they provide a basis for the structural and functional cell wall-plasma membrane-cytoskeleton (WMC) continuum spanning the whole cell. Here, the working of WMC contin-uum, and the participation of signalling molecules, like NO, would be presented in the context of plant responses to stress. In addition, the effects of the changing composition of WMC continuum will be considered, with particular attention paid to the modifications of the WMC components. Plant cells are normally adapted to changing osmotic conditions, resulting from variable wa-ter availability. The appearance of the osmotic stress activates adaptory mechanisms. If the strength of osmotic stress grows relatively slowly over longer period of time, the cells are able to adapt to conditions that are lethal to non-adapted cells. During stepwise adaptation of tobacco BY-2 suspension cells to the presence of various osmotically active agents, cells diverged into independent, osmoticum type-specific lines. In response to ionic agents (NaCl, KCl), the adhe-sive properties were increased and randomly dividing cells formed clumps, while cells adapted to nonionic osmotica (mannitol, sorbitol, PEG) revealed ordered pattern of precisely positioned cell divisions, resulting in the formation of long cell files. Changes in the growth patterns were accompanied by the alterations in the composition of wall proteins and polysaccharides. With respect to the cytoskeleton, in cells exposed to short-term osmotic stress significant rearrange-ments were observed. Surprisingly, the analyses of microfilaments and microtubules in adapted and in non-adapted, normal BY-2 cells, revealed no significant changes. It seems that upon prolonged exposure to osmotic stress conditions selective and adaptive alterations in wall com-position were occurring. Walls of cells grown in the presence of ionic agents were homogenous, while longitudinal walls and cross-walls in cells adapted to nonionic agents were significantly different. This might affect the anchorage of the cytoskeleton in the walls and modify the func-tioning of the whole WMC continuum. In this way, cell's mechanical balance restoration will be ensured and, in consequence, cells will be able to resist osmotic pressure and divide under severe stress conditions. In plants, cross-walls within cell files of axial organs exhibit specific properties that allow them to act as domains of contact and intense intercellular communica-tion, and the sites of the anchorage of cytoskeleton. As a further consequence, also cell-to-cell interactions would be affected. MM and RJ are students of biotechnology at Adam Mickiewicz University. The data coming from the authors' lab come from research supported by the DAAD scholarship to AK, and Alexander von Humboldt Research Fellowship and Polish Ministry of Science and Higher Edu-cation grants PBZ-KBN-110/P04/2004, N N303 294434, N N301 164435, and N N303 360735 to PW.

Imaging fluorescence detected linear dichroism of plant cell walls in laser scanning confocal microscope.

PubMed

Steinbach, Gábor; Pomozi, István; Zsiros, Ottó; Páy, Anikó; Horváth, Gábor V; Garab, Gyozo

2008-03-01

Anisotropy carries important information on the molecular organization of biological samples. Its determination requires a combination of microscopy and polarization spectroscopy tools. The authors constructed differential polarization (DP) attachments to a laser scanning microscope in order to determine physical quantities related to the anisotropic distribution of molecules in microscopic samples; here the authors focus on fluorescence-detected linear dichroism (FDLD). By modulating the linear polarization of the laser beam between two orthogonally polarized states and by using a demodulation circuit, the authors determine the associated transmitted and fluorescence intensity-difference signals, which serve the basis for LD (linear dichroism) and FDLD, respectively. The authors demonstrate on sections of Convallaria majalis root tissue stained with Acridin Orange that while (nonconfocal) LD images remain smeared and weak, FDLD images recorded in confocal mode reveal strong anisotropy of the cell wall. FDLD imaging is suitable for mapping the anisotropic distribution of transition dipoles in 3 dimensions. A mathematical model is proposed to account for the fiber-laminate ultrastructure of the cell wall and for the intercalation of the dye molecules in complex, highly anisotropic architecture. Copyright 2007 International Society for Analytical Cytology.

Method for delivery of small molecules and proteins across the cell wall of algae using molecular transporters

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

Geihe, Erika; Trantow, Brian; Wender, Paul

The introduction of tools to study, control or expand the inner-workings of algae has been slow to develop. Provided are embodiments of a molecular method based on guanidinium-rich molecular transporters (GR-MoTrs) for bringing molecular cargos into algal cells. The methods of the disclosure have been shown to work in wild-type algae that have an intact cell wall. Developed using Chlamydomonas reinhardtii, this method is also successful with less studied algae, including Neochloris oleoabundans and Scenedesmus dimorphus, thus providing a new and versatile tool for algal research and modification. The method of delivering a cargo compound to an algal cell comprisesmore » contacting an algal cell with a guanidinium-rich delivery vehicle comprising a guanidinium-rich molecular transporter (GR-MoTr) linked to a cargo compound desired to be delivered to the algal cell, whereby the guanidinium-rich molecular transporter can traverse the algal cell wall, thereby delivering the cargo compound to the algal cell.« less

Understanding Microbial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2017-10-01

pathogens and commensals. However, the technology available to track these molecules in host cells and tissues remains primitive. To address this...live, luminal bacteria into specific host intestinal immune cells and their subsequent degradation in host phagocytes. Notably, this approach also...click-chemistry, bacterial cell wall, bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide, inflammatory

Understanding Microbial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2017-10-01

both pathogens and commensals. However, the technology available to track these molecules in host cells and tissues remains primitive. To address this...from live, luminal bacteria into specific host intestinal immune cells and their subsequent degradation in host phagocytes. Notably, this approach...Bioorthogonal click-chemistry, bacterial cell wall, bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide

Plant cell wall sugars: sweeteners for a bio-based economy.

PubMed

Van de Wouwer, Dorien; Boerjan, Wout; Vanholme, Bartel

2018-02-12

Global warming and the consequent climate change is one of the major environmental challenges we are facing today. The driving force behind the rise in temperature is our fossil-based economy, which releases massive amounts of the greenhouse gas carbon dioxide into the atmosphere. In order to reduce greenhouse gas emission, we need to scale down our dependency on fossil resources, implying that we need other sources for energy and chemicals to feed our economy. Here, plants have an important role to play; by means of photosynthesis, plants capture solar energy to split water and fix carbon derived from atmospheric carbon dioxide. A significant fraction of the fixed carbon ends up as polysaccharides in the plant cell wall. Fermentable sugars derived from cell wall polysaccharides form an ideal carbon source for the production of bio-platform molecules. However, a major limiting factor in the use of plant biomass as feedstock for the bio-based economy is the complexity of the plant cell wall and its recalcitrance towards deconstruction. To facilitate the release of fermentable sugars during downstream biomass processing, the composition and structure of the cell wall can be engineered. Different strategies to reduce cell wall recalcitrance will be described in this review. The ultimate goal is to obtain a tailor-made biomass, derived from plants with a cell wall optimized for particular industrial or agricultural applications, without affecting plant growth and development. This article is protected by copyright. All rights reserved.

Peptidoglycan recognition proteins in Drosophila immunity.

PubMed

Kurata, Shoichiro

2014-01-01

Innate immunity is the front line of self-defense against infectious non-self in vertebrates and invertebrates. The innate immune system is mediated by germ-line encoding pattern recognition molecules (pathogen sensors) that recognize conserved molecular patterns present in the pathogens but absent in the host. Peptidoglycans (PGN) are essential cell wall components of almost all bacteria, except mycoplasma lacking a cell wall, which provides the host immune system an advantage for detecting invading bacteria. Several families of pattern recognition molecules that detect PGN and PGN-derived compounds have been indentified, and the role of PGRP family members in host defense is relatively well-characterized in Drosophila. This review focuses on the role of PGRP family members in the recognition of invading bacteria and the activation and modulation of immune responses in Drosophila. Copyright © 2013 Elsevier Ltd. All rights reserved.

Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

PubMed Central

Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; Edwards, Thayne L.; James, Conrad D.; Lidke, Keith A.

2016-01-01

We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet. PMID:27375939

LipidII: Just Another Brick in the Wall?

PubMed Central

Scheffers, Dirk-Jan; Tol, Menno B.

2015-01-01

Nearly all bacteria contain a peptidoglycan cell wall. The peptidoglycan precursor molecule is LipidII, containing the basic peptidoglycan building block attached to a lipid. Although the suitability of LipidII as an antibacterial target has long been recognized, progress on elucidating the role(s) of LipidII in bacterial cell biology has been slow. The focus of this review is on exciting new developments, both with respect to antibacterials targeting LipidII as well as the emerging role of LipidII in organizing the membrane and cell wall synthesis. It appears that on both sides of the membrane, LipidII plays crucial roles in organizing cytoskeletal proteins and peptidoglycan synthesis machineries. Finally, the recent discovery of no less than three different categories of LipidII flippases will be discussed. PMID:26679002

Differential metabolism of pectic galactan in tomato and strawberry fruit: detection of the LM26 branched galactan epitope in ripe strawberry fruit.

PubMed

Posé, Sara; Marcus, Susan E; Paul Knox, J

2018-04-24

Antibody-based approaches have been used to study cell wall architecture and modifications during the ripening process of two important fleshy fruit crops: tomato and strawberry. Cell wall polymers in both unripe and ripe fruits have been sequentially solubilized and fractions analysed with sets of monoclonal antibodies focusing on the pectic polysaccharides. We demonstrate the specific detection of the LM26 branched galactan epitope, associated with rhamnogalacturonan-I, in cell walls of ripe strawberry fruit. Analytical approaches confirm that the LM26 epitope is linked to sets of rhamnogalacturonan-I and homogalacturonan molecules. The cellulase-degradation of cellulose-rich residues that releases cell wall polymers intimately linked with cellulose microfibrils has been used to explore aspects of branched galactan occurrence and galactan metabolism. In situ analyses of ripe strawberry fruits indicate that the LM26 epitope is present in all primary cell walls and also particularly abundant in vascular tissues. The significance of the occurrence of branched galactan structures in the side chains of rhamnogalacturonan-I pectins in the context of ripening strawberry fruit is discussed. This article is protected by copyright. All rights reserved.

Detection of wood cell wall porosity using small carbohydrate molecules and confocal fluorescence microscopy.

PubMed

Donaldson, L A; Kroese, H W; Hill, S J; Franich, R A

2015-09-01

A novel approach to nanoscale detection of cell wall porosity using confocal fluorescence microscopy is described. Infiltration of cell walls with a range of nitrophenyl-substituted carbohydrates of different molecular weights was assessed by measuring changes in the intensity of lignin fluorescence, in response to the quenching effect of the 4-nitrophenyl group. The following carbohydrates were used in order of increasing molecular weight; 4-nitrophenyl β-D-glucopyrano-side (monosaccharide), 4-nitrophenyl β-D-lactopyranoside (disaccharide), 2-chloro-4-nitrophenyl β-D-maltotrioside (trisaccharide), and 4-nitrophenyl α-D-maltopentaoside (pentasaccharide). This technique was used to compare cell wall porosity in wood which had been dewatered to 40% moisture content using supercritical CO2, where cell walls remain fully hydrated, with kiln dried wood equilibrated to 12% moisture content. Infiltration of cell walls as measured by fluorescence quenching, was found to decrease with increasing molecular weight, with the pentasaccharide being significantly excluded compared to the monosaccharide. Porosity experiments were performed on blocks and sections to assess differences in cell wall accessibility. Dewatered and kiln dried wood infiltrated as blocks showed similar results, but greater infiltration was achieved by using sections, indicating that not all pores were easily accessible by infiltration from the lumen surface. In wood blocks infiltrated with 4-nitrophenyl α-D-maltopentaoside, quenching of the secondary wall was quite variable, especially in kiln dried wood, indicating limited connectivity of pores accessible from the lumen surface. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

From nano- to micrometer scale: the role of microwave-assisted acid and alkali pretreatments in the sugarcane biomass structure.

PubMed

Isaac, Augusta; de Paula, Jéssica; Viana, Carlos Martins; Henriques, Andréia Bicalho; Malachias, Angelo; Montoro, Luciano A

2018-01-01

To date, great strides have been made in elucidating the role of thermochemical pretreatments in the chemical and structural features of plant cell walls; however, there is no clear picture of the plant recalcitrance and its relationship to deconstruction. Previous studies precluded full answers due to the challenge of multiscale features of plant cell wall organization. Complementing the previous efforts, we undertook a systematic, multiscale, and integrated approach to track the effect of microwave-assisted H 2 SO 4 and NaOH treatments on the hierarchical structure of plants, i.e., from a nano- to micrometer scale. We focused on the investigation of the highly recalcitrant sclerenchyma cell walls from sugarcane bagasse. Through atomic force microscopy and X-ray diffraction analyses, remarkable details of the assembly of cellulose microfibrils not previously seen were revealed. Following the H 2 SO 4 treatment, we observed that cellulose microfibrils were almost double the width of the alkali pretreated sample at the temperature of 160 °C. Such enlargement led to a greater contact between cellulose chains, with a subsequent molecule alignment, as indicated by the X-ray diffraction (XRD) results with the conspicuous expansion of the average crystallite size. The delignification process had little effect on the local nanometer-sized arrangement of cellulose molecules. However, the rigidity and parallel alignment of cellulose microfibrils were partially degraded. The XRD analysis also agrees with these findings as evidenced by large momentum transfer vectors ( q  > 20 nm -1 ), interpreted as indicators of the long-range order of cell wall components, which were similar for all the studied samples except with application of the NaOH treatment at 160 °C. These changes were followed by the eventual swelling of the fiber cell walls. Based on an integrated approach, we presented multidimensional architectural models of cell wall deconstruction resulting from microwave-assisted pretreatments. We provided direct evidence supporting the idea that hemicellulose is the main barrier for the swelling of cellulose microfibrils, whereas lignin adds rigidity to cell walls. Our findings shed light on the design of more efficient strategies, not only for the conversion of biomass to fuels but also for the production of nanocellulose, which has great potential for several applications such as composites, rheology modifiers, and pharmaceuticals.

Bacterial immunostat: Mycobacterium tuberculosis lipids and their role in the host immune response.

PubMed

Queiroz, Adriano; Riley, Lee W

2017-01-01

The lipid-rich cell wall of Mycobacterium tuberculosis is a dynamic structure that is involved in the regulation of the transport of nutrients, toxic host-cell effector molecules, and anti-tuberculosis drugs. It is therefore postulated to contribute to the long-term bacterial survival in an infected human host. Accumulating evidence suggests that M. tuberculosis remodels the lipid composition of the cell wall as an adaptive mechanism against host-imposed stress. Some of these lipid species (trehalose dimycolate, diacylated sulphoglycolipid, and mannan-based lipoglycans) trigger an immunopathologic response, whereas others (phthiocerol dimycocerosate, mycolic acids, sulpholipid-1, and di-and polyacyltrehalose) appear to dampen the immune responses. These lipids appear to be coordinately expressed in the cell wall of M. tuberculosis during different phases of infection, ultimately determining the clinical fate of the infection. This review summarizes the current state of knowledge on the metabolism, transport, and homeostatic or immunostatic regulation of the cell wall lipids, and their orchestrated interaction with host immune responses that results in bacterial clearance, persistence, or tuberculosis.

Cellulose-Derived Oligomers Act as Damage-Associated Molecular Patterns and Trigger Defense-Like Responses1

PubMed Central

Li, Shundai; Zipfel, Cyril

2017-01-01

The plant cell wall, often the site of initial encounters between plants and their microbial pathogens, is composed of a complex mixture of cellulose, hemicellulose, and pectin polysaccharides as well as proteins. The concept of damage-associated molecular patterns (DAMPs) was proposed to describe plant elicitors like oligogalacturonides (OGs), which can be derived by the breakdown of the pectin homogalacturon by pectinases. OGs act via many of the same signaling steps as pathogen- or microbe-associated molecular patterns (PAMPs) to elicit defenses and provide protection against pathogens. Given both the complexity of the plant cell wall and the fact that many pathogens secrete a wide range of cell wall-degrading enzymes, we reasoned that the breakdown products of other cell wall polymers may be similarly biologically active as elicitors and may help to reinforce the perception of danger by plant cells. Our results indicate that oligomers derived from cellulose are perceived as signal molecules in Arabidopsis (Arabidopsis thaliana), triggering a signaling cascade that shares some similarities to responses to well-known elicitors such as chitooligomers and OGs. However, in contrast to other known PAMPs/DAMPs, cellobiose stimulates neither detectable reactive oxygen species production nor callose deposition. Confirming our idea that both PAMPs and DAMPs are likely to cooccur at infection sites, cotreatments of cellobiose with flg22 or chitooligomers led to synergistic increases in gene expression. Thus, the perception of cellulose-derived oligomers may participate in cell wall integrity surveillance and represents an additional layer of signaling following plant cell wall breakdown during cell wall remodeling or pathogen attack. PMID:28242654

Use of yeast spores for microencapsulation of enzymes.

PubMed

Shi, Libing; Li, Zijie; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

2014-08-01

Here, we report a novel method to produce microencapsulated enzymes using Saccharomyces cerevisiae spores. In sporulating cells, soluble secreted proteins are transported to the spore wall. Previous work has shown that the spore wall is capable of retaining soluble proteins because its outer layers work as a diffusion barrier. Accordingly, a red fluorescent protein (RFP) fusion of the α-galactosidase, Mel1, expressed in spores was observed in the spore wall even after spores were subjected to a high-salt wash in the presence of detergent. In vegetative cells, however, the cell wall cannot retain the RFP fusion. Although the spore wall prevents diffusion of proteins, it is likely that smaller molecules, such as sugars, pass through it. In fact, spores can contain much higher α-galactosidase activity to digest melibiose than vegetative cells. When present in the spore wall, the enzyme acquires resistance to environmental stresses including enzymatic digestion and high temperatures. The outer layers of the spore wall are required to retain enzymes but also decrease accessibility of the substrates. However, mutants with mild spore wall defects can retain and stabilize the enzyme while still permitting access to the substrate. In addition to Mel1, we also show that spores can retain the invertase. Interestingly the encapsulated invertase has significantly lower activity toward raffinose than toward sucrose.This suggests that substrate selectivity could be altered by the encapsulation.

Peptidoglycan and lipoteichoic acid, components of the streptococcal cell wall, have marked and differential effects on adhesion molecule expression and the production of reactive oxygen species in human whole blood leukocytes.

PubMed

Saetre, T; Kähler, H; Foster, S J; Lyberg, T

2000-07-01

To elucidate the pathophysiology of infections with Streptococcus pyogenes we applied flow cytometric techniques to study dose-response and time-related effects of the streptococcal cell-wall-derived components lipoteichoic acid (LTA 0.005 to 50 microg/ml) and peptidoglycan (10 and 100 microg/ml) on the expression of leukocyte adhesion molecules, the CD14 receptor, and the production of leukocyte reactive oxygen species (ROS). LTA (50 microg/ml, 1-2 h) markedly increased the expression of CD11b (approximately 5-fold), CD11c (approximately 2-fold) and CD11a. Concomitantly, CD62L was downregulated (60%). Peptidoglycan alone or in combination with LTA had little effect on adhesion molecules, except for an amplification of the downregulation of CD62L to 90%. Monocyte CD14 expression was doubled by LTA. Leukocyte ROS production was 10-fold and 5-fold increased by peptidoglycan in granulocytes and monocytes, respectively. LTA alone had no effect, while the combination of peptidoglycan with LTA doubled the increase in ROS caused by peptidoglycan. LTA and peptidoglycan had marked and differential effects: LTA caused mainly adhesion molecule modulation, whereas peptidoglycan mainly increased ROS production. These changes are important in inflammatory cell activation and recruitment, intracellular microbial killing and adverse tissue injury.

In tobacco BY-2 cells xyloglucan oligosaccharides alter the expression of genes involved in cell wall metabolism, signalling, stress responses, cell division and transcriptional control.

PubMed

González-Pérez, Lien; Perrotta, Lara; Acosta, Alexis; Orellana, Esteban; Spadafora, Natasha; Bruno, Leonardo; Bitonti, Beatrice M; Albani, Diego; Cabrera, Juan Carlos; Francis, Dennis; Rogers, Hilary J

2014-10-01

Xyloglucan oligosaccharides (XGOs) are breakdown products of XGs, the most abundant hemicelluloses of the primary cell walls of non-Poalean species. Treatment of cell cultures or whole plants with XGOs results in accelerated cell elongation and cell division, changes in primary root growth, and a stimulation of defence responses. They may therefore act as signalling molecules regulating plant growth and development. Previous work suggests an interaction with auxins and effects on cell wall loosening, however their mode of action is not fully understood. The effect of an XGO extract from tamarind (Tamarindus indica) on global gene expression was therefore investigated in tobacco BY-2 cells using microarrays. Over 500 genes were differentially regulated with similar numbers and functional classes of genes up- and down-regulated, indicating a complex interaction with the cellular machinery. Up-regulation of a putative XG endotransglycosylase/hydrolase-related (XTH) gene supports the mechanism of XGO action through cell wall loosening. Differential expression of defence-related genes supports a role for XGOs as elicitors. Changes in the expression of genes related to mitotic control and differentiation also support previous work showing that XGOs are mitotic inducers. XGOs also affected expression of several receptor-like kinase genes and transcription factors. Hence, XGOs have significant effects on expression of genes related to cell wall metabolism, signalling, stress responses, cell division and transcriptional control.

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

DOE PAGES

Hasim, Sahar; Allison, David P.; Retterer, Scott T.; ...

2016-11-14

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is alteredmore » or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in this paper in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. Finally, AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.« less

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

PubMed Central

Hasim, Sahar; Allison, David P.; Retterer, Scott T.; Hopke, Alex; Wheeler, Robert T.; Doktycz, Mitchel J.

2016-01-01

ABSTRACT Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system. PMID:27849179

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

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

Hasim, Sahar; Allison, David P.; Retterer, Scott T.

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is alteredmore » or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in this paper in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. Finally, AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.« less

Simulated microgravity does not alter epithelial cell adhesion to matrix and other molecules

NASA Technical Reports Server (NTRS)

Jessup, J. M.; Brown, K.; Ishii, S.; Ford, R.; Goodwin, T. J.; Spaulding, G.

1994-01-01

Microgravity has advantages for the cultivation of tissues with high fidelity; however, tissue formation requires cellular recognition and adhesion. We tested the hypothesis that simulated microgravity does not affect cell adhesion. Human colorectal carcinoma cells were cultured in the NASA Rotating Wall Vessel (RWV) under low shear stress with randomization of the gravity vector that simulates microgravity. After 6 - 7 days, cells were assayed for binding to various substrates and compared to cells grown in standard tissue culture flasks and static suspension cultures. The RWV cultures bound as well to basement membrane proteins and to Carcinoembryonic Antigen (CEA), an intercellular adhesion molecule, as control cultures did. Thus, microgravity does not alter epithelial cell adhesion and may be useful for tissue engineering.

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

PubMed

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

2017-10-01

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

Reactive oxygen species, essential molecules, during plant-pathogen interactions.

PubMed

Camejo, Daymi; Guzmán-Cedeño, Ángel; Moreno, Alexander

2016-06-01

Reactive oxygen species (ROS) are continually generated as a consequence of the normal metabolism in aerobic organisms. Accumulation and release of ROS into cell take place in response to a wide variety of adverse environmental conditions including salt, temperature, cold stresses and pathogen attack, among others. In plants, peroxidases class III, NADPH oxidase (NOX) locates in cell wall and plasma membrane, respectively, may be mainly enzymatic systems involving ROS generation. It is well documented that ROS play a dual role into cells, acting as important signal transduction molecules and as toxic molecules with strong oxidant power, however some aspects related to its function during plant-pathogen interactions remain unclear. This review focuses on the principal enzymatic systems involving ROS generation addressing the role of ROS as signal molecules during plant-pathogen interactions. We described how the chloroplasts, mitochondria and peroxisomes perceive the external stimuli as pathogen invasion, and trigger resistance response using ROS as signal molecule. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Advanced biorefinery in lower termite-effect of combined pretreatment during the chewing process

PubMed Central

2012-01-01

Background Currently the major barrier in biomass utilization is the lack of an effective pretreatment of plant cell wall so that the carbohydrates can subsequently be hydrolyzed into sugars for fermentation into fuel or chemical molecules. Termites are highly effective in degrading lignocellulosics and thus can be used as model biological systems for studying plant cell wall degradation. Results We discovered a combination of specific structural and compositional modification of the lignin framework and partial degradation of carbohydrates that occurs in softwood with physical chewing by the termite, Coptotermes formosanus, which are critical for efficient cell wall digestion. Comparative studies on the termite-chewed and native (control) softwood tissues at the same size were conducted with the aid of advanced analytical techniques such as pyrolysis gas chromatography mass spectrometry, attenuated total reflectance Fourier transform infrared spectroscopy and thermogravimetry. The results strongly suggest a significant increase in the softwood cellulose enzymatic digestibility after termite chewing, accompanied with utilization of holocellulosic counterparts and an increase in the hydrolysable capacity of lignin collectively. In other words, the termite mechanical chewing process combines with specific biological pretreatment on the lignin counterpart in the plant cell wall, resulting in increased enzymatic cellulose digestibility in vitro. The specific lignin unlocking mechanism at this chewing stage comprises mainly of the cleavage of specific bonds from the lignin network and the modification and redistribution of functional groups in the resulting chewed plant tissue, which better expose the carbohydrate within the plant cell wall. Moreover, cleavage of the bond between the holocellulosic network and lignin molecule during the chewing process results in much better exposure of the biomass carbohydrate. Conclusion Collectively, these data indicate the participation of lignin-related enzyme(s) or polypeptide(s) and/or esterase(s), along with involvement of cellulases and hemicellulases in the chewing process of C. formosanus, resulting in an efficient pretreatment of biomass through a combination of mechanical and enzymatic processes. This pretreatment could be mimicked for industrial biomass conversion. PMID:22390274

The xyloglucan-cellulose assembly at the atomic scale.

PubMed

Hanus, Jaroslav; Mazeau, Karim

2006-05-01

The assembly of cell wall components, cellulose and xyloglucan (XG), was investigated at the atomistic scale using molecular dynamics simulations. A molecular model of a cellulose crystal corresponding to the allomorph Ibeta and exhibiting a flexible complex external morphology was employed to mimic the cellulose microfibril. The xyloglucan molecules considered were the three typical basic repeat units, differing only in the size of one of the lateral chain. All the investigated XG fragments adsorb nonspecifically onto cellulose fiber; multiple arrangements are equally probable, and every cellulose surface was capable of binding the short XG molecules. The following structural effects emerged: XG molecules that do not have any long side chains tended to adapt themselves nicely to the topology of the microfibril, forming a flat, outstretched conformation with all the sugar residues interacting with the surface. In contrast, the XG molecules, which have long side chains, were not able to adopt a flat conformation that would enable the interaction of all the XG residues with the surface. In addition to revealing the fundamental atomistic details of the XG adsorption on cellulose, the present calculations give a comprehensive understanding of the way the XG molecules can unsorb from cellulose to create a network that forms the cell wall. Our revisited view of the adsorption features of XG on cellulose microfibrils is consistent with experimental data, and a model of the network is proposed. Copyright (c) 2006 Wiley Periodicals, Inc.

Chitinases in Pneumocystis carinii pneumonia

PubMed Central

Villegas, Leah R.; Kottom, Theodore J.

2014-01-01

Pneumocystis pneumonia remains an important complication of immune suppression. The cell wall of Pneumocystis has been demonstrated to potently stimulate host inflammatory responses, with most studies focusing on β-glucan components of the Pneumocystis cell wall. In the current study, we have elaborated the potential role of chitins and chitinases in Pneumocystis pneumonia. We demonstrated differential host mammalian chitinase expression during Pneumocystis pneumonia. We further characterized a chitin synthase gene in Pneumocystis carinii termed Pcchs5, a gene with considerable homolog to the fungal chitin biosynthesis protein Chs5. We also observed the impact of chitinase digestion on Pneumocystis-induced host inflammatory responses by measuring TNFα release and mammalian chitinase expression by cultured lung epithelial and macrophage cells stimulated with Pneumocystis cell wall isolates in the presence and absence of exogenous chitinase digestion. These findings provide evidence supporting a chitin biosynthetic pathway in Pneumocystis organisms and that chitinases modulate inflammatory responses in lung cells. We further demonstrate lung expression of chitinase molecules during Pneumocystis pneumonia. PMID:22535444

The Hygroscopic Nature of Wood,

DTIC Science & Technology

The cell walls of wood are organized as a structural system involving filamentous microfibrils , oriented essentially in the direction of the...longitudinal axis embedded in an amorphous matrix of noncrystalline cellulose , hemicelluloses, and lignin. The molecules in the amorphous regions, primarily

Oligosaccharins.

ERIC Educational Resources Information Center

Albersheim, Peter; Darvill, Alan G.

1985-01-01

Related history and laboratory work which lead to isolation of oligosaccharins, a new class of regulatory molecules found in plant cell walls. These substances function in growth, development, reproduction, and defense. Mixtures of oligosaccharins and other hormones can stimulate growth of an undifferentiated callus, roots, vegetative…

Imaging the distribution of individual platinum-based anticancer drug molecules attached to single-wall carbon nanotubes

PubMed Central

Bhirde, Ashwin A; Sousa, Alioscka A; Patel, Vyomesh; Azari, Afrouz A; Gutkind, J Silvio; Leapman, Richard D; Rusling, James F

2009-01-01

Aims To image the distribution of drug molecules attached to single-wall carbon nanotubes (SWNTs). Materials & methods Herein we report the use of scanning transmission electron microscopy (STEM) for atomic scale visualization and quantitation of single platinum-based drug molecules attached to SWNTs designed for targeted drug delivery. Fourier transform infrared spectroscopy and energy-dispersive x-ray spectroscopy were used for characterization of the SWNT drug conjugates. Results Z-contrast STEM imaging enabled visualization of the first-line anticancer drug cisplatin on the nanotubes at single molecule level. The identity and presence of cisplatin on the nanotubes was confirmed using energy-dispersive x-ray spectroscopy and Fourier transform infrared spectroscopy. STEM tomography was also used to provide additional insights concerning the nanotube conjugates. Finally, our observations provide a rationale for exploring the use of SWNT bioconjugates to selectively target and kill squamous cancer cells. Conclusion Z-contrast STEM imaging provides a means for direct visualization of heavy metal containing molecules (i.e., cisplatin) attached to surfaces of carbon SWNTs along with distribution and quantitation. PMID:19839812

Enemy at the gates: traffic at the plant cell pathogen interface.

PubMed

Hoefle, Caroline; Hückelhoven, Ralph

2008-12-01

The plant apoplast constitutes a space for early recognition of potentially harmful non-self. Basal pathogen recognition operates via dynamic sensing of conserved microbial patterns by pattern recognition receptors or of elicitor-active molecules released from plant cell walls during infection. Recognition elicits defence reactions depending on cellular export via SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex-mediated vesicle fusion or plasma membrane transporter activity. Lipid rafts appear also involved in focusing immunity-associated proteins to the site of pathogen contact. Simultaneously, pathogen effectors target recognition, apoplastic host proteins and transport for cell wall-associated defence. This microreview highlights most recent reports on the arms race for plant disease and immunity at the cell surface.

Inhibition of intracellular bacterial replication in fibroblasts is dependent on the perforin-like protein (Perforin-2) encoded by macrophage expressed gene 1

PubMed Central

McCormack, Ryan; de Armas, Lesley R.; Shiratsuchi, Motoaki; Ramos, Jay; Podack, Eckhard R.

2013-01-01

Fibroblasts are known to eliminate intracellular bacteria, but the lethal hit of the bactericidal mechanism has not been defined. We show that primary embryonic and established fibroblasts can be induced by interferons or by intracellular bacterial infection to express a perforin-like mRNA previously described as macrophage expressed gene 1 (mpeg1). The presence and level of the perforin-like mRNA correlate with the ability of primary mouse embryonic fibroblasts (MEF) to eliminate intracellular bacteria. In addition, siRNA knock-down of the perforin-like molecule abolishes bactericidal activity and allows intracellular bacterial replication. Complementation of MEF in which the endogenous perforin-like molecule has been knocked down with an RFP-tagged version restores bactericidal activity. The perforin-like molecule has broad bactericidal specificity for pathogenic and non-pathogenic bacteria including Gram positive, Gram negative and acid fast bacteria. The perforin-like molecule renders previously lysozyme-resistant bacteria sensitive to lysis by lysozyme suggesting physical damage of the outer cell wall by the perforin-like protein. MEFs damage cell walls of intracellular bacteria by insertion, polymerization and pore-formation of the perforin-like protein, analogous to pore-formers of complement and Perforin-1 of cytolytic lymphocytes. We propose the name Perforin-2. PMID:23257510

Evidence for land plant cell wall biosynthetic mechanisms in charophyte green algae

PubMed Central

Mikkelsen, Maria D.; Harholt, Jesper; Ulvskov, Peter; Johansen, Ida E.; Fangel, Jonatan U.; Doblin, Monika S.; Bacic, Antony; Willats, William G. T.

2014-01-01

Background and Aims The charophyte green algae (CGA) are thought to be the closest living relatives to the land plants, and ancestral CGA were unique in giving rise to the land plant lineage. The cell wall has been suggested to be a defining structure that enabled the green algal ancestor to colonize land. These cell walls provide support and protection, are a source of signalling molecules, and provide developmental cues for cell differentiation and elongation. The cell wall of land plants is a highly complex fibre composite, characterized by cellulose cross-linked by non-cellulosic polysaccharides, such as xyloglucan, embedded in a matrix of pectic polysaccharides. How the land plant cell wall evolved is currently unknown: early-divergent chlorophyte and prasinophyte algae genomes contain a low number of glycosyl transferases (GTs), while land plants contain hundreds. The number of GTs in CGA is currently unknown, as no genomes are available, so this study sought to give insight into the evolution of the biosynthetic machinery of CGA through an analysis of available transcriptomes. Methods Available CGA transcriptomes were mined for cell wall biosynthesis GTs and compared with GTs characterized in land plants. In addition, gene cloning was employed in two cases to answer important evolutionary questions. Key Results Genetic evidence was obtained indicating that many of the most important core cell wall polysaccharides have their evolutionary origins in the CGA, including cellulose, mannan, xyloglucan, xylan and pectin, as well as arabino-galactan protein. Moreover, two putative cellulose synthase-like D family genes (CSLDs) from the CGA species Coleochaete orbicularis and a fragment of a putative CSLA/K-like sequence from a CGA Spirogyra species were cloned, providing the first evidence that all the cellulose synthase/-like genes present in early-divergent land plants were already present in CGA. Conclusions The results provide new insights into the evolution of cell walls and support the notion that the CGA were pre-adapted to life on land by virtue of the their cell wall biosynthetic capacity. These findings are highly significant for understanding plant cell wall evolution as they imply that some features of land plant cell walls evolved prior to the transition to land, rather than having evolved as a result of selection pressures inherent in this transition. PMID:25204387

The solvation study of carbon, silicon and their mixed nanotubes in water solution.

PubMed

Hashemi Haeri, Haleh; Ketabi, Sepideh; Hashemianzadeh, Seyed Majid

2012-07-01

Nanotubes are believed to open the road toward different modern fields, either technological or biological. However, the applications of nanotubes have been badly impeded for the poor solubility in water which is especially essential for studies in the presence of living cells. Therefore, water soluble samples are in demand. Herein, the outcomes of Monte Carlo simulations of different sets of multiwall nanotubes immersed in water are reported. A number of multi wall nanotube samples, comprised of pure carbon, pure silicon and several mixtures of carbon and silicon are the subjects of study. The simulations are carried out in an (N,V,T) ensemble. The purpose of this report is to look at the effects of nanotube size (diameter) and nanotube type (pure carbon, pure silicon or a mixture of carbon and silicon) variation on solubility of multiwall nanotubes in terms of number of water molecules in shell volume. It is found that the solubility of the multi wall carbon nanotube samples is size independent, whereas multi wall silicon nanotube samples solubility varies with diameter of the inner tube. The higher solubility of samples containing silicon can be attributed to the larger atomic size of silicon atom which provides more direct contact with the water molecules. The other affecting factor is the bigger inter space (the space between inner and outer tube) in the case of silicon samples. Carbon type multi wall nanotubes appeared as better candidates for transporting water molecules through a multi wall nanotube structure, while in the case of water adsorption problems it is better to use multi wall silicon nanotubes or a mixture of multi wall carbon/ silicon nanotubes.

ZmXTH1, a new xyloglucan endotransglucosylase/hydrolase in maize, affects cell wall structure and composition in Arabidopsis thaliana.

PubMed

Genovesi, Valeria; Fornalé, Silvia; Fry, Stephen C; Ruel, Katia; Ferrer, Pau; Encina, Antonio; Sonbol, Fathi-Mohamed; Bosch, Josep; Puigdomènech, Pere; Rigau, Joan; Caparrós-Ruiz, David

2008-01-01

Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207 and/or EC 3.2.1.151) are enzymes involved in the modification of cell wall structure by cleaving and, often, also re-joining xyloglucan molecules in primary plant cell walls. Using a pool of antibodies raised against an enriched cell wall protein fraction, a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNA expression library obtained from the elongation zone of the maize root. The predicted protein has a putative N-terminal signal peptide and possesses the typical domains of this enzyme family, such as a catalytic domain that is homologous to that of Bacillus macerans beta-glucanase, a putative N-glycosylation motif, and four cysteine residues in the central and C terminal regions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1 reveals that it belongs to subgroup 4, so far only reported from Poaceae monocot species. ZmXTH1 has been expressed in Pichia pastoris (a methylotrophic yeast) and the recombinant enzyme showed xyloglucan endotransglucosylase but not xyloglucan endohydrolase activity, representing the first enzyme belonging to subgroup 4 characterized in maize so far. Expression data indicate that ZmXTH1 is expressed in elongating tissues, modulated by culture conditions, and induced by gibberellins. Transient expression assays in onion cells reveal that ZmXTH1 is directed to the cell wall, although weakly bound. Finally, Arabidopsis thaliana plants expressing ZmXTH1 show slightly increased xyloglucan endohydrolase activity and alterations in the cell wall structure and composition.

A mathematical model for expected time to extinction of pathogenic bacteria through antibiotic

NASA Astrophysics Data System (ADS)

Ghosh, M. K.; Nandi, S.; Roy, P. K.

2016-04-01

Application of antibiotics in human system to prevent bacterial diseases like Gastritis, Ulcers, Meningitis, Pneumonia and Gonorrhea are indispensable. Antibiotics saved innumerable lives and continue to be a strong support for therapeutic application against pathogenic bacteria. In human system, bacterial diseases occur when pathogenic bacteria gets into the body and begin to reproduce and crowd out healthy bacteria. In this process, immature bacteria releases enzyme which is essential for bacterial cell-wall biosynthesis. After complete formation of cell wall, immature bacteria are converted to mature or virulent bacteria which are harmful to us during bacterial infections. Use of antibiotics as drug inhibits the bacterial cell wall formation. After application of antibiotics within body, the released bacterial enzyme binds with antibiotic molecule instead of its functional site during the cell wall synthesis in a competitive inhibition approach. As a consequence, the bacterial cell-wall formation as well as maturation process of pathogenic bacteria is halted and the disease is cured with lysis of bacterial cells. With this idea, a mathematical model has been developed in the present research investigation to review the inhibition of biosynthesis of bacterial cell wall by the application of antibiotics as drug in the light of enzyme kinetics. This approach helps to estimate the expected time to extinction of the pathogenic bacteria. Our mathematical approach based on the enzyme kinetic model for finding out expected time to extinction contributes favorable results for understanding of disease dynamics. Analytical and numerical results based on simulated findings validate our mathematical model.

Hybrid materials and methods for producing the same

DOEpatents

Luzzi, David E [Wallingford, PA; Smith, Brian W [Schelton, CT

2003-04-08

A hybrid material is provided which comprises a first single-walled nanotube having a lumen, and a fill molecule contained within the lumen of the single-walled nanotube. A method for producing the hybrid material is also provided wherein a single-walled nanotube is contacted with a fill molecule to cause the fill molecule to enter the lumen of the single-walled nanotube.

Hybrid materials and methods for producing the same

DOEpatents

Luzzi, David E [Wallingford, PA; Smith, Brian W [Philadelphia, PA

2008-02-19

A hybrid material is provided which comprises a first single-walled nanotube having a lumen, and a fill molecule contained within the lumen of the single-walled nanotube. A method for producing the hybrid material is also provided wherein a single-walled nanotube is contacted with a fill molecule to cause the fill molecule to enter the lumen of the single-walled nanotube.

Stem Cell Therapy with Overexpressed VEGF and PDGF Genes Improves Cardiac Function in a Rat Infarct Model

PubMed Central

Das, Hiranmoy; George, Jon C.; Joseph, Matthew; Das, Manjusri; Abdulhameed, Nasreen; Blitz, Anna; Khan, Mahmood; Sakthivel, Ramasamy; Mao, Hai-Quan; Hoit, Brian D.; Kuppusamy, Periannan; Pompili, Vincent J.

2009-01-01

Background Therapeutic potential was evaluated in a rat model of myocardial infarction using nanofiber-expanded human cord blood derived hematopoietic stem cells (CD133+/CD34+) genetically modified with VEGF plus PDGF genes (VIP). Methods and Findings Myocardial function was monitored every two weeks up to six weeks after therapy. Echocardiography revealed time dependent improvement of left ventricular function evaluated by M-mode, fractional shortening, anterior wall tissue velocity, wall motion score index, strain and strain rate in animals treated with VEGF plus PDGF overexpressed stem cells (VIP) compared to nanofiber expanded cells (Exp), freshly isolated cells (FCB) or media control (Media). Improvement observed was as follows: VIP>Exp> FCB>media. Similar trend was noticed in the exercise capacity of rats on a treadmill. These findings correlated with significantly increased neovascularization in ischemic tissue and markedly reduced infarct area in animals in the VIP group. Stem cells in addition to their usual homing sites such as lung, spleen, bone marrow and liver, also migrated to sites of myocardial ischemia. The improvement of cardiac function correlated with expression of heart tissue connexin 43, a gap junctional protein, and heart tissue angiogenesis related protein molecules like VEGF, pNOS3, NOS2 and GSK3. There was no evidence of upregulation in the molecules of oncogenic potential in genetically modified or other stem cell therapy groups. Conclusion Regenerative therapy using nanofiber-expanded hematopoietic stem cells with overexpression of VEGF and PDGF has a favorable impact on the improvement of rat myocardial function accompanied by upregulation of tissue connexin 43 and pro-angiogenic molecules after infarction. PMID:19809493

Multiwalled carbon nanotubes effect on the bioavailability of artemisinin and its cytotoxity to cancerous cells

NASA Astrophysics Data System (ADS)

Rezaei, Behzad; Majidi, Najmeh; Noori, Shokoofe; Hassan, Zuhair M.

2011-12-01

Artemisinin regarded as one of the most promising anticancer drugs can bind to DNA with a binding constant of 1.04 × 104 M-1. The electrochemical experiments indicated that for longer incubation time periods, the reduction peak current of artemisinin on carbon nanotube modified electrode increases. Therefore, the uptake of drug molecules from a solution into CNTs will be achieved automatically by adsorption of 88.7% of artemisinin onto carbon nanotubes surface without alteration in drug properties. Hence, capability of carbon nanotubes to have synergistic effect on the bioavailability of artemisinin was investigated. Experimental tests on K562 cancer cell lines growth by MTT assay proved that multi-walled carbon nanotubes can enhance the cytotoxity of artemisinin to the targeted cancer cells with unprecedented accuracy and efficiency. The IC50 values were 65 and 35 μM for artemisinin and artemisinin loaded on multi-walled carbon nanotubes, respectively; demonstrating that artemisinin loaded on multi-walled carbon nanotubes is more effective in inhibition of cancer cell lines growth.

The lantibiotic mersacidin is a strong inducer of the cell wall stress response of Staphylococcus aureus

PubMed Central

Sass, Peter; Jansen, Andrea; Szekat, Christiane; Sass, Vera; Sahl, Hans-Georg; Bierbaum, Gabriele

2008-01-01

Background The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids that is ribosomally produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin acts by complexing the sugar phosphate head group of the peptidoglycan precursor lipid II, thereby inhibiting the transglycosylation reaction of peptidoglycan biosynthesis. Results Here, we studied the growth of Staphylococcus aureus in the presence of subinhibitory concentrations of mersacidin. Transcriptional data revealed an extensive induction of the cell wall stress response, which is partly controlled by the two-component regulatory system VraSR. In contrast to other cell wall-active antibiotics such as vancomycin, very low concentrations of mersacidin (0.15 × MIC) were sufficient for induction. Interestingly, the cell wall stress response was equally induced in vancomycin intermediately resistant S. aureus (VISA) and in a highly susceptible strain. Since the transcription of the VraDE ABC transporter genes was induced up to 1700-fold in our experiments, we analyzed the role of VraDE in the response to mersacidin. However, the deletion of the vraE gene did not result in an increased susceptibility to mersacidin compared to the wild type strain. Moreover, the efficacy of mersacidin was not affected by an increased cell wall thickness, which is part of the VISA-type resistance mechanism and functions by trapping the vancomycin molecules in the cell wall before they reach lipid II. Therefore, the relatively higher concentration of mersacidin at the membrane might explain why mersacidin is such a strong inducer of VraSR compared to vancomycin. Conclusion In conclusion, mersacidin appears to be a strong inducer of the cell wall stress response of S. aureus at very low concentrations, which reflects its general mode of action as a cell wall-active peptide as well as its use of a unique target site on lipid II. Additionally, mersacidin does not seem to be a substrate for the resistance transporter VraDE. PMID:18947397

Ultrastructure of potato tubers formed in microgravity under controlled environmental conditions

NASA Technical Reports Server (NTRS)

Cook, Martha E.; Croxdale, Judith G.; Tibbitts, T. W. (Principal Investigator)

2003-01-01

Previous spaceflight reports attribute changes in plant ultrastructure to microgravity, but it was thought that the changes might result from growth in uncontrolled environments during spaceflight. To test this possibility, potato explants were examined (a leaf, axillary bud, and small stem segment) grown in the ASTROCULTURETM plant growth unit, which provided a controlled environment. During the 16 d flight of space shuttle Columbia (STS-73), the axillary bud of each explant developed into a mature tuber. Upon return to Earth, tuber slices were examined by transmission electron microscopy. Results showed that the cell ultrastructure of flight-grown tubers could not be distinguished from that of tuber cells grown in the same growth unit on the ground. No differences were observed in cellular features such as protein crystals, plastids with starch grains, mitochondria, rough ER, or plasmodesmata. Cell wall structure, including underlying microtubules, was typical of ground-grown plants. Because cell walls of tubers formed in space were not required to provide support against the force due to gravity, it was hypothesized that these walls might exhibit differences in wall components as compared with walls formed in Earth-grown tubers. Wall components were immunolocalized at the TEM level using monoclonal antibodies JIM 5 and JIM 7, which recognize epitopes of pectins, molecules thought to contribute to wall rigidity and cell adhesion. No difference in presence, abundance or distribution of these pectin epitopes was seen between space- and Earth-grown tubers. This evidence indicates that for the parameters studied, microgravity does not affect the cellular structure of plants grown under controlled environmental conditions.

Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus

PubMed Central

Foster, Timothy J.; Geoghegan, Joan A.; Ganesh, Vannakambadi K.; Höök, Magnus

2014-01-01

Staphylococcus aureus is an important opportunistic pathogen and persistently colonizes about 20% of the human population. Its surface is ‘decorated’ with proteins that are covalently anchored to the cell wall peptidoglycan. Structural and functional analysis has identified four distinct classes of surface proteins, of which microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) are the largest class. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections. PMID:24336184

Molecular mechanisms of maternal vascular dysfunction in preeclampsia.

PubMed

Goulopoulou, Styliani; Davidge, Sandra T

2015-02-01

In preeclampsia, as a heterogeneous syndrome, multiple pathways have been proposed for both the causal as well as the perpetuating factors leading to maternal vascular dysfunction. Postulated mechanisms include imbalance in the bioavailability and activity of endothelium-derived contracting and relaxing factors and oxidative stress. Studies have shown that placenta-derived factors [antiangiogenic factors, microparticles (MPs), cell-free nucleic acids] are released into the maternal circulation and act on the vascular wall to modify the secretory capacity of endothelial cells and alter the responsiveness of vascular smooth muscle cells to constricting and relaxing stimuli. These molecules signal their deleterious effects on the maternal vascular wall via pathways that provide the molecular basis for novel and effective therapeutic interventions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Renew or die: The molecular mechanisms of peptidoglycan recycling and antibiotic resistance in Gram-negative pathogens.

PubMed

Domínguez-Gil, Teresa; Molina, Rafael; Alcorlo, Martín; Hermoso, Juan A

2016-09-01

Antimicrobial resistance is one of the most serious health threats. Cell-wall remodeling processes are tightly regulated to warrant bacterial survival and in some cases are directly linked to antibiotic resistance. Remodeling produces cell-wall fragments that are recycled but can also act as messengers for bacterial communication, as effector molecules in immune response and as signaling molecules triggering antibiotic resistance. This review is intended to provide state-of-the-art information about the molecular mechanisms governing this process and gather structural information of the different macromolecular machineries involved in peptidoglycan recycling in Gram-negative bacteria. The growing body of literature on the 3D structures of the corresponding macromolecules reveals an extraordinary complexity. Considering the increasing incidence and widespread emergence of Gram-negative multidrug-resistant pathogens in clinics, structural information on the main actors of the recycling process paves the way for designing novel antibiotics disrupting cellular communication in the recycling-resistance pathway. Copyright © 2016. Published by Elsevier Ltd.

A ReaxFF-based molecular dynamics study of the mechanisms of interactions between reactive oxygen plasma species and the Candida albicans cell wall

NASA Astrophysics Data System (ADS)

Zhao, T.; Shi, L.; Zhang, Y. T.; Zou, L.; Zhang, L.

2017-10-01

Atmospheric pressure non-equilibrium plasmas have attracted significant attention and have been widely used to inactivate pathogens, yet the mechanisms underlying the interactions between plasma-generated species and bio-organisms have not been elucidated clearly. In this paper, reactive molecular dynamics simulations are employed to investigate the mechanisms of interactions between reactive oxygen plasma species (O, OH, and O2) and β-1,6-glucan (a model for the C. albicans cell wall) from a microscopic point of view. Our simulations show that O and OH species can break structurally important C-C and C-O bonds, while O2 molecules exhibit only weak, non-bonded interactions with β-1,6-glucan. Hydrogen abstraction from hydroxyl or CH groups occurs first in all bond cleavage mechanisms. This is followed by a cascade of bond cleavage and double bond formation events. These lead to the destruction of the fungal cell wall. O and OH have similar effects related to their bond cleavage mechanisms. Our simulation results provide fundamental insights into the mechanisms underlying the interactions between reactive oxygen plasma species and the fungal cell wall of C. albicans at the atomic level.

Use of Phenylboronic Acids to Investigate Boron Function in Plants. Possible Role of Boron in Transvacuolar Cytoplasmic Strands and Cell-to-Wall Adhesion

PubMed Central

Bassil, Elias; Hu, Hening; Brown, Patrick H.

2004-01-01

The only defined physiological role of boron in plants is as a cross-linking molecule involving reversible covalent bonds with cis-diols on either side of borate. Boronic acids, which form the same reversible bonds with cis-diols but cannot cross-link two molecules, were used to selectively disrupt boron function in plants. In cultured tobacco (Nicotiana tabacum cv BY-2) cells, addition of boronic acids caused the disruption of cytoplasmic strands and cell-to-cell wall detachment. The effect of the boronic acids could be relieved by the addition of boron-complexing sugars and was proportional to the boronic acid-binding strength of the sugar. Experiments with germinating petunia (Petunia hybrida) pollen and boronate-affinity chromatography showed that boronic acids and boron compete for the same binding sites. The boronic acids appear to specifically disrupt or prevent borate-dependent cross-links important for the structural integrity of the cell, including the organization of transvacuolar cytoplasmic strands. Boron likely plays a structural role in the plant cytoskeleton. We conclude that boronic acids can be used to rapidly and reversibly induce boron deficiency-like responses and therefore are useful tools for investigating boron function in plants. PMID:15466241

Behaviour of nematic liquid crystals doped with ferroelectric nanoparticles in the presence of an electric field

NASA Astrophysics Data System (ADS)

Emdadi, M.; Poursamad, J. B.; Sahrai, M.; Moghaddas, F.

2018-06-01

A planar nematic liquid crystal cell (NLC) doped with spherical ferroelectric nanoparticles is considered. Polarisation of the nanoparticles are assumed to be along the NLC molecules parallel and antiparallel to the director with equal probability. The NLC molecules anchoring to the cell walls are considered to be strong, while soft anchoring at the nanoparticles surface is supposed. Behaviour of the NLC molecules and nanoparticles in the presence of a perpendicular electric field to the NLC cell is theoretically investigated. The electric field of the nanoparticles is taken into account in the calculations. Freedericksz transition (FT) threshold field in the presence of nanoparticles is found. Then, the director and particles reorientations for the electric fields larger than the threshold field are studied. Measuring the onset of the nanoparticles reorientation is proposed as a new method for the FT threshold measurement.

Lipoteichoic acids are embedded in cell walls during logarithmic phase, but exposed on membrane vesicles in Lactobacillus gasseri JCM 1131T.

PubMed

Shiraishi, T; Yokota, S; Sato, Y; Ito, T; Fukiya, S; Yamamoto, S; Sato, T; Yokota, A

2018-06-15

Lipoteichoic acid (LTA) is a cell surface molecule specific to Gram-positive bacteria. How LTA localises on the cell surface is a fundamental issue in view of recognition and immunomodulation in hosts. In the present study, we examined LTA localisation using strain JCM 1131T of Lactobacillus gasseri, which is a human intestinal lactic acid bacterium, during various growth phases by immunoelectron microscopy. We first evaluated the specificity of anti-LTA monoclonal antibody clone 55 used as a probe. The glycerophosphate backbone comprising almost intact size (20 to 30 repeating units) of LTA was required for binding. The antibody did not bind to other cellular components, including wall-teichoic acid. Immunoelectron microscopy indicated that LTA was embedded in the cell wall during the logarithmic phase, and was therefore not exposed on the cell surface. Similar results were observed for Lactobacillus fermentum ATCC 9338 and Lactobacillus rhamnosus ATCC 7469T. By contrast, membrane vesicles were observed in the logarithmic phase of L. gasseri with LTA exposed on their surface. In the stationary and death phases, LTA was exposed on cell wall-free cell membrane generated by autolysis. The dramatic alternation of localisation in different growth phases and exposure on the surface of membrane vesicles should relate with complicated interaction between bacteria and host.

Spermine Regulates Pollen Tube Growth by Modulating Ca2+-Dependent Actin Organization and Cell Wall Structure

PubMed Central

Aloisi, Iris; Cai, Giampiero; Faleri, Claudia; Navazio, Lorella; Serafini-Fracassini, Donatella; Del Duca, Stefano

2017-01-01

Proper growth of the pollen tube depends on an elaborate mechanism that integrates several molecular and cytological sub-processes and ensures a cell shape adapted to the transport of gametes. This growth mechanism is controlled by several molecules among which cytoplasmic and apoplastic polyamines. Spermine (Spm) has been correlated with various physiological processes in pollen, including structuring of the cell wall and modulation of protein (mainly cytoskeletal) assembly. In this work, the effects of Spm on the growth of pear pollen tubes were analyzed. When exogenous Spm (100 μM) was supplied to germinating pollen, it temporarily blocked tube growth, followed by the induction of apical swelling. This reshaping of the pollen tube was maintained also after growth recovery, leading to a 30–40% increase of tube diameter. Apical swelling was also accompanied by a transient increase in cytosolic calcium concentration and alteration of pH values, which were the likely cause for major reorganization of actin filaments and cytoplasmic organelle movement. Morphological alterations of the apical and subapical region also involved changes in the deposition of pectin, cellulose, and callose in the cell wall. Thus, results point to the involvement of Spm in cell wall construction as well as cytoskeleton organization during pear pollen tube growth. PMID:29033970

Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system.

PubMed

Liang, Xing-xiang; Wang, Bei-bei; Sun, Yu-fei; Lin, Ying; Han, Shuang-yan; Zheng, Sui-ping; Cui, Tang-bing

2013-03-01

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10(4) molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.

Orchestrating rapid long-distance signaling in plants with Ca2+ , ROS and electrical signals.

PubMed

Choi, Won-Gyu; Miller, Gad; Wallace, Ian; Harper, Jeffrey; Mittler, Ron; Gilroy, Simon

2017-05-01

Plants show a rapid systemic response to a wide range of environmental stresses, where the signals from the site of stimulus perception are transmitted to distal organs to elicit plant-wide responses. A wide range of signaling molecules are trafficked through the plant, but a trio of potentially interacting messengers, reactive oxygen species (ROS), Ca 2+ and electrical signaling ('trio signaling') appear to form a network supporting rapid signal transmission. The molecular components underlying this rapid communication are beginning to be identified, such as the ROS producing NAPDH oxidase RBOHD, the ion channel two pore channel 1 (TPC1), and glutamate receptor-like channels GLR3.3 and GLR3.6. The plant cell wall presents a plant-specific route for possible propagation of signals from cell to cell. However, the degree to which the cell wall limits information exchange between cells via transfer of small molecules through an extracellular route, or whether it provides an environment to facilitate transmission of regulators such as ROS or H + remains to be determined. Similarly, the role of plasmodesmata as both conduits and gatekeepers for the propagation of rapid cell-to-cell signaling remains a key open question. Regardless of how signals move from cell to cell, they help prepare distant parts of the plant for impending challenges from specific biotic or abiotic stresses. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

The Influence of Soft Layer Electrokinetics on Electroporation of Gram-positive Bacteria

NASA Astrophysics Data System (ADS)

Dingari, Naga Neehar; Moran, Jeffrey L.; Garcia, Paulo A.; Buie, Cullen R.

2016-11-01

Bacterial electroporation involves subjecting cells to intense ( 10 kV/cm) electric pulses, to open pores on the cell membrane for intracellular delivery of exogenous molecules. Its high efficiency in genetic transformation makes it an attractive tool for synthetic biology. While mammalian cell electroporation has received extensive theoretical and experimental investigation, bacterial electroporation has received markedly less attention. In this work, we develop a theoretical model of electroporation for gram-positive bacteria, taking into account the effect of the bacterial cell envelope on the cell's response to an electroporation pulse. We model the influence of the cell wall charge on the electrokinetic transport (and hence the pore properties) around the bacterial cell envelope using the Poisson-Nernst-Planck equations. Further, we account for the influence of the cell wall's mechanical elasticity on the pore radius evolution during electroporation, which is typically neglected in mammalian cell electroporation. This yields valuable information about favorable conditions for pore formation and will enable designing optimal platforms for bacteria electroporation.

CXCL16/CXCR6-mediated adhesion of human peripheral blood mononuclear cells to inflamed endothelium.

PubMed

Linke, Bona; Meyer Dos Santos, Sascha; Picard-Willems, Bettina; Keese, Michael; Harder, Sebastian; Geisslinger, Gerd; Scholich, Klaus

2017-06-21

The endothelial chemokine CXC motif ligand 16 (CXCL16) is involved in the recruitment and firm adhesion of CXCR6 + cells to the atherosclerosis-prone aortic vessel wall. Recently we showed that CXCR6 + platelets from flowing blood attach to CXCL16 expressed by activated endothelium on the luminal side of the blood vessel. With this study we supplement these findings with the observation that platelets bound to the inflamed endothelium are presenting CXCR6 to CXCL16-positive peripheral blood mononuclear cells (PBMCs) and, thus, are mediating an increased adhesion of PBMCs to the arterial wall. Furthermore we identified endothelial CXCL16 as an important adhesion molecule promoting the firm adhesion of CXCR6-positive PBMCs to inflamed endothelium. Our results demonstrate that endothelial CXCL16 as well as platelet CXCR6 are acting as potent PBMC-adhesion ligands, inducing PBMC-adhesion to the atherosclerosis-prone vessel wall and thus promoting the progression of atherosclerosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

The experimental vibrational infrared spectrum of lemon peel and simulation of spectral properties of the plant cell wall

NASA Astrophysics Data System (ADS)

Berezin, K. V.; Shagautdinova, I. T.; Chernavina, M. L.; Novoselova, A. V.; Dvoretskii, K. N.; Likhter, A. M.

2017-09-01

The experimental vibrational IR spectra of the outer part of lemon peel are recorded in the range of 3800-650 cm-1. The effect of artificial and natural dehydration of the peel on its vibrational spectrum is studied. It is shown that the colored outer layer of lemon peel does not have a noticeable effect on the vibrational spectrum. Upon 28-day storage of a lemon under natural laboratory conditions, only sequential dehydration processes are reflected in the vibrational spectrum of the peel. Within the framework of the theoretical DFT/B3LYP/6-31G(d) method, a model of a plant cell wall is developed consisting of a number of polymeric molecules of dietary fibers like cellulose, hemicellulose, pectin, lignin, some polyphenolic compounds (hesperetin glycoside-flavonoid), and a free water cluster. Using a supermolecular approach, the spectral properties of the wall of a lemon peel cell was simulated, and a detailed theoretical interpretation of the recorded vibrational spectrum is given.

Bio-inspired synthetic receptor molecules towards mimicry of vancomycin.

PubMed

Monnee, M C; Brouwer, A J; Verbeek, L M; van Wageningen, A M; Liskamp, R M

2001-06-18

A 512-member library of bio-inspired synthetic receptor molecules was prepared featuring a triazacyclophane scaffold. The purpose of this scaffold was to orient three (identical) peptide 'binding arms' in order to mimic an antibiotic binding cavity as is present in the vancomycin antibiotics. The library was screened with D-Ala-D-Ala and D-Ala-D-Lac containing ligands, which are present in the cell wall precursors of pathogenic bacteria. Screening and validation led to identification of a synthetic receptor capable of binding these ligands.

Direct Leukocyte Migration across Pulmonary Arterioles and Venules into the Perivascular Interstitium of Murine Lungs during Bleomycin Injury and Repair

PubMed Central

Wang, Ping M.; Kachel, Diane L.; Cesta, Mark F.; Martin, William J.

2011-01-01

During acute lung injury and repair, leukocytes are thought to enter the lung primarily across alveolar capillaries and postcapillary venules. We hypothesized that leukocytes also migrate across pulmonary arterioles and venules, which serve as alternative sites for leukocyte influx into the lung during acute lung injury and repair. Lung sections from C57BL/6J mice up to 14 days after intratracheal bleomycin (3.33 U/kg) or saline instillation were assessed by light, fluorescence, confocal, and transmission electron microscopy for evidence of inflammatory cell sequestration and transmigration at these sites. After bleomycin treatment, large numbers of leukocytes (including neutrophils, eosinophils, and monocytes) were present in the vascular lumina and in perivascular interstitia of pulmonary arterioles and venules, as well as within the vascular walls. Leukocytes were observed within well-defined pathways in arteriolar walls and much less structured pathways in venular walls, apparently in the process of transmigration. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were expressed at sites of leukocyte interaction with the luminal surface, especially in arterioles. Leukocytes appeared to exit from the vessels near collagen fibers into the perivascular interstitium. Results indicate that leukocytes can directly migrate across arteriolar and venular walls into the perivascular interstitium, which may represent an important but under-recognized pathway for leukocyte influx into the lung during injury and repair. PMID:21641381

Use of bacteriophage cell wall-binding proteins for rapid diagnostics of Listeria.

PubMed

Schmelcher, Mathias; Loessner, Martin J

2014-01-01

Diagnostic protocols for food-borne bacterial pathogens such as Listeria need to be sensitive, specific, rapid, and inexpensive. Conventional culture methods are hampered by lengthy enrichment and incubation steps. Bacteriophage-derived high-affinity binding molecules (cell wall-binding domains, CBDs) specific for Listeria cells have recently been introduced as tools for detection and differentiation of this pathogen in foods. When coupled with magnetic separation, these proteins offer advantages in sensitivity and speed compared to the standard diagnostic methods. Furthermore, fusion of CBDs to differently colored fluorescent reporter proteins enables differentiation of Listeria strains in mixed cultures. This chapter provides protocols for detection of Listeria in food by CBD-based magnetic separation and subsequent multiplexed identification of strains of different serotypes with reporter-CBD fusion proteins.

A density functional theory for association of fluid molecules with a functionalized surface: fluid-wall single and double bonding.

PubMed

Haghmoradi, Amin; Wang, Le; Chapman, Walter G

2017-02-01

In this manuscript we extend Wertheim's two-density formalism beyond its first order to model a system of fluid molecules with a single association site close to a planar hard wall with association sites on its surface in a density functional theory framework. The association sites of the fluid molecules are small enough that they can form only one bond, while the wall association sites are large enough to bond with more than one fluid molecule. The effects of temperature and of bulk fluid and wall site densities on the fluid density profile, extent of association, and competition between single and double bonding of fluid segments at the wall sites versus distance from the wall are presented. The theory predictions are compared with new Monte Carlo simulation results and they are in good agreement. The theory captures the surface coverage over wide ranges of temperature and bulk density by introducing the effect of steric hindrance in fluid association at a wall site.

Heparin-like entities from marine organisms.

PubMed

Colliec-Jouault, S; Bavington, C; Delbarre-Ladrat, C

2012-01-01

Polysaccharides are ubiquitous in animals and plant cells where they play a significant role in a number of physiological situations e.g. hydration, mechanical properties of cell walls and ionic regulation. This review concentrates on heparin-like entities from marine procaryotes and eukaryotes. Carbohydrates from marine prokaryotes offer a significant structural chemodiversity with novel material and biological properties. Cyanobacteria are Gram-negative photosynthetic prokaryotes considered as a rich source of novel molecules, and marine bacteria are a rich source of polysaccharides with novel structures, which may be a good starting point from which to synthesise heparinoid molecules. For example, some sulphated polysaccharides have been isolated from gamma-proteobacteria such as Alteromonas and Pseudoalteromonas sp. In contrast to marine bacteria, all marine algae contain sulphated wall polysaccharides, whereas such polymers are not found in terrestrial plants. In their native form, or after chemical modifications, a range of polysaccharides isolated from marine organisms have been described that have anticoagulant, anti-thrombotic, anti-tumour, anti-proliferative, anti-viral or anti-inflammatory activities.In spite of the enormous potential of sulphated oligosaccharides from marine sources, their technical and pharmaceutical usage is still limited because of the high complexity of these molecules. Thus, the production of tailor-made oligo- and polysaccharidic structures by biocatalysis is also a growing field of interest in biotechnology.

Characterization of a New β(1–3)-Glucan Branching Activity of Aspergillus fumigatus

PubMed Central

Gastebois, Amandine; Mouyna, Isabelle; Simenel, Catherine; Clavaud, Cécile; Coddeville, Bernadette; Delepierre, Muriel; Latgé, Jean-Paul; Fontaine, Thierry

2010-01-01

A new HPLC method was developed to separate linear from β(1–6)-branched β(1–3)-glucooligosaccharides. This methodology has permitted the isolation of the first fungal β(1–6)/β(1–3)-glucan branching transglycosidase using a cell wall autolysate of Aspergillus fumigatus (Af). The encoding gene, AfBGT2 is an ortholog of AfBGT1, another transglycosidase of A. fumigatus previously analyzed (Mouyna, I., Hartland, R. P., Fontaine, T., Diaquin, M., Simenel, C., Delepierre, M., Henrissat, B., and Latgé, J. P. (1998) Microbiology 144, 3171–3180). Both enzymes release laminaribiose from the reducing end of a β(1–3)-linked oligosaccharide and transfer the remaining chain to another molecule of the original substrate. The AfBgt1p transfer occurs at C-6 of the non-reducing end group of the acceptor, creating a kinked β(1–3;1–6) linear molecule. The AfBgt2p transfer takes place at the C-6 of an internal group of the acceptor, resulting in a β(1–3)-linked product with a β(1–6)-linked side branch. The single Afbgt2 mutant and the double Afbgt1/Afbgt2 mutant in A. fumigatus did not display any cell wall phenotype showing that these activities were not responsible for the construction of the branched β(1–3)-glucans of the cell wall. PMID:19948732

Identification of the UDP-glucose-4-epimerase required for galactofuranose biosynthesis and galactose metabolism in A. niger.

PubMed

Park, Joohae; Tefsen, Boris; Arentshorst, Mark; Lagendijk, Ellen; van den Hondel, Cees Amjj; van Die, Irma; Ram, Arthur Fj

2014-01-01

Galactofuranose (Gal f )-containing glycoconjugates are important to secure the integrity of the cell wall of filamentous fungi. Mutations that prevent the biosynthesis of Gal f -containing molecules compromise cell wall integrity. In response to cell wall weakening, the cell wall integrity (CWI)-pathway is activated to reinforce the strength of the cell wall. Activation of CWI-pathway in Aspergillus niger is characterized by the specific induction of the agsA gene, which encodes a cell wall α-glucan synthase. In this study, we screened a collection of cell wall mutants with an induced expression of agsA for defects in Gal f biosynthesis using a with anti-Gal f antibody (L10). From this collection of mutants, we previously identified mutants in the UDP-galactopyranose mutase encoding gene ( ugmA ). Here, we have identified six additional UDP-galactopyranose mutase ( ugmA ) mutants and one mutant (named mutant #41) in an additional complementation group that displayed strongly reduced Gal f -levels in the cell wall. By using a whole genome sequencing approach, 21 SNPs in coding regions were identified between mutant #41 and its parental strain which changed the amino acid sequence of the encoded proteins. One of these mutations was in gene An14g03820, which codes for a putative UDP-glucose-4-epimerase (UgeA). The A to G mutation in this gene causes an amino acid change of Asn to Asp at position 191 in the UgeA protein. Targeted deletion of ugeA resulted in an even more severe reduction of Gal f in N-linked glucans, indicating that the UgeA protein in mutant #41 is partially active. The ugeA gene is also required for growth on galactose despite the presence of two UgeA homologs in the A. niger genome. By using a classical mutant screen and whole genome sequencing of a new Gal f -deficient mutant, the UDP-glucose-4-epimerase gene ( ugeA ) has been identified. UgeA is required for the biosynthesis of Gal f as well as for galactose metabolism in Aspergillus niger .

The molecular chaperone HSP70 binds to and stabilizes NOD2, an important protein involved in Crohn disease.

PubMed

Mohanan, Vishnu; Grimes, Catherine Leimkuhler

2014-07-04

Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The Molecular Chaperone HSP70 Binds to and Stabilizes NOD2, an Important Protein Involved in Crohn Disease*

PubMed Central

Mohanan, Vishnu; Grimes, Catherine Leimkuhler

2014-01-01

Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand. PMID:24790089

Microvascular hemodynamics and in vivo evidence for the role of intercellular adhesion molecule-1 in the sequestration of infected red blood cells in a mouse model of lethal malaria.

PubMed

Kaul, D K; Liu, X D; Nagel, R L; Shear, H L

1998-02-01

The cytoadherence of infected red blood cells (IRBCs) to the vascular endothelium is the major cause of IRBC sequestration and vessel blockage in the cerebral form of human malaria. Among the rodent models of malaria, Plasmodium yoelii 17XL-infected mice show many similarities with the human cerebral malaria caused by P. falciparum. In both, the sequestration of IRBCs in the brain vessels is secondary to the cytoadherence of IRBCs to the vascular endothelium. Similar to P. falciparum infection in the human but in contrast to P. berghei ANKA infection in mice, P. yoelii 17XL results in little, if any, accumulation of monocytes in the brain. In vivo microcirculatory studies reported here were designed to further understand the hemodynamic aspects and mechanisms underlying cytoadherence of IRBCs in the P. yoelii model using the easily accessible cremaster muscle vasculature. The results show significant decreases in arteriovenous red blood cell velocities (Vrbc) and wall shear rates in the microcirculation of P. yoelii-infected mice, with a maximal decrease occurring in small-diameter postcapillary venules, the main sites of cytoadherence. This reflects contributions from IRBC cytoadherence as well as from increased rigidity of parasitized red blood cells. No cytoadherence is observed in arterioles of the infected mice despite decreased wall shear rates, indicating that endothelial receptors for cytoadherence are restricted to venules. Infusion of a monoclonal antibody (MAb) against the intercellular adhesion molecule-1 (ICAM-1) resulted in significant increases in both arteriolar and venular Vrbc and wall shear rates, accompanied by detachment of adhered IRBCs at some venular sites. The peripheral blood smears taken after the MAb infusion showed a distinct increase in the percentage of schizonts, again indicating detachment and/or prevention of cytoadherence. An MAb against the vascular cell adhesion molecule-1 (VCAM-1) as well as an irrelevant control antibody had no effect on these parameters. These results provide the first in vivo microcirculatory evidence indicating involvement of ICAM-1, but not of VCAM-1, in the sequestration of IRBCs in a rodent model of cerebral malaria.

HIV Aspartic Peptidase Inhibitors Modulate Surface Molecules and Enzyme Activities Involved with Physiopathological Events in Fonsecaea pedrosoi

PubMed Central

Palmeira, Vanila F.; Alviano, Daniela S.; Braga-Silva, Lys A.; Goulart, Fátima R. V.; Granato, Marcela Q.; Rozental, Sonia; Alviano, Celuta S.; Santos, André L. S.; Kneipp, Lucimar F.

2017-01-01

Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs) currently used in the treatment of human immunodeficiency virus (HIV) have shown anti-F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 μM) for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi. In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i) reduction on the conidial granularity; (ii) irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii) a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv) inhibition of ergosterol and lanosterol production; (v) reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi) significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics. PMID:28579986

HIV Aspartic Peptidase Inhibitors Modulate Surface Molecules and Enzyme Activities Involved with Physiopathological Events in Fonsecaea pedrosoi.

PubMed

Palmeira, Vanila F; Alviano, Daniela S; Braga-Silva, Lys A; Goulart, Fátima R V; Granato, Marcela Q; Rozental, Sonia; Alviano, Celuta S; Santos, André L S; Kneipp, Lucimar F

2017-01-01

Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs) currently used in the treatment of human immunodeficiency virus (HIV) have shown anti- F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 μM) for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi . In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i) reduction on the conidial granularity; (ii) irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii) a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv) inhibition of ergosterol and lanosterol production; (v) reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi) significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics.

Biocompatible Capsules and Methods of Making

NASA Technical Reports Server (NTRS)

Loftus, David J. (Inventor)

2017-01-01

Embodiments of the invention include capsules for containing medical implants and delivery systems for release of active biological substances into a host body. Delivery systems comprise a capsule comprising an interior enclosed by walls, and a source of active biological substances enclosed within the capsule interior, wherein the active biological substances are free to diffuse across the capsule walls. The capsule walls comprise a continuous mesh of biocompatible fibers and a seal region where two capsule walls overlap. The interior of the capsule is substantially isolated from the medium surrounding the capsule, except for diffusion of at least one species of molecule between the capsule interior and the ambient medium, and prevents cell migration into or out of the capsule. Methods for preparing and using the capsules and delivery systems are provided.

Cell-to-cell communication via plasmodesmata in vascular plants

PubMed Central

Sevilem, Iris; Miyashima, Shunsuke; Helariutta, Ykä

2013-01-01

In plant development, cell-to-cell signaling is mediated by mobile signals, including transcription factors and small RNA molecules. This communication is essential for growth and patterning. Short-range movement of signals occurs in the extracellular space via the apoplastic pathway or directly from cell-to-cell via the symplastic pathway. Symplastic transport is mediated by plant specific structures called plasmodesmata, which are plasma membrane-lined pores that traverse the cell walls of adjacent cells thus connecting their cytoplasms. However, a thorough understanding of molecules moving via plasmodesmata and regulatory networks relying on symplastic signaling is lacking. Traffic via plasmodesmata is highly regulated, and callose turnover is known to be one mechanism. In Arabidopsis, plasmodesmata apertures can be regulated in a spatially and temporally specific manner with the icals3m, an inducible vector system expressing the mutated CalS3 gene encoding a plasmodesmata localized callose synthase that increases callose deposition at plasmodesmata. We discuss strategies to use the icals3m system for global analyses on symplastic signaling in plants. PMID:23076211

Cellulose microfibrils in plants: biosynthesis, deposition, and integration into the cell wall.

PubMed

Brett, C T

2000-01-01

Cellulose occurs in all higher plants and some algae, fungi, bacteria, and animals. It forms microfibrils containing the crystalline allomorphs, cellulose I alpha and I beta. Cellulose molecules are 500-15,000 glucose units long. What controls molecular size is unknown. Microfibrils are elongated by particle rosettes in the plasma membrane (cellulose synthase complexes). The precursor, UDP-glucose, may be generated from sucrose at the site of synthesis. The biosynthetic mechanism may involve lipid-linked intermediates. Cellulose synthase has been purified from bacteria, but not from plants. In plants, disrupted cellulose synthase may form callose. Cellulose synthase genes have been isolated from bacteria and plants. Cellulose-deficient mutants have been characterised. The deduced amino acid sequence suggests possible catalytic mechanisms. It is not known whether synthesis occurs at the reducing or nonreducing end. Endoglucanase may play a role in synthesis. Nascent cellulose molecules associate by Van der Waals and hydrogen bonds to form microfibrils. Cortical microtubules control microfibril orientation, thus determining the direction of cell growth. Self-assembly mechanisms may operate. Microfibril integration into the wall occurs by interactions with matrix polymers during microfibril formation.

The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

PubMed Central

López-Fernández, Loida; Ruiz-Roldán, Carmen; Pareja-Jaime, Yolanda; Prieto, Alicia; Khraiwesh, Husam; Roncero, M. Isabel G.

2013-01-01

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity. PMID:24416097

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

PubMed

Kuwahara, Go; Hashimoto, Takuya; Tsuneki, Masayuki; Yamamoto, Kota; Assi, Roland; Foster, Trenton R; Hanisch, Jesse J; Bai, Hualong; Hu, Haidi; Protack, Clinton D; Hall, Michael R; Schardt, John S; Jay, Steven M; Madri, Joseph A; Kodama, Shohta; Dardik, Alan

2017-06-01

Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation. AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice. CD44 mRNA and protein expression was increased in wild-type AVF. CD44 knockout mice showed no increase in AVF wall thickness (8.9 versus 26.8 μm; P =0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared with control AVF. CD44 knockout mice also showed no increase in vascular cell adhesion molecule-1 expression, intercellular adhesion molecule-1 expression, and monocyte chemoattractant protein-1 expression in the AVF compared with controls; there were also no increased M2 macrophage markers (transglutaminase-2: 81.5-fold, P =0.0015; interleukin-10: 7.6-fold, P =0.0450) in CD44 knockout mice. Delivery of monocyte chemoattractant protein-1 to CD44 knockout mice rescued the phenotype with thicker AVF walls (27.2 versus 14.7 μm; P =0.0306), increased collagen density (2.4-fold; P =0.0432), and increased number of M2 macrophages (2.1-fold; P =0.0335). CD44 promotes accumulation of M2 macrophages, extracellular matrix deposition, and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation. © 2017 American Heart Association, Inc.

Intracellular Action of a Secreted Peptide Required for Fungal Virulence.

PubMed

Homer, Christina M; Summers, Diana K; Goranov, Alexi I; Clarke, Starlynn C; Wiesner, Darin L; Diedrich, Jolene K; Moresco, James J; Toffaletti, Dena; Upadhya, Rajendra; Caradonna, Ippolito; Petnic, Sarah; Pessino, Veronica; Cuomo, Christina A; Lodge, Jennifer K; Perfect, John; Yates, John R; Nielsen, Kirsten; Craik, Charles S; Madhani, Hiten D

2016-06-08

Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C. neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence. Copyright © 2016 Elsevier Inc. All rights reserved.

Oligosaccharins — a new class of signalling molecules in plants

NASA Astrophysics Data System (ADS)

Usov, Anatolii I.

1993-11-01

The review deals with oligosaccharins — biologically active oligosaccharides of the following structural types: the (1→6, 1→3)-β-D-glucan fragments of the fungal cell wall; the chitin and chitosan fragments; polygalacturonic acid fragments; the xyloglucan fragments of the plant cell wall; lipooligosaccharides synthesised by bacteria — symbionts of leguminous plants. The isolation, determination of the structure, and total syntheses of these complex compounds as well as the results of the study of their biological role as inductors of the immunity of plants to attack by pathogenic fungi and bacteria and as regulators of the morphogenesis in the development processes in plants and of interaction of the latter with symbiont bacteria are described. The bibliography includes 197 references.

Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function

PubMed Central

Montanier, Cedric; van Bueren, Alicia Lammerts; Dumon, Claire; Flint, James E.; Correia, Marcia A.; Prates, Jose A.; Firbank, Susan J.; Lewis, Richard J.; Grondin, Gilles G.; Ghinet, Mariana G.; Gloster, Tracey M.; Herve, Cecile; Knox, J. Paul; Talbot, Brian G.; Turkenburg, Johan P.; Kerovuo, Janne; Brzezinski, Ryszard; Fontes, Carlos M. G. A.; Davies, Gideon J.; Boraston, Alisdair B.; Gilbert, Harry J.

2009-01-01

Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-β-d-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or Δ4,5-anhydrogalaturonic acid (Δ4,5-GalA). Δ4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands. PMID:19218457

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.

Characterization of a yeast sporulation-specific P450 family protein, Dit2, using an in vitro assay to crosslink formyl tyrosine.

PubMed

Bemena, Leo D; Mukama, Omar; Wang, Ning; Gao, Xiao-Dong; Nakanishi, Hideki

2018-02-01

The outermost layer of the yeast Saccharomyces cerevisiae spore, termed the dityrosine layer, is primarily composed of bisformyl dityrosine. Bisformyl dityrosine is produced in the spore cytosol by crosslinking of two formyl tyrosine molecules, after which it is transported to the nascent spore wall and assembled into the dityrosine layer by an unknown mechanism. A P450 family protein, Dit2, is believed to mediate the crosslinking of bisformyl dityrosine molecules. To characterize Dit2 and gain insight into the biological process of dityrosine layer formation, we performed an in vitro assay to crosslink formyl tyrosine with using permeabilized cells. For an unknown reason, the production of bisformyl dityrosine could not be confirmed under our experimental conditions, but dityrosine was detected in acid hydrolysates of the reaction mixtures in a Dit2 dependent manner. Thus, Dit2 mediated the crosslinking of formyl tyrosine in vitro. Dityrosine was detected when formyl tyrosine, but not tyrosine, was used as a substrate and the reaction required NADPH as a cofactor. Intriguingly, apart from Dit2, we found that the spore wall, but not the vegetative cell wall, contains bisformyl dityrosine crosslinking activity. This activity may be involved in the assembly of the dityrosine layer. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

A rapid live-cell ELISA for characterizing antibodies against cell surface antigens of Chlamydomonas reinhardtii and its use in isolating algae from natural environments with related cell wall components.

PubMed

Jiang, Wenzhi; Cossey, Sarah; Rosenberg, Julian N; Oyler, George A; Olson, Bradley J S C; Weeks, Donald P

2014-09-25

Cell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors. In the case of eukaryotic algae, relatively little is known of the composition, structure or mechanisms of assembly of cell walls in individual species or between species and how these differences enable algae to inhabit a great diversity of environments. In this paper we describe the use of camelid antibody fragments (VHHs) and a streamlined ELISA assay as powerful new tools for obtaining mono-specific reagents for detecting individual algal cell wall components and for isolating algae that share a particular cell surface component. To develop new microalgal bioprospecting tools to aid in the search of environmental samples for algae that share similar cell wall and cell surface components, we have produced single-chain camelid antibodies raised against cell surface components of the single-cell alga, Chlamydomonas reinhardtii. We have cloned the variable-region domains (VHHs) from the camelid heavy-chain-only antibodies and overproduced tagged versions of these monoclonal-like antibodies in E. coli. Using these VHHs, we have developed an accurate, facile, low cost ELISA that uses live cells as a source of antigens in their native conformation and that requires less than 90 minutes to perform. This ELISA technique was demonstrated to be as accurate as standard ELISAs that employ proteins from cell lysates and that generally require >24 hours to complete. Among the cloned VHHs, VHH B11, exhibited the highest affinity (EC50 < 1 nM) for the C. reinhardtii cell surface. The live-cell ELISA procedure was employed to detect algae sharing cell surface components with C. reinhardtii in water samples from natural environments. In addition, mCherry-tagged VHH B11 was used along with fluorescence activated cell sorting (FACS) to select individual axenic isolates of presumed wild relatives of C. reinhardtii and other Chlorphyceae from the same environmental samples. Camelid antibody VHH domains provide a highly specific tool for detection of individual cell wall components of algae and for allowing the selection of algae that share a particular cell surface molecule from diverse ecosystems.

Cytotoxicity Effects of Different Surfactant Molecules Conjugated to Carbon Nanotubes on Human Astrocytoma Cells

NASA Astrophysics Data System (ADS)

Dong, Lifeng; Witkowski, Colette M.; Craig, Michael M.; Greenwade, Molly M.; Joseph, Katherine L.

2009-12-01

Phase contrast and epifluorescence microscopy were utilized to monitor morphological changes in human astrocytoma cells during a time-course exposure to single-walled carbon nanotube (SWCNT) conjugates with different surfactants and to investigate sub-cellular distribution of the nanotube conjugates, respectively. Experimental results demonstrate that cytotoxicity of the nanotube/surfactant conjugates is related to the toxicity of surfactant molecules attached on the nanotube surfaces. Both sodium dodecyl sulfate (SDS) and sodium dodecylbenzene sulfonate (SDBS) are toxic to cells. Exposure to CNT/SDS conjugates (0.5 mg/mL) for less than 5 min caused changes in cell morphology resulting in a distinctly spherical shape compared to untreated cells. In contrast, sodium cholate (SC) and CNT/SC did not affect cell morphology, proliferation, or growth. These data indicate that SC is an environmentally friendly surfactant for the purification and dispersion of SWCNTs. Epifluorescence microscopy analysis of CNT/DNA conjugates revealed distribution in the cytoplasm of cells and did not show adverse effects on cell morphology, proliferation, or viability during a 72-h incubation. These observations suggest that the SWCNTs could be used as non-viral vectors for diagnostic and therapeutic molecules across the blood-brain barrier to the brain and the central nervous system.

Rim Pathway-Mediated Alterations in the Fungal Cell Wall Influence Immune Recognition and Inflammation.

PubMed

Ost, Kyla S; Esher, Shannon K; Leopold Wager, Chrissy M; Walker, Louise; Wagener, Jeanette; Munro, Carol; Wormley, Floyd L; Alspaugh, J Andrew

2017-01-31

Compared to other fungal pathogens, Cryptococcus neoformans is particularly adept at avoiding detection by innate immune cells. To explore fungal cellular features involved in immune avoidance, we characterized cell surface changes of the C. neoformans rim101Δ mutant, a strain that fails to organize and shield immunogenic epitopes from host detection. These cell surface changes are associated with an exaggerated, detrimental inflammatory response in mouse models of infection. We determined that the disorganized strain rim101Δ cell wall increases macrophage detection in a contact-dependent manner. Using biochemical and microscopy methods, we demonstrated that the rim101Δ strain shows a modest increase in the levels of both cell wall chitin and chitosan but that it shows a more dramatic increase in chito-oligomer exposure, as measured by wheat germ agglutinin staining. We also created a series of mutants with various levels of cell wall wheat germ agglutinin staining, and we demonstrated that the staining intensity correlates with the degree of macrophage activation in response to each strain. To explore the host receptors responsible for recognizing the rim101Δ mutant, we determined that both the MyD88 and CARD9 innate immune signaling proteins are involved. Finally, we characterized the immune response to the rim101Δ mutant in vivo, documenting a dramatic and sustained increase in Th1 and Th17 cytokine responses. These results suggest that the Rim101 transcription factor actively regulates the C. neoformans cell wall to prevent the exposure of immune stimulatory molecules within the host. These studies further explored the ways in which immune cells detect C. neoformans and other fungal pathogens by mechanisms that include sensing N-acetylglucosamine-containing structures, such as chitin and chitosan. Infectious microorganisms have developed many ways to avoid recognition by the host immune system. For example, pathogenic fungi alter their cell surfaces to mask immunogenic epitopes. We have created a fungal strain with a targeted mutation in a pH response pathway that is unable to properly organize its cell wall, resulting in a dramatic immune reaction during infection. This mutant cell wall is defective in hiding important cell wall components, such as the chito-oligomers chitin and chitosan. By creating a series of cell wall mutants, we demonstrated that the degree of chito-oligomer exposure correlates with the intensity of innate immune cell activation. This activation requires a combination of host receptors to recognize and respond to these infecting microorganisms. Therefore, these experiments explored host-pathogen interactions that determine the degree of the subsequent inflammatory response and the likely outcome of infection. Copyright © 2017 Ost et al.

ORIENTATION AND LOCUS OF TROPIC PHOTORECEPTOR MOLECULES IN SPORES OF BOTRYTIS AND OSMUNDA

PubMed Central

Jaffe, Lionel; Etzold, Helmut

1962-01-01

Study of the tropic responses of Botrytis cinerea and Osmunda cinnamomea spores to blue light shows the photoreceptor molecules to be highly dichroic and oriented: in Botrytis their axes of maximum absorption lie perpendicular to the nearby cell surface; in Osmunda, parallel. The chief evidence lies in a comparison of their responses to plane polarized light—both germinate parallel to the vibration planes (defined by the axis of vibration of the electric vector and the axis of light propagation)—with those to partial illumination with unpolarized light: Botrytis grows from its brighter part; Osmunda, from its darker. The degree of orientation produced by polarized light corresponds, at high intensities, to that produced by the imposition of such large (about 100 per cent) intensity differences across a cell as to preclude all alternatives to oriented dichroic receptors. The photoreceptors of the Botrytis spore lie within the cell wall's inner half. The chief evidence lies in the component of its tropic responses to polarized light within the vibration plane: germination peaks about 10° off the vibration axis. This deviation arises from focusing which is effective only in the wall's inner half. At high intensities, anomalies appear in Botrytis which are interpreted as "centering," i.e., a tendency toward growth from the center of two or more equally illuminated points of a cell rather than from one of them. PMID:14450869

The Role of Exo-(1→4)-β-galactanase in the Mobilization of Polysaccharides from the Cotyledon Cell Walls of Lupinus angustifolius Following Germination

PubMed Central

BUCKERIDGE, MARCOS S.; HUTCHEON, IAN S.; REID, J. S. GRANT

2005-01-01

• Background and Aims The cotyledons of Lupinus angustifolius contain large amounts of cell wall storage polysaccharide (CWSP) composed mainly of (1→4)-β-linked d-galactose residues in the form of branches attached to a rhamnogalacturonan core molecule. An exo-(1→4)-β-galactanase with a very high specificity towards (1→4)-β-linked d-galactan has been isolated from L. angustifolius cotyledons, and shown to vary (activity and specific protein) in step with CWSP mobilization. This work aimed to confirm the hypothesis that galactan is the main polymer retrieved from the wall during mobilization at the ultrastructural level, using the purified exo-galactanase as a probe. • Methods Storage mesophyll cell walls (‘ghosts’) were isolated from the cotyledons of imbibed but ungerminated lupin seeds, and also from cotyledons of seedlings after the mobilization of the CWSP. The pure exo-(1→4)-β-galactanase was coupled to colloidal gold particles and shown to be a specific probe for (1→4)-β-d-galactan. They were used to localize galactan in ultrathin sections of L. angustifolius cotyledonary mesophyll tissue during CWSP mobilization. • Key Results On comparing the morphologies of isolated cell walls, the post-mobilization ‘ghosts’ did not have the massive wall-thickenings of pre-mobilization walls. Compositional analysis showed that the post-mobilization walls were depleted in galactose and, to a lesser extent, in arabinose. When pre-mobilization ghosts were treated with the pure exo-galactanase, they became morphologically similar to the post-mobilization ghosts. They were depleted of approximately 70% of the galactose residues that would have been mobilized in vivo, and retained all the other sugar residues originally present. Sharply defined electron-transparent wall zones or pockets are associated with CWSP mobilization, being totally free of galactan, whereas wall areas immediately adjacent to them were apparently undepleted. • Conclusions The exo-(1→4)-β-galactanase is the principal enzyme involved in CWSP mobilization in lupin cotyledons in vivo. The storage walls dramatically change their texture during mobilization as most of the galactan is hydrolysed during seedling development. PMID:15994843

The role of exo-(1-->4)-beta-galactanase in the mobilization of polysaccharides from the cotyledon cell walls of Lupinus angustifolius following germination.

PubMed

Buckeridge, Marcos S; Hutcheon, Ian S; Reid, J S Grant

2005-09-01

The cotyledons of Lupinus angustifolius contain large amounts of cell wall storage polysaccharide (CWSP) composed mainly of (1-->4)-beta-linked D-galactose residues in the form of branches attached to a rhamnogalacturonan core molecule. An exo-(1-->4)-beta-galactanase with a very high specificity towards (1-->4)-beta-linked D-galactan has been isolated from L. angustifolius cotyledons, and shown to vary (activity and specific protein) in step with CWSP mobilization. This work aimed to confirm the hypothesis that galactan is the main polymer retrieved from the wall during mobilization at the ultrastructural level, using the purified exo-galactanase as a probe. Storage mesophyll cell walls ('ghosts') were isolated from the cotyledons of imbibed but ungerminated lupin seeds, and also from cotyledons of seedlings after the mobilization of the CWSP. The pure exo-(1-->4)-beta-galactanase was coupled to colloidal gold particles and shown to be a specific probe for (1-->4)-beta-D-galactan. They were used to localize galactan in ultrathin sections of L. angustifolius cotyledonary mesophyll tissue during CWSP mobilization. On comparing the morphologies of isolated cell walls, the post-mobilization 'ghosts' did not have the massive wall-thickenings of pre-mobilization walls. Compositional analysis showed that the post-mobilization walls were depleted in galactose and, to a lesser extent, in arabinose. When pre-mobilization ghosts were treated with the pure exo-galactanase, they became morphologically similar to the post-mobilization ghosts. They were depleted of approximately 70% of the galactose residues that would have been mobilized in vivo, and retained all the other sugar residues originally present. Sharply defined electron-transparent wall zones or pockets are associated with CWSP mobilization, being totally free of galactan, whereas wall areas immediately adjacent to them were apparently undepleted. The exo-(1-->4)-beta-galactanase is the principal enzyme involved in CWSP mobilization in lupin cotyledons in vivo. The storage walls dramatically change their texture during mobilization as most of the galactan is hydrolysed during seedling development.

Abscisic acid ameliorates atherosclerosis by suppressing macrophage and CD4+ T cell recruitment into the aortic wall

PubMed Central

Guri, Amir J.; Misyak, Sarah A.; Hontecillas, Raquel; Hasty, Alyssa; Liu, Dongmin; Si, Hongwei; Bassaganya-Riera, Josep

2009-01-01

Abscisic acid (ABA) is a natural phytohormone which improves insulin sensitivity and reduces adipose tissue inflammation when supplemented into diets of obese mice. The objective of this study was to investigate the mechanisms by which abscisic acid (ABA) prevents or ameliorates atherosclerosis. Apolipoprotein E-deficient (ApoE −/−) mice were fed high-fat diets with or without ABA for 84 days. Systolic blood pressure was assessed on days 0, 28, 56, and 72. Gene expression, immune cell infiltration, and histological lesions were evaluated in the aortic root wall. Human aortic endothelial cells were used to examine the effect of ABA on 3’, 5’-cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) production in vitro. We report that ABA-treated mice had significantly improved systolic blood pressure and decreased accumulation of F4/80+CD11b+ macrophages and CD4+ T cells in aortic root walls. At the molecular level, ABA significantly enhanced aortic endothelial nitric oxide synthase (eNOS) and tended to suppress aortic vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) expression and plasma MCP-1 concentrations. ABA also caused a dose-dependent increase in intracellular concentrations of cAMP and NO and upregulated eNOS mRNA expression in human aortic endothelial cells. This is the first report showing that ABA prevents or ameliorates atherosclerosis-induced hypertension, immune cell recruitment into the aortic root wall, and upregulates aortic eNOS expression in ApoE−/− mice. PMID:20092994

Abscisic acid ameliorates atherosclerosis by suppressing macrophage and CD4+ T cell recruitment into the aortic wall.

PubMed

Guri, Amir J; Misyak, Sarah A; Hontecillas, Raquel; Hasty, Alyssa; Liu, Dongmin; Si, Hongwei; Bassaganya-Riera, Josep

2010-12-01

Abscisic acid (ABA) is a natural phytohormone which improves insulin sensitivity and reduces adipose tissue inflammation when supplemented into diets of obese mice. The objective of this study was to investigate the mechanisms by which ABA prevents or ameliorates atherosclerosis. apolipoprotein E-deficient (ApoE(-/-)) mice were fed high-fat diets with or without ABA for 84 days. Systolic blood pressure was assessed on Days 0, 28, 56 and 72. Gene expression, immune cell infiltration and histological lesions were evaluated in the aortic root wall. Human aortic endothelial cells were used to examine the effect of ABA on 3',5'-cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) production in vitro. We report that ABA-treated mice had significantly improved systolic blood pressure and decreased accumulation of F4/80(+)CD11b(+) macrophages and CD4(+) T cells in aortic root walls. At the molecular level, ABA significantly enhanced aortic endothelial nitric oxide synthase (eNOS) and tended to suppress aortic vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) expression and plasma MCP-1 concentrations. ABA also caused a dose-dependent increase in intracellular concentrations of cAMP and NO and up-regulated eNOS mRNA expression in human aortic endothelial cells. This is the first report showing that ABA prevents or ameliorates atherosclerosis-induced hypertension, immune cell recruitment into the aortic root wall and up-regulates aortic eNOS expression in ApoE(-/-) mice. Copyright © 2010 Elsevier Inc. All rights reserved.

New insights into pioneer root xylem development: evidence obtained from Populus trichocarpa plants grown under field conditions

PubMed Central

Bagniewska-Zadworna, Agnieszka; Arasimowicz-Jelonek, Magdalena; Smoliński, Dariusz J.; Stelmasik, Agnieszka

2014-01-01

Background and Aims Effective programmed xylogenesis is critical to the structural framework of the plant root system and its central role in the acquisition and long-distance transport of water and nutrients. The process of xylem differentiation in pioneer roots under field conditions is poorly understood. In this study it is hypothesized that xylogenesis, an example of developmental programmed cell death (PCD), in the roots of woody plants demonstrates a clearly defined sequence of events resulting in cell death. A comprehensive analysis was therefore undertaken to identify the stages of xylogenesis in pioneer roots from procambial cells to fully functional vessels with lignified cell walls and secondary cell wall thickenings. Methods Xylem differentiation was monitored in the pioneer roots of Populus trichocarpa at the cytological level using rhizotrons under field conditions. Detection and localization of the signalling molecule nitric oxide (NO) and hydrogen peroxide (H2O2) was undertaken and a detailed examination of nuclear changes during xylogenesis was conducted. In addition, analyses of the expression of genes involved in secondary cell wall synthesis were performed in situ. Key Results The primary event in initially differentiating tracheary elements (TEs) was a burst of NO in thin-walled cells, followed by H2O2 synthesis and the appearance of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei. The first changes in nuclear structure were observed in the early stages of xylogenesis of pioneer roots, prior to lignification; however, the nucleus was detectable under transmission electron microscopy in differentiating cells until the stage at which vacuole integrity was maintained, indicating that their degradation was slow and prolonged. The subsequent sequence of events involved secondary cell wall formation and autophagy. Potential gene markers from the cinnamyl alcohol dehydrogenase (CAD) gene family that were related to secondary wall synthesis were associated with primary xylogenesis, showing clear expression in cells that undergo differentiation into TEs and in the thin-walled cells adjacent to the xylem pole. Conclusions The early events of TE formation during pioneer root development are described, together with the timing of xylogenesis from signalling via NO, through secondary cell wall synthesis and autophagy events that are initiated long before lignification. This is the first work describing experiments conducted in planta on roots under field conditions demonstrating that the process of xylogenesis in vivo might be gradual and complex. PMID:24812251

Nanoneedle transistor-based sensors for the selective detection of intracellular calcium ions.

PubMed

Son, Donghee; Park, Sung Young; Kim, Byeongju; Koh, Jun Tae; Kim, Tae Hyun; An, Sangmin; Jang, Doyoung; Kim, Gyu Tae; Jhe, Wonho; Hong, Seunghun

2011-05-24

We developed a nanoneedle transistor-based sensor (NTS) for the selective detection of calcium ions inside a living cell. In this work, a single-walled carbon nanotube-based field effect transistor (swCNT-FET) was first fabricated at the end of a glass nanopipette and functionalized with Fluo-4-AM probe dye. The selective binding of calcium ions onto the dye molecules altered the charge state of the dye molecules, resulting in the change of the source-drain current of the swCNT-FET as well as the fluorescence intensity from the dye. We demonstrated the electrical and fluorescence detection of the concentration change of intracellular calcium ions inside a HeLa cell using the NTS.

Inhibition of fucosylation of cell wall components by 2-fluoro 2-deoxy-L-fucose induces defects in root cell elongation.

PubMed

Dumont, Marie; Lehner, Arnaud; Bardor, Muriel; Burel, Carole; Vauzeilles, Boris; Lerouxel, Olivier; Anderson, Charles T; Mollet, Jean-Claude; Lerouge, Patrice

2015-12-01

Screening of commercially available fluoro monosaccharides as putative growth inhibitors in Arabidopsis thaliana revealed that 2-fluoro 2-l-fucose (2F-Fuc) reduces root growth at micromolar concentrations. The inability of 2F-Fuc to affect an Atfkgp mutant that is defective in the fucose salvage pathway indicates that 2F-Fuc must be converted to its cognate GDP nucleotide sugar in order to inhibit root growth. Chemical analysis of cell wall polysaccharides and glycoproteins demonstrated that fucosylation of xyloglucans and of N-linked glycans is fully inhibited by 10 μm 2F-Fuc in Arabidopsis seedling roots, but genetic evidence indicates that these alterations are not responsible for the inhibition of root development by 2F-Fuc. Inhibition of fucosylation of cell wall polysaccharides also affected pectic rhamnogalacturonan-II (RG-II). At low concentrations, 2F-Fuc induced a decrease in RG-II dimerization. Both RG-II dimerization and root growth were partially restored in 2F-Fuc-treated seedlings by addition of boric acid, suggesting that the growth phenotype caused by 2F-Fuc was due to a deficiency of RG-II dimerization. Closer investigation of the 2F-Fuc-induced growth phenotype demonstrated that cell division is not affected by 2F-Fuc treatments. In contrast, the inhibitor suppressed elongation of root cells and promoted the emergence of adventitious roots. This study further emphasizes the importance of RG-II in cell elongation and the utility of glycosyltransferase inhibitors as new tools for studying the functions of cell wall polysaccharides in plant development. Moreover, supplementation experiments with borate suggest that the function of boron in plants might not be restricted to RG-II cross-linking, but that it might also be a signal molecule in the cell wall integrity-sensing mechanism. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.

PubMed

Morita, Chisato; Sumioka, Ryuichi; Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg(2+) and Ca(2+) for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.

Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

PubMed Central

Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg2+ and Ca2+ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression. PMID:25084357

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

Cell wall integrity, genotoxic injury and PCD dynamics in alfalfa saponin-treated white poplar cells highlight a complex link between molecule structure and activity.

PubMed

Paparella, Stefania; Tava, Aldo; Avato, Pinarosa; Biazzi, Elisa; Macovei, Anca; Biggiogera, Marco; Carbonera, Daniela; Balestrazzi, Alma

2015-03-01

In the present work, eleven saponins and three sapogenins purified from Medicago sativa were tested for their cytotoxicity against highly proliferating white poplar (Populus alba L.) cell suspension cultures. After preliminary screening, four saponins with different structural features in terms of aglycone moieties and sugar chains (saponin 3, a bidesmoside of hederagenin; saponins 4 and 5, monodesmoside and bidesmoside of medicagenic acid respectively, and saponin 10, a bidesmoside of zanhic acid) and different cytotoxicity were selected and used for further investigation on their structure-activity relationship. Transmission Electron Microscopy (TEM) analyses provided for the first time evidence of the effects exerted by saponins on plant cell wall integrity. Exposure to saponin 3 and saponin 10 resulted into disorganization of the outer wall layer and the effect was even more pronounced in white poplar cells treated with the two medicagenic acid derivatives, saponins 4 and 5. Oxidative burst and nitric oxide accumulation were common hallmarks of the response of white poplar cells to saponins. When DNA damage accumulation and DNA repair profiles were evaluated by Single Cell Gel Electrophoresis, induction of single and double strand breaks followed by effective repair was observed within 24h. The reported data are discussed in view of the current issues dealing with saponin structure-activity relationship. Copyright © 2015 Elsevier Ltd. All rights reserved.

Is the wall of a cellulose fiber saturated with liquid whether or not permeable with CO2 dissolved molecules? Application to bubble nucleation in champagne wines.

PubMed

Liger-Belair, Gérard; Topgaard, Daniel; Voisin, Cédric; Jeandet, Philippe

2004-05-11

In this paper, the transversal diffusion coefficient D perpendicular of CO2 dissolved molecules through the wall of a hydrated cellulose fiber was approached, from the liquid bulk diffusion coefficient of CO2 dissolved molecules modified by an obstruction factor. The porous network between the cellulose microfibrils of the fiber wall was assumed being saturated with liquid. We retrieved information from previous NMR experiments on the self-diffusion of water in cellulose fibers to reach an order of magnitude for the transversal diffusion coefficient of CO2 molecules through the fiber wall. A value of about D perpendicular approximately 0.2D0 was proposed, D0 being the diffusion coefficient of CO2 molecules in the liquid bulk. Because most of bubble nucleation sites in a glass poured with carbonated beverage are cellulose fibers cast off from paper or cloth which floated from the surrounding air, or remaining from the wiping process, this result directly applies to the kinetics of carbon dioxide bubble formation from champagne and sparkling wines. If the cellulose fiber wall was impermeable with regard to CO2 dissolved molecules, it was suggested that the kinetics of bubbling would be about three times less than it is.

Chitosan Mediates Germling Adhesion in Magnaporthe oryzae and Is Required for Surface Sensing and Germling Morphogenesis

PubMed Central

Geoghegan, Ivey A.; Gurr, Sarah J.

2016-01-01

The fungal cell wall not only plays a critical role in maintaining cellular integrity, but also forms the interface between fungi and their environment. The composition of the cell wall can therefore influence the interactions of fungi with their physical and biological environments. Chitin, one of the main polysaccharide components of the wall, can be chemically modified by deacetylation. This reaction is catalyzed by a family of enzymes known as chitin deacetylases (CDAs), and results in the formation of chitosan, a polymer of β1,4-glucosamine. Chitosan has previously been shown to accumulate in the cell wall of infection structures in phytopathogenic fungi. Here, it has long been hypothesized to act as a 'stealth' molecule, necessary for full pathogenesis. In this study, we used the crop pathogen and model organism Magnaporthe oryzae to test this hypothesis. We first confirmed that chitosan localizes to the germ tube and appressorium, then deleted CDA genes on the basis of their elevated transcript levels during appressorium differentiation. Germlings of the deletion strains showed loss of chitin deacetylation, and were compromised in their ability to adhere and form appressoria on artificial hydrophobic surfaces. Surprisingly, the addition of exogenous chitosan fully restored germling adhesion and appressorium development. Despite the lack of appressorium development on artificial surfaces, pathogenicity was unaffected in the mutant strains. Further analyses demonstrated that cuticular waxes are sufficient to over-ride the requirement for chitosan during appressorium development on the plant surface. Thus, chitosan does not have a role as a 'stealth' molecule, but instead mediates the adhesion of germlings to surfaces, thereby allowing the perception of the physical stimuli necessary to promote appressorium development. This study thus reveals a novel role for chitosan in phytopathogenic fungi, and gives further insight into the mechanisms governing appressorium development in M.oryzae. PMID:27315248

Neutron Scattering Studies of Nano-Scale Wood-Water Interactions

NASA Astrophysics Data System (ADS)

Plaza Rodriguez, Nayomi Z.

Understanding and controlling water in wood is critical to both improving forest products moisture durability and developing new sustainable forest products-based technologies. While wood is known to be hygroscopic, there is still a lack of understanding on the nanoscale wood-water interactions necessary for increased moisture-durability and dimensional stability. My PhD thesis focuses on the development and implementation of neutron scattering methods that can provide insight on both the structural and dynamical changes associated with these interactions so that products with improved moisture durability can be developed efficiently. Using small angle neutron scattering (SANS) and a custom-built in situ relative humidity chamber I studied the anisotropic moisture-induced swelling of wood nanostructure. First, I studied the effects of sample preparation by comparing SANS patterns of wiley milled wood and intact latewood cell walls, and found that scattering from intact wood provide more information about the spatial arrangement of the wood nanostructures inside the cell wall. Comparisons between SANS patterns from earlywood and latewood, also showed that the higher cell wall density of latewood cell walls results in patterns with more pronounced anisotropic features. Then, by measuring latewood loblolly pine sections obtained from the same growth ring and prepared in each of the primary wood planes, I tracked the cellulose elementary fibril spacing as a function of humidity in both intact and partially cut cell walls. These studies showed that even though swelling at the elementary fibril spacing is responsible for the majority of the transverse swelling observed at the S2 level, it is not primary plane dependent. Additionally, there were no differences in the elementary fibril spacing between partially-cut and intact cell walls, except at high humidity where the spacing in partially-cut cells was higher. SANS was also used to study the effects of two chemical modifications, namely, adhesive infiltration and acetylation, on the wood nanostructure as well as its moisture-induced swelling. Tangential-longitudinal latewood loblolly pine 0.5 mm thick sections were acetylated or treated with an adhesive (Phenol-formaldehyde (PF) or polymeric methylene diisocyanate (pMDI)) using deuterated or hydrogenated chemicals. Contrast variation experiments on wood modified with deuterated chemicals revealed that PF can infiltrate the regions between the elementary fibrils, while acetylation does not. The moisture-induced swelling of the chemically modified wood was studied, by studying the samples modified with hydrogenated chemicals using SANS and the previously built humidity chamber. These studies revealed that while both PF and pMDI can infiltrate the microfibrils, only PF reduced significantly the swelling at both the elementary fibril and bulk levels. In acetylated samples, the elementary fibril spacing was proportional to the moisture-content of the sample, which was reduced with increasing acetylation. This suggested that the acetylation treatment did not reduce the swelling at the elementary fibril but prevented water from entering the microfibril by modifying the regions surrounding the elementary fibrils. Using quasi-elastic neutron scattering (QENS) and a custom-built in situ relative humidity sample environment I measured experimentally the (5 - 400 ps) water dynamics inside wood cell walls for the first time and found that there are two types of bound water in the cell wall, namely, slow and fast water. The motion of both water types is well described by a jump-diffusion model, which corresponds to water molecules whose movement follows a stop and go process. Here, the slow water corresponds to water molecules that are highly associated to the wood polymers, whereas the fast water corresponds to water confined inside nanopores within the wood cell wall.

Impact of expressing p-coumaryl transferase in Medicago sativa L. on cell wall chemistry and digestibility

USDA-ARS?s Scientific Manuscript database

The addition of p-coumaric acid (pCA) to lignin molecules is frequently found in members of the grass family. The role of this addition is not clearly understood, but is thought to potentially aid in the formation of syringyl type lignin. This is because the incorporation is as a conjugate of pCA es...

Targeting chronic inflammation in cerebral aneurysms: focusing on NF-kappaB as a putative target of medical therapy.

PubMed

Aoki, Tomohiro; Nishimura, M

2010-03-01

Cerebral aneurysms (CAs) are the main cause of life-threatening subarachnoid hemorrhage. Given its prevalence and endpoint, CA treatment is a public health issue. Effective medical treatment of CAs is lacking because the detailed mechanisms of CA formation are incompletely understood. The aim of this contribution is to review recent articles about CA formation, to suggest the underlying mechanisms of CA formation, and to discuss potential therapeutic targets for treatment. Articles were collected by an internet search of PubMed using the keywords 'intracranial' or 'cerebral aneurysm'. A review of articles about the pathogenesis of CA formation focusing on inflammation. Recent articles demonstrate that inflammation-related-molecule induction and inflammatory cell infiltration in CA walls and the close relationship between inflammatory responses and CA formation. From studies in experimental models, chronic inflammation triggered primarily by NF-kappaB activation in endothelial cells and subsequent macrophage infiltration have critical roles in CA formation. Inhibition of inflammation-related molecules in CA walls results in the decreased incidence of CA formation. Agents with anti-inflammatory activity (particularly anti- NF-kappaB effects) have potential as therapeutic drugs for CAs.

Biomineralization of gold by Mucor plumbeus: The progress in understanding the mechanism of nanoparticles' formation.

PubMed

Maliszewska, Irena; Tylus, Włodzimierz; Chęcmanowski, Jacek; Szczygieł, Bogdan; Pawlaczyk-Graja, Izabela; Pusz, Wojciech; Baturo-Cieśniewska, Anna

2017-09-01

This contribution describes the deposition of gold nanoparticles by microbial reduction of Au(III) ions using the mycelium of Mucor plumbeus. Biosorption as the major mechanism of Au(III) ions binding by the fungal cells and the reduction of them to the form of Au(0) on/in the cell wall, followed by the transportation of the synthesized gold nanoparticles to the cytoplasm, is postulated. The probable mechanism behind the reduction of Au(III) ions is discussed, leading to the conclusion that this process is nonenzymatic one. Chitosan of the fungal cell wall is most likely to be the major molecule involved in biomineralization of gold by the mycelium of M. plumbeus. Separation of gold nanoparticles from the cells has been carried out by the ultrasonic disintegration and the obtained nanostructures were characterized by UV-vis spectroscopy and transmission electron micrograph analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1381-1392, 2017. © 2017 American Institute of Chemical Engineers.

How Many States Can the Motor Molecule, Prestin, Assume in an Electric Field?

PubMed Central

Scherer, Marc P.; Gummer, Anthony W.

2005-01-01

By using an analogy between the magnetization of a paramagnetic material in an external magnetic field and the electric polarization of the lateral wall of outer hair cells in response to the transmembrane potential, we show that, based on experimental data on the charge transfer across the membrane, it is impossible to make a statement about the number of possible conformational states of the motor molecule, prestin. Although the choice of model affects the values of derived parameters, such as total charge and motor charge, this is frequently overlooked in the literature. PMID:15764650

A novel screen-printed mast cell-based electrochemical sensor for detecting spoilage bacterial quorum signaling molecules (N-acyl-homoserine-lactones) in freshwater fish.

PubMed

Jiang, Donglei; Liu, Yan; Jiang, Hui; Rao, Shengqi; Fang, Wu; Wu, Mangang; Yuan, Limin; Fang, Weiming

2018-04-15

A novel screen-printed cell-based electrochemical sensor was developed to assess bacterial quorum signaling molecules, N-acylhomoserine lactones (AHLs). Screen-printed carbon electrode (SPCE), which possesses excellent properties such as low-cost, disposable and energy-efficient, was modified with multi-walled carbon nanotubes (MWNTs) to improve electrochemical signals and enhance the sensitivity. Rat basophilic leukemia (RBL-2H3) mast cells encapsulated in alginate/graphene oxide (NaAgl/GO) hydrogel were immobilized on the MWNTs/SPCE to serve as recognition element. Electrochemical impedance spectroscopy (EIS) was employed to record the cell impedance signal as-influenced by Pseudomonas aeruginosa quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC 12 -HSL). Experimental results show that 3OC 12 -HSL caused a significant decrease in cell viability in a dose dependent manner. The EIS value decreased with concentrations of 3OC 12 -HSL in the range of 0.1-1μM, and the detection limit for 3OC 12 -HSL was calculated to be 0.094μM. These results were confirmed via cell viability, SEM, TEM analysis. Next, the sensor was successfully applied to monitoring the production of AHLs by spoilage bacteria in three different freshwater fish juice samples which efficiently proved the practicability of this cell based method. Therefore, the proposed cell sensor may serve as an innovative and effective approach to the measurement of quorum signaling molecule and thus provides a new avenue for real-time monitoring the spoilage bacteria in freshwater fish production. Copyright © 2017 Elsevier B.V. All rights reserved.

Feasibility study on the use of liquid crystal/dye cells for digital signage

NASA Astrophysics Data System (ADS)

Itaya, Shunsuke; Azumi, Nada Dianah B. M.; Ohta, Masamichi; Ozawa, Shintaro; Fujieda, Ichiro

2016-03-01

Elongated dye molecules orient themselves with surrounding liquid crystal molecules. We propose to incorporate such a guest-host cell in a screen of a projection display. This configuration might be applied for digital signage to be installed on building walls. Dual-mode operation is realized by the bias applied to the cell. In display-mode, the dye molecules are oriented in parallel to the substrate of the cell. When excited by ultra-violet light, photoluminescence (PL) is generated. Because it is mostly perpendicular to the long axis of the molecule, it exits the cell efficiently. In powerharvesting mode, they are oriented vertically. The PL generated by ambient light is directed to edge surfaces where solar cells are mounted. In experiment, we fabricated a cell with commonly-available materials (coumarin 6 and a nematic liquid crystal). Anti-parallel alignment condition was adopted. We recorded PL spectra from the cell for the two excitation conditions. First, the center of the cell was irradiated by a 1.69mW blue laser beam. Second, the whole cell was uniformly exposed to the light from a fluorescent lamp at illuminance of 800lx. From the measured spectra for these cases, the contrast of luminance is calculated to be 3.2 ×105 . This factor is improved to 5 7.5×105 by attaching a polarizer sheet on the cell surface. The optical power reaching its edge surfaces is measured and it roughly agrees with the prediction by a simple model neglecting self-absorption. Development of phosphor materials with a large Stokes shift is desired to boost performance of the proposed system.

Compartmentalization of the outer hair cell demonstrated by slow diffusion in the extracisternal space.

PubMed

Gliko, Olga; Saggau, Peter; Brownell, William E

2009-08-19

In the outer hair cell (OHC), the extracisternal space (ECiS) is a conduit and reservoir of the molecular and ionic substrates of the lateral wall, including those necessary for electromotility. To determine the mechanisms through which molecules are transported in the ECiS of the OHC, we selectively imaged the time-dependent spatial distribution of fluorescent molecules in a <100 nm layer near the cell/glass interface of the recording chamber after their photolytic activation in a diffraction-limited volume. The effective diffusion coefficient was calculated using the analytical solution of the diffusion equation. It was found that diffusion in the ECiS is isotropic and not affected by depolarizing the OHC. Compared with free solution, the diffusion of 10 kDa dextran was slowed down in both the ECiS and the axial core by a factor of 4.6 and 1.6, respectively.

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

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

Dumont, Marie; Lehner, Arnaud; Bardor, Muriel

Screening of commercially available fluoro monosaccharides as putative growth inhibitors in Arabidopsis thaliana revealed that 2-fluoro 2-l-fucose (2F-Fuc) reduces root growth at micromolar concentrations. The inability of 2F-Fuc to affect an Atfkgp mutant that is defective in the fucose salvage pathway indicates that 2F-Fuc must be converted to its cognate GDP nucleotide sugar in order to inhibit root growth. Chemical analysis of cell wall polysaccharides and glycoproteins demonstrated that fucosylation of xyloglucans and of N-linked glycans is fully inhibited by 10 μm 2F-Fuc in Arabidopsis seedling roots, but genetic evidence indicates that these alterations are not responsible for the inhibitionmore » of root development by 2F-Fuc. Inhibition of fucosylation of cell wall polysaccharides also affected pectic rhamnogalacturonan-II (RG-II). At low concentrations, 2F-Fuc induced a decrease in RG-II dimerization. Both RG-II dimerization and root growth were partially restored in 2F-Fuc-treated seedlings by addition of boric acid, suggesting that the growth phenotype caused by 2F-Fuc was due to a deficiency of RG-II dimerization. Closer investigation of the 2F-Fuc-induced growth phenotype demonstrated that cell division is not affected by 2F-Fuc treatments. In contrast, the inhibitor suppressed elongation of root cells and promoted the emergence of adventitious roots. This study further emphasizes the importance of RG-II in cell elongation and the utility of glycosyltransferase inhibitors as new tools for studying the functions of cell wall polysaccharides in plant development. Moreover, supplementation experiments with borate suggest that the function of boron in plants might not be restricted to RG-II cross-linking, but that it might also be a signal molecule in the cell wall integrity-sensing mechanism.« less

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.

Distribution of protein motors along the lateral wall of the outer hair cell.

PubMed

Wada, H; Usukura, H; Takeuchi, S; Sugawara, M; Kakehata, S; Ikeda, K

2001-12-01

The outer hair cell (OHC) plays an important role in the normal functioning of the cochlea, and cochlear amplification is believed to be based on OHC electromotility. This electromotility putatively arises from a conformational change of molecules, i.e., 'protein motors', which would be distributed along the plasma membrane. Although it has been assumed that protein motors are distributed in a restricted area of the plasma membrane, details of such distribution remain unclarified. In this study, first, in order to understand the difference in the stiffness along the cell axis, the local deformation of the OHC in response to hypotonic stimulation is analyzed by measuring the displacement of microspheres attached randomly to the lateral wall of the cell. As a result, the stiffness is expected to be constant throughout the region except in the apical part of the cell, and the stiffness of the apical part is expected to be higher than that of the other regions. Then, the local elongation and contraction of the OHC in response to sinusoidal voltage stimulation are analyzed by measuring the displacement of the microspheres in the same way as in the case of the hypotonic stimulation. From the two measurements mentioned above, it is concluded that there are no motors in the apical and basal parts of the cell, and that the motors are equally distributed along the cell lateral wall in the middle part of the cell.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

PubMed

Formosa, C; Lachaize, V; Galés, C; Rols, M P; Martin-Yken, H; François, J M; Duval, R E; Dague, E

2015-01-01

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.

Sulforaphane suppresses vascular adhesion molecule-1 expression in TNF-α-stimulated mouse vascular smooth muscle cells: involvement of the MAPK, NF-κB and AP-1 signaling pathways.

PubMed

Kim, Ji-Yun; Park, Hye-Jin; Um, Sung Hee; Sohn, Eun-Hwa; Kim, Byung-Oh; Moon, Eun-Yi; Rhee, Dong-Kwon; Pyo, Suhkneung

2012-01-01

Atherosclerosis is a long-term inflammatory disease of the arterial wall. Increased expression of the cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) is associated with increased proliferation of vascular smooth muscle cells (VSMCs), leading to increased neointima or atherosclerotic lesion formation. Therefore, the functional inhibition of adhesion molecules could be a critical therapeutic target of inflammatory disease. In the present study, we investigate the effect of sulforaphane on the expression of VCAM-1 induced by TNF-α in cultured mouse vascular smooth muscle cell lines. Pretreatment of VSMCs for 2h with sulforaphane (1-5μg/ml) dose-dependently inhibited TNF-α-induced adhesion of THP-1 monocytic cells and protein expression of VCAM-1. Sulforaphane also suppressed TNF-α-induced production of intracellular reactive oxygen species (ROS) and activation of p38, ERK and JNK. Furthermore, sulforaphane inhibited NK-κB and AP-1 activation induced by TNF-α. Sulforaphane inhibited TNF-α-induced ΙκΒ kinase activation, subsequent degradation of ΙκΒα and nuclear translocation of p65 NF-κB and decreased c-Jun and c-Fos protein level. This study suggests that sulforaphane inhibits the adhesive capacity of VSMC and downregulates the TNF-α-mediated induction of VCAM-1 in VSMC by inhibiting the MAPK, NF-κB and AP-1 signaling pathways and intracellular ROS production. Thus, sulforaphane may have beneficial effects to suppress inflammation within the atherosclerotic lesion. Copyright © 2011 Elsevier Inc. All rights reserved.

Recognition, survival and persistence of Staphylococcus aureus in the model host Tenebrio molitor.

PubMed

Dorling, Jack; Moraes, Caroline; Rolff, Jens

2015-02-01

The degree of specificity of any given immune response to a parasite is governed by the complexity and variation of interactions between host and pathogen derived molecules. Here, we assess the extent to which recognition and immuno-resistance of cell wall mutants of the pathogen Staphylococcus aureus may contribute to establishment and maintenance of persistent infection in the model insect host, Tenebrio molitor. The cell surface of S. aureus is decorated with various molecules, including glycopolymers such as wall teichoic acid (WTA). WTA is covalently bound to peptidoglycan (PGN) and its absence has been associated with increased recognition of PGN by host receptors (PGRPs). WTA is also further modified by other molecules such as D-alanine (D-alanylation). Both the level of WTA expression and its D-alanylation were found to be important in the mediation of the host-parasite interaction in this model system. Specifically, WTA itself was seen to influence immune recognition, while D-alanylation of WTA was found to increase immuno-resistance and was associated with prolonged persistence of S. aureus in T. molitor. These results implicate WTA and its D-alanylation as important factors in the establishment and maintenance of persistent infection, affecting different critical junctions in the immune response; through potential evasion of recognition by PGRPs and resistance to humoral immune effectors during prolonged exposure to the immune system. This highlights a mechanism by which specificity in this host-parasite interaction may arise. Copyright © 2014 Elsevier Ltd. All rights reserved.

Transcriptomic and molecular genetic analysis of the cell wall salvage response of Aspergillus niger to the absence of galactofuranose synthesis.

PubMed

Park, Joohae; Hulsman, Mark; Arentshorst, Mark; Breeman, Matthijs; Alazi, Ebru; Lagendijk, Ellen L; Rocha, Marina C; Malavazi, Iran; Nitsche, Benjamin M; van den Hondel, Cees A M J J; Meyer, Vera; Ram, Arthur F J

2016-09-01

The biosynthesis of cell surface-located galactofuranose (Galf)-containing glycostructures such as galactomannan, N-glycans and O-glycans in filamentous fungi is important to secure the integrity of the cell wall. UgmA encodes an UDP-galactopyranose mutase, which is essential for the formation of Galf. Consequently, the ΔugmA mutant lacks Galf-containing molecules. Our previous work in Aspergillus niger work suggested that loss of function of ugmA results in activation of the cell wall integrity (CWI) pathway which is characterized by increased expression of the agsA gene, encoding an α-glucan synthase. In this study, the transcriptional response of the ΔugmA mutant was further linked to the CWI pathway by showing the induced and constitutive phosphorylation of the CWI-MAP kinase in the ΔugmA mutant. To identify genes involved in cell wall remodelling in response to the absence of galactofuranose biosynthesis, a genome-wide expression analysis was performed using RNAseq. Over 400 genes were higher expressed in the ΔugmA mutant compared to the wild-type. These include genes that encode enzymes involved in chitin (gfaB, gnsA, chsA) and α-glucan synthesis (agsA), and in β-glucan remodelling (bgxA, gelF and dfgC), and also include several glycosylphosphatidylinositol (GPI)-anchored cell wall protein-encoding genes. In silico analysis of the 1-kb promoter regions of the up-regulated genes in the ΔugmA mutant indicated overrepresentation of genes with RlmA, MsnA, PacC and SteA-binding sites. The importance of these transcription factors for survival of the ΔugmA mutant was analysed by constructing the respective double mutants. The ΔugmA/ΔrlmA and ΔugmA/ΔmsnA double mutants showed strong synthetic growth defects, indicating the importance of these transcription factors to maintain cell wall integrity in the absence of Galf biosynthesis. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

Elicitors and defense gene induction in plants with altered lignin compositions.

PubMed

Gallego-Giraldo, Lina; Posé, Sara; Pattathil, Sivakumar; Peralta, Angelo Gabriel; Hahn, Michael G; Ayre, Brian G; Sunuwar, Janak; Hernandez, Jonathan; Patel, Monika; Shah, Jyoti; Rao, Xiaolan; Knox, J Paul; Dixon, Richard A

2018-06-27

A reduction in the lignin content in transgenic plants induces the ectopic expression of defense genes, but the importance of altered lignin composition in such phenomena remains unclear. Two Arabidopsis lines with similar lignin contents, but strikingly different lignin compositions, exhibited different quantitative and qualitative transcriptional responses. Plants with lignin composed primarily of guaiacyl units overexpressed genes responsive to oomycete and bacterial pathogen attack, whereas plants with lignin composed primarily of syringyl units expressed a far greater number of defense genes, including some associated with cis-jasmone-mediated responses to aphids; these plants exhibited altered responsiveness to bacterial and aphid inoculation. Several of the defense genes were differentially induced by water-soluble extracts from cell walls of plants of the two lines. Glycome profiling, fractionation and enzymatic digestion studies indicated that the different lignin compositions led to differential extractability of a range of heterogeneous oligosaccharide epitopes, with elicitor activity originating from different cell wall polymers. Alteration of lignin composition affects interactions with plant cell wall matrix polysaccharides to alter the sequestration of multiple latent defense signal molecules with an impact on biotic stress responses. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Gravisensing, apoptosis, and drug recovery in Taxus cell suspensions

NASA Technical Reports Server (NTRS)

Durzan, D. J.

1999-01-01

Haploid and diploid cell suspensions of Taxus spp. were examined for their adaptive plasticity in response to simulated microgravity, unit gravity, and hypergravity. Cell suspensions produced the taxane, paclitaxel, (TAXOL (R)), which is useful for the treatment of various cancers. Amyloplasts contributed to taxane ring biosynthesis and to drug release at the cell wall. Drug-producing cells reacted as gravisensing osmotic tensiometers. In stressed cells, amyloplasts docked and fused in clusters to sites on the plasmalemma before taxane discharge into the culture medium. In simulated microgravity and compared to all other treatments, taxane production was reduced nearly 100-fold. The percent paclitaxel of total taxanes remained 3-to 6-fold greater, and biomass doubled. When p53-independent programmed cell death was induced, taxanes were released into the culture medium as free molecules (soluble and insoluble) or bound to membranes, nuclear fragments, xylan residues, and other particulate materials. Unit gravity and especially hypergravity promoted xylogenesis and significant drug overproduction. A model relating families of >touch = (TCH), taxane early response (TER), nuclear cycling, and apoptosis-regulating genes to gravisensing, cell wall modifications, and to taxane recovery accounted for most but not all of the observations.

Axisymmetric Implementation for 3D-Based DSMC Codes

NASA Technical Reports Server (NTRS)

Stewart, Benedicte; Lumpkin, F. E.; LeBeau, G. J.

2011-01-01

The primary objective in developing NASA s DSMC Analysis Code (DAC) was to provide a high fidelity modeling tool for 3D rarefied flows such as vacuum plume impingement and hypersonic re-entry flows [1]. The initial implementation has been expanded over time to offer other capabilities including a novel axisymmetric implementation. Because of the inherently 3D nature of DAC, this axisymmetric implementation uses a 3D Cartesian domain and 3D surfaces. Molecules are moved in all three dimensions but their movements are limited by physical walls to a small wedge centered on the plane of symmetry (Figure 1). Unfortunately, far from the axis of symmetry, the cell size in the direction perpendicular to the plane of symmetry (the Z-direction) may become large compared to the flow mean free path. This frequently results in inaccuracies in these regions of the domain. A new axisymmetric implementation is presented which aims to solve this issue by using Bird s approach for the molecular movement while preserving the 3D nature of the DAC software [2]. First, the computational domain is similar to that previously used such that a wedge must still be used to define the inflow surface and solid walls within the domain. As before molecules are created inside the inflow wedge triangles but they are now rotated back to the symmetry plane. During the move step, molecules are moved in 3D but instead of interacting with the wedge walls, the molecules are rotated back to the plane of symmetry at the end of the move step. This new implementation was tested for multiple flows over axisymmetric shapes, including a sphere, a cone, a double cone and a hollow cylinder. Comparisons to previous DSMC solutions and experiments, when available, are made.

The sensing mechanism of N-doped SWCNTs toward SF6 decomposition products: A first-principle study

NASA Astrophysics Data System (ADS)

Gui, Yingang; Tang, Chao; Zhou, Qu; Xu, Lingna; Zhao, Zhongyong; Zhang, Xiaoxing

2018-05-01

In order to monitor the insulation status of SF6-insulated equipment on-line, SOF2 and SO2F2, two typical decomposition products of SF6 under electric discharge condition, are chosen as the target gases to evaluate the type and severity of discharge. In this work, single N atom doping method is adopted to improve the gas sensitivity of single wall carbon nanotubes to SOF2 and SO2F2. Single and double gas molecules adsorptions are considered to completely analyze the adsorption properties of N-doped single wall carbon nanotubes. Calculation results show that N atom doping enhances the surface activity of carbon nanotubes. When gas molecules physically adsorbed on N-doped single wall carbon nanotubes, the weak interaction between gas molecules and N-doped single wall carbon nanotubes nearly not changes the electrical property according to analysis of the density of states and molecular orbitals. While the chemisorption between gas molecules and N-doped single wall carbon nanotubes distinctly decreases the conductivity of adsorption system.

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

PubMed Central

Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M.; Kumar, Manish; Nixon, B. Tracy; Bulone, Vincent; Zimmer, Jochen

2016-01-01

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils. PMID:27647898

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro.

PubMed

Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M; Kumar, Manish; Nixon, B Tracy; Bulone, Vincent; Zimmer, Jochen

2016-10-04

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

Whole-Teflon microfluidic chips

PubMed Central

Ren, Kangning; Dai, Wen; Zhou, Jianhua; Su, Jing; Wu, Hongkai

2011-01-01

Although microfluidics has shown exciting potential, its broad applications are significantly limited by drawbacks of the materials used to make them. In this work, we present a convenient strategy for fabricating whole-Teflon microfluidic chips with integrated valves that show outstanding inertness to various chemicals and extreme resistance against all solvents. Compared with other microfluidic materials [e.g., poly(dimethylsiloxane) (PDMS)] the whole-Teflon chip has a few more advantages, such as no absorption of small molecules, little adsorption of biomolecules onto channel walls, and no leaching of residue molecules from the material bulk into the solution in the channel. Various biological cells have been cultured in the whole-Teflon channel. Adherent cells can attach to the channel bottom, spread, and proliferate well in the channels (with similar proliferation rate to the cells in PDMS channels with the same dimensions). The moderately good gas permeability of the Teflon materials makes it suitable to culture cells inside the microchannels for a long time. PMID:21536918

Affinity Reagents for Multiplexed, Rapid Diagnosis of Bacterial Infections at the Point of Care using Diagnostic Magnetic Resonance

DTIC Science & Technology

2012-10-01

previously demonstrated that we can accurately identify bacteria, including Staphylococcus aureus and Mycobacterium tuberculosis , with startling speed... Mycobacterium tuberculosis , with moderate sensitivity and specificity. Existing antibodies perform poorly for identification of broad classes of...and test panel of existing small molecules with potential to bind to bacterial cell walls. 5. Assess technologies develop recombinant antibodies. 6

X-ray crystal structures of Staphylococcus aureus collagen adhesin and sortases

NASA Astrophysics Data System (ADS)

Zong, Yinong

For many gram-positive bacteria, adhesion to host tissues is the first critical step in developing an infection. The adhesion is mediated by a superfamily of bacterial surface proteins, called MSCRAMM (microbial surface components recognizing adhesive matrix molecules), which in most cases are covalently attached to the cell wall peptidoglycan. Collagen adhesin (CNA) from Staphylococcus aureus, one of the MSCRAMMs, is responsible for bacterial binding to collagen molecules. CNA and other MSCRAMMs are anchored to the cell wall by a transpeptidase, sortase. The knowledge about how bacterial surface proteins adhere to host molecules and how they are sorted onto the cell wall is crucial for the design of novel antibiotics against bacterial infections. The crystal structures of CNA31--344 (residue 31 to 334), a truncation of CNA's collagen binding region, and CNA31--344 in complex with a collagen peptide were determined. CNA31--344 contains two domains, and between them is a big hole formed by a loop connecting the two domains. In the structure of CNA31--344-collagen complex, the collagen peptide is locked in the hole formed by the two domains of CNA 31--344. We reason that the two domains of CNA31--344 are open in the physiological condition, and close up when binding to collagen. This binding mechanism may be common for other bacterial collagen adhesins. There are two known sortases in Staphylococcus aureus. Sortase A is responsible for anchoring most MSCRAMMs that have a LPXTG (X represents any amino acid) sorting motif and sortase B for a bacterial ion acquisition protein. The crystal structures of both sortases indicate that they share a common catalytic mechanism. Unlike typical cysteine transpeptidases, sortases may use a novel Cys-Arg catalytic dyad instead of a Cys-His pair. All other sortases found in gram-positive bacteria may have similar active site architecture and employ the same catalytic dyad because the critical residues are all conserved among them. The structures of sortases in complex with their substrates and inhibitors are also useful to explain their substrate specificity and catalytic kinetics.

Single- and double-walled carbon nanotubes enhance atherosclerogenesis by promoting monocyte adhesion to endothelial cells and endothelial progenitor cell dysfunction.

PubMed

Suzuki, Yuka; Tada-Oikawa, Saeko; Hayashi, Yasuhiko; Izuoka, Kiyora; Kataoka, Misa; Ichikawa, Shunsuke; Wu, Wenting; Zong, Cai; Ichihara, Gaku; Ichihara, Sahoko

2016-10-13

The use of carbon nanotubes has increased lately. However, the cardiovascular effect of exposure to carbon nanotubes remains elusive. The present study investigated the effects of pulmonary exposure to single-walled carbon nanotubes (SWCNTs) and double-walled carbon nanotubes (DWCNTs) on atherosclerogenesis using normal human aortic endothelial cells (HAECs) and apolipoprotein E-deficient (ApoE -/- ) mice, a model of human atherosclerosis. HAECs were cultured and exposed to SWCNTs or DWCNTs for 16 h. ApoE -/- mice were exposed to SWCNTs or DWCNTs (10 or 40 μg/mouse) once every other week for 10 weeks by pharyngeal aspiration. Exposure to CNTs increased the expression level of adhesion molecule (ICAM-1) and enhanced THP-1 monocyte adhesion to HAECs. ApoE -/- mice exposed to CNTs showed increased plaque area in the aorta by oil red O staining and up-regulation of ICAM-1 expression in the aorta, compared with vehicle-treated ApoE -/- mice. Endothelial progenitor cells (EPCs) are mobilized from the bone marrow into the circulation and subsequently migrate to the site of endothelial damage and repair. Exposure of ApoE -/- mice to high-dose SWCNTs or DWCNTs reduced the colony-forming units of EPCs in the bone marrow and diminished their migration function. The results suggested that SWCNTs and DWCNTs enhanced atherosclerogenesis by promoting monocyte adhesion to endothelial cells and inducing EPC dysfunction.

Analysis of fumonisin B1 removal by microorganisms in co-occurrence with aflatoxin B1 and the nature of the binding process.

PubMed

Pizzolitto, Romina P; Salvano, Mario A; Dalcero, Ana M

2012-06-01

The objectives of this investigation were to evaluate the ability of Saccharomyces cerevisiae CECT 1891 and Lactobacillus acidophilus 24 to remove fumonisin B(1) (FB(1)) from liquid medium; to determine the nature of the mechanism involved in FB(1)-microorganism interaction and to analyze whether the presence of aflatoxin B(1) (AFB(1)) interferes with the removal of FB(1) and vice versa. The results obtained indicated that: (i) both microorganisms were able to remove FB(1) from liquid medium; (ii) the removal was a fast and reversible process; (iii) cell viability was not necessary; (iv) the amount of FB(1) removed was both toxin- and microorganism concentration-dependent; (v) the process did not involve chemical modification of FB(1) molecules; and (vi) cell wall structural integrity of the microorganisms was required for FB(1) removal. Consequently, we propose that the mechanism involved in the removal of FB(1) is a physical adsorption (physisorption) of the toxin molecule to cell wall components of the microorganisms. It is highly probable that FB(1) and AFB(1) co-occur in contaminated foods, since the fungal genera Aspergillus and Fusarium frequently occur simultaneously. Therefore, we analyzed whether the presence of AFB(1) interferes with the removal of FB(1) by the microorganisms previously evaluated, and vice versa. Studies of co-occurrence of both mycotoxins clearly showed that they did not compete for binding sites on the microorganism cell wall and the presence of one toxin did not modify the efficiency of the organism in the removal of the other mycotoxin. These findings may be useful for optimization of mycotoxin binding and provide an important contribution to research on microorganisms with ability to remove these secondary metabolites. Copyright © 2012. Published by Elsevier B.V.

Single Molecule Detection in Living Biological Cells using Carbon Nanotube Optical Probes

NASA Astrophysics Data System (ADS)

Strano, Michael

2009-03-01

Nanoscale sensing elements offer promise for single molecule analyte detection in physically or biologically constrained environments. Molecular adsorption can be amplified via modulation of sharp singularities in the electronic density of states that arise from 1D quantum confinement [1]. Single-walled carbon nanotubes (SWNT), as single molecule optical sensors [2-3], offer unique advantages such as photostable near-infrared (n-IR) emission for prolonged detection through biological media, single-molecule sensitivity and, nearly orthogonal optical modes for signal transduction that can be used to identify distinct classes of analytes. Selective binding to the SWNT surface is difficult to engineer [4]. In this lecture, we will briefly review the immerging field of fluorescent diagnostics using band gap emission from SWNT. In recent work, we demonstrate that even a single pair of SWNT provides at least four optical modes that can be modulated to uniquely fingerprint chemical agents by the degree to which they alter either the emission band intensity or wavelength. We validate this identification method in vitro by demonstrating detection and identification of six genotoxic analytes, including chemotherapeutic drugs and reactive oxygen species (ROS), which are spectroscopically differentiated into four distinct classes. We also demonstrate single-molecule sensitivity in detecting hydrogen peroxide, one of the most common genotoxins and an important cellular signal. Finally, we employ our sensing and fingerprinting method of these analytes in real time within live 3T3 cells, demonstrating the first multiplexed optical detection from a nanoscale biosensor and the first label-free tool to optically discriminate between genotoxins. We will also discuss our recent efforts to fabricate biomedical sensors for real time detection of glucose and other important physiologically relevant analytes in-vivo. The response of embedded SWNT in a swellable hydrogel construct to osmotic pressure gradients will be discussed, as well as its potential as a unique transduction mechanism for a new class of implantable sensors. [4pt] [1] Saito, R., Dresselhaus, G. & Dresselhaus, M. S. Physical Properties of Carbon Nanotubes (Imperial College Press, London, 1998). [0pt] [2] Barone, P. W., Baik, S., Heller, D. A. & Strano, M. S. Near-Infrared Optical Sensors Based on Single-Walled Carbon Nanotubes. Nature Materials 4, 86-92 (2005). [0pt] [3] Jeng, E. S., Moll, A. E., Roy, A. C., Gastala, J. B. & Strano, M. S. Detection of DNA hybridization using the near infrared band-gap fluorescence of single-walled carbon nanotubes. Nano Letters 6, 371-375 (2006). [0pt] [4] Heller, D. A. et al. Optical detection of DNA conformational polymorphism on single-walled carbon nanotubes. Science 311, 508-511 (2006).

Small molecules targeting LapB protein prevent Listeria attachment to catfish muscle

PubMed Central

Das, Bhaskar; Lawrence, Mark

2017-01-01

Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listeriosis. L. monocytogenes lapB gene encodes a cell wall surface anchor protein, and mutation of this gene causes Listeria attenuation in mice. In this work, the potential role of Listeria LapB protein in catfish fillet attachment was investigated. To achieve this, boron-based small molecules designed to interfere with the active site of the L. monocytogenes LapB protein were developed, and their ability to prevent L. monocytogenes attachment to fish fillet was tested. Results indicated that seven out of nine different small molecules were effective in reducing the Listeria attachment to catfish fillets. Of these, three small molecules (SM3, SM5, and SM7) were highly effective in blocking Listeria attachment to catfish fillets. This study suggests an alternative strategy for reduction of L. monocytogenes contamination in fresh and frozen fish products. PMID:29253892

The role of endothelial cell attachment to elastic fibre molecules in the enhancement of monolayer formation and retention, and the inhibition of smooth muscle cell recruitment.

PubMed

Williamson, Matthew R; Shuttleworth, Adrian; Canfield, Ann E; Black, Richard A; Kielty, Cay M

2007-12-01

The endothelium is an essential modulator of vascular tone and thrombogenicity and a critical barrier between the vessel wall and blood components. In tissue-engineered small-diameter vascular constructs, endothelial cell detachment in flow can lead to thrombosis and graft failure. The subendothelial extracellular matrix provides stable endothelial cell anchorage through interactions with cell surface receptors, and influences the proliferation, migration, and survival of both endothelial cells and smooth muscle cells. We have tested the hypothesis that these desired physiological characteristics can be conferred by surface coatings of natural vascular matrix components, focusing on the elastic fiber molecules, fibrillin-1, fibulin-5 and tropoelastin. On fibrillin-1 or fibulin-5-coated surfaces, endothelial cells exhibited strong integrin-mediated attachment in static conditions (82% and 76% attachment, respectively) and flow conditions (67% and 78% cell retention on fibrillin-1 or fibulin-5, respectively, at 25 dynes/cm2), confluent monolayer formation, and stable functional characteristics. Adhesion to these two molecules also strongly inhibited smooth muscle cell migration to the endothelial monolayer. In contrast, on elastin, endothelial cells attached poorly, did not spread, and had markedly impaired functional properties. Thus, fibrillin-1 and fibulin-5, but not elastin, can be exploited to enhance endothelial stability, and to inhibit SMC migration within vascular graft scaffolds. These findings have important implications for the design of vascular graft scaffolds, the clinical performance of which may be enhanced by exploiting natural cell-matrix biology to regulate cell attachment and function.

Delivery of nitric oxide to the interior of mammalian cell by carbon nanotube: MD simulation.

PubMed

Raczyński, Przemysław; Górny, Krzysztof; Dawid, Aleksander; Gburski, Zygmunt

2014-07-15

Computer simulations have been performed to study the nanoindentation of phospholipid bilayer by the single-walled armchair carbon nanotube, filled with the nitric oxide molecules. The process has been simulated by means of molecular dynamics (MD) technique at physiological temperature T = 310 K with a constant pulling velocity of the nanotube. The force acting on the nanotube during membrane penetration has been calculated. We show that the indentation by carbon nanotube does not permanently destroy the membrane structure (self-sealing of the membrane occurs). The mobility of nitric oxide molecules during the membrane nanoindentation is discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

Preliminary X-ray characterization of a novel type of anchoring cohesin from the cellulosome of Ruminococcus flavefaciens

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

Alber, Orly; Noach, Ilit; Lamed, Raphael

2008-02-01

The cloning, expression, purification, crystallization and preliminary X-ray characterization of a novel class of cohesin module (type III) from the R. flavefaciens ScaE anchoring scaffoldin are described. Ruminococcus flavefaciens is an anaerobic bacterium that resides in the gastrointestinal tract of ruminants. It produces a highly organized multi-enzyme cellulosome complex that plays a key role in the degradation of plant cell walls. ScaE is one of the critical structural components of its cellulosome that serves to anchor the complex to the cell wall. The seleno-l-methionine-labelled derivative of the ScaE cohesin module has been cloned, expressed, purified and crystallized. The crystals belongmore » to space group C2, with unit-cell parameters a = 155.6, b = 69.3, c = 93.0 Å, β = 123.4°, and contain four molecules in the asymmetric unit. Diffraction data were phased to 1.95 Å using the anomalous signal from the Se atoms.« less

Octa-ammonium POSS-conjugated single-walled carbon nanotubes as vehicles for targeted delivery of paclitaxel

PubMed Central

Naderi, Naghmeh; Madani, Seyed Y.; Mosahebi, Afshin; Seifalian, Alexander M.

2015-01-01

Background Carbon nanotubes (CNTs) have unique physical and chemical properties. Furthermore, novel properties can be developed by attachment or encapsulation of functional groups. These unique properties facilitate the use of CNTs in drug delivery. We developed a new nanomedicine consisting of a nanocarrier, cell-targeting molecule, and chemotherapeutic drug and assessed its efficacy in vitro. Methods The efficacy of a single-walled carbon nanotubes (SWCNTs)-based nanoconjugate system is assessed in the targeted delivery of paclitaxel (PTX) to cancer cells. SWCNTs were oxidized and reacted with octa-ammonium polyhedral oligomeric silsesquioxanes (octa-ammonium POSS) to render them biocompatible and water dispersable. The functionalized SWCNTs were loaded with PTX, a chemotherapeutic agent toxic to cancer cells, and Tn218 antibodies for cancer cell targeting. The nanohybrid composites were characterized with transmission electron microscopy (TEM), Fourier transform infrared (FTIR), and ultraviolet–visible–near-infrared (UV–Vis–NIR). Additionally, their cytotoxic effects on Colon cancer cell (HT-29) and Breast cancer cell (MCF-7) lines were assessed in vitro. Results TEM, FTIR, and UV–Vis–NIR studies confirmed side-wall functionalization of SWCNT with COOH-groups, PTX, POSS, and antibodies. Increased cell death was observed with PTX–POSS–SWCNT, PTX–POSS–Ab–SWCNT, and free PTX compared to functionalized-SWCNT (f-SWCNT), POSS–SWCNT, and cell-only controls at 48 and 72 h time intervals in both cell lines. At all time intervals, there was no significant cell death in the POSS–SWCNT samples compared to cell-only controls. Conclusion The PTX-based nanocomposites were shown to be as cytotoxic as free PTX. This important finding indicates successful release of PTX from the nanocomposites and further reiterates the potential of SWCNTs to deliver drugs directly to targeted cells and tissues. PMID:26356347

Mechanics of receptor-mediated endocytosis

NASA Astrophysics Data System (ADS)

Gao, Huajian; Shi, Wendong; Freund, Lambert B.

2005-07-01

Most viruses and bioparticles endocytosed by cells have characteristic sizes in the range of tens to hundreds of nanometers. The process of viruses entering and leaving animal cells is mediated by the binding interaction between ligand molecules on the viral capid and their receptor molecules on the cell membrane. How does the size of a bioparticle affect receptor-mediated endocytosis? Here, we study how a cell membrane containing diffusive mobile receptors wraps around a ligand-coated cylindrical or spherical particle. It is shown that particles in the size range of tens to hundreds of nanometers can enter or exit cells via wrapping even in the absence of clathrin or caveolin coats, and an optimal particles size exists for the smallest wrapping time. This model can also be extended to include the effect of clathrin coat. The results seem to show broad agreement with experimental observations. Author contributions: H.G. and L.B.F. designed research; H.G., W.S., and L.B.F. performed research; and H.G., W.S., and L.B.F. wrote the paper.Abbreviations: CNT, carbon nanotube; SWNT, single-walled nanotube.

Layered Double Hydroxide Nanotransporter for Molecule Delivery to Intact Plant Cells

PubMed Central

Bao, Wenlong; Wang, Junya; Wang, Qiang; O’Hare, Dermot; Wan, Yinglang

2016-01-01

Here we report a powerful method that facilitates the transport of biologically active materials across the cell wall barrier in plant cells. Positively charged delaminated layered double hydroxide lactate nanosheets (LDH-lactate-NS) with a 0.5‒2 nm thickness and 30‒60 nm diameter exhibit a high adsorptive capacity for negatively charged biomolecules, including fluorescent dyes such as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate isomer I(FITC) and DNA molecules, forming neutral LDH-nanosheet conjugates. These neutral conjugates can shuttle the bound fluorescent dye into the cytosol of intact plant cell very efficiently. Furthermore, typical inhibitors of endocytosis and low temperature incubation did not prevent LDH-lactate-NS internalization, suggesting that LDH-lactate-NS penetrated the plasma membrane via non-endocytic pathways, which will widen the applicability to a variety of plant cells. Moreover, the absence of unwanted side effects in our cytological studies, and the nuclear localization of ssDNA-FITC suggest that nano-LDHs have potential application as a novel gene carrier to plants. PMID:27221055

Inactivation of the Ecs ABC transporter of Staphylococcus aureus attenuates virulence by altering composition and function of bacterial wall.

PubMed

Jonsson, Ing-Marie; Juuti, Jarmo T; François, Patrice; AlMajidi, Rana; Pietiäinen, Milla; Girard, Myriam; Lindholm, Catharina; Saller, Manfred J; Driessen, Arnold J M; Kuusela, Pentti; Bokarewa, Maria; Schrenzel, Jacques; Kontinen, Vesa P

2010-12-02

Ecs is an ATP-binding cassette (ABC) transporter present in aerobic and facultative anaerobic gram-positive Firmicutes. Inactivation of Bacillus subtilis Ecs causes pleiotropic changes in the bacterial phenotype including inhibition of intramembrane proteolysis. The molecule(s) transported by Ecs is (are) still unknown. In this study we mutated the ecsAB operon in two Staphylococcus aureus strains, Newman and LS-1. Phenotypic and functional characterization of these Ecs deficient mutants revealed a defect in growth, increased autolysis and lysostaphin sensitivity, altered composition of cell wall proteins including the precursor form of staphylokinase and an altered bacterial surface texture. DNA microarray analysis indicated that the Ecs deficiency changed expression of the virulence factor regulator protein Rot accompanied by differential expression of membrane transport proteins, particularly ABC transporters and phosphate-specific transport systems, protein A, adhesins and capsular polysaccharide biosynthesis proteins. Virulence of the ecs mutants was studied in a mouse model of hematogenous S. aureus infection. Mice inoculated with the ecs mutant strains developed markedly milder infections than those inoculated with the wild-type strains and had consequently lower mortality, less weight loss, milder arthritis and decreased persistence of staphylococci in the kidneys. The ecs mutants had higher susceptibility to ribosomal antibiotics and plant alkaloids chelerythrine and sanguinarine. Our results show that Ecs is essential for staphylococcal virulence and antimicrobial resistance probably since the transport function of Ecs is essential for the normal structure and function of the cell wall. Thus targeting Ecs may be a new approach in combating staphylococcal infection.

Biocentricity and economy of scale : hypothesis (and product) testing when wood is a part of an experimental system evaluating durability

Treesearch

Alex C. Wiedenhoeft

2009-01-01

Wood is a biological material, and its structure and organization are relicts of its biogenesis. From the hydrogen bonding of water molecules in the cell wall to extractives bleeding from knots in siding, the characteristics and behavior of wood are derived from its biological origin; this is my unashamedly biocentric view of wood structure. The structure of wood...

Therapeutic Potential of Modulating microRNAs in Atherosclerotic Vascular Disease

PubMed Central

Araldi, Elisa; Chamorro-Jorganes, Aranzazu; van Solingen, Coen; Fernández-Hernando, Carlos; Suárez, Yajaira

2013-01-01

Atherosclerosis (also known as arteriosclerotic vascular disease) is a chronic inflammatory disease of the arterial wall, characterized by the formation of lipid-laden lesions. The activation of endothelial cells at atherosclerotic lesion–prone sites in the arterial tree results in the up-regulation of cell adhesion molecules and chemokines, which mediate the recruitment of circulating monocytes. Accumulation of monocytes and monocyte-derived phagocytes in the wall of large arteries leads to chronic inflammation and the development and progression of atherosclerosis. The lesion experiences the following steps: foam cell formation, fatty streak accumulation, migration and proliferation of vascular smooth muscle cells, and fibrous cap formation. Finally, the rupture of the unstable fibrous cap causes thrombosis in complications of advanced lesions that leads to unstable coronary syndromes, myocardial infarction and stroke. MicroRNAs have recently emerged as a novel class of gene regulators at the post-transcriptional level. Several functions of vascular cells, such as cell differentiation, contraction, migration, proliferation and inflammation that are involved in angiogenesis, neointimal formation and lipid metabolism underlying various vascular diseases, have been found to be regulated by microRNAs and are described in the present review as well as their potential therapeutic application. PMID:23713860

Surfaceome and Proteosurfaceome in Parietal Monoderm Bacteria: Focus on Protein Cell-Surface Display

PubMed Central

Desvaux, Mickaël; Candela, Thomas; Serror, Pascale

2018-01-01

The cell envelope of parietal monoderm bacteria (archetypal Gram-positive bacteria) is formed of a cytoplasmic membrane (CM) and a cell wall (CW). While the CM is composed of phospholipids, the CW is composed at least of peptidoglycan (PG) covalently linked to other biopolymers, such as teichoic acids, polysaccharides, and/or polyglutamate. Considering the CW is a porous structure with low selective permeability contrary to the CM, the bacterial cell surface hugs the molecular figure of the CW components as a well of the external side of the CM. While the surfaceome corresponds to the totality of the molecules found at the bacterial cell surface, the proteinaceous complement of the surfaceome is the proteosurfaceome. Once translocated across the CM, secreted proteins can either be released in the extracellular milieu or exposed at the cell surface by associating to the CM or the CW. Following the gene ontology (GO) for cellular components, cell-surface proteins at the CM can either be integral (GO: 0031226), i.e., the integral membrane proteins, or anchored to the membrane (GO: 0046658), i.e., the lipoproteins. At the CW (GO: 0009275), cell-surface proteins can be covalently bound, i.e., the LPXTG-proteins, or bound through weak interactions to the PG or wall polysaccharides, i.e., the cell wall binding proteins. Besides monopolypeptides, some proteins can associate to each other to form supramolecular protein structures of high molecular weight, namely the S-layer, pili, flagella, and cellulosomes. After reviewing the cell envelope components and the different molecular mechanisms involved in protein attachment to the cell envelope, perspectives in investigating the proteosurfaceome in parietal monoderm bacteria are further discussed. PMID:29491848

Stimuli-responsive protamine-based biodegradable nanocapsules for enhanced bioavailability and intracellular delivery of anticancer agents

NASA Astrophysics Data System (ADS)

Radhakrishnan, Krishna; Thomas, Midhun B.; Pulakkat, Sreeranjini; Gnanadhas, Divya P.; Chakravortty, Dipshikha; Raichur, Ashok M.

2015-08-01

Enzyme- and pH-responsive polyelectrolyte nanocapsules having diameters in the range of 200 ± 20 nm were fabricated by means of Layer-by-Layer assembly of biopolymers, protamine, and heparin, and then loaded with anticancer drug doxorubicin. The incorporation of the FDA-approved peptide drug protamine as a wall component rendered the capsules responsive to enzyme stimuli. The stimuli-responsive drug release from these nanocapsules was evaluated, and further modulation of capsule permeability to avoid premature release was demonstrated by crosslinking the wall components. The interaction of the nanocapsules with cancer cells was studied using MCF-7 breast cancer cells. These capsules were readily internalized and disintegrated inside the cells, culminating in the release of the loaded doxorubicin and subsequent cell death as observed by confocal microscopy and MTT Assay. The bioavailability studies performed using BALB/c mice revealed that the encapsulated doxorubicin exhibited enhanced bioavailability compared to free doxorubicin. Our results indicate that this stimuli-responsive system fabricated from clinically used FDA-approved molecules and exhibiting minimal premature release has great potential for drug-delivery applications.

Biological construction of single-walled carbon nanotube electron transfer pathways in dye-sensitized solar cells.

PubMed

Inoue, Ippei; Watanabe, Kiyoshi; Yamauchi, Hirofumi; Ishikawa, Yasuaki; Yasueda, Hisashi; Uraoka, Yukiharu; Yamashita, Ichiro

2014-10-01

We designed and mass-produced a versatile protein supramolecule that can be used to manufacture a highly efficient dye-sensitized solar cell (DSSC). Twelve single-walled carbon-nanotube (SWNT)-binding and titanium-mineralizing peptides were genetically integrated on a cage-shaped dodecamer protein (CDT1). A process involving simple mixing of highly conductive SWNTs with CDT1 followed by TiO2 biomineralization produces a high surface-area/weight TiO2 -(anatase)-coated intact SWNT nanocomposite under environmentally friendly conditions. A DSSC with a TiO2 photoelectrode containing 0.2 wt % of the SWNT-TiO2 nanocomposite shows a current density improvement by 80% and a doubling of the photoelectric conversion efficiency. The SWNT-TiO2 nanocomposite transfers photon-generated electrons from dye molecules adsorbed on the TiO2 to the anode electrode swiftly. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A new methodology to obtain wine yeast strains overproducing mannoproteins.

PubMed

Quirós, Manuel; Gonzalez-Ramos, Daniel; Tabera, Laura; Gonzalez, Ramon

2010-04-30

Yeast mannoproteins are highly glycosylated proteins that are covalently bound to the beta-1,3-glucan present in the yeast cell wall. Among their outstanding enological properties, yeast mannoproteins contribute to several aspects of wine quality by protecting against protein haze, reducing astringency, retaining aroma compounds and stimulating growth of lactic-acid bacteria. The development of a non-recombinant method to obtain enological yeast strains overproducing mannoproteins would therefore be very useful. Our previous experience on the genetic determinants of the release of these molecules by Saccharomyces cerevisiae has allowed us to propose a new methodology to isolate and characterize wine yeast that overproduce mannoproteins. The described methodology is based on the resistance of the killer 9 toxin produced by Williopsis saturnus, a feature linked to an altered biogenesis of the yeast cell wall. Copyright 2010 Elsevier B.V. All rights reserved.

Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

DOE PAGES

Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; ...

2016-01-01

Here, we have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single moleculemore » super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.« less

Gum arabic glycoprotein is a twisted hairy rope. A new model based on O-galactosylhydroxyproline as the polysaccharide attachment site

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

Wu Qi; Fong, C.; Lamport, D.T.A.

1991-07-01

Separation of the wound exudate from Acacia senegal (L.) Willd., gum arabic, on a preparative Superose-6 column gave two major fractions: a high molecular weight gum arabic glyco-protein (GAGP) containing about 90% carbohydrate and a lower molecular weight heterogeneous gum arabic polysaccharide fraction. Hydrogen fluoride-deglycosylation of GAGP gave a large hydroxyproline-rich polypeptide backbone (dGAGP). Alkaline hydrolysis of GAGP showed that most of the carbohydrate was attached to the polypeptide backbone as small hydroxyproline (Hyp)-polysaccharide substituents. The data imply a rodlike molecule with numerous small polysaccharide substituents (attached to 24% of the Hyp residues), regularly arranged along a highly periodic polypeptidemore » backbone based, hypothetically, on a 10 to 12 residue repetitive peptide motif. Thus, a simple statistical model of the gum arabic glycoprotein predicts a repeating polysaccharide substituents will maximize intramolecular hydrogen bonding if aligned along the long axis of the molecule, forming in effect a twisted hairy rope. Electron micrographs of rotary shadowed GAGP molecules support that prediction and may also explain show such apparently large molecules can exit the cell by endwise reptation through the small pores of the primary cell wall.« less

Benzoxazolinone detoxification by N-Glucosylation: The multi-compartment-network of Zea mays L.

PubMed Central

Schulz, Margot; Filary, Barbara; Kühn, Sabine; Colby, Thomas; Harzen, Anne; Schmidt, Jürgen; Sicker, Dieter; Hennig, Lothar; Hofmann, Diana; Disko, Ulrich; Anders, Nico

2016-01-01

ABSTRACT The major detoxification product in maize roots after 24 h benzoxazolin-2(3H)-one (BOA) exposure was identified as glucoside carbamate resulting from rearrangement of BOA-N-glucoside, but the pathway of N-glucosylation, enzymes involved and the site of synthesis were previously unknown. Assaying whole cell proteins revealed the necessity of H2O2 and Fe2+ ions for glucoside carbamate production. Peroxidase produced BOA radicals are apparently formed within the extraplastic space of the young maize root. Radicals seem to be the preferred substrate for N-glucosylation, either by direct reaction with glucose or, more likely, the N-glucoside is released by glucanase/glucosidase catalyzed hydrolysis from cell wall components harboring fixed BOA. The processes are accompanied by alterations of cell wall polymers. Glucoside carbamate accumulation could be suppressed by the oxireductase inhibitor 2-bromo-4´-nitroacetophenone and by peroxidase inhibitor 2,3-butanedione. Alternatively, activated BOA molecules with an open heterocycle may be produced by microorganisms (e.g., endophyte Fusarium verticillioides) and channeled for enzymatic N-glucosylation. Experiments with transgenic Arabidopsis lines indicate a role of maize glucosyltransferase BX9 in BOA-N-glycosylation. Western blots with BX9 antibody demonstrate the presence of BX9 in the extraplastic space. Proteomic analyses verified a high BOA responsiveness of multiple peroxidases in the apoplast/cell wall. BOA incubations led to shifting, altered abundances and identities of the apoplast and cell wall located peroxidases, glucanases, glucosidases and glutathione transferases (GSTs). GSTs could function as glucoside carbamate transporters. The highly complex, compartment spanning and redox-regulated glucoside carbamate pathway seems to be mainly realized in Poaceae. In maize, carbamate production is independent from benzoxazinone synthesis. PMID:26645909

Benzoxazolinone detoxification by N-Glucosylation: The multi-compartment-network of Zea mays L.

PubMed

Schulz, Margot; Filary, Barbara; Kühn, Sabine; Colby, Thomas; Harzen, Anne; Schmidt, Jürgen; Sicker, Dieter; Hennig, Lothar; Hofmann, Diana; Disko, Ulrich; Anders, Nico

2016-01-01

The major detoxification product in maize roots after 24 h benzoxazolin-2(3H)-one (BOA) exposure was identified as glucoside carbamate resulting from rearrangement of BOA-N-glucoside, but the pathway of N-glucosylation, enzymes involved and the site of synthesis were previously unknown. Assaying whole cell proteins revealed the necessity of H2O2 and Fe(2+) ions for glucoside carbamate production. Peroxidase produced BOA radicals are apparently formed within the extraplastic space of the young maize root. Radicals seem to be the preferred substrate for N-glucosylation, either by direct reaction with glucose or, more likely, the N-glucoside is released by glucanase/glucosidase catalyzed hydrolysis from cell wall components harboring fixed BOA. The processes are accompanied by alterations of cell wall polymers. Glucoside carbamate accumulation could be suppressed by the oxireductase inhibitor 2-bromo-4´-nitroacetophenone and by peroxidase inhibitor 2,3-butanedione. Alternatively, activated BOA molecules with an open heterocycle may be produced by microorganisms (e.g., endophyte Fusarium verticillioides) and channeled for enzymatic N-glucosylation. Experiments with transgenic Arabidopsis lines indicate a role of maize glucosyltransferase BX9 in BOA-N-glycosylation. Western blots with BX9 antibody demonstrate the presence of BX9 in the extraplastic space. Proteomic analyses verified a high BOA responsiveness of multiple peroxidases in the apoplast/cell wall. BOA incubations led to shifting, altered abundances and identities of the apoplast and cell wall located peroxidases, glucanases, glucosidases and glutathione transferases (GSTs). GSTs could function as glucoside carbamate transporters. The highly complex, compartment spanning and redox-regulated glucoside carbamate pathway seems to be mainly realized in Poaceae. In maize, carbamate production is independent from benzoxazinone synthesis.

Interaction between hyaluronan and CD44 in the development of dimethylnitrosamine-induced liver cirrhosis.

PubMed

Satoh, T; Ichida, T; Matsuda, Y; Sugiyama, M; Yonekura, K; Ishikawa, T; Asakura, H

2000-04-01

A significant increase in serum hyaluronan (HA) levels has been reported in patients with liver cirrhosis. This mechanism is not yet clear, and receptors for HA have not been characterized. In this study, we examined the expression of both HA and its receptors, CD44 and intercellular adhesion molecule-1 (ICAM-1), in dimethylnitrosamine-induced liver cirrhosis. Using biotinylated HA binding protein, HA was detected in the area of periportal fibrosis and around the sinusoidal wall where hepatic fibrosis was developing. Electron microscopy revealed that HA was localized on Ito cells and sinusoidal endothelial cells (SEC). Conversely, CD44, which was only expressed weakly in normal liver, was present in large amounts in cirrhotic liver. The distribution pattern of CD44 was similar to that of HA, however, CD44 was mainly localized on the infiltrating lymphocytes and Kupffer cells. Moreover, CD44 was detected on part of factor VIII-positive SEC. Intercellular adhesion molecule-1, another receptor for HA, was detected on the surface of hepatocytes and around the sinusoidal wall in cirrhotic liver, but its distribution was not accompanied by expression of HA. With respect to CD44 isoforms, the standard form m-RNA predominated in both normal and cirrhotic liver. Variant pMeta-1 mRNA was detected at low levels. An interaction between HA and CD44 may play a role in the recruitment of numerous infiltrating cells and HA accumulation in hepatic sinusoids. Together with phenotypic changes in the SEC, these results may lead to a disturbance in the elimination of HA during the progression of liver cirrhosis.

Shear-induced desorption of isolated polymer molecules from a planar wall

NASA Astrophysics Data System (ADS)

Dutta, Sarit; Dorfman, Kevin; Kumar, Satish

2014-03-01

Shear-induced desorption of isolated polymer molecules is studied using Brownian dynamics simulations. The polymer molecules are modeled as freely jointed bead-spring chains interacting with a planar wall via a short-range potential. The simulations include both intrachain and chain-wall hydrodynamic interactions. Shear flow is found to cause chain flattening, resulting at low shear rates in an increased fraction of chain segments bound to the wall. However, above a critical shear rate the chains desorb completely. The desorption process is nucleated by random protrusions in the shear gradient direction which evolve under the combined effect of drag, hydrodynamic interaction, and vorticity-induced rotation, and subsequently lead to recapture. Above the critical shear rate, these protrusions grow in length until the entire chain is peeled off the wall. For free-draining chains, the protrusions are not sustained and no desorption is observed even at shear rates much higher than the critical value. These simulations can help in interpreting experiments on shear-induced desorption of polymer films and brushes.

Nano Sponges for Drug Delivery and Medicinal Applications

NASA Technical Reports Server (NTRS)

Tour, James M.; Lucente-Schultz, Rebecca; Leonard, Ashley; Kosynkin, Dimitry V.; Price, Brandi Katherine; Hudson, Jared L.; Conyers, Jodie L., Jr.; Moore, Valerie C.; Casscells, S. Ward; Myers, Jeffrey N.;

Differences in Cell Morphometry, Cell Wall Topography and Gp70 Expression Correlate with the Virulence of Sporothrix brasiliensis Clinical Isolates

PubMed Central

Castro, Rafaela A.; Kubitschek-Barreira, Paula H.; Teixeira, Pedro A. C.; Sanches, Glenda F.; Teixeira, Marcus M.; Quintella, Leonardo P.; Almeida, Sandro R.; Costa, Rosane O.; Camargo, Zoilo P.; Felipe, Maria S. S.; de Souza, Wanderley; Lopes-Bezerra, Leila M.

2013-01-01

Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes. PMID:24116065

Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses

NASA Astrophysics Data System (ADS)

Moon, James J.; Suh, Heikyung; Bershteyn, Anna; Stephan, Matthias T.; Liu, Haipeng; Huang, Bonnie; Sohail, Mashaal; Luo, Samantha; Ho Um, Soong; Khant, Htet; Goodwin, Jessica T.; Ramos, Jenelyn; Chiu, Wah; Irvine, Darrell J.

2011-03-01

Vaccines based on recombinant proteins avoid the toxicity and antivector immunity associated with live vaccine (for example, viral) vectors, but their immunogenicity is poor, particularly for CD8+ T-cell responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8+ T-cell responses comparable to those for live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. Interbilayer-crosslinked vesicles stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying vesicles form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to those for the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for therapeutic protein delivery.

Combined Cytological and Transcriptomic Analysis Reveals a Nitric Oxide Signaling Pathway Involved in Cold-Inhibited Camellia sinensis Pollen Tube Growth

PubMed Central

Wang, Weidong; Sheng, Xianyong; Shu, Zaifa; Li, Dongqin; Pan, Junting; Ye, Xiaoli; Chang, Pinpin; Li, Xinghui; Wang, Yuhua

2016-01-01

Nitric oxide (NO) as a signaling molecule plays crucial roles in many abiotic stresses in plant development processes, including pollen tube growth. Here, the signaling networks dominated by NO during cold stress that inhibited Camellia sinensis pollen tube growth are investigated in vitro. Cytological analysis show that cold-induced NO is involved in the inhibition of pollen tube growth along with disruption of the cytoplasmic Ca2+ gradient, increase in ROS content, acidification of cytoplasmic pH and abnormalities in organelle ultrastructure and cell wall component distribution in the pollen tube tip. Furthermore, differentially expressed genes (DEGs)-related to signaling pathway, such as NO synthesis, cGMP, Ca2+, ROS, pH, actin, cell wall, and MAPK cascade signal pathways, are identified and quantified using transcriptomic analyses and qRT-PCR, which indicate a potential molecular mechanism for the above cytological results. Taken together, these findings suggest that a complex signaling network dominated by NO, including Ca2+, ROS, pH, RACs signaling and the crosstalk among them, is stimulated in the C. sinensis pollen tube in response to cold stress, which further causes secondary and tertiary alterations, such as ultrastructural abnormalities in organelles and cell wall construction, ultimately resulting in perturbed pollen tube extension. PMID:27148289

A novel beta-glucosidase from the cell wall of maize (Zea mays L.): rapid purification and partial characterization

NASA Technical Reports Server (NTRS)

Nematollahi, W. P.; Roux, S. J.

1999-01-01

Plants have a variety of glycosidic conjugates of hormones, defense compounds, and other molecules that are hydrolyzed by beta-glucosidases (beta-D-glucoside glucohydrolases, E.C. 3.2.1.21). Workers have reported several beta-glucosidases from maize (Zea mays L.; Poaceae), but have localized them mostly by indirect means. We have purified and partly characterized a 58-Ku beta-glucosidase from maize, which we conclude from a partial sequence analysis, from kinetic data, and from its localization is not identical to any of those already reported. A monoclonal antibody, mWP 19, binds this enzyme, and localizes it in the cell walls of maize coleoptiles. An earlier report showed that mWP19 inhibits peroxidase activity in crude cell wall extracts and can immunoprecipitate peroxidase activity from these extracts, yet purified preparations of the 58 Ku protein had little or no peroxidase activity. The level of sequence similarity between beta-glucosidases and peroxidases makes it unlikely that these enzymes share epitopes in common. Contrary to a previous conclusion, these results suggest that the enzyme recognized by mWP19 is not a peroxidase, but there is a wall peroxidase closely associated with the 58 Ku beta-glucosidase in crude preparations. Other workers also have co-purified distinct proteins with beta-glucosidases. We found no significant charge in the level of immunodetectable beta-glucosidase in mesocotyls or coleoptiles that precedes the red light-induced changes in the growth rate of these tissues.

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

Bai, Rui; Yi, Shaoqiong; Zhang, Xuejie

Highlights: • We evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations. • AFM was used to measure single-molecule binding ability on living cells. • The SNP of ICAM-1 may induce changes in expressions rather than single-molecule binding ability. - Abstract: Atherosclerosis (As) is characterized by chronic inflammation and is a major cause of human mortality. ICAM-1-mediated adhesion of leukocytes in vessel walls plays an important role in the pathogenesis of atherosclerosis. Two single nucleotide polymorphisms (SNPs) of human intercellular adhesion molecule-1 (ICAM-1), G241R and K469E, are associated with a number of inflammatory diseases. SNP inducedmore » changes in ICAM-1 function rely not only on the expression level but also on the single-molecule binding ability which may be affected by single molecule conformation variations such as protein splicing and folding. Previous studies have shown associations between G241R/K469E polymorphisms and ICAM-1 gene expression. Nevertheless, few studies have been done that focus on the single-molecule forces of the above SNPs and their ligands. In the current study, we evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations – GK (G241/K469), GE (G241/E469), RK (R241/K469) and RE (R241/E469). No difference in adhesion ability was observed via cell adhesion assay or atomic force microscopy (AFM) measurement when comparing the GK, GE, RK, or RE genotypes of ICAM-1 to each other. On the other hand, flow cytometry suggested that there was significantly higher expression of GE genotype of ICAM-1 on transfected CHO cells. Thus, we concluded that genetic susceptibility to diseases related to ICAM-1 polymorphisms, G241R or K469E, might be due to the different expressions of ICAM-1 variants rather than to the single-molecule binding ability of ICAM-1.« less

Molecular mapping of the cell wall polysaccharides of the human pathogen Streptococcus agalactiae

NASA Astrophysics Data System (ADS)

Beaussart, Audrey; Péchoux, Christine; Trieu-Cuot, Patrick; Hols, Pascal; Mistou, Michel-Yves; Dufrêne, Yves F.

2014-11-01

The surface of many bacterial pathogens is covered with polysaccharides that play important roles in mediating pathogen-host interactions. In Streptococcus agalactiae, the capsular polysaccharide (CPS) is recognized as a major virulence factor while the group B carbohydrate (GBC) is crucial for peptidoglycan biosynthesis and cell division. Despite the important roles of CPS and GBC, there is little information available on the molecular organization of these glycopolymers on the cell surface. Here, we use atomic force microscopy (AFM) and transmission electron microscopy (TEM) to analyze the nanoscale distribution of CPS and GBC in wild-type (WT) and mutant strains of S. agalactiae. TEM analyses reveal that in WT bacteria, peptidoglycan is covered with a very thin (few nm) layer of GBC (the ``pellicle'') overlaid by a 15-45 nm thick layer of CPS (the ``capsule''). AFM-based single-molecule mapping with specific antibody probes shows that CPS is exposed on WT cells, while it is hardly detected on mutant cells impaired in CPS production (ΔcpsE mutant). By contrast, both TEM and AFM show that CPS is over-expressed in mutant cells altered in GBC expression (ΔgbcO mutant), indicating that the production of the two surface glycopolymers is coordinated in WT cells. In addition, AFM topographic imaging and molecular mapping with specific lectin probes demonstrate that removal of CPS (ΔcpsE), but not of GBC (ΔgbcO), leads to the exposure of peptidoglycan, organized into 25 nm wide bands running parallel to the septum. These results indicate that CPS forms a homogeneous barrier protecting the underlying peptidoglycan from environmental exposure, while the presence of GBC does not prevent peptidoglycan detection. This work shows that single-molecule AFM, combined with high-resolution TEM, represents a powerful platform for analysing the molecular arrangement of the cell wall polymers of bacterial pathogens.

Central Nervous System Fibrosis Is Associated with Fibrocyte-Like Infiltrates

PubMed Central

Aldrich, Amy; Kielian, Tammy

2011-01-01

Fibrotic wall formation is essential for limiting pathogen dissemination during brain abscess development. However, little is known about the regulation of fibrotic processes in the central nervous system (CNS). Most CNS injury responses are associated with hypertrophy of resident astrocytes, a process termed reactive gliosis. Studies of fibrosis outside the CNS have identified two bone marrow–derived cell types, fibrocytes and alternatively activated M2 macrophages, as key mediators of fibrosis. The current study used bone marrow chimeras generated from green fluorescent protein transgenic mice to evaluate the appearance of these cell types and whether bone marrow–derived cells were capable of acquiring fibrotic characteristics during brain abscess development. Immunofluorescence staining revealed partial overlap between green fluorescent protein, α-smooth muscle actin, and procollagen, suggesting that a population of cells forming the brain abscess capsule originate from a bone marrow precursor. In addition, the influx of fibrocyte-like cells into brain abscesses immediately preceded the onset of fibrotic encapsulation. Fibrotic wall formation was also associated with increased numbers of alternatively activated M2 microglia and macrophages. To our knowledge, this is the first study demonstrating that bone marrow–derived infiltrates are capable of expressing fibrotic molecules during CNS inflammation. PMID:22015460

Wood decay by brown-rot fungi : changes in pore structure and cell wall volume

Treesearch

Douglas S. Flournoy; T. Kent Kirk; T.L. Highley

1991-01-01

Sweetgum (Liquidambar styraciflua L.) wood blocks were decayed by Postia (= Poria) placenta in soilblock cultures. Decay was terminated at various weight losses, and the pore volumes available to four low molecular weight molecules, (water, 4 Å,; glucose, 8 Å,; maltose, 10 Å; and raffinose, 128,) and three dextrans (Mr 6,000, 38 Å; 11,200, 51 Å; nd 17,500, 61 Å) were...

Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases.

PubMed

Wheeler, Richard; Turner, Robert D; Bailey, Richard G; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A S; Hayhurst, Emma J; Horsburgh, Malcolm; Hobbs, Jamie K; Foster, Simon J

2015-07-28

Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. Understanding bacterial growth and division is a fundamental problem, and knowledge in this area underlies the treatment of many infectious diseases. Almost all bacteria are surrounded by a macromolecule of peptidoglycan that encloses the cell and maintains shape, and bacterial cells must increase the size of this molecule in order to enlarge themselves. This requires not only the insertion of new peptidoglycan monomers, a process targeted by antibiotics, including penicillin, but also breakage of existing bonds, a potentially hazardous activity for the cell. Using Staphylococcus aureus, we have identified a set of enzymes that are critical for cellular enlargement. We show that these enzymes are required for normal growth and define the mechanism through which cellular enlargement is accomplished, i.e., by breaking bonds in the peptidoglycan, which reduces the stiffness of the cell wall, enabling it to stretch and expand, a process that is likely to be fundamental to many bacteria. Copyright © 2015 Wheeler et al.

[Review on characteristics and detecting assay of bacterial endotoxin contamination in water environment].

PubMed

Zhang, Can; Liu, Wen-Jun; Zhang, Ming-Lu; Tian, Fang; Yang, Yi; An, Dai-Zhi

2014-04-01

Endotoxins, also known as lipopolysaccharide complexes, are anchored in the outer membrane cell wall of most Gram-negative bacteria and some cyanobacteria. They are continuously released to environment during cell decay. Being common pyrogens and highly immunogenic molecules, endotoxins are related to many human diseases. Due to the tolerances and thermo-stability of endotoxin molecules, they were hard to be removed by common methods. The health risk caused by the endotoxin contamination in drinking water and water environment by various exposure pathways have attracted more and more attention in recent years. In this paper, the physical and chemical properties, biological activities and detection assay of the endotoxin contamination were reviewed, and interfere factors of the main assay, the LAL/TAL (Limulus amebocyte lysate/Tachypleus amebocyte lysate) assay, for detecting endotoxin in water sample were investigated, and the development tendency of the endotoxin detection assay was analyzed.

The effect of carrier gas flow rate and source cell temperature on low pressure organic vapor phase deposition simulation by direct simulation Monte Carlo method

PubMed Central

Wada, Takao; Ueda, Noriaki

2013-01-01

The process of low pressure organic vapor phase deposition (LP-OVPD) controls the growth of amorphous organic thin films, where the source gases (Alq3 molecule, etc.) are introduced into a hot wall reactor via an injection barrel using an inert carrier gas (N2 molecule). It is possible to control well the following substrate properties such as dopant concentration, deposition rate, and thickness uniformity of the thin film. In this paper, we present LP-OVPD simulation results using direct simulation Monte Carlo-Neutrals (Particle-PLUS neutral module) which is commercial software adopting direct simulation Monte Carlo method. By estimating properly the evaporation rate with experimental vaporization enthalpies, the calculated deposition rates on the substrate agree well with the experimental results that depend on carrier gas flow rate and source cell temperature. PMID:23674843

Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

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

Zhang, Wenyao; Li, Xuezhong; Xu, Tong

Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferationmore » and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.« less

Mechanisms of Vascular Smooth Muscle Contraction and the Basis for Pharmacologic Treatment of Smooth Muscle Disorders

PubMed Central

Brozovich, F.V.; Nicholson, C.J.; Degen, C.V.; Gao, Yuan Z.; Aggarwal, M.

2016-01-01

The smooth muscle cell directly drives the contraction of the vascular wall and hence regulates the size of the blood vessel lumen. We review here the current understanding of the molecular mechanisms by which agonists, therapeutics, and diseases regulate contractility of the vascular smooth muscle cell and we place this within the context of whole body function. We also discuss the implications for personalized medicine and highlight specific potential target molecules that may provide opportunities for the future development of new therapeutics to regulate vascular function. PMID:27037223

Direct evidence for activated CD8+ T cell transmigration across portal vein endothelial cells in liver graft rejection.

PubMed

Kariya, Taro; Ueta, Hisashi; Xu, Xue-Dong; Koga, Daisuke; Ezaki, Taichi; Yu, Enqiao; Kusumi, Satoshi; Kitazawa, Yusuke; Sawanobori, Yasushi; Ushiki, Tatsuo; Issekutz, Thomas; Matsuno, Kenjiro

2016-10-01

Lymphocyte recruitment into the portal tract is crucial not only for homeostatic immune surveillance but also for many liver diseases. However, the exact route of entry for lymphocytes into portal tract is still obscure. We investigated this question using a rat hepatic allograft rejection model. A migration route was analyzed by immunohistological methods including a recently developed scanning electron microscopy method. Transmigration-associated molecules such as selectins, integrins, and chemokines and their receptors expressed by hepatic vessels and recruited T-cells were analyzed by immunohistochemistry and flow cytometry. The immunoelectron microscopic analysis clearly showed CD8β(+) cells passing through the portal vein (PV) endothelia. Furthermore, the migrating pathway seemed to pass through the endothelial cell body. Local vascular cell adhesion molecule-1 (VCAM-1) expression was induced in PV endothelial cells from day 2 after liver transplantation. Although intercellular adhesion molecule-1 (ICAM-1) expression was also upregulated, it was restricted to sinusoidal endothelia. Recipient T-cells in the graft perfusate were CD25(+)CD44(+)ICAM-1(+)CXCR3(+)CCR5(-) and upregulated α4β1 or αLβ2 integrins. Immunohistochemistry showed the expression of CXCL10 in donor MHCII(high) cells in the portal tract as well as endothelial walls of PV. We show for the first time direct evidence of T-cell transmigration across PV endothelial cells during hepatic allograft rejection. Interactions between VCAM-1 on endothelia and α4β1 integrin on recipient effector T-cells putatively play critical roles in adhesion and transmigration through endothelia. A chemokine axis of CXCL10 and CXCR3 also may be involved.

FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow

PubMed Central

Zhu, Shu; Travers, Richard J.; Morrissey, James H.

2015-01-01

Factor XIIa (FXIIa) and factor XIa (FXIa) contribute to thrombosis in animal models, whereas platelet-derived polyphosphate (polyP) may potentiate contact or thrombin-feedback pathways. The significance of these mediators in human blood under thrombotic flow conditions on tissue factor (TF) –bearing surfaces remains inadequately resolved. Human blood (corn trypsin inhibitor treated [4 μg/mL]) was tested by microfluidic assay for clotting on collagen/TF at TF surface concentration ([TF]wall) from ∼0.1 to 2 molecules per μm2. Anti-FXI antibodies (14E11 and O1A6) or polyP-binding protein (PPXbd) were used to block FXIIa-dependent FXI activation, FXIa-dependent factor IX (FIX) activation, or platelet-derived polyP, respectively. Fibrin formation was sensitive to 14E11 at 0 to 0.1 molecules per µm2 and sensitive to O1A6 at 0 to 0.2 molecules per µm2. However, neither antibody reduced fibrin generation at ∼2 molecules per µm2 when the extrinsic pathway became dominant. Interestingly, PPXbd reduced fibrin generation at low [TF]wall (0.1 molecules per µm2) but not at zero or high [TF]wall, suggesting a role for polyP distinct from FXIIa activation and requiring low extrinsic pathway participation. Regardless of [TF]wall, PPXbd enhanced fibrin sensitivity to tissue plasminogen activator and promoted clot retraction during fibrinolysis concomitant with an observed PPXbd-mediated reduction of fibrin fiber diameter. This is the first detection of endogenous polyP function in human blood under thrombotic flow conditions. When triggered by low [TF]wall, thrombosis may be druggable by contact pathway inhibition, although thrombolytic susceptibility may benefit from polyP antagonism regardless of [TF]wall. PMID:26136249

Influence of Wall Material on VUV Emission from Hydrogen Plasma in H- Source

NASA Astrophysics Data System (ADS)

Bacal, M.; Glass-Maujean, M.; Ivanov, A. A., Jr; Nishiura, M.; Sasao, M.; Wada, M.

2002-11-01

The study of VUV emission from a hydrogen plasma produced in a filament discharge in a magnetic multicusp device showed that the use of tantalum and tungsten filaments leads to significant differences in the spectra. The effect of the filament material is interpreted in terms of the fresh film of this material, deposited on the wall. The synthetic spectrum convoluted with our apparatus function for the conditions of this experiment (gas temperature 500 K, electron energy 100 eV) agrees roughly well with the spectrum obtained with tungsten covered walls, but not with the spectrum obtained with tantalum covered walls. We show that in the case of tungsten covered walls the E-V singlet excitation is indeed a two-step Franck-Condon transition, going through either B or C state from an initial H2 molecule with v"=0, added to a Franck-Condon transition to highly excited states cascading to the B or C states. The excitation process to high v" states in the case of tantalum covered walls is a three step process, in which the first step is the formation by recombinative desorption on the wall of a vibrationally excited molecule with v"=1 or 2, which serves as the initial molecule in the subsequent E-V excitation through the B state. The results indicate a larger recombination coefficient of atoms on the tantalum covered wall.

Contribution of vascular cell-derived cytokines to innate and inflammatory pathways in atherogenesis

PubMed Central

Loppnow, Harald; Buerke, Michael; Werdan, Karl; Rose-John, Stefan

2011-01-01

Abstract Inflammation is a central element of atherogenesis. Innate pathways contribute to vascular inflammation. However, the initial molecular process(es) starting atherogenesis remain elusive. The various risk factors, represented by particular compounds (activators), may cause altered cellular functions in the endothelium (e.g. vascular endothelial cell activation or -dysfunction), in invading cells (e.g. inflammatory mediator production) or in local vessel wall cells (e.g. inflammatory mediators, migration), thereby triggering the innate inflammatory process. The cellular components of innate immunology include granulocytes, natural killer cells and monocytes. Among the molecular innate constituents are innate molecules, such as the toll-like receptors or innate cytokines. Interleukin-1 (IL-1) and IL-6 are among the innate cytokines. Cytokines are potent activators of a great number of cellular functions relevant to maintain or commove homeostasis of the vessel wall. Within the vessel wall, vascular smooth muscle cells (SMCs) can significantly contribute to the cytokine-dependent inflammatory network by: (i) production of cytokines, (ii) response to cytokines and (iii) cytokine-mediated interaction with invading leucocytes. The cytokines IL-1 and IL-6 are involved in SMC-leucocyte interaction. The IL-6 effects are proposed to be mediated by trans-signalling. Dysregulated cellular functions resulting from dysregulated cytokine production may be the cause of cell accumulation, subsequent low-density lipoprotein accumulation and deposition of extracellular matrix (ECM). The deposition of ECM, increased accumulation of leucocytes and altered levels of inflammatory mediators may constitute an ‘innate-immunovascular-memory’ resulting in an ever-growing response to anew invasion. Thus, SMC-fostered inflammation, promoted by invading innate cells, may be a potent component for development and acceleration of atherosclerosis. PMID:21199323

Calculation of ground state rotational populations for kinetic gas homonuclear diatomic molecules including electron-impact excitation and wall collisions.

PubMed

Farley, David R

2010-09-07

A model has been developed to calculate the ground state rotational populations of homonuclear diatomic molecules in kinetic gases, including the effects of electron-impact excitation, wall collisions, and gas feed rate. The equations are exact within the accuracy of the cross sections used and of the assumed equilibrating effect of wall collisions. It is found that the inflow of feed gas and equilibrating wall collisions can significantly affect the rotational distribution in competition with nonequilibrating electron-impact effects. The resulting steady-state rotational distributions are generally Boltzmann for N≥3, with a rotational temperature between the wall and feed gas temperatures. The N=0,1,2 rotational level populations depend sensitively on the relative rates of electron-impact excitation versus wall collision and gas feed rates.

DNA-templated synthesis of Pt nanoparticles on single-walled carbon nanotubes.

PubMed

Dong, Lifeng

2009-11-18

A series of electron microscopy characterizations demonstrate that single-stranded deoxyribonucleic acid (ssDNA) can bind to nanotube surfaces and disperse bundled single-walled carbon nanotubes (SWCNTs) into individual tubes. The ssDNA molecules on the nanotube surfaces demonstrate various morphologies, such as aggregated clusters and spiral wrapping around a nanotube with different pitches and spaces, indicating that the morphology of the SWCNT/DNA hybrids is not related solely to the base sequence of the ssDNA or the chirality or the diameter of the nanotubes. In addition to serving as a non-covalent dispersion agent, the ssDNA molecules bonded to the nanotube surface can provide addresses for localizing Pt(II) complexes along the nanotubes. The Pt nanoparticles obtained by a reduction of the Pt2+-DNA adducts are crystals with a size of < or =1-2 nm. These results expand our understanding of the interactions between ssDNA and SWCNTs and provide an efficient approach for positioning Pt and other metal particles, with uniform sizes and without aggregations, along the nanotube surfaces for applications in direct ethanol/methanol fuel cells and nanoscale electronics.

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

PubMed Central

Pillai, Karthik; Navarro Arzate, Fernando; Zhang, Wei; Renneckar, Scott

2014-01-01

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall. PMID:24961302

Jasmonate-dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors.

PubMed

Taurino, Marco; Abelenda, Jose A; Río-Alvarez, Isabel; Navarro, Cristina; Vicedo, Begonya; Farmaki, Theodora; Jiménez, Pedro; García-Agustín, Pilar; López-Solanilla, Emilia; Prat, Salomé; Rojo, Enrique; Sánchez-Serrano, José J; Sanmartín, Maite

2014-02-01

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell-wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell-wall composition to reinforce this defensive barrier remains unknown. The enzyme 13-allene oxide synthase (13-AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13-AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13-AOS enzymes. Indeed, transgenic potato plants lacking both St13-AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound-responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild-type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell-wall pectin composition between wild-type and CoAOS1/2 plants. Importantly, wild-type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Assembly mechanism of FCT region type 1 pili in serotype M6 Streptococcus pyogenes.

PubMed

Nakata, Masanobu; Kimura, Keiji Richard; Sumitomo, Tomoko; Wada, Satoshi; Sugauchi, Akinari; Oiki, Eiji; Higashino, Miharu; Kreikemeyer, Bernd; Podbielski, Andreas; Okahashi, Nobuo; Hamada, Shigeyuki; Isoda, Ryutaro; Terao, Yutaka; Kawabata, Shigetada

2011-10-28

The human pathogen Streptococcus pyogenes produces diverse pili depending on the serotype. We investigated the assembly mechanism of FCT type 1 pili in a serotype M6 strain. The pili were found to be assembled from two precursor proteins, the backbone protein T6 and ancillary protein FctX, and anchored to the cell wall in a manner that requires both a housekeeping sortase enzyme (SrtA) and pilus-associated sortase enzyme (SrtB). SrtB is primarily required for efficient formation of the T6 and FctX complex and subsequent polymerization of T6, whereas proper anchoring of the pili to the cell wall is mainly mediated by SrtA. Because motifs essential for polymerization of pilus backbone proteins in other Gram-positive bacteria are not present in T6, we sought to identify the functional residues involved in this process. Our results showed that T6 encompasses the novel VAKS pilin motif conserved in streptococcal T6 homologues and that the lysine residue (Lys-175) within the motif and cell wall sorting signal of T6 are prerequisites for isopeptide linkage of T6 molecules. Because Lys-175 and the cell wall sorting signal of FctX are indispensable for substantial incorporation of FctX into the T6 pilus shaft, FctX is suggested to be located at the pilus tip, which was also implied by immunogold electron microscopy findings. Thus, the elaborate assembly of FCT type 1 pili is potentially organized by sortase-mediated cross-linking between sorting signals and the amino group of Lys-175 positioned in the VAKS motif of T6, thereby displaying T6 and FctX in a temporospatial manner.

Transcript profiling by microarray and marker analysis of the short cotton (Gossypium hirsutum L.) fiber mutant Ligon lintless-1 (Li1).

PubMed

Gilbert, Matthew K; Turley, Rickie B; Kim, Hee Jin; Li, Ping; Thyssen, Gregory; Tang, Yuhong; Delhom, Christopher D; Naoumkina, Marina; Fang, David D

2013-06-17

Cotton fiber length is very important to the quality of textiles. Understanding the genetics and physiology of cotton fiber elongation can provide valuable tools to the cotton industry by targeting genes or other molecules responsible for fiber elongation. Ligon Lintless-1 (Li1) is a monogenic mutant in Upland cotton (Gossypium hirsutum) which exhibits an early cessation of fiber elongation resulting in very short fibers (< 6 mm) at maturity. This presents an excellent model system for studying the underlying molecular and cellular processes involved with cotton fiber elongation. Previous reports have characterized Li1 at early cell wall elongation and during later secondary cell wall synthesis, however there has been very limited analysis of the transition period between these developmental time points. Physical and morphological measurements of the Li1 mutant fibers were conducted, including measurement of the cellulose content during development. Affymetrix microarrays were used to analyze transcript profiles at the critical developmental time points of 3 days post anthesis (DPA), the late elongation stage of 12 DPA and the early secondary cell wall synthesis stage of 16 DPA. The results indicated severe disruption to key hormonal and other pathways related to fiber development, especially pertaining to the transition stage from elongation to secondary cell wall synthesis. Gene Ontology enrichment analysis identified several key pathways at the transition stage that exhibited altered regulation. Genes involved in ethylene biosynthesis and primary cell wall rearrangement were affected, and a primary cell wall-related cellulose synthase was transcriptionally repressed. Linkage mapping using a population of 2,553 F2 individuals identified SSR markers associated with the Li1 genetic locus on chromosome 22. Linkage mapping in combination with utilizing the diploid G. raimondii genome sequences permitted additional analysis of the region containing the Li1 gene. The early termination of fiber elongation in the Li1 mutant is likely controlled by an early upstream regulatory factor resulting in the altered regulation of hundreds of downstream genes. Several elongation-related genes that exhibited altered expression profiles in the Li1 mutant were identified. Molecular markers closely associated with the Li1 locus were developed. Results presented here will lay the foundation for further investigation of the genetic and molecular mechanisms of fiber elongation.

Rapid culture-independent microbial analysis aboard the international space station (ISS) stage two: quantifying three microbial biomarkers.

PubMed

Morris, Heather C; Damon, Michael; Maule, Jake; Monaco, Lisa A; Wainwright, Norm

2012-09-01

Abstract A portable, rapid, microbial detection unit, the Lab-On-a-Chip Application Development Portable Test System (LOCAD-PTS), was launched to the International Space Station (ISS) as a technology demonstration unit in December 2006. Results from the first series of experiments designed to detect Gram-negative bacteria on ISS surfaces by quantifying a single microbial biomarker lipopolysaccharide (LPS) were reported in a previous article. Herein, we report additional technology demonstration experiments expanding the on-orbit capabilities of the LOCAD-PTS to detecting three different microbial biomarkers on ISS surfaces. Six different astronauts on more than 20 occasions participated in these experiments, which were designed to test the new beta-glucan (fungal cell wall molecule) and lipoteichoic acid (LTA; Gram-positive bacterial cell wall component) cartridges individually and in tandem with the existing Limulus Amebocyte Lysate (LAL; Gram-negative bacterial LPS detection) cartridges. Additionally, we conducted the sampling side by side with the standard culture-based detection method currently used on the ISS. Therefore, we present data on the distribution of three microbial biomarkers collected from various surfaces in every module present on the ISS at the time of sampling. In accordance with our previous experiments, we determined that spacecraft surfaces known to be frequently in contact with crew members demonstrated higher values of all three microbial molecules. Key Words: Planetary protection-Spaceflight-Microbiology-Biosensor. Astrobiology 12, 830-840.

The regulation of virulence in phytopathogenic Erwinia species: quorum sensing, antibiotics and ecological considerations.

PubMed

Whitehead, Neil A; Byers, Joseph T; Commander, Paul; Corbett, Mark J; Coulthurst, Sarah J; Everson, Lee; Harris, Abigail K P; Pemberton, Clare L; Simpson, Natalie J L; Slater, Holly; Smith, Debra S; Welch, Martin; Williamson, Neil; Salmond, George P C

2002-08-01

Erwinia carotovora is a Gram-negative bacterial phytopathogen that causes soft-rot disease and potato blackleg. The organism is environmentally widespread and exhibits an opportunistic plant pathogenesis. The ability to secrete multiple plant cell wall-degrading enzymes is a key virulence trait and exoenzyme production is responsive to multiple environmental and physiological cues. One important cue is the cell population density of the pathogen. Cell density is monitored via an acylated homoserine lactone (acyl HSL) signalling molecule, which is thought to diffuse between Erwinia cells in a process now commonly known as 'quorum sensing'. This molecule also acts as the chemical communication signal controlling production of a broad-spectrum beta-lactam antibiotic (1-carbapen-2-em-3-carboxylic acid; carbapenem) synthesised in concert with exoenzyme elaboration, possibly for niche defence. In antibiotic production control, quorum sensing acts at the level of transcriptional activation of the antibiotic biosynthetic cluster. This is achieved via a dedicated LuxR-type protein, CarR that is bound to the signalling molecule. The molecular relay connecting acyl HSL production and exoenzyme induction is not clear, despite the identification of a multitude of global regulatory genes, including those of the RsmA/rsmB system, impinging on enzyme synthesis. Quorum sensing control mediated by acyl HSLs is widespread in Gram-negative bacteria and is responsible for the regulation of diverse phenotypes. Although there is still a paucity of meaningful information on acyl HSL availability and in-situ biological function, there is growing evidence that such molecules play significant roles in microbial ecology.

Angiogenic and angiostatic factors in the molecular control of angiogenesis.

PubMed

Distler, J H W; Hirth, A; Kurowska-Stolarska, M; Gay, R E; Gay, S; Distler, O

2003-09-01

The vascular system that ensures an adequate blood flow is required to provide the cells with sufficient supply of nutrients and oxygen. Two different mechanisms of the formation of new vessels can be distinguished: vasculogenesis, the formation of the first primitive vascular plexus de novo and angiogenesis, the formation of new vessels from preexisting ones. Both processes are regulated by a delicate balance of pro- and anti-angiogenic factors. Physiologically, angiostatic mediators outweigh the angiogenic molecules and angiogenesis does not occur. Under certain conditions such as tumor formation or wound healing, the positive regulators of angiogenesis predominate and the endothelium becomes activated. Angiogenesis is initiated by vasodilatation and an increased permeability. After destabilization of the vessel wall, endothelial cells proliferate, migrate and form a tube, which is finally stabilized by pericytes and smooth muscle cells. Numerous soluble growth factors and inhibitors, cytokines and proteases as well as extracellular matrix proteins and adhesion molecules strictly control this multi-step process. The properties and interactions of angiogenic molecules such as VEGFs, FGFs, angiopoietins, PDGF, angiogenin, angiotropin, HGF, CXC chemokines with ELR motif, PECAM-1, integrins and VE-cadherin as well as angiostatic key players such as angiostatin, endostatin, thrombospondin, CXC chemokines without ELR motif, PEDF are discussed in this review with respect to their molecular impact on angiogenesis.

Inhibition of STAT3 phosphorylation by sulforaphane reduces adhesion molecule expression in vascular endothelial cell.

PubMed

Cho, Young S; Kim, Chan H; Ha, Tae S; Ahn, Hee Y

2015-11-18

Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) play key roles in the initiation of vascular inflammation. In this study, we explored whether sulforaphane, a dietary phytochemical, can inhibit the expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS), and the mechanisms involved. Sulforaphane prevented the LPS-mediated increase in ICAM-1 and VCAM-1 expression, (P < 0.01) in HUVEC. Sulforaphane also prevented the LPS-mediated increase in the phosphorylation of signal transducer and activator of transcription 3 (STAT3) (P < 0.01). Stattic, a STAT3 inhibitor, reduced the LPS-induced expression of ICAM-1 and VCAM-1, and STAT3 phosphorylation (P < 0.01). STAT3 small interfering RNA treatment reduced the LPS-induced expression of ICAM-1, VCAM-1, and STAT3 (P < 0.01). Sulforaphane reduced LPS-mediated THP-1 monocyte adhesion to HUVEC (P < 0.01). In C57BL/6 mice, injection of LPS increased aortic ICAM-1 and VCAM-1 expression, and this effect was prevented by sulforaphane. These data provide insight into the mechanism through which sulforaphane partly reduces the expression of ICAM-1 and VCAM-1 on the vascular wall by inhibiting STAT3 phosphorylation.

Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast

PubMed Central

G. Cortés, Juan C.; Pujol, Nuria; Sato, Mamiko; Pinar, Mario; Ramos, Mariona; Moreno, Belén; Osumi, Masako; Ribas, Juan Carlos; Pérez, Pilar

2015-01-01

In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall overgrowth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast. PMID:26132084

Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast.

PubMed

Cortés, Juan C G; Pujol, Nuria; Sato, Mamiko; Pinar, Mario; Ramos, Mariona; Moreno, Belén; Osumi, Masako; Ribas, Juan Carlos; Pérez, Pilar

2015-07-01

In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall overgrowth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast.

Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays

NASA Astrophysics Data System (ADS)

Landry, Markita Patricia; Ando, Hiroki; Chen, Allen Y.; Cao, Jicong; Kottadiel, Vishal Isaac; Chio, Linda; Yang, Darwin; Dong, Juyao; Lu, Timothy K.; Strano, Michael S.

2017-05-01

A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

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

Insights into hydrogen bond dynamics at the interface of the charged monolayer-protected Au nanoparticle from molecular dynamics simulation.

PubMed

Li, Yunzhi; Yang, Zhen; Hu, Na; Zhou, Rongfei; Chen, Xiangshu

2013-05-14

The structure and dynamics properties of water molecules at the interface of the charged monolayer-protected Au nanoparticle (MPAN) have been investigated in detail by using classical molecular dynamics simulation. The simulation results demonstrated clearly that a well-defined hydration layer is formed at the interface of MPAN and a stable "ion wall" consisting of terminal NH3 (+) groups and Cl(-) counterions exists at the outmost region of self-assembled monolayer (SAM) where the translational and rotational motions of water molecules slow considerably down compared to those in the bulk owing to the presence of SAM and ion wall. Furthermore, we found that the translational motions of interfacial water molecules display a subdiffusive behavior while their rotational motions exhibit a nonexponential feature. The unique behavior of interfacial water molecules around the MPAN can be attributed to the interfacial hydrogen bond (HB) dynamics. By comparison, the lifetime of NH3 (+)-Cl(-) HBs was found to be the longest, favoring the stability of ion wall. Meanwhile, the lifetime of H2O-H2O HBs shows an obvious increase when the water molecules approach the Au core, suggesting the enhanced H2O-H2O HBs around the charged MPAN, which is contrary to the weaken H2O-H2O HBs around the neutral MPAN. Moreover, the HB lifetimes between water molecules and the ion wall (i.e., the Cl(-)-H2O and NH3 (+)-H2O HBs) are much longer than that of interfacial H2O-H2O HBs, which leads to the increasing rotational relaxation time and residence time of water molecules surrounding the ion wall. In addition, the corresponding binding energies for different HB types obtained from the precise density functional theory are in excellent accordance with above simulation results. The detailed HB dynamics studied in this work provides insights into the unique behavior of water molecules at the interface of charged self-assemblies of nanoparticles as well as proteins.

Simulation of stimuli-triggered release of molecular species from halloysite nanotubes

NASA Astrophysics Data System (ADS)

Elumalai, Divya Narayan; Tully, Joshua; Lvov, Yuri; Derosa, Pedro A.

2016-10-01

A Monte Carlo model is used to study the effect of environmental variables (pH and temperature) on the transport and release of dexamethasone molecules from Halloysite Nanotubes (HNTs) in a dielectric fluid medium. The model used for this study was introduced elsewhere and it is based on basic physics interactions without experimental parameters for these interactions. An intermediate phase between the burst and saturation phase is found and explained. Molecules experience a 1-D diffusion process that is different from the diffusion in the burst phase or the surface diffusion experienced by molecules attached to the wall. It is predicted that this phase exists when the molecule-wall interaction is attractive but not always noticeable in the release profile. In this work, it is shown that an agreement with the experiment better than previously reported is obtained when simulated delivery curves are produced by the weighted average of the release profiles from a collection of HNTs with diameters and lengths distributed according to the experimental sample, highlighting the relevance of HNTs' morphology in the release. HNTs are suitable for environment-triggered release and thus the effect of temperature, molecule zeta potential, and pH is studied. It is observed that for temperatures that significantly differ from room temperature (by 100's of degrees), the release profile changes significantly, increasing the delivery speed at high temperature and reducing that speed at low temperature. Finally, it is observed that as the pH becomes more acidic, both the molecule and inner wall surface become more positive (or less negative) with both eventually becoming positive leading to a repulsive interaction; thus, molecules are pushed out by electrostatic repulsion. On the contrary, as the pH becomes more basic, positive molecules become more positive while the wall becomes less negative, but even at pH 12, the wall remains negative and the interaction is attractive. Changes in pH between different regions may act as a trigger for delivery or as a control in the delivery rate.

Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

PubMed

Fang, Kefeng; Gao, Sai; Zhang, Weiwei; Xing, Yu; Cao, Qingqin; Qin, Ling

2016-01-01

A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.

Quantitative description of ion transport via plasma membrane of yeast and small cells.

PubMed

Volkov, Vadim

2015-01-01

Modeling of ion transport via plasma membrane needs identification and quantitative understanding of the involved processes. Brief characterization of main ion transport systems of a yeast cell (Pma1, Ena1, TOK1, Nha1, Trk1, Trk2, non-selective cation conductance) and determining the exact number of molecules of each transporter per a typical cell allow us to predict the corresponding ion flows. In this review a comparison of ion transport in small yeast cell and several animal cell types is provided. The importance of cell volume to surface ratio is emphasized. The role of cell wall and lipid rafts is discussed in respect to required increase in spatial and temporary resolution of measurements. Conclusions are formulated to describe specific features of ion transport in a yeast cell. Potential directions of future research are outlined based on the assumptions.

Quantitative description of ion transport via plasma membrane of yeast and small cells

PubMed Central

Volkov, Vadim

2015-01-01

Modeling of ion transport via plasma membrane needs identification and quantitative understanding of the involved processes. Brief characterization of main ion transport systems of a yeast cell (Pma1, Ena1, TOK1, Nha1, Trk1, Trk2, non-selective cation conductance) and determining the exact number of molecules of each transporter per a typical cell allow us to predict the corresponding ion flows. In this review a comparison of ion transport in small yeast cell and several animal cell types is provided. The importance of cell volume to surface ratio is emphasized. The role of cell wall and lipid rafts is discussed in respect to required increase in spatial and temporary resolution of measurements. Conclusions are formulated to describe specific features of ion transport in a yeast cell. Potential directions of future research are outlined based on the assumptions. PMID:26113853

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

PubMed Central

Yamashita, Tetsuji; Hakizimana, Pierre; Wu, Siva; Hassan, Ahmed; Jacob, Stefan; Temirov, Jamshid; Fang, Jie; Mellado-Lagarde, Marcia; Gursky, Richard; Horner, Linda; Leibiger, Barbara; Leijon, Sara; Centonze, Victoria E.; Berggren, Per-Olof; Frase, Sharon; Auer, Manfred; Brownell, William E.; Fridberger, Anders; Zuo, Jian

2015-01-01

Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases. PMID:26352669

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.

FT-IR and FT-Raman characterization of non-cellulosic polysaccharides fractions isolated from plant cell wall.

PubMed

Chylińska, Monika; Szymańska-Chargot, Monika; Zdunek, Artur

2016-12-10

The purpose of this work was to reveal the structural changes of cell wall polysaccharides' fractions during tomato fruit development by analysis of spectral data. Mature green and red ripe tomato fruit were taken into consideration. The FT-IR spectra of water soluble pectin (WSP), imidazole soluble pectin (ISP) and diluted alkali soluble pectin (DASP) contained bands typical for pectins. Whereas for KOH fraction spectra bands typical for hemicelluloses were present. The FT-IR spectra showed the drop down of esterification degree of WSP and ISP polysaccharides during maturation. The changes in polysaccharides structure revealed by spectra were the most visible in the case of pectic polysaccharides. The WSP and DASP fraction pectins molecules length were shortened during tomato maturation and ripening. Whereas the ISP fraction spectra analysis showed that this fraction contained rhamnogalacturonan I, but also for red ripe was rich in pectic galactan comparing with ISP fraction from mature green. Copyright © 2016 Elsevier Ltd. All rights reserved.

Activity of Co-N multi walled carbon nanotubes electrocatalysts for oxygen reduction reaction in acid conditions

NASA Astrophysics Data System (ADS)

Osmieri, Luigi; Monteverde Videla, Alessandro H. A.; Specchia, Stefania

2015-03-01

Two catalysts are synthesized by wet impregnation of multi walled carbon nanotubes (MWCNT) with a complex formed between Co(II) ions and the nitrogen-containing molecule 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ), followed by one or two identical heat treatments in N2 atmosphere at 800 °C for 3 h. Catalysts are fully characterized by FESEM, EDX, BET, XRD, FTIR, TGA, XPS analyses, and electrochemical techniques. The electrocatalytic activity towards oxygen reduction reaction (ORR) of the catalysts in acid conditions is assessed by means of a rotating disk electrode (RDE) apparatus and a specific type of cell equipped with a gas diffusion working electrode (GDE). In both testing approaches, the catalyst heat-treated twice (Co-N/MWCNT-2) exhibits higher electroactivity than the catalyst heat-treated once (Co-N/MWCNT-1). Chronoamperometries both in RDE and GDE cell are also performed, showing less electroactivity decay and better current performance for the catalyst heat-treated twice.

Functionalized single-walled carbon nanotubes: cellular uptake, biodistribution and applications in drug delivery.

PubMed

Li, Zixian; de Barros, Andre Luis Branco; Soares, Daniel Cristian Ferreira; Moss, Sara Nicole; Alisaraie, Laleh

2017-05-30

The unique properties of single-walled carbon nanotubes (SWNTs) enable them to play important roles in many fields. One of their functional roles is to transport cargo into cell. SWNTs are able to traverse amphipathic cell membranes due to their large surface area, flexible interactions with cargo, customizable dimensions, and surface chemistry. The cargoes delivered by SWNTs include peptides, proteins, nucleic acids, as well as drug molecules for therapeutic purpose. The drug delivery functions of SWNTs have been explored over the past decade. Many breakthrough studies have shown the high specificity and potency of functionalized SWNT-based drug delivery systems for the treatment of cancers and other diseases. In this review, we discuss different aspects of drug delivery by functionalized SWNT carriers, diving into the cellular uptake mechanisms, biodistribution of the delivery system, and safety concerns on degradation of the carriers. We emphasize the delivery of several common drugs to highlight the recent achievements of SWNT-based drug delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Antibacterial Effect of (2E,2E)-4,4-Trisulfanediylbis(but-2-enoic acid) against Staphylococcus aureus.

PubMed

Wu, Tao; Huang, Yina; Chen, Yijun; Zhang, Min

2018-01-01

A new highly active molecule, (2E, 2E)-4,4-trisulfanediylbis(but-2-enoic acid) (TSDB), was designed and synthesized through comparative molecular field analysis with the diallyl trisulfide structure of garlic. TSDB exerted a strong inhibitory effect against Staphylococcus aureus, with minimal inhibitory and minimal bactericidal concentrations of 16 and 128 μg/mL, respectively. TSDB destructed the integrity of the S. aureus cell membrane but weakly damaged the bacterial cell wall. TSDB also increased the conductivity and protein expression in microbial broth but minimally influenced the level of extracellular alkaline phosphatase. TSDB could be a novel food preservative.

Antifungal Compounds against Candida Infections from Traditional Chinese Medicine

PubMed Central

2017-01-01

Infections caused by Candida albicans, often refractory and with high morbidity and mortality, cause a heavy burden on the public health while the current antifungal drugs are limited and are associated with toxicity and resistance. Many plant-derived molecules including compounds isolated from traditional Chinese medicine (TCM) are reported to have antifungal activity through different targets such as cell membrane, cell wall, mitochondria, and virulence factors. Here, we review the recent progress in the anti-Candida compounds from TCM, as well as their antifungal mechanisms. Considering the diverse targets and structures, compounds from TCM might be a potential library for antifungal drug development. PMID:29445739

Cell division and turgor mediate enhanced plant growth in Arabidopsis plants treated with the bacterial signalling molecule lumichrome.

PubMed

Pholo, Motlalepula; Coetzee, Beatrix; Maree, Hans J; Young, Philip R; Lloyd, James R; Kossmann, Jens; Hills, Paul N

2018-05-17

Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plant growth through a combination of enhanced cell division and cell enlargement, and possibly enhances photosynthesis. Lumichrome (7,8 dimethylalloxazine), a novel multitrophic signal molecule produced by Sinorhizobium meliloti bacteria, has previously been shown to elicit growth promotion in different plant species (Phillips et al. in Proc Natl Acad Sci USA 96:12275-12280, https://doi.org/10.1073/pnas.96.22.12275 , 1999). However, the molecular mechanisms that underlie this plant growth promotion remain obscure. Global transcript profiling using RNA-seq suggests that lumichrome enhances growth by inducing genes impacting on turgor driven growth and mitotic cell cycle that ensures the integration of cell division and expansion of developing leaves. The abundance of XTH9 and XPA4 transcripts was attributed to improved mediation of cell-wall loosening to allow turgor-driven cell enlargement. Mitotic CYCD3.3, CYCA1.1, SP1L3, RSW7 and PDF1 transcripts were increased in lumichrome-treated Arabidopsis thaliana plants, suggesting enhanced growth was underpinned by increased cell differentiation and expansion with a consequential increase in biomass. Synergistic ethylene-auxin cross-talk was also observed through reciprocal over-expression of ACO1 and SAUR54, in which ethylene activates the auxin signalling pathway and regulates Arabidopsis growth by both stimulating auxin biosynthesis and modulating the auxin transport machinery to the leaves. Decreased transcription of jasmonate biosynthesis and responsive-related transcripts (LOX2; LOX3; LOX6; JAL34; JR1) might contribute towards suppression of the negative effects of methyl jasmonate (MeJa) such as chlorophyll loss and decreases in RuBisCO and photosynthesis. This work contributes towards a deeper understanding of how lumichrome enhances plant growth and development.

Photoactivated bioconjugation between ortho-azidophenols and anilines: a facile approach to biomolecular photopatterning.

PubMed

El Muslemany, Kareem M; Twite, Amy A; ElSohly, Adel M; Obermeyer, Allie C; Mathies, Richard A; Francis, Matthew B

2014-09-10

Methods for the surface patterning of small molecules and biomolecules can yield useful platforms for drug screening, synthetic biology applications, diagnostics, and the immobilization of live cells. However, new techniques are needed to achieve the ease, feature sizes, reliability, and patterning speed necessary for widespread adoption. Herein, we report an easily accessible and operationally simple photoinitiated reaction that can achieve patterned bioconjugation in a highly chemoselective manner. The reaction involves the photolysis of 2-azidophenols to generate iminoquinone intermediates that couple rapidly to aniline groups. We demonstrate the broad functional group compatibility of this reaction for the modification of proteins, polymers, oligonucleotides, peptides, and small molecules. As a specific application, the reaction was adapted for the photolithographic patterning of azidophenol DNA on aniline glass substrates. The presence of the DNA was confirmed by the ability of the surface to capture living cells bearing the sequence complement on their cell walls or cytoplasmic membranes. Compared to other light-based DNA patterning methods, this reaction offers higher speed and does not require the use of a photoresist or other blocking material.

Quantum dots as strain- and metabolism-specific microbiological labels

NASA Technical Reports Server (NTRS)

Kloepfer, J. A.; Mielke, R. E.; Wong, M. S.; Nealson, K. H.; Stucky, G.; Nadeau, J. L.

2003-01-01

Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells. However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls. Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface. While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs. Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate.

Cell Permeability, Migration, and Reactive Oxygen Species Induced by Multi-Walled Carbon Nanotubes in Human Microvascular Endothelial Cells

PubMed Central

Pacurari, M; Qian, Y; Fu, W; Schwegler-Berry, D; Ding, M; Castranova, V; Guo, NL

2011-01-01

Multi-walled carbon nanotubes (MWCNT) have elicited great interest in biomedical applications due to their extraordinary physical, chemical, and optical properties. Intravenous administration of MWCNT-based medical imaging agents and drugs in animal models was utilized. However, the potential harmful health effects of MWCNT administration in humans have not yet been elucidated. Furthermore, to date, there are no apparent reports regarding the precise mechanisms of translocation of MWCNT into target tissues and organs from blood circulation. This study demonstrates that exposure to MWCNT leads to an increase in cell permeability in human microvascular endothelial cells (HMVEC). The results obtained from this study also showed that the MWCNT-induced rise in endothelial permeability is mediated by reactive oxygen species (ROS) production and actin filament remodeling. In addition, it was found that MWCNT promoted cell migration in HMVEC. Mechanistically, MWCNT exposure elevated the levels of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) in HMVEC. Taken together, these results provide new insights into the bioreactivity of MWCNT, which may have implications in the biomedical application of MWCNT in vascular targeting, imaging, and drug delivery. The results generated from this study also elucidate the potential adverse effects of MWCNT exposure on humans at the cellular level. PMID:22129238

Structure of the choline-binding domain of Spr1274 in Streptococcus pneumoniae.

PubMed

Zhang, Zhenyi; Li, Wenzhe; Frolet, Cecile; Bao, Rui; di Guilmi, Anne Marie; Vernet, Thierry; Chen, Yuxing

2009-08-01

Spr1274 is a putative choline-binding protein that is bound to the cell wall of Streptococcus pneumoniae through noncovalent interactions with the choline moieties of teichoic and lipoteichoic acids. Its function is still unknown. The crystal structure of the choline-binding domain of Spr1274 (residues 44-129) was solved at 2.38 A resolution with three molecules in the asymmetric unit. It may provide a structural basis for functional analysis of choline-binding proteins.

Identification and Characterization of uvrA, a DNA Repair Gene of Deinococcus radiodurans

DTIC Science & Technology

1996-01-01

and Classificalion I 2 . TheCellWall 4 3. Intracellular Molecules 7 4. Genetics _ _ _ _ _.. 8 a. DNA COntent. 8 b. Chromosomes 8 c. Plasmids 10 d...Summary 11 B. DNA Damaging Agenls 12 I. Visible Light and Low-Frequency UV Radiation 12 2 . High-frequency UV Radiation 13 a. Pyrimidine DiIners 13 b. The...23 a. Photoreactivation Repair 23 b. Repair of Spore Pholoproducts 27 2 . Repair by Methods Involving Single Proteins 27 a. Repair of

The structure of a β-(1→6)-d-glucan from yeast cell walls

PubMed Central

Manners, David J.; Masson, Alan J.; Patterson, James C.; Björndal, Håkan; Lindberg, Bengt

1973-01-01

By selective enzymolysis, or chemical fractionation, a minor polysaccharide component has been isolated from yeast (Saccharomyces cerevisiae) glucan. This minor component has a degree of polymerization of about 130–140, a highly branched structure, and a high proportion of β-(1→6)-glucosidic linkages. The molecules also contain a smaller proportion of β-(1→3)-glucosidic linkages that serve mainly as interchain linkages, but some may also be inter-residue linkages. PMID:4590991

Next Generation LOCAD-PTS Cartridge Development

NASA Technical Reports Server (NTRS)

Morris, H.; Nutter, D.; Weite, E.; Wells, M.; Maule, J.; Damon, M.; Monaco, L.; Steele, A.; Wainwright, N.

2008-01-01

Future astrobiology exploration missions will require rapid, point-of-use techniques for surface science experiments and contamination monitoring. The Lab-On-a-Chip Application Development (LOCAD) team is developing operational instruments that advance spaceflight technologies to molecular-based methods. Currently, LOCAD-Portable Test System (PTS) is quantifying levels of the bacterial molecule endotoxin onboard the Internatioal Space Station. Future research and development will focus on more sensitive molecular techniques that expand the number of compounds detected to include beta-glucan from fungal cell walls.

Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

PubMed Central

2013-01-01

Background Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison. Results Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed. Conclusions Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell wall. A new variety of lignin fluorescence states were accessed by two-photon excitation, which allowed an even broader, but complementary, optical characterization of lignocellulosic materials. These results suggest that the lignin arrangement in untreated bagasse fiber is based on a well-organized nanoenvironment that favors a very low level of interaction between the molecules. PMID:23548159

Biomedical Platforms Based on Composite Nanomaterials and Cellular Toxicity

NASA Astrophysics Data System (ADS)

Bellucci, Stefano; Bergamaschi, A.; Bottini, M.; Magrini, A.; Mustelin, T.

2007-03-01

Carbon nanotubes possess unique chemical, physical, optical, and magnetic properties, which make them suitable for many uses in industrial products and in the field of nanotechnology, including nanomedicine. We describe fluorescent nanocomposites for use in biosensors or nanoelectronics. Then we describe recent results on the issue of cytotoxicity of carbon nanotubes obtained in our labs. Silica nanoparticles have been widely used for biosensing and catalytic applications due to their large surface area-to-volume ratio, straightforward manufacture, and the compatibility of silica chemistry with covalent coupling of biomolecules. Carbon nanotubes-composite materials, such as those based on Carbon nanotubes bound to nanoparticles, are suitable, in order to tailor Carbon nanotubes properties for specific applications. We present a tunable synthesis of Multi Wall Carbon nanotubes-Silica nanoparticles. The control of the nanotube morphology and the bead size, coupled with the versatility of silica chemistry, makes these structures an excellent platform for the development of biosensors (optical, magnetic and catalytic applications). We describe the construction and characterization of supramolecular nanostructures consisting of ruthenium-complex luminophores, directly grafted onto short oxidized single-walled carbon nanotubes or physically entrapped in silica nanobeads, which had been covalently linked to short oxidized single-walled carbon nanotubes or hydrophobically adsorbed onto full-length multi-walled carbon nanotubes. These structures have been evaluated as potential electron-acceptor complexes for use in the fabrication of photovoltaic devices, and for their properties as fluorescent nanocomposites for use in biosensors or nanoelectronics. Finally, we compare the toxicity of pristine and oxidized Multi Walled Carbon nanotubes on human T cells - which would be among the first exposed cell types upon intravenous administration of Carbon nanotubes in therapeutic and diagnostic nanodevices. Our results suggest that carbon nanotubes indeed can be very toxic and induce massive loss of cell viability through programmed cell death at sufficiently high concentrations (>1ng/cell). The cytotoxicity of Carbon nanotubes does depend on many other factors than concentration, including their physical form, diameter, length, and the nature of attached molecules or nanomaterials: carbon black, for instance, is less toxic than pristine CNTs (what shows the relevance of structure and topology); oxidized CNTs are more toxic than pristine CNTs.

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.

FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow.

PubMed

Zhu, Shu; Travers, Richard J; Morrissey, James H; Diamond, Scott L

2015-09-17

Factor XIIa (FXIIa) and factor XIa (FXIa) contribute to thrombosis in animal models, whereas platelet-derived polyphosphate (polyP) may potentiate contact or thrombin-feedback pathways. The significance of these mediators in human blood under thrombotic flow conditions on tissue factor (TF) -bearing surfaces remains inadequately resolved. Human blood (corn trypsin inhibitor treated [4 μg/mL]) was tested by microfluidic assay for clotting on collagen/TF at TF surface concentration ([TF]wall) from ∼0.1 to 2 molecules per μm(2). Anti-FXI antibodies (14E11 and O1A6) or polyP-binding protein (PPXbd) were used to block FXIIa-dependent FXI activation, FXIa-dependent factor IX (FIX) activation, or platelet-derived polyP, respectively. Fibrin formation was sensitive to 14E11 at 0 to 0.1 molecules per µm(2) and sensitive to O1A6 at 0 to 0.2 molecules per µm(2). However, neither antibody reduced fibrin generation at ∼2 molecules per µm(2) when the extrinsic pathway became dominant. Interestingly, PPXbd reduced fibrin generation at low [TF]wall (0.1 molecules per µm(2)) but not at zero or high [TF]wall, suggesting a role for polyP distinct from FXIIa activation and requiring low extrinsic pathway participation. Regardless of [TF]wall, PPXbd enhanced fibrin sensitivity to tissue plasminogen activator and promoted clot retraction during fibrinolysis concomitant with an observed PPXbd-mediated reduction of fibrin fiber diameter. This is the first detection of endogenous polyP function in human blood under thrombotic flow conditions. When triggered by low [TF]wall, thrombosis may be druggable by contact pathway inhibition, although thrombolytic susceptibility may benefit from polyP antagonism regardless of [TF]wall. © 2015 by The American Society of Hematology.

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

Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.

Here, we have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single moleculemore » super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.« less

DNA Molecules in Microfluidic Oscillatory Flow

PubMed Central

Chen, Y.-L.; Graham, M.D.; de Pablo, J.J.; Jo, K.; Schwartz, D.C.

2008-01-01

The conformation and dynamics of a single DNA molecule undergoing oscillatory pressure-driven flow in microfluidic channels is studied using Brownian dynamics simulations, accounting for hydrodynamic interactions between segments in the bulk and between the chain and the walls. Oscillatory flow provides a scenario under which the polymers may remain in the channel for an indefinite amount of time as they are stretched and migrate away from the channel walls. We show that by controlling the chain length, flow rate and oscillatory flow frequency, we are able to manipulate the chain extension and the chain migration from the channel walls. The chain stretch and the chain depletion layer thickness near the wall are found to increase as the Weissenberg number increases and as the oscillatory frequency decreases. PMID:19057656

Extracellular vesicle-mediated export of fungal RNA

PubMed Central

Peres da Silva, Roberta; Puccia, Rosana; Rodrigues, Marcio L.; Oliveira, Débora L.; Joffe, Luna S.; César, Gabriele V.; Nimrichter, Leonardo; Goldenberg, Samuel; Alves, Lysangela R.

2015-01-01

Extracellular vesicles (EVs) play an important role in the biology of various organisms, including fungi, in which they are required for the trafficking of molecules across the cell wall. Fungal EVs contain a complex combination of macromolecules, including proteins, lipids and glycans. In this work, we aimed to describe and characterize RNA in EV preparations from the human pathogens Cryptococcus neoformans, Paracoccidiodes brasiliensis and Candida albicans, and from the model yeast Saccharomyces cerevisiae. The EV RNA content consisted mostly of molecules less than 250 nt long and the reads obtained aligned with intergenic and intronic regions or specific positions within the mRNA. We identified 114 ncRNAs, among them, six small nucleolar (snoRNA), two small nuclear (snRNA), two ribosomal (rRNA) and one transfer (tRNA) common to all the species considered, together with 20 sequences with features consistent with miRNAs. We also observed some copurified mRNAs, as suggested by reads covering entire transcripts, including those involved in vesicle-mediated transport and metabolic pathways. We characterized for the first time RNA molecules present in EVs produced by fungi. Our results suggest that RNA-containing vesicles may be determinant for various biological processes, including cell communication and pathogenesis. PMID:25586039

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.

Effect of PEGylation on ligand-based targeting of drug carriers to the vascular wall in blood flow.

PubMed

Onyskiw, Peter J; Eniola-Adefeso, Omolola

2013-09-03

The blood vessel wall plays a prominent role in the development of many life-threatening diseases and as such is an attractive target for treatment. To target diseased tissue, particulate drug carriers often have their surfaces modified with antibodies or epitopes specific to vascular wall-expressed molecules, along with poly(ethylene glycol) (PEG) to improve carrier blood circulation time. However, little is known about the effect of poly(ethylene glycol) on carrier adhesion dynamics-specifically in blood flow. Here we examine the influence of different molecular weight PEG spacers on particle adhesion in blood flow. Anti-ICAM-1 or Sialyl Lewis(a) were grafted onto polystyrene 2 μm and 500 nm spheres via PEG spacers and perfused in blood over activated endothelial cells at physiological shear conditions. PEG spacers were shown to improve, reduce, or have no effect on the binding density of targeted-carriers depending on the PEG surface conformation, shear rate, and targeting moiety.

A switch in disulfide linkage during minicollagen assembly in Hydra nematocysts.

PubMed

Engel, U; Pertz, O; Fauser, C; Engel, J; David, C N; Holstein, T W

2001-06-15

The smallest known collagens with only 14 Gly-X-Y repeats referred to as minicollagens are the main constituents of the capsule wall of nematocysts. These are explosive organelles found in Hydra, jellyfish, corals and other Cnidaria. Minicollagen-1 of Hydra recombinantly expressed in mammalian 293 cells contains disulfide bonds within its N- and C-terminal Cys-rich domains but no interchain cross-links. It is soluble and self-associates through non-covalent interactions to form 25-nm-long trimeric helical rod-like molecules. We have used a polyclonal antibody prepared against the recombinant protein to follow the maturation of minicollagens from soluble precursors present in the endoplasmic reticulum and post-Golgi vacuoles to the disulfide-linked insoluble assembly form of the wall. The switch from intra- to intermolecular disulfide bonds is associated with 'hardening' of the capsule wall and provides an explanation for its high tensile strength and elasticity. The process is comparable to disulfide reshuffling between the NC1 domains of collagen IV in mammalian basement membranes.

Endogenous Molecules Induced by a Pathogen-Associated Molecular Pattern (PAMP) Elicit Innate Immunity in Shrimp

PubMed Central

Chen, Yu-Yuan; Chen, Jiann-Chu; Lin, Yong-Chin; Kitikiew, Suwaree; Li, Hui-Fang; Bai, Jia-Chin; Tseng, Kuei-Chi; Lin, Bo-Wei; Liu, Po-Chun; Shi, Yin-Ze; Kuo, Yi-Hsuan; Chang, Yu-Hsuan

2014-01-01

Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), β-1,3-glucan (βG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, βG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-β, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-β, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-β, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity. PMID:25517999

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

Supramolecular organization of pi-conjugated molecules monitored by single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Alvarez, Laurent; Almadori, Yann; Belhboub, Anouar; Le Parc, Rozenn; Aznar, Raymond; Dieudonné-George, Philippe; Rahmani, Abdelali; Hermet, Patrick; Fossard, Frédéric; Loiseau, Annick; Jousselme, Bruno; Campidelli, Stéphane; Saito, Takeshi; Wang, Guillaume; Bantignies, Jean-Louis

2016-03-01

Photoactive pi-conjugated molecules (quaterthiophene and phthalocyanine) are either encapsulated into the hollow core of single-walled carbon nanotubes or noncovalently stacked at their outer surface in order to elaborate hybrid nanosystems with new physical properties, providing practical routes to fit different requirements for potential applications. We are interested in the relationship between the structure and the optoelectronic properties. The structural properties are investigated mainly by x-ray diffraction and/or transmission electron microscopy and Raman spectroscopy. We show that the supramolecular organizations of confined quaterthiophenes depend on the nanocontainer size, whereas phthalocyanine encapsulation leads to the formation of a one-dimensional phase for which the angle between the molecule ring and the nanotube axis is close to 32 deg. Confined phthalocyanine molecules display Raman spectra hardly altered with respect to the bulk phase, suggesting a rather weak interaction with the tubes. In contrast, the vibrational properties of the molecules stacked at the outer surface of tubes display important modifications. We assume a significant curvature of the phthalocyanine induced by the interaction with the tube walls and a change of the central atom position within the molecular ring, in good agreement with our density functional theory calculations.

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.

Fluorescence excitation and imaging of single molecules near dielectric-coated and bare surfaces: a theoretical study.

PubMed

Axelrod, Daniel

2012-08-01

Microscopic fluorescent samples of interest to cell and molecular biology are commonly embedded in an aqueous medium near a solid surface that is coated with a thin film such as a lipid multilayer, collagen, acrylamide, or a cell wall. Both excitation and emission of fluorescent single molecules near film-coated surfaces are strongly affected by the proximity of the coated surface, the film thickness, its refractive index and the fluorophore's orientation. For total internal reflection excitation, multiple reflections in the film can lead to resonance peaks in the evanescent intensity versus incidence angle curve. For emission, multiple reflections arising from the fluorophore's near field emission can create a distinct intensity pattern in both the back focal plane and the image plane of a high aperture objective. This theoretical analysis discusses how these features can be used to report film thickness and refractive index, and fluorophore axial position and orientation. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

Analyte concentration at the tip of a nanopipette.

PubMed

Calander, Nils

2009-10-15

Concentration of molecules within the tips of nanopipettes when applying a DC voltage is herein investigated using finite-element simulations. The ion concentrations and fluxes due to diffusion, electro-migration, and electro-osmotic flow, and the electric potential are determined by the simultaneous solution of the Nernst-Planck, Poisson, and Navier-Stokes equations within the water solution containing sodium and chloride ions and negatively charged molecules. The electric potential within the pipette glass wall is at the same time determined by the Poisson equation together with appropriate boundary conditions and accounts for a field effect through the wall. Fixed negative surface charge on both the internal and external glass surfaces of the nanopipette is included together with the field effect through the glass wall to account for the electric double layer and the electro-osmosis. The inclusion of the field effect through the pipette wall is new compared to previous modeling of similar structures and is shown to be crucial for the behavior at the tip. It is demonstrated that the concentration of molecules is a consequence of ionic charge accumulation at the tip screening the electric field, thereby slowing down the electrophoretic motion of the molecules, which is further slowed down or stopped by the oppositely directed electro-osmosis. It is also shown that the trapping is very sensitive to the properties of the molecule, that is, its electrophoretic mobility and diffusion coefficient, the properties of the pipette, the ionic strength of the solution, and the applied electric field.

The Extracellular Protein Factor Epf from Streptococcus pyogenes Is a Cell Surface Adhesin That Binds to Cells through an N-terminal Domain Containing a Carbohydrate-binding Module*

PubMed Central

Linke, Christian; Siemens, Nikolai; Oehmcke, Sonja; Radjainia, Mazdak; Law, Ruby H. P.; Whisstock, James C.; Baker, Edward N.; Kreikemeyer, Bernd

2012-01-01

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain. PMID:22977243

The extracellular protein factor Epf from Streptococcus pyogenes is a cell surface adhesin that binds to cells through an N-terminal domain containing a carbohydrate-binding module.

PubMed

Linke, Christian; Siemens, Nikolai; Oehmcke, Sonja; Radjainia, Mazdak; Law, Ruby H P; Whisstock, James C; Baker, Edward N; Kreikemeyer, Bernd

2012-11-02

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain.

Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

PubMed

Lee, Seung S; Roche, Philip Jr; Giannopoulos, Paresa N; Mitmaker, Elliot J; Tamilia, Michael; Paliouras, Miltiadis; Trifiro, Mark A

2017-03-01

Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.

Phosphodiesterase inhibitors suppress Lactobacillus casei cell-wall-induced NF-κB and MAPK activations and cell proliferation through protein kinase A--or exchange protein activated by cAMP-dependent signal pathway.

PubMed

Saito, Takekatsu; Sugimoto, Naotoshi; Ohta, Kunio; Shimizu, Tohru; Ohtani, Kaori; Nakayama, Yuko; Nakamura, Taichi; Hitomi, Yashiaki; Nakamura, Hiroyuki; Koizumi, Shoichi; Yachie, Akihiro

2012-01-01

Specific strains of Lactobacillus have been found to be beneficial in treating some types of diarrhea and vaginosis. However, a high mortality rate results from underlying immunosuppressive conditions in patients with Lactobacillus casei bacteremia. Cyclic AMP (cAMP) is a small second messenger molecule that mediates signal transduction. The onset and progression of inflammatory responses are sensitive to changes in steady-state cAMP levels. L. casei cell wall extract (LCWE) develops arteritis in mice through Toll-like receptor-2 signaling. The purpose of this study was to investigate whether intracellular cAMP affects LCWE-induced pathological signaling. LCWE was shown to induce phosphorylation of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and cell proliferation in mice fibroblast cells. Theophylline and phosphodiesterase inhibitor increased intracellular cAMP and inhibited LCWE-induced cell proliferation as well as phosphorylation of NF-κB and MAPK. Protein kinase A inhibitor H89 prevented cAMP-induced MAPK inhibition, but not cAMP-induced NF-κB inhibition. An exchange protein activated by cAMP (Epac) agonist inhibited NF-κB activation but not MAPK activation. These results indicate that an increase in intracellular cAMP prevents LCWE induction of pathological signaling pathways dependent on PKA and Epac signaling.

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.

Sulfated Hexasaccharides Attenuate Metastasis by Inhibition of P-selectin and Heparanase1

PubMed Central

Borsig, Lubor; Vlodavsky, Israel; Ishai-Michaeli, Rivka; Torri, Giangiacomo; Vismara, Elena

2011-01-01

Development of compounds that target both heparanase and selectins is emerging as a promising approach for cancer therapy. Selectins are vascular cell adhesion molecules that mediate tumor cell interactions with platelets, leukocytes, and the vascular endothelium. Heparanase is an endoglycosidase that degrades heparan sulfate in the tumor microenvironment, cell surfaces, and vessel wall. Acting together, these molecules facilitate tumor cell arrest, extravasation, and metastasis. Here, we report the preparation of novel semisynthetic sulfated tri mannose C-C-linked dimers (STMCs) endowed with heparanase and selectin inhibitory activity. The P-selectin specificity of the STMC was defined by the anomeric linkage of the C-C bond. This STMC hexasaccharide is an effective inhibitor of P-selectin in vivo. We show that selective inhibition of heparanase attenuates metastasis in B16-BL6 melanoma cells, expressing high levels of this endoglycosidase, but has no effect on the metastasis of MC-38 carcinoma cells that express little or no heparanase activity. P-selectin-specific STMC attenuated metastasis in both animal models, indicating that inhibition of tumor cell interaction with the vascular endothelium is critical for cancer dissemination. Thus, the small size, the stability of the C-C bond, and the chemically defined structure of the newly generated STMCs make them superior to heparin derivatives and signify STMCs as valuable candidates for further evaluation. PMID:21532885

Mobilization of lipids and fortification of cell wall and cuticle are important in host defense against Hessian fly

PubMed Central

2013-01-01

Background Wheat – Hessian fly interaction follows a typical gene-for-gene model. Hessian fly larvae die in wheat plants carrying an effective resistance gene, or thrive in susceptible plants that carry no effective resistance gene. Results Gene sets affected by Hessian fly attack in resistant plants were found to be very different from those in susceptible plants. Differential expression of gene sets was associated with differential accumulation of intermediates in defense pathways. Our results indicated that resources were rapidly mobilized in resistant plants for defense, including extensive membrane remodeling and release of lipids, sugar catabolism, and amino acid transport and degradation. These resources were likely rapidly converted into defense molecules such as oxylipins; toxic proteins including cysteine proteases, inhibitors of digestive enzymes, and lectins; phenolics; and cell wall components. However, toxicity alone does not cause immediate lethality to Hessian fly larvae. Toxic defenses might slow down Hessian fly development and therefore give plants more time for other types of defense to become effective. Conclusion Our gene expression and metabolic profiling results suggested that remodeling and fortification of cell wall and cuticle by increased deposition of phenolics and enhanced cross-linking were likely to be crucial for insect mortality by depriving Hessian fly larvae of nutrients from host cells. The identification of a large number of genes that were differentially expressed at different time points during compatible and incompatible interactions also provided a foundation for further research on the molecular pathways that lead to wheat resistance and susceptibility to Hessian fly infestation. PMID:23800119

Functional Implications of the Subcellular Localization of Ethylene-Induced Chitinase and [beta]-1,3-Glucanase in Bean Leaves.

PubMed Central

Mauch, F.; Staehelin, L. A.

1989-01-01

Plants respond to an attack by potentially pathogenic organisms and to the plant stress hormone ethylene with an increased synthesis of hydrolases such as chitinase and [beta]-1,3-glucanase. We have studied the subcellular localization of these two enzymes in ethylene-treated bean leaves by immunogold cytochemistry and by biochemical fractionation techniques. Our micrographs indicate that chitinase and [beta]-1,3-glucanase accumulate in the vacuole of ethylene-treated leaf cells. Within the vacuole label was found predominantly over ethylene-induced electron dense protein aggregates. A second, minor site of accumulation of [beta]-1,3-glucanase was the cell wall, where label was present nearly exclusively over the middle lamella surrounding intercellular air spaces. Both kinds of antibodies labeled Golgi cisternae of ethylene-treated tissue, suggesting that the newly synthesized chitinase and [beta]-1,3-glucanase are processed in the Golgi apparatus. Biochemical fractionation studies confirmed the accumulation in high concentrations of both chitinase and [beta]-1,3-glucanase in isolated vacuoles, and demonstrated that only [beta]-1,3-glucanase, but not chitinase, was present in intercellular washing fluids collected from ethylene-treated leaves. Based on these results and earlier studies, we propose a model in which the vacuole-localized chitinase and [beta]-1,3-glucanase are used as a last line of defense to be released when the attacked host cells lyse. The cell wall-localized [beta]-1,3-glucanase, on the other hand, would be involved in recognition processes, releasing defense activating signaling molecules from the walls of invading pathogens. PMID:12359894

Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

PubMed

Ogasawara, Shun; Shimada, Nao; Kawata, Takefumi

2009-02-01

Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

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

Antibacterial peptides from plants: what they are and how they probably work.

PubMed

Barbosa Pelegrini, Patrícia; Del Sarto, Rafael Perseghini; Silva, Osmar Nascimento; Franco, Octávio Luiz; Grossi-de-Sa, Maria Fátima

2011-01-01

Plant antibacterial peptides have been isolated from a wide variety of species. They consist of several protein groups with different features, such as the overall charge of the molecule, the content of disulphide bonds, and structural stability under environmental stress. Although the three-dimensional structures of several classes of plant peptides are well determined, the mechanism of action of some of these molecules is still not well defined. However, further studies may provide new evidences for their function on bacterial cell wall. Therefore, this paper focuses on plant peptides that show activity against plant-pathogenic and human-pathogenic bacteria. Furthermore, we describe the folding of several peptides and similarities among their three-dimensional structures. Some hypotheses for their mechanisms of action and attack on the bacterial membrane surface are also proposed.

Cancer cells remodel themselves and vasculature to overcome the endothelial barrier.

PubMed

Shenoy, Anitha K; Lu, Jianrong

2016-10-01

Metastasis refers to the spread of cancer cells from a primary tumor to distant organs mostly via the bloodstream. During the metastatic process, cancer cells invade blood vessels to enter circulation, and later exit the vasculature at a distant site. Endothelial cells that line blood vessels normally serve as a barrier to the movement of cells into or out of the blood. It is thus critical to understand how metastatic cancer cells overcome the endothelial barrier. Epithelial cancer cells acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT), which enables them to move toward vasculature. Cancer cells also express a variety of adhesion molecules that allow them to attach to vascular endothelium. Finally, cancer cells secrete or induce growth factors and cytokines to actively prompt vascular hyperpermeability that compromises endothelial barrier function and facilitates transmigration of cancer cells through the vascular wall. Elucidation of the mechanisms underlying metastatic dissemination may help develop new anti-metastasis therapeutics. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

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

Genetic alteration of UDP-rhamnose metabolism in Botrytis cinerea leads to the accumulation of UDP-KDG that adversely affects development and pathogenicity.

PubMed

Ma, Liang; Salas, Omar; Bowler, Kyle; Oren-Young, Liat; Bar-Peled, Maor; Sharon, Amir

2017-02-01

Botrytis cinerea is a model plant-pathogenic fungus that causes grey mould and rot diseases in a wide range of agriculturally important crops. A previous study has identified two enzymes and corresponding genes (bcdh, bcer) that are involved in the biochemical transformation of uridine diphosphate (UDP)-glucose, the major fungal wall nucleotide sugar precursor, to UDP-rhamnose. We report here that deletion of bcdh, the first biosynthetic gene in the metabolic pathway, or of bcer, the second gene in the pathway, abolishes the production of rhamnose-containing glycans in these mutant strains. Deletion of bcdh or double deletion of both bcdh and bcer has no apparent effect on fungal development or pathogenicity. Interestingly, deletion of the bcer gene alone adversely affects fungal development, giving rise to altered hyphal growth and morphology, as well as reduced sporulation, sclerotia production and virulence. Treatments with wall stressors suggest the alteration of cell wall integrity. Analysis of nucleotide sugars reveals the accumulation of the UDP-rhamnose pathway intermediate UDP-4-keto-6-deoxy-glucose (UDP-KDG) in hyphae of the Δbcer strain. UDP-KDG could not be detected in hyphae of the wild-type strain, indicating fast conversion to UDP-rhamnose by the BcEr enzyme. The correlation between high UDP-KDG and modified cell wall and developmental defects raises the possibility that high levels of UDP-KDG result in deleterious effects on cell wall composition, and hence on virulence. This is the first report demonstrating that the accumulation of a minor nucleotide sugar intermediate has such a profound and adverse effect on a fungus. The ability to identify molecules that inhibit Er (also known as NRS/ER) enzymes or mimic UDP-KDG may lead to the development of new antifungal drugs. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Dielectric polarization in the Planck theory of sonoluminescence.

PubMed

Prevenslik, T V

1998-11-01

Sonoluminescence observed in the cavitation of liquid H2O may be explained by the Planck theory of SL, which treats the bubbles as collapsing miniature masers having optical waves standing in resonance with the dimensions of the bubble cavity. Microwaves are shown to be created from the Planck energy of the standing waves, provided the bubble wall can be treated as a perfect blackbody surface. Liquid H2O is strongly absorbent in the ultraviolet and there the bubble approaches a Planck blackbody enclosure. The microwaves are created at frequencies proportional to the bubble collapse velocity only to be promptly absorbed by the rotation quantum states of the H2O and other bubble wall molecules. The microwaves are absorbed discretely at rotation line frequencies, or continuously by dipole rotation at frequencies from 1 to 30 GHz. In the liquid state, molecular rotation of the H2O molecule is hindered and the microwave energy is rapidly turned into bending energy by intermolecular collisions. Subsequently, the bubble wall molecules may thereby ionize and produce visible photons. The microwaves create intense electrical fields in the bubble wall by dielectric polarization. If the gases adjacent to the bubble wall undergo electrical breakdown, free electrons are created, thereby providing sonoluminescence with a magnetic field effect.

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

PubMed Central

Billaud, Marie; Lohman, Alexander W.; Johnstone, Scott R.; Biwer, Lauren A.; Mutchler, Stephanie; Isakson, Brant E.

2014-01-01

It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function. PMID:24671377

Regulation of cellular communication by signaling microdomains in the blood vessel wall.

PubMed

Billaud, Marie; Lohman, Alexander W; Johnstone, Scott R; Biwer, Lauren A; Mutchler, Stephanie; Isakson, Brant E

2014-01-01

It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function.

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

Antibacterial Effect of (2E,2E)-4,4-Trisulfanediylbis(but-2-enoic acid) against Staphylococcus aureus

PubMed Central

Chen, Yijun; Zhang, Min

2018-01-01

A new highly active molecule, (2E, 2E)-4,4-trisulfanediylbis(but-2-enoic acid) (TSDB), was designed and synthesized through comparative molecular field analysis with the diallyl trisulfide structure of garlic. TSDB exerted a strong inhibitory effect against Staphylococcus aureus, with minimal inhibitory and minimal bactericidal concentrations of 16 and 128 μg/mL, respectively. TSDB destructed the integrity of the S. aureus cell membrane but weakly damaged the bacterial cell wall. TSDB also increased the conductivity and protein expression in microbial broth but minimally influenced the level of extracellular alkaline phosphatase. TSDB could be a novel food preservative. PMID:29795597

Steady-state configurations and dynamics of the MreB helix within bacteria

NASA Astrophysics Data System (ADS)

Rutenberg, Andrew; Allard, Jun

2007-03-01

We present a quantitative model of the actin-like MreB cytoskeleton that is present in many prokaryotes. Individual MreB polymers are bundled into a supra-molecular array to make up helical cables. The cell wall imposes constraint forces through a global elasticity model. With variational techniques and stochastic simulations we obtain relationships between observable quantities such as the pitch of the helix, the total abundance of MreB molecules, and the thickness of the MreB cables. We address changes expected with slow cell growth, as well as turnover dynamics that are relevant to FRAP studies. We also address polarized macromolecular trafficking along the MreB cables without motor proteins.

Investigation of the mechanism for penetration of low density lipoprotein into the arterial wall

NASA Astrophysics Data System (ADS)

Glukhova, O. E.; Zyktin, A. A.; Slepchenkov, M. M.

2018-02-01

Currently, the pathology of the cardiovascular system is an extremely urgent problem of fundamental and clinical medicine. These diseases are caused, mainly, by atherosclerotic changes in the wall of blood vessels. The predominant role in the development of atherosclerosis is attributed to the penetration of various kinds of lipoproteins into the arterial intima. In this paper, we in silico investigated the dynamics of the penetration of low density lipoprotein (LDL) through the intercellular gap using molecular modeling methods. The simulation was carried out in the GROMACS software package using a coarse-grained MARTINI model. During investigation we carried out the LDL self-assembly for the first time. The coarse-grained model of LDL was collected from the following molecules: POPC (phosphatidylcholine) - 630 molecules, LPC (lysophosphatidylcholine) - 80 molecules CHOL (cholesterol) - 600 molecules CHYO (cholesteryl oleate) - 1600 molecules TOG (glycerol trioleate) 180 Molecules. The coarse-grained model of the intercellular endothelial gap was based on a model of lipid bilayer consisting of DPPC phospholipids and cholesterol in a percentage ratio of 70% and 30%, respectively. Based on the obtained results, we can predict the mechanism of LDL diffusion. Lipoproteins can be deformed so as to pass through narrow gaps. Our investigations open the way for the research of the behavior dynamics of LDL moving with the blood flow rate when interacting with the intercellular gaps of the endothelial layer of the vessel inner wall.

Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis.

PubMed

Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

2016-11-15

Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Embryonic development of pleuropodia of the cicada, Magicicada cassini

PubMed Central

Strauß, Johannes; Lakes-Harlan, Reinhard

2006-01-01

In many insects the first abdominal segment possesses embryonic appendages called pleuropodia. Here we show the embryogenesis of pleuropodial cells of the periodical cicada, Magicicada cassini (Fisher 1851) (Insecta, Homoptera, Cicadidae). An antibody, anti-horseradish perioxidase (HRP), that is usually neuron-specific strongly marked the pleuropodial anlagen and revealed their ectodermal origin shortly after limb bud formation. Thereafter the cells sank into the epidermis and their apical parts enlarged. A globular part protruded from the body wall. Filamentous structures were marked at the stem region and into the apical dilation. In later embryonic stages the pleuropodia degenerated. Despite the binding of anti-HRP the cells had no morphological neuronal characters and cannot be regarded as neurons. The binding indicates that glycosylated cell surface molecules contribute to the adhesion between the presumably glandular pleuropodial cells. In comparison, anti-HRP does not mark the pleuropodia of Orthoptera. PMID:19537987

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.

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

Modelling of adsorption and intercalation of hydrogen on/into tungsten disulphide multilayers and multiwall nanotubes.

PubMed

Martínez, José I; Laikhtman, Alex; Moon, Hoi Ri; Zak, Alla; Alonso, Julio A

2018-05-07

Understanding the interaction of hydrogen with layered materials is crucial in the fields of sensors, catalysis, fuel cells and hydrogen storage, among others. Density functional theory, improved by the introduction of van der Waals dispersion forces, provides an efficient and practical workbench to investigate the interaction of molecular and atomic hydrogen with WS 2 multilayers and nanotubes. We find that H 2 physisorbs on the surface of those materials on top of W atoms, while atomic H chemisorbs on top of S atoms. In the case of nanotubes, the chemisorption strength is sensitive to the nanotube diameter. Diffusion of H 2 on the surface of WS 2 encounters quite small activation barriers whose magnitude helps to explain previous and new experimental results for the observed dependence of the hydrogen concentration with temperature. Intercalation of H 2 between adjacent planar WS 2 layers reveals an endothermic character. Intercalating H atoms is energetically favorable, but the intercalation energy does not compensate for the cost of dissociating the molecules. When H 2 molecules are intercalated between the walls of a double wall nanotube, the rigid confinement induces the dissociation of the confined molecules. A remarkable result is that the presence of a full H 2 monolayer adsorbed on top of the first WS 2 layer of a WS 2 multilayer system strongly facilitates the intercalation of H 2 between WS 2 layers underneath. This opens up an additional gate to intercalation processes.

Smooth muscle cell phenotypic switching in stroke.

PubMed

Poittevin, Marine; Lozeron, Pierre; Hilal, Rose; Levy, Bernard I; Merkulova-Rainon, Tatiana; Kubis, Nathalie

2014-06-01

Disruption of cerebral blood flow after stroke induces cerebral tissue injury through multiple mechanisms that are not yet fully understood. Smooth muscle cells (SMCs) in blood vessel walls play a key role in cerebral blood flow control. Cerebral ischemia triggers these cells to switch to a phenotype that will be either detrimental or beneficial to brain repair. Moreover, SMC can be primarily affected genetically or by toxic metabolic molecules. After stroke, this pathological phenotype has an impact on the incidence, pattern, severity, and outcome of the cerebral ischemic disease. Although little research has been conducted on the pathological role and molecular mechanisms of SMC in cerebrovascular ischemic diseases, some therapeutic targets have already been identified and could be considered for further pharmacological development. We examine these different aspects in this review.

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.

[Glycation, glycoxidation and diabetes mellitus].

PubMed

Boulanger, Eric; Wautier, Jean-Luc; Dequiedt, Philippe; Schmidt, Anne-Marie

2006-01-01

Advanced glycation end-products (AGEs) result from a reaction between carbohydrates and the free amino groups of proteins, lipids, and DNA. Non enzymatic glycation, glycoxidation with glucose auto-oxidation and the polyol pathway are involved in glycated protein formation. AGEs also named glycotoxins are found in excess in pathological situations such as diabetes mellitus, renal failure, and aging or after absorption of food containing glycated products. Three major pathophysiological mechanisms are described to explain AGE toxicity, first AGEs can accumulate in the vessel wall and in collagen of different tissues; second in situ glycation is possible; third, AGEs bind to cell receptors inducing deleterious consequences. AGE receptor RAGE is a multiligand member of the immunoglobulin superfamily of cell surface molecules. AGE-receptor interaction can alter, macrophage, endothelial cell, mesangial and mesothelial cell functions and can induce inflammation. Oxidant stress, vascular hyperpermeability, vascular cell adhesion molecule-1 (VCAM-1) overexpression and monocytes chemotactic Protein-1 (MCP-1) production have been observed after cell activation by AGEs. AGEs appear to be involved in the genesis of diabetic macro but also microangiopathy such as retinopathy and glomerulosclerosis. New drugs are tested to prevent or break the AGE-protein cross-linkage, or to control the AGE-receptor interaction and their consequences. Dietary treatment, strict glycemic control and preservation of renal function remain the best approach for preventing AGE formation and limiting their deleterious effects.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

PubMed Central

Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X.

2008-01-01

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-κB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-κB activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1) and antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and contributes to innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. PMID:18191935

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2.

PubMed

Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X

2008-05-01

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappaB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappaB activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1), antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules.

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

Interaction of single-walled carbon nanotubes and saxitoxin: Ab initio simulations and biological responses in hippocampal cell line HT-22.

PubMed

Ramos, Patrícia; Schmitz, Marcos; Filgueira, Daza; Votto, Ana Paula; Durruthy, Michael; Gelesky, Marcos; Ruas, Caroline; Yunes, João; Tonel, Mariana; Fagan, Solange; Monserrat, José

2017-07-01

Saxitoxins (STXs) are potent neurotoxins that also induce cytotoxicity through the generation of reactive oxygen species. Carbon nanotubes (CNTs) are nanomaterials that can promote a Trojan horse effect, facilitating the entry of toxic molecules to cells when adsorbed to nanomaterials. The interaction of pristine single-walled (SW)CNTs and carboxylated (SWCNT-COOH) nanotubes with STX was evaluated by ab initio simulation and bioassays using the cell line HT-22. Cells (5 × 10 4  cells/mL) were exposed to SWCNT and SWCNT-COOH (5 μg mL -1 ), STX (200 μg L -1 ), SWCNT+STX, and SWCNT-COOH+STX for 30 min or 24 h. Results of ab initio simulation showed that the interaction between SWCNT and SWCNT-COOH with STX occurs in a physisorption. The interaction of SWCNT+STX induced a decrease in cell viability. Cell proliferation was not affected in any treatment after 30 min or 24 h of exposure (p > 0.05). Treatment with SWCNT-COOH induced high reactive oxygen species levels, an effect attenuated in SWCNT-COOH+STX treatment. In terms of cellular oxygen consumption, both CNTs when coexposed with STX antagonize the toxin effect. Based on these results, it can be concluded that the results obtained in vitro corroborate the semiempirical evidence found using density functional theory ab initio simulation. Environ Toxicol Chem 2017;36:1728-1737. © 2016 SETAC. © 2016 SETAC.

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.

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.

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.

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.

Rapid detection of food pathogens using RNA aptamers-immobilized slide.

PubMed

Maeng, Jin-Soo; Kim, Namsoo; Kim, Chong-Tai; Han, Seung Ryul; Lee, Young Ju; Lee, Seong-Wook; Lee, Myung-Hyun; Cho, Yong-Jin

2012-07-01

The purpose of this study was to develop a simple and rapid detection system for foodborne bacteria, which consisted of an optical microscope and its slide chip with artificial antibodies, or RNA aptamers. From an RNA pool, three each RNA aptamers were built by the method of SELEX (systematic evolution of ligands by exponential enrichment) for components of cell wall, LPS (lipopolysaccharide) from E. coli O157:H7, teichoic acid from Staphylococcus aureus and a cell membrane protein of OmpC from Salmonella typhimurium, respectively. These aptamers were hybridized with thiol-conjugated 16 dT-linker molecules in order to be immobilized on silver surface which was, in advance, fabricated on glass slide, using a spin-coating method. To confirm that each aptamers retained its specific binding activities to their antigenic live bacteria, microscopic view of bound cells immobilized on silver film were observed. Furthermore, we observed the fluorescence-emitting bacteria-aptamer complex immobilized on silver film after adding RNA aptamers hybridized with fluorophore, FAM-conjugated 16 dT-linker molecules. As a result, the RNA aptamers-immobilized slide system developed in this study was a useful new tool to rapidly monitor individual food pathogens.

Structural Model for Covalent Adhesion of the Streptococcus pyogenes Pilus through a Thioester Bond*

PubMed Central

Linke-Winnebeck, Christian; Paterson, Neil G.; Young, Paul G.; Middleditch, Martin J.; Greenwood, David R.; Witte, Gregor; Baker, Edward N.

2014-01-01

The human pathogen Streptococcus pyogenes produces pili that are essential for adhesion to host surface receptors. Cpa, the adhesin at the pilus tip, was recently shown to have a thioester-containing domain. The thioester bond is believed to be important in adhesion, implying a mechanism of covalent attachment analogous to that used by human complement factors. Here, we have characterized a second active thioester-containing domain on Cpa, the N-terminal domain of Cpa (CpaN). Expression of CpaN in Escherichia coli gave covalently linked dimers. These were shown by x-ray crystallography and mass spectrometry to comprise two CpaN molecules cross-linked by the polyamine spermidine following reaction with the thioester bonds. This cross-linked CpaN dimer provides a model for the covalent attachment of Cpa to target receptors and thus the streptococcal pilus to host cells. Similar thioester domains were identified in cell wall proteins of other Gram-positive pathogens, suggesting that thioester domains are more widely used and provide a mechanism of adhesion by covalent bonding to target molecules on host cells that mimics that used by the human complement system to eliminate pathogens. PMID:24220033

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

Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane

PubMed Central

2005-01-01

The effects of antimicrobial peptides on artificial membranes have been well-documented; however, reports on the ultrastructural effects on the membranes of micro-organisms are relatively scarce. We compared the effects of histatin 5 and LL-37, two antimicrobial peptides present in human saliva, on the functional and morphological properties of the Candida albicans cell membrane. Fluorescence microscopy and immunogold transmission electron microscopy revealed that LL-37 remained associated with the cell wall and cell membrane, whereas histatin 5 transmigrated over the membrane and accumulated intracellularly. Freeze-fracture electron microscopy revealed that LL-37 severely affected the membrane morphology, resulting in the disintegration of the membrane bilayer into discrete vesicles, and an instantaneous efflux of small molecules such as ATP as well as larger molecules such as proteins with molecular masses up to 40 kDa. The effects of histatin 5 on the membrane morphology were less pronounced, but still resulted in the efflux of nucleotides. As the morphological defects induced by histatin 5 are much smaller than those induced by LL-37, but the efflux of nucleotides is similar at comparable candidacidal concentrations, we suggest that the loss of nucleotides plays an important role in the killing process. PMID:15707390

Mechanosensory Genes Pkd1 and Pkd2 Contribute to the Planar Polarization of Brain Ventricular Epithelium

PubMed Central

Herranz-Pérez, Vicente; Nakatani, Jin; Boletta, Alessandra; García-Verdugo, José Manuel

2015-01-01

Directional beating of ependymal (E) cells' cilia in the walls of the ventricles in the brain is essential for proper CSF flow. E cells display two forms of planar cell polarity (PCP): rotational polarity of individual cilium and translational polarity (asymmetric positioning of cilia in the apical area). The orientation of individual E cells varies according to their location in the ventricular wall (location-specific PCP). It has been hypothesized that hydrodynamic forces on the apical surface of radial glia cells (RGCs), the embryonic precursors of E cells, could guide location-specific PCP in the ventricular epithelium. However, the detection mechanisms for these hydrodynamic forces have not been identified. Here, we show that the mechanosensory proteins polycystic kidney disease 1 (Pkd1) and Pkd2 are present in primary cilia of RGCs. Ablation of Pkd1 or Pkd2 in Nestin-Cre;Pkd1flox/flox or Nestin-Cre;Pkd2flox/flox mice, affected PCP development in RGCs and E cells. Early shear forces on the ventricular epithelium may activate Pkd1 and Pkd2 in primary cilia of RGCs to properly polarize RGCs and E cells. Consistently, Pkd1, Pkd2, or primary cilia on RGCs were required for the proper asymmetric localization of the PCP protein Vangl2 in E cells' apical area. Analyses of single- and double-heterozygous mutants for Pkd1 and/or Vangl2 suggest that these genes function in the same pathway to establish E cells' PCP. We conclude that Pkd1 and Pkd2 mechanosensory proteins contribute to the development of brain PCP and prevention of hydrocephalus. SIGNIFICANCE STATEMENT This study identifies key molecules in the development of planar cell polarity (PCP) in the brain and prevention of hydrocephalus. Multiciliated ependymal (E) cells within the brain ventricular epithelium generate CSF flow through ciliary beating. E cells display location-specific PCP in the orientation and asymmetric positioning of their cilia. Defects in this PCP can result in hydrocephalus. Hydrodynamic forces on radial glial cells (RGCs), the embryonic progenitors of E cells, have been suggested to guide PCP. We show that the mechanosensory proteins Pkd1 and Pkd2 localize to primary cilia in RGCs, and their ablation disrupts the development of PCP in E cells. Early shear forces on RGCs may activate Pkd1 and Pkd2 in RGCs' primary cilia to properly orient E cells. This study identifies key molecules in the development of brain PCP and prevention of hydrocephalus. PMID:26245976

Changes in gravity inhibit lymphocyte locomotion through type I collagen

NASA Technical Reports Server (NTRS)

Pellis, N. R.; Goodwin, T. J.; Risin, D.; McIntyre, B. W.; Pizzini, R. P.; Cooper, D.; Baker, T. L.; Spaulding, G. F.

1997-01-01

Immunity relies on the circulation of lymphocytes through many different tissues including blood vessels, lymphatic channels, and lymphoid organs. The ability of lymphocytes to traverse the interstitium in both nonlymphoid and lymphoid tissues can be determined in vitro by assaying their capacity to locomote through Type I collagen. In an attempt to characterize potential causes of microgravity-induced immunosuppression, we investigated the effects of simulated microgravity on human lymphocyte function in vitro using a specialized rotating-wall vessel culture system developed at the Johnson Space Center. This very low shear culture system randomizes gravitational vectors and provides an in vitro approximation of microgravity. In the randomized gravity of the rotating-wall vessel culture system, peripheral blood lymphocytes did not locomote through Type I collagen, whereas static cultures supported normal movement. Although cells remained viable during the entire culture period, peripheral blood lymphocytes transferred to unit gravity (static culture) after 6 h in the rotating-wall vessel culture system were slow to recover and locomote into collagen matrix. After 72 h in the rotating-wall vessel culture system and an additional 72 h in static culture, peripheral blood lymphocytes did not recover their ability to locomote. Loss of locomotory activity in rotating-wall vessel cultures appears to be related to changes in the activation state of the lymphocytes and the expression of adhesion molecules. Culture in the rotating-wall vessel system blunted the ability of peripheral blood lymphocytes to respond to polyclonal activation with phytohemagglutinin. Locomotory response remained intact when peripheral blood lymphocytes were activated by anti-CD3 antibody and interleukin-2 prior to introduction into the rotating-wall vessel culture system. Thus, in addition to the systemic stress factors that may affect immunity, isolated lymphocytes respond to gravitational changes by ceasing locomotion through model interstitium. These in vitro investigations suggest that microgravity induces non-stress-related changes in cell function that may be critical to immunity. Preliminary analysis of locomotion in true microgravity revealed a substantial inhibition of cellular movement in Type I collagen. Thus, the rotating-wall vessel culture system provides a model for analyzing the microgravity-induced inhibition of lymphocyte locomotion and the investigation of the mechanisms related to lymphocyte movement.

Efficient visible light induced synthesis of silver nanoparticles by Penicillium polonicum ARA 10 isolated from Chetomorpha antennina and its antibacterial efficacy against Salmonella enterica serovar Typhimurium.

PubMed

Neethu, Sahadevan; Midhun, Sebastian Jose; Sunil, M A; Soumya, Soman; Radhakrishnan, E K; Jyothis, Mathew

2018-03-01

The green synthesis of silver nanoparticles (AgNPs) using biological systems such as fungi has evolved to become an important area of nanobiotechnology. Herein, we report for the first time the light-induced extracellular synthesis of silver nanoparticles using algicolous endophytic fungus Penicillium polonicum ARA 10, isolated from the marine green alga Chetomorpha antennina. Parametric optimization, including the concentration of AgNO 3 , fungal biomass, ratio of cell filtrate and AgNO 3 , pH, reaction time and presence of light, was done for rapid AgNPs production. The obtained silver nanoparticles (AgNPs) were characterized by UV-Visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy and Transmission electron microscopy (HRTEM-EDAX). The AgNPs showed a characteristic UV-visible peak at 430 nm with an average size of 10-15 nm. The NH stretches in FTIR indicate the presence of protein molecules. The Raman vibrational bands suggest that the molecules responsible for the reduction and stability of AgNPs were extracellular proteins produced by P.polonicum. Antibacterial evaluation of AgNPs against the major foodborne bacterial pathogen Salmonella enterica serovar Typhimurium MTCC 1251, was assessed by well diffusion, Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assay. Killing kinetic studies revealed complete killing of the bacterial cells within 4 h and the bactericidal nature of synthesized nanoparticles was confirmed by fluorescent microscopy and scanning electron microscopy. Furthermore, the bactericidal studies with Transmission electron microscopy (TEM) at different time intervals explored the presence of AgNPs in the cell wall of S.Typhimurium at about 30 min and the complete bacterial lysis was found at 24 h. The current research opens an insight into the green synthesis of AgNPs and the mechanism of bacterial lysis by direct damage to the cell wall. Copyright © 2018 Elsevier B.V. All rights reserved.

Toward an improved model of maple sap exudation: the location and role of osmotic barriers in sugar maple, butternut and white birch.

PubMed

Cirelli, Damián; Jagels, Richard; Tyree, Melvin T

2008-08-01

Two theories have been proposed to explain how high positive pressures are developed in sugar maple stems when temperatures fluctuate around freezing. The Milburn-O'Malley theory proposes that pressure development is purely physical and does not require living cells or sucrose. The osmotic theory invokes the involvement of living cells and sucrose to generate an osmotic pressure difference between fibers and vessels, which are assumed to be separated by an osmotic barrier. We analyzed wood of Acer saccharum Marsh., Juglans cinerea L. and Betula papyrifera Marsh. (all generate positive pressures) examining three critical components of the osmotic model: pits in cell walls, selectivity of the osmotic barrier and stability of air bubbles under positive xylem pressure. We examined the distribution and type of pits directly by light and scanning electron microscopy (SEM), and indirectly by perfusion of branch segments with fluorescent dyes with molecular masses similar to sucrose. The latter approach allowed us to use osmotic surrogates for sucrose that could be tracked by epifluorescence. Infusion experiments were used to assess the compartmentalization of sucrose and to determine the behavior of gas bubbles as predicted by Fick's and Henry's laws. The SEM images of sugar maple revealed a lack of pitting between fibers and vessels but connections between fiber-tracheids and vessels were present. Fluorescein-perfusion experiments demonstrated that large molecules do not diffuse into libriform fibers but are confined within the domain of vessels, parenchyma and fiber-tracheids. Results of the infusion experiments were in agreement with those of the fluorescein perfusions and further indicated the necessity of a compartmentalized osmolyte to drive stem pressure, as well as the inability of air bubbles to maintain such pressure because of instability. These results support the osmotic model and demonstrate that the secondary cell wall is an effective osmotic barrier for molecules larger than 300 g mol(-1).

Progress Toward Sequestering Carbon Nanotubes in PmPV

NASA Technical Reports Server (NTRS)

Bley, Richard A.

2009-01-01

Sequestration of single-walled carbon nanotubes (SWNTs) in molecules of poly(m-phenylenevinylene-co-2,5-diocty-loxy-p-phenylenevinylene) [PmPV] is a candidate means of promoting dissolution of single-walled carbon nanotubes (SWNTs) into epoxies for making strong, lightweight epoxy-matrix/carbon-fiber composite materials. Bare SWNTs cannot be incorporated because they are not soluble in epoxies. In the present approach, one exploits the tendency of PmPV molecules to wrap themselves around SWNTs without chemically bonding to them.

Plasmodesmata in integrated cell signalling: insights from development and environmental signals and stresses

PubMed Central

Sager, Ross; Lee, Jung-Youn

2014-01-01

To survive as sedentary organisms built of immobile cells, plants require an effective intercellular communication system, both locally between neighbouring cells within each tissue and systemically across distantly located organs. Such a system enables cells to coordinate their intracellular activities and produce concerted responses to internal and external stimuli. Plasmodesmata, membrane-lined intercellular channels, are essential for direct cell-to-cell communication involving exchange of diffusible factors, including signalling and information molecules. Recent advances corroborate that plasmodesmata are not passive but rather highly dynamic channels, in that their density in the cell walls and gating activities are tightly linked to developmental and physiological processes. Moreover, it is becoming clear that specific hormonal signalling pathways play crucial roles in relaying primary cellular signals to plasmodesmata. In this review, we examine a number of studies in which plasmodesmal structure, occurrence, and/or permeability responses are found to be altered upon given cellular or environmental signals, and discuss common themes illustrating how plasmodesmal regulation is integrated into specific cellular signalling pathways. PMID:25262225

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.

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.

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

Vacuum template synthesis of multifunctional nanotubes with tailored nanostructured walls

NASA Astrophysics Data System (ADS)

Filippin, A. Nicolas; Macias-Montero, Manuel; Saghi, Zineb; Idígoras, Jesús; Burdet, Pierre; Barranco, Angel; Midgley, Paul; Anta, Juan A.; Borras, Ana

2016-02-01

A three-step vacuum procedure for the fabrication of vertical TiO2 and ZnO nanotubes with three dimensional walls is presented. The method combines physical vapor deposition of small-molecules, plasma enhanced chemical vapor deposition of inorganic functional thin films and layers and a post-annealing process in vacuum in order to remove the organic template. As a result, an ample variety of inorganic nanotubes are made with tunable length, hole dimensions and shapes and tailored wall composition, microstructure, porosity and structure. The fabrication of multishell nanotubes combining different semiconducting oxides and metal nanoparticles is as well explored. This method provides a feasible and reproducible route for the fabrication of high density arrays of vertically alligned nanotubes on processable substrates. The emptying mechanism and microstructure of the nanotubes have been elucidated through SEM, STEM, HAADF-STEM tomography and energy dispersive X-ray spectroscopy. In this article, as a proof of concept, it is presented the straightforward integration of ZnO nanotubes as photoanode in a photovoltaic cell and as a photonic oxygen gas sensor.

Vacuum template synthesis of multifunctional nanotubes with tailored nanostructured walls

PubMed Central

Filippin, A. Nicolas; Macias-Montero, Manuel; Saghi, Zineb; Idígoras, Jesús; Burdet, Pierre; Barranco, Angel; Midgley, Paul; Anta, Juan A.; Borras, Ana

2016-01-01

A three-step vacuum procedure for the fabrication of vertical TiO2 and ZnO nanotubes with three dimensional walls is presented. The method combines physical vapor deposition of small-molecules, plasma enhanced chemical vapor deposition of inorganic functional thin films and layers and a post-annealing process in vacuum in order to remove the organic template. As a result, an ample variety of inorganic nanotubes are made with tunable length, hole dimensions and shapes and tailored wall composition, microstructure, porosity and structure. The fabrication of multishell nanotubes combining different semiconducting oxides and metal nanoparticles is as well explored. This method provides a feasible and reproducible route for the fabrication of high density arrays of vertically alligned nanotubes on processable substrates. The emptying mechanism and microstructure of the nanotubes have been elucidated through SEM, STEM, HAADF-STEM tomography and energy dispersive X-ray spectroscopy. In this article, as a proof of concept, it is presented the straightforward integration of ZnO nanotubes as photoanode in a photovoltaic cell and as a photonic oxygen gas sensor. PMID:26860367

Genetic evidence supporting a critical role of endothelial caveolin-1 during the progression of atherosclerosis

PubMed Central

Fernández-Hernando, Carlos; Yu, Jun; Suárez, Yajaira; Rahner, Christoph; Dávalos, Alberto; Lasunción, Miguel A.; Sessa, William C.

2009-01-01

SUMMARY The accumulation of LDL-derived cholesterol in the artery wall is the initiating event that causes atherosclerosis. However, the mechanisms that lead to the initiation of atherosclerosis are still poorly understood. Here, by using endothelial cell-specific transgenesis of the caveolin-1 (Cav-1) gene in mice, we show the critical role of Cav-1 in promoting atherogenesis. Mice were generated lacking Cav-1 and apoE but expressing endothelial-specific Cav-1 in the double knockout background. Genetic ablation of Cav-1 on an apoE knockout background inhibits the progression of atherosclerosis while re-expression of Cav-1 in the endothelium promotes lesion expansion. Mechanistically, the loss of Cav-1 reduces LDL infiltration into the artery wall, promotes nitric oxide production and reduces the expression of leukocyte adhesion molecules, effects completely reversed in transgenic mice. In summary, this unique model provides physiological evidence supporting the important role of endothelial Cav-1 expression in regulating the entry of LDL into the vessel wall and the initiation of atherosclerosis. PMID:19583953

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.

Short peptides allowing preferential detection of Candida albicans hyphae.

PubMed

Kaba, Hani E J; Pölderl, Antonia; Bilitewski, Ursula

2015-09-01

Whereas the detection of pathogens via recognition of surface structures by specific antibodies and various types of antibody mimics is frequently described, the applicability of short linear peptides as sensor molecules or diagnostic tools is less well-known. We selected peptides which were previously reported to bind to recombinant S. cerevisiae cells, expressing members of the C. albicans Agglutinin-Like-Sequence (ALS) cell wall protein family. We slightly modified amino acid sequences to evaluate peptide sequence properties influencing binding to C. albicans cells. Among the selected peptides, decamer peptides with an "AP"-N-terminus were superior to shorter peptides. The new decamer peptide FBP4 stained viable C. albicans cells more efficiently in their mature hyphal form than in their yeast form. Moreover, it allowed distinction of C. albicans from other related Candida spp. and could thus be the basis for the development of a useful tool for the diagnosis of invasive candidiasis.

Redox-dependent impairment of vascular function in sickle cell disease.

PubMed

Aslan, Mutay; Freeman, Bruce A

2007-12-01

The vascular pathophysiology of sickle cell disease (SCD) is influenced by many factors, including adhesiveness of red and white blood cells to endothelium, increased coagulation, and homeostatic perturbation. The vascular endothelium is central to disease pathogenesis because it displays adhesion molecules for blood cells, balances procoagulant and anticoagulant properties of the vessel wall, and regulates vascular homeostasis by synthesizing vasoconstricting and vasodilating substances. The occurrence of intermittent vascular occlusion in SCD leads to reperfusion injury associated with granulocyte accumulation and enhanced production of reactive oxygen species. The participation of nitric oxide (NO) in oxidative reactions causes a reduction in NO bioavailability and contributes to vascular dysfunction in SCD. Therapeutic strategies designed to counteract endothelial, inflammatory, and oxidative abnormalities may reduce the frequency of hospitalization and blood transfusion, the incidence of pain, and the occurrence of acute chest syndrome and pulmonary hypertension in patients with SCD.

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

Signal Regulatory Protein α Negatively Regulates β2 Integrin-Mediated Monocyte Adhesion, Transendothelial Migration and Phagocytosis

PubMed Central

Liu, Dan-Qing; Li, Li-Min; Guo, Ya-Lan; Bai, Rui; Wang, Chen; Bian, Zhen; Zhang, Chen-Yu; Zen, Ke

2008-01-01

Background Signal regulate protein α (SIRPα) is involved in many functional aspects of monocytes. Here we investigate the role of SIRPα in regulating β2 integrin-mediated monocyte adhesion, transendothelial migration (TEM) and phagocytosis. Methodology/Principal Findings THP-1 monocytes/macropahges treated with advanced glycation end products (AGEs) resulted in a decrease of SIRPα expression but an increase of β2 integrin cell surface expression and β2 integrin-mediated adhesion to tumor necrosis factor-α (TNFα)–stimulated human microvascular endothelial cell (HMEC-1) monolayers. In contrast, SIRPα overexpression in THP-1 cells showed a significant less monocyte chemotactic protein-1 (MCP-1)–triggered cell surface expression of β2 integrins, in particular CD11b/CD18. SIRPα overexpression reduced β2 integrin-mediated firm adhesion of THP-1 cells to either TNFα–stimulated HMEC-1 monolayers or to immobilized intercellular adhesion molecule-1 (ICAM-1). SIRPα overexpression also reduced MCP-1–initiated migration of THP-1 cells across TNFα–stimulated HMEC-1 monolayers. Furthermore, β2 integrin-mediated THP-1 cell spreading and actin polymerization in response to MCP-1, and phagocytosis of bacteria were both inhibited by SIRPα overexpression. Conclusions/Significance SIRPα negatively regulates β2 integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis, thus may serve as a critical molecule in preventing excessive activation and accumulation of monocytes in the arterial wall during early stage of atherosclerosis. PMID:18820737

Magnetic resonance tells microbiology where to go; bacterial teichoic acid protects liquid water at sub-zero temperatures

NASA Astrophysics Data System (ADS)

Rice, Charles V.; Wickham, Jason R.; Eastman, Margaret A.; Harrison, William; Pereira, Mark P.; Brown, Eric D.

2008-08-01

Numerous chemical additives lower the freezing point of water, but life at sub-zero temperatures is sustained by a limited number of biological cryoprotectants. Antifreeze proteins in fish, plants, and insects provide protection to a few degrees below freezing. Microbes have been found to survive at even lower temperatures, although, with a few exceptions, antifreeze proteins are missing. Survival has been attributed to external factors, such as high salt concentration (brine veins) and adhesion to particulates or ice crystal defects. Teichoic acid is a phosphodiester polymer ubiquitous in Gram positive bacteria, composing 50% of the mass of the bacterial cell wall and excreted into the extracellular space of biofilm communities. We have found that when bound to the peptidoglycan cell wall (wall teichoic acid) or as a free molecule (lipoteichoic acid), teichoic acid is surrounded by liquid water at temperatures significantly below freezing. Using solid-state NMR, we are unable to collect 31P CPMAS spectra for frozen solutions of lipoteichoic acid at temperatures above -60 °C. For wall teichoic acid in D2O, signals are not seen above -30 °C. These results can be explained by the presence of liquid water, which permits rapid molecular motion to remove 1H/31P dipolar coupling. 2H quadrupole echo NMR spectroscopy reveals that both liquid and solid water are present. We suggest that teichoic acids could provide a shell of liquid water around biofilms and planktonic bacteria, removing the need for brine veins to prevent bacterial freezing.

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.

Recombinant Lactobacillus plantarum induces immune responses to cancer testis antigen NY-ESO-1 and maturation of dendritic cells

PubMed Central

Mobergslien, Anne; Vasovic, Vlada; Mathiesen, Geir; Fredriksen, Lasse; Westby, Phuong; Eijsink, Vincent GH; Peng, Qian; Sioud, Mouldy

2015-01-01

Given their safe use in humans and inherent adjuvanticity, Lactic Acid Bacteria may offer several advantages over other mucosal delivery strategies for cancer vaccines. The objective of this study is to evaluate the immune responses in mice after oral immunization with Lactobacillus (L) plantarum WCFS1 expressing a cell-wall anchored tumor antigen NY-ESO-1. And to investigate the immunostimulatory potency of this new candidate vaccine on human dendritic cells (DCs). L. plantarum displaying NY-ESO-1 induced NY-ESO-1 specific antibodies and T-cell responses in mice. By contrast, L. plantarum displaying conserved proteins such as heat shock protein-27 and galectin-1, did not induce immunity, suggesting that immune tolerance to self-proteins cannot be broken by oral administration of L. plantarum. With respect to immunomodulation, immature DCs incubated with wild type or L. plantarum-NY-ESO-1 upregulated the expression of co-stimulatory molecules and secreted a large amount of interleukin (IL)-12, TNF-α, but not IL-4. Moreover, they upregulated the expression of immunosuppressive factors such as IL-10 and indoleamine 2,3-dioxygenase. Although L. plantarum-matured DCs expressed inhibitory molecules, they stimulated allogeneic T cells in-vitro. Collectively, the data indicate that L. plantarum-NY-ESO-1 can evoke antigen-specific immunity upon oral administration and induce DC maturation, raising the potential of its use in cancer immunotherapies. PMID:26185907

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.

ETV5 transcription factor is overexpressed in ovarian cancer and regulates cell adhesion in ovarian cancer cells.

PubMed

Llauradó, Marta; Abal, Miguel; Castellví, Josep; Cabrera, Sílvia; Gil-Moreno, Antonio; Pérez-Benavente, Asumpció; Colás, Eva; Doll, Andreas; Dolcet, Xavier; Matias-Guiu, Xavier; Vazquez-Levin, Mónica; Reventós, Jaume; Ruiz, Anna

2012-04-01

Epithelial ovarian cancer is the most lethal gynecological malignancy and the fifth leading cause of cancer deaths in women in the Western world. ETS transcription factors are known to act as positive or negative regulators of the expression of genes that are involved in various biological processes, including those that control cellular proliferation, differentiation, apoptosis, tissue remodeling, angiogenesis and transformation. ETV5 belongs to the PEA3 subfamily. PEA3 subfamily members are able to activate the transcription of proteases, matrix metalloproteinases and tissue inhibitor of metalloproteases, which is central to both tumor invasion and angiogenesis. Here, we examined the role of the ETV5 transcription factor in epithelial ovarian cancer and we found ETV5 was upregulated in ovarian tumor samples compared to ovarian tissue controls. The in vitro inhibition of ETV5 decreased cell proliferation in serum-deprived conditions, induced EMT and cell migration and decreased cell adhesion to extracellular matrix components. ETV5 inhibition also decreased cell-cell adhesion and induced apoptosis in anchorage-independent conditions. Accordingly, upregulation of ETV5 induced the expression of cell adhesion molecules and enhanced cell survival in a spheroid model. Our findings suggest that the overexpression of ETV5 detected in ovarian cancer cells may contribute to ovarian tumor progression through the ability of ETV5 to enhance proliferation of ovarian cancer cells. In addition, upregulation of ETV5 would play a role in ovarian cancer cell dissemination and metastasis into the peritoneal cavity by protecting ovarian cancer cells from apoptosis and by increasing the adhesion of ovarian cancer cells to the peritoneal wall through the regulation of cell adhesion molecules. Copyright © 2011 UICC.

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.

The Effect of Water Molecules on Mechanical Properties of Cell Walls

NASA Astrophysics Data System (ADS)

Rahbar, Nima; Youssefian, Sina

The unique properties of bamboo fibers come from their natural composite structures that comprise mainly cellulose nanofibrils in a matrix of intertwined hemicellulose and lignin called lignin-carbohydrate complex (LCC). Here, we have utilized atomistic simulations to investigate the mechanical properties and mechanisms of interactions between these materials, in the presence of water molecules. The role of hemicellulose found to be enhancing the mechanical properties and lignin found to be providing the strength of bamboo fibers. The abundance of Hbonds in hemicellulose chains is responsible for improving the mechanical behavior of LCC. The strong van der Waals forces between lignin molecules and cellulose nanofibrils are responsible for higher adhesion energy between LCC/cellulose nanofibrils. We also found out that the amorphous regions of cellulose nanofibrils is the weakest interface in bamboo Microfibrils. In presence of water, the elastic modulus of lignin increases at low water content and decreases in higher water content, whereas the hemicellulose elastic modulus constantly decreases. The variations of Radial Distribution Function and Free Fractional Volume of these materials with water suggest that water molecules enhance the mechanical properties of lignin by filling voids in the system and creating Hbond bridges between polymer chains. For hemicellulose, however, the effect is always regressive due to the destructive effect of water molecules on the Hbond of its dense structure.

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.

Actin polymerization drives septation of Listeria monocytogenes namA hydrolase mutants, demonstrating host correction of a bacterial defect.

PubMed

Alonzo, Francis; McMullen, P David; Freitag, Nancy E

2011-04-01

The Gram-positive bacterial cell wall presents a structural barrier that requires modification for protein secretion and large-molecule transport as well as for bacterial growth and cell division. The Gram-positive bacterium Listeria monocytogenes adjusts cell wall architecture to promote its survival in diverse environments that include soil and the cytosol of mammalian cells. Here we provide evidence for the enzymatic flexibility of the murein hydrolase NamA and demonstrate that bacterial septation defects associated with a loss of NamA are functionally complemented by physical forces associated with actin polymerization within the host cell cytosol. L. monocytogenes ΔnamA mutants formed long bacterial chains during exponential growth in broth culture; however, normal septation could be restored if mutant cells were cocultured with wild-type L. monocytogenes bacteria or by the addition of exogenous NamA. Surprisingly, ΔnamA mutants were not significantly attenuated for virulence in mice despite the pronounced exponential growth septation defect. The physical force of L. monocytogenes-mediated actin polymerization within the cytosol was sufficient to sever ΔnamA mutant intracellular chains and thereby enable the process of bacterial cell-to-cell spread so critical for L. monocytogenes virulence. The inhibition of actin polymerization by cytochalasin D resulted in extended intracellular bacterial chains for which septation was restored following drug removal. Thus, despite the requirement for NamA for the normal septation of exponentially growing L. monocytogenes cells, the hydrolase is essentially dispensable once L. monocytogenes gains access to the host cell cytosol. This phenomenon represents a notable example of eukaryotic host cell complementation of a bacterial defect.

The impact of proteomics on the understanding of functions and biogenesis of fungal extracellular vesicles.

PubMed

Rodrigues, Marcio L; Nakayasu, Ernesto S; Almeida, Igor C; Nimrichter, Leonardo

2014-01-31

Several microbial molecules are released to the extracellular space in vesicle-like structures. In pathogenic fungi, these molecules include pigments, polysaccharides, lipids, and proteins, which traverse the cell wall in vesicles that accumulate in the extracellular space. The diverse composition of fungal extracellular vesicles (EV) is indicative of multiple mechanisms of cellular biogenesis, a hypothesis that was supported by EV proteomic studies in a set of Saccharomyces cerevisiae strains with defects in both conventional and unconventional secretory pathways. In the human pathogens Cryptococcus neoformans, Histoplasma capsulatum, and Paracoccidioides brasiliensis, extracellular vesicle proteomics revealed the presence of proteins with both immunological and pathogenic activities. In fact, fungal EV have been demonstrated to interfere with the activity of immune effector cells and to increase fungal pathogenesis. In this review, we discuss the impact of proteomics on the understanding of functions and biogenesis of fungal EV, as well as the potential role of these structures in fungal pathogenesis. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. Copyright © 2013 Elsevier B.V. All rights reserved.

Targeted Delivery of Glucan Particle Encapsulated Gallium Nanoparticles Inhibits HIV Growth in Human Macrophages

PubMed Central

Soto, Ernesto R.; O'Connell, Olivia; Dikengil, Fusun; Peters, Paul J.; Clapham, Paul R.

2016-01-01

Glucan particles (GPs) are hollow, porous 3–5 μm microspheres derived from the cell walls of Baker's yeast (Saccharomyces cerevisiae). The 1,3-β-glucan outer shell provides for receptor-mediated uptake by phagocytic cells expressing β-glucan receptors. GPs have been used for macrophage-targeted delivery of a wide range of payloads (DNA, siRNA, protein, small molecules, and nanoparticles) encapsulated inside the hollow GPs or bound to the surface of chemically derivatized GPs. Gallium nanoparticles have been proposed as an inhibitory agent against HIV infection. Here, macrophage targeting of gallium using GPs provides for more efficient delivery of gallium and inhibition of HIV infection in macrophages compared to free gallium nanoparticles. PMID:27965897

DSMC Simulations of High Mach Number Taylor-Couette Flow

NASA Astrophysics Data System (ADS)

Pradhan, Sahadev

2017-11-01

The main focus of this work is to characterise the Taylor-Couette flow of an ideal gas between two coaxial cylinders at Mach number Ma =(Uw /√{ kbTw / m }) in the range 0.01

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)

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.

Manipulating energy transfer in copolymer-based nanocomposites by their controlled nanocaging and release of an ionic styryl dye: a case of an ultrasensitive pH sensor.

PubMed

Manna, Anamika; Sahoo, Dibakar; Chakravorti, Sankar

2012-03-01

We report an interesting pH-tunable energy transfer between an acceptor ionic styryl dye 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide and a donor charge-transfer dye 1,8-naphthalimide in a vesicular medium. The polyethylene-b-polyethylene glycol block copolymer intercalates with the sodium dodecyl sulfate anionic surfactant to form self-aggregated nanocomposites. These nanocomposites interact with the donor molecules in aqueous solution to form "vesicles", and the donor molecules become attached on the outer wall by hydrogen bonding. The acceptor molecules are observed to be loaded in the vesicular interior. By controlling the spectral overlap of the donor and acceptor molecules by changing the pH of the medium, the energy-transfer efficiency in vesicles has been studied. The efficiency of energy transfer in vesicular media (55%) is found to be less compared to that in aqueous media (80%) at pH 7. The fall in efficiency has been attributed to the perturbation imparted by the vesicular wall due to the good matching of the donor-acceptor distance with the wall thickness. At low pH, the efficiency shows an abrupt increase (95%) due to the release of the acceptor molecules from the vesicular medium causing subsequent reduction of donor-acceptor separation and an increase of the spectral overlap at that pH.

Study of gas adsorption on as-produced and modified carbon nanotubes

NASA Astrophysics Data System (ADS)

Rawat, Dinesh Singh

Volumetric adsorption isotherm measurements were used to study the adsorption characteristics of Ethane (C2H6) and Butane (C 4H10) on as-produced single-walled carbon nanotubes. The binding energy of the adsorbed alkane molecule was found to increase with increasing carbon chain length. Two adsorption substeps were obtained for each alkane molecule. However, the size of the high pressure substep was found to be gradually smearing with the increase in size of the adsorbed molecule. This phenomenon is interpreted as size entropy effect for linear molecules. This interpretation was also verified by determining the specific surface area of the substrate using linear molecules of different sizes. Kinetics measurements of alkane adsorption on SWNTs were also conducted and their dependence on the molecular length was determined. Similar adsorption measurements were performed for Argon (Ar) on as-produced single-walled carbon nanotubes and nanotubes that were structurally modified using acid treatment. Enhancement of the sorptive capacity and the presence of two distinct kinetics of gas adsorption verified partial opening of single walled carbon nanotubes as a result of chemical treatment. Mutiwalled carbon nanotubes were exposed to oxygen plasma treatment for varying time periods. Afterwards, adsorption measurements of Methane (CH 4) were conducted on untreated and oxygen plasma treated tubes. The presence of an additional substep, after exposing multiwalled carbon nanotubes to oxygen plasma for varying time periods, suggested progressive cleaning of nanotube surface.

Expression of agrin, dystroglycan, and utrophin in normal renal tissue and in experimental glomerulopathies.

PubMed

Raats, C J; van den Born, J; Bakker, M A; Oppers-Walgreen, B; Pisa, B J; Dijkman, H B; Assmann, K J; Berden, J H

2000-05-01

The dystrophin-glycoprotein complex, which comprises alpha- and beta-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against alpha- and beta-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane.

Expression of Agrin, Dystroglycan, and Utrophin in Normal Renal Tissue and in Experimental Glomerulopathies

PubMed Central

Raats, C. J. Ilse; van den Born, Jacob; Bakker, Marinka A. H.; Oppers-Walgreen, Birgitte; Pisa, Brenda J. M.; Dijkman, Henry B. P. M.; Assmann, Karel J. M.; Berden, Jo H. M.

2000-01-01

The dystrophin-glycoprotein complex, which comprises α- and β-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against α- and β-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane. PMID:10793086

Anti-Candida albicans effectiveness of citral and investigation of mode of action.

PubMed

Lima, Igara Oliveira; de Medeiros Nóbrega, Fernanda; de Oliveira, Wylly Araújo; de Oliveira Lima, Edeltrudes; Albuquerque Menezes, Everardo; Cunha, Francisco Afrânio; Formiga Melo Diniz, Margareth de Fátima

2012-12-01

Candidiasis is a mycosis caused by Candida species, which is of clinical importance due to the increase in resistant yeasts. Candida infection has been a serious health problem due to the inappropriate use of antibiotics. Therefore, it is necessary to study molecules with an antifungal action. Citral is a monoterpene with known pharmacological properties, including antimicrobial action. The aim of this work was to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of citral and the probable mode of action. The MIC of citral was determined by the broth microdilution method using Sabouraud dextrose medium. Additionally, the interference of citral in cell wall (sorbitol assay) and the binding of citral to ergosterol and cholesterol were studied, carried out by broth microdilution method. The MIC and MFC of citral were 512 and 1024 µg/mL, respectively. The MIC of amphotericin B was 1 µg/mL. The mechanism of action did not involve either the cell wall or ergosterol. However, the presence of cholesterol increased the MIC of citral to 1024 µg/mL, indicating there is some interaction between citral and cholesterol. Amphotericin B was used as the positive control, and it showed a high MIC in the presence of ergosterol (32 µg/mL), while in the presence of cholesterol MIC increased to 4 µg/mL. Citral inhibits the growth of C. albicans. The probable mechanism of action did not involve the cell wall or ergosterol. Citral is able to interact with cholesterol. More studies are necessary to describe their effects completely.

Diffusion of macromolecules in self-assembled cellulose/hemicellulose hydrogels.

PubMed

Lopez-Sanchez, Patricia; Schuster, Erich; Wang, Dongjie; Gidley, Michael J; Strom, Anna

2015-05-28

Cellulose hydrogels are extensively applied in many biotechnological fields and are also used as models for plant cell walls. We synthesised model cellulosic hydrogels containing hemicelluloses, as a biomimetic of plant cell walls, in order to study the role of hemicelluloses on their mass transport properties. Microbial cellulose is able to self-assemble into composites when hemicelluloses, such as xyloglucan and arabinoxylan, are present in the incubation media, leading to hydrogels with different nano and microstructures. We investigated the diffusivities of a series of fluorescently labelled dextrans, of different molecular weight, and proteins, including a plant pectin methyl esterase (PME), using fluorescence recovery after photobleaching (FRAP). The presence of xyloglucan, known to be able to crosslink cellulose fibres, confirmed by scanning electron microscopy (SEM) and (13)C NMR, reduced mobility of macromolecules of molecular weight higher than 10 kDa, reflected in lower diffusion coefficients. Furthermore PME diffusion was reduced in composites containing xyloglucan, despite the lack of a particular binding motif in PME for this polysaccharide, suggesting possible non-specific interactions between PME and this hemicellulose. In contrast, hydrogels containing arabinoxylan coating cellulose fibres showed enhanced diffusivity of the molecules studied. The different diffusivities were related to the architectural features found in the composites as a function of polysaccharide composition. Our results show the effect of model hemicelluloses in the mass transport properties of cellulose networks in highly hydrated environments relevant to understanding the role of hemicelluloses in the permeability of plant cell walls and aiding design of plant based materials with tailored properties.

[Oxidative stress and vascular function].

PubMed

Urbański, Karol; Nowak, Michal; Guzik, Tomasz J

2013-01-01

The maintenance of blood vessel homeostasis is closely associated with Reactive Oxygen and Nitrogen Species (ROS and RNS) production in the blood vessel wall. The main molecules taking part in this process are nitric oxide (NO), superoxide anion (O2*-), hydrogen peroxide (H2O2) and their derivatives. The production of these factors occurs in health and disease, however the increased ROS release is often referred to as oxidative stress. While initially oxidative stress was considered systemically, recent data indicate that it occurs locally in subcellular spaces and may be a result of dysfunction of individual enzyme systems. Oxidative stress induces inflammation, proliferation and migration of vascular smooth muscle cells, may regulate apoptosis and the function of the cells of vascular wall, finally leading to dysfunction of endothelium, media and adventitia, leading to cardiovascular diseases such as atherosclerosis, hypertension or heart failure. It is believed that a family of NADPH oxidases is the main source of ROS in the vessel wall, but also in other organs and tissues. It consists of seven known and quite precisely characterized homologues (NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2) which often have very distinct activity and cellular localization and function. Besides harmful actions, we are beginning to understand the protective effects of ROS and RNS. They have many functions regulating redox-sensitive gene expression and influencing a proper function of cells and vessels. NOX4 has been particularly well characterized in this respect. Thus, the maintenance of the right homeostasis depends not only on ROS removing capabilities, but especially on preserving the adequate level of ROS production.

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.

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.

Plasmodesmal regulation during plant-pathogen interactions.

PubMed

Cheval, Cecilia; Faulkner, Christine

2018-01-01

Contents Summary 62 I. Introduction 62 II. Plasmodesmal regulation is an innate defence response 63 III. Reactive oxygen species regulate plasmodesmal function 63 IV. Plasmodesmal regulation by and of defence-associated small molecules 64 V. Plasmodesmata facilitate systemic defence signalling 64 VI. Virulent pathogens exploit plasmodesmata 66 VII. Outlook 66 Acknowledgements 66 References 66 SUMMARY: Plasmodesmata (PD) are plasma membrane-lined pores that connect neighbouring plant cells, bridging the cell wall and establishing cytoplasmic and membrane continuity between cells. PD are dynamic structures regulated by callose deposition in a variety of stress and developmental contexts. This process crudely controls the aperture of the pore and thus the flux of molecules between cells. During pathogen infection, plant cells initiate a range of immune responses and it was recently identified that, following perception of fungal and bacterial pathogens, plant cells initially close their PD. Systemic defence responses depend on the spread of signals between cells, raising questions about whether PD are in different functional states during different immune responses. It is well established that viral pathogens exploit PD to spread between cells, but it has more recently been identified that protein effectors secreted by fungal pathogens can spread between host cells via PD. It is possible that many classes of pathogens specifically target PD to aid infection, which would infer antagonistic regulation of PD by host and pathogen. How PD regulation benefits both host immune responses and pathogen infection is an important question and demands that we examine the multicellular nature of plant-pathogen interactions. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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.

c-di-AMP: An Essential Molecule in the Signaling Pathways that Regulate the Viability and Virulence of Gram-Positive Bacteria

PubMed Central

Fahmi, Tazin; Port, Gary C.

2017-01-01

Signal transduction pathways enable organisms to monitor their external environment and adjust gene regulation to appropriately modify their cellular processes. Second messenger nucleotides including cyclic adenosine monophosphate (c-AMP), cyclic guanosine monophosphate (c-GMP), cyclic di-guanosine monophosphate (c-di-GMP), and cyclic di-adenosine monophosphate (c-di-AMP) play key roles in many signal transduction pathways used by prokaryotes and/or eukaryotes. Among the various second messenger nucleotides molecules, c-di-AMP was discovered recently and has since been shown to be involved in cell growth, survival, and regulation of virulence, primarily within Gram-positive bacteria. The cellular level of c-di-AMP is maintained by a family of c-di-AMP synthesizing enzymes, diadenylate cyclases (DACs), and degradation enzymes, phosphodiesterases (PDEs). Genetic manipulation of DACs and PDEs have demonstrated that alteration of c-di-AMP levels impacts both growth and virulence of microorganisms. Unlike other second messenger molecules, c-di-AMP is essential for growth in several bacterial species as many basic cellular functions are regulated by c-di-AMP including cell wall maintenance, potassium ion homeostasis, DNA damage repair, etc. c-di-AMP follows a typical second messenger signaling pathway, beginning with binding to receptor molecules to subsequent regulation of downstream cellular processes. While c-di-AMP binds to specific proteins that regulate pathways in bacterial cells, c-di-AMP also binds to regulatory RNA molecules that control potassium ion channel expression in Bacillus subtilis. c-di-AMP signaling also occurs in eukaryotes, as bacterially produced c-di-AMP stimulates host immune responses during infection through binding of innate immune surveillance proteins. Due to its existence in diverse microorganisms, its involvement in crucial cellular activities, and its stimulating activity in host immune responses, c-di-AMP signaling pathway has become an attractive antimicrobial drug target and therefore has been the focus of intensive study in several important pathogens. PMID:28783096

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.

Proof-of-principle for SERS imaging of Aspergillus nidulans hyphae using in vivo synthesis of gold nanoparticles.

PubMed

Prusinkiewicz, Martin A; Farazkhorasani, Fatemeh; Dynes, James J; Wang, Jian; Gough, Kathleen M; Kaminskyj, Susan G W

2012-11-07

High spatial resolution methods to assess the physiology of growing cells should permit analysis of fungal biochemical composition. Whole colony methods cannot capture the details of physiology and organism-environment interaction, in part because the structure, function and composition of fungal hyphae vary within individual cells depending on their distance from the growing apex. Surface Enhanced Raman Scattering (SERS) can provide chemical information on materials that are in close contact with appropriate metal substrates, such as nanopatterned gold surfaces and gold nanoparticles (AuNPs). Since nanoparticles can be generated by living cells, we have created conditions for AuNP formation within and on the surface of Aspergillus nidulans hyphae in order to explore their potential for SERS analysis. AuNP distribution and composition have been assessed by UV-Vis spectroscopy, fluorescence light microscopy, transmission electron microscopy, and scanning transmission X-ray microscopy. AuNPs were often associated with hyphal walls, both in the peripheral cytoplasm and on the outer wall surface. Interpretation of SERS spectra is challenging, and will require validation for the diversity of organic molecules present. Here, we show proof-of-principle that it is possible to generate SERS spectra from nanoparticles grown in situ by living hyphae.

Ecological and agricultural applications of synchrotron IR microscopy

NASA Astrophysics Data System (ADS)

Raab, T. K.; Vogel, J. P.

2004-10-01

The diffraction-limited spot size of synchrotron-based IR microscopes provides cell-specific, spectrochemical imaging of cleared leaf, stem and root tissues of the model genetic organism Arabidopsis thaliana, and mutant plants created either by T-DNA insertional inactivation or chemical mutagenesis. Spectra in the wavelength region from 6 to 12 μm provide chemical and physical information on the cell wall polysaccharides of mutants lacking particular biosynthetic enzymes ("Cellulose synthase-like" genes). In parallel experiments, synchrotron IR microscopy delineates the role of Arabidopsis cell wall enzymes as susceptibility factors to the fungus Erysiphe cichoracearum, a causative agent of powdery mildew disease. Three genes, pmr4, pmr5, and pmr6 have been characterized by these methods, and biochemical relations between two of the genes suggested by IR spectroscopy and multivariate statistical techniques could not have been inferred through classical molecular biology. In ecological experiments, live plants can also be imaged in small microcosms with mid-IR transmitting ZnSe windows. Small exudate molecules may be spatially mapped in relation to root architecture at diffraction-limited resolution, and the effect of microbial symbioses on the quantity and quality of exudates inferred. Synchrotron IR microscopy provides a useful adjunct to molecular biological methods and underground observatories in the ongoing assessment of the role of root-soil-microbe communication.

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.

Theoretical analysis of the influence of flexoelectric effect on the defect site in nematic inversion walls

NASA Astrophysics Data System (ADS)

Gui-Li, Zheng; Hui, Zhang; Wen-Jiang, Ye; Zhi-Dong, Zhang; Hong-Wei, Song; Li, Xuan

2016-03-01

Based on the experimental phenomena of flexoelectric response at defect sites in nematic inversion walls conducted by Kumar et al., we gave the theoretical analysis using the Frank elastic theory. When a direct-current electric field normal to the plane of the substrate is applied to the parallel aligned nematic liquid crystal cell with weak anchoring, the rotation of ±1 defects in the narrow inversion walls can be exhibited. The free energy of liquid crystal molecules around the +1 and -1 defect sites in the nematic inversion walls under the electric field was formulated and the electric-field-driven structural changes at the defect site characterized by polar and azimuthal angles of the local director were simulated. The results reveal that the deviation of azimuthal angle induced by flexoelectric effect are consistent with the switching of extinction brushes at the +1 and -1 defects obtained in the experiment conducted by Kumar et al. Project supported by the National Natural Science Foundation of China (Grant Nos. 11374087, 11274088, and 11304074), the Natural Science Foundation of Hebei Province, China (Grant Nos. A2014202123 and A2016202282), the Research Project of Hebei Education Department, China (Grant Nos. QN2014130 and QN2015260), and the Key Subject Construction Project of Hebei Province University, China.

Cytotoxicity of doxrubicin loaded single-walled carbon nanotubes.

PubMed

Ünlü, Ayhan; Meran, Mehdi; Dinc, Bircan; Karatepe, Nilgün; Bektaş, Muhammet; Güner, F Seniha

2018-05-24

Carbon nanotube (CNTs) is a new alternative for efficient drug delivery and it has a great potential to change drug delivery system profile in pharmaceutical industry. One of the important advantage of CNTs is their needle-like, cylindrical shape. This shape provides a high surface area for multiple connections and adsorption onto for millions of therapeutic molecules. CNTs can be internalized by cells via endocytosis, passive diffusion and phagocytosis and release the drug with different effects like pH and temperature. The acidic nature of cancer cells and the susceptibility of CNTs to release the drug in the acidic environment have made it a promising area of research in cancer drug delivery. In this research, we investigated cell viability, cytotoxicity and drug delivery in breast cancer cell line by designing non-covalent single walled carbon nanotubes (SWNT)-doxorubicin (DOX) supramolecular complex that can be developed for cancer therapy. Applied high concentrations of DOX loaded SWNTs changed the actin structure of the cells and prevented the proliferation of the cells. It was showed that doxorubicin loaded SWNTs were more effective than free doxorubicin at relatively small concentrations. Once we applied same procedure for short and long (short: 1-1.3 µm; long: 2.5-4 µm) SWNTs and compared the results, more disrupted cell structure and reduction in cell proliferation were observed for long CNTs. DOX is bounded more to nanotubes in basic medium, less bound in acidic environment. Cancer cells were also examined for concentration at which they were effective by applying DOX and it was seen that 3.68 µM doxorubicin kills more than 55% of the cells.

Simulation of diatomic gas-wall interaction and accommodation coefficients for negative ion sources and accelerators.

PubMed

Sartori, E; Brescaccin, L; Serianni, G

2016-02-01

Particle-wall interactions determine in different ways the operating conditions of plasma sources, ion accelerators, and beams operating in vacuum. For instance, a contribution to gas heating is given by ion neutralization at walls; beam losses and stray particle production-detrimental for high current negative ion systems such as beam sources for fusion-are caused by collisional processes with residual gas, with the gas density profile that is determined by the scattering of neutral particles at the walls. This paper shows that Molecular Dynamics (MD) studies at the nano-scale can provide accommodation parameters for gas-wall interactions, such as the momentum accommodation coefficient and energy accommodation coefficient: in non-isothermal flows (such as the neutral gas in the accelerator, coming from the plasma source), these affect the gas density gradients and influence efficiency and losses in particular of negative ion accelerators. For ideal surfaces, the computation also provides the angular distribution of scattered particles. Classical MD method has been applied to the case of diatomic hydrogen molecules. Single collision events, against a frozen wall or a fully thermal lattice, have been simulated by using probe molecules. Different modelling approximations are compared.

Simulation of diatomic gas-wall interaction and accommodation coefficients for negative ion sources and accelerators

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

Sartori, E., E-mail: emanuele.sartori@igi.cnr.it; Serianni, G.; Brescaccin, L.

2016-02-15

Particle-wall interactions determine in different ways the operating conditions of plasma sources, ion accelerators, and beams operating in vacuum. For instance, a contribution to gas heating is given by ion neutralization at walls; beam losses and stray particle production—detrimental for high current negative ion systems such as beam sources for fusion—are caused by collisional processes with residual gas, with the gas density profile that is determined by the scattering of neutral particles at the walls. This paper shows that Molecular Dynamics (MD) studies at the nano-scale can provide accommodation parameters for gas-wall interactions, such as the momentum accommodation coefficient andmore » energy accommodation coefficient: in non-isothermal flows (such as the neutral gas in the accelerator, coming from the plasma source), these affect the gas density gradients and influence efficiency and losses in particular of negative ion accelerators. For ideal surfaces, the computation also provides the angular distribution of scattered particles. Classical MD method has been applied to the case of diatomic hydrogen molecules. Single collision events, against a frozen wall or a fully thermal lattice, have been simulated by using probe molecules. Different modelling approximations are compared.« less

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.

One and two hydrogen molecules in the large cage of the structure II clathrate hydrate: quantum translation-rotation dynamics close to the cage wall.

PubMed

Sebastianelli, Francesco; Xu, Minzhong; Kanan, Dalal K; Bacić, Zlatko

2007-07-19

We have performed a rigorous theoretical study of the quantum translation-rotation (T-R) dynamics of one and two H2 and D2 molecules confined inside the large hexakaidecahedral (5(12)6(4)) cage of the sII clathrate hydrate. For a single encapsulated H2 and D2 molecule, accurate quantum five-dimensional calculations of the T-R energy levels and wave functions are performed that include explicitly, as fully coupled, all three translational and the two rotational degrees of freedom of the hydrogen molecule, while the cage is taken to be rigid. In addition, the ground-state properties, energetics, and spatial distribution of one and two p-H2 and o-D2 molecules in the large cage are calculated rigorously using the diffusion Monte Carlo method. These calculations reveal that the low-energy T-R dynamics of hydrogen molecules in the large cage are qualitatively different from that inside the small cage, studied by us recently. This is caused by the following: (i) The large cage has a cavity whose diameter is about twice that of the small cage for the hydrogen molecule. (ii) In the small cage, the potential energy surface (PES) for H2 is essentially flat in the central region, while in the large cage the PES has a prominent maximum at the cage center, whose height exceeds the T-R zero-point energy of H2/D2. As a result, the guest molecule is excluded from the central part of the large cage, its wave function localized around the off-center global minimum. Peculiar quantum dynamics of the hydrogen molecule squeezed between the central maximum and the cage wall manifests in the excited T-R states whose energies and wave functions differ greatly from those for the small cage. Moreover, they are sensitive to the variations in the hydrogen-bonding topology, which modulate the corrugation of the cage wall.

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.

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.

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.

Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts.

PubMed

Kaipa, Balasankara Reddy; Shao, Huanjie; Schäfer, Gritt; Trinkewitz, Tatjana; Groth, Verena; Liu, Jianqi; Beck, Lothar; Bogdan, Sven; Abmayr, Susan M; Önel, Susanne-Filiz

2013-01-01

The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

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.

Single-walled carbon nanotube and graphene: Nano-delivery of Gambogic acid increases its cytotoxicty in various cancer cells

NASA Astrophysics Data System (ADS)

Saeed, Lamya M.

Nanomedicine is a new branch of medicine that has been developed due to the critical need to treat challenging diseases, especially cancer since it remains a significant cause of morbidity and mortality worldwide and the second most common cause of death after heart disease in the USA. One of the most important health care applications of nanomedicine concerns the development of drug delivery systems. Graphene (Gn), an atom-thick carbon monolayer of sp2- bonded carbon atoms arranged in a two dimensional (2D) honeycomb crystal lattice, and single-walled carbon nanotubes (SWCNTs) (1D, tubular) are among the most promising nanomaterials with the capability of delivering drugs or small therapeutic molecules to cancerous cells. For example, they have been used as vehicles for the anti-cancer, low-toxicity drug Gambogic acid (GA). Here, the cytotoxicity of GA in breast (MCF-7), pancreatic (PANC-1), cervical (HELA), ovarian (NCI/ADR), and prostate (PC3) cancer cells was assessed to determine what effect nanodelivery by either Gn or SWCNTs had on the efficacy of this promising drug. The nanomaterials showed no toxicity at the concentrations used. The inhibition of cell proliferation and apoptosis of the cells was due to the effects of GA which was significantly enhanced by nanodelivery. Such delivery of GA by either Gn or SWCNTs represents a first step toward assessing their effectiveness in more complex, targeted nano-delivery in vivo settings and signals their potential application in the treatment of cancer.

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.

Microbe Profile: Mycobacterium tuberculosis: Humanity's deadly microbial foe.

PubMed

Gordon, Stephen V; Parish, Tanya

2018-04-01

Mycobacterium tuberculosis is an expert and deadly pathogen, causing the disease tuberculosis (TB) in humans. It has several notable features: the ability to enter non-replicating states for long periods and cause latent infection; metabolic remodelling during chronic infection; a thick, waxy cell wall; slow growth rate in culture; and intrinsic drug resistance and antibiotic tolerance. As a pathogen, M. tuberculosis has a complex relationship with its host, is able to replicate inside macrophages, and expresses diverse immunomodulatory molecules. M. tuberculosis currently causes over 1.8 million deaths a year, making it the world's most deadly human pathogen.

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

Superdiffusive gas recovery from nanopores

NASA Astrophysics Data System (ADS)

Wu, Haiyi; He, Yadong; Qiao, Rui

2016-11-01

Understanding the recovery of gas from reservoirs featuring pervasive nanopores is essential for effective shale gas extraction. Classical theories cannot accurately predict such gas recovery and many experimental observations are not well understood. Here we report molecular simulations of the recovery of gas from single nanopores, explicitly taking into account molecular gas-wall interactions. We show that, in very narrow pores, the strong gas-wall interactions are essential in determining the gas recovery behavior both quantitatively and qualitatively. These interactions cause the total diffusion coefficients of the gas molecules in nanopores to be smaller than those predicted by kinetic theories, hence slowing down the rate of gas recovery. These interactions also lead to significant adsorption of gas molecules on the pore walls. Because of the desorption of these gas molecules during gas recovery, the gas recovery from the nanopore does not exhibit the usual diffusive scaling law (i.e., the accumulative recovery scales as R ˜t1 /2 ) but follows a superdiffusive scaling law R ˜tn (n >0.5 ), which is similar to that observed in some field experiments. For the system studied here, the superdiffusive gas recovery scaling law can be captured well by continuum models in which the gas adsorption and desorption from pore walls are taken into account using the Langmuir model.

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.

Effects of transplanted circulating endothelial progenitor cells and platelet microparticles in atherosclerosis development.

PubMed

Georgescu, Adriana; Alexandru, Nicoleta; Andrei, Eugen; Dragan, Emanuel; Cochior, Daniel; Dias, Sérgio

2016-08-01

Atherosclerosis is an inflammatory disease, in which risk factors such as hyperlipidemia and hypertension affect the arterial endothelium, resulting in dysfunction, cell damage or both. The number of circulating endothelial progenitor cells and microparticles provides invaluable outcome prediction for atherosclerosis disease. However, evidence for the therapeutic potential of endothelial progenitor cells and microparticles in atherosclerosis development is limited. Our study was designed to investigate the possible protective role of a cell therapy-based approach, using endothelial progenitor cells and the dual behaviour of circulating platelet microparticles, on atherosclerosis development in hypertensive-hypercholesterolemic hamster model. Consequently, control hamsters received four intravenous inoculations of: (1) 1×10(5) endothelial progenitor cells of healthy origins in one dose per month, during four months of diet-induced atherosclerosis, and after hypertensive-hypercholesterolemic diet for further four months; (2) in a second set of experiments, 1×10(5) endothelial progenitor cells of healthy origins or/and 1×10(5) platelet microparticles of atherosclerotic origins were inoculated every other month during hypertensive-hypercholesterolemic diet. Endothelial progenitor cell treatment had the following effects: (1) re-established plasmatic parameters: cholesterol and triglyceride concentrations, blood pressure, heart rate, cytokine and chemokine profiles, platelet microparticle pro-thrombotic activity and endothelial progenitor cell paracrine activity reflected by cytokine/chemokine detection; (2) reduced lipid, macrophage and microparticle accumulation in liver; (3) reduced atherosclerosis development, revealed by decreased lipid, macrophage and microparticle content of arterial wall; (4) induced the recruitment and incorporation of endothelial progenitor cells into liver and arterial wall; (5) improved arterial dysfunction by increasing contraction and relaxation; (6) reduced the protein expression of specific pro-inflammatory molecules in liver and arterial wall. Platelet microparticle transplantation aggravated the above-mentioned biomarkers and atherosclerosis process, which were partially reverted with co-inoculation of platelet microparticles and endothelial progenitor cells. With this study, we demonstrate in a hypertensive-hypercholesterolemic hamster model, that the endothelial progenitor cell-based therapy suppresses the development of atherosclerosis and reduces hepatic lipid and macrophage accumulation with the consequent alleviation of dyslipidaemia and hypertension. Our results support the notion that increasing the number of circulating endothelial progenitor cells by different ways could be a promising therapeutic tool for atherosclerosis. © 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

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.

Characterization of adhesive molecule with affinity to Caco-2 cells in Lactobacillus acidophilus by proteome analysis.

PubMed

Ashida, Nobuhisa; Yanagihara, Sae; Shinoda, Tadashi; Yamamoto, Naoyuki

2011-10-01

The adhesive activities of eight Lactobacillus acidophilus strains toward intestinal epithelial Caco-2 cells were studied to understand the probiotic characteristics of the L. acidophilus L-92 strain. Most of the strains, including L-92, showed high adhesive activity; CP23 showed the lowest adhesive activity. CP23 was selected for comparative analysis of cell wall-associated proteins versus the L-92 strain. Cell wall-associated proteins extracted from L-92 and CP23 were subjected to two-dimensional electrophoresis, and major spots observed in the former were compared to the corresponding spots in the latter. To understand the effects of key components of L-92 on its adhesion to Caco-2 cells, 18 spots with stronger signals in L-92 than those in CP23 were identified by a MALDI-TOF/TOF of Ultraflex analysis. Among the identified proteins of L-92, surface-layer protein A (SlpA) was considered strongly involved in adhesion in the eight L. acidophilus strains. To study the importance of SlpA in the adhesion of L. acidophilus, the amounts of SlpA proteins in LiCl extracts of the eight strains were compared by SDSpolyacrylamide gel electrophoresis. As a result, the adhesive abilities of L. acidophilus strains to Caco-2 cells correlated closely to the amount of SlpA in the cells and the productivity of IL-12, an inflammatory cytokine, in all eight strains. These results strongly suggested that SlpA in L. acidophilus might play a key role in its attachment to Caco-2 cells and in the release of IL-12 from dendritic cells.

(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.

Monitoring and quantifying the passive transport of molecules through patch-clamp suspended real and model cell membranes.

PubMed

Messina, Pierluca; Lemaître, Frédéric; Huet, François; Ngo, Kieu An; Vivier, Vincent; Labbé, Eric; Buriez, Olivier; Amatore, Christian

2014-03-17

Transport of active molecules across biological membranes is a central issue for the success of many pharmaceutical strategies. Herein, we combine the patch-clamp principle with amperometric detection for monitoring fluxes of redox-tagged molecular species across a suspended membrane patched from a macrophage. Solvent- and protein-free lipid bilayers (DPhPC, DOPC, DOPG) patched from single-wall GUV have been thoroughly investigated and the corresponding fluxes measurements quantified. The quality of the patches and their proper sealing were successfully characterized by electrochemical impedance spectroscopy. This procedure appears versatile and perfectly adequate to allow the investigation of transport and quantification of the transport properties through direct measurement of the coefficients of partition and diffusion of the compound in the membrane, thus offering insight on such important biological and pharmacological issues. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Supported lipid bilayer/carbon nanotube hybrids

NASA Astrophysics Data System (ADS)

Zhou, Xinjian; Moran-Mirabal, Jose M.; Craighead, Harold G.; McEuen, Paul L.

2007-03-01

Carbon nanotube transistors combine molecular-scale dimensions with excellent electronic properties, offering unique opportunities for chemical and biological sensing. Here, we form supported lipid bilayers over single-walled carbon nanotube transistors. We first study the physical properties of the nanotube/supported lipid bilayer structure using fluorescence techniques. Whereas lipid molecules can diffuse freely across the nanotube, a membrane-bound protein (tetanus toxin) sees the nanotube as a barrier. Moreover, the size of the barrier depends on the diameter of the nanotube-with larger nanotubes presenting bigger obstacles to diffusion. We then demonstrate detection of protein binding (streptavidin) to the supported lipid bilayer using the nanotube transistor as a charge sensor. This system can be used as a platform to examine the interactions of single molecules with carbon nanotubes and has many potential applications for the study of molecular recognition and other biological processes occurring at cell membranes.

Crystallographic snapshot of cellulose synthesis and membrane translocation.

PubMed

Morgan, Jacob L W; Strumillo, Joanna; Zimmer, Jochen

2013-01-10

Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.

Vertically aligned double wall carbon nanotube arrays adsorbent for pure and mixture adsorption of H2S, ethylbenzene and carbon monoxide, grand canonical Monte Carlo simulation.

PubMed

Tasharrofi, Saeideh; Taghdisian, Hossein; Golchoobi, Abdollah

2018-05-01

In this study, pure and ternary adsorption of hydrogen sulfide (H 2 S), ethylbenzene (EB), and carbon monoxide (CO) on different arrays of zigzag double wall carbon nanotube was investigated using grand canonical Monte Carlo simulations. The internal diameters of nanotube were fixed at 2r = 50.17 Å while nanotube wall distances were different values from d = 0 Å to d = 150 Å. Pure simulation results indicated that adsorption quantity of H 2 S and EB in low pressure ranges of P = 1.9 bar to P = 3.1 bar was at least 100% more than CO adsorption quantities. At high pressure ranges of P = 23.1 bar to P = 38.2 bar H 2 S adsorption was greater than EB and CO by about 200 molecules per unit cell (UC) at low nanotube distances. This was related to smaller kinetic diameter and greater dipole moment of H 2 S compared to EB and CO. At higher nanotube distance the effect of size however disappears and all three gases approach to adsorption quantity of about 800 molecules/UC. Graphical representation of adsorption areas showed that H 2 S and CO form multilayer adsorption around nanotube inner and outer walls while EB fill the whole space uniformly without any congestion around the walls. Ternary adsorption results EB/CO and H 2 S/CO selectivity are greater than EB/H 2 S selectivity. In addition, at smaller nanotube distances H 2 S/CO selectivity is generally higher than EB/CO selectivity, which at higher nanotube distance the order becomes revers suggesting that size dependent effects on adsorption vanishes. Isosteric heat of adsorption shows that the order of EB > H 2 S > CO suggesting that ethylbenzene interaction with nanotube arrays was strongest. Although H 2 S has a greater dipole moment and smaller molecular dimension, EB adsorption at higher nanotube distance is greater than H 2 S by at least 50% probably because EB is less volatile. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Phototransformation-Induced Aggregation of Functionalized Single-Walled Carbon Nanotubes: The Importance of Amorphous Carbon

EPA Science Inventory

Single-walled carbon nanotubes (SWCNTs) with proper functionalization are desirable for applications that require dispersion in aqueous and biological environments, and functionalized SWCNTs also serve as building blocks for conjugation with specific molecules in these applicatio...

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.

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.

The big CGRP flood - sources, sinks and signalling sites in the trigeminovascular system.

PubMed

Messlinger, Karl

2018-03-12

Calcitonin gene-related peptide (CGRP) has long been a focus of migraine research, since it turned out that inhibition of CGRP or CGRP receptors by antagonists or monoclonal IgG antibodies was therapeutic in frequent and chronic migraine. This contribution deals with the questions, from which sites CGRP is released, where it is drained and where it acts to cause its headache proliferating effects in the trigeminovascular system. The available literature suggests that the bulk of CGRP is released from trigeminal afferents both in meningeal tissues and at the first synapse in the spinal trigeminal nucleus. CGRP may be drained off into three different compartments, the venous blood plasma, the cerebrospinal fluid and possibly the glymphatic system. CGRP receptors in peripheral tissues are located on arterial vessel walls, mononuclear immune cells and possibly Schwann cells; within the trigeminal ganglion they are located on neurons and glial cells; in the spinal trigeminal nucleus they can be found on central terminals of trigeminal afferents. All these structures are potential signalling sites for CGRP, where CGRP mediates arterial vasodilatation but not direct activation of trigeminal afferents. In the spinal trigeminal nucleus a facilitating effect on synaptic transmission seems likely. In the trigeminal ganglion CGRP is thought to initiate long-term changes including cross-signalling between neurons and glial cells based on gene expression. In this way, CGRP may upregulate the production of receptor proteins and pro-nociceptive molecules. CGRP and other big molecules cannot easily pass the blood-brain barrier. These molecules may act in the trigeminal ganglion to influence the production of pronociceptive substances and receptors, which are transported along the central terminals into the spinal trigeminal nucleus. In this way peripherally acting therapeutics can have a central antinociceptive effect.

Tip-growing cells of the moss Ceratodon purpureus Are gravitropic in high-density media

NASA Technical Reports Server (NTRS)

Schwuchow, Jochen Michael; Kern, Volker Dieter; Sack, Fred David

2002-01-01

Gravity sensing in plants and algae is hypothesized to rely upon either the mass of the entire cell or that of sedimenting organelles (statoliths). Protonemata of the moss Ceratodon purpureus show upward gravitropism and contain amyloplasts that sediment. If moss sensing were whole-cell based, then media denser than the cell should prevent gravitropism or reverse its direction. Cells that were inverted or reoriented to the horizontal displayed distinct negative gravitropism in solutions of iodixanol with densities of 1.052 to 1.320 as well as in bovine serum albumin solutions with densities of 1.037 to 1.184 g cm(-3). Studies using tagged molecules of different sizes and calculations of diffusion times suggest that both types of media penetrate through the apical cell wall. Estimates of the density of the apical cell range from 1.004 to 1.085. Because protonemata grow upward when the cells have a density that is lower than the surrounding medium, gravitropic sensing probably utilizes an intracellular mass in moss protonemata. These data provide additional support for the idea that sedimenting amyloplasts function as statoliths in gravitropism.

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).

Cell Wall Modifications during Conidial Maturation of the Human Pathogenic Fungus Pseudallescheria boydii

PubMed Central

Ghamrawi, Sarah; Rénier, Gilles; Saulnier, Patrick; Cuenot, Stéphane; Zykwinska, Agata; Dutilh, Bas E.; Thornton, Christopher; Faure, Sébastien; Bouchara, Jean-Philippe

2014-01-01

Progress in extending the life expectancy of cystic fibrosis (CF) patients remains jeopardized by the increasing incidence of fungal respiratory infections. Pseudallescheria boydii (P. boydii), an emerging pathogen of humans, is a filamentous fungus frequently isolated from the respiratory secretions of CF patients. It is commonly believed that infection by this fungus occurs through inhalation of airborne conidia, but the mechanisms allowing the adherence of Pseudallescheria to the host epithelial cells and its escape from the host immune defenses remain largely unknown. Given that the cell wall orchestrates all these processes, we were interested in studying its dynamic changes in conidia as function of the age of cultures. We found that the surface hydrophobicity and electronegative charge of conidia increased with the age of culture. Melanin that can influence the cell surface properties, was extracted from conidia and estimated using UV-visible spectrophotometry. Cells were also directly examined and compared using electron paramagnetic resonance (EPR) that determines the production of free radicals. Consistent with the increased amount of melanin, the EPR signal intensity decreased suggesting polymerization of melanin. These results were confirmed by flow cytometry after studying the effect of melanin polymerization on the surface accessibility of mannose-containing glycoconjugates to fluorescent concanavalin A. In the absence of melanin, conidia showed a marked increase in fluorescence intensity as the age of culture increased. Using atomic force microscopy, we were unable to find rodlet-forming hydrophobins, molecules that can also affect conidial surface properties. In conclusion, the changes in surface properties and biochemical composition of the conidial wall with the age of culture highlight the process of conidial maturation. Mannose-containing glycoconjugates that are involved in immune recognition, are progressively masked by polymerization of melanin, an antioxidant that is commonly thought to allow fungal escape from the host immune defenses. PMID:24950099

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

Electrostatically controlled heat shutter

NASA Technical Reports Server (NTRS)

Derr, L. J. (Inventor)

1973-01-01

A heat transfer assembly for conducting thermal energy is described. The assembly includes a hermetically sealed container enclosing a quantity of inert gas such as nitrogen. Two opposed walls of the container have high thermal conducting characteristics while the connecting walls have low thermal conducting characteristics. Electrodes are positioned adjacent to the high thermal conducing walls and biased relative to the conducting walls to a corona potential for creating an ionic gas wind which must contact the conducting walls to be neutralized. The contact of the gas molecules permits the maximum thermal energy transfer between the walls. Baffles can be positioned adjacent to the electrodes to regulate gas flow between the high thermal conducting surfaces.

Combined effects of physiologically relevant disturbed wall shear stress and glycated albumin on endothelial cell functions associated with inflammation, thrombosis and cytoskeletal dynamics.

PubMed

Maria, Zahra; Yin, Wei; Rubenstein, David Alan

2014-07-01

Diabetes mellitus is a major risk factor in the development of cardiovascular diseases (CVDs). The presence of advanced glycation end-products (AGEs) promotes CVDs by upregulating endothelial cell (EC) inflammatory and thrombotic responses, in a similar manner as disturbed shear stress. However, the combined effect of disturbed shear stress and AGEs on EC function has yet to be determined. Our goal was to evaluate these effects on EC responses. ECs were incubated with AGEs for 5 days. ECs were then subjected to physiological or pathological shear stress. Cell metabolic activity, surface expression of intercellular adhesion molecule-1, thrombomodulin, connexin-43 and caveolin-1, and cytoskeleton organization were quantified. The results show that irreversibly glycated albumin and pathological shear stress increased EC metabolic activity, and upregulated and downregulated the EC surface expression of intercellular adhesion molecule-1 and thrombomodulin, respectively. Expression of connexin-43, caveolin-1 and cytoskeletal organization was independent of shear stress; however, the presence of irreversibly glycated AGEs markedly increased connexin-43, and decreased caveolin-1 expression and actin cytoskeletal connectivity. Our data suggest that irreversibly glycated albumin and disturbed shear stress could promote CVD pathogenesis by enhancing EC inflammatory and thrombotic responses, and through the deterioration of the cytoskeletal organization.

Synthesis and characterization of carbon nanotube from coconut shells activated carbon

NASA Astrophysics Data System (ADS)

Melati, A.; Hidayati, E.

2016-03-01

Carbon nanotubes (CNTs) have been explored in almost every single cancer treatment modality, including drug delivery, lymphatic targeted chemotherapy, photodynamic therapy, and gene therapy. They are considered as one of the most promising nanomaterial with the capability of both detecting the cancerous cells and delivering drugs or small therapeutic molecules to the cells. CNTs have unique physical and chemical properties such as high aspect ratio, ultralight weight, high mechanical strength, high electrical conductivity, and high thermal conductivity. Coconut Shell was researched as active carbon source on 500 - 600°C. These activated carbon was synthesized becomes carbon nanotube and have been proposed as a promising tool for detecting the expression of indicative biological molecules at early stage of cancer. Clinically, biomarkers cancer can be detected by CNT Biosensor. We are using pyrolysis methods combined with CVD process or Wet Chemical Process on 600°C. Our team has successfully obtained high purity, and aligned MWCNT (Multi Wall Nanotube) bundles on synthesis CNT based on coconut shells raw materials. CNTs can be used to cross the mammalian cell membrane by endocytosis or other mechanisms. SEM characterization of these materials have 179 nm bundles on phase 83° and their materials compound known by using FTIR characterization.

Rational design of adjuvants targeting the C-type lectin Mincle.

PubMed

Decout, Alexiane; Silva-Gomes, Sandro; Drocourt, Daniel; Barbe, Sophie; André, Isabelle; Cueto, Francisco J; Lioux, Thierry; Sancho, David; Pérouzel, Eric; Vercellone, Alain; Prandi, Jacques; Gilleron, Martine; Tiraby, Gérard; Nigou, Jérôme

2017-03-07

The advances in subunit vaccines development have intensified the search for potent adjuvants, particularly adjuvants inducing cell-mediated immune responses. Identification of the C-type lectin Mincle as one of the receptors underlying the remarkable immunogenicity of the mycobacterial cell wall, via recognition of trehalose-6,6'-dimycolate (TDM), has opened avenues for the rational design of such molecules. Using a combination of chemical synthesis, biological evaluation, molecular dynamics simulations, and protein mutagenesis, we gained insight into the molecular bases of glycolipid recognition by Mincle. Unexpectedly, the fine structure of the fatty acids was found to play a key role in the binding of a glycolipid to the carbohydrate recognition domain of the lectin. Glucose and mannose esterified at O -6 by a synthetic α-ramified 32-carbon fatty acid showed agonist activity similar to that of TDM, despite their much simpler structure. Moreover, they were seen to stimulate proinflammatory cytokine production in primary human and murine cells in a Mincle-dependent fashion. Finally, they were found to induce strong Th1 and Th17 immune responses in vivo in immunization experiments in mice and conferred protection in a murine model of Mycobacterium tuberculosis infection. Here we describe the rational development of new molecules with powerful adjuvant properties.

Chronic features of allergic asthma are enhanced in the absence of resistin-like molecule-beta.

PubMed

LeMessurier, Kim S; Palipane, Maneesha; Tiwary, Meenakshi; Gavin, Brian; Samarasinghe, Amali E

2018-05-04

Asthma is characterized by inflammation and architectural changes in the lungs. A number of immune cells and mediators are recognized as initiators of asthma, although therapeutics based on these are not always effective. The multifaceted nature of this syndrome necessitate continued exploration of immunomodulators that may play a role in pathogenesis. We investigated the role of resistin-like molecule-beta (RELM-β), a gut antibacterial, in the development and pathogenesis of Aspergillus-induced allergic airways disease. Age and gender matched C57BL/6J and Retnlb -/- mice rendered allergic to Aspergillus fumigatus were used to measure canonical markers of allergic asthma at early and late time points. Inflammatory cells in airways were similar, although Retnlb -/- mice had reduced tissue inflammation. The absence of RELM-β elevated serum IgA and pro-inflammatory cytokines in the lungs at homeostasis. Markers of chronic disease including goblet cell numbers, Muc genes, airway wall remodelling, and hyperresponsiveness were greater in the absence RELM-β. Specific inflammatory mediators important in antimicrobial defence in allergic asthma were also increased in the absence of RELM-β. These data suggest that while characteristics of allergic asthma develop in the absence of RELM-β, that RELM-β may reduce the development of chronic markers of allergic airways disease.

Highly Selective Photothermal Therapy by a Phenoxylated-Dextran-Functionalized Smart Carbon Nanotube Platform.

PubMed

Han, Seungmin; Kwon, Taeyun; Um, Jo-Eun; Haam, Seungjoo; Kim, Woo-Jae

2016-05-01

Near-infrared (NIR) photothermal therapy using biocompatible single-walled carbon nanotubes (SWNTs) is advantageous because as-produced SWNTs, without additional size control, both efficiently absorb NIR light and demonstrate high photothermal conversion efficiency. In addition, covalent attachment of receptor molecules to SWNTs can be used to specifically target infected cells. However, this technique interrupts SWNT optical properties and inevitably lowers photothermal conversion efficiency and thus remains major hurdle for SWNT applications. This paper presents a smart-targeting photothermal therapy platform for inflammatory disease using newly developed phenoxylated-dextran-functionalized SWNTs. Phenoxylated dextran is biocompatible and efficiently suspends SWNTs by noncovalent π-π stacking, thereby minimizing SWNT bundle formations and maintaining original SWNT optical properties. Furthermore, it selectively targets inflammatory macrophages by scavenger-receptor binding without any additional receptor molecules; therefore, its preparation is a simple one-step process. Herein, it is experimentally demonstrated that phenoxylated dextran-SWNTs (pD-SWNTs) are also biocompatible, selectively penetrate inflammatory macrophages over normal cells, and exhibit high photothermal conversion efficiency. Consequently, NIR laser-triggered macrophage treatment can be achieved with high accuracy by pD-SWNT without damaging receptor-free cells. These smart targeting materials can be a novel photothermal agent candidate for inflammatory disease. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy

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

Verhertbruggen, Yves; Walker, Jesse L.; Guillon, Fabienne

Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of thesemore » three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy.« less

A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy

DOE PAGES

Verhertbruggen, Yves; Walker, Jesse L.; Guillon, Fabienne; ...

2017-08-29

Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of thesemore » three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy.« less

A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy

PubMed Central

Verhertbruggen, Yves; Walker, Jesse L.; Guillon, Fabienne; Scheller, Henrik V.

2017-01-01

Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of these three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy. PMID:28900439

Combined Secretomics and Transcriptomics Revealed Cancer-Derived GDF15 is Involved in Diffuse-Type Gastric Cancer Progression and Fibroblast Activation.

PubMed

Ishige, Takayuki; Nishimura, Motoi; Satoh, Mamoru; Fujimoto, Mai; Fukuyo, Masaki; Semba, Toshihisa; Kado, Sayaka; Tsuchida, Sachio; Sawai, Setsu; Matsushita, Kazuyuki; Togawa, Akira; Matsubara, Hisahiro; Kaneda, Atsushi; Nomura, Fumio

2016-02-19

Gastric cancer is classified into two subtypes, diffuse and intestinal. The diffuse-type gastric cancer (DGC) has poorer prognosis, and the molecular pathology is not yet fully understood. The purpose of this study was to identify functional secreted molecules involved in DGC progression. We integrated the secretomics of six gastric cancer cell lines and gene expression analysis of gastric cancer tissues with publicly available microarray data. Hierarchical clustering revealed characteristic gene expression differences between diffuse- and intestinal-types. GDF15 was selected as a functional secreted molecule owing to high expression only in fetal tissues. Protein expression of GDF15 was higher in DGC cell lines and tissues. Serum levels of GDF15 were significant higher in DGC patients as compared with healthy individuals and chronic gastritis patients, and positively correlated with wall invasion and lymph node metastasis. In addition, the stimulation of GDF15 on NIH3T3 fibroblast enhanced proliferation and up-regulated expression of extracellular matrix genes, which were similar to TGF-β stimulation. These results indicate that GDF15 contributes to fibroblast activation. In conclusion, this study revealed that GDF15 may be a novel functional secreted molecule for DGC progression, possibly having important roles for cancer progression via the affecting fibroblast function, as well as TGF-β.

Comparative chemical characterization of pigmented and less pigmented cell walls of Alternaria tenuissima.

PubMed

Kishore, Kankipati Hara; Kanjilal, Sanjit; Misra, Sunil; Reddy, Chinnathimma Rajagopal; Murty, Upadyayula Suryanarayana

2005-12-01

Alternaria tenuissima, the parasitic fungus, was obtained from the pruned upper-cut surfaces of mulberry stems. This fungus contains dark pigment because of the presence of melanin in the cell wall. To obtain less-pigmented cell walls, this fungus was grown under dark condition. When the pigmented and less-pigmented cell walls were chemically analyzed, no differences were observed in amino-acid composition, hexoses, or pentoses. However, in pigmented cell walls, higher contents of melanin (2.6%) were found than in less-pigmented cell walls (0.3%). Interestingly, a significant difference was observed in the relative fatty-acid compositions between these two types of cell walls. Among the major fatty acids, there were increased concentrations of tetradecanoic acid (C14:0), hexadecanoic acid (C16:0), 9-hexadecenoic acid (C16: 1,Delta 9), and 9-octadecanoic acid (C18:1,Delta 9) and a concomitant decrease in 9,12-octadecadienoic acid (C18:2,Delta 9,12) in less-pigmented compared with pigmented cell walls. This difference in fatty-acid composition may be related to the higher percentage of melanin in the pigmented than the less-pigmented cell walls. Lesser amounts of 9,12-octadecadienoic acid in less-pigmented cell walls may have been caused by the growth of the fungus under environmental stress conditions. An interesting observation was the presence in pigmented cell walls only of methyl-substituted fatty acids with carbon numbers C14 to C17, but their occurrence could not be ascertained in the present study.

The Complexity of Fungal β-Glucan in Health and Disease: Effects on the Mononuclear Phagocyte System

PubMed Central

Camilli, Giorgio; Tabouret, Guillaume; Quintin, Jessica

2018-01-01

β-glucan, the most abundant fungal cell wall polysaccharide, has gained much attention from the scientific community in the last few decades for its fascinating but not yet fully understood immunobiology. Study of this molecule has been motivated by its importance as a pathogen-associated molecular pattern upon fungal infection as well as by its promising clinical utility as biological response modifier for the treatment of cancer and infectious diseases. Its immune effect is attributed to the ability to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes, macrophages, neutrophils, and natural killer cells. The characteristics of the immune responses generated depend on the cell types and receptors involved. Size and biochemical composition of β-glucans isolated from different sources affect their immunomodulatory properties. The variety of studies using crude extracts of fungal cell wall rather than purified β-glucans renders data difficult to interpret. A better understanding of the mechanisms of purified fungal β-glucan recognition, downstream signaling pathways, and subsequent immune regulation activated, is, therefore, essential not only to develop new antifungal therapy but also to evaluate β-glucan as a putative anti-infective and antitumor mediator. Here, we briefly review the complexity of interactions between fungal β-glucans and mononuclear phagocytes during fungal infections. Furthermore, we discuss and present available studies suggesting how different fungal β-glucans exhibit antitumor and antimicrobial activities by modulating the biologic responses of mononuclear phagocytes, which make them potential candidates as therapeutic agents. PMID:29755450

The Complexity of Fungal β-Glucan in Health and Disease: Effects on the Mononuclear Phagocyte System.

PubMed

Camilli, Giorgio; Tabouret, Guillaume; Quintin, Jessica

2018-01-01

β-glucan, the most abundant fungal cell wall polysaccharide, has gained much attention from the scientific community in the last few decades for its fascinating but not yet fully understood immunobiology. Study of this molecule has been motivated by its importance as a pathogen-associated molecular pattern upon fungal infection as well as by its promising clinical utility as biological response modifier for the treatment of cancer and infectious diseases. Its immune effect is attributed to the ability to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes, macrophages, neutrophils, and natural killer cells. The characteristics of the immune responses generated depend on the cell types and receptors involved. Size and biochemical composition of β-glucans isolated from different sources affect their immunomodulatory properties. The variety of studies using crude extracts of fungal cell wall rather than purified β-glucans renders data difficult to interpret. A better understanding of the mechanisms of purified fungal β-glucan recognition, downstream signaling pathways, and subsequent immune regulation activated, is, therefore, essential not only to develop new antifungal therapy but also to evaluate β-glucan as a putative anti-infective and antitumor mediator. Here, we briefly review the complexity of interactions between fungal β-glucans and mononuclear phagocytes during fungal infections. Furthermore, we discuss and present available studies suggesting how different fungal β-glucans exhibit antitumor and antimicrobial activities by modulating the biologic responses of mononuclear phagocytes, which make them potential candidates as therapeutic agents.

Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption

PubMed Central

Luo, Ying; Wang, Jianguo; Liu, Bin; Wang, Zhouli; Yuan, Yahong; Yue, Tianli

2015-01-01

The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry. PMID:26295574

Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption.

PubMed

Luo, Ying; Wang, Jianguo; Liu, Bin; Wang, Zhouli; Yuan, Yahong; Yue, Tianli

2015-01-01

The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry.

Changes in cell wall properties coincide with overexpression of extensin fusion proteins in suspension cultured tobacco cells.

PubMed

Tan, Li; Pu, Yunqiao; Pattathil, Sivakumar; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G; Ragauskas, Arthur J; Kieliszewski, Marcia J

2014-01-01

Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.

Inhibition of cell surface expression of endothelial adhesion molecules by ursolic acid prevents intimal hyperplasia of venous bypass grafts in rats

PubMed Central

Zeller, Iris; Wiedemann, Dominik; Schwaiger, Stefan; Stelzmüller, Marlies; Kreutmayer, Simone; Leberfing, Oliver; Stuppner, Hermann; Bernhard, David

2012-01-01

OBJECTIVES Despite rapid progress in surgical techniques, there is still a significant lack of surgery-supportive pharmacological treatments. The aim of this study was to test the hypothesis that ursolic acid (UA) may prevent intimal hyperplasia of venous bypass grafts. METHODS The hypothesis was tested by means of primary cell isolation and culture followed by real-time polymerase chain reaction, western blotting, fluorescence microscopy and fluorescence-activated cell sorting analyses, as well as an in vivo rat model for intimal hyperplasia of venous bypass grafts and immunohistochemistry and histochemistry. RESULTS The local application of UA significantly inhibited intimal hyperplasia in vivo (intimal thickness control: 25 μm, UA group: 18 μM–8 weeks after surgery). The UA treatment of grafts significantly resulted in reduced endothelial vascular cell adhesion molecule-1 (VCAM-1) expression, reduced infiltration of the grafts vessel wall by CD45-positive cells and increased smooth muscle cell (SMC) death. In in vitro condition, it could be shown that UA inhibits VCAM-1 expression downstream of NFκB and is likely to interfere with VCAM-1 protein synthesis in endothelial cells. Quantification of cell death in vascular smooth muscle cells treated with UA indicated that UA is a potent inducer of SMC apoptosis. CONCLUSIONS Our results suggest that UA-mediated inhibition of endothelial VCAM-1 expression reduces the infiltration of venous bypass grafts by CD45-positive cells and inhibits intimal hyperplasia. Apoptosis induction in SMCs may be another method in which UA reduces intimal thickening. UA may constitute a surgery-supportive pharmacon that reduces intimal hyperplasia of vein grafts. PMID:22551965

Mating-Induced Shedding of Cell Walls, Removal of Walls from Vegetative Cells, and Osmotic Stress Induce Presumed Cell Wall Genes in Chlamydomonas1

PubMed Central

Hoffmann, Xenia-Katharina; Beck, Christoph F.

2005-01-01

The first step in sexual differentiation of the unicellular green alga Chlamydomonas reinhardtii is the formation of gametes. Three genes, GAS28, GAS30, and GAS31, encoding Hyp-rich glycoproteins that presumably are cell wall constituents, are expressed in the late phase of gametogenesis. These genes, in addition, are activated by zygote formation and cell wall removal and by the application of osmotic stress. The induction by zygote formation could be traced to cell wall shedding prior to gamete fusion since it was seen in mutants defective in cell fusion. However, it was absent in mutants defective in the initial steps of mating, i.e. in flagellar agglutination and in accumulation of adenosine 3′,5′-cyclic monophosphate in response to this agglutination. Induction of the three GAS genes was also observed when cultures were exposed to hypoosmotic or hyperosmotic stress. To address the question whether the induction seen upon cell wall removal from both gametes and vegetative cells was elicited by osmotic stress, cell wall removal was performed under isosmotic conditions. Also under such conditions an activation of the genes was observed, suggesting that the signaling pathway(s) is (are) activated by wall removal itself. PMID:16183845

Modifying lignin to improve bioenergy feedstocks: strengthening the barrier against pathogens?

USDA-ARS?s Scientific Manuscript database

Lignin is a ubiquitous polymer present in cell walls of all vascular plants, where it rigidifies and strengthens the cell wall structure through covalent cross-linkages to cell wall polysaccharides. The presence of lignin makes the cell wall recalcitrant to conversion into fermentable sugars for bi...

Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock.

PubMed

Van Amersfoort, Edwin S; Van Berkel, Theo J C; Kuiper, Johan

2003-07-01

Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins.

Cytotoxicity, cytokine release and ER stress-autophagy gene expression in endothelial cells and alveolar-endothelial co-culture exposed to pristine and carboxylated multi-walled carbon nanotubes.

PubMed

Chang, Shiwei; Zhao, Xuqi; Li, Siyu; Liao, Tuqiang; Long, Jimin; Yu, Zhiqiang; Cao, Yi

2018-06-18

Recently we found that direct exposure of human umbilical vein endothelial cells (HUVECs) to multi-walled carbon nanotubes (MWCNTs) might induce toxicological responses through the modulation of ER stress gene expression, but whether this signal could be transferred from other cells to endothelial cells (ECs) is unknown. This study investigated the toxicity of pristine and carboxylated MWCNTs to HUVECs and alveolar-endothelial co-culture, the later of which could mimic the possible signaling communications between ECs and MWCNT exposed alveolar cells. The results showed that direct contact with high levels of MWCNTs induced cytotoxicity and modulated expression of genes associated with ER stress (HSPA5, DDIT3 and XBP-1s) and autophagy (BECN1 and ATG12) both in A549-THP-1 macrophages cultured in the upper chambers as well as HUVECs. However, most of these responses were minimal or negligible in HUVECs cultured in the lower chambers. Moreover, significantly increased cytokine release (interleukin-6 and soluble vascular cell adhesion molecule-1) was only observed in MWCNT exposed HUVECs (p < 0.01) but not HUVECs cultured in the lower chambers (p > 0.05). The minimal or even absent response was likely due to relatively low translocation of MWCNTs from upper chambers to lower chambers, whereas A549-macrophages cultured in the upper chambers internalized large amount MWCNTs. The results indicated that ER stress-autophagy signaling might not be able to transfer from alveolar cells to endothelial cells unless sufficient MWCNTs are translocated. Copyright © 2018 Elsevier Inc. All rights reserved.

Probing Prokaryotic Social Behaviors with Bacterial “Lobster Traps”

PubMed Central

Connell, Jodi L.; Wessel, Aimee K.; Parsek, Matthew R.; Ellington, Andrew D.; Whiteley, Marvin; Shear, Jason B.

2010-01-01

Bacteria are social organisms that display distinct behaviors/phenotypes when present in groups. These behaviors include the abilities to construct antibiotic-resistant sessile biofilm communities and to communicate with small signaling molecules (quorum sensing [QS]). Our understanding of biofilms and QS arises primarily from in vitro studies of bacterial communities containing large numbers of cells, often greater than 108 bacteria; however, in nature, bacteria often reside in dense clusters (aggregates) consisting of significantly fewer cells. Indeed, bacterial clusters containing 101 to 105 cells are important for transmission of many bacterial pathogens. Here, we describe a versatile strategy for conducting mechanistic studies to interrogate the molecular processes controlling antibiotic resistance and QS-mediated virulence factor production in high-density bacterial clusters. This strategy involves enclosing a single bacterium within three-dimensional picoliter-scale microcavities (referred to as bacterial “lobster traps”) defined by walls that are permeable to nutrients, waste products, and other bioactive small molecules. Within these traps, bacteria divide normally into extremely dense (1012 cells/ml) clonal populations with final population sizes similar to that observed in naturally occurring bacterial clusters. Using these traps, we provide strong evidence that within low-cell-number/high-density bacterial clusters, QS is modulated not only by bacterial density but also by population size and flow rate of the surrounding medium. We also demonstrate that antibiotic resistance develops as cell density increases, with as few as ~150 confined bacteria exhibiting an antibiotic-resistant phenotype similar to biofilm bacteria. Together, these findings provide key insights into clinically relevant phenotypes in low-cell-number/high-density bacterial populations. PMID:21060734

Skin integrated with perfusable vascular channels on a chip.

PubMed

Mori, Nobuhito; Morimoto, Yuya; Takeuchi, Shoji

2017-02-01

This paper describes a method for fabricating perfusable vascular channels coated with endothelial cells within a cultured skin-equivalent by fixing it to a culture device connected to an external pump and tubes. A histological analysis showed that vascular channels were constructed in the skin-equivalent, which showed a conventional dermal/epidermal morphology, and the endothelial cells formed tight junctions on the vascular channel wall. The barrier function of the skin-equivalent was also confirmed. Cell distribution analysis indicated that the vascular channels supplied nutrition to the skin-equivalent. Moreover, the feasibility of a skin-equivalent containing vascular channels as a model for studying vascular absorption was demonstrated by measuring test molecule permeation from the epidermal layer into the vascular channels. The results suggested that this skin-equivalent can be used for skin-on-a-chip applications including drug development, cosmetics testing, and studying skin biology. Copyright © 2016 Elsevier Ltd. All rights reserved.

A wide range optical pH sensor for living cells using Au@Ag nanoparticles functionalized carbon nanotubes based on SERS signals.

PubMed

Chen, Peng; Wang, Zhuyuan; Zong, Shenfei; Chen, Hui; Zhu, Dan; Zhong, Yuan; Cui, Yiping

2014-10-01

p-Aminothiophenol (pATP) functionalized multi-walled carbon nanotubes (MWCNTs) have been demonstrated as an efficient pH sensor for living cells. The proposed sensor employs gold/silver core-shell nanoparticles (Au@Ag NPs) functionalized MWCNTs hybrid structure as the surface-enhanced Raman scattering (SERS) substrate and pATP molecules as the SERS reporters, which possess a pH-dependent SERS performance. By using MWCNTs as the substrate to be in a state of aggregation, the pH sensing range could be extended to pH 3.0∼14.0, which is much wider than that using unaggregated Au@Ag NPs without MWCNTs. Furthermore, the pH-sensitive performance was well retained in living cells with a low cytotoxicity. The developed SERS-active MWCNTs-based nanocomposite is expected to be an efficient intracellular pH sensor for bio-applications.

Novel Materials Containing Single-Wall Carbon Nanotubes Wrapped in Polymer Molecules

NASA Technical Reports Server (NTRS)

Smalley, Richard E.; O'Connell, Michael J.; Smith, Kenneth; Colbert, Daniel T.

2009-01-01

In this design, single-wall carbon nanotubes (SWNTs) have been coated in polymer molecules to create a new type of material that has low electrical conductivity, but still contains individual nanotubes, and small ropes of individual nanotubes, which are themselves good electrical conductors and serve as small conducting rods immersed in an electrically insulating matrix. The polymer is attached through weak chemical forces that are primarily non-covalent in nature, caused primarily through polarization rather than the sharing of valence electrons. Therefore, the electronic structure of the SWNT involved is substantially the same as that of free, individual (and small ropes of) SWNT. Their high conductivity makes the individual nanotubes extremely electrically polarizable, and materials containing these individual, highly polarizable molecules exhibit novel electrical properties including a high dielectric constant.

Lower cell wall pectin solubilisation and galactose loss during early fruit development in apple (Malus x domestica) cultivar 'Scifresh' are associated with slower softening rate.

PubMed

Ng, Jovyn K T; Schröder, Roswitha; Brummell, David A; Sutherland, Paul W; Hallett, Ian C; Smith, Bronwen G; Melton, Laurence D; Johnston, Jason W

2015-03-15

Substantial differences in softening behaviour can exist between fruit even within the same species. Apple cultivars 'Royal Gala' and 'Scifresh' soften at different rates despite having a similar genetic background and producing similar amounts of ethylene during ripening. An examination of cell wall metabolism from the fruitlet to the ripe stages showed that in both cultivars pectin solubilisation increased during cell expansion, declined at the mature stage and then increased again during ripening. This process was much less pronounced in the slower softening 'Scifresh' than in 'Royal Gala' at every developmental stage examined, consistent with less cell separation and softening in this cultivar. Both cultivars also exhibited a progressive loss of pectic galactan and arabinan side chains during development. The cell wall content of arabinose residues was similar in both cultivars, but the galactose residue content in 'Scifresh' remained higher than that of 'Royal Gala' at every developmental stage. The higher content of cell wall galactose residue in 'Scifresh' cell walls correlated with a lower β-galactosidase activity and more intense immunolabelling of RG-I galactan side chains in both microscopy sections and glycan microarrays. A high cell wall galactan content has been associated with reduced cell wall porosity, which may restrict access of cell wall-modifying enzymes and thus maintain better structural integrity later in development. The data suggest that the composition and structure of the cell wall at very early development stages may influence subsequent cell wall loosening, and may even predispose the wall's ensuing properties. Copyright © 2014 Elsevier GmbH. All rights reserved.

Cell wall integrity modulates RHO1 activity via the exchange factor ROM2.

PubMed Central

Bickle, M; Delley, P A; Schmidt, A; Hall, M N

1998-01-01

The essential phosphatidylinositol kinase homologue TOR2 of Saccharomyces cerevisiae controls the actin cytoskeleton by activating a GTPase switch consisting of RHO1 (GTPase), ROM2 (GEF) and SAC7 (GAP). We have identified two mutations, rot1-1 and rot2-1, that suppress the loss of TOR2 and are synthetic-lethal. The wild-type ROT1 and ROT2 genes and a multicopy suppressor, BIG1, were isolated by their ability to rescue the rot1-1 rot2-1 double mutant. ROT2 encodes glucosidase II, and ROT1 and BIG1 encode novel proteins. We present evidence that cell wall defects activate RHO1. First, rot1, rot2, big1, cwh41, gas1 and fks1 mutations all confer cell wall defects and suppress tor2(ts). Second, destabilizing the cell wall by supplementing the growth medium with 0.005% SDS also suppresses a tor2(ts) mutation. Third, disturbing the cell wall with SDS or a rot1, rot2, big1, cwh41, gas1 or fks1 mutation increases GDP/GTP exchange activity toward RHO1. These results suggest that cell wall defects suppress a tor2 mutation by activating RHO1 independently of TOR2, thereby inducing TOR2-independent polarization of the actin cytoskeleton and cell wall synthesis. Activation of RHO1, a subunit of the cell wall synthesis enzyme glucan synthase, by a cell wall alteration would ensure that cell wall synthesis occurs only when and where needed. The mechanism of RHO1 activation by a cell wall alteration is via the exchange factor ROM2 and could be analogous to signalling by integrin receptors in mammalian cells. PMID:9545237

Enhanced cell uptake via non-covalent decollation of a single-walled carbon nanotube-DNA hybrid with polyethylene glycol-grafted poly(l-lysine) labeled with an Alexa-dye and its efficient uptake in a cancer cell

NASA Astrophysics Data System (ADS)

Fujigaya, Tsuyohiko; Yamamoto, Yuki; Kano, Arihiro; Maruyama, Atsushi; Nakashima, Naotoshi

2011-10-01

The use of single-walled carbon nanotubes (SWNTs) for biomedical applications is a promising approach due to their unique outer optical stimuli response properties, such as a photothermal response triggered by near-IR laser irradiation. The challenging task in order to realize such applications is to render the SWNTs biocompatible. For this purpose, the stable and homogeneous functionalization of the SWNTs with a molecule carrying a biocompatible group is very important. Here, we describe the design and synthesis of a polyanionic SWNT/DNA hybrid combined with a cationic poly(l-lysine) grafted by polyethylene glycol (PLL-g-PEG) to provide a supramolecular SWNT assembly. A titration experiment revealed that the assembly undergoes an approximately 1 : 1 reaction of the SWNT/DNA with PLL-g-PEG. We also found that SWNT/DNA is coated with PLL-g-PEG very homogeneously that avoids the non-specific binding of proteins on the SWNT surface. The experiment using the obtained supramolecular hybrid was carried out in vitro and a dramatic enhancement in the cell uptake efficiency compared to that of the SWNT/DNA hybrid without PLL-g-PEG was found.The use of single-walled carbon nanotubes (SWNTs) for biomedical applications is a promising approach due to their unique outer optical stimuli response properties, such as a photothermal response triggered by near-IR laser irradiation. The challenging task in order to realize such applications is to render the SWNTs biocompatible. For this purpose, the stable and homogeneous functionalization of the SWNTs with a molecule carrying a biocompatible group is very important. Here, we describe the design and synthesis of a polyanionic SWNT/DNA hybrid combined with a cationic poly(l-lysine) grafted by polyethylene glycol (PLL-g-PEG) to provide a supramolecular SWNT assembly. A titration experiment revealed that the assembly undergoes an approximately 1 : 1 reaction of the SWNT/DNA with PLL-g-PEG. We also found that SWNT/DNA is coated with PLL-g-PEG very homogeneously that avoids the non-specific binding of proteins on the SWNT surface. The experiment using the obtained supramolecular hybrid was carried out in vitro and a dramatic enhancement in the cell uptake efficiency compared to that of the SWNT/DNA hybrid without PLL-g-PEG was found. Electronic supplementary information (ESI) available: Additional absorption spectra, DLS plots and PL spectra. See DOI: 10.1039/c1nr10635j

DISTRIBUTION OF RADIOACTIVITY IN AUTOLYZED CELL WALL OF BACILLUS CEREUS DURING SPHEROPLAST FORMATION1

PubMed Central

Kronish, Donald P.; Mohan, Raam R.; Schwartz, Benjamin S.

1964-01-01

Kronish, Donald P. (Warner-Lambert Research Institute, Morris Plains, N.J.), Raam R. Mohan, and Benjamin S. Schwartz. Distribution of radioactivity in autolyzed cell wall of Bacillus cereus during spheroplast formation. J. Bacteriol. 87:581–587. 1964.—Spheroplasts of Bacillus cereus strain T were produced from cells grown in the presence of uniformly labeled C14-glucose. At regular intervals during spheroplast formation, enzymatically degraded cell wall was isolated by a new procedure. Radioactivity of solubilized cell wall in cell-free material increased from 2.5 to 42% of the total incorporated label during spheroplast formation. The rate of cell-wall degradation as measured by increase in radioactivity was biphasic with relative slopes of 2.0 and 5.0. During autolytic depolymerization of B. cereus cell wall, two major components were solubilized at different rates. Chemical fractionation revealed these to be a peptide and a mucopeptide. The possibility of two enzymes being involved in spheroplast formation and cell-wall degradation is discussed. Images PMID:14127573

Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space

PubMed Central

Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki; Kotake, Toshihisa; Yamazaki, Takashi; Higashibata, Akira; Ishioka, Noriaki; Shimazu, Toru; Fukui, Keiji; Osada, Ikuko; Kasahara, Haruo; Kamada, Motoshi

2015-01-01

Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions. PMID:26378793

Microanalysis of plant cell wall polysaccharides.

PubMed

Obel, Nicolai; Erben, Veronika; Schwarz, Tatjana; Kühnel, Stefan; Fodor, Andrea; Pauly, Markus

2009-09-01

Oligosaccharide Mass Profiling (OLIMP) allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry (MALDI-TOF MS). The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first isolating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apoplastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.

Short-Term Boron Deprivation Inhibits Endocytosis of Cell Wall Pectins in Meristematic Cells of Maize and Wheat Root Apices1

PubMed Central

Yu, Qin; Hlavacka, Andrej; Matoh, Toru; Volkmann, Dieter; Menzel, Diedrik; Goldbach, Heiner E.; Baluška, František

2002-01-01

By using immunofluorescence microscopy, we observed rapidly altered distribution patterns of cell wall pectins in meristematic cells of maize (Zea mays) and wheat (Triticum aestivum) root apices. This response was shown for homogalacturonan pectins characterized by a low level (up to 40%) of methylesterification and for rhamnogalacturonan II pectins cross-linked by a borate diol diester. Under boron deprivation, abundance of these pectins rapidly increased in cell walls, whereas their internalization was inhibited, as evidenced by a reduced and even blocked accumulation of these cell wall pectins within brefeldin A-induced compartments. In contrast, root cells of species sensitive to the boron deprivation, like zucchini (Cucurbita pepo) and alfalfa (Medicago sativa), do not internalize cell wall pectins into brefeldin A compartments and do not show accumulation of pectins in their cell walls under boron deprivation. For maize and wheat root apices, we favor an apoplastic target for the primary action of boron deprivation, which signals deeper into the cell via endocytosis-mediated pectin signaling along putative cell wall-plasma membrane-cytoskeleton continuum. PMID:12226520

Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases.

PubMed

Kováčová, Kristína; Farkaš, Vladimír

2016-04-01

Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated.

Cell wall composition profiling of parasitic giant dodder (Cuscuta reflexa) and its hosts: a priori differences and induced changes.

PubMed

Johnsen, Hanne R; Striberny, Bernd; Olsen, Stian; Vidal-Melgosa, Silvia; Fangel, Jonatan U; Willats, William G T; Rose, Jocelyn K C; Krause, Kirsten

2015-08-01

Host plant penetration is the gateway to survival for holoparasitic Cuscuta and requires host cell wall degradation. Compositional differences of cell walls may explain why some hosts are amenable to such degradation while others can resist infection. Antibody-based techniques for comprehensive profiling of cell wall epitopes and cell wall-modifying enzymes were applied to several susceptible hosts and a resistant host of Cuscuta reflexa and to the parasite itself. Infected tissue of Pelargonium zonale contained high concentrations of de-esterified homogalacturonans in the cell walls, particularly adjacent to the parasite's haustoria. High pectinolytic activity in haustorial extracts and high expression levels of pectate lyase genes suggest that the parasite contributes directly to wall remodeling. Mannan and xylan concentrations were low in P. zonale and in five susceptible tomato introgression lines, but high in the resistant Solanum lycopersicum cv M82, and in C. reflexa itself. Knowledge of the composition of resistant host cell walls and the parasite's own cell walls is useful in developing strategies to prevent infection by parasitic plants. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

The chaotrope-soluble glycoprotein GP1 is a constituent of the insoluble glycoprotein framework of the Chlamydomonas cell wall.

PubMed

Voigt, Jürgen; Frank, Ronald; Wöstemeyer, Johannes

2009-02-01

Chlamydomonas reinhardtii wild-type cells are surrounded by the insoluble cell wall component, a sac-like framework of cross-linked glycoproteins containing 22% hydroxyproline. The chaotrope-soluble cell wall glycoprotein GP1 is the only polypeptide with an even higher proportion of hydroxyproline (35%) occurring in vegetative C. reinhardtii cells. Mass spectrometric analyses of peptides released from the purified insoluble cell wall fraction by trypsin treatment and epitope analyses of polyclonal antibodies raised against different deglycosylation products of this particular wall fraction using 181 chemically synthesized GP1-derived pentadecapeptides revealed evidence that GP1 is indeed a constituent of the insoluble wall component.

Calcium deprivation disrupts enlargement of Chara corallina cells: further evidence for the calcium pectate cycle.

PubMed

Proseus, Timothy E; Boyer, John S

2012-06-01

Pectin is a normal constituent of cell walls of green plants. When supplied externally to live cells or walls isolated from the large-celled green alga Chara corallina, pectin removes calcium from load-bearing cross-links in the wall, loosening the structure and allowing it to deform more rapidly under the action of turgor pressure. New Ca(2+) enters the vacated positions in the wall and the externally supplied pectin binds to the wall, depositing new wall material that strengthens the wall. A calcium pectate cycle has been proposed for these sub-reactions. In the present work, the cycle was tested in C. corallina by depriving the wall of external Ca(2+) while allowing the cycle to run. The prediction is that growth would eventually be disrupted by a lack of adequate deposition of new wall. The test involved adding pectate or the calcium chelator EGTA to the Ca(2+)-containing culture medium to bind the calcium while the cycle ran in live cells. After growth accelerated, turgor and growth eventually decreased, followed by an abrupt turgor loss and growth cessation. The same experiment with isolated walls suggested the walls of live cells became unable to support the plasma membrane. If instead the pectate or EGTA was replaced with fresh Ca(2+)-containing culture medium during the initial acceleration in live cells, growth was not disrupted and returned to the original rates. The operation of the cycle was thus confirmed, providing further evidence that growth rates and wall biosynthesis are controlled by these sub-reactions in plant cell walls.

Measurements of Gas-Wall Partitioning of Oxidized Species in Environmental Smog Chambers and Teflon Sampling Lines

NASA Astrophysics Data System (ADS)

Krechmer, J.; Pagonis, D.; Ziemann, P. J.; Jimenez, J. L.

2015-12-01

Environmental "smog" chambers have played an integral role in atmospheric aerosol research for decades. Recently, many works have demonstrated that the loss of gas-phase material to fluorinated ethylene propylene (FEP) chamber walls can have significant effects on secondary organic aerosol (SOA) yield results. The effects of gas-wall partitioning on highly oxidized species is still controversial, however. In this work we performed a series of experiments examining the losses of oxidized gas-phase compounds that were generated in-situ­ in an environmental chamber. The loss of species to the walls was measured using three chemical ionization mass spectrometry techniques: proton-transfer-reaction (PTR), nitrate (NO3-) ion, and iodide (I-). Many oxidized species have wall loss timescales ranging between 15 to 45 minutes and scale according to the molecule's estimated saturation concentration c* and functional groups. By comparing results of the different techniques, and in particular by the use of the "wall-less" NO3- source, we find that measuring species with high chamber wall-loss rates is complicated by the use of a standard ion-molecule reaction (IMR) region, as well as long Teflon sampling lines, which can be important sinks for gas-phase species. This effect is observed even for semi-volatile species and could have significant effects on ambient sampling techniques that make highly time-resolved measurements using long sampling lines, such as eddy covariance measurements.

Branched Pectic Galactan in Phloem-Sieve-Element Cell Walls: Implications for Cell Mechanics.

PubMed

Torode, Thomas A; O'Neill, Rachel; Marcus, Susan E; Cornuault, Valérie; Pose, Sara; Lauder, Rebecca P; Kračun, Stjepan K; Rydahl, Maja Gro; Andersen, Mathias C F; Willats, William G T; Braybrook, Siobhan A; Townsend, Belinda J; Clausen, Mads H; Knox, J Paul

2018-02-01

A major question in plant biology concerns the specification and functional differentiation of cell types. This is in the context of constraints imposed by networks of cell walls that both adhere cells and contribute to the form and function of developing organs. Here, we report the identification of a glycan epitope that is specific to phloem sieve element cell walls in several systems. A monoclonal antibody, designated LM26, binds to the cell wall of phloem sieve elements in stems of Arabidopsis ( Arabidopsis thaliana ), Miscanthus x giganteus , and notably sugar beet ( Beta vulgaris ) roots where phloem identification is an important factor for the study of phloem unloading of Suc. Using microarrays of synthetic oligosaccharides, the LM26 epitope has been identified as a β-1,6-galactosyl substitution of β-1,4-galactan requiring more than three backbone residues for optimized recognition. This branched galactan structure has previously been identified in garlic ( Allium sativum ) bulbs in which the LM26 epitope is widespread throughout most cell walls including those of phloem cells. Garlic bulb cell wall material has been used to confirm the association of the LM26 epitope with cell wall pectic rhamnogalacturonan-I polysaccharides. In the phloem tissues of grass stems, the LM26 epitope has a complementary pattern to that of the LM5 linear β-1,4-galactan epitope, which is detected only in companion cell walls. Mechanical probing of transverse sections of M x giganteus stems and leaves by atomic force microscopy indicates that phloem sieve element cell walls have a lower indentation modulus (indicative of higher elasticity) than companion cell walls. © 2018 The author(s). All Rights Reserved.

Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation

PubMed Central

Hayot, Céline M.; Forouzesh, Elham; Goel, Ashwani; Avramova, Zoya; Turner, Joseph A.

2012-01-01

Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall. PMID:22291130

Reconstitution of a secondary cell wall in a secondary cell wall-deficient Arabidopsis mutant.

PubMed

Sakamoto, Shingo; Mitsuda, Nobutaka

2015-02-01

The secondary cell wall constitutes a rigid frame of cells in plant tissues where rigidity is required. Deposition of the secondary cell wall in fiber cells contributes to the production of wood in woody plants. The secondary cell wall is assembled through co-operative activities of many enzymes, and their gene expression is precisely regulated by a pyramidal cascade of transcription factors. Deposition of a transmuted secondary cell wall in empty fiber cells by expressing selected gene(s) in this cascade has not been attempted previously. In this proof-of-concept study, we expressed chimeric activators of 24 transcription factors that are preferentially expressed in the stem, in empty fiber cells of the Arabidopsis nst1-1 nst3-1 double mutant, which lacks a secondary cell wall in fiber cells, under the control of the NST3 promoter. The chimeric activators of MYB46, SND2 and ANAC075, as well as NST3, reconstituted a secondary cell wall with different characteristics from those of the wild type in terms of its composition. The transgenic lines expressing the SND2 or ANAC075 chimeric activator showed increased glucose and xylose, and lower lignin content, whereas the transgenic line expressing the MYB46 chimeric activator showed increased mannose content. The expression profile of downstream genes in each transgenic line was also different from that of the wild type. This study proposed a new screening strategy to identify factors of secondary wall formation and also suggested the potential of the artificially reconstituted secondary cell walls as a novel raw material for production of bioethanol and other chemicals. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Changes in levels of cell wall constituents in wheat seedlings grown under continuous hypergravity conditions

NASA Astrophysics Data System (ADS)

Wakabayashi, K.; Soga, K.; Kamisaka, S.; Hoson, T.

Effects of continuous hypergravity stimuli on the amounts and composition of cell wall constituents were investigated in wheat shoots. Hypergravity (300 g) treatment for three days after germination increased the net amount of cell wall polysaccharides such as hemicellulose and cellulose, but reduced the shoot elongation. As a result, the amount of cell wall polysaccharides per unit length of shoot increased under hypergravity. The hemicellulose fraction contained polysaccharides in the middle and low molecular mass range (5 kDa-1 MDa) and increased in response to hypergravity. Also, the amounts of arabinose (Ara) and xylose (Xyl), the major sugar components of the hemicellulose fraction, increased under hypergravity conditions. In addition to wall polysaccharides, hypergravity increased the amounts of cell wall-bound phenolic acids, such as ferulic acid (FA) and diferulic acid (DFA). Furthermore, the activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was enhanced under hypergravity conditions. These results suggest that continuous hypergravity stimulates the synthesis of cell wall constituents, especially hemicellulosic arabinoxylans and cell wall-bound FA and DFA in wheat shoots. The increased PAL activity may promote the formation of FA and DFA. These changes in cell wall architecture may be involved in making rigid and tough cell walls under hypergravity conditions and thereby contribute to the ability of plant to sustain their structures against gravitational stimuli.

Electron Tomography of Cryo-Immobilized Plant Tissue: A Novel Approach to Studying 3D Macromolecular Architecture of Mature Plant Cell Walls In Situ

PubMed Central

Sarkar, Purbasha; Bosneaga, Elena; Yap, Edgar G.; Das, Jyotirmoy; Tsai, Wen-Ting; Cabal, Angelo; Neuhaus, Erica; Maji, Dolonchampa; Kumar, Shailabh; Joo, Michael; Yakovlev, Sergey; Csencsits, Roseann; Yu, Zeyun; Bajaj, Chandrajit; Downing, Kenneth H.; Auer, Manfred

2014-01-01

Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls. PMID:25207917

Tools to Understand Structural Property Relationships for Wood Cell Walls

Treesearch

Joseph E. Jakes; Daniel J. Yelle; Charles R. Frihart

2011-01-01

Understanding structure-property relationships for wood cell walls has been hindered by the complex polymeric structures comprising these cell walls and the difficulty in assessing meaningful mechanical property measurements of individual cell walls. To help overcome these hindrances, we have developed two experimental methods: 1) two-dimensional solution state nuclear...

The plant cell wall integrity maintenance mechanism--a case study of a cell wall plasma membrane signaling network.

PubMed

Hamann, Thorsten

2015-04-01

Some of the most important functions of plant cell walls are protection against biotic/abiotic stress and structural support during growth and development. A prerequisite for plant cell walls to perform these functions is the ability to perceive different types of stimuli in both qualitative and quantitative manners and initiate appropriate responses. The responses in turn involve adaptive changes in cellular and cell wall metabolism leading to modifications in the structures originally required for perception. While our knowledge about the underlying plant mechanisms is limited, results from Saccharomyces cerevisiae suggest the cell wall integrity maintenance mechanism represents an excellent example to illustrate how the molecular mechanisms responsible for stimulus perception, signal transduction and integration can function. Here I will review the available knowledge about the yeast cell wall integrity maintenance system for illustration purposes, summarize the limited knowledge available about the corresponding plant mechanism and discuss the relevance of the plant cell wall integrity maintenance mechanism in biotic stress responses. Copyright © 2014 Elsevier Ltd. All rights reserved.

Building and degradation of secondary cell walls: are there common patterns of lamellar assembly of cellulose microfibrils and cell wall delamination?

PubMed

De Micco, Veronica; Ruel, Katia; Joseleau, Jean-Paul; Aronne, Giovanna

2010-08-01

During cell wall formation and degradation, it is possible to detect cellulose microfibrils assembled into thicker and thinner lamellar structures, respectively, following inverse parallel patterns. The aim of this study was to analyse such patterns of microfibril aggregation and cell wall delamination. The thickness of microfibrils and lamellae was measured on digital images of both growing and degrading cell walls viewed by means of transmission electron microscopy. To objectively detect, measure and classify microfibrils and lamellae into thickness classes, a method based on the application of computerized image analysis combined with graphical and statistical methods was developed. The method allowed common classes of microfibrils and lamellae in cell walls to be identified from different origins. During both the formation and degradation of cell walls, a preferential formation of structures with specific thickness was evidenced. The results obtained with the developed method allowed objective analysis of patterns of microfibril aggregation and evidenced a trend of doubling/halving lamellar structures, during cell wall formation/degradation in materials from different origin and which have undergone different treatments.

Model of Fission Yeast Cell Shape Driven by Membrane-Bound Growth Factors and the Cytoskeleton

PubMed Central

Drake, Tyler; Vavylonis, Dimitrios

2013-01-01

Fission yeast serves as a model for how cellular polarization machinery consisting of signaling molecules and the actin and microtubule cytoskeleton regulates cell shape. In this work, we develop mathematical models to investigate how these cells maintain a tubular shape of approximately constant diameter. Many studies identify active Cdc42, found in a cap at the inner membrane of growing cell tips, as an important regulator of local cell wall remodeling, likely through control of exocyst tethering and the targeting of other polarity-enhancing structures. First, we show that a computational model with Cdc42-dependent local cell wall remodeling under turgor pressure predicts a relationship between spatial extent of growth signal and cell diameter that is in agreement with prior experiments. Second, we model the consequences of feedback between cell shape and distribution of Cdc42 growth signal at cell tips. We show that stability of cell diameter over successive cell divisions places restrictions on their mutual dependence. We argue that simple models where the spatial extent of the tip growth signal relies solely on geometrical alignment of confined microtubules might lead to unstable width regulation. Third, we study a computational model that combines a growth signal distributed over a characteristic length scale (as, for example, by a reaction-diffusion mechanism) with an axis-sensing microtubules system that places landmarks at positions where microtubule tips touch the cortex. A two-dimensional implementation of this model leads to stable cell diameter for a wide range of parameters. Changes to the parameters of this model reproduce straight, bent, and bulged cell shapes, and we discuss how this model is consistent with other observed cell shapes in mutants. Our work provides an initial quantitative framework for understanding the regulation of cell shape in fission yeast, and a scaffold for understanding this process on a more molecular level in the future. PMID:24146607

Cell wall-bound silicon optimizes ammonium uptake and metabolism in rice cells.

PubMed

Sheng, Huachun; Ma, Jie; Pu, Junbao; Wang, Lijun

2018-05-16

Turgor-driven plant cell growth depends on cell wall structure and mechanics. Strengthening of cell walls on the basis of an association and interaction with silicon (Si) could lead to improved nutrient uptake and optimized growth and metabolism in rice (Oryza sativa). However, the structural basis and physiological mechanisms of nutrient uptake and metabolism optimization under Si assistance remain obscure. Single-cell level biophysical measurements, including in situ non-invasive micro-testing (NMT) of NH4+ ion fluxes, atomic force microscopy (AFM) of cell walls, and electrolyte leakage and membrane potential, as well as whole-cell proteomics using isobaric tags for relative and absolute quantification (iTRAQ), were performed. The altered cell wall structure increases the uptake rate of the main nutrient NH4+ in Si-accumulating cells, whereas the rate is only half in Si-deprived counterparts. Rigid cell walls enhanced by a wall-bound form of Si as the structural basis stabilize cell membranes. This, in turn, optimizes nutrient uptake of the cells in the same growth phase without any requirement for up-regulation of transmembrane ammonium transporters. Optimization of cellular nutrient acquisition strategies can substantially improve performance in terms of growth, metabolism and stress resistance.