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Sample records for cell wall mutants

  1. [Composition of cell walls of 2 mutant strains of Streptomyces chrysomallus].

    Science.gov (United States)

    Zaretskaia, M Sh; Nefelova, M V; Baratova, L A; Polin, A N

    1984-12-01

    The cell walls and peptidoglycans of two mutant strains, Streptomyces chrysomallus var. carotenoides and Streptomyces chrysomallus var. macrotetrolidi, were studied. The strains are organisms producing carotenes and antibiotics of the macrotetrolide group. By the qualitative composition of the peptidoglycans the mutants belong to Streptomyces and are similar. Their glycan portion consists of equimolar quantities of N-acetyl glucosamine and muramic acid. The peptide subunit is presented by glutamic acid, L, L-diaminopimelic acid, glycine and alanine. The molar ratio of alanine is 1.2-1.3. The mutant strains differ in the content of carbohydrates, total phosphorus and phosphorus belonging to teichoic acids. Teichoic acids of the cell walls of the both strains are of the ribitolhosphate nature. The cell walls of the mutants contain polysaccharides differing from teichoic acids and consisting of glucose, galactose, arabinose and fucose. The influence of the cell wall composition of the mutant strains on their morphology and metabolism and comparison of the data relative to the mutant strains with those relative to the starting strain are discussed.

  2. Rice Brittleness Mutants: A Way to Open the 'Black Box' of Monocot Cell Wall Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Baocai Zhang; Yihua Zhou

    2011-01-01

    Rice is a model organism for studying the mechanism of cell wall biosynthesis and remolding in Gramineae.Mechanical strength is an important agronomy trait of rice(Oryza sativa L.)plants that affects crop lodging and grain yield.As a prominent physical property of cell walls,mechanical strength reflects upon the structure of different wall polymers and how they interact.Studies on the mechanisms that regulate the mechanical strength therefore consequently results in uncovering the genes functioning in cell wall biosynthesis and remodeling.Our group focuses on the study of isolation of brittle culm(bc)mutants and characterization of their corresponding genes.To date,several bc mutants have been reported.The identified genes have covered several pathways of cell wall biosynthesis,revealing many secrets of monocot cell wall biosynthesis.Here,we review the progress achieved in this research field and also highlight the perspectives in expectancy.All of those lend new insights into mechanisms of cell wall formation and are helpful for harnessing the waste rice straws for biofuel production.

  3. Detection of Cell Wall Chemical Variation in Zea Mays Mutants Using Near-Infrared Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Buyck, N.; Thomas, S.

    2001-01-01

    Corn stover is regarded as the prime candidate feedstock material for commercial biomass conversion in the United States. Variations in chemical composition of Zea mays cell walls can affect biomass conversion process yields and economics. Mutant lines were constructed by activating a Mu transposon system. The cell wall chemical composition of 48 mutant families was characterized using near-infrared (NIR) spectroscopy. NIR data were analyzed using a multivariate statistical analysis technique called Principal Component Analysis (PCA). PCA of the NIR data from 349 maize leaf samples reveals 57 individuals as outliers on one or more of six Principal Components (PCs) at the 95% confidence interval. Of these, 19 individuals from 16 families are outliers on either PC3 (9% of the variation) or PC6 (1% of the variation), the two PCs that contain information about cell wall polymers. Those individuals for which altered cell wall chemistry is confirmed with wet chemical analysis will then be subjected to fermentation analysis to determine whether or not biomass conversion process kinetics, yields and/or economics are significantly affected. Those mutants that provide indications for a decrease in process cost will be pursued further to identify the gene(s) responsible for the observed changes in cell wall composition and associated changes in process economics. These genes will eventually be incorporated into maize breeding programs directed at the development of a truly dual use crop.

  4. The Tomato Fruit Cell Wall : II. Polyuronide Metabolism in a Nonsoftening Tomato Mutant.

    Science.gov (United States)

    Koch, J L; Nevins, D J

    1990-03-01

    A nonsoftening tomato (Lycopersicon esculentum L.) variety, dg, was examined to assess the physiological basis for its inability to soften during ripening. Total uronic acid levels, 18 milligrams uronic acid/100 milligrams wall, and the extent of pectin esterification, 60 mole%, remained constant throughout fruit development in this mutant. The proportion of uronic acid susceptible to polygalacturonase in vitro also remained constant. Pretreatment of heat-inactivated dg fruit cell walls with tomato pectinmethylesterase enhances polygalacturonase susceptibility at all ripening stages. Pectinesterase activity of cell wall protein extracts from red ripe dg fruit was half that in extracts from analogous tissue of VF145B. Polygalacturonase activities of cell wall extracts, however, were similar in both varieties. Diffusion of uronic acid from tissue discs of both varieties increased beginning at the turning stage to a maximum of 2.0 milligrams uronic acid released/gram fresh weight at the ripe stage. The increased quantity of hydrolytic products released during ripening suggests the presence of in situ polygalacturonase activity. Low speed centrifugation was employed to induce efflux of uronide components from the cell wall tree space. In normal fruit, at the turning stage, 2.1 micrograms uronic acid/gram fresh weight was present in the eluant after 1 hour, and this value increased to a maximum of 8.2 micrograms uronic acid/gram fresh weight at the red ripe stage. However, centrifuge-aided extraction of hydrolytic products failed to provide evidence for in situ polygalacturonase activity in dg fruit. We conclude that pectinesterase and polygalacturonase enzymes are not active in situ during the ripening of dg fruit. This could account for the maintenance of firmness in ripe fruit tissue.

  5. Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis.

    Science.gov (United States)

    Cantrell, Sally A; Leavell, Michael D; Marjanovic, Olivera; Iavarone, Anthony T; Leary, Julie A; Riley, Lee W

    2013-10-01

    The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen's persistence.

  6. Aberrant Expression of Critical Genes during Secondary Cell Wall Biogenesis in a Cotton Mutant, Ligon Lintless-1 (Li-1

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    James J. Bolton

    2009-01-01

    Full Text Available Over ninety percent of the value of cotton comes from its fiber; however, the genetic mechanisms governing fiber development are poorly understood. Due to their biochemical and morphological diversity in fiber cells cotton fiber mutants have been useful in examining fiber development; therefore, using the Ligon Lintless (Li-1 mutant, a monogenic dominant cotton mutant with very short fibers, we employed the high throughput approaches of microarray technology and real time PCR to gain insights into what genes were critical during the secondary cell wall synthesis stage. Comparative transcriptome analysis of the normal TM-1 genotype and the near isogenic Li-1 revealed that over 100 transcripts were differentially expressed at least 2-fold during secondary wall biogenesis, although the genetic profile of the expansion phase showed no significant differences in the isolines. Of particular note, we identified three candidate gene families-expansin, sucrose synthase, and tubulin—whose expression in Li-1 deviates from normal expression patterns of its parent, TM-1. These genes may contribute to retarded growth of fibers in Li-1 since they are fiber-expressed structural and metabolic genes. This work provides more details into the mechanisms of fiber development, and suggests the Li gene is active during the later stages of fiber development.

  7. Structural characterization of a mixed-linkage glucan deficient mutant reveals alteration in cellulose microfibril orientation in rice coleoptile mesophyll cell walls

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    Andreia Michelle Smith-Moritz

    2015-08-01

    Full Text Available The CELLULOSE SYNTHASE-LIKE F6 (CslF6 gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG, a cell wall polysaccharide that is hypothesized to be a tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to test the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of three day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell was of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion.

  8. Genetic background impacts soluble and cell wall-bound aromatics in brown midrib mutants of sorghum

    Science.gov (United States)

    To evaluate the effects that genetic background has on two sorghum brown midrib (bmr) mutants, plant phenolics, lignin biosynthetic enzymes and stem anatomy were evaluated in wild-type (WT), bmr-6, bmr-12 and double-mutants (bmr-6 and bmr-12) in near isogenic , RTx430 and Wheatland backgrounds. The...

  9. Arabidopsis thaliana T-DNA Mutants Implicate GAUT Genes in the Biosynthesis of Pectin and Xylan in Cell Walls and Seed Testa

    Institute of Scientific and Technical Information of China (English)

    Kerry H. Caffall; Sivakumar Pattathil; Sarah E. Phillips; Michael G. Hahn; Debra Mohnen

    2009-01-01

    Galacturonosyltransferase 1 (GAUT1) is an α1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5'-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan (Sterling et al., 2006). The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like (GATL) proteins with, respectively, 56-84 and 42-53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades: A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades: clade A-1 (GAUTs 1 to 3); A-2 (GAUT4); A-3 (GAUTs 5 and 6); A-4 (GAUT7); B-1(GAUTs 8 and 9); B-2 (GAUTs 10 and 11); and clade C (GAUTs 12 to 15). The Arabidopsis GAUTs have a distribution com-parable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with eitherArabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUTgenes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as dem-onstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mu-tant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUTgenes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.

  10. Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core.

    Science.gov (United States)

    Alderwick, Luke J; Radmacher, Eva; Seidel, Mathias; Gande, Roland; Hitchen, Paul G; Morris, Howard R; Dell, Anne; Sahm, Hermann; Eggeling, Lothar; Besra, Gurdyal S

    2005-09-16

    The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG

  11. The deoxyhypusine synthase mutant dys1-1 reveals the association of eIF5A and Asc1 with cell wall integrity.

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    Fabio Carrilho Galvão

    Full Text Available The putative eukaryotic translation initiation factor 5A (eIF5A is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1 and deoxyhypusine hydroxylase (Lia1 catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1 and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of m

  12. Genome-wide Expression Profiling in Seedlings of the Arabidopsis Mutant uro that is Defective in the Secondary Cell Wall Formation

    Institute of Scientific and Technical Information of China (English)

    Zheng Yuan; Xuan Yao; Dabing Zhang; Yue Sun; Hai Huang

    2007-01-01

    Plant secondary growth is of tremendous importance, not only for plant growth and development but also for economic usefulness.Secondary tissues such as xylem and phloem are the conducting tissues in plant vascular systems, essentially for water and nutrient transport, respectively.On the other hand, products of plant secondary growth are important raw materials and renewable sources of energy.Although advances have been recently made towards describing molecular mechanisms that regulate secondary growth, the genetic control for this process is not yet fully understood.Secondary cell wall formation in plants shares some common mechanisms with other plant secondary growth processes.Thus, studies on the secondary cell wall formation using Arabidopsis may help to understand the regulatory mechanisms for plant secondary growth.We previously reported phenotypic characterizations of an Arabidopsis semi-dominant mutant,upright rosette (uro), which is defective in secondary cell wall growth and has an unusually soft stem.Here, we show that lignification in the secondary cell wall in uro is aberrant by analyzing hypocotyl and stem.We also show genome-wide expression profiles of uro seedlings, using the Affymetrix GeneChip that contains approximately 24 000 Arabidopsis genes.Genes identified with altered expression levels include those that function in plant hormone biosynthesis and signaling,cell division and plant secondary tissue growth.These results provide useful information for further characterizations of the regulatory network in plant secondary cell wall formation.

  13. Aspergillus nidulans cell wall composition and function change in response to hosting several Aspergillus fumigatus UDP-galactopyranose mutase activity mutants.

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    Md Kausar Alam

    Full Text Available Deletion or repression of Aspergillus nidulans ugmA (AnugmA, involved in galactofuranose biosynthesis, impairs growth and increases sensitivity to Caspofungin, a β-1,3-glucan synthesis antagonist. The A. fumigatus UgmA (AfUgmA crystal structure has been determined. From that study, AfUgmA mutants with altered enzyme activity were transformed into AnugmA▵ to assess their effect on growth and wall composition in A. nidulans. The complemented (AnugmA::wild type AfugmA strain had wild type phenotype, indicating these genes had functional homology. Consistent with in vitro studies, AfUgmA residues R182 and R327 were important for its function in vivo, with even conservative amino (RK substitutions producing AnugmA? phenotype strains. Similarly, the conserved AfUgmA loop III histidine (H63 was important for Galf generation: the H63N strain had a partially rescued phenotype compared to AnugmA▵. Collectively, A. nidulans strains that hosted mutated AfUgmA constructs with low enzyme activity showed increased hyphal surface adhesion as assessed by binding fluorescent latex beads. Consistent with previous qPCR results, immunofluorescence and ELISA indicated that AnugmA▵ and AfugmA-mutated A. nidulans strains had increased α-glucan and decreased β-glucan in their cell walls compared to wild type and AfugmA-complemented strains. Like the AnugmA▵ strain, A. nidulans strains containing mutated AfugmA showed increased sensitivity to antifungal drugs, particularly Caspofungin. Reduced β-glucan content was correlated with increased Caspofungin sensitivity. Aspergillus nidulans wall Galf, α-glucan, and β-glucan content was correlated in A. nidulans hyphal walls, suggesting dynamic coordination between cell wall synthesis and cell wall integrity.

  14. Aspergillus nidulans cell wall composition and function change in response to hosting several Aspergillus fumigatus UDP-galactopyranose mutase activity mutants.

    Science.gov (United States)

    Alam, Md Kausar; van Straaten, Karin E; Sanders, David A R; Kaminskyj, Susan G W

    2014-01-01

    Deletion or repression of Aspergillus nidulans ugmA (AnugmA), involved in galactofuranose biosynthesis, impairs growth and increases sensitivity to Caspofungin, a β-1,3-glucan synthesis antagonist. The A. fumigatus UgmA (AfUgmA) crystal structure has been determined. From that study, AfUgmA mutants with altered enzyme activity were transformed into AnugmA▵ to assess their effect on growth and wall composition in A. nidulans. The complemented (AnugmA::wild type AfugmA) strain had wild type phenotype, indicating these genes had functional homology. Consistent with in vitro studies, AfUgmA residues R182 and R327 were important for its function in vivo, with even conservative amino (RK) substitutions producing AnugmA? phenotype strains. Similarly, the conserved AfUgmA loop III histidine (H63) was important for Galf generation: the H63N strain had a partially rescued phenotype compared to AnugmA▵. Collectively, A. nidulans strains that hosted mutated AfUgmA constructs with low enzyme activity showed increased hyphal surface adhesion as assessed by binding fluorescent latex beads. Consistent with previous qPCR results, immunofluorescence and ELISA indicated that AnugmA▵ and AfugmA-mutated A. nidulans strains had increased α-glucan and decreased β-glucan in their cell walls compared to wild type and AfugmA-complemented strains. Like the AnugmA▵ strain, A. nidulans strains containing mutated AfugmA showed increased sensitivity to antifungal drugs, particularly Caspofungin. Reduced β-glucan content was correlated with increased Caspofungin sensitivity. Aspergillus nidulans wall Galf, α-glucan, and β-glucan content was correlated in A. nidulans hyphal walls, suggesting dynamic coordination between cell wall synthesis and cell wall integrity.

  15. Global Proteomic Profiling of the Secretome of Candida albicans ecm33 Cell Wall Mutant Reveals the Involvement of Ecm33 in Sap2 Secretion.

    Science.gov (United States)

    Gil-Bona, Ana; Monteoliva, Lucía; Gil, Concha

    2015-10-02

    Candida albicans secretes numerous proteins related to cell wall remodeling, adhesion, nutrient acquisition and host interactions. Also, extracellular vesicles containing cytoplasmic proteins are secreted into the medium. The C. albicans ecm33/ecm33 mutant (RML2U) presents an altered cell wall and is avirulent. The proteomic analysis of proteins secreted by RML2U cells identified a total of 170 proteins: 114 and 154 of which correspond to the vesicle-free secretome and extracellular vesicles, respectively. Notably, 98 proteins were common to both samples, and the groups most represented were metabolic and cell wall-related proteins. The results of this study showed that RML2U had an altered pattern of proteins secreted by the classical secretion pathway as well as the formation of extracellular vesicles, including their size, quantity, and protein composition. Specifically, the secretion of aspartic protease 2 (Sap2) was compromised but not its intracellular expression, with bovine serum albumin (BSA) degradation by RML2U being altered when BSA was used as the sole nitrogen source. Furthermore, as recent research links the expression of Sap2 to the TOR (Target Of Rapamycin) signaling pathway, the sensitivity of RML2U to rapamycin (the inhibitor of TOR kinase) was tested and found to be enhanced, connecting Ecm33 with this pathway.

  16. The role of cell wall-based defences in the early restriction of non-pathogenic hrp mutant bacteria in Arabidopsis.

    Science.gov (United States)

    Mitchell, Kathy; Brown, Ian; Knox, Paul; Mansfield, John

    2015-04-01

    We have investigated the cause of the restricted multiplication of hrp mutant bacteria in leaves of Arabidopsis. Our focus was on early interactions leading to differentiation between virulent wild-type and non-pathogenic hrpA mutant strains of Pseudomonas syringae pv. tomato. An initial drop in recoverable bacteria detected 0-4 h after inoculation with either strain was dependent on a functional FLS2 receptor and H2O2 accumulation in challenged leaves. Wild-type bacteria subsequently multiplied rapidly whereas the hrpA mutant was restricted within 6 h. Despite the early restriction, the hrpA mutant was still viable several days after inoculation. Analysis of intercellular washing fluids (IWFs), showed that high levels of nutrients were readily available to bacteria in the apoplast and that no diffusible inhibitors were produced in response to bacterial challenge. Histochemical and immunocytochemical methods were used to detect changes in polysaccharides (callose, two forms of cellulose, and pectin), arabinogalactan proteins (AGPs), H2O2 and peroxidase. Quantitative analysis showed very similar changes in localisation of AGPs, cellulose epitopes and callose 2 and 4 h after inoculation with either strain. However from 6 to 12 h after inoculation papillae expanded only next to the hrp mutant. In contrast to the similar patterns of secretory activity recorded from mesophyll cells, accumulation of H2O2 and peroxidase was significantly greater around the hrpA mutant within the first 4h after inoculation. A striking differential accumulation of H2O2 was also found in chloroplasts in cells next to the mutant. Ascorbate levels were lower in the IWFs recovered from sites inoculated with the hrp mutant than with wild-type bacteria. The critical response, observed at the right time and place to explain the observed differential behaviour of wild-type and hrpA mutant bacteria was the accumulation of H2O2, probably generated through Type III peroxidase activity and in

  17. The Lamportian cell wall

    Energy Technology Data Exchange (ETDEWEB)

    Keiliszewski, M.; Lamport, D. (Michigan State Univ. Plant Research Lab., East Lansing (United States))

    1991-05-01

    The Lamportian Warp-Weft hypothesis suggests a cellulose-extensin interpenetrating network where extensin mechanically couples the load-bearing cellulose microfibrils in a wall matrix that is best described as a microcomposite. This model is based on data gathered from the extensin-rich walls of tomato and sycamore cell suspension culture, wherein extensin precursors are insolubilized into the wall by undefined crosslinks. The authors recent work with cell walls isolated from intact tissue as well as walls from suspension cultured cells of the graminaceous monocots maize and rice, the non-graminaceous monocot asparagus, the primitive herbaceous dicot sugar beet, and the gymnosperm Douglas Fir indicate that although extensins are ubiquitous to all plant species examined, they are not the major structural protein component of most walls examined. Amino acid analyses of intact and HF-treated walls shows a major component neither an HRGP, nor directly comparable to the glycine-rich wall proteins such as those associated with seed coat walls or the 67 mole% glycine-rich proteins cloned from petunia and soybean. Clearly, structural wall protein alternatives to extensin exist and any cell wall model must take that into account. If we assume that extracellular matrices are a priori network structures, then new Hypless' structural proteins in the maize cell wall raise questions about the sort of network these proteins create: the kinds of crosslinks involved; how they are formed; and the roles played by the small amounts of HRGPs.

  18. Cell Wall Proteome

    OpenAIRE

    Boudart, Georges; Minic, Zoran; Albenne, Cécile; Canut, Hervé; Jamet, Elisabeth; Pont-Lezica, Rafael F

    2007-01-01

    In this chapter, we will focus on the contribution of proteomics to the identification and determination of the structure and function of CWPs as well as discussing new perspectives in this area. The great variety of proteins found in the plant cell wall is described. Some families, such as glycoside hydrolases, proteases, lectins, and inhibitors of cell wall modifying enzymes, are discussed in detail. Examples of the use of proteomic techniques to elucidate the structure of various cell wall...

  19. Identification of Novel Cell Wall Components

    Energy Technology Data Exchange (ETDEWEB)

    Michelle Momany

    2009-10-26

    Our DOE Biosciences-funded work focused on the fungal cell wall and morphogenesis. We are especially interested in how new cell wall material is targeted to appropriate areas for polar (asymmetric) growth. Polar growth is the only way that filamentous fungi explore the environment to find suitable substrates to degrade. Work funded by this grant has resulted in a total of twenty peer-reviewed publications. In work funded by this grant, we identified nine Aspergillus nidulans temperature-sensitive (ts) mutants that fail to send out a germ tube and show a swollen cell phenotype at restrictive temperature, the swo mutants. In other organisms, a swollen cell phenotype is often associated with misdirected growth or weakened cell walls. Our work shows that several of the A. nidulans swo mutants have defects in the establishment and maintenance of polarity. Cloning of several swo genes by complementation also showed that secondary modification of proteins seems is important in polarity. We also investigated cell wall biosynthesis and branching based on leads in literature from other organisms and found that branching and nuclear division are tied and that the cell wall reorganizes during development. In our most recent work we have focused on gene expression during the shift from isotropic to polar growth. Surprisingly we found that genes previously thought to be involved only in spore formation are important in early vegetative growth as well.

  20. Function of laccases in cell wall biosynthesis

    DEFF Research Database (Denmark)

    Larsen, Anders; Holm, Preben Bach; Andersen, Jeppe Reitan

    2011-01-01

    substrate specificities and expression patterns. As part of the strategic research centre Bio4Bio, the present project deals with laccase functions in relation to cell wall formation in grasses based on a study of the model species Brachypodium distachyon. Thirty-one isozymes have been retrieved from......Laccases are multicopper oxidases capable of polymerizing monolignols. Histochemical assays have shown temporal and spatial correlation with secondary cell wall formation in both herbs and woody perennials. However, in plants laccases constitutes a relatively large group of isoenzymes with unique...... hybridization. Specific isozymes that show high correlation with the process of secondary cell wall formation will be further studied in a reverse genetic study in which candidates will be knocked out using RNA interference. Phenotypes of knock-out mutants are to be described in relation to cell wall...

  1. Staphylococcus aureus mutants lacking cell wall-bound protein A found in isolates from bacteraemia, MRSA infection and a healthy nasal carrier.

    Science.gov (United States)

    Sørum, Marit; Sangvik, Maria; Stegger, Marc; Olsen, Renate S; Johannessen, Mona; Skov, Robert; Sollid, Johanna U E

    2013-02-01

    Staphylococcus aureus is a major human pathogen and a multitude of virulence factors enables it to cause infections, from superficial lesions to life-threatening systemic conditions. Staphylococcal protein A (SpA) is a surface protein contributing to S. aureus pathogenesis by interfering with immune responses and activating inflammation. Seven isolates with frameshift mutations in the spa repeat region were investigated to determine whether these mutations lead to truncation and secretion of SpA into the extracellular environment. Five isolates originated from blood cultures, one from an MRSA infection and one from a persistent nasal carrier. Full-length spa genes from the seven isolates were sequenced, and Western blot experiments were performed to localize SpA. Three isolates had identical deviating 25-bp spa repeats, but all isolates displayed different repeat successions. The DNA sequence revealed that the frameshift mutations created premature stop codons in all seven isolates, resulting in truncated SpA of different lengths, however, all lacking the XC region with the C-terminal sorting signal. SpA was detected by Western blot in six of the seven isolates, mainly extracellularly. Our findings demonstrate that S. aureus isolates with truncated SpA, not anchored to the cell wall, can still be found in bacteraemia, infection and among carriers.

  2. An emerging role of pectic rhamnogalacturonanII for cell wall integrity.

    Science.gov (United States)

    Reboul, Rebecca; Tenhaken, Raimund

    2012-02-01

    The plant cell wall is a complex network of different polysaccharides and glycoproteins, showing high diversity in nature. The essential components, tethering cell wall are under debate, as novel mutants challenge established models. The mutant ugd2,3 with a reduced supply of the important wall precursor UDP-glucuronic acid reveals the critical role of the pectic compound rhamnogalacturonanII for cell wall stability. This polymer seems to be more important for cell wall integrity than the previously favored xyloglucan.

  3. Cell Wall Biology: Perspectives from Cell Wall Imaging

    Institute of Scientific and Technical Information of China (English)

    Kieran J.D.Lee; Susan E.Marcus; J.Paul Knox

    2011-01-01

    Polysaccharide-rich plant cell walls are important biomaterials that underpin plant growth,are major repositories for photosynthetically accumulated carbon,and,in addition,impact greatly on the human use of plants. Land plant cell walls contain in the region of a dozen major polysaccharide structures that are mostly encompassed by cellulose,hemicelluloses,and pectic polysaccharides. During the evolution of land plants,polysaccharide diversification appears to have largely involved structural elaboration and diversification within these polysaccharide groups. Cell wall chemistry is well advanced and a current phase of cell wall science is aimed at placing the complex polysaccharide chemistry in cellular contexts and developing a detailed understanding of cell wall biology. Imaging cell wall glycomes is a challenging area but recent developments in the establishment of cell wall molecular probe panels and their use in high throughput procedures are leading to rapid advances in the molecular understanding of the spatial heterogeneity of individual cell walls and also cell wall differences at taxonomic levels. The challenge now is to integrate this knowledge of cell wall heterogeneity with an understanding of the molecular and physiological mechanisms that underpin cell wall properties and functions.

  4. Interconnections between cell wall polymers, wall mechanics, and cortical microtubules: Teasing out causes and consequences.

    Science.gov (United States)

    Xiao, Chaowen; Anderson, Charles T

    2016-09-01

    In plants, cell wall components including cellulose, hemicelluloses, and pectins interact with each other to form complex extracellular network structures that control cell growth and maintain cell shape. However, it is still not clear exactly how different wall polymers interact, how the conformations and interactions of cell wall polymers relate to wall mechanics, and how these factors impinge on intracellular structures such as the cortical microtubule cytoskeleton. Here, based on studies of Arabidopsis thaliana xxt1 xxt2 mutants, which lack detectable xyloglucan in their walls and display aberrant wall mechanics, altered cellulose patterning and biosynthesis, and reduced cortical microtubule stability, we discuss the potential relationships between cell wall biosynthesis, wall mechanics, and cytoskeletal dynamics in an effort to better understand their roles in controlling plant growth and morphogenesis.

  5. 灰葡萄孢T-DNA插入细胞壁缺陷突变体的筛选%Isolation of the cell wall defect mutants by T-DNA insertion in Botrytis cinerea

    Institute of Scientific and Technical Information of China (English)

    雷娜; 张为宏; 朱廷恒; 汪琨; 崔志峰

    2011-01-01

    用荧光增白剂Calcofluor White(CFW)对234株T-DNA插入灰葡萄孢突变株进行筛选,获得了3株对CFW敏感性(B-117,B-169,D-9)和1株对CFW抗性(B-62)的突变株.1.2 mol· L- Sorbito对D-9的生长缓慢有挽救作用.在SDS培养基上D-9和野生型的差异不大,但在NaCl培养基上野生型的生长受到抑制,D-9则不受NaCl影响.在孢子萌发试验中,D-9突变株也与野生型明显不同,表现为D-9的孢子膨大、菌丝较粗、长度增加而且不弯曲.在番茄感染试验中,D-9突变株侵染番茄的毒力大幅度减弱.由此推测D-9突变株与细胞壁缺损以及致病性有关.%234 T-DNA insertion mutants of Botrytis cinerea were screened by using Calcofluor White, and three sensitive mutants(B-117, B-169, D-9)and one resistant mutant ( B-62) were obtained. In the test with isotonic PDA plates containing 1. 2 mol·L-1 sorbitol, the growth of D-9 was not affected or rescued, while the growth of wild type was suppressed. Also, the growth of wild type was suppressed by 0. 6 mol·L-1 NaCl but D-9 was not affected. No obvious difference was found between wild type and D-9 in the SDS medium. The spores of D-9 appeared swollen, its hyphae were thicker, and the length increased without bending, which was obviously different from that of wild type in the spore germination assay. In the experiment of tomato infection, the virulence of D-9 mutant was significantly reduced , almost no infection phenomenon can be observed in the period of six days. These results indicated that D-9 mutant was probably related to cell wall defect and pathogenicity.

  6. Stiff mutant genes of phycomyces affect turgor pressure and wall mechanical properties to regulate elongation growth rate.

    Science.gov (United States)

    Ortega, Joseph K E; Munoz, Cindy M; Blakley, Scott E; Truong, Jason T; Ortega, Elena L

    2012-01-01

    Regulation of cell growth is paramount to all living organisms. In plants, algae and fungi, regulation of expansive growth of cells is required for development and morphogenesis. Also, many sensory responses of stage IVb sporangiophores of Phycomyces blakesleeanus are produced by regulating elongation growth rate (growth responses) and differential elongation growth rate (tropic responses). "Stiff" mutant sporangiophores exhibit diminished tropic responses and are found to be defective in at least five genes; madD, E, F, G, and J. Prior experimental research suggests that the defective genes affect growth regulation, but this was not verified. All the growth of the single-celled stalk of the stage IVb sporangiophore occurs in a short region termed the "growth zone." Prior experimental and theoretical research indicates that elongation growth rate of the stage IVb sporangiophore can be regulated by controlling the cell wall mechanical properties within the growth zone and the magnitude of the turgor pressure. A quantitative biophysical model for elongation growth rate is required to elucidate the relationship between wall mechanical properties and turgor pressure during growth regulation. In this study, it is hypothesized that the mechanical properties of the wall within the growth zone of stiff mutant sporangiophores are different compared to wild type (WT). A biophysical equation for elongation growth rate is derived for fungal and plant cells with a growth zone. Two strains of stiff mutants are studied, C149 madD120 (-) and C216 geo- (-). Experimental results demonstrate that turgor pressure is larger but irreversible wall deformation rates within the growth zone and growth zone length are smaller for stiff mutant sporangiophores compared to WT. These findings can explain the diminished tropic responses of the stiff mutant sporangiophores. It is speculated that the defective genes affect the amount of wall-building material delivered to the inner cell wall.

  7. Mechanical Properties of Plant Cell Walls Probed by Relaxation Spectra

    DEFF Research Database (Denmark)

    Hansen, Steen Laugesen; Ray, Peter Martin; Karlsson, Anders Ola

    2011-01-01

    Transformants and mutants with altered cell wall composition are expected to display a biomechanical phenotype due to the structural role of the cell wall. It is often quite difficult, however, to distinguish the mechanical behavior of a mutant's or transformant's cell walls from that of the wild...... type. This may be due to the plant’s ability to compensate for the wall modification or because the biophysical method that is often employed, determination of simple elastic modulus and breakstrength, lacks the resolving power necessary for detecting subtle mechanical phenotypes. Here, we apply...... a method, determination of relaxation spectra, which probes, and can separate, the viscoelastic properties of different cell wall components (i.e. those properties that depend on the elastic behavior of load-bearing wall polymers combined with viscous interactions between them). A computer program, Bayes...

  8. Altered cell wall disassembly during ripening of Cnr tomato fruit : implications for cell wall adhesion and fruit softening

    NARCIS (Netherlands)

    Orfila, C.; Huisman, M.M.H.; Willats, W.G.T.; Alebeek, van G.J.W.M.; Schols, H.A.; Seymour, G.B.; Knox, J.P.

    2002-01-01

    The Cnr (Colourless non-ripening) tomato (Lycopersicon esculentum Mill.) mutant has an aberrant fruit-ripening phenotype in which fruit do not soften and have reduced cell adhesion between pericarp cells. Cell walls from Cnr fruit were analysed in order to assess the possible contribution of pectic

  9. Isolation of the Cell Wall.

    Science.gov (United States)

    Canut, Hervé; Albenne, Cécile; Jamet, Elisabeth

    2017-01-01

    This chapter describes a method allowing the purification of the cell wall for studying both polysaccharides and proteins. The plant primary cell wall is mainly composed of polysaccharides (90-95 % in mass) and of proteins (5-10 %). At the end of growth, specialized cells may synthesize a lignified secondary wall composed of polysaccharides (about 65 %) and lignin (about 35 %). Due to its composition, the cell wall is the cellular compartment having the highest density and this property is used for its purification. It plays critical roles during plant development and in response to environmental constraints. It is largely used in the food and textile industries as well as for the production of bioenergy. All these characteristics and uses explain why its study as a true cell compartment is of high interest. The proposed method of purification can be used for large amount of material but can also be downscaled to 500 mg of fresh material. Tools for checking the quality of the cell wall preparation, such as protein analysis and microscopy observation, are also provided.

  10. Catalysts of plant cell wall loosening

    OpenAIRE

    Cosgrove, Daniel J.

    2016-01-01

    The growing cell wall in plants has conflicting requirements to be strong enough to withstand the high tensile forces generated by cell turgor pressure while selectively yielding to those forces to induce wall stress relaxation, leading to water uptake and polymer movements underlying cell wall expansion. In this article, I review emerging concepts of plant primary cell wall structure, the nature of wall extensibility and the action of expansins, family-9 and -12 endoglucanases, family-16 xyl...

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

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Zhaohua PEng [Mississippi State University; Ronald, Palmela [UC-Davis; Wang, Guo-Liang [The Ohio State University

    2013-04-26

    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 each 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

  12. Shape dynamics of growing cell walls

    CERN Document Server

    Banerjee, Shiladitya; Dinner, Aaron R

    2015-01-01

    We introduce a general theoretical framework to study the shape dynamics of actively growing and remodeling surfaces. Using this framework we develop a physical model for growing bacterial cell walls and study the interplay of cell shape with the dynamics of growth and constriction. The model allows us to derive constraints on cell wall mechanical energy based on the observed dynamics of cell shape. We predict that exponential growth in cell size requires a constant amount of cell wall energy to be dissipated per unit volume. We use the model to understand and contrast growth in bacteria with different shapes such as spherical, ellipsoidal, cylindrical and toroidal morphologies. Coupling growth to cell wall constriction, we predict a discontinuous shape transformation, from partial constriction to cell division, as a function of the chemical potential driving cell-wall synthesis. Our model for cell wall energy and shape dynamics relates growth kinetics with cell geometry, and provides a unified framework to d...

  13. Polypeptone induces dramatic cell lysis in ura4 deletion mutants of fission yeast.

    Directory of Open Access Journals (Sweden)

    Yuzy Matsuo

    Full Text Available Polypeptone is widely excluded from Schizosaccharomyces pombe growth medium. However, the reasons why polypeptone should be avoided have not been documented. Polypeptone dramatically induced cell lysis in the ura4 deletion mutant when cells approached the stationary growth phase, and this phenotype was suppressed by supplementation of uracil. To determine the specificity of this cell lysis phenotype, we created deletion mutants of other genes involved in de novo biosynthesis of uridine monophosphate (ura1, ura2, ura3, and ura5. Cell lysis was not observed in these gene deletion mutants. In addition, concomitant disruption of ura1, ura2, ura3, or ura5 in the ura4 deletion mutant suppressed cell lysis, indicating that cell lysis induced by polypeptone is specific to the ura4 deletion mutant. Furthermore, cell lysis was also suppressed when the gene involved in coenzyme Q biosynthesis was deleted. This is likely because Ura3 requires coenzyme Q for its activity. The ura4 deletion mutant was sensitive to zymolyase, which mainly degrades (1,3-beta-D glucan, when grown in the presence of polypeptone, and cell lysis was suppressed by the osmotic stabiliser, sorbitol. Finally, the induction of cell lysis in the ura4 deletion mutant was due to the accumulation of orotidine-5-monophosphate. Cell wall integrity was dramatically impaired in the ura4 deletion mutant when grown in the presence of polypeptone. Because ura4 is widely used as a selection marker in S. pombe, caution needs to be taken when evaluating phenotypes of ura4 mutants.

  14. Phenotypic screening of Arabidopsis T-DNA insertion lines for cell wall mechanical properties revealed ANTHOCYANINLESS2, a cell wall-related gene.

    Science.gov (United States)

    Mabuchi, Atsushi; Soga, Kouichi; Wakabayashi, Kazuyuki; Hoson, Takayuki

    2016-02-01

    We performed a phenotypic screening of confirmed homozygous T-DNA insertion lines in Arabidopsis for cell wall extensibility, in an attempt to identify genes involved in the regulation of cell wall mechanical properties. Seedlings of each line were cultivated and the cell wall extensibility of their hypocotyls was measured with a tensile tester. Hypocotyls of lines with known cell wall-related genes showed higher or lower extensibility than those of the wild-type at high frequency, indicating that the protocol used was effective. In the first round of screening of randomly selected T-DNA insertion lines, we identified ANTHOCYANINLESS2 (ANL2), a gene involved in the regulation of cell wall mechanical properties. In the anl2 mutant, the cell wall extensibility of hypocotyls was significantly lower than that of the wild-type. Levels of cell wall polysaccharides per hypocotyl, particularly cellulose, increased in anl2. Microarray analysis showed that in anl2, expression levels of the major peroxidase genes also increased. Moreover, the activity of ionically wall-bound peroxidases clearly increased in anl2. The activation of peroxidases as well as the accumulation of cell wall polysaccharides may be involved in decreased cell wall extensibility. The approach employed in the present study could contribute to our understanding of the mechanisms underlying the regulation of cell wall mechanical properties.

  15. Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

    DEFF Research Database (Denmark)

    Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha;

    2013-01-01

    . The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco) mannan, and xyloglucan as well as overall cell wall acetylation is affected differently...

  16. Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

    Science.gov (United States)

    Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

    1990-12-01

    Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

  17. Microanalysis of Plant Cell Wall Polysaccharides

    Institute of Scientific and Technical Information of China (English)

    Nicolai Obel; Veronika Erben; Tatjana Schwarz; Stefan Kühne; Andrea Fodor; Markus Pauly

    2009-01-01

    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 iso-lating 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 apo-plastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.

  18. [The cell wall of Coelastrum (Chlorophycees)].

    Science.gov (United States)

    Reymond, O

    1975-01-01

    The cell wall of Coelastrum is usually composed of three layers. The outermost layer was studied most extensively. It consists of erect tubules which often bear long bristles whose function may be to stabilize the algae in its enviroment. The cell wall can modify its morphology according to the enviroment.

  19. Isolation of plant cell wall proteins

    OpenAIRE

    Jamet, Elisabeth; Boudart, Georges; Borderies, Gisèle; Charmont, Stéphane; Lafitte, Claude; Rossignol, Michel; Canut, Hervé; Pont-Lezica, Rafael F

    2007-01-01

    The quality of a proteomic analysis of a cell compartment strongly depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific drawbacks: (i) the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP) during the isolation procedure; (ii) polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins; (iii) the presence of proteins ...

  20. Isolation of plant cell wall proteins.

    Science.gov (United States)

    Jamet, Elisabeth; Boudart, Georges; Borderies, Giséle; Charmont, Stephane; Lafitte, Claude; Rossignol, Michel; Canut, Herve; Pont-Lezica, Rafael

    2008-01-01

    The quality of a proteomic analysis of a cell compartment strongly depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific drawbacks: (1) the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP) during the isolation procedure; (2) polysaccharide networks of cellulose, hemicelluloses, and pectins form potential traps for contaminants such as intracellular proteins; (3) the presence of proteins interacting in many different ways with the polysaccharide matrix require different procedures to elute them from the cell wall. Three categories of CWP are distinguished: labile proteins that have little or no interactions with cell wall components, weakly bound proteins extractable with salts, and strongly bound proteins. Two alternative protocols are decribed for cell wall proteomics: (1) nondestructive techniques allowing the extraction of labile or weakly bound CWP without damaging the plasma membrane; (2) destructive techniques to isolate cell walls from which weakly or strongly bound CWP can be extracted. These protocols give very low levels of contamination by intracellular proteins. Their application should lead to a realistic view of the cell wall proteome at least for labile and weakly bound CWP extractable by salts.

  1. Induced mutants from dihaploid potatoes after pollen mother cell treatment.

    Science.gov (United States)

    Przewoźny, T; Schieder, O; Wenzel, G

    1980-05-01

    Microspore mother cells of dihaploid Solanum tuberosum plants were mutagenically treated during the stage of meiosis. Mutagenesis was performed either by irradiation with x- or γ-rays or by the application of nitrosomethylurethane or methylnitronitrosoguanidine. Then, by use of the anther culture technique, 913 functional plants and 442 untreated control plants were regenerated. From the exposed plants seven distinct mutants could be isolated, predominantly chlorophyll deficient lines, while from the controls no clear-cut mutants arose. One mutant turned out to be photomorphogenetic in addition to having a chlorophyll defect. In addition to the production of mutants the treatments significantly increased the frequency of multicellular structure formation from microspores.

  2. Accelerating forward genetics for cell wall deconstruction

    Directory of Open Access Journals (Sweden)

    Danielle eVidaurre

    2012-06-01

    Full Text Available One of the biggest challenges of cell wall biology is the elucidation of the genes involved the cell wall and their function due to the recalcitrance of the cell wall. Through traditional genetic approaches, many simple yet elegant screens have been able to identify components of the cell wall and their networks. Despite progress in the identification of several genes of the cell wall, there remain many unknown players whose function has yet to be determined. Exhausting the genetic toolbox by performing secondary screens on a genetically mutated background, chemical genetics using small molecules and improved cell wall imaging hold promise for new gene discovery and function. With the recent introduction of next-generation sequencing technologies, it is now possible to quickly and efficiently map and clone genes of interest in Arabidopsis and any model organism with a completed genome sequence. The combination of a classical genetics approach and cutting edge technology will propel cell wall biology of Arabidopsis and other useful crops forward into the future.

  3. Recent advances in plant cell wall proteomics.

    Science.gov (United States)

    Jamet, Elisabeth; Albenne, Cécile; Boudart, Georges; Irshad, Muhammad; Canut, Hervé; Pont-Lezica, Rafael

    2008-02-01

    The plant extracellular matrix contains typical polysaccharides such as cellulose, hemicelluloses, and pectins that interact to form dense interwoven networks. Plant cell walls play crucial roles during development and constitute the first barrier of defense against invading pathogens. Cell wall proteomics has greatly contributed to the description of the protein content of a compartment specific to plants. Around 400 cell wall proteins (CWPs) of Arabidopsis, representing about one fourth of its estimated cell wall proteome, have been described. The main points to note are that: (i) the diversity of enzymes acting on polysaccharides suggests a great plasticity of cell walls; (ii) CWPs such as proteases, polysaccharide hydrolytic enzymes, and lipases may contribute to the generation of signals; (iii) proteins of unknown functions were identified, suggesting new roles for cell walls. Recently, the characterization of PTMs such as N- and O-glycosylations improved our knowledge of CWP structure. The presence of many glycoside hydrolases and proteases suggests a complex regulation of CWPs involving various types of post-translational events. The first 3-D structures to be resolved gave clues about the interactions between CWPs, or between CWPs and polysaccharides. Future work should include: extracting and identifying CWPs still recalcitrant to proteomics, describing the cell wall interactome, improving quantification, and unraveling the roles of each of the CWPs.

  4. Molecular regulation of plant cell wall extensibility

    Science.gov (United States)

    Cosgrove, D. J.

    1998-01-01

    Gravity responses in plants often involve spatial and temporal changes in cell growth, which is regulated primarily by controlling the ability of the cell wall to extend. The wall is thought to be a cellulose-hemicellulose network embedded in a hydrated matrix of complex polysaccharides and a small amount of structural protein. The wall extends by a form of polymer creep, which is mediated by expansins, a novel group of wall-loosening proteins. Expansins were discovered during a molecular dissection of the "acid growth" behavior of cell walls. Expansin alters the rheology of plant walls in profound ways, yet its molecular mechanism of action is still uncertain. It lacks detectable hydrolytic activity against the major components of the wall, but it is able to disrupt noncovalent adhesion between wall polysaccharides. The discovery of a second family of expansins (beta-expansins) sheds light on the biological role of a major group of pollen allergens and implies that expansins have evolved for diverse developmental functions. Finally, the contribution of other processes to wall extensibility is briefly summarized.

  5. 2003 Plant Cell Walls Gordon Conference

    Energy Technology Data Exchange (ETDEWEB)

    Daniel J. Cosgrove

    2004-09-21

    This conference will address recent progress in many aspects of cell wall biology. Molecular, genetic, and genomic approaches are yielding major advances in our understanding of the composition, synthesis, and architecture of plant cell walls and their dynamics during growth, and are identifying the genes that encode the machinery needed to make their biogenesis possible. This meeting will bring together international scientists from academia, industry and government labs to share the latest breakthroughs and perspectives on polysaccharide biosynthesis, wood formation, wall modification, expansion and interaction with other organisms, and genomic & evolutionary analyses of wall-related genes, as well as to discuss recent ''nanotechnological'' advances that take wall analysis to the level of a single cell.

  6. Regulation of plant cells, cell walls and development by mechanical signals

    Energy Technology Data Exchange (ETDEWEB)

    Meyerowitz, Elliot M. [California Inst. of Technology (CalTech), Pasadena, CA (United States)

    2016-06-14

    The overall goal of the revised scope of work for the final year of funding was to characterize cell wall biosynthesis in developing cotyledons and in the shoot apical meristem of Arabidopsis thaliana, as a way of learning about developmental control of cell wall biosynthesis in plants, and interactions between cell wall biosynthesis and the microtubule cytoskeleton. The proposed work had two parts – to look at the effect of mutation in the SPIRAL2 gene on microtubule organization and reorganization, and to thoroughly characterize the glycosyltransferase genes expressed in shoot apical meristems by RNA-seq experiments, by in situ hybridization of the RNAs expressed in the meristem, and by antibody staining of the products of the glycosyltransferases in meristems. Both parts were completed; the spiral2 mutant was found to speed microtubule reorientation after ablation of adjacent cells, supporting our hypothesis that reorganization correlates with microtubule severing, the rate of which is increased by the mutation. The glycosyltransferase characterization was completed and published as Yang et al. (2016). Among the new things learned was that primary cell wall biosynthesis is strongly controlled both by cell type, and by stage of cell cycle, implying not only that different, even adjacent, cells can have different sugar linkages in their (nonshared) walls, but also that a surprisingly large proportion of glycosyltransferases is regulated in the cell cycle, and therefore that the cell cycle regulates wall maturation to a degree previously unrecognized.

  7. Cell wall proteins: a new insight through proteomics.

    Science.gov (United States)

    Jamet, Elisabeth; Canut, Hervé; Boudart, Georges; Pont-Lezica, Rafael F

    2006-01-01

    Cell wall proteins are essential constituents of plant cell walls; they are involved in modifications of cell wall components, wall structure, signaling and interactions with plasma membrane proteins at the cell surface. The application of proteomic approaches to the cell wall compartment raises important questions: are there technical problems specific to cell wall proteomics? What kinds of proteins can be found in Arabidopsis walls? Are some of them unexpected? What sort of post-translational modifications have been characterized in cell wall proteins to date? The purpose of this review is to discuss the experimental results obtained to date using proteomics, as well as some of the new questions challenging future research.

  8. Residence-time dependent cell wall deformation of different Staphylococcus aureus strains on gold measured using surface-enhanced-fluorescence

    NARCIS (Netherlands)

    Li, Jiuyi; Busscher, Henk J.; Swartjes, Jan J. T. M.; Chen, Yun; Harapanahalli, Akshay K.; Norde, Willem; van der Mei, Henny C.; Sjollema, Jelmer

    2014-01-01

    Bacterial adhesion to surfaces is accompanied by cell wall deformation that may extend to the lipid membrane with an impact on the antimicrobial susceptibility of the organisms. Nanoscale cell wall deformation upon adhesion is difficult to measure, except for Delta pbp4 mutants, deficient in peptido

  9. Modes of deformation of walled cells.

    Science.gov (United States)

    Dumais, Jacques

    2013-11-01

    The bewildering morphological diversity found in cells is one of the starkest illustrations of life's ability to self-organize. Yet the morphogenetic mechanisms that produce the multifarious shapes of cells are still poorly understood. The shared similarities between the walled cells of prokaryotes, many protists, fungi, and plants make these groups particularly appealing to begin investigating how morphological diversity is generated at the cell level. In this review, I attempt a first classification of the different modes of surface deformation used by walled cells. Five modes of deformation were identified: inextensional bending, equi-area shear, elastic stretching, processive intussusception, and chemorheological growth. The two most restrictive modes-inextensional and equi-area deformations-are embodied in the exine of pollen grains and the wall-like pellicle of euglenoids, respectively. For these modes, it is possible to express the deformed geometry of the cell explicitly in terms of the undeformed geometry and other easily observable geometrical parameters. The greatest morphogenetic power is reached with the processive intussusception and chemorheological growth mechanisms that underlie the expansive growth of walled cells. A comparison of these two growth mechanisms suggests a possible way to tackle the complexity behind wall growth.

  10. Mutant alpha-synuclein and autophagy in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Kangyong Liu; Chunfeng Liu; Chuancheng Ren; Yaping Yang; Liwei Shen; Xuezhong Li; Fen Wang; Zhenghong Qin

    2011-01-01

    Several studies have demonstrated that overexpression of mutant α-synuclein in PC12 cells is related to occurrence of autophagy.The present study established mutant a-synuclein (A30P)-transfected PC12 cells and treated them with the autophagy inducer rapamycin and autophagy inhibitor wortmannin, respectively.Results demonstrated that mutant o-synuclein resulted in cell death via autophagy and involved α-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity.Mutant α-synuclein (A30P) also mediated toxicity of1-methyl-4-phenylpyridinium ion.Moreover, rapamycin inhibited a-synuclein aggregation, while wortmannin promoted o-synuclein aggregation and cell death.To further determine the role of autophagy due to mutant a-synuclein, the present study measured expression of microtubule-associated protein light chain 3.Results revealed that wortmannin and 1-methyl-4-phenylpyridinium ion inhibited expression of microtubule-associated protein light chain 3,while rapamycin promoted its expression.These findings suggested that abnormal aggregation of a-synuclein induced autophagic programmed cell death in PC12 cells.

  11. "Steiner trees" between cell walls of sisal

    Institute of Scientific and Technical Information of China (English)

    LI GuanShi; YIN YaJun; LI Yan; ZHONG Zheng

    2009-01-01

    Through careful analysis on the cross-section of sisal fibers,it is found that the middle lamellae between the cell walls have clear geometric characteristics:between the cell walls of three neighboring cells,the middle lamellae form a three-way junction with 120°symmetry. If the neighboring three-way junctions are connected,a network of Steiner tree with angular symmetry and topological invariability is formed. If more and more Steiner trees are connected,a network of Steiner rings is generated. In another word,idealized cell walls and the middle lamellae are dominated by the Steiner geometry. This geometry not only depicts the geometric symmetry,the topological invariability and minimal property of the middle lamellae,but also controls the mechanics of sisal fibers.

  12. Anhydrobiosis in yeast: cell wall mannoproteins are important for yeast Saccharomyces cerevisiae resistance to dehydration.

    Science.gov (United States)

    Borovikova, Diana; Teparić, Renata; Mrša, Vladimir; Rapoport, Alexander

    2016-08-01

    The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms' viability in these conditions have been revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to the cell wall, and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wild-type strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment; this phenomenon is explained by the compensatory synthesis of chitin. The results clearly indicate that the cell wall structure/composition relates to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage to cell wall proteins during yeast desiccation can be an important factor leading to cell death. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Antisense downregulation of mutant huntingtin in a cell model

    DEFF Research Database (Denmark)

    Hasholt, L.; Abell, K.; Norremolle, A.;

    2003-01-01

    of specific neurons in the brains of HD patients correlate with the expression of mutant huntingtin. Therefore, we have studied whether mutant huntingtin expression can be downregulated by antisense technique. Methods NT2 precursor cells and differentiated postmitotic NT2-N neurons, respectively, were...... transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection...... or by addition to the culture medium. Results Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression...

  14. Nanoformulated cell-penetrating survivin mutant and its dual actions

    Directory of Open Access Journals (Sweden)

    Sriramoju B

    2014-07-01

    Full Text Available Bhasker Sriramoju, Rupinder K Kanwar, Jagat R Kanwar Nanomedicine Laboratory of Immunology and Molecular Biomedical Research (NLIMBR, School of Medicine, Faculty of Health, Deakin University, Geelong, Australia Abstract: In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells, and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome. Keywords: survivin mutant, neurological disorders, protein therapeutics, inhibitor of apoptosis protein family, poly(lactic-co-glycolic acid

  15. Atkinesin-13A modulates cell-wall synthesis and cell expansion in Arabidopsis thaliana via the THESEUS1 pathway.

    Directory of Open Access Journals (Sweden)

    Ushio Fujikura

    2014-09-01

    Full Text Available Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate here that the internal-motor kinesin AtKINESIN-13A (AtKIN13A limits cell expansion and cell size in Arabidopsis thaliana, with loss-of-function atkin13a mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of the two genes. AtKIN13A is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent with this function, AtKIN13A interacts genetically with ANGUSTIFOLIA, encoding a regulator of Golgi dynamics. Reduced AtKIN13A activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling via the THESEUS1-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the atkin13a mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion.

  16. Cell wall oxalate oxidase modifies the ferulate metabolism in cell walls of wheat shoots.

    Science.gov (United States)

    Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki

    2011-11-01

    Oxalate oxidase (OXO) utilizes oxalate to generate hydrogen peroxide, and thereby acts as a source of hydrogen peroxide. The present study was carried out to investigate whether apoplastic OXO modifies the metabolism of cell wall-bound ferulates in wheat seedlings. Histochemical staining of OXO showed that cell walls were strongly stained, indicating the presence of OXO activity in shoot walls. When native cell walls prepared from shoots were incubated with oxalate or hydrogen peroxide, the levels of ester-linked diferulic acid (DFA) isomers were significantly increased. On the other hand, the level of ester-linked ferulic acid (FA) was substantially decreased. The decrease in FA level was accounted neither by the increases in DFA levels nor by the release of FA from cell walls during the incubation. After the extraction of ester-linked ferulates, considerable ultraviolet absorption remained in the hemicellulosic and cellulose fractions, which was increased by the treatment with oxalate or hydrogen peroxide. Therefore, a part of FA esters may form tight linkages within cell wall architecture. These results suggest that cell wall OXO is capable of modifying the metabolism of ester-linked ferulates in cell walls of wheat shoots by promoting the peroxidase action via supply of hydrogen peroxide.

  17. Enzymatic Modification of Plant Cell Wall Polysaccharides

    DEFF Research Database (Denmark)

    Øbro, Jens; Hayashi, Takahisa; Mikkelsen, Jørn Dalgaard

    2011-01-01

    fibres, hydrocolloids, paper,textile, animal feeds or biofuels. Classical microbial-based fermentation systems could in the future face serious competition from plant-based expression systems for enzyme production. Plant expressed enzymes can either be targeted to specific cellular compartments......Plant cell walls are intricate structures with remarkable properties, widely used in almost every aspect of our life. Cell walls consist largely of complex polysaccharides and there is often a need for chemical and biochemical processing before industrial use. There is an increasing demand...... for sustainable processes that replace chemical treatments with white biotechnology. Plants can contribute significantly to this sustainable process by producing plant or microbialenzymes in planta that are necessary for plant cell wall modification or total degradation. This will give rise to superior food...

  18. Modeling dynamics of mutants in heterogeneous stem cell niche

    Science.gov (United States)

    Shahriyari, L.; Mahdipour-Shirayeh, A.

    2017-02-01

    Studying the stem cell (SC) niche architecture is a crucial step for investigating the process of oncogenesis and obtaining an effective stem cell therapy for various cancers. Recently, it has been observed that there are two groups of SCs in the SC niche collaborating with each other to maintain tissue homeostasis: border stem cells (BSCs), which are responsible in controlling the number of non-stem cells as well as stem cells, and central stem cells (CeSCs), which regulate the SC niche. Here, we develop a bi-compartmental stochastic model for the SC niche to study the spread of mutants within the niche. The analytic calculations and numeric simulations, which are in perfect agreement, reveal that in order to delay the spread of mutants in the SC niche, a small but non-zero number of SC proliferations must occur in the CeSC compartment. Moreover, the migration of BSCs to CeSCs delays the spread of mutants. Furthermore, the fixation probability of mutants in the SC niche is independent of types of SC division as long as all SCs do not divide fully asymmetrically. Additionally, the progeny of CeSCs have a much higher chance than the progeny of BSCs to take over the entire niche.

  19. Arabidopsis Heterotrimeric G-protein Regulates Cell Wall Defense and Resistance to Necrotrophic Fungi

    Institute of Scientific and Technical Information of China (English)

    Magdalena Delcado-Cerezo; Paul Schulze-Lefert; Shauna Somerville; José Manuel Estevez; Staffan Persson; Antonio Molina; Clara Sánchez-Rodríguez; Viviana Escudero; Eva Miedes; Paula Virginia Fernández; Lucía Jordá; Camilo Hernández-Blanco; Andrea Sánchez-Vallet; Pawel Bednarek

    2012-01-01

    The Arabidopsis heterotrimeric G-protein controls defense responses to necrotrophic and vascular fungi.The agb1 mutant impaired in the Gβ subunit displays enhanced susceptibility to these pathogens.Gβ/AGB1 forms an obligate dimer with either one of the Arabidopsis Gγ subunits (γ1/AGG1 and γ2/AGG2).Accordingly,we now demonstrate that the agg1 agg2 double mutant is as susceptible as agb1 plants to the necrotrophic fungus Plectosphaerella cucumerina.To elucidate the molecular basis of heterotrimeric G-protein-mediated resistance,we performed a comparative transcriptomic analysis of agb1-1 mutant and wild-type plants upon inoculation with P cucumerina.This analysis,together with metabolomic studies,demonstrated that G-protein-mediated resistance was independent of defensive pathways required for resistance to necrotrophic fungi,such as the salicylic acid,jasmonic acid,ethylene,abscisic acid,and tryptophan-derived metabolites signaling,as these pathways were not impaired in agb1 and agg1 agg2 mutants.Notably,many mis-regulated genes in agb1 plants were related with cell wall functions,which was also the case in agg1 agg2 mutant.Biochemical analyses and Fourier Transform InfraRed (FTIR) spectroscopy of cell walls from G-protein mutants revealed that the xylose content was lower in agb1 and agg1 agg2 mutants than in wild-type plants,and that mutant walls had similar FTIR spectratypes,which differed from that of wild-type plants.The data presented here suggest a canonical functionality of the Gβ and Gγ1/γ2 subunits in the control of Arabidopsis immune responses and the regulation of cell wall composition.

  20. Xyloglucan endotransglucosylase and cell wall extensibility.

    Science.gov (United States)

    Miedes, E; Zarra, I; Hoson, T; Herbers, K; Sonnewald, U; Lorences, E P

    2011-02-15

    Transgenic tomato hypocotyls with altered levels of an XTH gene were used to study how XET activity could affect the hypocotyl growth and cell wall extensibility. Transgenic hypocotyls showed significant over-expression (line 13) or co-suppression (line 33) of the SlXTH1 in comparison with the wild type, with these results being correlated with the results on specific soluble XET activity, suggesting that SlXTH1 translates mainly for a soluble XET isoenzyme. A relationship between XET activity and cell wall extensibility was found, and the highest total extensibility was located in the apical hypocotyl segment of the over-expressing SlXTH1 line, where the XET-specific activity and hypocotyl growth were also highest compared with the wild line. Also, in the co-suppression SlXTH1 line, total extensibility values were lower than in the wild type line. The study of linkages between cell wall polysaccharides by FTIR showed that hypocotyls over-expressing SlXTH1 and having a higher XET-specific activity, were grouped away from the wild line, indicating that the linkages between pectins and between cellulose and xyloglucans might differ. These results suggested that the action of the increased XET activity in the transgenic line could be responsible for the cell wall structural changes, and therefore, alter the cell wall extensibility. On the other hand, results on xyloglucan oligosaccharides composition of the xyloglucan by MALDI TOF-MS showed no differences between lines, indicating that the xyloglucan structure was not affected by the XET action. These results provide evidences that XTHs from group I are involved mainly in the restructuring of the cell wall during growth and development, but they are not the limiting factor for plant growth.

  1. The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans

    Directory of Open Access Journals (Sweden)

    Girolamo Antonietta

    2011-05-01

    Full Text Available Abstract Background The MP65 gene of Candida albicans (orf19.1779 encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation. Results The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p, a high level of expression of two stress-related genes (DDR48 and SOD5, and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion. Conclusions We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

  2. Participation of Candida albicans transcription factor RLM1 in cell wall biogenesis and virulence.

    Science.gov (United States)

    Delgado-Silva, Yolanda; Vaz, Catarina; Carvalho-Pereira, Joana; Carneiro, Catarina; Nogueira, Eugénia; Correia, Alexandra; Carreto, Laura; Silva, Sónia; Faustino, Augusto; Pais, Célia; Oliveira, Rui; Sampaio, Paula

    2014-01-01

    Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources.

  3. Participation of Candida albicans transcription factor RLM1 in cell wall biogenesis and virulence.

    Directory of Open Access Journals (Sweden)

    Yolanda Delgado-Silva

    Full Text Available Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol, confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213% and reduction in mannans (60%, in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources.

  4. Altered cell wall disassembly during ripening of Cnr tomato fruit: implications for cell adhesion and fruit softening

    DEFF Research Database (Denmark)

    Orfila, C.; Huisman, M.M.H.; Willats, William George Tycho;

    2002-01-01

    The Cnr (Colourless non-ripening) tomato (Lycopersicon esculentum Mill.) mutant has an aberrant fruit-ripening phenotype in which fruit do not soften and have reduced cell adhesion between pericarp cells. Cell walls from Cnr fruit were analysed in order to assess the possible contribution of pectic...... polysaccharides to the non-softening and altered cell adhesion phenotype. Cell wall material (CWM) and solubilised fractions of mature green and red ripe fruit were analysed by chemical, enzymatic and immunochemical techniques. No major differences in CWM sugar composition were detected although differences were...... that was chelator-soluble was 50% less in Cnr cell walls at both the mature green and red ripe stages. Chelator-soluble material from ripe-stage Cnr was more susceptible to endo-polygalacturonase degradation than the corresponding material from wild-type fruit. In addition, cell walls from Cnr fruit contained...

  5. Metabolic reprogramming in mutant IDH1 glioma cells.

    Directory of Open Access Journals (Sweden)

    Jose L Izquierdo-Garcia

    Full Text Available Mutations in isocitrate dehydrogenase (IDH 1 have been reported in over 70% of low-grade gliomas and secondary glioblastomas. IDH1 is the enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate while mutant IDH1 catalyzes the conversion of α-ketoglutarate into 2-hydroxyglutarate. These mutations are associated with the accumulation of 2-hydroxyglutarate within the tumor and are believed to be one of the earliest events in the development of low-grade gliomas. The goal of this work was to determine whether the IDH1 mutation leads to additional magnetic resonance spectroscopy (MRS-detectable changes in the cellular metabolome.Two genetically engineered cell models were investigated, a U87-based model and an E6/E7/hTERT immortalized normal human astrocyte (NHA-based model. For both models, wild-type IDH1 cells were generated by transduction with a lentiviral vector coding for the wild-type IDH1 gene while mutant IDH1 cells were generated by transduction with a lentiviral vector coding for the R132H IDH1 mutant gene. Metabolites were extracted from the cells using the dual-phase extraction method and analyzed by 1H-MRS. Principal Component Analysis was used to analyze the MRS data.Principal Component Analysis clearly discriminated between wild-type and mutant IDH1 cells. Analysis of the loading plots revealed significant metabolic changes associated with the IDH1 mutation. Specifically, a significant drop in the concentration of glutamate, lactate and phosphocholine as well as the expected elevation in 2-hydroxyglutarate were observed in mutant IDH1 cells when compared to their wild-type counterparts.The IDH1 mutation leads to several, potentially translatable MRS-detectable metabolic changes beyond the production of 2-hydroxyglutarate.

  6. Neural network analyses of infrared spectra for classifying cell wall architectures.

    Science.gov (United States)

    McCann, Maureen C; Defernez, Marianne; Urbanowicz, Breeanna R; Tewari, Jagdish C; Langewisch, Tiffany; Olek, Anna; Wells, Brian; Wilson, Reginald H; Carpita, Nicholas C

    2007-03-01

    About 10% of plant genomes are devoted to cell wall biogenesis. Our goal is to establish methodologies that identify and classify cell wall phenotypes of mutants on a genome-wide scale. Toward this goal, we have used a model system, the elongating maize (Zea mays) coleoptile system, in which cell wall changes are well characterized, to develop a paradigm for classification of a comprehensive range of cell wall architectures altered during development, by environmental perturbation, or by mutation. Dynamic changes in cell walls of etiolated maize coleoptiles, sampled at one-half-d intervals of growth, were analyzed by chemical and enzymatic assays and Fourier transform infrared spectroscopy. The primary walls of grasses are composed of cellulose microfibrils, glucuronoarabinoxylans, and mixed-linkage (1 --> 3),(1 --> 4)-beta-D-glucans, together with smaller amounts of glucomannans, xyloglucans, pectins, and a network of polyphenolic substances. During coleoptile development, changes in cell wall composition included a transient appearance of the (1 --> 3),(1 --> 4)-beta-D-glucans, a gradual loss of arabinose from glucuronoarabinoxylans, and an increase in the relative proportion of cellulose. Infrared spectra reflected these dynamic changes in composition. Although infrared spectra of walls from embryonic, elongating, and senescent coleoptiles were broadly discriminated from each other by exploratory principal components analysis, neural network algorithms (both genetic and Kohonen) could correctly classify infrared spectra from cell walls harvested from individuals differing at one-half-d interval of growth. We tested the predictive capabilities of the model with a maize inbred line, Wisconsin 22, and found it to be accurate in classifying cell walls representing developmental stage. The ability of artificial neural networks to classify infrared spectra from cell walls provides a means to identify many possible classes of cell wall phenotypes. This classification

  7. Immuno and affinity cytochemical analysis of cell wall composition in the moss Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Berry

    2016-03-01

    Full Text Available In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalacturonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogeneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants.

  8. Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens.

    Science.gov (United States)

    Berry, Elizabeth A; Tran, Mai L; Dimos, Christos S; Budziszek, Michael J; Scavuzzo-Duggan, Tess R; Roberts, Alison W

    2016-01-01

    In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants.

  9. Cell Wall Diversity in Forage Maize

    NARCIS (Netherlands)

    Torres, A.F.; Noordam-Boot, C.M.M.; Dolstra, Oene; Weijde, van der Tim; Combes, Eliette; Dufour, Philippe; Vlaswinkel, Louis; Visser, R.G.F.; Trindade, L.M.

    2015-01-01

    Genetic studies are ideal platforms for assessing the extent of genetic diversity, inferring the genetic architecture, and evaluating complex trait interrelations for cell wall compositional and bioconversion traits relevant to bioenergy applications. Through the characterization of a forage maiz

  10. Microanalysis of Plant Cell Wall Polysaccharides

    NARCIS (Netherlands)

    Obel, N.; Erben, V.; Schwarz, T.; Kühnel, S.; Fodor, A.; Pauly, M.

    2009-01-01

    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 s

  11. Cell Wall Heterogeneity in Root Development of Arabidopsis

    Science.gov (United States)

    Somssich, Marc; Khan, Ghazanfar Abbas; Persson, Staffan

    2016-01-01

    Plant cell walls provide stability and protection to plant cells. During growth and development the composition of cell walls changes, but provides enough strength to withstand the turgor of the cells. Hence, cell walls are highly flexible and diverse in nature. These characteristics are important during root growth, as plant roots consist of radial patterns of cells that have diverse functions and that are at different developmental stages along the growth axis. Young stem cell daughters undergo a series of rapid cell divisions, during which new cell walls are formed that are highly dynamic, and that support rapid anisotropic cell expansion. Once the cells have differentiated, the walls of specific cell types need to comply with and support different cell functions. For example, a newly formed root hair needs to be able to break through the surrounding soil, while endodermal cells modify their walls at distinct positions to form Casparian strips between them. Hence, the cell walls are modified and rebuilt while cells transit through different developmental stages. In addition, the cell walls of roots readjust to their environment to support growth and to maximize nutrient uptake. Many of these modifications are likely driven by different developmental and stress signaling pathways. However, our understanding of how such pathways affect cell wall modifications and what enzymes are involved remain largely unknown. In this review we aim to compile data linking cell wall content and re-modeling to developmental stages of root cells, and dissect how root cell walls respond to certain environmental changes. PMID:27582757

  12. Induced mutations in tomato SlExp1 alter cell wall metabolism and delay fruit softening.

    Science.gov (United States)

    Minoia, Silvia; Boualem, Adnane; Marcel, Fabien; Troadec, Christelle; Quemener, Bernard; Cellini, Francesco; Petrozza, Angelo; Vigouroux, Jacqueline; Lahaye, Marc; Carriero, Filomena; Bendahmane, Abdelhafid

    2016-01-01

    Fruit ripening and softening are key traits for many fleshy fruit. Since cell walls play a key role in the softening process, expansins have been investigated to control fruit over ripening and deterioration. In tomato, expression of Expansin 1 gene, SlExp1, during fruit ripening was associated with fruit softening. To engineer tomato plants with long shelf life, we screened for mutant plants impaired in SlExp1 function. Characterization of two induced mutations, Slexp1-6_W211S, and Slexp1-7_Q213Stop, showed that SlExp1 loss of function leads to enhanced fruit firmness and delayed fruit ripening. Analysis of cell wall polysaccharide composition of Slexp1-7_Q213Stop mutant pointed out significant differences for uronic acid, neutral sugar and total sugar contents. Hemicelluloses chemistry analysis by endo-β-1,4-d-glucanase hydrolysis and MALDI-TOF spectrometry revealed that xyloglucan structures were affected in the fruit pericarp of Slexp1-7_Q213Stop mutant. Altogether, these results demonstrated that SlExp1 loss of function mutants yield firmer and late ripening fruits through modification of hemicellulose structure. These SlExp1 mutants represent good tools for breeding long shelf life tomato lines with contrasted fruit texture as well as for the understanding of the cell wall polysaccharide assembly dynamics in fleshy fruits.

  13. Arrangement of peptidoglycan in the cell wall of Staphylococcus spp.

    OpenAIRE

    Amako, K.; Umeda, A; Murata, K

    1982-01-01

    The arrangement of peptidoglycan in the cell wall of Staphylococcus was observed with the newly developed freeze-fracture technique, using n-octanol instead of water as the freezing medium. The replica of the trichloroacetic acid-extracted cell wall (TCA-wall) showed two areas. One of them has a concentric circular structure, a characteristic surface structure of the staphylococcal cell wall, and the other showed an irregular and rough surface. The chemical analysis of the wall revealed that ...

  14. Genetic modification of plant cell walls to enhance biomass yield and biofuel production in bioenergy crops.

    Science.gov (United States)

    Wang, Yanting; Fan, Chunfen; Hu, Huizhen; Li, Ying; Sun, Dan; Wang, Youmei; Peng, Liangcai

    2016-01-01

    Plant cell walls represent an enormous biomass resource for the generation of biofuels and chemicals. As lignocellulose property principally determines biomass recalcitrance, the genetic modification of plant cell walls has been posed as a powerful solution. Here, we review recent progress in understanding the effects of distinct cell wall polymers (cellulose, hemicelluloses, lignin, pectin, wall proteins) on the enzymatic digestibility of biomass under various physical and chemical pretreatments in herbaceous grasses, major agronomic crops and fast-growing trees. We also compare the main factors of wall polymer features, including cellulose crystallinity (CrI), hemicellulosic Xyl/Ara ratio, monolignol proportion and uronic acid level. Furthermore, the review presents the main gene candidates, such as CesA, GH9, GH10, GT61, GT43 etc., for potential genetic cell wall modification towards enhancing both biomass yield and enzymatic saccharification in genetic mutants and transgenic plants. Regarding cell wall modification, it proposes a novel groove-like cell wall model that highlights to increase amorphous regions (density and depth) of the native cellulose microfibrils, providing a general strategy for bioenergy crop breeding and biofuel processing technology.

  15. Plant Cell Wall Matrix Polysaccharide Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Ajay Pal S. Sandhu; Gursharn S. Randhawa; Kanwarpal S. Dhugga

    2009-01-01

    The wall of an expanding plant cell consists primarily of cellulose microfibrils embedded in a matrix of hemi-cellulosic and pectic polysaccharides along with small amounts of structural and enzymatic proteins. Matrix polysacchar-ides are synthesized in the Golgi and exported to the cell wall by exocytosis, where they intercalate among cellulose microfibrUs, which are made at the plasma membrane and directly deposited into the cell wall. Involvement of Golgi glucan synthesis in auxin-induced cell expansion has long been recognized; however, only recently have the genes corresponding to glucan synthases been identified. Biochemical purification was unsuccessful because of the labile nature and very low abundance of these enzymes. Mutational genetics also proved fruitless. Expression of candidate genes identified through gene expression profiling or comparative genomics in heterologous systems followed by functional characterization has been relatively successful. Several genes from the cellulose synthase-like (Cs/) family have been found to be involved in the synthesis of various hemicellulosic glycans. The usefulness of this approach, however, is limited to those enzymes that probably do not form complexes consisting of unrelated proteins. Nonconventional approaches will continue to incre-mentally unravel the mechanisms of Golgi polysaccharide biosynthesis.

  16. Alfalfa stem tissues: Cell wall deposition, composition, and degradability

    NARCIS (Netherlands)

    Jung, H.G.; Engels, F.M.

    2002-01-01

    Declining cell wall degradability of alfalfa (Medicago sativa L.) stems with maturation limits the nutritional value of alfalfa for ruminants. This study characterized changes in cell wall concentration, composition, and degradability by rumen microbes resulting from alfalfa stem tissue proliferatio

  17. [Structure and function of fungal cell wall].

    Science.gov (United States)

    Ohno, Naohito

    2008-12-01

    Cell wall glycans of fungi/yeasts are reviewed. Fungi/yeasts produce various kinds of polysaccharides. As part of the cell wall they are interlinked with other components forming a huge network. The insolubility and complex with multiple components makes the research very tough. Studies on beta-glucan have been performed from various views, such as chemistry, conformation, solubility, tissue distribution and metabolism, biological activity, clinical application, receptor, biosynthesis, and antibody. Studies on mannan focus on immunotoxicity, such as anaphylactoid reaction and coronary arteritis induction. alpha-glucan, chitin, and capsular polysaccharide were also mentioned in relation to structure and genes. Compared with human and animal polysaccharides, fungi/yeasts polysaccharides have very characteristic properties.

  18. An ethanolamine kinase Eki1 affects radial growth and cell wall integrity in Trichoderma reesei.

    Science.gov (United States)

    He, Ronglin; Guo, Wei; Zhang, Dongyuan

    2015-09-01

    Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1.82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The functions of eki genes that encode ethanolamine kinase have been intensively studied in mammalian cells, fruit flies and yeast. However, the role of the eki gene has not yet been characterized in filamentous fungi. In this study, Treki1, an ortholog of Saccharomyces cerevisiae EKI1, was identified and functionally characterized using a target gene deletion strategy in Trichoderma reesei. A Treki deletion mutant was less sensitive to cell wall stressors calcofluor white and Congo red and released fewer protoplasts during cell wall digestion than the parent strain QM9414. Further transcription analysis showed that the expression levels of five genes that encode chitin synthases were drastically increased in the ΔTreki1 mutant. The chitin content was also increased in the null mutant of Treki1 comparing to the parent strain. In addition, the ΔTreki1 mutant exhibited defects in radial growth, conidiation and the accumulation of ethanolamine. The results indicate that Treki1 plays a key role in growth and development and in the maintenance of cell wall integrity in T. reesei.

  19. Association Mapping of Cell Wall Synthesis Regulatory Genes and Cell Wall Quality in Switchgrass

    Energy Technology Data Exchange (ETDEWEB)

    Bartley, Laura [Univ. of Oklahoma, Norman, OK (United States). Dept. of Microbiology and Plant Biology; Wu, Y. [Oklahoma State Univ., Stillwater, OK (United States); Zhu, L. [Oklahoma State Univ., Stillwater, OK (United States); Brummer, E. C. [Noble Foundation, Ardmore, OK (United States); Saha, M. [Noble Foundation, Ardmore, OK (United States)

    2016-05-31

    Inefficient conversion of biomass to biofuels is one of the main barriers for biofuel production from such materials. Approximately half of polysaccharides in biomass remain unused by typical biochemical conversion methods. Conversion efficiency is influenced by the composition and structure of cell walls of biomass. Grasses such as wheat, maize, and rice, as well as dedicated perennial bioenergy crops, like switchgrass, make up ~55% of biomass that can be produced in the United States. Grass cell walls have a different composition and patterning compared with dicotyledonous plants, including the well-studied model plant, Arabidopsis. This project identified genetic determinants of cell wall composition in grasses using both naturally occurring genetic variation of switchgrass and gene network reconstruction and functional assays in rice. In addition, the project linked functional data in rice and other species to switchgrass improvement efforts through curation of the most abundant class of regulators in the switchgrass genome. Characterizing natural diversity of switchgrass for variation in cell wall composition and properties, also known as quality, provides an unbiased avenue for identifying biologically viable diversity in switchgrass cell walls. To characterizing natural diversity, this project generated cell wall composition and enzymatic deconstruction data for ~450 genotypes of the Switchgrass Southern Association Collection (SSAC), a diverse collection composed of 36 switchgrass accessions from the southern U.S. distribution of switchgrass. Comparing these data with other measures of cell wall quality for the same samples demonstrated the complementary nature of the diverse characterization platforms now being used for biomass characterization. Association of the composition data with ~3.2K single nucleotide variant markers identified six significant single nucleotide variant markers co-associated with digestibility and another compositional trait. These

  20. Protein overexport in a Saccharomyces cerevisiae mutant is not due to facilitated release of cell-surface proteins.

    Science.gov (United States)

    Alexieva, K I; Venkov, P V

    2000-01-01

    Saccharomyces cerevisiae strain MW11 is a temperature-sensitive mutant which exports twenty times more proteins at 37 degrees C than parental or wild-type strains do. To understand the mechanism underlying the protein overexport in the mutant the possibility of an altered cell-wall structure leading to facilitated release of cell-surface proteins was studied. Data on calcofluor white and zymolyase sensitivities, resistance to killer 1 toxin and determination of exported acid phosphatase and invertase did not provide evidence for alterations in the cell-wall structure that could explain the protein overexport phenotype. The results were obtained in experiments when transcription of mutated gene was discontinued which permits the full expression of the protein overexport phenotype.

  1. Association Mapping of Cell Wall Synthesis Regulatory Genes and Cell Wall Quality in Switchgrass

    Energy Technology Data Exchange (ETDEWEB)

    Bartley, Laura [Univ. of Oklahoma, Norman, OK (United States). Dept. of Microbiology and Plant Biology; Wu, Y. [Oklahoma State Univ., Stillwater, OK (United States); Zhu, L. [Oklahoma State Univ., Stillwater, OK (United States); Brummer, E. C. [Noble Foundation, Ardmore, OK (United States); Saha, M. [Noble Foundation, Ardmore, OK (United States)

    2016-05-31

    Inefficient conversion of biomass to biofuels is one of the main barriers for biofuel production from such materials. Approximately half of polysaccharides in biomass remain unused by typical biochemical conversion methods. Conversion efficiency is influenced by the composition and structure of cell walls of biomass. Grasses such as wheat, maize, and rice, as well as dedicated perennial bioenergy crops, like switchgrass, make up ~55% of biomass that can be produced in the United States. Grass cell walls have a different composition and patterning compared with dicotyledonous plants, including the well-studied model plant, Arabidopsis. This project identified genetic determinants of cell wall composition in grasses using both naturally occurring genetic variation of switchgrass and gene network reconstruction and functional assays in rice. In addition, the project linked functional data in rice and other species to switchgrass improvement efforts through curation of the most abundant class of regulators in the switchgrass genome. Characterizing natural diversity of switchgrass for variation in cell wall composition and properties, also known as quality, provides an unbiased avenue for identifying biologically viable diversity in switchgrass cell walls. To characterizing natural diversity, this project generated cell wall composition and enzymatic deconstruction data for ~450 genotypes of the Switchgrass Southern Association Collection (SSAC), a diverse collection composed of 36 switchgrass accessions from the southern U.S. distribution of switchgrass. Comparing these data with other measures of cell wall quality for the same samples demonstrated the complementary nature of the diverse characterization platforms now being used for biomass characterization. Association of the composition data with ~3.2K single nucleotide variant markers identified six significant single nucleotide variant markers co-associated with digestibility and another compositional trait. These

  2. The organization pattern of root border-like cells of Arabidopsis is dependent on cell wall homogalacturonan.

    Science.gov (United States)

    Durand, Caroline; Vicré-Gibouin, Maïté; Follet-Gueye, Marie Laure; Duponchel, Ludovic; Moreau, Myriam; Lerouge, Patrice; Driouich, Azeddine

    2009-07-01

    Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain attached to each other rather than completely detached from each other, as is usually observed in border cells of many species. Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing less homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1 than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein epitopes, in which the cells are trapped in the vicinity of the root tip.

  3. Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence

    Directory of Open Access Journals (Sweden)

    María J. Navarro-Arias

    2016-12-01

    Full Text Available The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast Candida albicans, Pmr1 acts as a Golgi-resident ion pump that provides cofactors to mannosyltransferases, regulating the synthesis of mannans attached to glycoproteins. To gain insight into a putative conservation of such a crucial process within opportunistic yeasts, we were particularly interested in studying the role of the PMR1 homolog in a low-virulent species that rarely causes candidiasis, Candida guilliermondii. We disrupted C. guilliermondii PMR1 and found that loss of Pmr1 affected cell growth and morphology, biofilm formation, susceptibility to cell wall perturbing agents, mannan levels, and the wall composition and organization. Despite there was a significant increment in the amount of β1,3-glucan exposed at the wall surface, this positively influenced only the ability of the mutant to stimulate IL-10 production by human monocytes, suggesting that recognition of both mannan and β1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate C. guilliermondii sensing by monocytes was critically dependent on the recognition of N-linked mannans and β1,3-glucan, as reported in other Candida species. In addition, chemical remotion of cell wall O-linked mannans was found to positively influence the recognition of C. guilliermondii by human monocytes, suggesting that O-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in C. albicans. Finally, mice infected with C. guilliermondii pmr1 null mutant cells had significantly lower fungal burdens compared to animals challenged with the parental strain. Accordingly, the null mutant showed inability to kill larvae in the Galleria mellonella infection model. This study thus demonstrates that mannans are relevant for the C. guilliermondii-host interaction, with

  4. Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence

    Science.gov (United States)

    Navarro-Arias, María J.; Defosse, Tatiana A.; Dementhon, Karine; Csonka, Katalin; Mellado-Mojica, Erika; Dias Valério, Aline; González-Hernández, Roberto J.; Courdavault, Vincent; Clastre, Marc; Hernández, Nahúm V.; Pérez-García, Luis A.; Singh, Dhirendra K.; Vizler, Csaba; Gácser, Attila; Almeida, Ricardo S.; Noël, Thierry; López, Mercedes G.; Papon, Nicolas; Mora-Montes, Héctor M.

    2016-01-01

    The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast Candida albicans, Pmr1 acts as a Golgi-resident ion pump that provides cofactors to mannosyltransferases, regulating the synthesis of mannans attached to glycoproteins. To gain insight into a putative conservation of such a crucial process within opportunistic yeasts, we were particularly interested in studying the role of the PMR1 homolog in a low-virulent species that rarely causes candidiasis, Candida guilliermondii. We disrupted C. guilliermondii PMR1 and found that loss of Pmr1 affected cell growth and morphology, biofilm formation, susceptibility to cell wall perturbing agents, mannan levels, and the wall composition and organization. Despite the significant increment in the amount of β1,3-glucan exposed at the wall surface, this positively influenced only the ability of the mutant to stimulate IL-10 production by human monocytes, suggesting that recognition of both mannan and β1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate C. guilliermondii sensing by monocytes was critically dependent on the recognition of N-linked mannans and β1,3-glucan, as reported in other Candida species. In addition, chemical remotion of cell wall O-linked mannans was found to positively influence the recognition of C. guilliermondii by human monocytes, suggesting that O-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in C. albicans. Finally, mice infected with C. guilliermondii pmr1Δ null mutant cells had significantly lower fungal burdens compared to animals challenged with the parental strain. Accordingly, the null mutant showed inability to kill larvae in the Galleria mellonella infection model. This study thus demonstrates that mannans are relevant for the C. guilliermondii-host interaction, with an atypical role for O

  5. Beyond growth: novel functions for bacterial cell wall hydrolases.

    Science.gov (United States)

    Wyckoff, Timna J; Taylor, Jennifer A; Salama, Nina R

    2012-11-01

    The peptidoglycan cell wall maintains turgor pressure and cell shape of most bacteria. Cell wall hydrolases are essential, together with synthases, for growth and daughter cell separation. Recent work in diverse organisms has uncovered new cell wall hydrolases that act autonomously or on neighboring cells to modulate invasion of prey cells, cell shape, innate immune detection, intercellular communication, and competitor lysis. The hydrolases involved in these processes catalyze the cleavage of bonds throughout the sugar and peptide moities of peptidoglycan. Phenotypes associated with these diverse hydrolases reveal new functions of the bacterial cell wall beyond growth and division.

  6. Identification and genetic characterization of maize cell wall variation for improved biorefinery feedstock characteristics

    Energy Technology Data Exchange (ETDEWEB)

    Pauly, Markus [UC Berkeley; Hake, Sarah [USDA Albany

    2013-10-31

    The objectives of this program are to 1) characterize novel maize mutants with altered cell walls for enhanced biorefinery characteristics and 2) find quantitative trait loci (QTLs) related to biorefinery characteristics by taking advantage of the genetic diversity of maize. As a result a novel non-transgenic maize plant (cal1) has been identified, whose stover (leaves and stalk) contain more glucan in their walls leading to a higher saccharification yield, when subjected to a standard enzymatic digestion cocktail. Stacking this trait with altered lignin mutants yielded evene higher saccharification yields. Cal-1 mutants do not show a loss of kernel and or biomass yield when grown in the field . Hence, cal1 biomass provides an excellent feedstock for the biofuel industry.

  7. Plant cell wall proteomics: the leadership of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Cécile eALBENNE

    2013-05-01

    Full Text Available 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, cells 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 ten 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.

  8. Plant cell wall proteomics: the leadership of Arabidopsis thaliana.

    Science.gov (United States)

    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.

  9. Molecular Imaging Of Metabolic Reprogramming In Mutant IDH Cells

    Directory of Open Access Journals (Sweden)

    Pavithra eViswanath

    2016-03-01

    Full Text Available Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG. Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG. In turn, 2-HG, which has been termed an oncometabolite, inhibits key α-KG- dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprogramming that extends beyond 2-HG production, and this reprogramming often differs from what has been previously reported in other cancer types. In this review we will discuss in detail what is known to date about the metabolic reprogramming of mutant IDH cells and how this reprogramming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

  10. An apoptotic cell cycle mutant in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Villadsen, Ingrid

    1996-01-01

    The simple eukaryote Saccharomyces cerevisiae has proved to be a useful organism for elucidating the mechanisms that govern cell cycle progression in eukaryotic cells. The excellent in vivo system permits a cell cycle study using temperature sensitive mutants. In addition, it is possible to study...... many genes and gene products from higher eukaryotes in Saccharomyces cerevisiae because many genes and biological processes are homologous or similar in lower and in higher eukaryotes. The highly developed methods of genetics and molecular biology greatly facilitates studies of higher eukaryotic...... processes.Programmmed cell death with apoptosis plays a major role in development and homeostatis in most, if not all, animal cells. Apoptosis is a morphologically distinct form of death, that requires the activation of a highly regulated suicide program. Saccharomyces cerevisiae provides a new system...

  11. Fermentation of the endosperm cell walls of monocotyledon and dicotyledon plant species: The relationship between cell wall characteristics and fermentability

    NARCIS (Netherlands)

    Laar, van H.; Tamminga, S.; Williams, B.A.; Verstegen, M.W.A.

    2000-01-01

    Cell walls from the endosperm of four monocotyledons (maize, wheat, rye, and rice) and four dicotyledons (soya bean, lupin, faba bean, and pea) seeds were studied to relate cell wall composition and structure with fermentation characteristics. Cell wall material was isolated from the endosperm of th

  12. Mutant γPKC that causes spinocerebellar ataxia type 14 upregulates Hsp70, which protects cells from the mutant's cytotoxicity.

    Science.gov (United States)

    Ogawa, Kota; Seki, Takahiro; Onji, Tomoya; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2013-10-11

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is misfolded, susceptible to aggregation and cytotoxic. Molecular chaperones assist the refolding and degradation of misfolded proteins and prevention of the proteins' aggregation. In the present study, we found that the expression of mutant γPKC-GFP increased the levels of heat-shock protein 70 (Hsp70) in SH-SY5Y cells. To elucidate the role of this elevation, we investigated the effect of siRNA-mediated knockdown of Hsp70 on the aggregation and cytotoxicity of mutant γPKC. Knockdown of Hsp70 exacerbated the aggregation and cytotoxicity of mutant γPKC-GFP by inhibiting this mutant's degradation. These findings suggest that mutant γPKC increases the level of Hsp70, which protects cells from the mutant's cytotoxicity by enhancing its degradation.

  13. Connexin mutant embryonic stem cells and human diseases

    Institute of Scientific and Technical Information of China (English)

    Kiyomasa; Nishii; Yosaburo; Shibata; Yasushi; Kobayashi

    2014-01-01

    Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin(Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells(ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

  14. Grass Cell Walls: A Story of Cross-Linking

    Science.gov (United States)

    Hatfield, Ronald D.; Rancour, David M.; Marita, Jane M.

    2017-01-01

    Cell wall matrices are complex composites mainly of polysaccharides, phenolics (monomers and polymers), and protein. We are beginning to understand the synthesis of these major wall components individually, but still have a poor understanding of how cell walls are assembled into complex matrices. Valuable insight has been gained by examining intact components to understand the individual elements that make up plant cell walls. Grasses are a prominent group within the plant kingdom, not only for their important roles in global agriculture, but also for the complexity of their cell walls. Ferulate incorporation into grass cell wall matrices (C3 and C4 types) leads to a cross-linked matrix that plays a prominent role in the structure and utilization of grass biomass compared to dicot species. Incorporation of p-coumarates as part of the lignin structure also adds to the complexity of grass cell walls. Feruoylation results in a wall with individual hemicellulosic polysaccharides (arabinoxylans) covalently linked to each other and to lignin. Evidence strongly suggests that ferulates not only cross-link arabinoxylans, but may be important factors in lignification of the cell wall. Therefore, the distribution of ferulates on arabinoxylans could provide a means of structuring regions of the matrix with the incorporation of lignin and have a significant impact upon localized cell wall organization. The role of other phenolics in cell wall formation such as p-coumarates (which can have concentrations higher than ferulates) remains unknown. It is possible that p-coumarates assist in the formation of lignin, especially syringyl rich lignin. The uniqueness of the grass cell wall compared to dicot sepcies may not be so much in the gross composition of the wall, but how the distinctive individual components are organized into a functional wall matrix. These features are discussed and working models are provided to illustrate how changing the organization of feruoylation and p

  15. Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

    DEFF Research Database (Denmark)

    Couchman, J R; Austria, R; Woods, A;

    1988-01-01

    In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin......-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased...

  16. Genes involved in cell wall localization and side chain formation of rhamnose-glucose polysaccharide in Streptococcus mutans.

    Science.gov (United States)

    Yamashita, Y; Tsukioka, Y; Tomihisa, K; Nakano, Y; Koga, T

    1998-11-01

    We identified in Streptococcus mutans six new genes (rgpA through rgpF), whose disruption results in a loss of serotype-specific antigenicity, specified by the glucose side chains of rhamnose-glucose polysaccharide from the cell wall. Rhamnose and glucose content of the cell wall decreased drastically in all these disruption mutants, except that in the rgpE mutant only the glucose content decreased. RgpC and RgpD are homologous to ATP-binding cassette transporter components and may be involved in polysaccharide export, whereas RgpE may be a transferase of side chain glucose.

  17. Genes Involved in Cell Wall Localization and Side Chain Formation of Rhamnose-Glucose Polysaccharide in Streptococcus mutans

    OpenAIRE

    Yamashita, Yoshihisa; Tsukioka, Yuichi; Tomihisa, Kiyotaka; Nakano, Yoshio; Koga, Toshihiko

    1998-01-01

    We identified in Streptococcus mutans six new genes (rgpA through rgpF), whose disruption results in a loss of serotype-specific antigenicity, specified by the glucose side chains of rhamnose-glucose polysaccharide from the cell wall. Rhamnose and glucose content of the cell wall decreased drastically in all these disruption mutants, except that in the rgpE mutant only the glucose content decreased. RgpC and RgpD are homologous to ATP-binding cassette transporter components and may be involve...

  18. Enzymes and other agents that enhance cell wall extensibility

    Science.gov (United States)

    Cosgrove, D. J.

    1999-01-01

    Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

  19. Disruption of cell walls for enhanced lipid recovery

    Science.gov (United States)

    Knoshaug, Eric P; Donohoe, Bryon S; Gerken, Henri; Laurens, Lieve; Van Wychen, Stefanie Rose

    2015-03-24

    Presented herein are methods of using cell wall degrading enzymes for recovery of internal lipid bodies from biomass sources such as algae. Also provided are algal cells that express at least one exogenous gene encoding a cell wall degrading enzyme and methods for recovering lipids from the cells.

  20. Combining multivariate analysis and monosaccharide composition modeling to identify plant cell wall variations by Fourier Transform Near Infrared spectroscopy

    Directory of Open Access Journals (Sweden)

    Smith-Moritz Andreia M

    2011-08-01

    Full Text Available Abstract We outline a high throughput procedure that improves outlier detection in cell wall screens using FT-NIR spectroscopy of plant leaves. The improvement relies on generating a calibration set from a subset of a mutant population by taking advantage of the Mahalanobis distance outlier scheme to construct a monosaccharide range predictive model using PLS regression. This model was then used to identify specific monosaccharide outliers from the mutant population.

  1. Two endogenous proteins that induce cell wall extension in plants

    Science.gov (United States)

    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.

  2. Features of the primary wall CESA complex in wild type and cellulose-deficient mutants of Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Jian; Elliott, Janet E; Williamson, Richard E

    2008-01-01

    Evidence from genetics, co-precipitation and bimolecular fluorescence complementation suggest that three CESAs implicated in making primary wall cellulose in Arabidopsis thaliana form a complex. This study shows the complex has a M(r) of approximately 840 kDa in detergent extracts and that it has undergone distinctive changes when extracts are prepared from some cellulose-deficient mutants. The mobility of CESAs 1, 3, and 6 in a Triton-soluble microsomal fraction subject to blue native polyacrylamide gel electrophoresis was consistent with a M(r) of about 840 kDa. An antibody specific to any one CESA pulled down all three CESAs consistent with their occupying the same 840 kDa complex. In rsw1, a CESA1 missense mutant, extracts of seedlings grown at the permissive temperature have an apparently normal CESA complex that was missing from extracts of seedlings grown at the restrictive temperature where CESAs precipitated independently. In prc1-19, with no CESA6, CESAs 1 and 3 were part of a 420 kDa complex in extracts of light-grown seedlings that was absent from extracts of dark-grown seedlings where the CESAs precipitated independently. Two CESA3 missense mutants retained apparently normal CESA complexes as did four cellulose-deficient mutants defective in proteins other than CESAs. The 840 kDa complex could contain six CESA subunits and, since loss of plasma membrane rosettes accompanies its loss in rsw1, the complex could form one of the six particles which electron microscopy reveals in rosettes.

  3. Structural basis of cell wall cleavage by a staphylococcal autolysin.

    Directory of Open Access Journals (Sweden)

    Sebastian Zoll

    2010-03-01

    Full Text Available The major autolysins (Atl of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis from the major autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several alpha-helices surrounding a central beta-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.

  4. The Role of Pectin Acetylation in the Organization of Plant Cell Walls

    DEFF Research Database (Denmark)

    Fimognari, Lorenzo

    adopt defined 3D organization to allow their composition/interactions to be tweaked upon developmental need. Failure to build functional cell wall architecture will affect plant growth and resistance to stresses. In this PhD dissertation I explored the role of pectin acetylation in controlling...... reduction in pectin and hemicellulose acetylation. We found that the increased resistance to pathogens in this mutant was due to the constitutive upregulation of defenses responses and the concomitant loss of integrity in the cell wall. Based on the results obtained in Manuscript I, we hypothesized...... that the loss of structural integrity in the cell wall was the underlying cause for triggering defenses response. This hypothesis was tested in Manuscript II. Through a suppressor screen of 30.000 Arabidopsis rwa2 plants and mapping of mutations by next generation sequencing, we pinpointed pectin deacetylation...

  5. Modification of potato cell wall pectin by the introduction of rhamnogalacturonan lyase and β-galactosidase transgenes and their side effects

    NARCIS (Netherlands)

    Huang, Jie Hong; Kortstee, Anne; Dees, Dianka C.T.; Trindade, Luisa M.; Schols, Henk A.; Gruppen, Harry

    2016-01-01

    Genes encoding pectic enzymes were introduced to wild-type potato Karnico. Cell wall materials were extracted from Karnico and transgenic lines expressing β-galactosidase (β-Gal-14 mutant) or rhamnogalacturonan lyase (RGL-18 mutant). After sequential extraction, β-Gal-14 hot buffer-soluble solids

  6. Multidimensional solid-state NMR spectroscopy of plant cell walls.

    Science.gov (United States)

    Wang, Tuo; Phyo, Pyae; Hong, Mei

    2016-09-01

    Plant biomass has become an important source of bio-renewable energy in modern society. The molecular structure of plant cell walls is difficult to characterize by most atomic-resolution techniques due to the insoluble and disordered nature of the cell wall. Solid-state NMR (SSNMR) spectroscopy is uniquely suited for studying native hydrated plant cell walls at the molecular level with chemical resolution. Significant progress has been made in the last five years to elucidate the molecular structures and interactions of cellulose and matrix polysaccharides in plant cell walls. These studies have focused on primary cell walls of growing plants in both the dicotyledonous and grass families, as represented by the model plants Arabidopsis thaliana, Brachypodium distachyon, and Zea mays. To date, these SSNMR results have shown that 1) cellulose, hemicellulose, and pectins form a single network in the primary cell wall; 2) in dicot cell walls, the protein expansin targets the hemicellulose-enriched region of the cellulose microfibril for its wall-loosening function; and 3) primary wall cellulose has polymorphic structures that are distinct from the microbial cellulose structures. This article summarizes these key findings, and points out future directions of investigation to advance our fundamental understanding of plant cell wall structure and function.

  7. Advanced technologies for plant cell wall evolution and diversity

    DEFF Research Database (Denmark)

    Fangel, Jonatan Ulrik

    Plant cell walls consist of polysaccharides, glycoproteins and phenolic polymers interlinked together in a highly complex network. The detailed analysis of cell walls is challenging because of their inherent complexity and heterogeneity. Also, complex carbohydrates, unlike proteins and nucleotides...... probes (monoclonal antibodies mAbs and carbohydrate binding modules, CBMs) to rapidly profile polysaccharides across a sample set. During my PhD I have further developed the CoMPP technique and used it for cell wall analysis within the context of a variety of applied and fundamental projects. The data...... produced has provided new insight into cell wall evolution and biosynthesis and has contributed to the commercial development of cell wall materials. A major focus of the work has been the wide scale sampling of cell wall diversity across the plant kingdom, from unicellular algae to highly evolved...

  8. Critical roles for lipomannan and lipoarabinomannan in cell wall integrity of mycobacteria and pathogenesis of tuberculosis.

    Science.gov (United States)

    Fukuda, Takeshi; Matsumura, Takayuki; Ato, Manabu; Hamasaki, Maho; Nishiuchi, Yukiko; Murakami, Yoshiko; Maeda, Yusuke; Yoshimori, Tamotsu; Matsumoto, Sohkichi; Kobayashi, Kazuo; Kinoshita, Taroh; Morita, Yasu S

    2013-02-19

    Lipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. In Mycobacterium smegmatis, structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to β-lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalent Mycobacterium tuberculosis mutants became more sensitive to β-lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis. IMPORTANCE Tuberculosis (TB) is a global burden, affecting millions of people worldwide. Mycobacterium tuberculosis is a causative agent of TB, and understanding the biology of M. tuberculosis is essential for tackling this devastating disease. The cell wall of M. tuberculosis is highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in all Mycobacterium species, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a

  9. AtPGL3 is an Arabidopsis BURP domain protein that is localized to the cell wall and promotes cell enlargement

    Directory of Open Access Journals (Sweden)

    Jiyoung ePark

    2015-06-01

    Full Text Available The BURP domain is a plant-specific protein domain that has been identified in secretory proteins, and some of these are involved in cell wall remodeling. Among Arabidopsis BURP domain proteins, three proteins exhibit strong amino acid similarities with the tomato polygalacturonase 1 beta (PG1β protein that interacts with a pectin-digesting enzyme. To investigate biological roles of the Arabidopsis PG1β-like proteins (AtPGLs, we generated Arabidopsis lines in which expression of AtPGLs is altered. Among the three AtPGLs, AtPGL3 exhibited highest transcriptional activity throughout all developmental stages. When tissue-specific expression pattern of AtPGL3 was examined, the gene was observed to be active in epidermal cell layers of rosette leaves and in the trichomes. AtPGL triple mutant plants were smaller than wild type plants because cells were smaller in the mutant plants. Interestingly, when we overexpressed AtPGL3 using a 35S promoter, cells in transgenic plants grew larger than those of the wild type, suggesting that AtPGL3 plays a role in cell expansion. A C-terminal GFP fusion protein of AtPGL3 complemented phenotypes of the triple mutant plants and localized to the cell wall. A truncated AtPGL3-GFP fusion protein that lacks the BURP domain failed to rescue the mutant phenotypes even though the GFP protein was targeted to the cell wall, indicating that the BURP domain is required for its effect on cell expansion. Quantitative RT-PCR and immunoblot analyses indicated that 2 α-expansin genes are down-regulated and up-regulated in the triple mutant and overexpressor lines, respectively. Taken together, AtPGL3 is a cell wall protein required for normal cell expansion and the coexpression results suggest that AtPGLs regulate cell wall loosening, in conjunction with α-expansins, to promote cell growth.

  10. Cell wall degradation in the autolysis of filamentous fungi.

    Science.gov (United States)

    Perez-Leblic, M I; Reyes, F; Martinez, M J; Lahoz, R

    1982-12-27

    A systematic study on autolysis of the cell walls of fungi has been made on Neurospora crassa, Botrytis cinerea, Polystictus versicolor, Aspergillus nidulans, Schizophyllum commune, Aspergillus niger, and Mucor mucedo. During autolysis each fungus produces the necessary lytic enzymes for its autodegradation. From autolyzed cultures of each fungus enzymatic precipitates were obtained. The degree of lysis of the cell walls, obtained from non-autolyzed mycelia, was studied by incubating these cell walls with and without a supply of their own lytic enzymes. The degree of lysis increased with the incubation time and generally was higher with a supply of lytic enzymes. Cell walls from mycelia of different ages were obtained. A higher degree of lysis was always found, in young cell walls than in older cell walls, when exogenous lytic enzymes were present. In all the fungi studied, there is lysis of the cell walls during autolysis. This is confirmed by the change of the cell wall structure as well as by the degree of lysis reached by the cell wall and the release of substances, principally glucose and N-acetylglucosamine in the medium.

  11. The cell wall sensor Wsc1p is involved in reorganization of actin cytoskeleton in response to hypo-osmotic shock in Saccharomyces cerevisiae.

    Science.gov (United States)

    Gualtieri, Tania; Ragni, Enrico; Mizzi, Luca; Fascio, Umberto; Popolo, Laura

    2004-10-15

    The cell wall is essential to preserve osmotic integrity of yeast cells. Some phenotypic traits of cell wall mutants suggest that, as a result of a weakening of the cell wall, hypo-osmotic stress-like conditions are created. Consequent expansion of the cell wall and stretching of the plasma membrane trigger a complex response to prevent cell lysis. In this work we examined two conditions that generate a cell wall and membrane stress: one is represented by the cell wall mutant gas1Delta and the other by a hypo-osmotic shock. We examined the actin cytoskeleton and the role of the cell wall sensors Wsc1p and Mid2p in these stress conditions. In the gas1 null mutant cells, which lack a beta(1,3)-glucanosyltransferase activity required for cell wall assembly, a constitutive marked depolarization of actin cytoskeleton was found. In a hypo-osmotic shock wild-type cells showed a transient depolarization of actin cytoskeleton. The percentage of depolarized cells was maximal at 30 min after the shift and then progressively decreased until cells reached a new steady-state condition. The maximal response was proportional to the magnitude of the difference in the external osmolarity before and after the shift within a given range of osmolarities. Loss of Wsc1p specifically delayed the repolarization of the actin cytoskeleton, whereas Wsc1p and Mid2p were essential for the maintenance of cell integrity in gas1Delta cells. The control of actin cytoskeleton is an important element in the context of the compensatory response to cell wall weakening. Wsc1p appears to be an important regulator of the actin network rearrangements in conditions of cell wall expansion and membrane stretching.

  12. Pinoresinol reductase 1 impacts lignin distribution during secondary cell wall biosynthesis in Arabidopsis.

    Science.gov (United States)

    Zhao, Qiao; Zeng, Yining; Yin, Yanbin; Pu, Yunqiao; Jackson, Lisa A; Engle, Nancy L; Martin, Madhavi Z; Tschaplinski, Timothy J; Ding, Shi-You; Ragauskas, Arthur J; Dixon, Richard A

    2015-04-01

    Pinoresinol reductase (PrR) catalyzes the conversion of the lignan (-)-pinoresinol to (-)-lariciresinol in Arabidopsis thaliana, where it is encoded by two genes, PrR1 and PrR2, that appear to act redundantly. PrR1 is highly expressed in lignified inflorescence stem tissue, whereas PrR2 expression is barely detectable in stems. Co-expression analysis has indicated that PrR1 is co-expressed with many characterized genes involved in secondary cell wall biosynthesis, whereas PrR2 expression clusters with a different set of genes. The promoter of the PrR1 gene is regulated by the secondary cell wall related transcription factors SND1 and MYB46. The loss-of-function mutant of PrR1 shows, in addition to elevated levels of pinoresinol, significantly decreased lignin content and a slightly altered lignin structure with lower abundance of cinnamyl alcohol end groups. Stimulated Raman scattering (SRS) microscopy analysis indicated that the lignin content of the prr1-1 loss-of-function mutant is similar to that of wild-type plants in xylem cells, which exhibit a normal phenotype, but is reduced in the fiber cells. Together, these data suggest an association of the lignan biosynthetic enzyme encoded by PrR1 with secondary cell wall biosynthesis in fiber cells.

  13. Small molecule probes for plant cell wall polysaccharide imaging

    Directory of Open Access Journals (Sweden)

    Ian eWallace

    2012-05-01

    Full Text Available Plant cell walls are composed of interlinked polymer networks consisting of cellulose, hemicelluloses, pectins, proteins, and lignin. The ordered deposition of these components is a dynamic process that critically affects the development and differentiation of plant cells. However, our understanding of cell wall synthesis and remodeling, as well as the diverse cell wall architectures that result from these processes, has been limited by a lack of suitable chemical probes that are compatible with live-cell imaging. In this review, we summarize the currently available molecular toolbox of probes for cell wall polysaccharide imaging in plants, with particular emphasis on recent advances in small molecule-based fluorescent probes. We also discuss the potential for further development of small molecule probes for the analysis of cell wall architecture and dynamics.

  14. Thermal radiosensitization in radiation-sensitive mutant mouse leukemic cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Toshikazu (Hiroshima Univ. (Japan). School of Dentistry)

    1994-06-01

    This study investigated thermal, radiation, and combined thermal radiation sensitization of mouse leukemic cells, L5178Y, and radiation-sensitive mutant cells, LX830. Radiation sensitivity (D[sub 0]) values were 0.41 Gy for LX830 and 1.39 Gy for L5178Y, with the ratio of D[sub 0] values in LX830 to in L5178Y being 3.4. Thus, LX830 was more radiosensitive than L5178Y. LX830 showed no shouldered survival curves. Although sublethal damage (SLD) repair was seen to the almost same degree in both LX830 and L5178Y, potential lethal damage (PLD) repair was scarcely observed in LX830. Both cell lines were similar in thermal sensitivity (T[sub 0]). Eosine staining suggested that cell killing due to hyperthermia had occurred in the interphase in both LX830 and L5178Y. L5178Y showed thermal sensitivity low in the G1 phase and high in the S phase; on the contrary, LX830 showed it high in the G1 phase and low in the S phase. Thermal radiosensitization was similar in both cell lines, although there was a great difference in radiation sensitivity between the cell lines. The difference in radiation sensitivity (D[sub 0]) between L5178Y and LX830 became small when radiation was given at the time of the maximum thermal resistance. This seemed to contribute to a decrease in radiation sensitivity in LX830. It can be concluded that thermal radiosensitization depends on thermal sensitivity and that radiation sensitivity decreases in radiation-sensitive cells when exposed to irradiation at the time of thermal resistance. (N.K.).

  15. Transcriptional dysregulation in NIPBL and cohesin mutant human cells.

    Directory of Open Access Journals (Sweden)

    Jinglan Liu

    2009-05-01

    Full Text Available Cohesin regulates sister chromatid cohesion during the mitotic cell cycle with Nipped-B-Like (NIPBL facilitating its loading and unloading. In addition to this canonical role, cohesin has also been demonstrated to play a critical role in regulation of gene expression in nondividing cells. Heterozygous mutations in the cohesin regulator NIPBL or cohesin structural components SMC1A and SMC3 result in the multisystem developmental disorder Cornelia de Lange Syndrome (CdLS. Genome-wide assessment of transcription in 16 mutant cell lines from severely affected CdLS probands has identified a unique profile of dysregulated gene expression that was validated in an additional 101 samples and correlates with phenotypic severity. This profile could serve as a diagnostic and classification tool. Cohesin binding analysis demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor.

  16. Structural Studies of Complex Carbohydrates of Plant Cell Walls

    Energy Technology Data Exchange (ETDEWEB)

    Darvill, Alan [Univ. of Georgia, Athens, GA (United States); Hahn, Michael G. [Univ. of Georgia, Athens, GA (United States); O' Neill, Malcolm A. [Univ. of Georgia, Athens, GA (United States); York, William S. [Univ. of Georgia, Athens, GA (United States)

    2015-02-17

    Most of the solar energy captured by land plants is converted into the polysaccharides (cellulose, hemicellulose, and pectin) that are the predominant components of the cell wall. These walls, which account for the bulk of plant biomass, have numerous roles in the growth and development of plants. Moreover, these walls have a major impact on human life as they are a renewable source of biomass, a source of diverse commercially useful polymers, a major component of wood, and a source of nutrition for humans and livestock. Thus, understanding the molecular mechanisms that lead to wall assembly and how cell walls and their component polysaccharides contribute to plant growth and development is essential to improve and extend the productivity and value of plant materials. The proposed research will develop and apply advanced analytical and immunological techniques to study specific changes in the structures and interactions of the hemicellulosic and pectic polysaccharides that occur during differentiation and in response to genetic modification and chemical treatments that affect wall biosynthesis. These new techniques will make it possible to accurately characterize minute amounts of cell wall polysaccharides so that subtle changes in structure that occur in individual cell types can be identified and correlated to the physiological or developmental state of the plant. Successful implementation of this research will reveal fundamental relationships between polysaccharide structure, cell wall architecture, and cell wall functions.

  17. Characterising the cellulose synthase complexes of cell walls

    NARCIS (Netherlands)

    Mansoori Zangir, N.

    2012-01-01

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the c

  18. Hemicellulose biosynthesis and degradation in tobacco cell walls

    NARCIS (Netherlands)

    Compier, M.G.M.

    2005-01-01

    Natural fibres have a wide range of technological applications, such as in paper and textile industries. The basic properties and the quality of plant fibres are determined by the composition of the plant cell wall. Characteristic for fibres are thick secondary cell walls, which consist of cellulose

  19. Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation

    Directory of Open Access Journals (Sweden)

    Kouki eYoshida

    2013-10-01

    Full Text Available Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs can regulate secondary wall formation in rice (Oryza sativa and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S has very low transcriptional activation ability, but the longer protein (OsSWN2L and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications.

  20. On-Off Switches for Secondary Cell Wall Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Huan-Zhong Wang; Richard A.Dixon

    2012-01-01

    Secondary cell walls provide plants with rigidity and strength to support their body weight and ensure water and nutrient transport.They also provide textiles,timber,and potentially second-generation biofuels for human use.Genes responsible for synthesis of the different cell wall components,namely cellulose,hemicelluloses,and lignin,are coordinately expressed and under transcriptional regulation.In the past several years,cell wall-related NAC and MYB transcription factors have been intensively investigated in different species and shown to be master switches of secondary cell wall biosynthesis.Positive and negative regulators,which function upstream of NAC master switches,have also been identified in different plant tissues.Further elucidation of the regulatory mechanisms of cell wall synthesis will facilitate the engineering of plant feedstocks suitable for biofuel production.

  1. Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls.

    Science.gov (United States)

    Kotake, Toshihisa; Aohara, Tsutomu; Hirano, Ko; Sato, Ami; Kaneko, Yasuko; Tsumuraya, Yoichi; Takatsuji, Hiroshi; Kawasaki, Shinji

    2011-03-01

    The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.

  2. Dynamic metabolic flux analysis of plant cell wall synthesis.

    Science.gov (United States)

    Chen, Xuewen; Alonso, Ana P; Shachar-Hill, Yair

    2013-07-01

    The regulation of plant cell wall synthesis pathways remains poorly understood. This has become a bottleneck in designing bioenergy crops. The goal of this study was to analyze the regulation of plant cell wall precursor metabolism using metabolic flux analysis based on dynamic labeling experiments. Arabidopsis T87 cells were cultured heterotrophically with (13)C labeled sucrose. The time course of ¹³C labeling patterns in cell wall precursors and related sugar phosphates was monitored using liquid chromatography tandem mass spectrometry until steady state labeling was reached. A kinetic model based on mass action reaction mechanisms was developed to simulate the carbon flow in the cell wall synthesis network. The kinetic parameters of the model were determined by fitting the model to the labeling time course data, cell wall composition, and synthesis rates. A metabolic control analysis was performed to predict metabolic regulations that may improve plant biomass composition for biofuel production. Our results describe the routes and rates of carbon flow from sucrose to cell wall precursors. We found that sucrose invertase is responsible for the entry of sucrose into metabolism and UDP-glucose-4-epimerase plays a dominant role in UDP-Gal synthesis in heterotrophic Aradidopsis cells under aerobic conditions. We also predicted reactions that exert strong regulatory influence over carbon flow to cell wall synthesis and its composition.

  3. The Transcriptional Targets of Mutant FOXL2 in Granulosa Cell Tumours

    OpenAIRE

    2012-01-01

    BACKGROUND: Despite their distinct biology, granulosa cell tumours (GCTs) are treated the same as other ovarian tumours. Intriguingly, a recurring somatic mutation in the transcription factor Forkhead Box L2 (FOXL2) 402C>G has been found in nearly all GCTs examined. This investigation aims to identify the pathogenicity of mutant FOXL2 by studying its altered transcriptional targets. METHODS: The expression of mutant FOXL2 was reduced in the GCT cell line KGN, and wildtype and mutant FOXL2 wer...

  4. High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells.

    Science.gov (United States)

    Xing, James Z; Gabos, Stephan; Huang, Biao; Pan, Tianhong; Huang, Min; Chen, Jie

    2012-10-01

    The mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell's kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p > 0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays.

  5. Maize development: cell wall changes in leaves and sheaths

    Science.gov (United States)

    Developmental changes occur in maize (Zea mays L.) as it transitions from juvenile stages to the mature plant. Changes also occur as newly formed cells mature into adult cells. Maize leaf blades, including the midribs and sheaths, undergo cell wall changes as cells transition to fully mature cell ty...

  6. 2009 Plant Cell Walls Gordon Research Conference-August 2-7,2009

    Energy Technology Data Exchange (ETDEWEB)

    Debra Mohnen

    2009-08-07

    Plant cell walls are a complex cellular compartment essential for plant growth, development and response to biotic and abiotic stress and a major biological resource for meeting our future bioenergy and natural product needs. The goal of the 2009 Plant Cell Walls Gordon Research Conference is to summarize and critically evaluate the current level of understanding of the structure, synthesis and function of the whole plant extracellular matrix, including the polysaccharides, proteins, lignin and waxes that comprise the wall, and the enzymes and regulatory proteins that drive wall synthesis and modification. Innovative techniques to study how both primary and secondary wall polymers are formed and modified throughout plant growth will be emphasized, including rapid advances taking place in the use of anti-wall antibodies and carbohydrate binding proteins, comparative and evolutionary wall genomics, and the use of mutants and natural variants to understand and identify wall structure-function relationships. Discussions of essential research advances needed to push the field forward toward a systems biology approach will be highlighted. The meeting will include a commemorative lecture in honor of the career and accomplishments of the late Emeritus Professor Bruce A. Stone, a pioneer in wall research who contributed over 40 years of outstanding studies on plant cell wall structure, function, synthesis and remodeling including emphasis on plant cell wall beta-glucans and arabinogalactans. The dwindling supply of fossil fuels will not suffice to meet our future energy and industrial product needs. Plant biomass is the renewable resource that will fill a large part of the void left by vanishing fossil fuels. It is therefore critical that basic research scientists interact closely with industrial researchers to critically evaluate the current state of knowledge regarding how plant biomass, which is largely plant cell walls, is synthesized and utilized by the plant. A final

  7. Suppression of Arabidopsis peroxidase 72 alters cell wall and phenylpropanoid metabolism.

    Science.gov (United States)

    Fernández-Pérez, Francisco; Pomar, Federico; Pedreño, María A; Novo-Uzal, Esther

    2015-10-01

    Class III peroxidases are glycoproteins with a major role in cell wall maturation such as lignin formation. Peroxidases are usually present in a high number of isoenzymes, which complicates to assign specific functions to individual peroxidase isoenzymes. Arabidopsis genome encodes for 73 peroxidases, among which AtPrx72 has been shown to participate in lignification. Here, we report by using knock out peroxidase mutants how the disruption of AtPrx72 causes thinner secondary walls in interfascicular fibres but not in the xylem of the stem. This effect is also age-dependent, and AtPrx72 function seems to be particularly important when lignification prevails over elongation processes. Finally, the suppression AtPrx72 leads to the down-regulation of lignin biosynthesis pathway, as well as genes and transcription factors involved in secondary wall thickening.

  8. TAE226, a Bis-Anilino Pyrimidine Compound, Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Hiroki Otani

    Full Text Available TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK and insulin-like growth factor-I receptor (IGF-IR. In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC, especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750 mutant, and the reduced affinity of ATP to the L858R (or delE746_A750 mutant resulted in good responsiveness of the L858R (or delE746_A750 mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation.

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

    Science.gov (United States)

    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.

  10. Cell wall glucomannan in Arabidopsis is synthesised by CSLA glycosyltransferases, and influences the progression of embryogenesis.

    Science.gov (United States)

    Goubet, Florence; Barton, Christopher J; Mortimer, Jennifer C; Yu, Xiaolan; Zhang, Zhinong; Miles, Godfrey P; Richens, Jenny; Liepman, Aaron H; Seffen, Keith; Dupree, Paul

    2009-11-01

    Mannans are hemicellulosic polysaccharides that have previously been implicated as structural constituents of cell walls and as storage reserves but which may serve other functions during plant growth and development. Several members of the Arabidopsis cellulose synthase-like A (CSLA) family have previously been shown to synthesise mannan polysaccharides in vitro when heterologously expressed. It has also been found that CSLA7 is essential for embryogenesis, suggesting a role for the CSLA7 product in development. To determine whether the CSLA proteins are responsible for glucomannan synthesis in vivo, we characterised insertion mutants in each of the nine Arabidopsis CSLA genes and several double and triple mutant combinations. csla9 mutants showed substantially reduced glucomannan, and triple csla2csla3csla9 mutants lacked detectable glucomannan in stems. Nevertheless, these mutants showed no alteration in stem development or strength. Overexpression of CSLA2, CSLA7 and CSLA9 increased the glucomannan content in stems. Increased glucomannan synthesis also caused defective embryogenesis, leading to delayed development and occasional embryo death. The embryo lethality of csla7 was complemented by overexpression of CSLA9, suggesting that the glucomannan products are similar. We conclude that CSLA2, CSLA3 and CSLA9 are responsible for the synthesis of all detectable glucomannan in Arabidopsis stems, and that CSLA7 synthesises glucomannan in embryos. These results are inconsistent with a substantial role for glucomannan in wall strength in Arabidopsis stems, but indicate that glucomannan levels affect embryogenesis. Together with earlier heterologous expression studies, the glucomannan deficiency observed in csla mutant plants demonstrates that the CSLA family encodes glucomannan synthases.

  11. Agronomic traits and RAPD analysis of two mutants derived from rice somatic cell culturing

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Genetic variation, including agronomic trait variation, often occurs in somatic cell culturing. In this study, we compared the main agronomic traits of two rice mutants, M3 and M14, which were derived from Shenxiangjing 5 somatic cell culturing. Significant differences were found between the two mutants and the wild rice Shenxiangjing 5 (Table 1). Results were as follows:

  12. Regulation of genes involved in cell wall synthesis and structure during Ustilago maydis dimorphism.

    Science.gov (United States)

    Robledo-Briones, Mariana; Ruiz-Herrera, José

    2013-02-01

    The cell wall is the structure that provides the shape to fungal cells and protects them from the difference in osmotic pressure existing between the cytosol and the external medium. Accordingly, changes in structure and composition of the fungal wall must occur during cell differentiation, including the dimorphic transition of fungi. We analyzed, by use of microarrays, the transcriptional regulation of the 639 genes identified to be involved in cell wall synthesis and structure plus the secretome of the Basidiomycota species Ustilago maydis during its dimorphic transition induced by a change in pH. Of these, 189 were differentially expressed during the process, and using as control two monomorphic mutants, one yeast like and the other mycelium constitutive, 66 genes specific of dimorphism were identified. Most of these genes were up-regulated in the mycelial phase. These included CHS genes, genes involved in β-1,6-glucan synthesis, N-glycosylation, and proteins containing a residue of glycosylphosphatidylinositol, and a number of genes from the secretome. The possible significance of these data on cell wall plasticity is discussed.

  13. The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans

    Science.gov (United States)

    Thevissen, Karin; de Mello Tavares, Patricia; Xu, Deming; Blankenship, Jill; Vandenbosch, Davy; Idkowiak-Baldys, Jolanta; Govaert, Gilmer; Bink, Anna; Rozental, Sonia; de Groot, Piet W.J.; Davis, Talya R.; Kumamoto, Carol A.; Vargas, Gabriele; Nimrichter, Leonardo; Coenye, Tom; Mitchell, Aaron; Roemer, Terry; Hannun, Yusuf A.; Cammue, Bruno P.A.

    2012-01-01

    Summary The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2,868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2-hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast-to-hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analyzed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24-ceramides in membranes of RsAFP2-treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation. PMID:22384976

  14. Transmission Fourier transform infrared microspectroscopy allows simultaneous assessment of cutin and cell-wall polysaccharides of Arabidopsis petals.

    Science.gov (United States)

    Mazurek, Sylwester; Mucciolo, Antonio; Humbel, Bruno M; Nawrath, Christiane

    2013-06-01

    A procedure for the simultaneous analysis of cell-wall polysaccharides, amides and aliphatic polyesters by transmission Fourier transform infrared microspectroscopy (FTIR) has been established for Arabidopsis petals. The combination of FTIR imaging with spectra derivatization revealed that petals, in contrast to other organs, have a characteristic chemical zoning with high amount of aliphatic compounds and esters in the lamina and of polysaccharides in the stalk of the petal. The hinge region of petals was particular rich in amides as well as in vibrations potentially associated with hemicellulose. In addition, a number of other distribution patterns have been identified. Analyses of mutants in cutin deposition confirmed that vibrations of aliphatic compounds and esters present in the lamina were largely associated with the cuticular polyester. Calculation of spectrotypes, including the standard deviation of intensities, allowed detailed comparison of the spectral features of various mutants. The spectrotypes not only revealed differences in the amount of polyesters in cutin mutants, but also changes in other compound classes. For example, in addition to the expected strong deficiencies in polyester content, the long-chain acyl CoA synthase 2 mutant showed increased intensities of vibrations in a wavelength range that is typical for polysaccharides. Identical spectral features were observed in quasimodo2, a cell-wall mutant of Arabidopsis with a defect in pectin formation that exhibits increased cellulose synthase activity. FTIR thus proved to be a convenient method for the identification and characterization of mutants affected in the deposition of cutin in petals.

  15. Non-acid-fastness in Mycobacterium tuberculosis ΔkasB mutant correlates with the cell envelope electron density.

    Science.gov (United States)

    Yamada, Hiroyuki; Bhatt, Apoorva; Danev, Radostin; Fujiwara, Nagatoshi; Maeda, Shinji; Mitarai, Satoshi; Chikamatsu, Kinuyo; Aono, Akio; Nitta, Koji; Jacobs, William R; Nagayama, Kuniaki

    2012-07-01

    The acid-fastness is the most important and the most specific characteristics in mycobacteria, the mechanism of which is not clear but may be attributed to the lipid rich cell wall of this bacterium. While the exact component(s) responsible for this staining method remained unidentified, a Mycobacterium tuberculosis mutant, attenuated strain that produced shorter mycolic acids with defects in trans-cyclopropanation was shown to be acid fast negative. In this study, we examined the ultrastructure of the cell envelope (CE) of the mutant strain ΔkasB (missing a beta-ketoacyl-ACP synthase involved in mycolic acid biosynthesis), the parental CDC1551 (wild type strain) and kasB complemented strain, and compared ultrastructural differences among them with conventional transmission electron microscopy (TEM) and cryo-transmission electron microscopy (CEM). Conventional TEM revealed that there were no detectable differences in the thickness of the cell envelope among three strains (wild-type: 43.35 ± 6.13 nm; ΔkasB: 45.98 ± 11.32 nm; complement: 40.71 ± 6.3 nm). However, CEM data demonstrated that the region between the inner and outer membranes of the mutant strain, which is composed mainly of cell wall anchored mycolic acids (MA), showed a significant decrease in electron density as compared to the wild type and kasB complement strain (567.1 ± 372.7 vs. 301.4 ± 262.1, or vs. 235.2 ± 174.9, p tuberculosis cell envelope, resulting in a reduced electron density of this layer as seen by CEM and loss of acid-fastness in light microscopical observation, and we propose a novel model of the cell envelope structure in tubercle bacilli.

  16. A role for katanin in plant cell division: microtubule organization in dividing root cells of fra2 and lue1Arabidopsis thaliana mutants.

    Science.gov (United States)

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Voulgari, Georgia; Papadopoulou, Galini

    2011-07-01

    Severing of microtubules by katanin has proven to be crucial for cortical microtubule organization in elongating and differentiating plant cells. On the contrary, katanin is currently not considered essential during cell division in plants as it is in animals. However, defects in cell patterning have been observed in katanin mutants, implying a role for it in dividing plant cells. Therefore, microtubule organization was studied in detail by immunofluorescence in dividing root cells of fra2 and lue1 katanin mutants of Arabidopsis thaliana. In both, early preprophase bands consisted of poorly aligned microtubules, prophase spindles were multipolar, and the microtubules of expanding phragmoplasts were elongated, bended toward and connected to the surface of daughter nuclei. Accordingly, severing by katanin seems to be necessary for the proper organization of these microtubule arrays. In both fra2 and lue1, metaphase/anaphase spindles and initiating phragmoplasts exhibited typical organization. However, they were obliquely oriented more frequently than in the wild type. It is proposed that this oblique orientation may be due to prophase spindle multipolarity and results in a failure of the cell plate to follow the predetermined division plane, during cytokinesis, producing oblique cell walls in the roots of both mutants. It is therefore concluded that, like in animal cells, katanin is important for plant cell division, influencing the organization of several microtubule arrays. Moreover, failure in microtubule severing indirectly affects the orientation of the division plane.

  17. Cell Wall Metabolism in Response to Abiotic Stress

    Directory of Open Access Journals (Sweden)

    Hyacinthe Le Gall

    2015-02-01

    Full Text Available This review focuses on the responses of the plant cell wall to several abiotic stresses including drought, flooding, heat, cold, salt, heavy metals, light, and air pollutants. The effects of stress on cell wall metabolism are discussed at the physiological (morphogenic, transcriptomic, proteomic and biochemical levels. The analysis of a large set of data shows that the plant response is highly complex. The overall effects of most abiotic stress are often dependent on the plant species, the genotype, the age of the plant, the timing of the stress application, and the intensity of this stress. This shows the difficulty of identifying a common pattern of stress response in cell wall architecture that could enable adaptation and/or resistance to abiotic stress. However, in most cases, two main mechanisms can be highlighted: (i an increased level in xyloglucan endotransglucosylase/hydrolase (XTH and expansin proteins, associated with an increase in the degree of rhamnogalacturonan I branching that maintains cell wall plasticity and (ii an increased cell wall thickening by reinforcement of the secondary wall with hemicellulose and lignin deposition. Taken together, these results show the need to undertake large-scale analyses, using multidisciplinary approaches, to unravel the consequences of stress on the cell wall. This will help identify the key components that could be targeted to improve biomass production under stress conditions.

  18. Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis.

    Science.gov (United States)

    Liu, Xiaokun; Grabherr, Heini M; Willmann, Roland; Kolb, Dagmar; Brunner, Frédéric; Bertsche, Ute; Kühner, Daniel; Franz-Wachtel, Mirita; Amin, Bushra; Felix, Georg; Ongena, Marc; Nürnberger, Thorsten; Gust, Andrea A

    2014-06-23

    Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites.

  19. 2D-immunoblotting analysis of Sporothrix schenckii cell wall

    Directory of Open Access Journals (Sweden)

    Estela Ruiz-Baca

    2011-03-01

    Full Text Available We utilized two-dimensional gel electrophoresis and immunoblotting (2D-immunoblotting with anti-Sporothrix schenckii antibodies to identify antigenic proteins in cell wall preparations obtained from the mycelial and yeast-like morphologies of the fungus. Results showed that a 70-kDa glycoprotein (Gp70 was the major antigen detected in the cell wall of both morphologies and that a 60-kDa glycoprotein was present only in yeast-like cells. In addition to the Gp70, the wall from filament cells showed four proteins with molecular weights of 48, 55, 66 and 67 kDa, some of which exhibited several isoforms. To our knowledge, this is the first 2D-immunoblotting analysis of the S. schenckii cell wall.

  20. Up against the wall: is yeast cell wall integrity ensured by mechanosensing in plasma membrane microdomains?

    Science.gov (United States)

    Kock, Christian; Dufrêne, Yves F; Heinisch, Jürgen J

    2015-02-01

    Yeast cell wall integrity (CWI) signaling serves as a model of the regulation of fungal cell wall synthesis and provides the basis for the development of antifungal drugs. A set of five membrane-spanning sensors (Wsc1 to Wsc3, Mid2, and Mtl1) detect cell surface stress and commence the signaling pathway upon perturbations of either the cell wall structure or the plasma membrane. We here summarize the latest advances in the structure/function relationship primarily of the Wsc1 sensor and critically review the evidence that it acts as a mechanosensor. The relevance and physiological significance of the information obtained for the function of the other CWI sensors, as well as expected future developments, are discussed.

  1. Glycoprotein processing in mutants of HSV-1 that induce cell fusion

    Energy Technology Data Exchange (ETDEWEB)

    Person, S.; Kousoulas, K.G.; Knowles, R.W.; Read, G.S.; Holland, T.C.; Keller, P.M.; Warner, S.C.

    1982-01-01

    The synthesis of viral-specified glycoproteins, and their appearance on cell surfaces, were compared for cells infected either with syncytial mutants of HSV-1 or with the parental strains from which the mutants were derived. The mutants MP and tsB5, representatives of two different viral genes that affect fusion, were employed in these studies. Cells infected with either mutant gave rise to reduce surface labeling of major viral-specified glycoproteins throughout infection relative to the parental strains. Putative glycoprotein precursors were synthesized in similar amounts throughout infection in mutant and wild-type infected cells. However, during a chase period after a pulse of labeling, fully glycosylated species of glycoproteins accumulated more slowly in mutant than in parent infected cells. The effect was clearly visible early in MP-infected cells and by 9 hr after infection for tsB5-infected cells, and increased thereafter. Apparently the decrease in appearance of glycoproteins on the cell surface of mutant-infected cells is due to a decrease in their rate of processing. 26 references, 7 figures.

  2. Cell wall-associated malate dehydrogenase activity from maize roots.

    Science.gov (United States)

    Hadži-Tašković Šukalović, Vesna; Vuletić, Mirjana; Marković, Ksenija; Vučinić, Zeljko

    2011-10-01

    Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn(2+) and Cu(2+) in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn(2+) and Cu(2+) in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.

  3. Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR

    Science.gov (United States)

    Simmons, Thomas J.; Mortimer, Jenny C.; Bernardinelli, Oigres D.; Pöppler, Ann-Christin; Brown, Steven P.; Deazevedo, Eduardo R.; Dupree, Ray; Dupree, Paul

    2016-12-01

    Exploitation of plant lignocellulosic biomass is hampered by our ignorance of the molecular basis for its properties such as strength and digestibility. Xylan, the most prevalent non-cellulosic polysaccharide, binds to cellulose microfibrils. The nature of this interaction remains unclear, despite its importance. Here we show that the majority of xylan, which forms a threefold helical screw in solution, flattens into a twofold helical screw ribbon to bind intimately to cellulose microfibrils in the cell wall. 13C solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, supported by in silico predictions of chemical shifts, shows both two- and threefold screw xylan conformations are present in fresh Arabidopsis stems. The twofold screw xylan is spatially close to cellulose, and has similar rigidity to the cellulose microfibrils, but reverts to the threefold screw conformation in the cellulose-deficient irx3 mutant. The discovery that induced polysaccharide conformation underlies cell wall assembly provides new principles to understand biomass properties.

  4. Substitution of L-fucose by L-galactose in cell walls of arabidopsis mur1

    Energy Technology Data Exchange (ETDEWEB)

    Zablackis, E.; York, W.S.; Pauly, M. [Univ. of Georgia, Athens (United States)

    1996-06-21

    An Arabidopsis thaliana mutant (mur1) has less than 2 percent of the normal amounts of L-fucose in the primary cell walls of aerial portions of the plant. The survival of mur1 plants challenged the hypothesis that fucose is a required component of biologically active oligosaccharides derived from cell wall xyloglucan. However, the replacement of L-fucose (that is, 6-deoxyl-L-galactose) by L-galactose does not detectably alter the biological activity of the oligosaccharides derived from xyloglucan. Thus, essential structural and conformational features of xyloglucan and xyloglucan-derived oligosaccharides are retained when L-galactose replaces L-fucose. 29 refs., 2 figs., 2 tabs.

  5. Large-scale co-expression approach to dissect secondary cell wall formation across plant species

    Directory of Open Access Journals (Sweden)

    Colin eRuprecht

    2011-07-01

    Full Text Available Plant cell walls are complex composites largely consisting of carbohydrate-based polymers, and are generally divided into primary and secondary walls based on content and characteristics. Cellulose microfibrils constitute a major component of both primary and secondary cell walls and are synthesized at the plasma membrane by cellulose synthase (CESA complexes. Several studies in Arabidopsis have demonstrated the power of co-expression analyses to identify new genes associated with secondary wall cellulose biosynthesis. However, across-species comparative co-expression analyses remain largely unexplored. Here, we compared co-expressed gene vicinity networks of primary and secondary wall CESAs in Arabidopsis, barley, rice, poplar, soybean, Medicago and wheat, and identified gene families that are consistently co-regulated with cellulose biosynthesis. In addition to the expected polysaccharide acting enzymes, we also found many gene families associated with cytoskeleton, signaling, transcriptional regulation, oxidation and protein degradation. Based on these analyses, we selected and biochemically analyzed T-DNA insertion lines corresponding to approximately twenty genes from gene families that re-occur in the co-expressed gene vicinity networks of secondary wall CESAs across the seven species. We developed a statistical pipeline using principal component analysis (PCA and optimal clustering based on silhouette width to analyze sugar profiles. One of the mutants, corresponding to a pinoresinol reductase gene, displayed disturbed xylem morphology and held lower levels of lignin molecules. We propose that this type of large-scale co-expression approach, coupled with statistical analysis of the cell wall contents, will be useful to facilitate rapid knowledge transfer across plant species.

  6. Patterns of expression of cell wall related genes in sugarcane

    Directory of Open Access Journals (Sweden)

    Lima D.U.

    2001-01-01

    Full Text Available Our search for genes related to cell wall metabolism in the sugarcane expressed sequence tag (SUCEST database (http://sucest.lbi.dcc.unicamp.br resulted in 3,283 reads (1% of the total reads which were grouped into 459 clusters (potential genes with an average of 7.1 reads per cluster. To more clearly display our correlation coefficients, we constructed surface maps which we used to investigate the relationship between cell wall genes and the sugarcane tissues libraries from which they came. The only significant correlations that we found between cell wall genes and/or their expression within particular libraries were neutral or synergetic. Genes related to cellulose biosynthesis were from the CesA family, and were found to be the most abundant cell wall related genes in the SUCEST database. We found that the highest number of CesA reads came from the root and stem libraries. The genes with the greatest number of reads were those involved in cell wall hydrolases (e.g. beta-1,3-glucanases, xyloglucan endo-beta-transglycosylase, beta-glucosidase and endo-beta-mannanase. Correlation analyses by surface mapping revealed that the expression of genes related to biosynthesis seems to be associated with the hydrolysis of hemicelluloses, pectin hydrolases being mainly associated with xyloglucan hydrolases. The patterns of cell wall related gene expression in sugarcane based on the number of reads per cluster reflected quite well the expected physiological characteristics of the tissues. This is the first work to provide a general view on plant cell wall metabolism through the expression of related genes in almost all the tissues of a plant at the same time. For example, developing flowers behaved similarly to both meristematic tissues and leaf-root transition zone tissues. Besides providing a basis for future research on the mechanisms of plant development which involve the cell wall, our findings will provide valuable tools for plant engineering in the

  7. The Organization Pattern of Root Border-Like Cells of Arabidopsis Is Dependent on Cell Wall Homogalacturonan12[C][W

    Science.gov (United States)

    Durand, Caroline; Vicré-Gibouin, Maïté; Follet-Gueye, Marie Laure; Duponchel, Ludovic; Moreau, Myriam; Lerouge, Patrice; Driouich, Azeddine

    2009-01-01

    Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain attached to each other rather than completely detached from each other, as is usually observed in border cells of many species. Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing less homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1 than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein epitopes, in which the cells are trapped in the vicinity of the root tip. PMID:19448034

  8. Pectin, a versatile polysaccharide present in plant cell walls

    NARCIS (Netherlands)

    Voragen, A.G.J.; Coenen, G.J.; Verhoef, R.P.; Schols, H.A.

    2009-01-01

    Pectin or pectic substances are collective names for a group of closely associated polysaccharides present in plant cell walls where they contribute to complex physiological processes like cell growth and cell differentiation and so determine the integrity and rigidity of plant tissue. They also pla

  9. How the deposition of cellulose microfibrils builds cell wall architecture

    NARCIS (Netherlands)

    Emons, A.M.C.; Mulder, B.M.

    2000-01-01

    Cell walls, the extracytoplasmic matrices of plant cells, consist of an ordered array of cellulose microfibrils embedded in a matrix of polysaccharides and glycoproteins. This construction is reminiscent of steel rods in reinforced concrete. How a cell organizes these ordered textures around itself,

  10. Cell wall deposition during morphogenesis in fucoid algae.

    Science.gov (United States)

    Bisgrove, S R; Kropf, D L

    2001-04-01

    Cell was deposition was investigated during morphogenesis in zygotes of Pelvetia compressa (J. Agardh) De Toni. Young zygotes are spherical and wall is deposited uniformly, but at germination (about 10 h after fertilization) wall deposition becomes localized to the apex of the tip-growing rhizoid. Wall deposition was investigated before and after the initiation of tip growth by disrupting cytoskeleton, secretion or cellulose deposition; effects on wall strength and structure were examined. All three were involved in generating wall strength in both spherical and tip-growing zygotes, but their relative importance were different at the two developmental stages. Much of the wall strength in young zygotes was dependent on F-actin, whereas cellulose and a sulfated component, probably a fucan (F2), were most important in tip growing zygotes. Some treatments had contrasting effects at the two developmental stages; for example, disruption of F-actin or inhibition of secretion weakened walls in spherical zygotes but strengthened those in tip-growing zygotes. Transmission electron microscopic analysis showed that most treatments that altered wall strength induced modifications of internal wall structure.

  11. Calpains are involved in asexual and sexual development, cell wall integrity and pathogenicity of the rice blast fungus.

    Science.gov (United States)

    Liu, Xiao-Hong; Ning, Guo-Ao; Huang, Lu-Yao; Zhao, Ya-Hui; Dong, Bo; Lu, Jian-Ping; Lin, Fu-Cheng

    2016-08-09

    Calpains are ubiquitous and well-conserved proteins that belong to the calcium-dependent, non-lysosomal cysteine protease family. In this study, 8 putative calpains were identified using Pfam domain analysis and BlastP searches in M. oryzae. Three single gene deletion mutants (ΔMocapn7, ΔMocapn9 and ΔMocapn14) and two double gene deletion mutants (ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7) were obtained using the high-throughput gene knockout system. The calpain disruption mutants showed defects in colony characteristics, conidiation, sexual reproduction and cell wall integrity. The mycelia of the ΔMocapn7, ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7 mutants showed reduced pathogenicity on rice and barley.

  12. Autosomal mutants of proton-exposed kidney cells display frequent loss of heterozygosity on nonselected chromosomes.

    Science.gov (United States)

    Grygoryev, Dmytro; Dan, Cristian; Gauny, Stacey; Eckelmann, Bradley; Ohlrich, Anna P; Connolly, Marissa; Lasarev, Michael; Grossi, Gianfranco; Kronenberg, Amy; Turker, Mitchell S

    2014-05-01

    High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton-exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome.

  13. The transcriptional targets of mutant FOXL2 in granulosa cell tumours.

    Directory of Open Access Journals (Sweden)

    Roseanne Rosario

    Full Text Available BACKGROUND: Despite their distinct biology, granulosa cell tumours (GCTs are treated the same as other ovarian tumours. Intriguingly, a recurring somatic mutation in the transcription factor Forkhead Box L2 (FOXL2 402C>G has been found in nearly all GCTs examined. This investigation aims to identify the pathogenicity of mutant FOXL2 by studying its altered transcriptional targets. METHODS: The expression of mutant FOXL2 was reduced in the GCT cell line KGN, and wildtype and mutant FOXL2 were overexpressed in the GCT cell line COV434. Total RNA was hybridised to Affymetrix U133 Plus 2 microarrays. Comparisons were made between the transcriptomes of control cells and cells altered by FOXL2 knockdown and overexpression, to detect potential transcriptional targets of mutant FOXL2. RESULTS: The overexpression of wildtype and mutant FOXL2 in COV434, and the silencing of mutant FOXL2 expression in KGN, has shown that mutant FOXL2 is able to differentially regulate the expression of many genes, including two well known FOXL2 targets, StAR and CYP19A. We have shown that many of the genes regulated by mutant FOXL2 are clustered into functional annotations of cell death, proliferation, and tumourigenesis. Furthermore, TGF-β signalling was found to be enriched when using the gene annotation tools GATHER and GeneSetDB. This enrichment was still significant after performing a robust permutation analysis. CONCLUSION: Given that many of the transcriptional targets of mutant FOXL2 are known TGF-β signalling genes, we suggest that deregulation of this key antiproliferative pathway is one way mutant FOXL2 contributes to the pathogenesis of adult-type GCTs. We believe this pathway should be a target for future therapeutic interventions, if outcomes for women with GCTs are to improve.

  14. Loss of Cell Adhesion Increases Tumorigenic Potential of Polarity Deficient Scribble Mutant Cells.

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    Indrayani Waghmare

    Full Text Available Epithelial polarity genes are important for maintaining tissue architecture, and regulating growth. The Drosophila neoplastic tumor suppressor gene scribble (scrib belongs to the basolateral polarity complex. Loss of scrib results in disruption of its growth regulatory functions, and downregulation or mislocalization of Scrib is correlated to tumor growth. Somatic scribble mutant cells (scrib- surrounded by wild-type cells undergo apoptosis, which can be prevented by introduction of secondary mutations that provide a growth advantage. Using genetic tools in Drosophila, we analyzed the phenotypic effects of loss of scrib in different growth promoting backgrounds. We investigated if a central mechanism that regulates cell adhesion governs the growth and invasive potential of scrib mutant cells. Here we show that increased proliferation, and survival abilities of scrib- cells in different genetic backgrounds affect their differentiation, and intercellular adhesion. Further, loss of scrib is sufficient to cause reduced cell survival, activation of the JNK pathway and a mild reduction of cell adhesion. Our data show that for scrib cells to induce aggressive tumor growth characterized by loss of differentiation, cell adhesion, increased proliferation and invasion, cooperative interactions that derail signaling pathways play an essential role in the mechanisms leading to tumorigenesis. Thus, our study provides new insights on the effects of loss of scrib and the modification of these effects via cooperative interactions that enhance the overall tumorigenic potential of scrib deficient cells.

  15. Role of the plant cell wall in gravity resistance.

    Science.gov (United States)

    Hoson, Takayuki; Wakabayashi, Kazuyuki

    2015-04-01

    Gravity resistance, mechanical resistance to the gravitational force, is a principal graviresponse in plants, comparable to gravitropism. The cell wall is responsible for the final step of gravity resistance. The gravity signal increases the rigidity of the cell wall via the accumulation of its constituents, polymerization of certain matrix polysaccharides due to the suppression of breakdown, stimulation of cross-link formation, and modifications to the wall environment, in a wide range of situations from microgravity in space to hypergravity. Plants thus develop a tough body to resist the gravitational force via an increase in cell wall rigidity and the modification of growth anisotropy. The development of gravity resistance mechanisms has played an important role in the acquisition of responses to various mechanical stresses and the evolution of land plants.

  16. Sorption of volatile phenols by yeast cell walls

    Directory of Open Access Journals (Sweden)

    Nerea Jiménez-Moreno

    2009-01-01

    Full Text Available Nerea Jiménez-Moreno, Carmen Ancín-AzpilicuetaDepartment of Applied Chemistry, Universidad Pública de Navarra, Pamplona, SpainAbstract: Yeast walls can retain different wine compounds and so its use is interesting in order to eliminate harmful substances from the must which affect alcoholic fermentation (medium chain fatty acids or which affect wine quality in a negative way (ethyl phenols, ochratoxin A. The aim of this study was to examine the capacity of commercial yeast cell walls in eliminating volatile phenols (4-ethylphenol and 4-ethylguaiacol from a synthetic wine that contained 1 mg/L of each one of these compounds. The binding of these compounds to the wall was quite fast which would seem to indicate that the yeast wall-volatile compound union is produced in the outer surface layers of this enological additive. The cell walls used reduced the concentration of 4-ethylphenol and 4-ethylguaiacol, although it would seem that on modifying the matrix of the wine the number of free binding sites on the walls is also modified.Keywords: volatile phenols, yeast cell walls, wine, sorption

  17. Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls

    Directory of Open Access Journals (Sweden)

    Canut Hervé

    2006-05-01

    Full Text Available Abstract Background The ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments. Reaching such a goal closely depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific difficulties: (i the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP during the isolation procedure, (ii polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins. Several reported procedures to isolate cell walls for proteomic analyses led to the isolation of a high proportion (more than 50% of predicted intracellular proteins. Since isolated cell walls should hold secreted proteins, one can imagine alternative procedures to prepare cell walls containing a lower proportion of contaminant proteins. Results The rationales of several published procedures to isolate cell walls for proteomics were analyzed, with regard to the bioinformatic-predicted subcellular localization of the identified proteins. Critical steps were revealed: (i homogenization in low ionic strength acid buffer to retain CWP, (ii purification through increasing density cushions, (iii extensive washes with a low ionic strength acid buffer to retain CWP while removing as many cytosolic proteins as possible, and (iv absence of detergents. A new procedure was developed to prepare cell walls from etiolated hypocotyls of Arabidopsis thaliana. After salt extraction, a high proportion of proteins predicted to be secreted was released (73%, belonging to the same functional classes as proteins identified using previously described protocols. Finally, removal of intracellular proteins was obtained using detergents, but their amount represented less than 3% in mass of the total protein extract, based on protein quantification. Conclusion The

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

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    Lori B Huberman

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  20. Another brick in the cell wall: biosynthesis dependent growth model.

    Science.gov (United States)

    Barbacci, Adelin; Lahaye, Marc; Magnenet, Vincent

    2013-01-01

    Expansive growth of plant cell is conditioned by the cell wall ability to extend irreversibly. This process is possible if (i) a tensile stress is developed in the cell wall due to the coupling effect between turgor pressure and the modulation of its mechanical properties through enzymatic and physicochemical reactions and if (ii) new cell wall elements can be synthesized and assembled to the existing wall. In other words, expansive growth is the result of coupling effects between mechanical, thermal and chemical energy. To have a better understanding of this process, models must describe the interplay between physical or mechanical variable with biological events. In this paper we propose a general unified and theoretical framework to model growth in function of energy forms and their coupling. This framework is based on irreversible thermodynamics. It is then applied to model growth of the internodal cell of Chara corallina modulated by changes in pressure and temperature. The results describe accurately cell growth in term of length increment but also in term of cell pectate biosynthesis and incorporation to the expanding wall. Moreover, the classical growth model based on Lockhart's equation such as the one proposed by Ortega, appears as a particular and restrictive case of the more general growth equation developed in this paper.

  1. Another brick in the cell wall: biosynthesis dependent growth model.

    Directory of Open Access Journals (Sweden)

    Adelin Barbacci

    Full Text Available Expansive growth of plant cell is conditioned by the cell wall ability to extend irreversibly. This process is possible if (i a tensile stress is developed in the cell wall due to the coupling effect between turgor pressure and the modulation of its mechanical properties through enzymatic and physicochemical reactions and if (ii new cell wall elements can be synthesized and assembled to the existing wall. In other words, expansive growth is the result of coupling effects between mechanical, thermal and chemical energy. To have a better understanding of this process, models must describe the interplay between physical or mechanical variable with biological events. In this paper we propose a general unified and theoretical framework to model growth in function of energy forms and their coupling. This framework is based on irreversible thermodynamics. It is then applied to model growth of the internodal cell of Chara corallina modulated by changes in pressure and temperature. The results describe accurately cell growth in term of length increment but also in term of cell pectate biosynthesis and incorporation to the expanding wall. Moreover, the classical growth model based on Lockhart's equation such as the one proposed by Ortega, appears as a particular and restrictive case of the more general growth equation developed in this paper.

  2. The actin-related protein Sac1 is required for morphogenesis and cell wall integrity in Candida albicans.

    Science.gov (United States)

    Zhang, Bing; Yu, Qilin; Jia, Chang; Wang, Yuzhou; Xiao, Chenpeng; Dong, Yijie; Xu, Ning; Wang, Lei; Li, Mingchun

    2015-08-01

    Candida albicans is a common pathogenic fungus and has aroused widespread attention recently. Actin cytoskeleton, an important player in polarized growth, protein secretion and organization of cell shape, displays irreplaceable role in hyphal development and cell integrity. In this study, we demonstrated a homologue of Saccharomyces cerevisiae Sac1, in C. albicans. It is a potential PIP phosphatase with Sac domain which is related to actin organization, hyphal development, biofilm formation and cell wall integrity. Deletion of SAC1 did not lead to insitiol-auxotroph phenotype in C. albicans, but this gene rescued the growth defect of S. cerevisiae sac1Δ in the insitiol-free medium. Hyphal induction further revealed the deficiency of sac1Δ/Δ in hyphal development and biofilm formation. Fluorescence observation and real time PCR (RT-PCR) analysis suggested both actin and the hyphal cell wall protein Hwp1 were overexpressed and mislocated in this mutant. Furthermore, cell wall integrity (CWI) was largely affected by deletion of SAC1, due to the hypersensitivity to cell wall stress, changed content and distribution of chitin in the mutant. As a result, the virulence of sac1Δ/Δ was seriously attenuated. Taken together, this study provides evidence that Sac1, as a potential PIP phosphatase, is essential for actin organization, hyphal development, CWI and pathogenicity in C. albicans.

  3. Mutant p53 and mTOR/PKM2 regulation in cancer cells.

    Science.gov (United States)

    Dando, Ilaria; Cordani, Marco; Donadelli, Massimo

    2016-09-01

    Mutations of TP53 gene are the most common feature in aggressive malignant cells. In addition to the loss of the tumor suppressive role of wild-type p53, hotspot mutant p53 isoforms display oncogenic proprieties notoriously referred as gain of functions (GOFs) which result in chemoresistance to therapies, genomic instability, aberrant deregulation of cell cycle progression, invasiveness and enhanced metastatic potential, and finally, in patient poor survival rate. The identification of novel functional oncogenic pathways regulated by mutant p53 represent and intriguing topic for emerging therapies against a broad spectrum of cancer types bearing mutant TP53 gene. Mammalian target of rapamycin (mTOR), as well as pyruvate kinase isoform M2 (PKM2) are master regulators of cancer growth, metabolism, and cell proliferation. Herein, we report that GOF mutant R175H and R273H p53 proteins trigger PKM2 phosphorylation on Tyr 105 through the involvement of mTOR signaling. Our data, together with the newly discovered connection between mutant p53 and mTOR stimulation, raise important implications for the potential therapeutic use of synthetic drugs inhibiting mTOR/PKM2 axis in cancer cells bearing mutant TP53 gene. We further hypothesize that mTOR/PKM2 pathway stimulation serves to sustain the oncogenic activity of mutant p53 through both the enhancement of chemoresistance and of aerobic glycolysis of cancer cells. © 2016 IUBMB Life, 68(9):722-726, 2016.

  4. GDP-mannose pyrophosphorylase is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus.

    Science.gov (United States)

    Jiang, Hechun; Ouyang, Haomiao; Zhou, Hui; Jin, Cheng

    2008-09-01

    GDP-mannose pyrophosphorylase (GMPP) catalyses the synthesis of GDP-mannose, which is the precursor for the mannose residues in glycoconjugates, using mannose 1-phosphate and GTP as substrates. Repression of GMPP in yeast leads to phenotypes including cell lysis, defective cell wall, and failure of polarized growth and cell separation. Although several GMPPs have been isolated and characterized in filamentous fungi, the physiological consequences of their actions are not clear. In this study, Afsrb1, which is a homologue of yeast SRB1/PSA1/VIG9, was identified in the Aspergillus fumigatus genome. The Afsrb1 gene was expressed in Escherichia coli, and recombinant AfSrb1 was functionally confirmed as a GMPP. By the replacement of the native Afsrb1 promoter with an inducible Aspergillus nidulans alcA promoter, the conditional inactivation mutant strain YJ-gmpp was constructed. The presence of 3 % glucose completely blocked transcription of P(alcA)-Afsrb1, and was lethal to strain YJ-gmpp. Repression of Afsrb1 expression in strain YJ-gmpp led to phenotypes including hyphal lysis, defective cell wall, impaired polarity maintenance, and branching site selection. Also, rapid germination and reduced conidiation were documented. However, in contrast to yeast, strain YJ-gmpp retained the ability to direct polarity establishment and septation. Our results showed that the Afsrb1 gene is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus.

  5. Primary Cell Wall Structure in the Evolution of Land Plants

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Investigation of the primary cell walls of lower plants improves our understanding of the cell biology of these organisms but also has the potential to improve our understanding of cell wall structure and function in angiosperms that evolved from lower plants. Cell walls were prepared from eight species, ranging from a moss to advanced gymnosperms, and subjected to sequential chemical extraction to separate the main polysaccharide fractions. The glycosyl compositions of these fractions were then determined by gas chromatography. The results were compared among the eight plants and among data from related studies reported in the existing published reports to identify structural features that have been either highly conserved or clearly modified during evolution. Among the highly conserved features are the presence of a cellulose framework, the presence of certain hemicelluloses such as xyloglucan, and the presence of rhamnogalacturonan Ⅱ, a domain in pectic polysaccharides. Among the modified features are the abundance of mannosyl-containing hemicelluloses and the presence of methylated sugars.

  6. Magnetic domain wall conduits for single cell applications

    DEFF Research Database (Denmark)

    Donolato, Marco; Torti, A.; Kostesha, Natalie;

    2011-01-01

    The ability to trap, manipulate and release single cells on a surface is important both for fundamental studies of cellular processes and for the development of novel lab-on-chip miniaturized tools for biological and medical applications. In this paper we demonstrate how magnetic domain walls...... generated in micro- and nano-structures fabricated on a chip surface can be used to handle single yeast cells labeled with magnetic beads. In detail, first we show that the proposed approach maintains the microorganism viable, as proven by monitoring the division of labeled yeast cells trapped by domain...... walls over 16 hours. Moreover, we demonstrate the controlled transport and release of individual yeast cells via displacement and annihilation of individual domain walls in micro- and nano-sized magnetic structures. These results pave the way to the implementation of magnetic devices based on domain...

  7. Simulated microgravity inhibits cell wall regeneration of Penicillium decumbens protoplasts

    Science.gov (United States)

    Zhao, C.; Sun, Y.; Yi, Z. C.; Rong, L.; Zhuang, F. Y.; Fan, Y. B.

    2010-09-01

    This work compares cell wall regeneration from protoplasts of the fungus Penicillium decumbens under rotary culture (simulated microgravity) and stationary cultures. Using an optimized lytic enzyme mixture, protoplasts were successfully released with a yield of 5.3 × 10 5 cells/mL. Under simulated microgravity conditions, the protoplast regeneration efficiency was 33.8%, lower than 44.9% under stationary conditions. Laser scanning confocal microscopy gave direct evidence for reduced formation of polysaccharides under simulated conditions. Scanning electron microscopy showed the delayed process of cell wall regeneration by simulated microgravity. The delayed regeneration of P. decumbens cell wall under simulated microgravity was likely caused by the inhibition of polysaccharide synthesis. This research contributes to the understanding of how gravitational loads affect morphological and physiological processes of fungi.

  8. Analyzing Cell Wall Elasticity After Hormone Treatment: An Example Using Tobacco BY-2 Cells and Auxin.

    Science.gov (United States)

    Braybrook, Siobhan A

    2017-01-01

    Atomic force microscopy, and related nano-indentation techniques, is a valuable tool for analyzing the elastic properties of plant cell walls as they relate to changes in cell wall chemistry, changes in development, and response to hormones. Within this chapter I will describe a method for analyzing the effect of the phytohormone auxin on the cell wall elasticity of tobacco BY-2 cells. This general method may be easily altered for different experimental systems and hormones of interest.

  9. Diffusion of an organic cation into root cell walls.

    Science.gov (United States)

    Meychik, N R; Yermakov, I P; Prokoptseva, O S

    2003-07-01

    Uptake of a cationic dye (methylene blue) by isolated root cell walls, roots of whole transpiring seedlings, and excised roots was investigated using 7-day-old seedlings of cucumber, maize, and wheat. The number of ionogenic groups per 1 g dry and wet weight of the root cell walls, their swelling capacity (K(cw)), time-dependence of methylene blue (M(cw)) ion exchange capacity, and diffusion coefficients of the cation diffusion in the polymer matrix of the cell walls (D(cw)) were determined. The M(cw) value depended on pH (or carboxyl group dissociation); it changed in accordance with the number of carboxyl groups per 1 g cell wall dry weight. This parameter decreased in the order: cucumber > wheat > maize. For description of experimental kinetic curves and calculation of cation diffusion coefficients, the equation for ion diffusion into a cylinder of infinite length was used. The chosen model adequately described cation diffusion in cell walls and roots. Diffusion coefficient values for cucumber, wheat, and maize were 3.1*10(-8), 1.3*10(-8), and 8.4*10(-8) cm(2)/sec, respectively. There was a statistically significant linear dependence between K(cw) and D(cw) values, which characterize the same property of the polymer matrix, rigidity of its polymer structure or the degree of cross-linkage or permeability. This also confirms the right choice of the model selected for calculation of methylene blue diffusion coefficients, because K(cw) and D(cw) values were obtained in independent experiments. The coefficients determined for methylene blue diffusion in transpiring seedling roots (D(ts)) and excised roots (D(er)) depended on the plant species. The rate of methylene blue diffusion into the excised roots was either 1.5-fold lower (cucumber) or 3-4-times lower (maize, wheat) than in cell walls. The values of diffusion coefficients in roots of whole seedlings were comparable which those for the cell walls. On the basis of the experimental data and results of calculations

  10. Growth and cell wall changes in rice roots during spaceflight.

    Science.gov (United States)

    Hoson, Takayuki; Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Tanimoto, Eiichi

    2003-08-01

    We analyzed the changes in growth and cell wall properties of roots of rice (Oryza sativa L. cv. Koshihikari) grown for 68.5, 91.5, and 136 h during the Space Shuttle STS-95 mission. In space, most of rice roots elongated in a direction forming a constant mean angle of about 55 degrees with the perpendicular base line away from the caryopsis in the early phase of growth, but later the roots grew in various directions, including away from the agar medium. In space, elongation growth of roots was stimulated. On the other hand, some of elasticity moduli and viscosity coefficients were higher in roots grown in space than on the ground, suggesting that the cell wall of space-grown roots has a lower capacity to expand than the controls. The levels of both cellulose and the matrix polysaccharides per unit length of roots decreased greatly, whereas the ratio of the high molecular mass polysaccharides in the hemicellulose fraction increased in space-grown roots. The prominent thinning of the cell wall could overwhelm the disadvantageous changes in the cell wall mechanical properties, leading to the stimulation of elongation growth in rice roots in space. Thus, growth and the cell wall properties of rice roots were strongly modified under microgravity conditions during spaceflight.

  11. The Structure of Plant Cell Walls: II. The Hemicellulose of the Walls of Suspension-cultured Sycamore Cells.

    Science.gov (United States)

    Bauer, W D; Talmadge, K W; Keegstra, K; Albersheim, P

    1973-01-01

    The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed "amyloid" xyloglucans.Xyloglucan-or fragments of xyloglucan-and acidic fragments of the pectic polysaccharides are released from endopolygalacturonase-pretreated sycamore walls by treatment of these walls with 8 m urea, endoglucanase, or 0.5 n NaOH. Some of the xyloglucan thus released is found to cochromatograph with the acidic pectic fragments on diethylaminoethyl Sephadex. The chemical or enzymic treatments required for the release of xyloglucan from the walls and the cochromatography of xyloglucan with the acidic pectic fragments indicate that xyloglucan is covalently linked to the pectic polysaccharides and is noncovalently bound to the cellulose fibrils of the sycamore cell wall.The molecular structure of sycamore xyloglucan was characterized by methylation analysis of the oligosaccharides obtained by endoglucanase treatment of the polymer. The structure of the polymer is based on a repeating heptasaccharide unit which consists of 4 residues of beta-1-4-linked glucose and 3 residues of terminal xylose. A single xylose residue is glycosidically linked to carbon 6 of 3 of the glucosyl residues.

  12. Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG.

    Directory of Open Access Journals (Sweden)

    Ingmar J J Claes

    Full Text Available Lactobacillus rhamnosus GG (LGG produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75 and Msp2 (LGG_00031 or p40, which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.

  13. Glycosylation of a Fasciclin-Like Arabinogalactan-Protein (SOS5) Mediates Root Growth and Seed Mucilage Adherence via a Cell Wall Receptor-Like Kinase (FEI1/FEI2) Pathway in Arabidopsis.

    Science.gov (United States)

    Basu, Debarati; Tian, Lu; Debrosse, Tayler; Poirier, Emily; Emch, Kirk; Herock, Hayley; Travers, Andrew; Showalter, Allan M

    2016-01-01

    Fundamental processes that underpin plant growth and development depend crucially on the action and assembly of the cell wall, a dynamic structure that changes in response to both developmental and environmental cues. While much is known about cell wall structure and biosynthesis, much less is known about the functions of the individual wall components, particularly with respect to their potential roles in cellular signaling. Loss-of-function mutants of two arabinogalactan-protein (AGP)-specific galactosyltransferases namely, GALT2 and GALT5, confer pleiotropic growth and development phenotypes indicating the important contributions of carbohydrate moieties towards AGP function. Notably, galt2galt5 double mutants displayed impaired root growth and root tip swelling in response to salt, likely as a result of decreased cellulose synthesis. These mutants phenocopy a salt-overly sensitive mutant called sos5, which lacks a fasciclin-like AGP (SOS5/FLA4) as well as a fei1fei2 double mutant, which lacks two cell wall-associated leucine-rich repeat receptor-like kinases. Additionally, galt2gal5 as well as sos5 and fei2 showed reduced seed mucilage adherence. Quintuple galt2galt5sos5fei1fei2 mutants were produced and provided evidence that these genes act in a single, linear genetic pathway. Further genetic and biochemical analysis of the quintuple mutant demonstrated involvement of these genes with the interplay between cellulose biosynthesis and two plant growth regulators, ethylene and ABA, in modulating root cell wall integrity.

  14. Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant

    KAUST Repository

    Hudik, Elodie

    2014-07-18

    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.

  15. Chloroplast dysfunction causes multiple defects in cell cycle progression in the Arabidopsis crumpled leaf mutant.

    Science.gov (United States)

    Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2014-09-01

    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.

  16. Transcriptional Wiring of Cell Wall-Related Genes in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Marek Mutwil; Colin Ruprecht; Federico M. Giorgi; Martin Bringmann; Bj(o)rn Usadel; Staffan Persson

    2009-01-01

    Transcriptional coordination, or co-expression, of genes may signify functional relatedness of the correspond-ing proteins. For example, several genes involved in secondary cell wall cellulose biosynthesis are co-expressed with genes engaged in the synthesis of xylan, which is a major component of the secondary cell wall. To extend these types of anal-yses, we investigated the co-expression relationships of all Carbohydrate-Active enZYmes (CAZy)-related genes for Arabidopsis thaliana. Thus, the intention was to transcriptionally link different cell wall-related processes to each other, and also to other biological functions. To facilitate easy manual inspection, we have displayed these interactions as networks and matrices, and created a web-based interface (http://aranet.mpimp-golm.mpg.de/corecarb) containing downloadable files for all the transcriptional associations.

  17. Fluorescent Probes for Exploring Plant Cell Wall Deconstruction: A Review

    Directory of Open Access Journals (Sweden)

    Gabriel Paës

    2014-07-01

    Full Text Available Plant biomass is a potential resource of chemicals, new materials and biofuels that could reduce our dependency on fossil carbon, thus decreasing the greenhouse effect. However, due to its chemical and structural complexity, plant biomass is recalcitrant to green biological transformation by enzymes, preventing the establishment of integrated bio-refineries. In order to gain more knowledge in the architecture of plant cell wall to facilitate their deconstruction, many fluorescent probes bearing various fluorophores have been devised and used successfully to reveal the changes in structural motifs during plant biomass deconstruction, and the molecular interactions between enzymes and plant cell wall polymers. Fluorescent probes are thus relevant tools to explore plant cell wall deconstruction.

  18. The role of the cell wall in fungal pathogenesis.

    Science.gov (United States)

    Arana, David M; Prieto, Daniel; Román, Elvira; Nombela, César; Alonso-Monge, Rebeca; Pla, Jesús

    2009-05-01

    Fungal infections are a serious health problem. In recent years, basic research is focusing on the identification of fungal virulence factors as promising targets for the development of novel antifungals. The wall, as the most external cellular component, plays a crucial role in the interaction with host cells mediating processes such as adhesion or phagocytosis that are essential during infection. Specific components of the cell wall (called PAMPs) interact with specific receptors in the immune cell (called PRRs), triggering responses whose molecular mechanisms are being elucidated. We review here the main structural carbohydrate components of the fungal wall (glucan, mannan and chitin), how their biogenesis takes place in fungi and the specific receptors that they interact with. Different model fungal pathogens are chosen to illustrate the functional consequences of this interaction. Finally, the identification of the key components will have important consequences in the future and will allow better approaches to treat fungal infections.

  19. Identification of a novel arabinofuranosyltransferase (AftA) involved in cell wall arabinan biosynthesis in Mycobacterium tuberculosis.

    Science.gov (United States)

    Alderwick, Luke J; Seidel, Mathias; Sahm, Hermann; Besra, Gurdyal S; Eggeling, Lothar

    2006-06-09

    The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. Following a detailed bioinformatics analysis of genes surrounding the conserved emb locus, we present the identification and characterization of a novel arabinofuranosyltransferase AftA (Rv3792). The enzyme catalyzes the addition of the first key arabinofuranosyl residue from the sugar donor beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to the galactan domain of the cell wall, thus "priming" the galactan for further elaboration by the arabinofuranosyltransferases. Because aftA is an essential gene in M. tuberculosis, we deleted its orthologue in Corynebacterium glutamicum to produce a slow growing but viable mutant. Analysis of its cell wall revealed the complete absence of arabinose resulting in a truncated cell wall structure possessing only a galactan core with a concomitant loss of cell wall-bound mycolates. Complementation of the mutant was fully restored to the wild type phenotype by Cg-aftA. In addition, by developing an in vitro assay using recombinant Escherichia coli expressing Mt-aftA and use of cell wall galactan as an acceptor, we demonstrated the transfer of arabinose from beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to galactan, and unlike the Mt-Emb proteins, Mt-AftA was not inhibited by ethambutol. This newly discovered glycosyltransferase represents an attractive drug target for further exploitation by chemotherapeutic intervention.

  20. A novel role of the ferric reductase Cfl1 in cell wall integrity, mitochondrial function, and invasion to host cells in Candida albicans.

    Science.gov (United States)

    Yu, Qilin; Dong, Yijie; Xu, Ning; Qian, Kefan; Chen, Yulu; Zhang, Biao; Xing, Laijun; Li, Mingchun

    2014-11-01

    Candida albicans is an important opportunistic pathogen, causing both superficial mucosal infections and life-threatening systemic diseases. Iron acquisition is an important factor for pathogen-host interaction and also a significant element for the pathogenicity of this organism. Ferric reductases, which convert ferric iron into ferrous iron, are important components of the high-affinity iron uptake system. Sequence analyses have identified at least 17 putative ferric reductase genes in C. albicans genome. CFL1 was the first ferric reductase identified in C. albicans. However, little is known about its roles in C. albicans physiology and pathogenicity. In this study, we found that disruption of CFL1 led to hypersensitivity to chemical and physical cell wall stresses, activation of the cell wall integrity (CWI) pathway, abnormal cell wall composition, and enhanced secretion, indicating a defect in CWI in this mutant. Moreover, this mutant showed abnormal mitochondrial activity and morphology, suggesting a link between ferric reductases and mitochondrial function. In addition, this mutant displayed decreased ability of adhesion to both the polystyrene microplates and buccal epithelial cells and invasion of host epithelial cells. These findings revealed a novel role of C. albicans Cfl1 in maintenance of CWI, mitochondrial function, and interaction between this pathogen and the host.

  1. Cdc48 and cofactors Npl4-Ufd1 are important for G1 progression during heat stress by maintaining cell wall integrity in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Meng-Ti Hsieh

    Full Text Available The ubiquitin-selective chaperone Cdc48, a member of the AAA (ATPase Associated with various cellular Activities ATPase superfamily, is involved in many processes, including endoplasmic reticulum-associated degradation (ERAD, ubiquitin- and proteasome-mediated protein degradation, and mitosis. Although Cdc48 was originally isolated as a cell cycle mutant in the budding yeast Saccharomyces cerevisiae, its cell cycle functions have not been well appreciated. We found that temperature-sensitive cdc48-3 mutant is largely arrested at mitosis at 37°C, whereas the mutant is also delayed in G1 progression at 38.5°C. Reporter assays show that the promoter activity of G1 cyclin CLN1, but not CLN2, is reduced in cdc48-3 at 38.5°C. The cofactor npl4-1 and ufd1-2 mutants also exhibit G1 delay and reduced CLN1 promoter activity at 38.5°C, suggesting that Npl4-Ufd1 complex mediates the function of Cdc48 at G1. The G1 delay of cdc48-3 at 38.5°C is a consequence of cell wall defect that over-activates Mpk1, a MAPK family member important for cell wall integrity in response to stress conditions including heat shock. cdc48-3 is hypersensitive to cell wall perturbing agents and is synthetic-sick with mutations in the cell wall integrity signaling pathway. Our results suggest that the cell wall defect in cdc48-3 is exacerbated by heat shock, which sustains Mpk1 activity to block G1 progression. Thus, Cdc48-Npl4-Ufd1 is important for the maintenance of cell wall integrity in order for normal cell growth and division.

  2. Characters of Fractal Ultrastructure in Wood Cell Wall

    Institute of Scientific and Technical Information of China (English)

    LI Beimei; ZHAO Guangjie

    2006-01-01

    Fractal theory was introduced in order to describe the ultrastructure of wood cell wall in this paper.The cellulose chain clusters around nano-scale were viewed as a fractal object that consists of many fibrillar structural units with different scales including microfibrils.On the basis of the morphological data of wood cell wall.fractal dimensions of multi-level fibrillar structural units were calculated by fractal-geometry approach,and then the morphological and structural characteristics of fibers as well as the influences on wood properties were investigated according to the dimensions.Besides,the fractal self-nesting character of the ultrastruture was also analyzed.

  3. Hematopoietic Stem Cells Expansionin Rotating Wall Vessel

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    1 IntroductionClinical trials have demonstrated that ex vivo expanded hematopoietic stem cells (HSCs) and progenitors offer great promise in reconstituting in vivo hematopoiesis in patients who have undergone intensive chemotherapy. It is therefore necessary to develop a clinical-scale culture system to provide the expanded HSCs and progenitors. Static culture systems such as T-flasks and gas-permeable blood bags are the most widely used culture devices for expanding hematopoietic cells. But they reveal sev...

  4. Meiotic and Mitotic Cell Cycle Mutants Involved in Gametophyte Development in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Jingjing Liu; Li-Jia Qu

    2008-01-01

    The alternation between diploid and haploid generations is fundamentalin the life cycles of both animals and plants.The meiotic cell cycle is common to both animals and plants gamete formation, but in animals the products of meiosis are gametes,whereas for most plants,subsequent mitotic cell cycles are needed for their formation. Clarifying the regulatory mechanisms of mitotic cell cycle progression during gametophyte development will help understanding of sexual reproduction in plants.Many mutants defective in gametophyte development and,in particular,many meiotic and mitotic cell cycle mutants in Arabidopsis male and female gametophyte development were identified through both forward and reverse genetics approaches.

  5. Ligand dependent restoration of human TLR3 signaling and death in p53 mutant cells.

    Science.gov (United States)

    Menendez, Daniel; Lowe, Julie M; Snipe, Joyce; Resnick, Michael A

    2016-09-20

    Diversity within the p53 transcriptional network can arise from a matrix of changes that include target response element sequences and p53 expression level variations. We previously found that wild type p53 (WT p53) can regulate expression of most innate immune-related Toll-like-receptor genes (TLRs) in human cells, thereby affecting immune responses. Since many tumor-associated p53 mutants exhibit change-of-spectrum transactivation from various p53 targets, we examined the ability of twenty-five p53 mutants to activate endogenous expression of the TLR gene family in p53 null human cancer cell lines following transfection with p53 mutant expression vectors. While many mutants retained the ability to drive TLR expression at WT levels, others exhibited null, limited, or change-of-spectrum transactivation of TLR genes. Using TLR3 signaling as a model, we show that some cancer-associated p53 mutants amplify cytokine, chemokine and apoptotic responses after stimulation by the cognate ligand poly(I:C). Furthermore, restoration of WT p53 activity for loss-of-function p53 mutants by the p53 reactivating drug RITA restored p53 regulation of TLR3 gene expression and enhanced DNA damage-induced apoptosis via TLR3 signaling. Overall, our findings have many implications for understanding the impact of WT and mutant p53 in immunological responses and cancer therapy.

  6. Functional rescue of a kidney anion exchanger 1 trafficking mutant in renal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Carmen Y S Chu

    Full Text Available Mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1 can cause distal renal tubular acidosis (dRTA, a disease often due to mis-trafficking of the mutant protein. In this study, we investigated whether trafficking of a Golgi-retained dRTA mutant, G701D kAE1, or two dRTA mutants retained in the endoplasmic reticulum, C479W and R589H kAE1, could be functionally rescued to the plasma membrane of Madin-Darby Canine Kidney (MDCK cells. Treatments with DMSO, glycerol, the corrector VX-809, or low temperature incubations restored the basolateral trafficking of G701D kAE1 mutant. These treatments had no significant rescuing effect on trafficking of the mis-folded C479W or R589H kAE1 mutants. DMSO was the only treatment that partially restored G701D kAE1 function in the plasma membrane of MDCK cells. Our experiments show that trafficking of intracellularly retained dRTA kAE1 mutants can be partially restored, and that one chemical treatment rescued both trafficking and function of a dRTA mutant. These studies provide an opportunity to develop alternative therapeutic solutions for dRTA patients.

  7. Phagocytic properties of lung alveolar wall cells

    Directory of Open Access Journals (Sweden)

    Tanaka,Akisuke

    1974-04-01

    Full Text Available For the purpose to define the mechanism of heavy metal intoxication by inhalation, morphologic observations were made on rat lungs after nasal instillation of iron colloid particles of positive and negative electric charges. Histochemical observation was also made on the liver and spleen of these animals. The instilled iron colloid particles reach the alveolar cavity easily, as can be seen in the tissue sections stained by Prussian blue reaction. Alveolar macrophages do take up them avidly both of positive and negative charges, though much less the positive particles than negative ones. In contrast, the alveolar epithelial cells take up solely positive particles by phagocytosis but not negative ones. Electron microscope observation revealed that the positive particles are ingested by Type I epithelial cells by pinocytosis and by Type II cells by phagocytosis as well. Then the iron colloid particles are transferred into the basement membrane by exocytosis. Travelling through the basement membrane they are again taken up by capillary endothelial cells by phagocytosis. Some particles were found in the intercellular clefts of capillary endothelial cells but not any iron colloid particles in the intercellular spaces of epithelial cells and in the capillary lumen. However, the liver and spleen tissues of the animals given iron colloid showed a strong positive iron reaction. On the basis of these observations, the mechanism of acute intoxication by inhaling heavy metal dusts like lead fume is discussed from the view point of selective uptake of alveolar epithelial and capillary endothelial cells for the particles of the positive electric cha'rge.

  8. Cell wall pectic arabinans influence the mechanical properties of Arabidopsis thaliana inflorescence stems and their response to mechanical stress.

    Science.gov (United States)

    Verhertbruggen, Yves; Marcus, Susan E; Chen, Jianshe; Knox, J Paul

    2013-08-01

    Little is known of the dynamics of plant cell wall matrix polysaccharides in response to the impact of mechanical stress on plant organs. The capacity of the imposition of a mechanical stress (periodic brushing) to reduce the height of the inflorescence stem of Arabidopsis thaliana seedlings has been used to study the role of pectic arabinans in the mechanical properties and stress responsiveness of a plant organ. The arabinan-deficient-1 (arad1) mutation that affects arabinan structures in epidermal cell walls of inflorescence stems is demonstrated to reduce the impact on inflorescence stem heights caused by mechanical stress. The arabinan-deficient-2 (arad2) mutation, that does not have detectable impact on arabinan structures, is also shown to reduce the impact on stem heights caused by mechanical stress. The LM13 linear arabinan epitope is specifically detected in epidermal cell walls of the younger, flexible regions of inflorescence stems and increases in abundance at the base of inflorescence stems in response to an imposed mechanical stress. The strain (percentage deformation) of stem epidermal cells in the double mutant arad1 × arad2 is lower in unbrushed plants than in wild-type plants, but rises to wild-type levels in response to brushing. The study demonstrates the complexity of arabinan structures within plant cell walls and also that their contribution to cell wall mechanical properties is a factor influencing responsiveness to mechanical stress.

  9. Bst1 is required for Candida albicans infecting host via facilitating cell wall anchorage of Glycosylphosphatidyl inositol anchored proteins

    Science.gov (United States)

    Liu, Wei; Zou, Zui; Huang, Xin; Shen, Hui; He, Li Juan; Chen, Si Min; Li, Li Ping; Yan, Lan; Zhang, Shi Qun; Zhang, Jun Dong; Xu, Zheng; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

    2016-01-01

    Glycosylphosphatidyl inositol anchored proteins (GPI-APs) on fungal cell wall are essential for invasive infections. While the function of inositol deacylation of GPI-APs in mammalian cells has been previously characterized the impact of inositol deacylation in fungi and implications to host infection remains largely unexplored. Herein we describe our identification of BST1, an inositol deacylase of GPI-Aps in Candida albicans, was critical for GPI-APs cell wall attachment and host infection. BST1-deficient C. albicans (bst1Δ/Δ) was associated with severely impaired cell wall anchorage of GPI-APs and subsequen unmasked β-(1,3)-glucan. Consistent with the aberrant cell wall structures, bst1Δ/Δ strain did not display an invasive ability and could be recognized more efficiently by host immune systems. Moreover, BST1 null mutants or those expressing Bst1 variants did not display inositol deacylation activity and exhibited severely attenuated virulence and reduced organic colonization in a murine systemic candidiasis model. Thus, Bst1 can facilitate cell wall anchorage of GPI-APs in C. albicans by inositol deacylation, and is critical for host invasion and immune escape. PMID:27708385

  10. Gravity resistance, another graviresponse in plants - role of microtubule-membrane-cell wall continuum

    Science.gov (United States)

    Hoson, T.; Saito, Y.; Usui, S.; Soga, K.; Wakabayashi, K.

    Resistance to the gravitational force has been a serious problem for plants to survive on land, after they first went ashore more than 400 million years ago. Thus, gravity resistance is the principal graviresponse in plants comparable to gravitropism. Nevertheless, only limited information has been obtained for this second gravity response. We have examined the mechanism of gravity resistance using hypergravity conditions produced by centrifugation. The results led a hypothesis on the mechanism of plant resistance to the gravitational force that the plant constructs a tough body by increasing the cell wall rigidity, which are brought about by modification of the cell wall metabolism and cell wall environment, especially pH. The hypothesis was further supported by space experiments during the Space Shuttle STS-95 mission. On the other hand, we have shown that gravity signal may be perceived by mechanoreceptors (mechanosensitive ion channels) on the plasma membrane and amyloplast sedimentation in statocytes is not involved in gravity resistance. Moreover, hypergravity treatment increased the expression levels of genes encoding alpha-tubulin, a component of microtubules and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of terpenoids such as membrane sterols. The expression of HMGR and alpha- and beta-tubulin genes increased within several hours after hypergravity treatment, depending on the magnitude of gravity. The determination of levels of gene products as well as the analysis with knockout mutants of these genes by T-DNA insertions in Arabidopsis supports the involvement of both membrane sterols and microtubules in gravity resistance. These results suggest that structural or physiological continuum of microtubule-cell membrane-cell wall is responsible for plant resistance to the gravitational force.

  11. Control of the C. albicans cell wall damage response by transcriptional regulator Cas5.

    Directory of Open Access Journals (Sweden)

    Vincent M Bruno

    2006-03-01

    Full Text Available The fungal cell wall is vital for growth, development, and interaction of cells with their environment. The response to cell wall damage is well understood from studies in the budding yeast Saccharomyces cerevisiae, where numerous cell wall integrity (CWI genes are activated by transcription factor ScRlm1. Prior evidence suggests the hypothesis that both response and regulation may be conserved in the major fungal pathogen Candida albicans. We have tested this hypothesis by using a new C. albicans genetic resource: we have screened mutants defective in putative transcription factor genes for sensitivity to the cell wall biosynthesis inhibitor caspofungin. We find that the zinc finger protein CaCas5, which lacks a unique ortholog in S. cerevisiae, governs expression of many CWI genes. CaRlm1 has a modest role in this response. The transcriptional coactivator CaAda2 is also required for expression of many CaCas5-dependent genes, as expected if CaCas5 recruits CaAda2 to activate target gene transcription. Many caspofungin-induced C. albicans genes specify endoplasmic reticulum and secretion functions. Such genes are not induced in S. cerevisiae, but promote its growth in caspofungin. We have used a new resource to identify a key C. albicans transcriptional regulator of CWI genes and antifungal sensitivity. Our gene expression findings indicate that both divergent and conserved response genes may have significant functional roles. Our strategy may be broadly useful for identification of pathogen-specific regulatory pathways and critical response genes.

  12. Mitochondrial mutant cells are hypersensitive to ionizing radiation, phleomycin and mitomycin C

    Energy Technology Data Exchange (ETDEWEB)

    Kulkarni, Rohan; Reither, Adrian; Thomas, Robert A. [Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Suite 1370, Detroit, MI 48202 (United States); Tucker, James D., E-mail: jtucker@biology.biosci.wayne.edu [Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Suite 1370, Detroit, MI 48202 (United States)

    2009-04-26

    Mitochondrial DNA (mtDNA) is an important contributor to the ATP-generating oxidative phosphorylation complex. Single nucleotide mutations in mitochondrial genes involved in ATP synthesis result in a broad range of diseases. Leber optic atrophy and Leigh's syndrome are two such diseases arising from point mutations in the mitochondrial genome. Here, ionizing radiation, phleomycin and mitomycin C (MMC) were used to induce structural chromosomal aberrations in Leber's and Leigh's cells to investigate how these mitochondrial mutations affect the cell's DNA repair processes. Because of the energy deprivation that results from mitochondrial mutations, we hypothesized that these mutant cells would demonstrate hypersensitivity when exposed to oxidative and genotoxic stress and we also expected that these cells would not be able to repair nuclear DNA damage as efficiently as normal cells. As a consequence, these mutant cells are expected to show increased levels of DNA damage, longer cell cycle delays and increased levels of cell death. Following acute radiation exposure these mutant cells showed an increase in the number of chromosomal aberrations and decreased mitotic indices when compared with normal human lymphoblastoid cells with wild-type mtDNA. When exposed to phleomycin or MMC, the mitochondrial mutant cells again showed hypersensitivity and decreased mitotic indices compared to normal cells. These results suggest that Leber's and Leigh's cells have an impaired ability to cope with oxidative and genotoxic stress. These observations may help explain the role of ATP generation in understanding the enhanced sensitivity of mitochondrial mutant cells to cancer therapeutic agents and to adverse environmental exposure, suggesting that individuals with mtDNA mutations may be at a greater risk for cancer and other diseases that result from an accumulation of nuclear DNA damage.

  13. Bacterial Cell Wall Growth, Shape and Division

    NARCIS (Netherlands)

    Derouaux, A.; Terrak, M.; den Blaauwen, T.; Vollmer, W.; Remaut, H.; Fronzes, R.

    2014-01-01

    The shape of a bacterial cell is maintained by its peptidoglycan sacculus that completely surrounds the cytoplasmic membrane. During growth the sacculus is enlarged by peptidoglycan synthesis complexes that are controlled by components linked to the cytoskeleton and, in Gram-negative bacteria, by ou

  14. Cell wall modification in grapevine cells in response to UV stress investigated by atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lesniewska, E.; Adrian, M.; Klinguer, A.; Pugin, A

    2004-08-15

    Despite cell wall reinforcement being a well-known defence mechanism of plants, it remains poorly characterized from a physical point of view. The objective of this work was to further describe this mechanism. Vitis vinifera cv Gamay cells were treated with UV-light (254 nm), a well-known elicitor of defence mechanisms in grapevines, and physical cell wall modifications were observed using the atomic force microscopy (AFM) under native conditions. The grapevine cell suspensions were continuously observed in their culture medium from 30 min to 24 h after elicitation. In the beginning, cellulose fibrils covered by a matrix surrounded the control and treated cells. After 3 h, the elicited cells displayed sprouted expansions around the cell wall that correspond to pectin chains. These expansions were not observed on untreated grapevine cells. The AFM tip was used to determine the average surface elastic modulus of cell wall that account for cell wall mechanical properties. The elasticity is diminished in UV-treated cells. In a comparative study, grapevine cells showed the same decrease in cell wall elasticity when treated with a fungal biotic elicitor of defence response. These results demonstrate cell wall strengthening by UV stress.

  15. Cell wall structure and function in lactic acid bacteria.

    Science.gov (United States)

    Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius

    2014-08-29

    The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.

  16. In planta modification of the potato tuber cell wall

    NARCIS (Netherlands)

    Oomen, R.J.F.J.

    2003-01-01

    Apart from its well known uses in the human diet a large amount of the grown potatoes (about one third in the Netherlands) is used for the isolation of starch which is used in several food and non-food applications. The cell wall fibres comprise a large portion of the waste material remaining after

  17. Characterisation of cell wall polysaccharides in bilberries and black currants

    NARCIS (Netherlands)

    Hilz, H.

    2007-01-01

    During berry juice production, polysaccharides are released from the cell walls and cause thickening and high viscosity when the berries are mashed. Consequences are a low juice yield and a poor colour. This can be prevented by the use of enzymes that degrade these polysaccharides. To use these enzy

  18. Analyzing the complex machinery of cell wall biosynthesis

    NARCIS (Netherlands)

    Timmers, J.F.P.

    2009-01-01

    The plant cell wall polymers make up most of the plant biomass and provide the raw material for many economically important products including food, feed, bio-materials, chemicals, textiles, and biofuel. This broad range of functions and applications make the biosynthesis of these polysaccharides a

  19. Evidence for a Melanin Cell Wall Component in Pneumocystis carinii

    OpenAIRE

    Icenhour, Crystal R.; Kottom, Theodore J.; Limper, Andrew H

    2003-01-01

    Fluorescein isothiocyanate-labeled monoclonal antibodies specific for fungal melanin were used in this study to visualize melanin-like components of the Pneumocystis carinii cell wall. A colorimetric enzyme assay confirmed these findings. This is the first report of melanin-like pigments in Pneumocystis.

  20. The role of the cell wall in plant immunity

    DEFF Research Database (Denmark)

    Malinovsky, Frederikke Gro; Fangel, Jonatan Ulrik; Willats, William George Tycho

    2014-01-01

    The battle between plants and microbes is evolutionarily ancient, highly complex, and often co-dependent. A primary challenge for microbes is to breach the physical barrier of host cell walls whilst avoiding detection by the plant's immune receptors. While some receptors sense conserved microbial...

  1. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides

    NARCIS (Netherlands)

    Vries, de R.P.; Visser, J.

    2001-01-01

    Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a m

  2. Characterisation of cell-wall polysaccharides from mandarin segment membranes

    NARCIS (Netherlands)

    Coll-Almela, L.; Saura-Lopez, D.; Laencina-Sanchez, J.; Schols, H.A.; Voragen, A.G.J.; Ros-García, J.M.

    2015-01-01

    In an attempt to develop a process of enzymatic peeling of mandarin segments suitable for use on an industrial scale, the cell wall fraction of the segment membrane of Satsuma mandarin fruits was extracted to obtain a chelating agent-soluble pectin fraction (ChSS), a dilute sodium hydroxide-soluble

  3. Polymer mobility in cell walls of cucumber hypocotyls

    Science.gov (United States)

    Fenwick, K. M.; Apperley, D. C.; Cosgrove, D. J.; Jarvis, M. C.

    1999-01-01

    Cell walls were prepared from the growing region of cucumber (Cucumis sativus) hypocotyls and examined by solid-state 13C NMR spectroscopy, in both enzymically active and inactivated states. The rigidity of individual polymer segments within the hydrated cell walls was assessed from the proton magnetic relaxation parameter, T2, and from the kinetics of cross-polarisation from 1H to 13C. The microfibrils, including most of the xyloglucan in the cell wall, as well as cellulose, behaved as very rigid solids. A minor xyloglucan fraction, which may correspond to cross-links between microfibrils, shared a lower level of rigidity with some of the pectic galacturonan. Other pectins, including most of the galactan side-chain residues of rhamnogalacturonan I, were much more mobile and behaved in a manner intermediate between the solid and liquid states. The only difference observed between the enzymically active and inactive cell walls, was the loss of a highly mobile, methyl-esterified galacturonan fraction, as the result of pectinesterase activity.

  4. The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis

    DEFF Research Database (Denmark)

    Thygesen, Lisbeth Garbrecht; E. Thybring, Emil; Johansen, Katja Salomon;

    2014-01-01

    . Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry......, particularly when it comes to up-scaling of processes based on insoluble feed stocks....

  5. Roles of tRNA in cell wall biosynthesis

    DEFF Research Database (Denmark)

    Dare, Kiley; Ibba, Michael

    2012-01-01

    Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families...

  6. The digestion of yeast cell wall polysaccharides in veal calves

    NARCIS (Netherlands)

    Gaillard, B.D.E.; Weerden, van E.J.

    1976-01-01

    1. The digestibility of the cell wall polysaccharides of an alkane-grown yeast in different parts of the digestive tract of two veal calves fitted with re-entrant cannulas at the end of the ileum was studied by replacing part of the skim-milk powder of their ‘normal’, milk-substitute (all-milk-prote

  7. Action of xyloglucan hydrolase within the native cell wall architecture and its effect on cell wall extensibility in azuki bean epicotyls.

    Science.gov (United States)

    Kaku, Tomomi; Tabuchi, Akira; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Hoson, Takayuki

    2002-01-01

    Xyloglucan hydrolase (XGH) has recently been purified from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls as a new type of xyloglucan-degrading enzyme [Tabuchi et al. (2001) Plant Cell Physiol. 42: 154]. In the present study, the effects of XGH on the mechanical properties of the cell wall and on the level and the molecular size of xyloglucans within the native wall architecture were examined in azuki bean epicotyls. When the epidermal tissue strips from the growing regions of azuki bean epicotyls were incubated with XGH, the mechanical extensibility of the cell wall dramatically increased. XGH exogenously applied to cell wall materials (homogenates) or epidermal tissue strips decreased the amount of xyloglucans via the solubilization of the polysaccharides. Also, XGH substantially decreased the molecular mass of xyloglucans in both materials. These results indicate that XGH is capable of hydrolyzing xyloglucans within the native cell wall architecture and thereby increasing the cell wall extensibility in azuki bean epicotyls.

  8. Molecular deformation mechanisms of the wood cell wall material.

    Science.gov (United States)

    Jin, Kai; Qin, Zhao; Buehler, Markus J

    2015-02-01

    Wood is a biological material with outstanding mechanical properties resulting from its hierarchical structure across different scales. Although earlier work has shown that the cellular structure of wood is a key factor that renders it excellent mechanical properties at light weight, the mechanical properties of the wood cell wall material itself still needs to be understood comprehensively. The wood cell wall material features a fiber reinforced composite structure, where cellulose fibrils act as stiff fibers, and hemicellulose and lignin molecules act as soft matrix. The angle between the fiber direction and the loading direction has been found to be the key factor controlling the mechanical properties. However, how the interactions between theses constitutive molecules contribute to the overall properties is still unclear, although the shearing between fibers has been proposed as a primary deformation mechanism. Here we report a molecular model of the wood cell wall material with atomistic resolution, used to assess the mechanical behavior under shear loading in order to understand the deformation mechanisms at the molecular level. The model includes an explicit description of cellulose crystals, hemicellulose, as well as lignin molecules arranged in a layered nanocomposite. The results obtained using this model show that the wood cell wall material under shear loading deforms in an elastic and then plastic manner. The plastic regime can be divided into two parts according to the different deformation mechanisms: yielding of the matrix and sliding of matrix along the cellulose surface. Our molecular dynamics study provides insights of the mechanical behavior of wood cell wall material at the molecular level, and paves a way for the multi-scale understanding of the mechanical properties of wood.

  9. Structure of Plant Cell Walls: XI. GLUCURONOARABINOXYLAN, A SECOND HEMICELLULOSE IN THE PRIMARY CELL WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS.

    Science.gov (United States)

    Darvill, J E; McNeil, M; Darvill, A G; Albersheim, P

    1980-12-01

    The isolation, purification, and partial characterization of a glucuronoarabinoxylan, a previously unobserved component of the primary cell walls of dicotyledonous plants, are described. The glucuronoarabinoxylan constitutes approximately 5% of the primary walls of suspension-cultured sycamore cells. This glucuronoarabinoxylan possesses many of the structural characteristics of analogous polysaccharides that have been isolated from the primary and secondary cell walls of monocots as well as from the secondary cell walls of dicots. The glucuronoarabinoxylan of primary dicot cell walls has a linear beta-1,4-linked d-xylopyranosyl backbone with both neutral and acidic sidechains attached at intervals along its length. The acidic sidechains are terminated with glucuronosyl or 4-O-methyl glucuronosyl residues, whereas the neutral sidechains are composed of arabinosyl and/or xylosyl residues.

  10. Genetic Screening for Bacterial Mutants in Liquid Growth Media By Fluorescence-Activated Cell Sorting

    Science.gov (United States)

    Abuaita, Basel H.; Withey, Jeffrey H.

    2010-01-01

    Many bacterial pathogens have defined in vitro virulence inducing conditions in liquid media which lead to production of virulence factors important during an infection. Identifying mutants that no longer respond to virulence inducing conditions will increase our understanding of bacterial pathogenesis. However, traditional genetic screens require growth on solid media. Bacteria in a single colony are in every phase of the growth curve, which complicates the analysis and make screens for growth phase-specific mutants problematic. Here, we utilize fluorescence-activated cell sorting in conjunction with random transposon mutagenesis to isolate bacteria grown in liquid media that are defective in virulence activation. This method permits analysis of an entire bacterial population in real time and selection of individual bacterial mutants with the desired gene expression profile at any time point after induction. We have used this method to identify Vibrio cholerae mutants defective in virulence induction. PMID:21094189

  11. Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

    Science.gov (United States)

    Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

    2015-02-01

    Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line.

  12. Studying biomolecule localization by engineering bacterial cell wall curvature.

    Directory of Open Access Journals (Sweden)

    Lars D Renner

    Full Text Available In this article we describe two techniques for exploring the relationship between bacterial cell shape and the intracellular organization of proteins. First, we created microchannels in a layer of agarose to reshape live bacterial cells and predictably control their mean cell wall curvature, and quantified the influence of curvature on the localization and distribution of proteins in vivo. Second, we used agarose microchambers to reshape bacteria whose cell wall had been chemically and enzymatically removed. By combining microstructures with different geometries and fluorescence microscopy, we determined the relationship between bacterial shape and the localization for two different membrane-associated proteins: i the cell-shape related protein MreB of Escherichia coli, which is positioned along the long axis of the rod-shaped cell; and ii the negative curvature-sensing cell division protein DivIVA of Bacillus subtilis, which is positioned primarily at cell division sites. Our studies of intracellular organization in live cells of E. coli and B. subtilis demonstrate that MreB is largely excluded from areas of high negative curvature, whereas DivIVA localizes preferentially to regions of high negative curvature. These studies highlight a unique approach for studying the relationship between cell shape and intracellular organization in intact, live bacteria.

  13. Putrescine Alleviates Iron Deficiency via NO-Dependent Reutilization of Root Cell-Wall Fe in Arabidopsis.

    Science.gov (United States)

    Zhu, Xiao Fang; Wang, Bin; Song, Wen Feng; Zheng, Shao Jian; Shen, Ren Fang

    2016-01-01

    Plants challenged with abiotic stress show enhanced polyamines levels. Here, we show that the polyamine putrescine (Put) plays an important role to alleviate Fe deficiency. The adc2-1 mutant, which is defective in Put biosynthesis, was hypersensitive to Fe deficiency compared with wild type (Col-1 of Arabidopsis [Arabidopsis thaliana]). Exogenous Put decreased the Fe bound to root cell wall, especially to hemicellulose, and increased root and shoot soluble Fe content, thus alleviating the Fe deficiency-induced chlorosis. Intriguingly, exogenous Put induced the accumulation of nitric oxide (NO) under both Fe-sufficient (+Fe) and Fe-deficient (-Fe) conditions, although the ferric-chelate reductase (FCR) activity and the expression of genes related to Fe uptake were induced only under -Fe treatment. The alleviation of Fe deficiency by Put was diminished in the hemicellulose-level decreased mutant-xth31 and in the noa1 and nia1nia2 mutants, in which the endogenous NO levels are reduced, indicating that both NO and hemicellulose are involved in Put-mediated alleviation of Fe deficiency. However, the FCR activity and the expression of genes related to Fe uptake were still up-regulated under -Fe+Put treatment compared with -Fe treatment in xth31, and Put-induced cell wall Fe remobilization was abolished in noa1 and nia1nia2, indicating that Put-regulated cell wall Fe reutilization is dependent on NO. From our results, we conclude that Put is involved in the remobilization of Fe from root cell wall hemicellulose in a process dependent on NO accumulation under Fe-deficient condition in Arabidopsis.

  14. Isolation of carotenoid hyperproducing mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by flow cytometry and cell sorting.

    Science.gov (United States)

    Brehm-Stecher, Byron F; Johnson, Eric A

    2012-01-01

    Approaches for improving astaxanthin yields in Xanthophyllomyces dendrorhous include optimization of fermentation conditions and generation of hyperproducing mutants through random mutagenesis using chemical or physical means. A key limitation of classical mutagenesis is the labor-intensive nature of the screening processes required to find relatively rare mutants having increased carotenoid content, as these are present against a high background of low-interest cells. Here, flow cytometry is described as a high-throughput, single-cell method for primary enrichment of mutagenized cells expressing high levels of astaxanthin. This approach improves the speed and productivity of classical strain selection, enhancing the chances for isolating the carotenoid hyperproducing mutants (CHMs) needed to enable high-titer, economical production of natural astaxanthin.

  15. Diverse functions for six glycosyltransferases in Caulobacter crescentus cell wall assembly.

    Science.gov (United States)

    Yakhnina, Anastasiya A; Gitai, Zemer

    2013-10-01

    The essential process of peptidoglycan synthesis requires two enzymatic activities, transpeptidation and transglycosylation. While the PBP2 and PBP3 transpeptidases perform highly specialized functions that are widely conserved, the specific roles of different glycosyltransferases are poorly understood. For example, Caulobacter crescentus encodes six glycosyltransferase paralogs of largely unknown function. Using genetic analyses, we found that Caulobacter glycosyltransferases are primarily redundant but that PbpX is responsible for most of the essential glycosyltransferase activity. Cells containing PbpX as their sole glycosyltransferase are viable, and the loss of pbpX leads to a general defect in the integrity of the cell wall structure even in the presence of the other five glycosyltransferases. However, neither PbpX nor any of its paralogs is required for the specific processes of cell elongation or division, while the cell wall synthesis required for stalk biogenesis is only partially disrupted in several of the glycosyltransferase mutants. Despite their genetic redundancy, Caulobacter glycosyltransferases exhibit different subcellular localizations. We suggest that these enzymes have specialized roles and normally function in distinct subcomplexes but retain the ability to substitute for one another so as to ensure the robustness of the peptidoglycan synthesis process.

  16. Cell wall staining with Trypan Blue enables quantitative analysis of morphological changes in yeast cells

    Directory of Open Access Journals (Sweden)

    Johannes eLiesche

    2015-02-01

    Full Text Available 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.

  17. Resistance to antibiotics targeted to the bacterial cell wall.

    Science.gov (United States)

    Nikolaidis, I; Favini-Stabile, S; Dessen, A

    2014-03-01

    Peptidoglycan is the main component of the bacterial cell wall. It is a complex, three-dimensional mesh that surrounds the entire cell and is composed of strands of alternating glycan units crosslinked by short peptides. Its biosynthetic machinery has been, for the past five decades, a preferred target for the discovery of antibacterials. Synthesis of the peptidoglycan occurs sequentially within three cellular compartments (cytoplasm, membrane, and periplasm), and inhibitors of proteins that catalyze each stage have been identified, although not all are applicable for clinical use. A number of these antimicrobials, however, have been rendered inactive by resistance mechanisms. The employment of structural biology techniques has been instrumental in the understanding of such processes, as well as the development of strategies to overcome them. This review provides an overview of resistance mechanisms developed toward antibiotics that target bacterial cell wall precursors and its biosynthetic machinery. Strategies toward the development of novel inhibitors that could overcome resistance are also discussed.

  18. Dislocation-mediated growth of bacterial cell walls

    CERN Document Server

    Amir, Ariel

    2012-01-01

    Recent experiments have illuminated a remarkable growth mechanism of rod-shaped bacteria: proteins associated with cell wall extension move at constant velocity in circles oriented approximately along the cell circumference (Garner et al., Science (2011), Dominguez-Escobar et al. Science (2011), van Teeffelen et al. PNAS (2011). We view these as dislocations in the partially ordered peptidoglycan structure, activated by glycan strand extension machinery, and study theoretically the dynamics of these interacting defects on the surface of a cylinder. Generation and motion of these interacting defects lead to surprising effects arising from the cylindrical geometry, with important implications for growth. We also discuss how long range elastic interactions and turgor pressure affect the dynamics of the fraction of actively moving dislocations in the bacterial cell wall.

  19. Targeted and non-targeted effects in cell wall polysaccharides from transgenetically modified potato tubers

    NARCIS (Netherlands)

    Huang, J.H.

    2016-01-01

    The plant cell wall is a chemically complex network composed mainly of polysaccharides. Cell wall polysaccharides surround and protect plant cells and are responsible for the stability and rigidity of plant tissue. Pectin is a major component of primary cell wall and the middle lamella of plants. Ho

  20. Pectic arabinan side chains are essential for pollen cell wall integrity during pollen development

    NARCIS (Netherlands)

    Cankar, K.; Kortstee, A.J.; Toonen, M.A.J.; Wolters-Arts, M.; Houbein, R.; Mariani, C.; Ulvskov, P.; Jorgensen, B.; Schols, H.A.; Visser, R.G.F.; Trindade, L.M.

    2014-01-01

    Pectin is a complex polysaccharide and an integral part of the primary plant cell wall and middle lamella, contributing to cell wall mechanical strength and cell adhesion. To understand the structure–function relationships of pectin in the cell wall, a set of transgenic potato lines with altered pec

  1. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Salazar, E.P.; Brookman, K.W.; Collins, C.C.; Stewart, S.A.; Busch, D.B.; Weber, C.A.

    1986-04-02

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs.

  2. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures

    Institute of Scientific and Technical Information of China (English)

    Hugo Melida; Antonio Encina; Asier Largo-Gosens; Esther Novo-Uzal; Rogelio Santiago; Federico Pomar; Pedro Garca; Penelope Garca-Angulo; Jose Luis Acebes; Jesus Alvarez

    2015-01-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.

  3. Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells.

    Science.gov (United States)

    Invernizzi, F; D'Amato, I; Jensen, P B; Ravaglia, S; Zeviani, M; Tiranti, V

    2012-03-01

    Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders.

  4. The transcriptional repressor TupA in Aspergillus niger is involved in controlling gene expression related to cell wall biosynthesis, development, and nitrogen source availability.

    Directory of Open Access Journals (Sweden)

    Doreen Schachtschabel

    Full Text Available The Tup1-Cyc8 (Ssn6 complex is a well characterized and conserved general transcriptional repressor complex in eukaryotic cells. Here, we report the identification of the Tup1 (TupA homolog in the filamentous fungus Aspergillus niger in a genetic screen for mutants with a constitutive expression of the agsA gene. The agsA gene encodes a putative alpha-glucan synthase, which is induced in response to cell wall stress in A. niger. Apart from the constitutive expression of agsA, the selected mutant was also found to produce an unknown pigment at high temperatures. Complementation analysis with a genomic library showed that the tupA gene could complement the phenotypes of the mutant. Screening of a collection of 240 mutants with constitutive expression of agsA identified sixteen additional pigment-secreting mutants, which were all mutated in the tupA gene. The phenotypes of the tupA mutants were very similar to the phenotypes of a tupA deletion strain. Further analysis of the tupA-17 mutant and the ΔtupA mutant revealed that TupA is also required for normal growth and morphogenesis. The production of the pigment at 37°C is nitrogen source-dependent and repressed by ammonium. Genome-wide expression analysis of the tupA mutant during exponential growth revealed derepression of a large group of diverse genes, including genes related to development and cell wall biosynthesis, and also protease-encoding genes that are normally repressed by ammonium. Comparison of the transcriptome of up-regulated genes in the tupA mutant showed limited overlap with the transcriptome of caspofungin-induced cell wall stress-related genes, suggesting that TupA is not a general suppressor of cell wall stress-induced genes. We propose that TupA is an important repressor of genes related to development and nitrogen metabolism.

  5. Tomato Fruit Cell Wall Synthesis during Development and Senescence : In Vivo Radiolabeling of Wall Fractions Using [C]Sucrose.

    Science.gov (United States)

    Mitcham, E J; Gross, K C; Ng, T J

    1989-02-01

    The pedicel of tomato fruit (Lycopersicon esculentum Mill., cv ;Rutgers') of different developmental stages from immature-green (IG) to red was injected on the vine with 7 microcuries [(14)C(U)]sucrose and harvested after 18 hours. Cell walls were isolated from outer pericarp and further fractionated yielding ionically associated pectin, covalently bound pectin, hemicellulosic fraction I, hemicellulosic fraction II, and cellulosic fraction II. The dry weight of the total cell wall and of each cell wall fraction per gram fresh weight of pericarp tissue decreased after the mature-green (MG) stage of development. Incorporation of radiolabeled sugars into each fraction decreased from the IG to MG3 (locules jellied but still green) stage. Incorporation in all fractions increased from MG3 to breaker and turning (T) and then decreased from T to red. Data indicate that cell wall synthesis continues throughout ripening and increases transiently from MG4 (locules jellied and yellow to pink in color) to T, corresponding to the peak in respiration and ethylene synthesis during the climacteric. Synthesis continued at a time when total cell wall fraction dry weight decreased indicating the occurrence of cell wall turnover. Synthesis and insertion of a modified polymer with removal of other polymers may produce a less rigid cell wall and allow softening of the tissue integrity during ripening.

  6. IDENTIFYING GENES CONTROLLING FERULATE CROSS-LINKING FORMATION IN GRASS CELL WALLS

    Energy Technology Data Exchange (ETDEWEB)

    de O Buanafina, Marcia Maria

    2013-10-16

    DESCRIPTION/ABSTRACT This proposal focuses on cell wall feruloylation and our long term goal is to identify and isolate novel genes controlling feruloylation and to characterize the phenotype of mutants in this pathway, with a spotlight on cell wall properties. Currently, the genes underlying AX feruloylation have not been identified and the isolation of such genes could be of great importance in manipulating ferulates accretion to the wall. Mutation of the feruloyl transferase gene(s) should lead to less ferulates secreted to the cell wall and reduced ferulate cross-linking. Our current research is based on the hypothesis that controlling the level of total feruloylation will have a direct impact on the level of cross-linking and in turn impact biomass utility for forage and biofuel production. Our results/accomplishments for this project so far include: 1. Mutagenised Brachypodium population. We have developed EMS mutagenized populations of model grass species Brachypodium distachyon. EMS populations have been developed from over 28,000 mutagenized seeds generating 5,184 M2 families. A total of 20,793 plants have been screened and 1,233 were originally selected. 2. Selected Brachypodium mutants: Potential mutants on their levels of cell wall ferulates and cell wall AX ? have been selected from 708 M2 families. A total of 303 back-crosses to no-mutagenized parental stock have been done, followed by selfing selected genotypes in order to confirm heritability of traits and to remove extraneous mutations generated by EMS mutagenesis. We are currently growing 12 F5 and F6 populations in order to assess CW composition. If low level of ferulates are confirmed in the candidate lines selected the mutation could be altered in different in one or several kinds of genes such as genes encoding an AX feruloyl transferase; genes encoding the arabinosyl transferase; genes encoding the synthesis of the xylan backbone; genes encoding enzymes of the monolignol pathway affecting FA

  7. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    Energy Technology Data Exchange (ETDEWEB)

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M. [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany); Wehnert, Manfred [Institute of Human Genetics, University of Greifswald, Greifswald (Germany); Huebner, Stefan, E-mail: stefan.huebner@mail.uni-wuerzburg.de [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)

    2009-08-15

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

  8. Analysis of the soluble cell wall proteome of gymnosperms.

    Science.gov (United States)

    Uzal, Esther Novo; Gómez-Ros, Laura V; Hernández, Jose A; Pedreño, María A; Cuello, Juan; Ros Barceló, Alfonso

    2009-05-15

    We analyzed the cell wall proteome of lignifying suspension cell cultures (SCCs) from four gymnosperms that differ in evolution degree. This analysis showed the presence of "peptide sequence tags" (PSTs) corresponding to glucan endo-1,3-beta-D-glucosidase, xyloglucan-endotrans-glucosylase/hydrolase, chitinases, thaumatin-like proteins and proteins involved in lignin/lignan biosynthesis, such as dirigent-like proteins and peroxidases. Surprisingly, and given the abundance of peroxidases in the cell wall proteome of these gymnosperms, PSTs corresponding to peroxidases were only detected in tryptic fragments of the cell wall proteome of Cycas revoluta. The current lack of knowledge regarding C. revoluta peroxidases led us to purify, characterize and partially sequence the peroxidases responsible for lignin biosynthesis in this species. This yielded three peroxidase-enriched fractions: CrPrx 1, CrPrx 2 and CrPrx 3. Analyses of tryptic peptides of CrPrx 2 (32kDa) and CrPrx 3 (26kDa) suggest that CrPrx 3 arises from CrPrx 2 by protein truncation, and that CrPrx 3 apparently constitutes a post-translational modification of CrPrx 2. That CrPrx 2 and CrPrx 3 are apparently the same enzyme was also deduced from the similarity between the k(cat) shown by both peroxidases for the three monolignols. These results emphasize the analogies between the cell wall proteome of gymnosperms and angiosperms, the complexity of the peroxidase proteome, and the difficulties involved in establishing fine structure-function relationships.

  9. Orbital wall infarction in child with sickle cell disease.

    Science.gov (United States)

    Janssens, C; Claeys, L; Maes, P; Boiy, T; Wojciechowski, M

    2015-12-01

    We present the case of a 17-year-old boy, known with homozygous sickle cell disease, who was admitted because of generalised pain. He developed bilateral periorbital oedema and proptosis, without pain or visual disturbances. In addition to hyperhydration, oxygen and analgesia IV antibiotics were started, to cover a possible osteomyelitis. Patients with sickle cell disease are at risk for vaso-occlusive crises, when the abnormally shaped red blood cells aggregate and block the capillaries. Such a crisis typically presents at a location with high bone marrow activity, as the vertebrae and long bones. At an early age, the bone marrow is still active at other sites, for example the orbital wall, and thus infarction can also occur there. Thus, in young persons with sickle cell disease, it is important to consider orbital wall infarction in the differential diagnosis, since the approach is different from osteomyelitis. If the disease is complicated by an orbital compression syndrome, corticosteroids or surgical intervention may be necessary to preserve the vision. In our patient, an MRI of the orbitae demonstrated periorbital oedema with bone anomalies in the orbital and frontal bones, confirming orbital wall infarction. Ophthalmological examination revealed no signs of pressure on the nervus opticus. The patient recovered gradually with conservative treatment.

  10. Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD

    Science.gov (United States)

    Møller, Svenning Rune; Yi, Xueying; Velásquez, Silvia Melina; Gille, Sascha; Hansen, Pernille Louise Munke; Poulsen, Christian P.; Olsen, Carl Erik; Rejzek, Martin; Parsons, Harriet; Zhang, Yang; Wandall, Hans H.; Clausen, Henrik; Field, Robert A.; Pauly, Markus; Estevez, Jose M.; Harholt, Jesper; Ulvskov, Peter; Petersen, Bent Larsen

    2017-01-01

    Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp4, serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1–5 β- or α-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-β1,4Araf-β1,2Araf-β1,2Araf) side chains in an α-linkage, to yield Hyp-Araf4 which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its’ product, Hyp-Araf4, point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae. PMID:28358137

  11. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells

    DEFF Research Database (Denmark)

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    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...

  12. Complementation of mutant phenotypes and genotypes of cultured mammalian cells

    NARCIS (Netherlands)

    A.J.R. de Jonge

    1985-01-01

    textabstractThis dissertation describes experiments aimed at the complementation of a genetic mutation in cultured mammalian cells in order to investigate several aspects of the structure and functioning of the human genome. Complementation is indicated by the correction of a biochemical function in

  13. Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Bierbaum Gabriele

    2007-09-01

    Full Text Available Abstract Background Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore β-lactam susceptibility in methicillin-resistant S. aureus (MRSA. Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. Results In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. Conclusion Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors.

  14. Electron tomography of cryo-immobilized plant tissue: a novel approach to studying 3D macromolecular architecture of mature plant cell walls in situ.

    Science.gov (United States)

    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

    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.

  15. Electron tomography of cryo-immobilized plant tissue: a novel approach to studying 3D macromolecular architecture of mature plant cell walls in situ.

    Directory of Open Access Journals (Sweden)

    Purbasha Sarkar

    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.

  16. Gain-of-function of mutant p53: mutant p53 enhances cancer progression by inhibiting KLF17 expression in invasive breast carcinoma cells.

    Science.gov (United States)

    Ali, Amjad; Shah, Abdus Saboor; Ahmad, Ayaz

    2014-11-01

    Kruppel-like-factor 17 (KLF17) is a negative regulator of metastasis and epithelial-mesenchymal-transition (EMT). However, its expression is downregulated in metastatic breast cancer that contains p53 mutations. Here, we show that mutant-p53 plays a key role to suppress KLF17 and thereby enhances cancer progression, which defines novel gain-of-function (GOF) of mutant-p53. Mutant-p53 interacts with KLF17 and antagonizes KLF17 mediated EMT genes transcription. Depletion of KLF17 promotes cell viability, decreases apoptosis and induces drug resistance in metastatic breast cancer cells. KLF17 suppresses cell migration and invasion by decreasing CD44, PAI-1 and Cyclin-D1 expressions. Taken together, our results show that KLF17 is important for the suppression of metastasis and could be a potential therapeutic target during chemotherapy.

  17. Cytoplasmic streaming in plant cells: the role of wall slip.

    Science.gov (United States)

    Wolff, K; Marenduzzo, D; Cates, M E

    2012-06-01

    We present a computer simulation study, via lattice Boltzmann simulations, of a microscopic model for cytoplasmic streaming in algal cells such as those of Chara corallina. We modelled myosin motors tracking along actin lanes as spheres undergoing directed motion along fixed lines. The sphere dimension takes into account the fact that motors drag vesicles or other organelles, and, unlike previous work, we model the boundary close to which the motors move as walls with a finite slip layer. By using realistic parameter values for actin lane and myosin density, as well as for endoplasmic and vacuole viscosity and the slip layer close to the wall, we find that this simplified view, which does not rely on any coupling between motors, cytoplasm and vacuole other than that provided by viscous Stokes flow, is enough to account for the observed magnitude of streaming velocities in intracellular fluid in living plant cells.

  18. Cell wall bound anionic peroxidases from asparagus byproducts.

    Science.gov (United States)

    Jaramillo-Carmona, Sara; López, Sergio; Vazquez-Castilla, Sara; Jimenez-Araujo, Ana; Rodriguez-Arcos, Rocio; Guillen-Bejarano, Rafael

    2014-10-08

    Asparagus byproducts are a good source of cationic soluble peroxidases (CAP) useful for the bioremediation of phenol-contaminated wastewaters. In this study, cell wall bound peroxidases (POD) from the same byproducts have been purified and characterized. The covalent forms of POD represent >90% of the total cell wall bound POD. Isoelectric focusing showed that whereas the covalent fraction is constituted primarily by anionic isoenzymes, the ionic fraction is a mixture of anionic, neutral, and cationic isoenzymes. Covalently bound peroxidases were purified by means of ion exchange chromatography and affinity chromatography. In vitro detoxification studies showed that although CAP are more effective for the removal of 4-CP and 2,4-DCP, anionic asparagus peroxidase (AAP) is a better option for the removal of hydroxytyrosol (HT), the main phenol present in olive mill wastewaters.

  19. Plant cell walls: New insights from ancient species

    DEFF Research Database (Denmark)

    Sørensen, Iben; Willats, William George Tycho

    2008-01-01

    Cell walls are a defining feature of plants and have numerous crucial roles in growth and development. They are also the largest source of terrestrial biomass and have many important industrial applications - ranging from bulk products to functional food ingredients. There is considerable interest......¿4)-linked ß-D-Glcp are joined by occasional (1¿3)-linkages. This mixed linkage glucan (MLG) has been the subject of extensive research because of the economic importance of several Poales species including rice, barley and wheat and because MLG has proven health benefits. The recent discovery of MLG......-D-glucan is not unique to the Poales and is an abundant component of Equisetum arvense cell walls. Plant J 2008; 54:510-21....

  20. Selective knockdown of mutant SOD1 in Schwann cells ameliorates disease in G85R mutant SOD1 transgenic mice.

    Science.gov (United States)

    Wang, Lijun; Pytel, Peter; Feltri, M Laura; Wrabetz, Lawrence; Roos, Raymond P

    2012-10-01

    Mutants of superoxide dismutase type 1 (mtSOD1) that have full dismutase activity (e.g., G37R) as well as none (e.g., G85R) cause familial amyotrophic lateral sclerosis (FALS), indicating that mtSOD1-induced FALS results from a toxicity rather than loss in SOD1 enzymatic activity. Still, it has remained unclear whether mtSOD1 dismutase activity can influence disease. A previous study demonstrated that Cre-mediated knockdown of G37R expression in Schwann cells (SCs) of G37R transgenic mice shortened the late phase of disease and survival. These results suggested that the neuroprotective effect of G37R expressed in SCs was greater than its toxicity, presumably because its dismutase activity counteracted reactive oxygen species (ROS). In order to further investigate this, we knocked down G85R in SCs by crossing G85R(flox) mice with myelin-protein-zero (P(0)):Cre mice, which express Cre recombinase in SCs. Knockdown of G85R in SCs of G85R mice delayed disease onset and extended survival indicating that G85R expression in SCs is neurotoxic. These results demonstrate differences in the effect on disease of dismutase active vs. inactive mtSOD1 suggesting that both a loss as well as gain in function of mtSOD1 influence FALS pathogenesis. The results suggest that mtSOD1-induced FALS treatment may have to be adjusted depending on the cell type targeted and particular mtSOD1 involved.

  1. Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells

    OpenAIRE

    2012-01-01

    Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitat...

  2. Life behind cell walls: paradigm lost, paradigm regained.

    Science.gov (United States)

    Lamport, D T

    2001-09-01

    This review of the living cell wall and its protein components is in two parts. The first is anecdotal. A personal account spanning over 40 years research may perhaps be an antidote to one stereotypical view of scientists as detached and humorless. The second part deals with the meaning of function, particularly as it applies to hydroxyproline-rich glycoproteins. Function is a difficult word to define objectively. However, with help from such luminaries as Humpty Dumpty: "A word means what I want it to mean, neither more nor less," and Wittgenstein: "Giving examples of usage ... is the only way to talk about meaning," it is possible to construct a ziggurat representing increasingly complex levels of organization from molecular structure to ecology. Forty years ago I suggested that hydroxyproline-rich structural proteins played a key role in cell wall functioning. But because the bulk of the wall is carbohydrate, there has been an understandable resistance to paradigm change. Expansins, paradoxically, contribute greatly to this resistance because their modus operandi as cell-wall-loosening proteins is based on the idea that they break hydrogen bonds between polysaccharide chains allowing slippage. However, this view is not consistent with the recent discovery [Grobe et al. (1999) Eur. J. Biochem 263: 33-40] that beta-expansins may be proteases, as it implies that the extensin network is not a straightjacket but a substrate for expansin in muro. Such a direct role for extensins in both negative and positive regulation of cell expansion and elongation may constitute a major morphogenetic mechanism operating at all levels of plant growth and development.

  3. A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus.

    Science.gov (United States)

    Meier, Elizabeth L; Razavi, Shiva; Inoue, Takanari; Goley, Erin D

    2016-07-01

    In most bacteria, the tubulin-like GTPase FtsZ forms an annulus at midcell (the Z-ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z-ring assembly and early FtsZ-directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C-terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane-anchored FtsZ in the regulation of cell wall hydrolysis.

  4. Cell wall proteins in seedling cotyledons of Prosopis chilensis.

    Science.gov (United States)

    Rodríguez, J G; Cardemil, L

    1994-01-01

    Four cell wall proteins of cotyledons of Prosopis chilensis seedlings were characterized by PAGE and Western analyses using a polyclonal antibody, generated against soybean seed coat extensin. These proteins had M(r)s of 180,000, 126,000, 107,000 and 63,000, as determined by SDS-PAGE. The proteins exhibited a fluorescent positive reaction with dansylhydrazine suggesting that they are glycoproteins; they did not show peroxidase activity. The cell wall proteins were also characterized by their amino acid composition and by their amino-terminal sequence. These analyses revealed that there are two groups of related cell wall proteins in the cotyledons. The first group comprises the proteins of M(r)s 180,000, 126,000, 107,000 which are rich in glutamic acid/glutamine and aspartic acid/asparagine and they have almost identical NH2-terminal sequences. The second group comprises the M(r) 63,000 protein which is rich in proline, glycine, valine and tyrosine, with an NH2-terminal sequence which was very similar to that of soybean proline-rich proteins.

  5. Progress Towards the Tomato Fruit Cell Wall Proteome

    Directory of Open Access Journals (Sweden)

    Eliel eRuiz May

    2013-05-01

    Full Text Available The plant cell wall (CW compartment, or apoplast, is host to a highly dynamic proteome, comprising large numbers of both enzymatic and structural proteins. This reflects its importance as the interface between adjacent cells and the external environment, the presence of numerous extracellular metabolic and signaling pathways, and the complex nature of wall structural assembly and remodeling during cell growth and differentiation. Tomato fruit ontogeny, with its distinct phases of rapid growth and ripening, provides a valuable experimental model system for CW proteomic studies, in that it involves substantial wall assembly, remodeling and coordinated disassembly. Moreover, diverse populations of secreted proteins must be deployed to resist microbial infection and protect against abiotic stresses. Tomato fruits also provide substantial amounts of biological material, which is a significant advantage for many types of biochemical analyses, and facilitates the detection of lower abundance proteins. In this review we describe a variety of orthogonal techniques that have been applied to identify CW localized proteins from tomato fruit, including approaches that: target the proteome of the CW and the overlying cuticle; functional ‘secretome’ screens; lectin affinity chromatography; and computational analyses to predict proteins that enter the secretory pathway. Each has its merits and limitations, but collectively they are providing important insights into CW proteome composition and dynamics, as well as some potentially controversial issues, such as the prevalence of non-canonical protein secretion.

  6. Cell sorting enriches Escherichia coli mutants that rely on peptidoglycan endopeptidases to suppress highly aberrant morphologies.

    Science.gov (United States)

    Laubacher, Mary E; Melquist, Amy L; Chandramohan, Lakshmi; Young, Kevin D

    2013-02-01

    Bacterial morphology imparts physiological advantages to cells in different environments and, judging by the fidelity with which shape is passed to daughter cells, is a tightly regulated characteristic. Surprisingly, only in the past 10 to 15 years has significant headway been made in identifying the mechanisms by which cells create and maintain particular shapes. One reason for this is that the relevant discoveries have relied heavily on the arduous, somewhat subjective process of manual microscopy. Here, we show that flow cytometry, coupled with the sorting capability of fluorescence-activated cell sorting (FACS), can detect, quantify, and enrich bacteria with morphological alterations. The light scattering properties of several highly aberrant morphological mutants of Escherichia coli were characterized by flow cytometry. Cells from a region that overlapped the distribution of normal rod-shaped cells were collected by FACS and reincubated. After 4 to 15 iterations of this enrichment process, suppressor mutants were isolated that returned almost all the population to a near-normal shape. Suppressors were successfully isolated from strains lacking three or four penicillin binding proteins (PBPs) but not from a mutant lacking a total of seven PBPs. The peptidoglycan endopeptidase, AmpH, was identified as being important for the suppression process, as was a related endopeptidase, MepA. The results validate the use of cell sorting as a means for studying bacterial morphology and identify at least one new class of enzymes required for the suppression of cell shape defects.

  7. Adsorption of polycyclic aromatic hydrocarbons (PAHs) on Rhizopus oryzae cell walls: application of cosolvent models for validating the cell wall-water partition coefficient.

    Science.gov (United States)

    Ma, Bin; Xu, Minmin; Wang, Jiaojiao; Chen, Huaihai; He, Yan; Wu, Laosheng; Wang, Haizhen; Xu, Jianming

    2011-11-01

    The cell wall-cosolvent partition coefficients (Km) of polycyclic aromatic hydrocarbons (PAHs) were determined for Rhizopus oryzae cell walls by controlling the volume fraction of methanol (f) ranging from 0.1 to 0.5. Five cosolvent models were employed for extrapolating the cell wall-water partition coefficients (Kw) in pure water. The extrapolated Kw values of four PAHs on R. oryzae cell walls were ranged from 2.9 to 5.1. Comparison of various Kw values of pyrene generated from extrapolation and the QSPR model, together with predicted different (PD), mean percentage deviations (MPD), and root mean square errors (RSE), revealed that the performance of the LL and Bayesian models were the best among all five tested cosolvent models. This study suggests that R. oryzae cell walls play an important role in the partitioning of PAHs during bioremediation because of the high Kw of fungal cell walls.

  8. Weaver mutant mouse cerebellar granule cells respond normally to chronic depolarization

    DEFF Research Database (Denmark)

    Bjerregaard, Annette; Mogensen, Helle Smidt; Hack, N;

    1997-01-01

    We studied the effects of chronic K(+)-induced membrane depolarization and treatment with N-methyl-D-aspartate (NMDA) on cerebellar granule cells (CGCs) from weaver mutant mice and non-weaver litter-mates. The weaver mutation is a Gly-to-Ser substitution in a conserved region of the Girk2 G prote...

  9. THERMAL RADIOSENSITIZATION IN HEAT-SENSITIVE AND RADIATION-SENSITIVE MUTANTS OF CHO CELLS

    NARCIS (Netherlands)

    KAMPINGA, HH; KANON, B; KONINGS, AWT; STACKHOUSE, MA; BEDFORD, JS

    1993-01-01

    Recently, it has been hypothesized (Iliakis and Seaner 1990) that DNA double-strand break (dsb) repair proficiency is a prerequisite for heat radiosensitization on the basis of the finding that the radiosensitive and dsb-repair-deficient mutant xrs-5 cell line shows no significant heat-induced radio

  10. Cell surface physico chemistry alters biofilm development of Pseudomonas aeruginosa lipopolysaccharide mutants

    NARCIS (Netherlands)

    Flemming, CA; Palmer, RJ; Arrage, AA; Van der Mei, HC; White, DC

    1999-01-01

    The hydrophobic and electrostatic characteristics of bacterial cell surfaces were compared with attachment proclivity and biomass accumulation over time between wildtype Pseudomonas aeruginosa serotype O6 (possesses A and B band LPS), and three LPS-deficient mutants, vi;. A28 (A(+)B(-)), R5 (A(+)B(-

  11. Cell wall composition and candidate biosynthesis gene expression during rice development

    DEFF Research Database (Denmark)

    Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra

    2016-01-01

    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 compone...

  12. Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall Precursor Lipid II

    DEFF Research Database (Denmark)

    Schneider, Tanja; Kruse, Thomas; Wimmer, Reinhard

    2010-01-01

    that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular...

  13. The Transcriptional Repressor TupA in Aspergillus niger Is Involved in Controlling Gene Expression Related to Cell Wall Biosynthesis, Development, and Nitrogen Source Availability

    DEFF Research Database (Denmark)

    Schachtschabel, Doreen; Arentshorst, Mark; Nitsche, Benjamin M

    2013-01-01

    The Tup1-Cyc8 (Ssn6) complex is a well characterized and conserved general transcriptional repressor complex in eukaryotic cells. Here, we report the identification of the Tup1 (TupA) homolog in the filamentous fungus Aspergillus niger in a genetic screen for mutants with a constitutive expression...... of the agsA gene. The agsA gene encodes a putative alpha-glucan synthase, which is induced in response to cell wall stress in A. niger. Apart from the constitutive expression of agsA, the selected mutant was also found to produce an unknown pigment at high temperatures. Complementation analysis...

  14. Glycoside Hydrolase MoGls2 Controls Asexual/Sexual Development, Cell Wall Integrity and Infectious Growth in the Rice Blast Fungus

    Science.gov (United States)

    Li, Mengying; Liu, Xinyu; Liu, Zhixi; Sun, Yi; Liu, Muxing; Wang, Xiaoli; Zhang, Haifeng; Zheng, Xiaobo; Zhang, Zhengguang

    2016-01-01

    N-linked glycosylation is a way of glycosylation for newly synthesized protein, which plays a key role in the maturation and transport of proteins. Glycoside hydrolases (GHs) are essential in this process, and are involved in processing of N-linked glycoproteins or degradation of carbohydrate structures. Here, we identified and characterized MoGls2 in Magnaporthe oryzae, which is a yeast glucosidase II homolog Gls2 and is required for trimming the final glucose in N-linked glycans and normal cell wall synthesis. Target deletion of MoGLS2 in M. oryzae resulted in a reduced mycelial growth, an increased conidial production, delayed conidial germination and loss the ability of sexual reproduction. Pathogenicity assays revealed that the ΔMogls2 mutant showed significantly decreased in virulence and infectious growth. Further studies showed that the mutant was less sensitive to salt and osmotic stress, and increased sensitivity to cell wall stresses. Additionally, the ΔMogls2 mutant showed a defect in cell wall integrity. Our results indicate that MoGls2 is a key protein for the growth and development of M. oryzae, involving in the regulation of asexual/sexual development, stress response, cell wall integrity and infectious growth. PMID:27607237

  15. Down-regulation of UDP-glucuronic Acid Biosynthesis Leads to Swollen Plant Cell Walls and Severe Developmental Defects Associated with Changes in Pectic Polysaccharides*

    Science.gov (United States)

    Reboul, Rebecca; Geserick, Claudia; Pabst, Martin; Frey, Beat; Wittmann, Doris; Lütz-Meindl, Ursula; Léonard, Renaud; Tenhaken, Raimund

    2011-01-01

    UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation. PMID:21949134

  16. Down-regulation of UDP-glucuronic acid biosynthesis leads to swollen plant cell walls and severe developmental defects associated with changes in pectic polysaccharides.

    Science.gov (United States)

    Reboul, Rebecca; Geserick, Claudia; Pabst, Martin; Frey, Beat; Wittmann, Doris; Lütz-Meindl, Ursula; Léonard, Renaud; Tenhaken, Raimund

    2011-11-18

    UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.

  17. Effect of Wall Charge on Striation in Plasma Display Cells

    Institute of Scientific and Technical Information of China (English)

    HE Feng; OUYANG Jiting; CAO Jing; FENG Shuo; MIAO Jinsong; WANG Jianqi

    2007-01-01

    Different configurations and driving voltages have been employed to investigate the effect of the wall charge on the striations in macroscopic plasma display panel (PDP) cells.The experimental results show that a discharge channel near the dielectric layer is indispensable to striation occurring in the anode area during a discharge,while the pre-accumulated charge on the dielectric layer and the surface state are not important.The origin of the striation is related only to the physical process in the cell.The dielectric layer acts as a charge collector during a PDP discharge.

  18. Stress analysis for wall structure in mobile hot cell design

    Energy Technology Data Exchange (ETDEWEB)

    Bahrin, Muhammad Hannan, E-mail: hannan@nuclearmalaysia.gov.my; Rahman, Anwar Abdul, E-mail: anwar@nuclearmalaysia.gov.my; Hamzah, Mohd Arif, E-mail: arif@nuclearmalaysia.gov.my; Mamat, Mohd Rizal; Azman, Azraf; Hasan, Hasni [Prototype and Plant Development Centre, Technical Services Division, Malaysian Nuclear Agency (Malaysia)

    2016-01-22

    Malaysian Nuclear Agency is developing a Mobile Hot Cell (MHC) in order to handle and manage Spent High Activity Radioactive Sources (SHARS) such as teletherapy heads and irradiators. At present, there are only two units of MHC in the world, in South Africa and China. Malaysian Mobile Hot cell is developed by Malaysian Nuclear Agency with the assistance of IAEA expert, based on the design of South Africa and China, but with improved features. Stress analysis has been performed on the design in order to fulfil the safety requirement in operation of MHC. This paper discusses the loading analysis effect from the sand to the MHC wall structure.

  19. Evidence for 'silicon' within the cell walls of suspension-cultured rice cells.

    Science.gov (United States)

    He, Congwu; Wang, Lijun; Liu, Jian; Liu, Xin; Li, Xiuli; Ma, Jie; Lin, Yongjun; Xu, Fangsen

    2013-11-01

    Despite the ubiquity and beneficial role of silicon (Si) in plant biology, structural and chemical mechanisms operating at the single-cell level have not been extensively studied. To obtain insights regarding the effect of Si on individual cells, we cultivated suspended rice (Oryza sativa) cells in the absence and presence of Si and analyzed single cells using a combination of physical techniques including atomic force microscopy (AFM). Si is naturally present as a constituent of the cell walls, where it is firmly bound to the cell wall matrix rather than occurring within intra- or extracellular silica deposition, as determined by using inductively coupled plasma mass spectrometry (ICP-MS) and X-ray photoelectron spectroscopy (XPS). This species of Si, linked with the cell wall matrix, improves the structural stability of cell walls during their expansion and subsequent cell division. Maintaining cell shape is thereby enhanced, which may be crucial for the function and survival of cells. This study provides further evidence that organosilicon is present in plant cell walls, which broadens our understanding of the chemical nature of 'anomalous Si' in plant biology.

  20. In situ analysis of cell wall polymers associated with phloem fibre cells in stems of hemp, Cannabis sativa L.

    Science.gov (United States)

    Blake, Anthony W; Marcus, Susan E; Copeland, James E; Blackburn, Richard S; Knox, J Paul

    2008-06-01

    A study of stem anatomy and the sclerenchyma fibre cells associated with the phloem tissues of hemp (Cannabis sativa L.) plants is of interest for both understanding the formation of secondary cell walls and for the enhancement of fibre utility as industrial fibres and textiles. Using a range of molecular probes for cell wall polysaccharides we have surveyed the presence of cell wall components in stems of hemp in conjunction with an anatomical survey of stem and phloem fibre development. The only polysaccharide detected to occur abundantly throughout the secondary cell walls of phloem fibres was cellulose. Pectic homogalacturonan epitopes were detected in the primary cell walls/intercellular matrices between the phloem fibres although these epitopes were present at a lower level than in the surrounding parenchyma cell walls. Arabinogalactan-protein glycan epitopes displayed a diversity of occurrence in relation to fibre development and the JIM14 epitope was specific to fibre cells, binding to the inner surface of secondary cell walls, throughout development. Xylan epitopes were found to be present in the fibre cells (and xylem secondary cell walls) and absent from adjacent parenchyma cell walls. Analysis of xylan occurrence in the phloem fibre cells of hemp and flax indicated that xylan epitopes were restricted to the primary cell walls of fibre cells and were not present in the secondary cell walls of these cells.

  1. Isolation of anti-T cell receptor scFv mutants by yeast surface display.

    Science.gov (United States)

    Kieke, M C; Cho, B K; Boder, E T; Kranz, D M; Wittrup, K D

    1997-11-01

    Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.

  2. Distinct Rayleigh scattering from hot spot mutant p53 proteins reveals cancer cells.

    Science.gov (United States)

    Jun, Ho Joon; Nguyen, Anh H; Kim, Yeul Hong; Park, Kyong Hwa; Kim, Doyoun; Kim, Kyeong Kyu; Sim, Sang Jun

    2014-07-23

    The scattering of light redirects and resonances when an electromagnetic wave interacts with electrons orbits in the hot spot core protein and oscillated electron of the gold nanoparticles (AuNP). This report demonstrates convincingly that resonant Rayleigh scattering generated from hot spot mutant p53 proteins is correspondence to cancer cells. Hot spot mutants have unique local electron density changes that affect specificity of DNA binding affinity compared with wild types. Rayleigh scattering changes introduced by hot-spot mutations were monitored by localized surface plasmon resonance (LSPR) shift changes. The LSPR λmax shift for hot-spot mutants ranged from 1.7 to 4.2 nm for mouse samples and from 0.64 nm to 2.66 nm for human samples, compared to 9.6 nm and 15 nm for wild type and mouse and human proteins, respectively with a detection sensitivity of p53 concentration at 17.9 nM. It is interesting that hot-spot mutants, which affect only interaction with DNA, launches affinitive changes as considerable as wild types. These changes propose that hot-spot mutants p53 proteins can be easily detected by local electron density alterations that disturbs the specificity of DNA binding of p53 core domain on the surface of the DNA probed-nanoplasmonic sensor.

  3. Clear Cell Adenocarcinoma Arising from Abdominal Wall Endometriosis

    Directory of Open Access Journals (Sweden)

    Thouraya Achach

    2008-01-01

    Full Text Available Endometriosis is a frequent benign disorder. Malignancy arising in extraovarian endometriosis is a rare event. A 49-year-old woman is presented with a large painful abdominal wall mass. She underwent a myomectomy, 20 years before, for uterus leiomyoma. Computed tomography suggested that this was a desmoid tumor and she underwent surgery. Histological examination showed a clear cell adenocarcinoma associated with endometriosis foci. Pelvic ultrasound, computed tomography, and endometrial curettage did not show any malignancy or endometriosis in the uterus and ovaries. Adjuvant chemotherapy was recommended, but the patient was lost to follow up. Six months later, she returned with a recurrence of the abdominal wall mass. She was given chemotherapy and then she was reoperated.

  4. A genetic screen to isolate Toxoplasma gondii host-cell egress mutants.

    Science.gov (United States)

    Coleman, Bradley I; Gubbels, Marc-Jan

    2012-02-08

    The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca(2+), a signal that is also critical for host cell invasion. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology, only recently has genetic mapping of mutations underlying the phenotypes become routine. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 °C), but fail to proliferate at the restrictive temperature (40 °C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants

  5. Pressure Dependent Wall Relaxation in Polarized $^3$He Gaseous Cells

    CERN Document Server

    Peng, C; Chu, P -H; Gao, H; Zhang, Y

    2013-01-01

    Pressure dependence of longitudinal relaxation time (T$_1$) due to the cell wall was observed previously at both room temperature and low temperature in valved Rb-coated refillable $^3$He gaseous cells in \\cite{Zheng2}. The diffusion of $^3$He from measurement cell through a capillary tube to the valve and the subsequent depolarization on the surface of the valve was proposed to possibly explain such a pressure dependence at room temperature \\cite{Saam}. In this paper, we investigate this diffusion effect through measurements of T$_1$ with newly designed Rb-coated Pyrex glass cells at 295 K as well as finite element analysis (FEA) studies. Both the experimental results and FEA studies show that the diffusion effect is insufficient to explain the observed linear pressure-dependent behavior of T$_1$.

  6. Effect of diglycine mutant FAT10 on the proliferation and apoptosis of cervical cancer cells

    Directory of Open Access Journals (Sweden)

    Cui LI

    2015-01-01

    Full Text Available Objective To investigate the effects of FAT10ΔGG, a carboxyl-terminal diglycine deficient mutant, on the proliferation and apoptosis of cervical cancer cell line HeLa. Methods Specimens of cervical carcinoma in situ and normal cervix tissue, 5 each, were collected. The expressive levels of FAT10 protein in these specimens were detected by Western blotting. Sitedirected mutagenesis was applied to construct the mutant pcDNA3.0-flag-FAT10ΔGG plasmid. The HeLa cells were then transiently transfected with wild-type FAT10, FAT10ΔGG and empty vector (used as negative control, and the wild-type HeLa cells served as blank control. The transfection efficiency of FAT10 or FAT10ΔGG was detected by Western blotting, and cell proliferation was determined by CCK-8 assay. Cisplatin was used to induce cell apoptosis after cells were transfected for 24h, and the cell apoptotic rates of all groups were determined by flow cytometry. Results Western blotting showed a significantly increased expression of FAT10 protein in cervical carcinoma tissues compared with that in normal cervical tissue. Over-expression of wild FAT10 in HeLa cells obviously promoted cell proliferation, but this promotion was significantly inhibited in cells transfected with its diglycine mutant. Compared with blank control group (22.7%±4.2% and negative control group (24.1%±3.8%, the apoptotic rate was significantly reduced in wild FAT10 group (10.9%±2.0%, P0.05. Conclusion FAT10 can promote cell proliferation and inhibit cell apoptosis through its carboxyl-terminal diglycine motif, and it may play an essential role in carcinogenesis and development of cancer. DOI: 10.11855/j.issn.0577-7402.2014.12.01

  7. Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase.

    Science.gov (United States)

    Mathias, Jonathan R; Zhang, Zhanying; Saxena, Meera T; Mumm, Jeff S

    2014-04-01

    Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology.

  8. A fluorescence-activated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants.

    Science.gov (United States)

    Terashima, Mia; Freeman, Elizabeth S; Jinkerson, Robert E; Jonikas, Martin C

    2015-01-01

    There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high-lipid sorting (CHiLiS) strategy, which enables enrichment of high-lipid mutants by fluorescence-activated cell sorting (FACS) of pooled mutants stained with the lipid-sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high-lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20-fold enrichment of the known high-lipid mutant sta1 from a mixture of sta1 and wild-type cells. We then applied CHiLiS to sort thousands of high-lipid cells from a pool of about 60,000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high-lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole-cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems-level understanding of green algal lipid biology by enabling genome-saturating isolation of mutants in key genes.

  9. Cloning of the Zygosaccharomyces bailii GAS1 homologue and effect of cell wall engineering on protein secretory phenotype

    Directory of Open Access Journals (Sweden)

    Dato Laura

    2010-01-01

    Full Text Available Abstract Background Zygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a β-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion. Results The GAS1 homologue of Z. bailii was cloned by PCR, and when expressed in a S. cerevisiae GAS1 null mutant was able to restore the parental phenotype. The respective Z. bailii Δgas1 deleted strain was obtained by targeted deletion of both alleles of the ZbGAS1 gene with deletion cassettes having flanking regions of ~400 bp. The morphological and physiological characterization of the Z. bailii null mutant resulted very similar to that of the corresponding S. cerevisiae mutant. As for S. cerevisiae, in the Z. bailii Δgas1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the Candida rugosa lipase CRL1 and of the Yarrowia lipolytica protease XPR2, while for human IL-1β secretion disruption had no relevant effect. Conclusions The data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that Z. bailii is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as

  10. Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice

    Science.gov (United States)

    Fu, Chun; Begum, Khurshida; Jordan, Philip W.; He, Yan; Overbeek, Paul A.

    2016-01-01

    After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance) protein. Fanconi anemia (FA) proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2–3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line. PMID:27486799

  11. Change in wall composition of transfer and aleurone cells during wheat grain development.

    Science.gov (United States)

    Robert, P; Jamme, F; Barron, C; Bouchet, B; Saulnier, L; Dumas, P; Guillon, F

    2011-02-01

    In addition to the starchy endosperm, a specialized tissue accumulating storage material, the endosperm of wheat grain, comprises the aleurone layer and the transfer cells next to the crease. The transfer cells, located at the ventral region of the grain, are involved in nutrient transfer from the maternal tissues to the developing endosperm. Immunolabeling techniques, Raman spectroscopy, and synchrotron infrared micro-spectroscopy were used to study the chemistry of the transfer cell walls during wheat grain development. The kinetic depositions of the main cell wall polysaccharides of wheat grain endosperm, arabinoxylan, and (1-3)(1-4)-β-glucan in transfer cell walls were different from kinetics previously observed in the aleurone cell walls. While (1-3)(1-4)-β-glucan appeared first in the aleurone cell walls at 90°D, arabinoxylan predominated in the transfer cell walls from 90 to 445°D. Both aleurone and transfer cell walls were enriched in (1-3)(1-4)-β-glucan at the mature stage of wheat grain development. Arabinoxylan was more substituted in the transfer cell walls than in the aleurone walls. However, arabinoxylan was more feruloylated in the aleurone than in the transfer cell walls, whatever the stage of grain development. In the transfer cells, the ferulic acid was less abundant in the outer periclinal walls while para-coumarate was absent. Possible implications of such differences are discussed.

  12. Stimulation of elongation growth and cell wall loosening in rice coleoptiles under microgravity conditions in space.

    Science.gov (United States)

    Hoson, Takayuki; Soga, Kouichi; Mori, Ryuji; Saiki, Mizue; Nakamura, Yukiko; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro

    2002-09-01

    We analyzed the growth rate and the cell wall properties of coleoptiles of rice seedlings grown at 23.6 degrees C for 68.5, 91.5 and 136 h during the Space Shuttle STS-95 mission. In space, elongation growth of coleoptiles was stimulated and the cell wall extensibility increased. Also, the levels of the cell wall polysaccharides per unit length of coleoptiles and the relative content of the high molecular mass matrix polysaccharides decreased in space. These differences in the cell wall polysaccharides could be involved in increasing the cell wall extensibility, leading to growth stimulation of rice coleoptiles in space.

  13. Principles of Bacterial Cell-Size Determination Revealed by Cell-Wall Synthesis Perturbations

    Directory of Open Access Journals (Sweden)

    Carolina Tropini

    2014-11-01

    Full Text Available Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cytoskeleton. We quantified the biochemical and biophysical properties of the cell wall across a wide range of cell sizes. We find that, although cell-wall chemical composition is unaltered, MreB dynamics, cell twisting, and cellular mechanics exhibit systematic large-scale changes consistent with altered chirality and a more isotropic cell wall. This multiscale analysis enabled identification of distinct roles for MreB and PBP2, despite having similar morphological effects when depleted. Altogether, our results highlight the robustness of cell-wall synthesis and physical principles dictating cell-size control.

  14. Measuring the Mechanical Properties of Plant Cell Walls

    Directory of Open Access Journals (Sweden)

    Hannes Vogler

    2015-03-01

    Full Text Available The size, shape and stability of a plant depend on the flexibility and integrity of its cell walls, which, at the same time, need to allow cell expansion for growth, while maintaining mechanical stability. Biomechanical studies largely vanished from the focus of plant science with the rapid progress of genetics and molecular biology since the mid-twentieth century. However, the development of more sensitive measurement tools renewed the interest in plant biomechanics in recent years, not only to understand the fundamental concepts of growth and morphogenesis, but also with regard to economically important areas in agriculture, forestry and the paper industry. Recent advances have clearly demonstrated that mechanical forces play a crucial role in cell and organ morphogenesis, which ultimately define plant morphology. In this article, we will briefly review the available methods to determine the mechanical properties of cell walls, such as atomic force microscopy (AFM and microindentation assays, and discuss their advantages and disadvantages. But we will focus on a novel methodological approach, called cellular force microscopy (CFM, and its automated successor, real-time CFM (RT-CFM.

  15. [Hydroxyproline: Rich glycoproteins of the plant and cell wall

    Energy Technology Data Exchange (ETDEWEB)

    Varner, J.E.

    1993-01-01

    Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a number of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H[sub 2]O[sub 2] production reinforce the earlier ideas of others that H[sub 2]O[sub 2] is involved in normal lignification.

  16. Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines

    Science.gov (United States)

    Chandhok, Gursimran; Horvath, Judit; Aggarwal, Annu; Bhatt, Mohit; Zibert, Andree; Schmidt, Hartmut HJ

    2016-01-01

    AIM: To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7B mutations in cell culture. METHODS: The most common Wilson disease (WD) mutations p.H1069Q, p.R778L and p.C271*, found in the ATP7B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7B was used to stably express WD mutants. mRNA and protein expression of mutant ATP7B, survival of cells, apoptosis, and protein trafficking were determined. RESULTS: Low temperature increased ATP7B protein expression in several mutants. Intracellular ATP7B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069Q and to a lesser extent p.C271* improved by D-penicillamine (DPA) treatment, while mutant p.R778L showed a pronounced response to zinc (Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability. CONCLUSION: The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines. PMID:27122662

  17. Comparison between medium-chain acyl-CoA dehydrogenase mutant proteins overexpressed in bacterial and mammalian cells

    DEFF Research Database (Denmark)

    Jensen, T G; Bross, P; Andresen, B S;

    1995-01-01

    Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a potentially lethal inherited defect in the beta-oxidation of fatty acids. By comparing the behaviour of five missense MCAD mutant proteins expressed in COS cells and in Escherichia coli, we can define some of these as "pure folding mutants......." Upon expression in E. coli, these mutant proteins produce activity levels in the range of the wild-type enzyme only if the chaperonins GroESL are co-overproduced. When overexpressed in COS cells, the pure folding mutants display enzyme activities comparable to the wild-type enzyme. The results suggest...

  18. Synthesis of vanillic acid using whole cell nitrilase of wild and mutant Gordonia terrae.

    Science.gov (United States)

    Bhalla, Tek Chand; Prashant; Kumari, Nisha; Kumar, Vijay; Kumar, Virender; Savitri

    2016-01-01

    The resting cells of Gordonia terrae mutant E9 having enhanced nitrilase activity were used for biotransformation of 4-hydroxy-3-methoxybenzonitrile into vanillic acid. The maximum conversion was observed in 0.1 M phosphate buffer (pH 8.0), using 60 mM substrate and 0.75 mgDCW resting cells in 1 mL reaction at 40 °C. Km of the whole cell nitrilase of wild and mutant strains of G. terrae for this substrate were 20 and 16.6 mM, and Vmax were 0.19 and 0.95 Umg(-1)(DCW), respectively. Fed batch reaction for transformation of 4-hydroxy-3-methoxybenzonitrile using whole cell nitrilase of wild G. terrae resulted in 2.36 g of vanillic acid in 5 h with a catalytic and volumetric productivity of 0.78 gg(-1)(DCW) h(-1) and 4.72 gL(-1)h(-1), respectively. The whole cell nitrilase of G. terrae mutant E9 resulted in higher catalytic and volumetric productivity, i.e., 1.68 gg(-1)DCW h(-1) and 10 gL(-1)h(-1). A total 5.04 g of vanillic acid with 99% purity were accumulated in 100 mL of reaction after 5 h.

  19. IDENTIFYING GENES CONTROLLING FERULATE CROSS-LINKING FORMATION IN GRASS CELL WALLS

    Energy Technology Data Exchange (ETDEWEB)

    de O Buanafina, Marcia Maria

    2013-10-16

    DESCRIPTION/ABSTRACT This proposal focuses on cell wall feruloylation and our long term goal is to identify and isolate novel genes controlling feruloylation and to characterize the phenotype of mutants in this pathway, with a spotlight on cell wall properties. Currently, the genes underlying AX feruloylation have not been identified and the isolation of such genes could be of great importance in manipulating ferulates accretion to the wall. Mutation of the feruloyl transferase gene(s) should lead to less ferulates secreted to the cell wall and reduced ferulate cross-linking. Our current research is based on the hypothesis that controlling the level of total feruloylation will have a direct impact on the level of cross-linking and in turn impact biomass utility for forage and biofuel production. Our results/accomplishments for this project so far include: 1. Mutagenised Brachypodium population. We have developed EMS mutagenized populations of model grass species Brachypodium distachyon. EMS populations have been developed from over 28,000 mutagenized seeds generating 5,184 M2 families. A total of 20,793 plants have been screened and 1,233 were originally selected. 2. Selected Brachypodium mutants: Potential mutants on their levels of cell wall ferulates and cell wall AX ? have been selected from 708 M2 families. A total of 303 back-crosses to no-mutagenized parental stock have been done, followed by selfing selected genotypes in order to confirm heritability of traits and to remove extraneous mutations generated by EMS mutagenesis. We are currently growing 12 F5 and F6 populations in order to assess CW composition. If low level of ferulates are confirmed in the candidate lines selected the mutation could be altered in different in one or several kinds of genes such as genes encoding an AX feruloyl transferase; genes encoding the arabinosyl transferase; genes encoding the synthesis of the xylan backbone; genes encoding enzymes of the monolignol pathway affecting FA

  20. RAF Suppression Synergizes with MEK Inhibition in KRAS Mutant Cancer Cells

    Directory of Open Access Journals (Sweden)

    Simona Lamba

    2014-09-01

    Full Text Available KRAS is the most frequently mutated oncogene in human cancer, yet no therapies are available to treat KRAS mutant cancers. We used two independent reverse genetic approaches to identify components of the RAS-signaling pathways required for growth of KRAS mutant tumors. Small interfering RNA (siRNA screening of 37 KRAS mutant colorectal cancer cell lines showed that RAF1 suppression was synthetic lethal with MEK inhibition. An unbiased kinome short hairpin RNA (shRNA-based screen confirmed this synthetic lethal interaction in colorectal as well as in lung cancer cells bearing KRAS mutations. Compounds targeting RAF kinases can reverse resistance to the MEK inhibitor selumetinib. MEK inhibition induces RAS activation and BRAF-RAF1 dimerization and sustains MEK-ERK signaling, which is responsible for intrinsic resistance to selumetinib. Prolonged dual blockade of RAF and MEK leads to persistent ERK suppression and efficiently induces apoptosis. Our data underlie the relevance of developing combinatorial regimens of drugs targeting the RAF-MEK pathway in KRAS mutant tumors.

  1. Insulin and IGF-1 regularize energy metabolites in neural cells expressing full-length mutant huntingtin.

    Science.gov (United States)

    Naia, Luana; Ribeiro, Márcio; Rodrigues, Joana; Duarte, Ana I; Lopes, Carla; Rosenstock, Tatiana R; Hayden, Michael R; Rego, A Cristina

    2016-08-01

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder linked to the expression of mutant huntingtin. Bioenergetic dysfunction has been described to contribute to HD pathogenesis. Thus, treatment paradigms aimed to ameliorate energy deficits appear to be suitable candidates in HD. In previous studies, we observed protective effects of insulin growth factor-1 (IGF-1) in YAC128 and R6/2 mice, two HD mouse models, whereas IGF-1 and/or insulin halted mitochondrial-driven oxidative stress in mutant striatal cells and mitochondrial dysfunction in HD human lymphoblasts. Here, we analyzed the effect of IGF-1 versus insulin on energy metabolic parameters using striatal cells derived from HD knock-in mice and primary cortical cultures from YAC128 mice. STHdh(Q111/Q111) cells exhibited decreased ATP/ADP ratio and increased phosphocreatine levels. Moreover, pyruvate levels were increased in mutant cells, most probably in consequence of a decrease in pyruvate dehydrogenase (PDH) protein expression and increased PDH phosphorylation, reflecting its inactivation. Insulin and IGF-1 treatment significantly decreased phosphocreatine levels, whereas IGF-1 only decreased pyruvate levels in mutant cells. In a different scenario, primary cortical cultures derived from YAC128 mice also displayed energetic abnormalities. We observed a decrease in both ATP/ADP and phosphocreatine levels, which were prevented following exposure to insulin or IGF-1. Furthermore, decreased lactate levels in YAC128 cultures occurred concomitantly with a decline in lactate dehydrogenase activity, which was ameliorated with both insulin and IGF-1. These data demonstrate differential HD-associated metabolic dysfunction in striatal cell lines and primary cortical cultures, both of which being alleviated by insulin and IGF-1.

  2. Profiling the Hydrolysis of Isolated Grape Berry Skin Cell Walls by Purified Enzymes.

    Science.gov (United States)

    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.

  3. Direct measurement of cell wall stress-stiffening and turgor pressure in live bacterial cells

    CERN Document Server

    Deng, Yi; Shaevitz, Joshua W

    2011-01-01

    The mechanical properties of gram-negative bacteria are governed by a rigid peptidoglycan (PG) cell wall and the turgor pressure generated by the large concentration of solutes in the cytoplasm. The elasticity of the PG has been measured in bulk and in isolated sacculi and shown to be compliant compared to the overall stiffness of the cell itself. However, the stiffness of the cell wall in live cells has not been measured. In particular, the effects that pressure-induced stress might have on the stiffness of the mesh-like PG network have not been addressed even though polymeric materials often exhibit large amounts of stress-stiffening. We study bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. We find strong evidence of power-law stress-stiffening in the E. coli cell wall, with an exponent of $1.07 \\pm 0.25$, such that the wall is significantly stiffer in live cells ($E\\sim32\\pm10$ MPa) than in unpres...

  4. Multi-Walled Carbon Nanotubes Inhibit Breast Cancer Cell Migration.

    Science.gov (United States)

    Graham, Elizabeth G; Wailes, Elizabeth M; Levi-Polyachenko, Nicole H

    2016-02-01

    According to the American Cancer Society, breast cancer is the second leading cause of cancer death in the US. Cancerous cells may have inadequate adhesions to the extracellular matrix and adjacent cells. Previous work has suggested that restoring these contacts may negate the cancer phenotype. This work aims to restore those contacts using multi-walled carbon nanotubes (MWNTs). Varying concentrations of carboxylated MWNTs in water, with or without type I collagen, were dried to create a thin film upon which one of three breast cell lines were seeded: cancerous and metastatic MDA- MB-231 cells, cancerous but non-metastatic MCF7 cells, or non-cancerous MCF10A cells. Proliferation, adhesion, scratch and autophagy assays, western blots, and immunochemical staining were used to assess adhesion and E-cadherin expression. Breast cancer cells grown on a MWNT-collagen coated surface displayed increased adhesion and decreased migration which correlated with an increase in E-cadherin. This work suggests an alternative approach to cancer treatment by physically mediating the cells' microenvironment.

  5. Construction of novel tumor necrosis factor-alpha mutants with reduced toxicity and higher cytotoxicity on human tumor cells

    Institute of Scientific and Technical Information of China (English)

    LIU; Hui; (刘; 惠); LU; Fang; (卢; 芳); CHEN; Jianjun; (陈建军); REN; Hongyu; (任红玉); CHEN; Changqing(陈常庆)

    2003-01-01

    Two tumor necrosis factor-( mutants MT1 (32Trp157Phe) and MT2 (2Lys30Ser- 32Trp157Phe) were constructed by site-directed mutagenesis. These mutants were soluble and over-expressed in E. coli. The purity of purified mutants was above 95% by serial chromatography. The results of Western blot indicated that these mutants could be cross-reactive with monoclonal antibody against native hTNF-α. Compared to parent hTNF-α, the cytotoxicity of these mutants on murine fibrosarcoma L929 cell lines reduced 4-5 orders of magnitude but was equivalent to that of native hTNF-α on human tumor cell lines. The LD50 of mutant MT1 was reduced to 0.34% of wild type and the dose of MT2 that resulted in 30% death of mice reduced to less than 1/700 that of parent hTNF-α.

  6. Transient sedimentation in a cell with top and bottom walls

    Science.gov (United States)

    Dance, Sarah; Maxey, Martin

    2002-11-01

    Wall boundary conditions may play a role in the screening of particle velocity fluctuations in Stokes suspensions. Using a Force-Coupling Method (Maxey and Patel, Int. J. Multiphase Flow 27 (2001)) we simulate transient sedimentation. The numerical scheme is a mixed Fourier-spectral element method, based on the Uzawa algorithm for Stokes flows. The sedimentation cell has top and bottom wall boundaries and periodic boundaries in the horizontal. These boundaries are chosen both for computational convenience, and to determine the relative importance of bottom and side walls in screening the velocity fluctuations. We consider several different box sizes, in an attempt to elucidate the connection between particle velocity fluctuation levels and box width. We quantify the evolution of particle mean velocities and fluctuations as well as the particle microstructure. In each case we observe an initial growth, followed by a decay in both the mean particle velocity and fluctuations. We also observe that a stable stratification develops. We suggest that the stratification is important in the evolution of the bulk mean velocity. We propose a mechanism involving particle cluster dynamics to explain the behaviour of the velocity fluctuations.

  7. Mutant p53 promotes ovarian cancer cell adhesion to mesothelial cells via integrin β4 and Akt signals.

    Science.gov (United States)

    Lee, Jong-Gyu; Ahn, Ji-Hye; Jin Kim, Tae; Ho Lee, Jae; Choi, Jung-Hye

    2015-07-30

    Missense mutations in the TP53 gene resulting in the accumulation of mutant proteins are extremely common in advanced ovarian cancer, which is characterised by peritoneal metastasis. Attachment of cancer cells to the peritoneal mesothelium is regarded as an initial, key step for the metastatic spread of ovarian cancer. In the present study, we investigated the possible role of a p53 mutant in the mesothelial adhesion of ovarian cancer cells. We found that OVCAR-3 cells with the R248 TP53 mutation (p53(R248)) were more adhesive to mesothelial Met5A cells than were A2780 cells expressing wild-type p53. In addition, ectopic expression of p53(R248) in p53-null SKOV-3 cells significantly increased adhesion to Met5A cells. Knockdown of mutant p53 significantly compromised p53(R248)-induced cell adhesion to Met5A cells. Microarray analysis revealed that several adhesion-related genes, including integrin β4, were markedly up-regulated, and certain signalling pathways, including PI3K/Akt, were activated in p53(R248) transfectants of SKOV-3 cells. Inhibition of integrin β4 and Akt signalling using blocking antibody and the inhibitor LY294002, respectively, significantly attenuated p53(R248)-mediated ovarian cancer-mesothelial adhesion. These data suggest that the p53(R248) mutant endows ovarian cancer cells with increased adhesiveness and that integrin β4 and Akt signalling are associated with the mutation-enhanced ovarian cancer-mesothelial cell adhesion.

  8. Cellulose-hemicellulose interaction in wood secondary cell-wall

    Science.gov (United States)

    Zhang, Ning; Li, Shi; Xiong, Liming; Hong, Yu; Chen, Youping

    2015-12-01

    The wood cell wall features a tough and relatively rigid fiber reinforced composite structure. It acts as a pressure vessel, offering protection against mechanical stress. Cellulose microfibrils, hemicellulose and amorphous lignin are the three major components of wood. The structure of secondary cell wall could be imagined as the same as reinforced concrete, in which cellulose microfibrils acts as reinforcing steel bar and hemicellulose-lignin matrices act as the concrete. Therefore, the interface between cellulose and hemicellulose/lignin plays a significant role in determine the mechanical behavior of wood secondary cell wall. To this end, we present a molecular dynamics (MD) simulation study attempting to quantify the strength of the interface between cellulose microfibrils and hemicellulose. Since hemicellulose binds with adjacent cellulose microfibrils in various patterns, the atomistic models of hemicellulose-cellulose composites with three typical binding modes, i.e. bridge, loop and random binding modes are constructed. The effect of the shape of hemicellulose chain on the strength of hemicellulose-cellulose composites under shear loadings is investigated. The contact area as well as hydrogen bonds between cellulose and hemicellulose, together with the covalent bonds in backbone of hemicellulose chain are found to be the controlling parameters which determine the strength of the interfaces in the composite system. For the bridge binding model, the effect of shear loading direction on the strength of the cellulose material is also studied. The obtained results suggest that the shear strength of wood-inspired engineering composites can be optimized through maximizing the formations of the contributing hydrogen bonds between cellulose and hemicellulose.

  9. Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells

    Science.gov (United States)

    Goh, John SY; Liu, Yingwei; Chan, Kah Fai; Wan, Corrine; Teo, Gavin; Zhang, Peiqing; Zhang, Yuanxing; Song, Zhiwei

    2014-01-01

    Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors. PMID:24911584

  10. Modification of chemical properties of cell walls by silicon and its role in regulation of the cell wall extensibility in oat leaves.

    Science.gov (United States)

    Hossain, Mohammad Talim; Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Fujii, Shuhei; Yamamoto, Ryoichi; Hoson, Takayuki

    2007-04-01

    Effects of silicon on the mechanical and chemical properties of cell walls in the second leaf of oat (Avena sativa L.) seedlings were investigated. The cell wall extensibility in the basal region of the second leaf was considerably higher than that in the middle and subapical regions. Externally applied silicon increased the cell wall extensibility in the basal region, but it did not affect the extensibility in the middle and subapical regions. The amounts of cell wall polysaccharides and phenolic compounds, such as diferulic acid (DFA) and ferulic acid (FA), per unit length were lower in the basal region than in the middle and subapical regions of the leaf, and silicon altered these amounts in the basal region. In this region, silicon decreased the amounts of matrix polymers and cellulose per unit length and of DFA and FA, both per unit length and unit matrix polymer content. Silicon treatment also lowered the activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) in the basal region. In contrast, the amount of silicon in cell walls increased in response to silicon treatment in three regions. These results suggest that in the basal region, silicon reduces the net wall mass and the formation of phenolic acid-mediated cross-linkages between wall polysaccharides. Such modifications of wall architecture may be responsible for the silicon-induced increase in the cell wall extensibility in oat leaves.

  11. A radioimmunoassay for lignin in plant cell walls

    Energy Technology Data Exchange (ETDEWEB)

    Dawley, R.M.

    1989-01-01

    Lignin detection and determination in herbaceous tissue requires selective, specific assays which are not currently available. A radioimmunoassay (RIA) was developed to study lignin metabolism in these tissues. A {beta}-aryl ether lignin model compound was synthesized, linked to keyhole limpet hemocyanin using a water-soluble carbodiimide, and injected into rabbits. The highest titer of the antiserum obtained was 34 {eta}g/mL of model derivatized BSA. An in vitro system was developed to characterize the RIA. The model compound was linked to amino activated polyacrylamide beads to mimic lignin in the cell walls. {sup 125}I Radiolabelled protein A was used to detect IgG antibody binding. The RIA was shown in the in vitro system to exhibit saturable binding. The amount of antibody bound decreased when the serum was diluted. Immunoelectrophoresis and competitive binding experiments confirmed that both aromatic rings of the lignin model compound had been antigenic. Chlorogenic acid, a phenolic known to be present in plant cells, did not compete for antibody binding. The RIA was used to measure lignin in milled plant samples and barley seedlings. Antiserum binding to wheat cell walls and stressed barley segments was higher than preimmune serum binding. Antibody binding to stressed barley tissue decreased following NaClO{sub 2} delignification. The RIA was found to be less sensitive than expected, so several avenues for improving the method are discussed.

  12. A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence

    Science.gov (United States)

    Bertolo, Lisa; Monteiro, Mario A.; Agellon, Al; Viswanathan, V. K.; Vedantam, Gayatri

    2016-01-01

    Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA- and lcpB- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA- and lcpB- strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence. PMID:27741317

  13. Chitosan Obtained from Cell Wall of Aspergillus Niger Mycelium

    Institute of Scientific and Technical Information of China (English)

    HUANG Hui-li; LIN Wen-luan; LIN Jian-ming

    2004-01-01

    Chitin from cell walls of Aspergillus Niger mycelium was prepared. A new method for the preparation of high deacetylation degree chitosan was studied in a dilute sodium hydroxide solution at a high pressure. The experimental results indicate that the deacetylation degree of the chitosan can reach 80% under the condition of a 5.00 mol/L sodium hydroxide solution at 0.1 MPa of pressure for 1 h. This method shows the advantages of the applications in the industry production and environment protection.

  14. Mass spectrometry for characterizing plant cell wall polysaccharides

    Directory of Open Access Journals (Sweden)

    Stefan eBauer

    2012-03-01

    Full Text Available Mass spectrometry is a selective and powerful technique to obtain identification and structural information on compounds present in complex mixtures. Since it requires only small sample amount it is an excellent tool for researchers interested in detecting changes in composition of complex carbohydrates of plants. This mini-review gives an overview of common mass spectrometry techniques applied to the analysis of plant cell wall carbohydrates. It presents examples in which mass spectrometry has been used to elucidate the structure of oligosaccharides derived from hemicelluloses and pectins and illustrates how information on sequence, linkages, branching and modifications are obtained from characteristic fragmentation patterns.

  15. Mutants for Drosophila Isocitrate Dehydrogenase 3b Are Defective in Mitochondrial Function and Larval Cell Death

    Science.gov (United States)

    Duncan, Dianne M.; Kiefel, Paula; Duncan, Ian

    2017-01-01

    The death of larval salivary gland cells during metamorphosis in Drosophila melanogaster has been a key system for studying steroid controlled programmed cell death. This death is induced by a pulse of the steroid hormone ecdysone that takes place at the end of the prepupal period. For many years, it has been thought that the ecdysone direct response gene Eip93F (E93) plays a critical role in initiating salivary gland cell death. This conclusion was based largely on the finding that the three “type” alleles of E93 cause a near-complete block in salivary gland cell death. Here, we show that these three mutations are in fact allelic to Idh3b, a nearby gene that encodes the β subunit of isocitrate dehydrogenase 3, a mitochondrial enzyme of the tricarboxylic acid (TCA) cycle. The strongest of the Idh3b alleles appears to cause a near-complete block in oxidative phosphorylation, as mitochondria are depolarized in mutant larvae, and development arrests early during cleavage in embryos from homozygous-mutant germline mothers. Idh3b-mutant larval salivary gland cells fail to undergo mitochondrial fragmentation, which normally precedes the death of these cells, and do not initiate autophagy, an early step in the cell death program. These observations suggest a close relationship between the TCA cycle and the initiation of larval cell death. In normal development, tagged Idh3b is released from salivary gland mitochondria during their fragmentation, suggesting that Idh3b may be an apoptogenic factor that functions much like released cytochrome c in mammalian cells. PMID:28104670

  16. Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands.

    Directory of Open Access Journals (Sweden)

    Carlos Bueno

    Full Text Available Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the

  17. Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands

    Science.gov (United States)

    Bueno, Carlos; Tabares-Seisdedos, Rafael; Moraleda, Jose M.; Martinez, Salvador

    2016-01-01

    Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the idea that MeCP2 may

  18. Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands.

    Science.gov (United States)

    Bueno, Carlos; Tabares-Seisdedos, Rafael; Moraleda, Jose M; Martinez, Salvador

    2016-01-01

    Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the idea that MeCP2 may

  19. Role of Fimbriae, Flagella and Cellulose on the Attachment of Salmonella Typhimurium ATCC 14028 to Plant Cell Wall Models

    Science.gov (United States)

    Tan, Michelle S. F.; White, Aaron P.; Rahman, Sadequr

    2016-01-01

    Cases of foodborne disease caused by Salmonella are frequently associated with the consumption of minimally processed produce. Bacterial cell surface components are known to be important for the attachment of bacterial pathogens to fresh produce. The role of these extracellular structures in Salmonella attachment to plant cell walls has not been investigated in detail. We investigated the role of flagella, fimbriae and cellulose on the attachment of Salmonella Typhimurium ATCC 14028 and a range of isogenic deletion mutants (ΔfliC fljB, ΔbcsA, ΔcsgA, ΔcsgA bcsA and ΔcsgD) to bacterial cellulose (BC)-based plant cell wall models [BC-Pectin (BCP), BC-Xyloglucan (BCX) and BC-Pectin-Xyloglucan (BCPX)] after growth at different temperatures (28°C and 37°C). We found that all three cell surface components were produced at 28°C but only the flagella was produced at 37°C. Flagella appeared to be most important for attachment (reduction of up to 1.5 log CFU/cm2) although both cellulose and fimbriae also aided in attachment. The csgD deletion mutant, which lacks both cellulose and fimbriae, showed significantly higher attachment as compared to wild type cells at 37°C. This may be due to the increased expression of flagella-related genes which are also indirectly regulated by the csgD gene. Our study suggests that bacterial attachment to plant cell walls is a complex process involving many factors. Although flagella, cellulose and fimbriae all aid in attachment, these structures are not the only mechanism as no strain was completely defective in its attachment. PMID:27355584

  20. Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses

    DEFF Research Database (Denmark)

    Nafisi, Majse; Stranne, Maria; Fimognari, Lorenzo;

    2015-01-01

    The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants...... to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall...... acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis....

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

    Energy Technology Data Exchange (ETDEWEB)

    Yotis, W.W.; Zeb, M.; McNulty, J.; Kirchner, F.; Reilly, C.; Glendenin, L.

    1983-07-01

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

  2. Identification and characterization of glycosyltransferases involved in the synthesis of the side chains of the cell wall pectic polysaccharide rhamnogalacturonan II

    Energy Technology Data Exchange (ETDEWEB)

    O' Neill, Malcolm [Univ. of Georgia, Athens, GA (United States)

    2015-08-31

    Our goal was to gain insight into the genes and proteins involved in the biosynthesis of rhamnogalacturonan II (RG-II), a borate cross-linked and structurally conserved pectic polysaccharide present in the primary cell walls of all vascular plants. The research conducted during the funding period established that (i) Avascular plants have the ability to synthesize UDP-apiose but lack the glycosyltransferase machinery required to synthesize RG-II or other apiose-containing cell wall glycans. (ii) RG-II structure is highly conserved in the Lemnaceae (duckweeds and relatives). However, the structures of other wall pectins and hemicellulose have changed substantial during the diversification of the Lemnaceae. This supports the notion that a precise structure of RG-II must be maintained to allow borate cross-linking to occur in a controlled manner. (iii) Enzymes involved in the conversion of UDP-GlcA to UDP-Api, UDP-Xyl, and UDP-Ara may have an important role in controlling the composition of duckweed cell walls. (iv) RG-II exists as the borate ester cross-linked dimer in the cell walls of soybean root hairs and roots. Thus, RG-II is present in the walls of plants cells that grow by tip or by expansive growth. (v) A reduction in RG-II cross-linking in the maize tls1 mutant, which lacks a borate channel protein, suggests that the growth defects observed in the mutant are, at least in part, due to defects in the cell wall.

  3. AtPME3, a ubiquitous cell wall pectin methylesterase of Arabidopsis thaliana, alters the metabolism of cruciferin seed storage proteins during post-germinative growth of seedlings.

    Science.gov (United States)

    Guénin, Stéphanie; Hardouin, Julie; Paynel, Florence; Müller, Kerstin; Mongelard, Gaëlle; Driouich, Azeddine; Lerouge, Patrice; Kermode, Allison R; Lehner, Arnaud; Mollet, Jean-Claude; Pelloux, Jérôme; Gutierrez, Laurent; Mareck, Alain

    2017-02-01

    AtPME3 (At3g14310) is a ubiquitous cell wall pectin methylesterase. Atpme3-1 loss-of-function mutants exhibited distinct phenotypes from the wild type (WT), and were characterized by earlier germination and reduction of root hair production. These phenotypical traits were correlated with the accumulation of a 21.5-kDa protein in the different organs of 4-day-old Atpme3-1 seedlings grown in the dark, as well as in 6-week-old mutant plants. Microarray analysis showed significant down-regulation of the genes encoding several pectin-degrading enzymes and enzymes involved in lipid and protein metabolism in the hypocotyl of 4-day-old dark grown mutant seedlings. Accordingly, there was a decrease in proteolytic activity of the mutant as compared with the WT. Among the genes specifying seed storage proteins, two encoding CRUCIFERINS were up-regulated. Additional analysis by RT-qPCR showed an overexpression of four CRUCIFERIN genes in the mutant Atpme3-1, in which precursors of the α- and β-subunits of CRUCIFERIN accumulated. Together, these results provide evidence for a link between AtPME3, present in the cell wall, and CRUCIFERIN metabolism that occurs in vacuoles.

  4. Listeria monocytogenes is resistant to lysozyme through the regulation, not the acquisition, of cell wall-modifying enzymes.

    Science.gov (United States)

    Burke, Thomas P; Loukitcheva, Anastasia; Zemansky, Jason; Wheeler, Richard; Boneca, Ivo G; Portnoy, Daniel A

    2014-11-01

    Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood.

  5. Mutant huntingtin fragmentation in immune cells tracks Huntington’s disease progression

    OpenAIRE

    Weiss, Andreas; Träger, Ulrike; Wild, Edward J.; Grueninger, Stephan; Farmer, Ruth; Landles, Christian; Scahill, Rachael I.; Lahiri, Nayana; Haider, Salman; Macdonald, Douglas; Frost, Chris; Bates, Gillian P.; Bilbe, Graeme; Kuhn, Rainer; Andre, Ralph

    2012-01-01

    Huntington’s disease (HD) is a fatal, inherited neurodegenerative disorder caused by an expanded CAG repeat in the gene encoding huntingtin (HTT). Therapeutic approaches to lower mutant HTT (mHTT) levels are expected to proceed to human trials, but noninvasive quantification of mHTT is not currently possible. The importance of the peripheral immune system in neurodegenerative disease is becoming increasingly recognized. Peripheral immune cells have been implicated in HD pathogenesis, but HTT ...

  6. Properties of lead deposits in cell walls of radish (Raphanus sativus) roots.

    Science.gov (United States)

    Inoue, Hiroshi; Fukuoka, Daisuke; Tatai, Yuri; Kamachi, Hiroyuki; Hayatsu, Manabu; Ono, Manami; Suzuki, Suechika

    2013-01-01

    Various mechanisms are involved in detoxification of heavy metals such as lead (Pb) in plant cells. Most of the Pb taken up by plants accumulates in their roots. However, the detailed properties of Pb complexes in roots remain unclear. We have investigated the properties of Pb deposits in root cell walls of radish (Raphanus sativus L.) seedlings grown on glass beads bed containing Pb pellets, which are the source of Pb-contamination in shooting range soils. Pb deposits were tightly bound to cell walls. Cell wall fragments containing about 50,000 ppm Pb were prepared from the roots. After extracting Pb from the cell wall fragments using HCl, Pb ions were recombined with the Pb-extracted cell wall fragments in a solution containing Pb acetate. When the cell wall fragments were treated with pectinase (E.C. 3.2.1.15) and were chemically modified with 1-ethyl-3-dimethylamino-propylcarboimide, the Pb-rebinding ability of the treated cell wall fragments decreased. When acid-treated cell wall fragments were incubated in a solution containing Pb(2+) and excess amounts of a chelating agent, Pb recombined with the cell wall fragments were measured to estimate the affinity between Pb(2+) and the cell wall fragments. Our data show that Pb(2+) binds to carboxyl groups of cell walls. The source of the carboxyl groups is suggested to be pectic compounds. A stability constant of the Pb-cell wall complex was estimated to be about 10(8). The role of root cell walls in the mechanism underlying heavy metal tolerance was discussed.

  7. Low expression level of glnA1 accounts for absence of cell wall associated poly-l-glutamate/glutamine in Mycobacterium smegmatis.

    Science.gov (United States)

    Tripathi, Deeksha; Kant, Sashi; Garg, Rajni; Bhatnagar, Rakesh

    2015-03-01

    Cell wall associated poly-l-glutamine (PLG) layer synthesis is directly linked to glutamine synthetase (GS) encoded by glnA1 in tuberculosis causing mycobacteria. Avirulent Mycobacterium smegmatis (M. smegmatis) despite of having a glnA1 homolog lacks cell wall associated PLG layer. In the present study, we complemented a ΔglnA1 mutant of Mycobacterium bovis (lack PLG in cell wall) with M. smegmatis glnA1 cloned under M. bovis glnA1 promoter. PLG synthesis was restored in the cell wall of complemented strain. The complemented strain also showed increased resistance to physical stresses such as lysozyme, SDS and increased survival in THP-1 macrophages in comparison to the knockout. Further, in β-galactosidase reporter assay M. smegmatis glnA1 promoter showed ten times less activity as compared to M. bovis glnA1 promoter. GACT-8-11 → TGAC mutations in the M. smegmatis glnA1 promoter restored its activity by 60% as compared to the activity of glnA1 promoter of M. bovis. This mutation also showed increased GS expression and produced cell wall associated PLG in M. smegmatis. The results of this study demonstrate that glnA1 promoter of M. smegmatis accounts for low expression level of GS and apparently responsible for absence of cell wall associated PLG layer.

  8. Cellulose binding protein from the parasitic nematode Heterodera schachtii interacts with Arabidopsis pectin methylesterase: cooperative cell wall modification during parasitism.

    Science.gov (United States)

    Hewezi, Tarek; Howe, Peter; Maier, Tom R; Hussey, Richard S; Mitchum, Melissa Goellner; Davis, Eric L; Baum, Thomas J

    2008-11-01

    Plant-parasitic cyst nematodes secrete a complex of cell wall-digesting enzymes, which aid in root penetration and migration. The soybean cyst nematode Heterodera glycines also produces a cellulose binding protein (Hg CBP) secretory protein. To determine the function of CBP, an orthologous cDNA clone (Hs CBP) was isolated from the sugar beet cyst nematode Heterodera schachtii, which is able to infect Arabidopsis thaliana. CBP is expressed only in the early phases of feeding cell formation and not during the migratory phase. Transgenic Arabidopsis expressing Hs CBP developed longer roots and exhibited enhanced susceptibility to H. schachtii. A yeast two-hybrid screen identified Arabidopsis pectin methylesterase protein 3 (PME3) as strongly and specifically interacting with Hs CBP. Transgenic plants overexpressing PME3 also produced longer roots and exhibited increased susceptibility to H. schachtii, while a pme3 knockout mutant showed opposite phenotypes. Moreover, CBP overexpression increases PME3 activity in planta. Localization studies support the mode of action of PME3 as a cell wall-modifying enzyme. Expression of CBP in the pme3 knockout mutant revealed that PME3 is required but not the sole mechanism for CBP overexpression phenotype. These data indicate that CBP directly interacts with PME3 thereby activating and potentially targeting this enzyme to aid cyst nematode parasitism.

  9. The involvement of mutant Rac1 in the formation of invadopodia in cultured melanoma cells.

    Science.gov (United States)

    Revach, Or-Yam; Winograd-Katz, Sabina E; Samuels, Yardena; Geiger, Benjamin

    2016-04-10

    In this article, we discuss the complex involvement of a Rho-family GTPase, Rac1, in cell migration and in invadopodia-mediated matrix degradation. We discuss the involvement of invadopodia in invasive cell migration, and their capacity to promote cancer metastasis. Considering the regulation of invadopodia formation, we describe studies that demonstrate the role of Rac1 in the metastatic process, and the suggestion that this effect is attributable to the capacity of Rac1 to promote invadopodia formation. This notion is demonstrated here by showing that knockdown of Rac1 in melanoma cells expressing a wild-type form of this GTPase, reduces invadopodia-dependent matrix degradation. Interestingly, we also show that excessive activity of Rac1, displayed by the P29S, hyperactive, "fast cycling" mutant of Rac1, which is present in 5-10% of melanoma tumors, inhibits invadopodia function. Moreover, knockdown of this hyperactive mutant enhanced matrix degradation, indicating that excessive Rac1 activity by this mutant can negatively regulate invadopodia formation and function.

  10. Dental pulp response to bacterial cell wall material.

    Science.gov (United States)

    Warfvinge, J; Dahlén, G; Bergenholtz, G

    1985-08-01

    Lipopolysaccharides (LPS) from Bacteroides oralis and Veillonella parvula and cell wall material from Lactobacillus casei were studied for their capacity to induce leukocyte migration in the dental pulp and in an implanted wound chamber. Three adult monkeys were challenged using lyophilized material sealed into buccal Class V cavities prepared in dentin. Pulp tissue responses were observed histologically eight and 72 hours after initiation of the experiment. Subjacent to cut dentinal tubules, bacterial materials induced polymorphonuclear leukocyte (PMN's) infiltration in the pulp tissue of the majority of test teeth examined. Responses were similar for the three bacterial test materials at both time periods. Topical applications of bovine serum albumin (BSA), used as a control, induced significantly less accumulation of PMN's. Assessments of induced exudate volumes and leukocyte densities in chambers implanted in rats showed comparable rankings with pulpal experiment between test (i.e., bacterial) and control (BSA) materials. Analysis of the data indicates that high-molecular-weight complexes of bacterial cell walls may adversely affect pulpal tissue across freshly exposed dentin.

  11. Chemical Profiling of the Plant Cell Wall through Raman Microspectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ju; Singh, Seema; Sun, Lan; Simmons, Blake; Auer, Manfred; Parvin, Bahram

    2010-03-02

    This paper presents a computational framework for chemical pro.ling of the plant cell wall through the Raman spectroscopy. The system enables query of known spectral signatures and clustering of spectral data based on intrinsic properties. As a result, presence and relative concentration of speci.c chemical bonds can be quanti.ed. The primary contribution of this paper is in representation of raman pro.le in terms of .uorescence background and multiscale peak detection at each grid point (voxel). Such a representation allows ef.cient spatial segmentation based on the coupling between high-level salient properties and low-level symbolic representation at each voxel. The high-level salient properties refer to preferred peaks and their attributes for the entire image. The low-level symbolic representations are based on .uorescence background, spectral peak locations, and their attributes. We present results on a corn stover tissue section that is imaged through Raman microscopy, and the results are consistent with the literature. In addition, automatic clustering indicates several distinct layers of the cell walls with different spectral signatures.

  12. Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib.

    Science.gov (United States)

    Miller, Rowan E; Brough, Rachel; Bajrami, Ilirjana; Williamson, Chris T; McDade, Simon; Campbell, James; Kigozi, Asha; Rafiq, Rumana; Pemberton, Helen; Natrajan, Rachel; Joel, Josephine; Astley, Holly; Mahoney, Claire; Moore, Jonathan D; Torrance, Chris; Gordan, John D; Webber, James T; Levin, Rebecca S; Shokat, Kevan M; Bandyopadhyay, Sourav; Lord, Christopher J; Ashworth, Alan

    2016-07-01

    New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1-S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472-84. ©2016 AACR.

  13. Murein and pseudomurein cell wall binding domains of bacteria and archaea-a comparative view

    NARCIS (Netherlands)

    Visweswaran, Ganesh Ram R.; Dijkstra, Bauke W.; Kok, Jan

    2011-01-01

    The cell wall, a major barrier protecting cells from their environment, is an essential compartment of both bacteria and archaea. It protects the organism from internal turgor pressure and gives a defined shape to the cell. The cell wall serves also as an anchoring surface for various proteins and a

  14. Fasciclin-like arabinogalactan proteins: specialization for stem biomechanics and cell wall architecture in Arabidopsis and Eucalyptus.

    Science.gov (United States)

    MacMillan, Colleen P; Mansfield, Shawn D; Stachurski, Zbigniew H; Evans, Rob; Southerton, Simon G

    2010-05-01

    The ancient cell adhesion fasciclin (FAS) domain is found in bacteria, fungi, algae, insects and animals, and occurs in a large family of fasciclin-like arabinogalactan proteins (FLAs) in higher plants. Functional roles for FAS-containing proteins have been determined for insects, algae and vertebrates; however, the biological functions of the various higher-plant FLAs are not clear. Expression of some FLAs has been correlated with the onset of secondary-wall cellulose synthesis in Arabidopsis stems, and also with wood formation in the stems and branches of trees, suggesting a biological role in plant stems. We examined whether FLAs contribute to plant stem biomechanics. Using phylogenetic, transcript abundance and promoter-GUS fusion analyses, we identified a conserved subset of single FAS domain FLAs (group A FLAs) in Eucalyptus and Arabidopsis that have specific and high transcript abundance in stems, particularly in stem cells undergoing secondary-wall deposition, and that the phylogenetic conservation appears to extend to other dicots and monocots. Gene-function analyses revealed that Arabidopsis T-DNA knockout double mutant stems had altered stem biomechanics with reduced tensile strength and a reduced tensile modulus of elasticity, as well as altered cell-wall architecture and composition, with increased cellulose microfibril angle and reduced arabinose, galactose and cellulose content. Using materials engineering concepts, we relate the effects of these FLAs on cell-wall composition with stem biomechanics. Our results suggest that a subset of single FAS domain FLAs contributes to plant stem strength by affecting cellulose deposition, and to the stem modulus of elasticity by affecting the integrity of the cell-wall matrix.

  15. Plant Cell Wall Proteins: A Large Body of Data, but What about Runaways?

    Science.gov (United States)

    Albenne, Cécile; Canut, Hervé; Hoffmann, Laurent; Jamet, Elisabeth

    2014-04-17

    Plant cell wall proteomics has been a very dynamic field of research for about fifteen years. A full range of strategies has been proposed to increase the number of identified proteins and to characterize their post-translational modifications. The protocols are still improving to enlarge the coverage of cell wall proteomes. Comparisons between these proteomes have been done based on various working strategies or different physiological stages. In this review, two points are highlighted. The first point is related to data analysis with an overview of the cell wall proteomes already described. A large body of data is now available with the description of cell wall proteomes of seventeen plant species. CWP contents exhibit particularities in relation to the major differences in cell wall composition and structure between these plants and between plant organs. The second point is related to methodology and concerns the present limitations of the coverage of cell wall proteomes. Because of the variety of cell wall structures and of the diversity of protein/polysaccharide and protein/protein interactions in cell walls, some CWPs can be missing either because they are washed out during the purification of cell walls or because they are covalently linked to cell wall components.

  16. Plant Cell Wall Proteins: A Large Body of Data, but What about Runaways?

    Directory of Open Access Journals (Sweden)

    Cécile Albenne

    2014-04-01

    Full Text Available Plant cell wall proteomics has been a very dynamic field of research for about fifteen years. A full range of strategies has been proposed to increase the number of identified proteins and to characterize their post-translational modifications. The protocols are still improving to enlarge the coverage of cell wall proteomes. Comparisons between these proteomes have been done based on various working strategies or different physiological stages. In this review, two points are highlighted. The first point is related to data analysis with an overview of the cell wall proteomes already described. A large body of data is now available with the description of cell wall proteomes of seventeen plant species. CWP contents exhibit particularities in relation to the major differences in cell wall composition and structure between these plants and between plant organs. The second point is related to methodology and concerns the present limitations of the coverage of cell wall proteomes. Because of the variety of cell wall structures and of the diversity of protein/polysaccharide and protein/protein interactions in cell walls, some CWPs can be missing either because they are washed out during the purification of cell walls or because they are covalently linked to cell wall components.

  17. Molecular Mechanisms for Vascular Development and Secondary Cell Wall Formation

    Science.gov (United States)

    Yang, Jung Hyun; Wang, Huanzhong

    2016-01-01

    Vascular tissues are important for transporting water and nutrients throughout the plant and as physical support of upright growth. The primary constituents of vascular tissues, xylem, and phloem, are derived from the meristematic vascular procambium and cambium. Xylem cells develop secondary cell walls (SCWs) that form the largest part of plant lignocellulosic biomass that serve as a renewable feedstock for biofuel production. For the last decade, research on vascular development and SCW biosynthesis has seen rapid progress due to the importance of these processes to plant biology and to the biofuel industry. Plant hormones, transcriptional regulators and peptide signaling regulate procambium/cambium proliferation, vascular patterning, and xylem differentiation. Transcriptional regulatory pathways play a pivot role in SCW biosynthesis. Although most of these discoveries are derived from research in Arabidopsis, many genes have shown conserved functions in biofuel feedstock species. Here, we review the recent advances in our understanding of vascular development and SCW formation and discuss potential biotechnological uses. PMID:27047525

  18. MreB: pilot or passenger of cell wall synthesis?

    Science.gov (United States)

    White, Courtney L; Gober, James W

    2012-02-01

    The discovery that the bacterial cell shape determinant MreB is related to actin spurred new insights into bacterial morphogenesis and development. The trafficking and mechanical roles of the eukaryotic cytoskeleton were hypothesized to have a functional ancestor in MreB based on evidence implicating MreB as an organizer of cell wall synthesis. Genetic, biochemical and cytological studies implicate MreB as a coordinator of a large multi-protein peptidoglycan (PG) synthesizing holoenzyme. Recent advances in microscopy and new biochemical evidence, however, suggest that MreB may function differently than previously envisioned. This review summarizes our evolving knowledge of MreB and attempts to refine the generalized model of the proteins organizing PG synthesis in bacteria. This is generally thought to be conserved among eubacteria and the majority of the discussion will focus on studies from a few well-studied model organisms.

  19. Cell wall proteins of Sporothrix schenckii as immunoprotective agents.

    Science.gov (United States)

    Alba-Fierro, Carlos A; Pérez-Torres, Armando; López-Romero, Everardo; Cuéllar-Cruz, Mayra; Ruiz-Baca, Estela

    2014-01-01

    Sporothrix schenckii is the etiological agent of sporotrichosis, an endemic subcutaneous mycosis in Latin America. Cell wall (CW) proteins located on the cell surface are inducers of cellular and humoral immune responses, potential candidates for diagnosis purposes and to generate vaccines to prevent fungal infections. This mini-review emphasizes the potential use of S. schenckii CW proteins as protective and therapeutic immune response inducers against sporotrichosis. A number of pathogenic fungi display CW components that have been characterized as inducers of protective cellular and humoral immune responses against the whole pathogen from which they were originally purified. The isolation and characterization of immunodominant protein components of the CW of S. schenckii have become relevant because of their potential in the development of protective and therapeutic immune responses against sporotrichosis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

  20. Over-expression of p53 mutants in LNCaP cells alters tumor growth and angiogenesis in vivo

    DEFF Research Database (Denmark)

    Perryman, L A; Blair, J M; Kingsley, E A;

    2006-01-01

    This study has investigated the impact of three specific dominant-negative p53 mutants (F134L, M237L, and R273H) on tumorigenesis by LNCaP prostate cancer cells. Mutant p53 proteins were associated with an increased subcutaneous "take rate" in NOD-SCID mice, and increased production of PSA. Tumors...

  1. Soya beans and Maize : The effect of chemical and physical structure of cell wall polysaccharides on fermentation kinetics

    OpenAIRE

    Laar, van de, P.

    2000-01-01

    The analysis of the relationship between cell wall composition and fermentation of endosperm cell walls of soya beans and maize was approached from three different angles. Firstly, the fermentation (rate and extent of fermentation, the sugar degradation pattern, and volatile fatty acid production) of soya bean and maize cell walls was analysed, both in situ and in vitro. This analysis revealed that the physical structure of the cell wall (particle size and cell wall thickness) influences cell...

  2. The connection of cytoskeletal network with plasma membrane and the cell wall

    Institute of Scientific and Technical Information of China (English)

    Zengyu Liu; Staffan Persson; Yi Zhang

    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 biosyn-thesis and modifications, and aim to provide a platform for further studies in this field.

  3. Mutant presenilin2 promotes apoptosis through the p53/miR-34a axis in neuronal cells.

    Science.gov (United States)

    Li, Liu-Hong; Tu, Qiu-Yun; Deng, Xiao-Hua; Xia, Jian; Hou, De-Ren; Guo, Ke; Zi, Xiao-Hong

    2017-02-08

    Neurodegenerative disorders have attracted attention in last decades due to their high incidence in the world. The p53/miR-34a axis triggers apoptosis and suppresses viability in multiple types of cells, but little is known about its role in neurodegenerative diseases. In this study, we showed that presenilin (PS)-2, a major gene associated with familial Alzheimer's disease (AD) could trigger the apoptosis through the p53/miR-34a axis in PC12 cells. First we found that PC12 cell viability was downregulated by PS-2 and mutant PS-2 overexpression, especially by mutant PS-2 overexpression. Then, we established a mutant PS-2-overexpressing PC12 cell line and confirmed that mutant PS-2 induced not only p53 but also miR-34a expression. The transfection of miR-34a inhibitor reversed PS-2-induced effects on cellular viability and apoptosis. Mutant PS-2 overexpression promoted caspase-3 expression, reduced Sirt1 and Bcl-2 expression, all of which were miR-34a downstream genes related with cell apoptosis. Moreover, mutant PS-2 also activated the p53/miR-34a axis and induced apoptosis in AD transgenic mice brain. These results implied that mutant PS-2 might promote the apoptosis of neuronal cells through triggering the p53/miR-34a axis. Altogether our results provide a novel insight into neurodegenerative disease and deepen our understandings of AD pathogenic processes.

  4. Hematopoietic Stem Cells Expansion in Rotating Wall Vessel

    Institute of Scientific and Technical Information of China (English)

    Yang LIU; Tian-Qing LIU; Xiu-Bo FAN; Dan GE; Zhan-Feng CUI; Xue-Hu MA

    2005-01-01

    @@ 1 Introduction Clinical trials have demonstrated that ex vivo expanded hematopoietic stem cells (HSCs) and progenitors offer great promise in reconstituting in vivo hematopoiesis in patients who have undergone intensive chemotherapy.It is therefore necessary to develop a clinical-scale culture system to provide the expanded HSCs and progenitors.Static culture systems such as T-flasks and gas-permeable blood bags are the most widely used culture devices for expanding hematopoietic cells. But they reveal several inherent limitations: ineffective mixing, lack of control options for dissolved oxygen and pH and difficulty in continuous feeding, which restricts the usefulness of static systems. Several advanced bioreactors have been used in the field of HSCs expansion. But hematopoietic cells are extremely sensitive to shear, so cells in bioreactors such as stirred and perfusion culture systems may suffer physical damage. This problem will be improved by applying the rotating wall vessel (RWV) bioreactor in clinic because of its low shear and unique structure. In this research, cord blood (CB) HSCs were expanded by means of a cell-dilution feeding protocol in RWV.

  5. Mutant p53 confers chemoresistance in non-small cell lung cancer by upregulating Nrf2.

    Science.gov (United States)

    Tung, Min-Che; Lin, Po-Lin; Wang, Yao-Chen; He, Tsung-Ying; Lee, Ming-Ching; Yeh, Sauh D; Chen, Chih-Yi; Lee, Huei

    2015-12-08

    Nrf2 is a key transcription factor for genes coding for antioxidants, detoxification enzymes, and multiple drug resistance and it also confers resistance to anticancer drugs. Here, we hypothesized that mutant p53 could upregulate Nrf2 expression at the transcriptional level, thereby conferring cisplatin resistance in non-small cell lung cancer (NSCLC). Luciferase reporter assays and real-time PCR analysis indicated that the Nrf2 promoter activity and its mRNA levels were markedly suppressed by wild-type p53, but not by mutant p53. Chromatin immunoprecipitation (ChIP) further confirmed that wild-type p53 binds at the p53 putative binding site to block Sp1 binding to the Nrf2 promoter and consequently to suppress the Nrf2 promoter activity. The MTT assay indicated that an increase in Nrf2 expression by mutant p53 is responsible for cisplatin resistance. Among the Nrf2 downstream genes, Bcl-2 and Bcl-xL contribute more strongly to Nrf2-mediated cisplatin resistance when compared with heme oxygenase 1 (HO-1). Cox regression analysis showed that patients with high-Nrf2, high-Bcl-2, high-Bcl-xL mRNA tumors were more commonly occurred unfavorable response to cisplatin-based chemotherapy than their counterparts. The prognostic significance of Nrf2 mRNA levels on OS and RFS was also observed in patients who have received cisplatin-based chemotherapy, particularly in p53-mutant patients. Collectively, mutant p53 may confer cisplatin resistance via upregulation of Nrf2 expression, and Nrf2 mRNA level may predict chemotherapeutic response and outcomes in NSCLC.

  6. Isocitrate dehydrogenase 1 mutant R132H sensitizes glioma cells to BCNU-induced oxidative stress and cell death.

    Science.gov (United States)

    Mohrenz, Isabelle Vanessa; Antonietti, Patrick; Pusch, Stefan; Capper, David; Balss, Jörg; Voigt, Sophia; Weissert, Susanne; Mukrowsky, Alicia; Frank, Jan; Senft, Christian; Seifert, Volker; von Deimling, Andreas; Kögel, Donat

    2013-11-01

    Isocitrate dehydrogenase 1 (IDH1) decarboxylates isocitrate to α-ketoglutarate (α-KG) leading to generation of NADPH, which is required to regenerate reduced glutathione (GSH), the major cellular ROS scavenger. Mutation of R132 of IDH1 abrogates generation of α-KG and leads to conversion of α-KG to 2-hydroxyglutarate. We hypothesized that glioma cells expressing mutant IDH1 have a diminished antioxidative capacity and therefore may encounter an ensuing loss of cytoprotection under conditions of oxidative stress. Our study was performed with LN229 cells stably overexpressing IDH1 R132H and wild type IDH1 or with a lentiviral IDH1 knockdown. Quantification of GSH under basal conditions and following treatment with the glutathione reductase inhibitor BCNU revealed significantly lower GSH levels in IDH1 R132H expressing cells and IDH1 KD cells compared to their respective controls. FACS analysis of cell death and ROS production also demonstrated an increased sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to BCNU, but not to temozolomide. The sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to ROS induction and cell death was further enhanced with the transaminase inhibitor aminooxyacetic acid and under glutamine free conditions, indicating that these cells were more addicted to glutaminolysis. Increased sensitivity to BCNU-induced ROS production and cell death was confirmed in HEK293 cells inducibly expressing the IDH1 mutants R132H, R132C and R132L. Based on these findings we propose that in addition to its established pro-tumorigenic effects, mutant IDH1 may also limit the resistance of gliomas to specific death stimuli, therefore opening new perspectives for therapy.

  7. Establishment of HeLa cell mutants deficient in sphingolipid-related genes using TALENs.

    Directory of Open Access Journals (Sweden)

    Toshiyuki Yamaji

    Full Text Available Sphingolipids are essential components in eukaryotes and have various cellular functions. Recent developments in genome-editing technologies have facilitated gene disruption in various organisms and cell lines. We here show the disruption of various sphingolipid metabolic genes in human cervical carcinoma HeLa cells by using transcription activator-like effector nucleases (TALENs. A TALEN pair targeting the human CERT gene (alternative name COL4A3BP encoding a ceramide transport protein induced a loss-of-function phenotype in more than 60% of HeLa cells even though the cell line has a pseudo-triploid karyotype. We have isolated several loss-of-function mutant clones for CERT, UGCG (encoding glucosylceramide synthase, and B4GalT5 (encoding the major lactosylceramide synthase, and also a CERT/UGCG double-deficient clone. Characterization of these clones supported previous proposals that CERT primarily contributes to the synthesis of SM but not GlcCer, and that B4GalT5 is the major LacCer synthase. These newly established sphingolipid-deficient HeLa cell mutants together with our previously established stable transfectants provide a 'sphingolipid-modified HeLa cell panel,' which will be useful to elucidate the functions of various sphingolipid species against essentially the same genomic background.

  8. RNAi knockdown of PIK3CA preferentially inhibits invasion of mutant PIK3CA cells

    Institute of Scientific and Technical Information of China (English)

    Xin-Ke Zhou; Sheng-Song Tang; Gao Yi; Min Hou; Jin-Hui Chen; Bo Yang; Ji-Fang Liu; Zhi-Min He

    2011-01-01

    AIM: To explore the effects of siRNA silencing of PIK3CA on proliferation, migration and invasion of gastric cancer cells and to investigate the underlying mechanisms.METHODS: The mutation of PIK3CA in exons 9 and 20 of gastric cancer cell lines HGC-27, SGC-7901, BGC-823, MGC-803 and MKN-45 was screened by poly-merase chain reaction (PCR) followed by sequencing. BGC-823 cells harboring no mutations in either of the exons, and HGC-27 cells containing PIK3CA mutations were employed in the current study. siRNA targeting PIK3CA was chemically synthesized and was transfect-ed into these two cell lines in vitro. mRNA and protein expression of PIK3CA were detected by real-time PCR and Western blotting, respectively. We also measured phosphorylation of a serine/threonine protein kinase (Akt) using Western blotting. The proliferation, migra-tion and invasion of these cells were examined sepa-rately by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT), wound healing and Transwell chambers assay.RESULTS: The siRNA directed against PIK3CA effec-tively led to inhibition of both endogenous mRNA and protein expression of PIK3CA, and thus significantly down-regulated phosphorylation of Akt (P < 0.05). Furthermore, simultaneous silencing of PIK3CA result-ed in an obvious reduction in tumor cell proliferation activity, migration and invasion potential (P < 0.01). Intriguing, mutant HGC-27 cells exhibited stronger invasion ability than that shown by wild-type BGC-823 cells. Knockdown of PIK3CA in mutant HGC-27 cells contributed to a reduction in cell invasion to a greater extent than in non-mutant BGC-823 cells.CONCLUSION: siRNA mediated targeting of PIK3CA may specifically knockdown the expression of PIK3CA in gastric cancer cells, providing a potential implication for therapy of gastric cancer.

  9. Role of the cell wall integrity and filamentous growth mitogen-activated protein kinase pathways in cell wall remodeling during filamentous growth.

    Science.gov (United States)

    Birkaya, Barbara; Maddi, Abhiram; Joshi, Jyoti; Free, Stephen J; Cullen, Paul J

    2009-08-01

    Many fungal species including pathogens exhibit filamentous growth (FG) as a means of foraging for nutrients. Genetic screens were performed to identify genes required for FG in the budding yeast Saccharomyces cerevisiae. Genes encoding proteins with established functions in transcriptional activation (MCM1, MATalpha2, PHD1, MSN2, SIR4, and HMS2), cell wall integrity (MPT5, WSC2, and MID2), and cell polarity (BUD5) were identified as potential regulators of FG. The transcription factors MCM1 and MATalpha2 induced invasive growth by promoting diploid-specific bipolar budding in haploid cells. Components of the cell wall integrity pathway including the cell surface proteins Slg1p/Wsc1p, Wsc2p, Mid2p, and the mitogen-activated protein kinase (MAPK) Slt2p/Mpk1p contributed to multiple aspects of the FG response including cell elongation, cell-cell adherence, and agar invasion. Mid2p and Wsc2p stimulated the FG MAPK pathway through the signaling mucin Msb2p and components of the MAPK cascade. The FG pathway contributed to cell wall integrity in parallel with the cell wall integrity pathway and in opposition with the high osmolarity glycerol response pathway. Mass spectrometry approaches identified components of the filamentous cell wall including the mucin-like proteins Msb2p, Flo11p, and subtelomeric (silenced) mucin Flo10p. Secretion of Msb2p, which occurs as part of the maturation of the protein, was inhibited by the ss-1,3-glucan layer of the cell wall, which highlights a new regulatory aspect to cell wall remodeling in this organism. Disruption of ss-1,3-glucan linkages induced mucin shedding and resulted in defects in cell-cell adhesion and invasion of cells into the agar matrix.

  10. A comparative proteome analysis of Escherichia coli DeltarelA mutant cells

    Directory of Open Access Journals (Sweden)

    Sónia Carneiro

    2016-10-01

    Full Text Available The bacterial RelA-dependent stringent response exerts a strong influence over a wide variety of processes. In this work, the impact of the relA gene mutation in E. coli cells was evaluated by a quantitative proteomics analysis, employing stable-isotope labelling and high resolution mass spectrometry. Chemostat cultures of E. coli W3110 and ∆relA mutant strains were performed at two dilution rates (0.1 and 0.2 h-1 to assess the influence of the relA gene mutation in steady-state protein levels. A total of 121 proteins showed significant alterations in their abundance when comparing the proteome of mutant to wild-type cells. The relA gene mutation induced changes on key cellular processes, including the amino acids and nucleotide biosynthesis, the lipid metabolism, transport activities, transcription and translation processes and responses to stress. Furthermore, some of those changes were more pronounced under specific growth conditions, as the most significant differences in protein ratios were observed at one of the dilution rates. An effect of the relA gene mutation in the acetate overflow was also observed, which confers interesting characteristics to this mutant strain that could be useful in the production of recombinant proteins. Overall, these results provide a valuable insight into the E. coli stringent response under defined steady-state conditions, suggesting that this stress response might influence multiple metabolic processes like the acetate overflow or the catabolite repression.

  11. Comparison of phenotypes between different vangl2 mutants demonstrates dominant effects of the Looptail mutation during hair cell development.

    Directory of Open Access Journals (Sweden)

    Haifeng Yin

    Full Text Available Experiments utilizing the Looptail mutant mouse, which harbors a missense mutation in the vangl2 gene, have been essential for studies of planar polarity and linking the function of the core planar cell polarity proteins to other developmental signals. Originally described as having dominant phenotypic traits, the molecular interactions underlying the Looptail mutant phenotype are unclear because Vangl2 protein levels are significantly reduced or absent from mutant tissues. Here we introduce a vangl2 knockout mouse and directly compare the severity of the knockout and Looptail mutant phenotypes by intercrossing the two lines and assaying the planar polarity of inner ear hair cells. Overall the vangl2 knockout phenotype is milder than the phenotype of compound mutants carrying both the Looptail and vangl2 knockout alleles. In compound mutants a greater number of hair cells are affected and changes in the orientation of individual hair cells are greater when quantified. We further demonstrate in a heterologous cell system that the protein encoded by the Looptail mutation (Vangl2(S464N disrupts delivery of Vangl1 and Vangl2 proteins to the cell surface as a result of oligomer formation between Vangl1 and Vangl2(S464N, or Vangl2 and Vangl2(S464N, coupled to the intracellular retention of Vangl2(S464N. As a result, Vangl1 protein is missing from the apical cell surface of vestibular hair cells in Looptail mutants, but is retained at the apical cell surface of hair cells in vangl2 knockouts. Similarly the distribution of Prickle-like2, a putative Vangl2 interacting protein, is differentially affected in the two mutant lines. In summary, we provide evidence for a direct physical interaction between Vangl1 and Vangl2 through a combination of in vitro and in vivo approaches and propose that this interaction underlies the dominant phenotypic traits associated with the Looptail mutation.

  12. Human Lysozyme Synergistically Enhances Bactericidal Dynamics and Lowers the Resistant Mutant Prevention Concentration for Metronidazole to Helicobacter pylori by Increasing Cell Permeability

    Directory of Open Access Journals (Sweden)

    Xiaolin Zhang

    2016-10-01

    Full Text Available Metronidazole (MNZ is an effective agent that has been employed to eradicate Helicobacter pylori (H. pylori. The emergence of broad MNZ resistance in H. pylori has affected the efficacy of this therapeutic agent. The concentration of MNZ, especially the mutant prevention concentration (MPC, plays an important role in selecting or enriching resistant mutants and regulating therapeutic effects. A strategy to reduce the MPC that can not only effectively treat H. pylori but also prevent resistance mutations is needed. H. pylori is highly resistant to lysozyme. Lysozyme possesses a hydrolytic bacterial cell wall peptidoglycan and a cationic dependent mode. These effects can increase the permeability of bacterial cells and promote antibiotic absorption into bacterial cells. In this study, human lysozyme (hLYS was used to probe its effects on the integrity of the H. pylori outer and inner membranes using as fluorescent probe hydrophobic 1-N-phenyl-naphthylamine (NPN and the release of aspartate aminotransferase. Further studies using a propidium iodide staining method assessed whether hLYS could increase cell permeability and promote cell absorption. Finally, we determined the effects of hLYS on the bactericidal dynamics and MPC of MNZ in H. pylori. Our findings indicate that hLYS could dramatically increase cell permeability, reduce the MPC of MNZ for H. pylori, and enhance its bactericidal dynamic activity, demonstrating that hLYS could reduce the probability of MNZ inducing resistance mutations.

  13. Penium margaritaceum as a model organism for cell wall analysis of expanding plant cells

    DEFF Research Database (Denmark)

    Rydahl, Maja Gro; Fangel, Jonatan Ulrik; Mikkelsen, Maria Dalgaard

    2015-01-01

    organization of the polymeric networks of the cell wall around the protoplast also contributes to the direction of growth, the shape of the cell, and the proper positioning of the cell in a tissue. In essence, plant cell expansion represents the foundation of development. Most studies of plant cell expansion...... have focused primarily upon late divergent multicellular land plants and specialized cell types (e.g., pollen tubes, root hairs). Here, we describe a unicellular green alga, Penium margaritaceum (Penium), which can serve as a valuable model organism for understanding cell expansion and the underlying...

  14. Whole organ, venation and epidermal cell morphological variations are correlated in the leaves of Arabidopsis mutants.

    Science.gov (United States)

    Pérez-Pérez, José Manuel; Rubio-Díaz, Silvia; Dhondt, Stijn; Hernández-Romero, Diana; Sánchez-Soriano, Joaquín; Beemster, Gerrit T S; Ponce, María Rosa; Micol, José Luis

    2011-12-01

    Despite the large number of genes known to affect leaf shape or size, we still have a relatively poor understanding of how leaf morphology is established. For example, little is known about how cell division and cell expansion are controlled and coordinated within a growing leaf to eventually develop into a laminar organ of a definite size. To obtain a global perspective of the cellular basis of variations in leaf morphology at the organ, tissue and cell levels, we studied a collection of 111 non-allelic mutants with abnormally shaped and/or sized leaves, which broadly represent the mutational variations in Arabidopsis thaliana leaf morphology not associated with lethality. We used image-processing techniques on these mutants to quantify morphological parameters running the gamut from the palisade mesophyll and epidermal cells to the venation, whole leaf and rosette levels. We found positive correlations between epidermal cell size and leaf area, which is consistent with long-standing Avery's hypothesis that the epidermis drives leaf growth. In addition, venation parameters were positively correlated with leaf area, suggesting that leaf growth and vein patterning share some genetic controls. Positional cloning of the genes affected by the studied mutations will eventually establish functional links between genotypes, molecular functions, cellular parameters and leaf phenotypes.

  15. The serine/threonine phosphatase DhSIT4 modulates cell cycle, salt tolerance and cell wall integrity in halo tolerant yeast Debaryomyces hansenii.

    Science.gov (United States)

    Chawla, Srishti; Kundu, Debasree; Randhawa, Anmoldeep; Mondal, Alok K

    2017-03-30

    The highly conserved family of Phosphoprotein phosphatases (PPP) regulates several major physiological processes in yeast. However, very little is known about the PPP orthologs from the yeast species inhabiting extreme environmental niches. In the present study we have identified DhSIT4, a member of PPP6 class of serine threonine phosphatases from the halotolerant yeast Debaryomyces hansenii. Deletion of DhSIT4 in D. hansenii was not lethal but the mutant exhibited reduced growth due to its effect on the cell cycle. The knock out mutant Dhsit4Δ showed sensitivity towards Li(+), Na(+) and cell wall damaging agents. The expression of DhSit4p rescued salt, caffeine and calcofluor white sensitivity of Dhmpk1Δ strain and thereby indicating a genetic interaction of this phosphatase with the cell wall integrity pathway in this species. Our study also demonstrated the antagonistic roles of DhSit4p and DhPpz1p in maintaining the cell cycle and ion homeostasis in D. hansenii.

  16. Area Expansivity Moduli of Regenerating Plant Protoplast Cell Walls Exposed to Shear Flows

    Science.gov (United States)

    Fujimura, Yuu; Iino, Masaaki; Watanabe, Ugai

    2005-05-01

    To control the elasticity of the plant cell wall, protoplasts isolated from cultured Catharanthus roseus cells were regenerated in shear flows of 115 s-1 (high shear) and 19.2 s-1 (low shear, as a control). The surface area expansivity modulus and the surface breaking strength of these regenerating protoplasts were measured by a micropipette aspiration technique. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye. High shear exposure for 3 h doubled both the surface area modulus and breaking strength observed under low shear, significantly decreased cell wall synthesis, and roughly quadrupled the moduli of the cell wall. Based on the cell wall synthesis data, we estimated the three-dimensional modulus of the cell wall to be 4.1± 1.2 GPa for the high shear, and 0.35± 0.2 GPa for the low shear condition, using the surface area expansivity modulus divided by the cell wall thickness, which is identical with the Young’s modulus divided by 2(1-σ), where σ is Poisson's ratio. We concluded that high shear exposure considerably strengthens the newly synthesized cell wall.

  17. Human T-cell leukemia virus I tax protein sensitizes p53-mutant cells to DNA damage.

    Science.gov (United States)

    Mihaylova, Valia T; Green, Allison M; Khurgel, Moshe; Semmes, Oliver J; Kupfer, Gary M

    2008-06-15

    Mutations in p53 are a common cause of resistance of cancers to standard chemotherapy and, thus, treatment failure. Reports have shown that Tax, a human T-cell leukemia virus type I encoded protein that has been associated with genomic instability and perturbation of transcription and cell cycle, sensitizes HeLa cells to UV treatment. The extent to which Tax can sensitize cells and the mechanism by which it exerts its effect are unknown. In this study, we show that Tax sensitizes p53-mutant cells to a broad range of DNA-damaging agents, including mitomycin C, a bifunctional alkylator, etoposide, a topoisomerase II drug, and UV light, but not ionizing radiation, a double-strand break agent, or vinblastine, a tubulin poison. Tax caused hypersensitivity in all p53-deleted cell lines and several, but not all, mutant-expressed p53-containing cell lines, while unexpectedly being protective in p53 wild-type (wt) cells. The effect observed in p53-deleted lines could be reversed for this by transfection of wt p53. We also show that Tax activates a p53-independent proapoptotic program through decreased expression of the retinoblastoma protein and subsequent increased E2F1 expression. The expression of several proapoptotic proteins was also induced by Tax, including Puma and Noxa, culminating in a substantial increase in Bax dimerization. Our results show that Tax can sensitize p53-mutant cells to DNA damage while protecting p53 wt cells, a side benefit that might result in reduced toxicity in normal cells. Such studies hold the promise of a novel adjunctive therapy that could make cancer chemotherapy more effective.

  18. SpyB, a small heme-binding protein, affects the composition of the cell wall in Streptococcus pyogenes

    Directory of Open Access Journals (Sweden)

    Rebecca J Edgar

    2016-10-01

    Full Text Available Streptococcus pyogenes (Group A Streptococcus or GAS is a haemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C3-like ADP-ribosyltransferase SpyA. In this study we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to β-lactams of the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Thus, our data suggest the possibility that SpyB activity is regulated by heme.

  19. bcpmr1 encodes a P-type Ca(2+)/Mn(2+)-ATPase mediating cell-wall integrity and virulence in the phytopathogen Botrytis cinerea.

    Science.gov (United States)

    Plaza, Verónica; Lagües, Yanssuy; Carvajal, Mauro; Pérez-García, Luis A; Mora-Montes, Hector M; Canessa, Paulo; Larrondo, Luis F; Castillo, Luis

    2015-03-01

    The cell wall of fungi is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. These usually contain both N- and O-linked oligosaccharides, coupled to the proteins by stepwise addition of mannose residues by mannosyltransferases in the endoplasmic reticulum and the Golgi apparatus. In yeast, an essential luminal cofactor for these mannosyltransferases is Mn(2+) provided by the Ca(2+)/Mn(2+)-ATPase known as Pmr1. In this study, we have identified and characterized the Botrytis cinerea pmr1 gene, the closest homolog of yeast PMR1. We hypothesized that bcpmr1 also encodes a Ca(2+)/Mn(2+)-ATPase that plays an important role in the protein glycosylation pathway. Phenotypic analysis showed that bcpmr1 null mutants displayed a significant reduction in conidial production, radial growth and diameter of sclerotia. Significant alterations in hyphal cell wall composition were observed including a 83% decrease of mannan levels and an increase in the amount of chitin and glucan. These changes were accompanied by a hypersensitivity to cell wall-perturbing agents such as Calcofluor white, Congo red and zymolyase. Importantly, the Δbcpmr1 mutant showed reduced virulence in tomato (leafs and fruits) and apple (fruits) and reduced biofilm formation. Together, our results highlight the importance of bcpmr1 for protein glycosylation, cell wall structure and virulence of B. cinerea.

  20. Cyclic di-AMP Is Critical for Listeria monocytogenes Growth, Cell Wall Homeostasis, and Establishment of Infection

    Science.gov (United States)

    Witte, Chelsea E.; Whiteley, Aaron T.; Burke, Thomas P.; Sauer, John-Demian; Portnoy, Daniel A.; Woodward, Joshua J.

    2013-01-01

    ABSTRACT Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection. PMID:23716572

  1. Composition and architecture of the cell walls of grasses and the mechanisms of synthesis of cell wall polysaccharides. Final report for period September 1, 1988 - April 30, 2001

    Energy Technology Data Exchange (ETDEWEB)

    Carpita, Nicholas C.

    2001-10-18

    This program was devoted toward complete understanding of the polysaccharide structure and architecture of the primary cell walls grasses and cereals, and the biosynthesis of the mixed-linkage beta-glucane, a cellulose interacting polymer that is synthesized uniquely by grass species and close relatives. With these studies as focal point, the support from DOE was instrumental in the development of new analytical means that enabled us to characterize carbohydrate structure, to reveal new features of cell wall dynamics during cell growth, and to apply these techniques in other model organisms. The support by DOE in these basic studies was acknowledged on numerous occasions in review articles covering current knowledge of cell wall structure, architecture, dynamics, biosynthesis, and in all genes related to cell wall biogenesis.

  2. Pectinous cell wall thickenings formation - A common defense strategy of plants to cope with Pb.

    Science.gov (United States)

    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.

  3. Cell Wall Microstructure Analysis Implicates Hemicellulose Polysaccharides in Cell Adhesion in Tomato Fruit Pericarp Parenchyma

    Institute of Scientific and Technical Information of China (English)

    Jose J. Ordaz-Ortiz; Susan E. Marcus; J. Paul Knox

    2009-01-01

    Methods developed to isolate intact cells from both unripe and ripe tomato fruit pericarp parenchyma have allowed the cell biological analysis of polysaccharide epitopes at the surface of separated cells. The LM7 pectic homoga-lacturonan epitope is a marker of the junctions of adhesion planes and intercellular spaces in parenchyma systems. The LM7 epitope persistently marked the former edge of adhesion planes at the surface of cells separated from unripe and ripened tomato fruit and also from fruits with the Cnr mutation. The LM 11 xylan epitope was associated, in sections, with cell walls lining intercellular space but the epitope was not detected at the surface of isolated cells, being lost during cell isolation. The LM15 xyloglucan epitope was present at the surface of cells isolated from unripe fruit in a pattern reflecting the former edge of cell adhesion planes/intercellular space but with gaps and apparent breaks, An equivalent pattern ofLM15 epitope occurrence was revealed at the surface of cells isolated by pectate lyase action but was not present in cells isolated from ripe fruit or from Cnr fruit. In contrast to wild-type cells, the LM5 galactan and LM21 mannan epitopes oc-curred predominantly in positions reflecting intercellular space in Cnr, suggesting a concerted alteration in cell wall mi-crostructure in response to this mutation. Galactanase and mannanase, along with pectic homogalacturonan-degrading enzymes, were capable of releasing cells from unripe fruit parenchyma. These observations indicate that hemicellulose polymers are present in architectural contexts reflecting cell adhesion and that several cell wall polysaccharide classes are likely to contribute to cell adhesion/cell separation in tomato fruit pericarp parenchyma.

  4. Plant cell walls throughout evolution: towards a molecular understanding of their design principles

    Energy Technology Data Exchange (ETDEWEB)

    Sarkar, Purbasha; Bosneaga, Elena; Auer, Manfred

    2009-02-16

    Throughout their life, plants typically remain in one location utilizing sunlight for the synthesis of carbohydrates, which serve as their sole source of energy as well as building blocks of a protective extracellular matrix, called the cell wall. During the course of evolution, plants have repeatedly adapted to their respective niche,which is reflected in the changes of their body plan and the specific design of cell walls. Cell walls not only changed throughout evolution but also are constantly remodelled and reconstructed during the development of an individual plant, and in response to environmental stress or pathogen attacks. Carbohydrate-rich cell walls display complex designs, which together with the presence of phenolic polymers constitutes a barrier for microbes, fungi, and animals. Throughout evolution microbes have co-evolved strategies for efficient breakdown of cell walls. Our current understanding of cell walls and their evolutionary changes are limited as our knowledge is mainly derived from biochemical and genetic studies, complemented by a few targeted yet very informative imaging studies. Comprehensive plant cell wall models will aid in the re-design of plant cell walls for the purpose of commercially viable lignocellulosic biofuel production as well as for the timber, textile, and paper industries. Such knowledge will also be of great interest in the context of agriculture and to plant biologists in general. It is expected that detailed plant cell wall models will require integrated correlative multimodal, multiscale imaging and modelling approaches, which are currently underway.

  5. Antioxidant properties of cell wall polysaccharides of Stevia rebaudiana leaves

    Directory of Open Access Journals (Sweden)

    Mediesse Kengne Francine

    2014-12-01

    Full Text Available Objective: To examine the total phenolic and protein contents, and the antioxidant activities of cell wall polysaccharide fractions of Stevia rebaudiana leaves. Methods: Three different polysaccharide-enriched fractions, namely FPE (extract with 50 mmol/ L ethylene diamine tetra acetic acid, FPK (extract with 0.05 mol/L KOH and FH (extract with 4 mol/L KOH were extracted from Stevia rebaudiana leaves. The antioxidant activity of these fractions was evaluated based on their ability to scavenge DPPH (1, 1-diphenyl-2-picryl hydrazyl free radical, to reduce ferric power, to chelate ferrous ion and to protect human DNA. Results: The results indicated that protein content was found to be higher in FPK polysaccharide enriched fraction (47.48 µg per mg of FPK. Furthermore, the phenolic compound analysis according to the Folin-Ciocalteu method was higher in FPK (17.71 µg ferulic acid. The DPPH maximal inhibition percentage of the three polysaccharide-enriched fractions at 400 µg/mL was 27.66%, 59.90% and 23.21% respectively for FPE, FPK and FH. All the polysaccharide fractions exhibited a ferric reducing power except the FH one. The three fractions also exhibited lipid peroxidation inhibition, and they completely reverted the DNA damage induced by H2O2/FeCl2. FPK showed the strongest scavenging activity against the DPPH radical, the best chelating ability and lipid peroxidation inhibition. Conclusions: Stevia cell wall polysaccharide fractions are potent protective agents against oxidative stress. The analysis revealed major differences in the antioxidant activity in the three polysaccharides fractions. However, the 0.05 mol/L KOH pectin fraction (FPK showed better antioxidant activity.

  6. Antioxidant properties of cell wall polysaccharides of Stevia rebaudiana leaves

    Institute of Scientific and Technical Information of China (English)

    Mediesse Kengne Francine; Woguia Alice Louise; Fogue Souopgui Pythagore; Atogho-Tiedeu Barbara; Simo Gustave; Thadde Boudjeko

    2014-01-01

    Objective: To examine the total phenolic and protein contents, and the antioxidant activities of cell wall polysaccharide fractions of Stevia rebaudiana leaves.Methods:L ethylene diamine tetra acetic acid), FPK (extract with 0.05 mol/L KOH) and FH (extract with 4 mol/L KOH) were extracted from Stevia rebaudiana leaves. The antioxidant activity of these fractions was evaluated based on their ability to scavenge DPPH (1, 1-diphenyl-2-picryl hydrazyl) free radical, to reduce ferric power, to chelate ferrous ion and to protect human DNA. Three different polysaccharide-enriched fractions, namely FPE (extract with 50 mmol/Results: The results indicated that protein content was found to be higher in FPK polysaccharide enriched fraction (47.48 µg per mg of FPK). Furthermore, the phenolic compound analysis according to the Folin-Ciocalteu method was higher in FPK (17.71 µg ferulic acid). The DPPH maximal inhibition percentage of the three polysaccharide-enriched fractions at 400 µg/mL was 27.66%, 59.90% and 23.21% respectively for FPE, FPK and FH. All the polysaccharide fractions exhibited a ferric reducing power except the FH one. The three fractions also exhibited lipid peroxidation inhibition, and they completely reverted the DNA damage induced by H2O2/FeCl2. FPK showed the strongest scavenging activity against the DPPH radical, the best chelating ability and lipid peroxidation inhibition.Conclusions: Stevia cell wall polysaccharide fractions are potent protective agents against oxidative stress. The analysis revealed major differences in the antioxidant activity in the three polysaccharides fractions. However, the 0.05 mol/L KOH pectin fraction (FPK) showed better antioxidant activity.

  7. Evidence that pulsed electric field treatment enhances the cell wall porosity of yeast cells.

    Science.gov (United States)

    Ganeva, Valentina; Galutzov, Bojidar; Teissie, Justin

    2014-02-01

    The application of rectangular electric pulses, with 0.1-2 ms duration and field intensity of 2.5-4.5 kV/cm, to yeast suspension mediates liberation of cytoplasmic proteins without cell lysis. The aim of this study was to evaluate the effect of pulsed electric field with similar parameters on cell wall porosity of different yeast species. We found that electrically treated cells become more susceptible to lyticase digestion. In dependence on the strain and the electrical conditions, cell lysis was obtained at 2-8 times lower enzyme concentration in comparison with control untreated cells. The increase of the maximal lysis rate was between two and nine times. Furthermore, when applied at low concentration (1 U/ml), the lyticase enhanced the rate of protein liberation from electropermeabilized cells without provoking cell lysis. Significant differences in the cell surface of control and electrically treated cells were revealed by scanning electron microscopy. Data presented in this study allow us to conclude that electric field pulses provoke not only plasma membrane permeabilization, but also changes in the cell wall structure, leading to increased wall porosity.

  8. CELL-WALL GROWTH AND PROTEIN SECRETION IN FUNGI

    NARCIS (Netherlands)

    SIETSMA, JH; WOSTEN, HAB; WESSELS, JGH

    1995-01-01

    Secretion of proteins is a vital process in fungi. Because hyphal walls form a diffusion barrier for proteins, a mechanism different from diffusion probably exist to transport proteins across the wall. In Schizophyllum commune, evidence has been obtained for synthesis at the hyphal apex of wall comp

  9. Wall extensibility: its nature, measurement and relationship to plant cell growth

    Science.gov (United States)

    Cosgrove, D. J.

    1993-01-01

    Expansive growth of plant cells is controlled principally by processes that loosen the wall and enable it to expand irreversibly. The central role of wall relaxation for cell expansion is reviewed. The most common methods for assessing the extension properties of plant cell walls ( wall extensibility') are described, categorized and assessed critically. What emerges are three fundamentally different approaches which test growing cells for their ability (a) to enlarge at different values of turgor, (b) to induce wall relaxation, and (c) to deform elastically or plastically in response to an applied tensile force. Analogous methods with isolated walls are similarly reviewed. The results of these different assays are related to the nature of plant cell growth and pertinent biophysical theory. I argue that the extensibilities' measured by these assays are fundamentally different from one another and that some are more pertinent to growth than others.

  10. Apoptosis mechanisms of human gastric cancer cell line MKN-45 infected with human mutant p27

    Institute of Scientific and Technical Information of China (English)

    Jin-Shui Zhu; Long Wang; Guo-Qiang Cheng; Qin Li; Zu-Ming Zhu; Li Zhu

    2005-01-01

    AIM: To explore the inducing effect of human mutant p27 gene on the apoptosis of the human gastric cancer cell line MKN-45 and its associated mechanisms. METHODS: The recombinant adenovirus Ad-p27mt was constructed to infect the human gastric cancer cell line MKN-45. Using flow cytometry, TUNEL assay and DNA fragment analysis, we measured the apoptotic effect of Ad-p27mt on the human gastric cancer cells. RESULTS: Ad-p27mt was successfully constructed and the infection efficiency reached 100%. After 18 h of infection, we observed an apoptotic hypodiploid peak on the flow cytometer before G1-S and apoptotic characteristic bands in the DNA electrophoresis. The apoptotic rate detected by TUNEL method was significantly higher in the Ad-p27mt group (89.4±3.12%)compared to the control group (3.12±0.13%, P < 0.01).CONCLUSION: Human mutant p27 can induce apoptosis of the human gastric cancer cells in vitro.

  11. DBIO Best Thesis Award: Mechanics, Dynamics, and Organization of the Bacterial Cytoskeleton and Cell Wall

    Science.gov (United States)

    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.

  12. Absence of fks1p in lager brewing yeast results in aberrant cell wall composition and improved beer flavor stability.

    Science.gov (United States)

    Wang, Jin-jing; Xu, Wei-na; Li, Xin'er; Li, Jia; Li, Qi

    2014-06-01

    The flavor stability during storage is very important to the freshness and shelf life of beer. However, beer fermented with a yeast strain which is prone to autolyze will significantly affect the flavor of product. In this study, the gene encoding β-1,3-glucan synthetase catalytic subunit (fks1) of the lager yeast was destroyed via self-clone strategy. β-1,3-glucan is the principle cell wall component, so fks1 disruption caused a decrease in β-1,3-glucan level and increase in chitin level in cell wall, resulting in the increased cell wall thickness. Comparing with wild-type strain, the mutant strain had 39.9 and 63.41 % less leakage of octanoic acid and decanoic acid which would significantly affect the flavor of beer during storage. Moreover, the results of European Brewery Convention tube fermentation test showed that the genetic manipulation to the industrial brewing yeast helped with the anti-staling ability, rather than affecting the fermentation ability. The thiobarbituric acid value reduced by 65.59 %, and the resistant staling value increased by 26.56 %. Moreover, the anti-staling index of the beer fermented with mutant strain increased by 2.64-fold than that from wild-type strain respectively. China has the most production and consumption of beer around the world, so the quality of beer has a significant impact on Chinese beer industry. The result of this study could help with the improvement of the quality of beer in China as well as around the world.

  13. Mycobacterium tuberculosis CwsA overproduction modulates cell division and cell wall synthesis.

    Science.gov (United States)

    Plocinski, P; Martinez, L; Sarva, K; Plocinska, R; Madiraju, M; Rajagopalan, M

    2013-12-01

    We recently showed that two small membrane proteins of Mycobacterium tuberculosis, CwsA and CrgA, interact with each other, and that loss of CwsA in M. smegmatis is associated with defects in the cell division and cell wall synthesis processes. Here we show that CwsA overproduction also affected growth, cell division and cell shape of M. smegmatis and M. tuberculosis. CwsA overproduction in M. tuberculosis led to increased sensitivity to cefsulodin, a penicillin-binding protein (PBP) 1A/1B targeting beta (β) -lactam, but was unaffected by other β-lactams and vancomycin. A M. smegmatis cwsA overexpressing strain showed bulgy cells, increased fluorescent vancomycin staining and altered localization of Wag31-mCherry fusion protein. However, the levels of phosphorylated Wag31, important for optimal peptidoglycan synthesis and growth in mycobacteria, were not affected. Interestingly, CwsA overproduction in E. coli led to the formation of large rounded cells that eventually lysed whereas the overproduction of FtsZ along with CwsA reversed this phenotype. Together, our results emphasize that optimal levels of CwsA are required for regulated cell wall synthesis, hence maintenance of cell shape, and that CwsA likely interacts with and modulates the activities of other cell wall synthetic components including PBPs.

  14. A shift to organismal stress resistance in programmed cell death mutants.

    Directory of Open Access Journals (Sweden)

    Meredith E Judy

    Full Text Available Animals have many ways of protecting themselves against stress; for example, they can induce animal-wide, stress-protective pathways and they can kill damaged cells via apoptosis. We have discovered an unexpected regulatory relationship between these two types of stress responses. We find that C. elegans mutations blocking the normal course of programmed cell death and clearance confer animal-wide resistance to a specific set of environmental stressors; namely, ER, heat and osmotic stress. Remarkably, this pattern of stress resistance is induced by mutations that affect cell death in different ways, including ced-3 (cell death defective mutations, which block programmed cell death, ced-1 and ced-2 mutations, which prevent the engulfment of dying cells, and progranulin (pgrn-1 mutations, which accelerate the clearance of apoptotic cells. Stress resistance conferred by ced and pgrn-1 mutations is not additive and these mutants share altered patterns of gene expression, suggesting that they may act within the same pathway to achieve stress resistance. Together, our findings demonstrate that programmed cell death effectors influence the degree to which C. elegans tolerates environmental stress. While the mechanism is not entirely clear, it is intriguing that animals lacking the ability to efficiently and correctly remove dying cells should switch to a more global animal-wide system of stress resistance.

  15. Induction of 8-azaguanine resistant mutants in human cultured cells exposed to 31 MeV protons

    Energy Technology Data Exchange (ETDEWEB)

    Fuhrman Conti, A.M.; Francone, G.; Volonte, M.; Gallini, R.E.

    1988-03-01

    The authors report results on the induction of 8-azaguanine (8-AG)-resistant mutants in cultured human cells (EUE) exposed to 31 MeV protons. The spontaneous frequency of mutants was 5.6 +- 0.7 x 10/sup -6/ per viable cell. Gamma rays were taken as reference radiation. Expression times giving the highest frequency of mutants after 31 MeV protons and gamma irradiation were found to be about 10 days for both radiations. The dose-response relationship for mutant induction by protons, as determined at the optimal expression time, was compared to that obtained after gamma rays. The relative biological effectiveness (RBE) is 2.4 +- 0.5, this value being higher than the RBE value determined for cell survival.

  16. Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin

    Directory of Open Access Journals (Sweden)

    Seok-Hyung Kim

    2011-03-01

    Tuberous sclerosis complex (TSC is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin or TSC2 (encodes tuberin genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242 in the tsc2 gene. This tsc2vu242 allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2vu242 is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2vu242/vu242 homozygous zebrafish, and is moderately increased in tsc2vu242/+ heterozygotes. Forebrain neurons are poorly organized in tsc2vu242/vu242 homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2vu242/vu242 mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

  17. Cell wall dynamics modulate acetic acid-induced apoptotic cell death of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    António Rego

    2014-08-01

    Full Text Available Acetic acid triggers apoptotic cell death in Saccharomyces cerevisiae, similar to mammalian apoptosis. To uncover novel regulators of this process, we analyzed whether impairing MAPK signaling affected acetic acid-induced apoptosis and found the mating-pheromone response and, especially, the cell wall integrity pathways were the major mediators, especially the latter, which we characterized further. Screening downstream effectors of this pathway, namely targets of the transcription factor Rlm1p, highlighted decreased cell wall remodeling as particularly important for acetic acid resistance. Modulation of cell surface dynamics therefore emerges as a powerful strategy to increase acetic acid resistance, with potential application in industrial fermentations using yeast, and in biomedicine to exploit the higher sensitivity of colorectal carcinoma cells to apoptosis induced by acetate produced by intestinal propionibacteria.

  18. Stem cell-like ALDHbright cellular states in EGFR-mutant non-small cell lung cancer

    Science.gov (United States)

    Corominas-Faja, Bruna; Oliveras-Ferraros, Cristina; Cuyàs, Elisabet; Segura-Carretero, Antonio; Joven, Jorge; Martin-Castillo, Begoña; Barrajón-Catalán, Enrique; Micol, Vicente; Bosch-Barrera, Joaquim; Menendez, Javier A

    2013-01-01

    The enrichment of cancer stem cell (CSC)-like cellular states has not previously been considered to be a causative mechanism in the generalized progression of EGFR-mutant non-small cell lung carcinomas (NSCLC) after an initial response to the EGFR tyrosine kinase inhibitor erlotinib. To explore this possibility, we utilized a pre-clinical model of acquired erlotinib resistance established by growing NSCLC cells containing a TKI-sensitizing EGFR exon 19 deletion (ΔE746-A750) in the continuous presence of high doses of erlotinib. Genome-wide analyses using Agilent 44K Whole Human Genome Arrays were evaluated via bioinformatics analyses through GSEA-based screening of the KEGG pathway database to identify the molecular circuitries that were over-represented in the transcriptomic signatures of erlotinib-refractory cells. The genomic spaces related to erlotinib resistance included a preponderance of cell cycle genes (E2F1, -2, CDC2, -6) and DNA replication-related genes (MCM4, -5, -6, -7), most of which are associated with early lung development and poor prognosis. In addition, metabolic genes such as ALDH1A3 (a candidate marker for lung cancer cells with CSC-like properties) were identified. Thus, we measured the proportion of erlotinib-resistant cells expressing very high levels of aldehyde dehydrogenase (ALDH) activity attributed to ALDH1/3 isoforms. Using flow cytometry and the ALDEFLUOR® reagent, we confirmed that erlotinib-refractory cell populations contained drastically higher percentages (>4500%) of ALDHbright cells than the parental erlotinib-responsive cells. Notably, strong decreases in the percentages of ALDHbright cells were observed following incubation with silibinin, a bioactive flavonolignan that can circumvent erlotinib resistance in vivo. The number of lung cancer spheres was drastically suppressed by silibinin in a dose-dependent manner, thus confirming the ability of this agent to inhibit the self-renewal of erlotinib-refractory CSC-like cells

  19. Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant1[C][W

    Science.gov (United States)

    Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2014-01-01

    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants. PMID:25037213

  20. Overproduction of stomatal lineage cells in Arabidopsis mutants defective in active DNA demethylation.

    Science.gov (United States)

    Yamamuro, Chizuko; Miki, Daisuke; Zheng, Zhimin; Ma, Jun; Wang, Jing; Yang, Zhenbiao; Dong, Juan; Zhu, Jian-Kang

    2014-06-05

    DNA methylation is a reversible epigenetic mark regulating genome stability and function in many eukaryotes. In Arabidopsis, active DNA demethylation depends on the function of the ROS1 subfamily of genes that encode 5-methylcytosine DNA glycosylases/lyases. ROS1-mediated DNA demethylation plays a critical role in the regulation of transgenes, transposable elements and some endogenous genes; however, there have been no reports of clear developmental phenotypes in ros1 mutant plants. Here we report that, in the ros1 mutant, the promoter region of the peptide ligand gene EPF2 is hypermethylated, which greatly reduces EPF2 expression and thereby leads to a phenotype of overproduction of stomatal lineage cells. EPF2 gene expression in ros1 is restored and the defective epidermal cell patterning is suppressed by mutations in genes in the RNA-directed DNA methylation pathway. Our results show that active DNA demethylation combats the activity of RNA-directed DNA methylation to influence the initiation of stomatal lineage cells.

  1. The phenotype of FancB-mutant mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu Lingchuan [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245 (United States); Hasty, Paul, E-mail: hastye@uthscsa.edu [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245 (United States)

    2011-07-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslinking agent mitomycin C (MMC), increased spontaneous and MMC-induced chromosomal abnormalities, reduced spontaneous sister chromatid exchanges (SCEs), reduced gene targeting, reduced MMC-induced Rad51 foci and absent MMC-induced FancD2 foci. Since FancB is on the X chromosome and since ES cells are typically XY, FancB is an excellent target for an epistatic analysis to elucidate FA's role in ICL repair.

  2. Range of cell-wall alterations enhance saccharification in Brachypodium distachyon mutants

    DEFF Research Database (Denmark)

    Marriott, Poppy E; Sibout, Richard; Lapierre, Catherine

    2014-01-01

    Lignocellulosic plant biomass is an attractive feedstock for the production of sustainable biofuels, but the commercialization of such products is hampered by the high costs of processing this material into fermentable sugars (saccharification). One approach to lowering these costs is to produce ...

  3. Combinatorial BTK and MALT1 inhibition augments killing of CD79 mutant diffuse large B cell lymphoma.

    Science.gov (United States)

    Nagel, Daniel; Bognar, Miriam; Eitelhuber, Andrea C; Kutzner, Kerstin; Vincendeau, Michelle; Krappmann, Daniel

    2015-12-08

    Survival of activated B cell-subtype (ABC) of diffuse large B cell lymphoma (DLBCL) is driven by chronic B cell receptor (BCR) signaling that activates the canonical NF-κB pathway. Inhibition of BTK by Ibrutinib has been shown to kill ABC DLBCL cells that carry activating mutations in the BCR adaptor CD79. However, mutations in BTK or in downstream components such as CARMA1/CARD11 can render lymphomas Ibrutinib resistant. Therefore, we assessed here the simultaneous inhibition of BTK and the protease MALT1 that acts downstream of CARMA1 and is essential for ABC DLBCL tumor growth. We show that in CD79 mutant cells BTK is a crucial upstream regulator of MALT1, but dispensable in CARMA1 mutant ABC DLBCL. Combined inhibition of BTK by Ibrutinib and MALT1 by S-Mepazine additively impaired MALT1 cleavage activity and expression of NF-κB pro-survival factors. Thereby, combinatorial Ibrutinib and S-Mepazine treatment enhanced killing of CD79 mutant ABC DLBCL cells. Moreover, while expression of oncogenic CARMA1 in CD79 mutant cells conferred Ibrutinib resistance, double mutant cells were still sensitive to MALT1 inhibition by S-Mepazine. Thus, based on the genetic background combinatorial BTK and MALT1 inhibition may improve effectiveness of therapeutic treatment and reduce the chances for the development of drug resistances.

  4. Osmotic Stress Suppresses Cell Wall Stiffening and the Increase in Cell Wall-Bound Ferulic and Diferulic Acids in Wheat Coleoptiles.

    Science.gov (United States)

    Wakabayashi, K.; Hoson, T.; Kamisaka, S.

    1997-01-01

    The relationship between the mechanical properties of cell walls and the levels of wall-bound ferulic (FA) and diferulic (DFA) acids was investigated in wheat (Triticum aestivum L.) coleoptiles grown under osmotic stress (60 mM polyethylene glycol [PEG] 4000) conditions. The cell walls of stressed coleoptiles remained extensible compared with those of the unstressed ones. The contents of wall-bound FA and DFA increased under unstressed conditions, but the increase was substantially reduced by osmotic stress. In response to PEG removal, these contents increased and reached almost the same levels as those of the unstressed coleoptiles. A close correlation was observed between the contents of FA and DFA and the mechanical properties of cell walls. The activities of phenylalanine ammonia-lyase and tyrosine ammonia-lyase increased rapidly under unstressed conditions. Osmotic stress substantially reduced the increases in enzyme activities. When PEG was removed, however, the enzyme activities increased rapidly. There was a close correlation between the FA levels and enzyme activities. These results suggest that in osmotically stressed wheat coleoptiles, reduced rates of increase in phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities suppress phenylpropanoid biosynthesis, resulting in the reduced level of wall-bound FA that, in turn, probably causes the reduced level of DFA and thereby maintains cell wall extensibility. PMID:12223657

  5. Cell-free layer and wall shear stress variation in microvessels.

    Science.gov (United States)

    Yin, Xuewen; Zhang, Junfeng

    2012-01-01

    In this study, we simulated multiple red blood cells flowing through straight microvessels with the immersed-boundary lattice-Boltzmann model to examine the shear stress variation on the microvessel surface and its relation to the properties of cell-free layer. Significant variation in shear stress has been observed due to the irregular configuration of blood cells flowing near the microvessel wall. A low shear stress is typically found at locations where there is a cell flowing close to the wall, and a large shear stress at locations with a relatively wide gap between cell and wall. This relationship between the shear stress magnitude and the distance between cell and wall has been attributed to the reverse pressure difference developed between the front and rear sides of a cell flowing near the vessel wall. We further studied the effects of several hemodynamic factors on the variation of shear stress, including the cell deformability, the flow rate, and the aggregation among red blood cells. These simulations show that the shear stress variation is less profound in situations with wider cell-free layers, since the reverse pressure difference around the edge cells is less evident, and the influence of this pressure difference on wall shear stress becomes weaker. This study also demonstrates the complexity of the flow field in the gap between cell and wall. More precise experimental techniques are required accurately measure such shear stress variation in microcirculation.

  6. 5-Fluorouracil preferentially sensitizes mutant KRAS non-small cell lung carcinoma cells to TRAIL-induced apoptosis.

    Science.gov (United States)

    Wang, Haizhen; Yang, Tao; Wu, Xiangwei

    2015-11-01

    Mutations in the KRAS gene are very common in non-small cell lung cancer (NSCLC), but effective therapies targeting KRAS have yet to be developed. Interest in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent inducer of cell death, has increased following the observation that TRAIL can selectively kill a wide variety of human cancer cells without killing normal cells both in vitro and in xenograft models. However, results from clinical trials of TRAIL-based therapy are disappointingly modest at best and many have demonstrated a lack of therapeutic benefit. Current research has focused on selecting a subpopulation of cancer patients who may benefit from TRAIL-based therapy and identifying best drugs to work with TRAIL. In the current study, we found that NSCLC cells with a KRAS mutation were highly sensitive to treatment with TRAIL and 5-fluorouracil (5FU). Compared with other chemotherapeutic agents, 5FU displayed the highest synergy with TRAIL in inducing apoptosis in mutant KRAS NSCLC cells. We also found that, on a mechanistic level, 5FU preferentially repressed survivin expression and induced expression of TRAIL death receptor 5 to sensitize NSCLC cells to TRAIL. The combination of low-dose 5FU and TRAIL strongly inhibited xenograft tumor growth in mice. Our results suggest that the combination of TRAIL and 5FU may be beneficial for patients with mutant KRAS NSCLC.

  7. Primary abdominal wall clear cell carcinoma arising from incisional endometriosis

    Institute of Scientific and Technical Information of China (English)

    Burcu Gundogdu; Isin Ureyen; Gunsu Kimyon; Hakan Turan; Nurettin Boran; Gokhan Tulunay; Dilek Bulbul; Taner Turan; M Faruk Kose

    2013-01-01

    A 49 year-old patient with the complaint of a mass located in the caesarean scar was admitted. There was a fixed mass 30í30 mm in diameter with regular contour located at the right corner of the pfannenstiel incision. Computed tomography revealed a (40í50í50) mm solid mass lesion with margins that cannot be distinguished from the uterus, bladder and small intestines and a heterogeneous mass lesion (50í45í55) mm in diameter, located in the right side of the anterior abdominal wall. Cytoreductive surgery including total abdominal hysterectomy and bilateral salpingo-oophorectomy was performed. Final pathology was clear cell carcinoma. Clear cell carcinoma arising from an extraovarian endometriotic focus was diagnosed and the patient received 6 cycles paclitaxel-carboplatin chemotherapy as adjuvant treatment. The patient who was lost to follow-up applied to our clinic 2 years after surgery with a recurrent mass in the left inguinal region. After 3 cycles of chemotherapy, the patient's tumoral mass in the left inguinal region was excised. The result of the pathology was carcinoma metastasis. It is decided that the following treatment of the patient should be palliative radiation therapy. The patient who underwent palliative radiation therapy died of disease after 4 months of the second operation.

  8. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

    Directory of Open Access Journals (Sweden)

    Shayla K Shorter

    Full Text Available T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL, have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4 are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

  9. Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls

    Directory of Open Access Journals (Sweden)

    Pedersen Henriette L

    2008-05-01

    Full Text Available Abstract Background 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. Results 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. Conclusion 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

  10. Modification of antioxidant systems in cell walls of maize roots by different nitrogen sources

    Directory of Open Access Journals (Sweden)

    Vesna Hadži-Tašković Šukalović

    2016-12-01

    Full Text Available Antioxidant systems of maize root cell walls grown on different nitrogen sources were evaluated. Plants were grown on a medium containing only NO3- or the mixture of NO3-+NH4+, in a 2:1 ratio. Eleven-day old plants, two days after the initiation of lateral roots, were used for the experiments. Cell walls were isolated from lateral roots and primary root segments, 2-7 cm from tip to base, representing zones of intense or decreased growth rates, respectively. Protein content and the activity of enzymes peroxidase, malate dehydrogenase and ascorbate oxidase ionically or covalently bound to the walls, as well as cell wall phenolic content and antioxidant capacity, were determined. Cell walls of plants grown on mixed N possess more developed enzymatic antioxidant systems and lower non-enzymatic antioxidant defenses than cell walls grown on NO3-. Irrespective of N treatment, the activities of all studied enzymes and protein content were higher in cell walls of lateral compared to primary roots. Phenolic content of cell walls isolated from lateral roots was higher in NO3--grown than in mixed N grown plants. No significant differences could be observed in the isozyme patterns of cell wall peroxidases isolated from plants grown on different nutrient solution. Our results indicate that different N treatments modify the antioxidant systems of root cell walls. Treatment with NO3- resulted in an increase of constitutive phenolic content, while the combination of NO3-+NH4+ elevated the redox enzyme activities in root cell walls.

  11. ErbB2 inhibition by lapatinib promotes degradation of mutant p53 protein in cancer cells.

    Science.gov (United States)

    Li, Dun; Marchenko, Natalia D

    2017-01-24

    Mutations in the p53 tumor suppressor gene are the most prevalent genetic events in human Her2-positive breast cancer and are associated with poor prognosis and survival. Human clinical data and our in vitro and in vivo studies strongly suggest potent oncogenic cooperation between mutant p53 and Her2 (ErbB2). Yet, the translational significance of mutant p53 in Her2 positive breast cancer, especially with respect to Her2-targeted therapies, has not been evaluated. Our previous work identified novel oncogenic activity of mutant p53 whereby mutp53 amplifies ErbB2 signaling via the mutp53-HSF1-ErbB2 feed-forward loop. Here we report that pharmacological interception of this circuit by ErbB2 inhibitor lapatinib downregulates mutant p53 in vitro and in vivo. We found that ErbB2 inhibition by lapatinib inhibits transcription factor HSF1, and its target Hsp90, followed by mutant p53 degradation in MDM2 dependent manner. Thus, our data suggest that mutant p53 sensitizes cancer cells to lapatinib via two complementary mechanisms: mutant p53 mediated amplification of ErbB2 signaling, and simultaneous annihilation of both potent oncogenic drivers, ErbB2 and mutant p53. Hence, our study could provide valuable information for the optimization of therapeutic protocols to achieve superior clinical effects in the treatment of Her2 positive breast cancer.

  12. Role of the Group B antigen of Streptococcus agalactiae: a peptidoglycan-anchored polysaccharide involved in cell wall biogenesis.

    Directory of Open Access Journals (Sweden)

    Élise Caliot

    Full Text Available Streptococcus agalactiae (Group B streptococcus, GBS is a leading cause of infections in neonates and an emerging pathogen in adults. The Lancefield Group B carbohydrate (GBC is a peptidoglycan-anchored antigen that defines this species as a Group B Streptococcus. Despite earlier immunological and biochemical characterizations, the function of this abundant glycopolymer has never been addressed experimentally. Here, we inactivated the gene gbcO encoding a putative UDP-N-acetylglucosamine-1-phosphate:lipid phosphate transferase thought to catalyze the first step of GBC synthesis. Indeed, the gbcO mutant was unable to synthesize the GBC polymer, and displayed an important growth defect in vitro. Electron microscopy study of the GBC-depleted strain of S. agalactiae revealed a series of growth-related abnormalities: random placement of septa, defective cell division and separation processes, and aberrant cell morphology. Furthermore, vancomycin labeling and peptidoglycan structure analysis demonstrated that, in the absence of GBC, cells failed to initiate normal PG synthesis and cannot complete polymerization of the murein sacculus. Finally, the subcellular localization of the PG hydrolase PcsB, which has a critical role in cell division of streptococci, was altered in the gbcO mutant. Collectively, these findings show that GBC is an essential component of the cell wall of S. agalactiae whose function is reminiscent of that of conventional wall teichoic acids found in Staphylococcus aureus or Bacillus subtilis. Furthermore, our findings raise the possibility that GBC-like molecules play a major role in the growth of most if not all beta-hemolytic streptococci.

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

    Science.gov (United States)

    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.

  14. CELL WALL CARBOHYDRATE EPITOPES IN THE GREEN ALGA OEDOGONIUM BHARUCHAE F. MINOR (OEDOGONIALES, CHLOROPHYTA)(1).

    Science.gov (United States)

    Estevez, José M; Leonardi, Patricia I; Alberghina, Josefina S

    2008-10-01

    Cell wall changes in vegetative and suffultory cells (SCs) and in oogonial structures from Oedogonium bharuchae N. D. Kamat f. minor Vélez were characterized using monoclonal antibodies against several carbohydrate epitopes. Vegetative cells and SCs develop only a primary cell wall (PCW), whereas mature oogonial cells secrete a second wall, the oogonium cell wall (OCW). Based on histochemical and immunolabeling results, (1→4)-β-glucans in the form of crystalline cellulose together with a variable degree of Me-esterified homogalacturonans (HGs) and hydroxyproline-rich glycoprotein (HRGP) epitopes were detected in the PCW. The OCW showed arabinosides of the extensin type and low levels of arabinogalactan-protein (AGP) glycans but lacked cellulose, at least in its crystalline form. Surprisingly, strong colabeling in the cytoplasm of mature oogonia cells with three different antibodies (LM-5, LM-6, and CCRC-M2) was found, suggesting the presence of rhamnogalacturonan I (RG-I)-like structures. Our results are discussed relating the possible functions of these cell wall epitopes with polysaccharides and O-glycoproteins during oogonium differentiation. This study represents the first attempt to characterize these two types of cell walls in O. bharuchae, comparing their similarities and differences with those from other green algae and land plants. This work represents a contribution to the understanding of how cell walls have evolved from simple few-celled to complex multicelled organisms.

  15. Transcriptome profiling identifies genes and pathways deregulated upon floxuridine treatment in colorectal cancer cells harboring GOF mutant p53

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    Arindam Datta

    2016-06-01

    Full Text Available Mutation in TP53 is a common genetic alteration in human cancers. Certain tumor associated p53 missense mutants acquire gain-of-function (GOF properties and confer oncogenic phenotypes including enhanced chemoresistance. The colorectal cancers (CRC harboring mutant p53 are generally aggressive in nature and difficult to treat. To identify a potential gene expression signature of GOF mutant p53-driven acquired chemoresistance in CRC, we performed transcriptome profiling of floxuridine (FUdR treated SW480 cells expressing mutant p53R273H (GEO#: GSE77533. We obtained several genes differentially regulated between FUdR treated and untreated cells. Further, functional characterization and pathway analysis revealed significant enrichment of crucial biological processes and pathways upon FUdR treatment in SW480 cells. Our data suggest that in response to chemotherapeutics treatment, cancer cells with GOF mutant p53 can modulate key cellular pathways to withstand the cytotoxic effect of the drugs. The genes and pathways identified in the present study can be further validated and targeted for better chemotherapy response in colorectal cancer patients harboring mutant p53.

  16. Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa.

    Science.gov (United States)

    Ruiz-Baca, Estela; Hernández-Mendoza, Gustavo; Cuéllar-Cruz, Mayra; Toriello, Conchita; López-Romero, Everardo; Gutiérrez-Sánchez, Gerardo

    2014-07-01

    The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa.

  17. CONSTITUTIVE MELANIN IN THE CELL WALL OF THE ETIOLOGIC AGENT OF LOBO'S DISEASE

    Directory of Open Access Journals (Sweden)

    TABORDA Valeria B.A.

    1999-01-01

    Full Text Available Lobo's disease is a chronic granulomatous disease caused by the obligate pathogenic fungus, whose cell walls contain constitutive melanin. In contrast, melanin does not occur in the cell walls of Paracoccidioides brasiliensis when stained by the Fontana-Masson stain.

  18. Modification of cell wall architecture of wheat coleoptiles grown under hypergravity conditions.

    Science.gov (United States)

    Wakabayashi, Kazuyuki; Soga, Kouichi; Kamisaka, Seiichiro; Hoson, Takayuki

    2003-10-01

    Cell wall structure of wheat coleoptiles grown under continuous hypergravity (300 g) conditions was investigated. Length of coleoptiles exposed to hypergravity for 2-4 days from germination stage was 60-70% of that of 1 g control. The amounts of cell wall polysaccharides substantially increased during the incubation period both in 1 g control and hypergravity-treated coleoptiles. As a results, the levels of cell wall polysaccharides per unit length of coleoptile, which mean the thickness of cell walls, largely increased under hypergravity conditions. The major sugar components of the hemicellulose fraction, a polymer fraction extracted from cell walls with strong alkali, were arabinose (Ara), xylose (Xyl) and glucose (Glc). The molar ratios of Ara and Xyl to Glc in hypergravity-treated coleoptiles were higher than those in control coleoptiles. Furthermore, the fractionation of hemicellulosic polymers into the neutral and acidic polymers by the anion-exchange column showed that the levels of acidic polymers in cell walls of hypergravity-treated coleoptiles were higher than those of control coleoptiles. These results suggest that hypergravity stimuli bias the synthesis of hemicellulosic polysaccharides and increase the proportion of acidic polymers, such as arabinoxylans, in cell walls of wheat coleoptiles. These structural changes in cell walls may contribute to plant resistance to hypergravity stimuli.

  19. Cell wall composition as a maize defense mechanism against corn borers.

    Science.gov (United States)

    Barros-Rios, Jaime; Malvar, Rosa A; Jung, Hans-Joachim G; Santiago, Rogelio

    2011-04-01

    European and Mediterranean corn borers are two of the most economically important insect pests of maize (Zea mays L.) in North America and southern Europe, respectively. Cell wall structure and composition were evaluated in pith and rind tissues of resistant and susceptible inbred lines as possible corn borer resistance traits. Composition of cell wall polysaccharides, lignin concentration and composition, and cell wall bound forms of hydroxycinnamic acids were measured. As expected, most of the cell wall components were found at higher concentrations in the rind than in the pith tissues, with the exception of galactose and total diferulate esters. Pith of resistant inbred lines had significantly higher concentrations of total cell wall material than susceptible inbred lines, indicating that the thickness of cell walls could be the initial barrier against corn borer larvae attack. Higher concentrations of cell wall xylose and 8-O-4-coupled diferulate were found in resistant inbreds. Stem tunneling by corn borers was negatively correlated with concentrations of total diferulates, 8-5-diferulate and p-coumarate esters. Higher total cell wall, xylose, and 8-coupled diferulates concentrations appear to be possible mechanisms of corn borer resistance.

  20. In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth

    NARCIS (Netherlands)

    Muller, K.; Linkies, A.; Vreeburg, R.A.M.; Fry, S.C.; Krieger-Liszkay, A.; Leubner-Metzger, G.

    2009-01-01

    Loosening of cell walls is an important developmental process in key stages of the plant life cycle, including seed germination, elongation growth, and fruit ripening. Here, we report direct in vivo evidence for hydroxyl radical (·OH)-mediated cell wall loosening during plant seed germination and se

  1. Cell wall growth during elongation and division : one ring to bind them?

    NARCIS (Netherlands)

    Scheffers, Dirk-Jan

    2007-01-01

    The role of the cell division protein FtsZ in bacterial cell wall (CW) synthesis is believed to be restricted to localizing proteins involved in the synthesis of the septal wall. Elsewhere, compelling evidence is provided that in Caulobacter crescentus, FtsZ plays an additional role in CW synthesis

  2. Establishment and mitotic characterization of new Drosophila acentriolar cell lines from DSas-4 mutant

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    Nicolas Lecland

    2013-01-01

    In animal cells the centrosome is commonly viewed as the main cellular structure driving microtubule (MT assembly into the mitotic spindle apparatus. However, additional pathways, such as those mediated by chromatin and augmin, are involved in the establishment of functional spindles. The molecular mechanisms involved in these pathways remain poorly understood, mostly due to limitations inherent to current experimental systems available. To overcome these limitations we have developed six new Drosophila cell lines derived from Drosophila homozygous mutants for DSas-4, a protein essential for centriole biogenesis. These cells lack detectable centrosomal structures, astral MT, with dispersed pericentriolar proteins D-PLP, Centrosomin and γ-tubulin. They show poorly focused spindle poles that reach the plasma membrane. Despite being compromised for functional centrosome, these cells could successfully undergo mitosis. Live-cell imaging analysis of acentriolar spindle assembly revealed that nascent MTs are nucleated from multiple points in the vicinity of chromosomes. These nascent MTs then grow away from kinetochores allowing the expansion of fibers that will be part of the future acentriolar spindle. MT repolymerization assays illustrate that acentriolar spindle assembly occurs “inside-out” from the chromosomes. Colchicine-mediated depolymerization of MTs further revealed the presence of a functional Spindle Assembly Checkpoint (SAC in the acentriolar cells. Finally, pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly.

  3. An internal ribosome entry site (IRES mutant library for tuning expression level of multiple genes in mammalian cells.

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    Esther Y C Koh

    Full Text Available A set of mutated Encephalomyocarditis virus (EMCV internal ribosome entry site (IRES elements with varying strengths is generated by mutating the translation initiation codons of 10(th, 11(th, and 12(th AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells.

  4. Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides.

    Science.gov (United States)

    Ishii, T; Thomas, J; Darvill, A; Albersheim, P

    1989-02-01

    Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-alpha-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-alpha-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na(2)CO(3) at 1 and 22 degrees C. These previously uncharacterized polysaccharides accounted for approximately 4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO(3)-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na(2)CO(3) at 1 degrees C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells.

  5. Degenerative hairlets on the vestibular sensory cells in mutant bustling (BUS/Idr) mice.

    Science.gov (United States)

    Moriyama, K; Hashimoto, R; Hanai, A; Yoshizaki, N; Yonezawa, S; Otani, H

    1997-01-01

    The bustling mouse (BUS/Idr: bus) is a mutant mouse strain which exhibits deafness, bustling/hyperkinetic behaviour and functional disorders seemingly related to the vestibular system. This phenotype develops in homozygous (bus/bus) mice and has been shown from cross experiments to be genetically induced by a single autosomal recessive gene. We previously detected, with light and electron microscopy, post-natal degeneration of the inner ear sensory cells in homozygotes. In the present study, we examined, by electron microscopy, the development of pathological changes in the sensory epithelia of the macula acustica and crista ampullaris of homozygous mice of various ages, paying special attention to the detailed morphology of the sensory hairlets. The homozygous mice exhibited specific pathological changes: a decrease in the number of hairs; disarrangement of the kinocilium-stereocilia pattern; and, fused and/or very large stereocilia. Homozygotes also frequently exhibited apical cytoplasmic herniation, or bleb of hair cells, as well as a degenerated kinocilium in the sensory epithelium. Heterozygotes showed similar changes, but to a lesser degree and frequency. As for the vestibular organs, similar pathological changes had developed at day, 17 of gestation. These pathological findings and onset suggest that the BUS mouse may be a mutant mouse strain distinct from other reported strains which display similar behaviour, and may be a useful animal model for the study of human degenerative vestibular disorders.

  6. Antimicrobial action and cell agglutination by the eosinophil cationic protein are modulated by the cell wall lipopolysaccharide structure.

    Science.gov (United States)

    Pulido, David; Moussaoui, Mohammed; Andreu, David; Nogués, M Victòria; Torrent, Marc; Boix, Ester

    2012-05-01

    Antimicrobial proteins and peptides (AMPs) are essential effectors of innate immunity, acting as a first line of defense against bacterial infections. Many AMPs exhibit high affinity for cell wall structures such as lipopolysaccharide (LPS), a potent endotoxin able to induce sepsis. Hence, understanding how AMPs can interact with and neutralize LPS endotoxin is of special relevance for human health. Eosinophil cationic protein (ECP) is an eosinophil secreted protein with high activity against both Gram-negative and Gram-positive bacteria. ECP has a remarkable affinity for LPS and a distinctive agglutinating activity. By using a battery of LPS-truncated E. coli mutant strains, we demonstrate that the polysaccharide moiety of LPS is essential for ECP-mediated bacterial agglutination, thereby modulating its antimicrobial action. The mechanism of action of ECP at the bacterial surface is drastically affected by the LPS structure and in particular by its polysaccharide moiety. We have also analyzed an N-terminal fragment that retains the whole protein activity and displays similar cell agglutination behavior. Conversely, a fragment with further minimization of the antimicrobial domain, though retaining the antimicrobial capacity, significantly loses its agglutinating activity, exhibiting a different mechanism of action which is not dependent on the LPS composition. The results highlight the correlation between the protein's antimicrobial activity and its ability to interact with the LPS outer layer and promote bacterial agglutination.

  7. Trans-Golgi Network-An Intersection of Trafficking Cell Wall Components

    Institute of Scientific and Technical Information of China (English)

    Natasha Worden; Eunsook Park; Georgia Drakakaki

    2012-01-01

    The cell wall,a crucial cell compartment,is composed of a network of polysaccharides and proteins,providing structural support and protection from external stimuli.While the cell wall structure and biosynthesis have been extensively studied,very little is known about the transport of polysaccharides and other components into the developing cell wall.This review focuses on endomembrane trafficking pathways involved in cell wall deposition.Cellulose synthase complexes are assembled in the Golgi,and are transported in vesicles to the plasma membrane.Non-cellulosic polysaccharides are synthesized in the Golgi apparatus,whereas cellulose is produced by enzyme complexes at the plasma membrane.Polvsaccharides and enzymes that are involved in cell wall modification and assembly are transported by distinct vesicle types to their destinations; however,the precise mechanisms involved in selection,sorting and delivery remain to be identified.The endomembrane system orchestrates the delivery of Golgi-derived and possibly endocytic vesicles carrying cell wall and cell membrane components to the newly-formed cell plate.However,the nature of these vesicles,their membrane compositions,and the timing of their delivery are largely unknown.Emerging technologies such as chemical genomics and proteomics are promising avenues to gain insight into the trafficking of cell wall components.

  8. Clinostation influence on regeneration of cell wall in Solanum Tuberosum L. protoplasts

    Science.gov (United States)

    Nedukha, Elena M.; Sidorov, V. A.; Samoylov, V. M.

    1994-08-01

    Regeneration of cell walls in protoplasts was investigated using light- and electronmicroscopic methods. The protoplasts were isolated from mesophyll of Solanum tuberosum leaves and were cultivated on the horizontal low rotating clinostat (2 rpm) and in control for 10 days. Using a fluorescent method (with Calcofluor white) it was demonstrated that changes in vector gravity results in an regeneration inhibition of cell wall. With electron-microscopical and electro-cytochemical methods (staining with alcianum blue) dynamics of the regeneration of cell walls in protoplasts was studied; carbohydrate matrix of cell walls is deposited at the earliest stages of this process. The influence of microgravity on the cell wall regeneration is discussed in higher plants.

  9. Interactions between grape skin cell wall material and commercial enological tannins. Practical implications.

    Science.gov (United States)

    Bautista-Ortín, Ana Belén; Cano-Lechuga, Mario; Ruiz-García, Yolanda; Gómez-Plaza, Encarna

    2014-01-01

    Commercial enological tannins were used to investigate the role that cell wall material plays in proanthocyanidin adsorption. Insoluble cell wall material, prepared from the skin of Vitis vinifera L. cv. Monastrell berries, was combined with solutions containing six different commercial enological tannins (proanthocyanidin-type tannins). Analysis of the proanthocyanidins in the solution, after fining with cell wall material, using phloroglucinolysis and size exclusion chromatography, provided quantitative and qualitative information on the non-adsorbed compounds. Cell wall material showed strong affinity for the proanthocyanidins, one of the commercial tannins being bound up to 61% in the experiment. Comparison of the molecular mass distribution of the commercial enological tannins in solution, before and after fining, suggested that cell walls affinity for proanthocyanidins was more related with the proanthocyanidin molecular mass than with their percentage of galloylation. These interactions may have some enological implications, especially as regards the time of commercial tannins addition to the must/wine.

  10. B-RAF mutant alleles associated with Langerhans cell histiocytosis, a granulomatous pediatric disease.

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    Takeshi Satoh

    Full Text Available BACKGROUND: Langerhans cell histiocytosis (LCH features inflammatory granuloma characterised by the presence of CD1a+ dendritic cells or 'LCH cells'. Badalian-Very et al. recently reported the presence of a canonical (V600EB-RAF mutation in 57% of paraffin-embedded biopsies from LCH granuloma. Here we confirm their findings and report the identification of two novel B-RAF mutations detected in LCH patients. METHODS AND RESULTS: Mutations of B-RAF were observed in granuloma samples from 11 out of 16 patients using 'next generation' pyrosequencing. In 9 cases the mutation identified was (V600EB-RAF. In 2 cases novel polymorphisms were identified. A somatic (600DLATB-RAF insertion mimicked the structural and functional consequences of the (V600EB-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The (600DLATB-RAF and (V600EB-RAF mutations were found enriched in DNA and mRNA from the CD1a+ fraction of granuloma. They were absent from the blood and monocytes of 58 LCH patients, with a lower threshold of sequencing sensitivity of 1%-2% relative mutation abundance. A novel germ line (T599AB-RAF mutant allele was detected in one patient, at a relative mutation abundance close to 50% in the LCH granuloma, blood monocytes and lymphocytes. However, (T599AB-RAF did not destabilize the inactive conformation of the B-RAF kinase, and did not induce increased ERK phosphorylation or C-RAF transactivation. CONCLUSIONS: Our data confirmed presence of the (V600EB-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that (V600EB-RAF and (600DLATB-RAF mutations are somatic mutants enriched in LCH CD1a(+ cells and absent from the patient blood. Further studies are needed to assess the functional consequences of the germ-line (T599AB-RAF allele.

  11. Generation of hydroxyl radical in isolated pea root cell wall, and the role of cell wall-bound peroxidase, Mn-SOD and phenolics in their production.

    Science.gov (United States)

    Kukavica, Biljana; Mojovic, Milos; Vuccinic, Zeljko; Maksimovic, Vuk; Takahama, Umeo; Jovanovic, Sonja Veljovic

    2009-02-01

    The hydroxyl radical produced in the apoplast has been demonstrated to facilitate cell wall loosening during cell elongation. Cell wall-bound peroxidases (PODs) have been implicated in hydroxyl radical formation. For this mechanism, the apoplast or cell walls should contain the electron donors for (i) H(2)O(2) formation from dioxygen; and (ii) the POD-catalyzed reduction of H(2)O(2) to the hydroxyl radical. The aim of the work was to identify the electron donors in these reactions. In this report, hydroxyl radical (.OH) generation in the cell wall isolated from pea roots was detected in the absence of any exogenous reductants, suggesting that the plant cell wall possesses the capacity to generate .OH in situ. Distinct POD and Mn-superoxide dismutase (Mn-SOD) isoforms different from other cellular isoforms were shown by native gel electropho-resis to be preferably bound to the cell walls. Electron paramagnetic resonance (EPR) spectroscopy of cell wall isolates containing the spin-trapping reagent, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), was used for detection of and differentiation between .OH and the superoxide radical (O(2)(-).). The data obtained using POD inhibitors confirmed that tightly bound cell wall PODs are involved in DEPMPO/OH adduct formation. A decrease in DEPMPO/OH adduct formation in the presence of H(2)O(2) scavengers demonstrated that this hydroxyl radical was derived from H(2)O(2). During the generation of .OH, the concentration of quinhydrone structures (as detected by EPR spectroscopy) increased, suggesting that the H(2)O(2) required for the formation of .OH in isolated cell walls is produced during the reduction of O(2) by hydroxycinnamic acids. Cell wall isolates in which the proteins have been denaturated (including the endogenous POD and SOD) did not produce .OH. Addition of exogenous H(2)O(2) again induced the production of .OH, and these were shown to originate from the Fenton reaction with tightly bound metal ions

  12. The Cell Wall Lipid PDIM Contributes to Phagosomal Escape and Host Cell Exit of Mycobacterium tuberculosis

    Science.gov (United States)

    Quigley, Jeff; Hughitt, V. Keith; Velikovsky, Carlos A.; Mariuzza, Roy A.

    2017-01-01

    ABSTRACT The cell wall of Mycobacterium tuberculosis is composed of unique lipids that are important for pathogenesis. Indeed, the first-ever genetic screen in M. tuberculosis identified genes involved in the biosynthesis and transport of the cell wall lipid PDIM (phthiocerol dimycocerosates) as crucial for the survival of M. tuberculosis in mice. Here we show evidence for a novel molecular mechanism of the PDIM-mediated virulence in M. tuberculosis. We characterized the DNA interaction and the regulon of Rv3167c, a transcriptional repressor that is involved in virulence regulation of M. tuberculosis, and discovered that it controls the PDIM operon. A loss-of-function genetic approach showed that PDIM levels directly correlate with the capacity of M. tuberculosis to escape the phagosome and induce host cell necrosis and macroautophagy. In conclusion, our study attributes a novel role of the cell wall lipid PDIM in intracellular host cell modulation, which is important for host cell exit and dissemination of M. tuberculosis. PMID:28270579

  13. Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions.

    Science.gov (United States)

    Schafer, I A; Jamieson, A M; Petrelli, M; Price, B J; Salzman, G C

    1979-01-01

    Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.

  14. Decreased Polysaccharide Feruloylation Compromises Plant Cell Wall Integrity and Increases Susceptibility to Necrotrophic Fungal Pathogens

    Directory of Open Access Journals (Sweden)

    Nathan T Reem

    2016-05-01

    Full Text Available The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity and function remains unclear. Modifications of cell wall composition can induce plant responses known as Cell Wall Integrity control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, increased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant cell wall integrity, which contributes to plant resistance to necrotrophic pathogens.

  15. Auxin and Cell Wall Invertase Related Signaling during Rice Grain Development

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    Sarah Russell French

    2014-02-01

    Full Text Available Indole-3-acetic acid (IAA synthesis is required for grain-fill in maize and appears to be regulated by cell-wall invertase (CWIN activity. OsYUC12 is one of three IAA biosynthesis genes we previously reported as expressed during early rice grain development, correlating with a large increase in IAA content of the grain. This work aimed to investigate further the role of OsYUC12 and its relationship to CWIN activity and invertase inhibitors (INVINH. The analysis shows a brief peak of OsYUC12 expression early in endosperm development. Meta-analysis of microarray data, confirmed by quantitative expression analysis, revealed that OsYUC12 is coexpressed with OsIAA29, which encodes an unusual AUX/IAA transcription factor previously reported as poorly expressed. Maximum expression of OsYUC12 and OsIAA29 coincided with maximum CWIN activity, but also with a peak in INVINH expression. Unlike ZmYUC1, OsYUC12 expression is not reduced in the rice CWIN mutant, gif1. Several reports have investigated CWIN expression in rice grains but none has reported on expression of INVINH in this species. We show that rice has 54 genes encoding putative invertase/pectin methylesterase inhibitors, seven of which are expressed exclusively during grain development. Our results suggest a more complex relationship between IAA, CWIN, and INVINH than previously proposed.

  16. Immunoprofiling reveals unique cell-specific patterns of wall epitopes in the expanding Arabidopsis stem.

    Science.gov (United States)

    Hall, Hardy C; Cheung, Jingling; Ellis, Brian E

    2013-04-01

    The Arabidopsis inflorescence stem undergoes rapid directional growth, requiring massive axial cell-wall extension in all its tissues, but, at maturity, these tissues are composed of cell types that exhibit markedly different cell-wall structures. It is not clear whether the cell-wall compositions of these cell types diverge rapidly following axial growth cessation, or whether compositional divergence occurs at earlier stages in differentiation, despite the common requirement for cell-wall extensibility. To examine this question, seven cell types were assayed for the abundance and distribution of 18 major cell-wall glycan classes at three developmental stages along the developing inflorescence stem, using a high-throughput immunolabelling strategy. These stages represent a phase of juvenile growth, a phase displaying the maximum rate of stem extension, and a phase in which extension growth is ceasing. The immunolabelling patterns detected demonstrate that the cell-wall composition of most stem tissues undergoes pronounced changes both during and after rapid extension growth. Hierarchical clustering of the immunolabelling signals identified cell-specific binding patterns for some antibodies, including a sub-group of arabinogalactan side chain-directed antibodies whose epitope targets are specifically associated with the inter-fascicular fibre region during the rapid cell expansion phase. The data reveal dynamic, cell type-specific changes in cell-wall chemistry across diverse cell types during cell-wall expansion and maturation in the Arabidopsis inflorescence stem, and highlight the paradox between this structural diversity and the uniform anisotropic cell expansion taking place across all tissues during stem growth.

  17. [Heterocysts with reduced cell walls in populations of cycad cyanobionts].

    Science.gov (United States)

    Baulina, O I; Lobakova, E S

    2003-01-01

    The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycads circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. In addition to heterocysts with the typical ultrastructure, the cyanobiont microcolonies also contained altered heterocysts with reduced cell walls, which might dominate in all regions of the coralloid roots. The altered heterocysts represented a protoplast enclosed in a heterocyst-specific envelope with additional layers. Some heterocysts contained an additional reticular protoplast-enclosing sheath below the heterocyst-specific envelope, whereas the other heterocysts contained an additional electron-opaque outer layer. The substance of the inner sheath of the former heterocysts resembled the polysaccharides of mucilage, which fills the intercellular space of plant tissues, whereas the electron-opaque outer layer of the latter heterocysts probably had a protein nature. The substances that constitute the sheath and the outer layer are likely to be synthesized intracellularly and then released with the aid of membrane-bounded vesicles or by channels in the cytoplasmic membrane.

  18. Modifications of Saccharomyces pastorianus cell wall polysaccharides with brewing process.

    Science.gov (United States)

    Bastos, Rita; Coelho, Elisabete; Coimbra, Manuel A

    2015-06-25

    The cell wall polysaccharides of brewers spent yeast Saccharomyces pastorianus (BSY) and the inoculum yeast (IY) were studied in order to understand the changes induced by the brewing process. The hot water and alkali extractions performed solubilized mainly mannoproteins, more branched for BSY than those of IY. Also, (31)P solid state NMR showed that the BSY mannoproteins were 3 times more phosphorylated. By electron microscopy it was observed that the final residues of alkali sequential extraction until 4M KOH preserved the yeast three-dimensional structure. The final residues, composed mainly by glucans (92%), showed that the BSY, when compared with IY, contained higher amount of (1→4)-linked Glc (43% for BSY and 16% for IY) and lower (1→3)-linked Glc (17% for BSY and 42% for IY). The enzymatic treatment of final residue showed that both BSY and IY had (α1→4)-linked Glc and (β1→4)-linked Glc, in a 2:1 ratio, showing that S. pastorianus increases their cellulose-like linkages with the brewing process.

  19. Serologic response to cell wall mannoproteins and proteins of Candida albicans.

    Science.gov (United States)

    Martínez, J P; Gil, M L; López-Ribot, J L; Chaffin, W L

    1998-01-01

    The cell wall of Candida albicans not only is the structure in which many biological functions essential for the fungal cells reside but also is a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both the carbohydrate and protein moieties are able to trigger immune responses. Although cell-mediated immunity is often considered to be the most important line of defense against candidiasis, cell wall protein and glycoprotein components also elicit a potent humoral response from the host that may include some protective antibodies. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to influence profoundly the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins for host ligands. In this review, the various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo are examined. Although a number of proteins have been shown to stimulate an antibody response, for some of these species the response is not universal. On the other hand, some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidasis, particularly the disseminated form. In addition, recent studies have focused on the potential for antibodies to cell wall protein determinants to protect the host against infection. Hence, a better understanding of the humoral response to cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures

  20. B-RAF Mutant Alleles Associated with Langerhans Cell Histiocytosis, a Granulomatous Pediatric Disease

    Science.gov (United States)

    Lu, Hui-chun; Mian, Sophie; Trouillet, Celine; Mufti, Ghulam; Emile, Jean-Francois; Fraternali, Franca; Donadieu, Jean; Geissmann, Frederic

    2012-01-01

    Background Langerhans cell histiocytosis (LCH) features inflammatory granuloma characterised by the presence of CD1a+ dendritic cells or ‘LCH cells’. Badalian-Very et al. recently reported the presence of a canonical V600EB-RAF mutation in 57% of paraffin-embedded biopsies from LCH granuloma. Here we confirm their findings and report the identification of two novel B-RAF mutations detected in LCH patients. Methods and Results Mutations of B-RAF were observed in granuloma samples from 11 out of 16 patients using ‘next generation’ pyrosequencing. In 9 cases the mutation identified was V600EB-RAF. In 2 cases novel polymorphisms were identified. A somatic 600DLATB-RAF insertion mimicked the structural and functional consequences of the V600EB-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The 600DLATB-RAF and V600EB-RAF mutations were found enriched in DNA and mRNA from the CD1a+ fraction of granuloma. They were absent from the blood and monocytes of 58 LCH patients, with a lower threshold of sequencing sensitivity of 1%–2% relative mutation abundance. A novel germ line T599AB-RAF mutant allele was detected in one patient, at a relative mutation abundance close to 50% in the LCH granuloma, blood monocytes and lymphocytes. However, T599AB-RAF did not destabilize the inactive conformation of the B-RAF kinase, and did not induce increased ERK phosphorylation or C-RAF transactivation. Conclusions Our data confirmed presence of the V600EB-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that V600EB-RAF and 600DLATB-RAF mutations are somatic mutants enriched in LCH CD1a+ cells and absent from the patient blood. Further studies are needed to assess the functional consequences of the germ-line T599AB-RAF allele. PMID:22506009

  1. Changes in Cell Wall Composition during Ripening of Grape Berries1

    Science.gov (United States)

    Nunan, Kylie J.; Sims, Ian M.; Bacic, Antony; Robinson, Simon P.; Fincher, Geoffrey B.

    1998-01-01

    Cell walls were isolated from the mesocarp of grape (Vitis vinifera L.) berries at developmental stages from before veraison through to the final ripe berry. Fluorescence and light microscopy of intact berries revealed no measurable change in cell wall thickness as the mesocarp cells expanded in the ripening fruit. Isolated walls were analyzed for their protein contents and amino acid compositions, and for changes in the composition and solubility of constituent polysaccharides during development. Increases in protein content after veraison were accompanied by an approximate 3-fold increase in hydroxyproline content. The type I arabinogalactan content of the pectic polysaccharides decreased from approximately 20 mol % of total wall polysaccharides to about 4 mol % of wall polysaccharides during berry development. Galacturonan content increased from 26 to 41 mol % of wall polysaccharides, and the galacturonan appeared to become more soluble as ripening progressed. After an initial decrease in the degree of esterification of pectic polysaccharides, no further changes were observed nor were there large variations in cellulose (30–35 mol % of wall polysaccharides) or xyloglucan (approximately 10 mol % of wall polysaccharides) contents. Overall, the results indicate that no major changes in cell wall polysaccharide composition occurred during softening of ripening grape berries, but that significant modification of specific polysaccharide components were observed, together with large changes in protein composition. PMID:9808722

  2. Immunohistochemical detection of mutant p53 protein in small-cell lung cancer: relationship to treatment outcome.

    Science.gov (United States)

    Gemba, K; Ueoka, H; Kiura, K; Tabata, M; Harada, M

    2000-07-01

    We investigated the expression of mutant p53 proteins in small-cell lung cancer (SCLC) immunohistochemically, by identification of stabilized mutant p53 proteins with a much longer half-life than the wild-type protein. Of 103 tumor specimens obtained by transbronchial tumor biopsy for histologic diagnosis, 52 (50%) showed positive staining for p53 protein with a p53 monoclonal antibody, DO-1. Positive staining for p53 protein was not correlated with age, sex, performance status, lifetime cigarette consumption, serum concentration of neuron-specific enolase and extent of disease. Complete response rates in patients with a mutant p53 protein-positive tumor were significantly lower than those in p53-negative patients (25% versus 59%; P=0.0005, by chi-square test). Similarly, survival periods in patients with a mutant p53 protein-positive tumor were significantly shorter than those in mutant p53-protein-negative patients (10.8 months versus 20.6 months; P=0.0001, by generalized Wilcoxon test). Multivariate analysis using Cox's proportional hazards model revealed that the presence of mutant p53 protein is an independent factor associated with differences in overall survival (hazards ratio=2.72; 95% confidence interval, 1.71-4.34; P=0.0001). These observations suggest that the expression of mutant p53 proteins in SCLC may be an important factor predicting poor prognosis.

  3. Oncogene mutations, copy number gains and mutant allele specific imbalance (MASI frequently occur together in tumor cells.

    Directory of Open Access Journals (Sweden)

    Junichi Soh

    Full Text Available BACKGROUND: Activating mutations in one allele of an oncogene (heterozygous mutations are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI has been observed in tumors and cell lines harboring mutations of oncogenes. METHODOLOGY/PRINCIPAL FINDINGS: We determined 1 mutational status, 2 copy number gains (CNGs and 3 relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20% in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1 MASI with CNG, either complete or partial; and 2 MASI without CNG (uniparental disomy; UPD, due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75% and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%, was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival. CONCLUSIONS: MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

  4. Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells.

    Science.gov (United States)

    Moore, P J; Darvill, A G; Albersheim, P; Staehelin, L A

    1986-11-01

    PLANT CELL WALLS SERVE SEVERAL FUNCTIONS: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.

  5. Lipid Transfer Proteins Enhance Cell Wall Extension in TobaccoW⃞

    Science.gov (United States)

    Nieuwland, Jeroen; Feron, Richard; Huisman, Bastiaan A.H.; Fasolino, Annalisa; Hilbers, Cornelis W.; Derksen, Jan; Mariani, Celestina

    2005-01-01

    Plant cells are enclosed by a rigid cell wall that counteracts the internal osmotic pressure of the vacuole and limits the rate and direction of cell enlargement. When developmental or physiological cues induce cell extension, plant cells increase wall plasticity by a process called loosening. It was demonstrated previously that a class of proteins known as expansins are mediators of wall loosening. Here, we report a type of cell wall–loosening protein that does not share any homology with expansins but is a member of the lipid transfer proteins (LTPs). LTPs are known to bind a large range of lipid molecules to their hydrophobic cavity, and we show here that this cavity is essential for the cell wall–loosening activity of LTP. Furthermore, we show that LTP-enhanced wall extension can be described by a logarithmic time function. We hypothesize that LTP associates with hydrophobic wall compounds, causing nonhydrolytic disruption of the cell wall and subsequently facilitating wall extension. PMID:15937228

  6. HDAC inhibitors show differential epigenetic regulation and cell survival strategies on p53 mutant colon cancer cells.

    Science.gov (United States)

    R, Mahalakshmi; P, Husayn Ahmed; Mahadevan, Vijayalakshmi

    2017-03-06

    Besides inactivating tumour suppressor activity in cells, mutations in p53 confer significant oncogenic functions and promote metastasis and resistance to anti cancer therapy. A variety of therapies involving genetic and epigenetic signalling events regulate tumorogenesis and progression in such cases. Pharmacological interventions with HDAC inhibitors have shown promise in therapy. This work explores the changes in efficacy of the four HDAC inhibitors SAHA, MS-275, valproic acid and sodium butyrate on a panel of colon cancer cell lines - HCT116 (p53 wt), HCT116 p53-/-, HT29 and SW480 (with mutations in p53). Clonogenic assays, gene profiling and epigenetic expression done on these cells point to p53 dependent differential activity of the 4 HDAC inhibitors which also elevate methylation levels in p53 mutant cell lines. In silico modelling establishes the alterations in interactions that lead to such differential activity of valproic acid, one of the inhibitors considered for the work. Molecular Dynamic simulations carried out on the valproic acid complex ensure stability of the complex. This work establishes a p53 dependent epigenetic signalling mechanism triggered by HDAC inhibition expanding the scope of HDAC inhibitors in adjuvant therapy for p53 mutant tumours.

  7. Aggregates of mutant CFTR fragments in airway epithelial cells of CF lungs: new pathologic observations.

    Science.gov (United States)

    Du, Kai; Karp, Philip H; Ackerley, Cameron; Zabner, Joseph; Keshavjee, Shaf; Cutz, Ernest; Yeger, Herman

    2015-03-01

    Cystic fibrosis (CF) is caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene resulting in a loss of Cl(-) channel function, disrupting ion and fluid homeostasis, leading to severe lung disease with airway obstruction due to mucus plugging and inflammation. The most common CFTR mutation, F508del, occurs in 90% of patients causing the mutant CFTR protein to misfold and trigger an endoplasmic reticulum based recycling response. Despite extensive research into the pathobiology of CF lung disease, little attention has been paid to the cellular changes accounting for the pathogenesis of CF lung disease. Here we report a novel finding of intracellular retention and accumulation of a cleaved fragment of F508del CFTR in concert with autophagic like phagolysosomes in the airway epithelium of patients with F508del CFTR. Aggregates consisting of poly-ubiquitinylated fragments of only the N-terminal domain of F508del CFTR but not the full-length molecule accumulate to appreciable levels. Importantly, these undegraded intracytoplasmic aggregates representing the NT-NBD1 domain of F508del CFTR were found in ciliated, in basal, and in pulmonary neuroendocrine cells. Aggregates were found in both native lung tissues and ex-vivo primary cultures of bronchial epithelial cells from CF donors, but not in normal control lungs. Our findings present a new, heretofore, unrecognized innate CF gene related cell defect and a potential contributing factor to the pathogenesis of CF lung disease. Mutant CFTR intracytoplasmic aggregates could be analogous to the accumulation of misfolded proteins in other degenerative disorders and in pulmonary "conformational protein-associated" diseases. Consequently, potential alterations to the functional integrity of airway epithelium and regenerative capacity may represent a critical new element in the pathogenesis of CF lung disease.

  8. Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

    Directory of Open Access Journals (Sweden)

    Lijuan Han

    2016-05-01

    Full Text Available Abstract Background Somatic calreticulin (CALR, Janus kinase 2 (JAK2, and thrombopoietin receptor (MPL mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN, suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated. Methods The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment. Results The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Conclusions These

  9. Phenylalanine ammonia-lyase and cell wall peroxidase are cooperatively involved in the extensive formation of ferulate network in cell walls of developing rice shoots.

    Science.gov (United States)

    Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki

    2012-02-15

    The relationship between the formation of cell wall-bound ferulic acid (FA) and diferulic acid (DFA) and the change in activities of phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) was studied in rice shoots. The length and the fresh mass of shoots increased during the growth period from day 4 to 6, while coleoptiles ceased elongation growth on day 5. The amounts of FA and DFA isomers as well as cell wall polysaccharides continued to increase during the whole period. The activities of PAL and CW-PRX greatly increased in the same manner during the period. There were close correlations between the PAL activity and ferulate content or between the CW-PRX activity and DFA content. The expression levels of investigated genes for PAL and putative CW-PRX showed good accordance with the activities of these enzymes. These results suggest that increases in PAL and CW-PRX activities are cooperatively involved in the formation of ferulate network in cell walls of rice shoots and that investigated genes may be, at least in part, associated with the enzyme activities. The substantial increase in such network probably causes the maturation of cell walls and thus the cessation of elongation growth of coleoptiles.

  10. A new picture of cell wall protein dynamics in elongating cells of Arabidopsis thaliana: Confirmed actors and newcomers

    Directory of Open Access Journals (Sweden)

    Jamet Elisabeth

    2008-09-01

    Full Text Available Abstract Background Cell elongation in plants requires addition and re-arrangements of cell wall components. Even if some protein families have been shown to play roles in these events, a global picture of proteins present in cell walls of elongating cells is still missing. A proteomic study was performed on etiolated hypocotyls of Arabidopsis used as model of cells undergoing elongation followed by growth arrest within a short time. Results Two developmental stages (active growth and after growth arrest were compared. A new strategy consisting of high performance cation exchange chromatography and mono-dimensional electrophoresis was established for separation of cell wall proteins. This work allowed identification of 137 predicted secreted proteins, among which 51 had not been identified previously. Apart from expected proteins known to be involved in cell wall extension such as xyloglucan endotransglucosylase-hydrolases, expansins, polygalacturonases, pectin methylesterases and peroxidases, new proteins were identified such as proteases, proteins related to lipid metabolism and proteins of unknown function. Conclusion This work highlights the CWP dynamics that takes place between the two developmental stages. The presence of proteins known to be related to cell wall extension after growth arrest showed that these proteins may play other roles in cell walls. Finally, putative regulatory mechanisms of protein biological activity are discussed from this global view of cell wall proteins.

  11. Growth regulation mechanisms in higher plants under microgravity conditions - changes in cell wall metabolism.

    Science.gov (United States)

    Hoson, T; Kamisaka, S; Wakabayashi, K; Soga, K; Tabuchi, A; Tokumoto, H; Okamura, K; Nakamura, Y; Mori, R; Tanimoto, E; Takeba, G; Nishitani, K; Izumi, R; Ishioka, N; Kamigaichi, S; Aizawa, S; Yoshizaki, I; Shimazu, T; Fukui, K

    2000-06-01

    During Space Shuttle STS-95 mission, we cultivated seedlings of rice (Oryza sativa L. cv. Koshihikari and cv. Tan-ginbozu) and Arabidopsis (Arabidopsis thaliana L. cv. Columbia and cv. etr1-1) for 68.5, 91.5, and 136 hr on board, and then analyzed changes in the nature of their cell walls, growth, and morphogenesis under microgravity conditions. In space, elongation growth of both rice coleoptiles and Arabidopsis hypocotyls was stimulated. Also, the increase in the cell wall extensibility, especially that in the irreversible extensibility, was observed for such materials. The analyses of the amounts, the structure, and the physicochemical properties of the cell wall constituents indicated that the decreases in levels and molecular masses of cell wall polysaccharides were induced under microgravity conditions, which appeared to contribute to the increase in the wall extensibility. The activity of certain wall enzymes responsible for the metabolic turnover of the wall polysaccharides was increased in space. By the space flight, we also confirmed the occurrence of automorphogenesis of both seedlings under microgravity conditions; rice coleoptiles showed an adaxial bending, whereas Arabidopsis hypocotyls elongated in random directions. Furthermore, it was shown that spontaneous curvatures of rice coleoptiles in space were brought about uneven modifications of cell wall properties between the convex and the concave sides.

  12. Critical cell wall hole size for lysis in Gram-positive bacteria

    Science.gov (United States)

    Mitchell, Gabriel; Wiesenfeld, Kurt; Nelson, Daniel; Weitz, Joshua

    2013-03-01

    Gram-positive bacteria transport molecules necessary for their survival through holes in their cell wall. The holes in cell walls need to be large enough to let critical nutrients pass through. However, the cell wall must also function to prevent the bacteria's membrane from protruding through a large hole into the environment and lysing the cell. As such, we hypothesize that there exists a range of cell wall hole sizes that allow for molecule transport but prevent membrane protrusion. Here we develop and analyze a biophysical theory of the response of a Gram-positive cell's membrane to the formation of a hole in the cell wall. We predict a critical hole size in the range 15-24nm beyond which lysis occurs. To test our theory, we measured hole sizes in Streptococcus pyogenes cells undergoing enzymatic lysis via transmission electron microscopy. The measured hole sizes are in strong agreement with our theoretical prediction. Together, the theory and experiments provide a means to quantify the mechanisms of death of Gram-positive cells via enzymatically mediated lysis and provides insight into the range of cell wall hole sizes compatible with bacterial homeostasis.

  13. LytR-CpsA-Psr enzymes as determinants of Bacillus anthracis secondary cell wall polysaccharide assembly.

    Science.gov (United States)

    Liszewski Zilla, Megan; Chan, Yvonne G Y; Lunderberg, Justin Mark; Schneewind, Olaf; Missiakas, Dominique

    2015-01-01

    Bacillus anthracis, the causative agent of anthrax, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and bind to the secondary cell wall polysaccharide (SCWP). In this study, we identified the B. anthracis lcpD mutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, the B. anthracis lcpB3 variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. In other bacteria, LytR-CpsA-Psr (LCP) proteins attach wall teichoic acid (WTA) and polysaccharide capsule to peptidoglycan. B. anthracis does not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed in S. aureus, promote WTA attachment. We propose a model whereby B. anthracis LCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan.

  14. Superoxide generation in extracts from isolated plant cell walls is regulated by fungal signal molecules.

    Science.gov (United States)

    Kiba, A; Miyake, C; Toyoda, K; Ichinose, Y; Yamada, T; Shiraishi, T

    1997-08-01

    ABSTRACT Fractions solubilized with NaCl from cell walls of pea and cowpea plants catalyzed the formation of blue formazan from nitroblue tetrazolium. Because superoxide dismutase decreased formazan production by over 90%, superoxide anion (O(2) ) may participate in the formation of formazan in the solubilized cell wall fractions. The formazan formation in the fractions solubilized from pea and cowpea cell walls was markedly reduced by exclusion of NAD(P)H, manganese ion, or p-coumaric acid from the reaction mixture. The formazan formation was severely inhibited by salicylhydroxamic acid and catalase, but not by imidazole, pyridine, quinacrine, and diphenyleneiodonium. An elicitor preparation from the pea pathogen Mycosphaerella pinodes enhanced the activities of formazan formation nonspecifically in both pea and cowpea fractions. The suppressor preparation from M. pinodes inhibited the activity in the pea fraction in the presence or absence of the elicitor. In the cowpea fraction, however, the suppressor did not inhibit the elicitor-enhanced activity, and the suppressor alone stimulated formazan formation. These results indicated that O(2) generation in the fractions solubilized from pea and cowpea cell walls seems to be catalyzed by cell wall-bound peroxidase(s) and that the plant cell walls alone are able to respond to the elicitor non-specifically and to the suppressor in a species-specific manner, suggesting the plant cell walls may play an important role in determination of plant-fungal pathogen specificity.

  15. Altered cell wall properties are responsible for ammonium-reduced aluminium accumulation in rice roots.

    Science.gov (United States)

    Wang, Wei; Zhao, Xue Qiang; Chen, Rong Fu; Dong, Xiao Ying; Lan, Ping; Ma, Jian Feng; Shen, Ren Fang

    2015-07-01

    The phytotoxicity of aluminium (Al) ions can be alleviated by ammonium (NH4(+)) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al-binding assay revealed that neither NH4(+) nor NO3(-) altered the Al-binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH4(+)-supplied roots displayed lower Al-binding capacity than those from NO3(-)-supplied roots when grown in non-buffered solutions. Fourier-transform infrared microspectroscopy analysis revealed that, compared with NO3(-)-supplied roots, NH4(+)-supplied roots possessed fewer Al-binding groups (-OH and COO-) and lower contents of pectin and hemicellulose. However, when grown in pH-buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al-binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH4(+)-reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH4(+) uptake rather than direct competition for the cell wall binding sites between Al(3+) and NH4(+).

  16. Malignant transformation of ectopic pancreatic cells in the duodenal wall

    Institute of Scientific and Technical Information of China (English)

    Roberto; Bini; Paolo; Voghera; Alberto; Tapparo; Raffaele; Nunziata; Andrea; Demarchi; Matteo; Capocefalo; Renzo; Leli

    2010-01-01

    Ectopic pancreas (EP) is the relatively uncommon presence of pancreatic tissue outside the normal location of the pancreas. This condition is usually asymptomatic and rarely complicated by pancreatitis and malignant transformation. A few cases of neoplastic phenomena that developed from EP into the duodenal wall are described in the literature. Herein we report a case of gastric outlet obstruction due to adenocarcinoma arising from EP of the duodenal wall. The patient underwent a Whipple's procedure and had...

  17. Navigating the transcriptional roadmap regulating plant secondary cell wall deposition

    Directory of Open Access Journals (Sweden)

    Steven Grant Hussey

    2013-08-01

    Full Text Available The current status of lignocellulosic biomass as an invaluable resource in industry, agriculture and health has spurred increased interest in understanding the transcriptional regulation of secondary cell wall (SCW biosynthesis. The last decade of research has revealed an extensive network of NAC, MYB and other families of transcription factors regulating Arabidopsis SCW biosynthesis, and numerous studies have explored SCW-related transcription factors in other dicots and monocots. Whilst the general structure of the Arabidopsis network has been a topic of several reviews, they have not comprehensively represented the detailed protein-DNA and protein-protein interactions described in the literature, and an understanding of network dynamics and functionality has not yet been achieved for SCW formation. Furthermore the methodologies employed in studies of SCW transcriptional regulation have not received much attention, especially in the case of non-model organisms. In this review, we have reconstructed the most exhaustive literature-based network representations to date of SCW transcriptional regulation in Arabidopsis. We include a manipulable Cytoscape representation of the Arabidopsis SCW transcriptional network to aid in future studies, along with a list of supporting literature for each documented interaction. Amongst other topics, we discuss the various components of the network, its evolutionary conservation in plants, putative modules and dynamic mechanisms that may influence network function, and the approaches that have been employed in network inference. Future research should aim to better understand network function and its response to dynamic perturbations, whilst the development and application of genome-wide approaches such as ChIP-seq and systems genetics are in progress for the study of SCW transcriptional regulation in non-model organisms.

  18. Mutant AKT1-E17K is oncogenic in lung epithelial cells

    Science.gov (United States)

    De Marco, Carmela; Malanga, Donatella; Rinaldo, Nicola; De Vita, Fernanda; Scrima, Marianna; Lovisa, Sara; Fabris, Linda; Carriero, Maria Vincenza; Franco, Renato; Rizzuto, Antonia; Baldassarre, Gustavo; Viglietto, Giuseppe

    2015-01-01

    The hotspot E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6–2% of human lung cancers. In this manuscript, we sought to determine whether this AKT1 variant is a bona-fide activating mutation and plays a role in the development of lung cancer. Here we report that in immortalized human bronchial epithelial cells (BEAS-2B cells) mutant AKT1-E17K promotes anchorage-dependent and -independent proliferation, increases the ability to migrate, invade as well as to survive and duplicate in stressful conditions, leading to the emergency of cells endowed with the capability to form aggressive tumours at high efficiency. We provide also evidence that the molecular mechanism whereby AKT1-E17K is oncogenic in lung epithelial cells involves phosphorylation and consequent cytoplasmic delocalization of the cyclin-dependent kinase (cdk) inhibitor p27. In agreement with these results, cytoplasmic p27 is preferentially observed in primary NSCLCs with activated AKT and predicts poor survival. PMID:26053093

  19. Activation of Notch in lgd mutant cells requires the fusion of late endosomes with the lysosome.

    Science.gov (United States)

    Schneider, Markus; Troost, Tobias; Grawe, Ferdi; Martinez-Arias, Alfonso; Klein, Thomas

    2013-01-15

    The tumour suppressor Lethal (2) giant discs (Lgd) is a regulator of endosomal trafficking of the Notch signalling receptor as well as other transmembrane proteins in Drosophila. The loss of its function results in an uncontrolled ligand-independent activation of the Notch signalling receptor. Here, we investigated the consequences of loss of lgd function and the requirements for the activation of Notch. We show that the activation of Notch in lgd cells is independent of Kuz and dependent on γ-secretase. We found that the lgd cells have a defect that delays degradation of transmembrane proteins, which are residents of the plasma membrane. Furthermore, our results show that the activation of Notch in lgd cells occurs in the lysosome. By contrast, the pathway is activated at an earlier phase in mutants of the gene that encodes the ESCRT-III component Shrub, which is an interaction partner of Lgd. We further show that activation of Notch appears to be a general consequence of loss of lgd function. In addition, electron microscopy of lgd cells revealed that they contain enlarged multi-vesicular bodies. The presented results further elucidate the mechanism of uncontrolled Notch activation upon derailed endocytosis.

  20. Selection of functional T cell receptor mutants from a yeast surface-display library.

    Science.gov (United States)

    Kieke, M C; Shusta, E V; Boder, E T; Teyton, L; Wittrup, K D; Kranz, D M

    1999-05-11

    The heterodimeric alphabeta T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues. These residues are located in two regions of the TCR, at the interface of the alpha- and beta-chains and in the beta-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeast-displayed mutant TCRs bind specifically to the peptide/MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.

  1. Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Bin Huang

    Full Text Available Huntingtin (Htt is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q and mutant (46Q and 128Q Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.

  2. Lycopene in the prevention of renal cell cancer in the TSC2 mutant Eker rat model.

    Science.gov (United States)

    Sahin, Kazim; Cross, Brian; Sahin, Nurhan; Ciccone, Karina; Suleiman, Shadeah; Osunkoya, Adeboye O; Master, Viraj; Harris, Wayne; Carthon, Bradley; Mohammad, Ramzi; Bilir, Birdal; Wertz, Karin; Moreno, Carlos S; Walker, Cheryl L; Kucuk, Omer

    2015-04-15

    Renal cell carcinoma (RCC) is the most frequent upper urinary tract cancer in humans and accounts for 80-85% of malignant renal tumors. Eker rat represents a unique animal model to study RCC since these rats develop spontaneous renal tumors and leiomyoma, which may be due to tuberous sclerosis 2 (TSC2) mutation resulting in the activation of the mammalian target of rapamycin (mTOR) pathway. This study examines the role of a lycopene-rich diet in the development of RCC in the TSC2 mutant Eker rat model. Ten-week old female Eker rats (n=90) were assigned in equal numbers to receive 0, 100 or 200mg/kg of lycopene as part of their daily diet. After 18 months the rats were sacrificed and the kidneys were removed. Immunohistochemical staining with antibodies against mTOR, phospho-S6 and EGFR were performed, as well as hematoxylin-eosin staining for histologic examination of the tumors. Tumors were counted and measured in individual kidneys. Presence of tumor decreased from 94% in control animals to 65% in the experimental group, but the difference was not statistically significant (Plycopene-treated rats (Plycopene group, tumor numbers decreased (Plycopene increased from 0 to 200. Control rats fed only basal diet had a greater length of tumors (23.98 mm) than rats fed lycopene supplement groups (12.90 mm and 11.07 mm) (Plycopene increased from 0 to 200mg/kg. All tumors showed strong staining with antibodies against mTOR, phospho-S6 and EGFR. In conclusion, dietary supplementation with lycopene attenuates the development of renal cell cancers in the predisposed TSC2 mutant Eker rat model. These results suggest that lycopene may play a role in the prevention of RCC.

  3. L-Ascorbic acid can abrogate SVCT-2-dependent cetuximab resistance mediated by mutant KRAS in human colon cancer cells.

    Science.gov (United States)

    Jung, Soo-A; Lee, Dae-Hee; Moon, Jai-Hee; Hong, Seung-Woo; Shin, Jae-Sik; Hwang, Ih Yeon; Shin, Yu Jin; Kim, Jeong Hee; Gong, Eun-Yeung; Kim, Seung-Mi; Lee, Eun Young; Lee, Seul; Kim, Jeong Eun; Kim, Kyu-Pyo; Hong, Yong Sang; Lee, Jung Shin; Jin, Dong-Hoon; Kim, TaeWon; Lee, Wang Jae

    2016-06-01

    Colon cancer patients with mutant KRAS are resistant to cetuximab, an antibody directed against the epidermal growth factor receptor, which is an effective clinical therapy for patients with wild-type KRAS. Numerous combinatorial therapies have been tested to overcome the resistance to cetuximab. However, no combinations have been found that can be used as effective therapeutic strategies. In this study, we demonstrate that L-ascorbic acid partners with cetuximab to induce killing effects, which are influenced by sodium-dependent vitamin C transporter 2 (SVCT-2) in human colon cancer cells with a mutant KRAS. L-Ascorbic acid treatment of human colon cancer cells that express a mutant KRAS differentially and synergistically induced cell death with cetuximab in a SVCT-2-dependent manner. The ectopic expression of SVCT-2 induced sensitivity to L-ascorbic acid treatment in human colon cancer cells that do not express SVCT-2, whereas the knockdown of endogenous SVCT-2 induced resistance to L-ascorbic acid treatment in SVCT-2-positive cells. Moreover, tumor regression via the administration of L-ascorbic acid and cetuximab in mice bearing tumor cell xenografts corresponded to SVCT-2 protein levels. Interestingly, cell death induced by the combination of L-ascorbic acid and cetuximab resulted in both apoptotic and necrotic cell death. These cell death mechanisms were related to a disruption of the ERK pathway and were represented by the impaired activation of RAFs and the activation of the ASK-1-p38 pathway. Taken together, these results suggest that resistance to cetuximab in human colon cancer patients with a mutant KRAS can be bypassed by L-ascorbic acid in an SVCT-2-dependent manner. Furthermore, SVCT-2 in mutant KRAS colon cancer may act as a potent marker for potentiating L-ascorbic acid co-treatment with cetuximab.

  4. Comparative characterization of stromal vascular cells derived from three types of vascular wall and adipose tissue.

    Science.gov (United States)

    Yang, Santsun; Eto, Hitomi; Kato, Harunosuke; Doi, Kentaro; Kuno, Shinichiro; Kinoshita, Kahori; Ma, Hsu; Tsai, Chi-Han; Chou, Wan-Ting; Yoshimura, Kotaro

    2013-12-01

    Multipotent stem/progenitor cells localize perivascularly in many organs and vessel walls. These tissue-resident stem/progenitor cells differentiate into vascular endothelial cells, pericytes, and other mesenchymal lineages, and participate in physiological maintenance and repair of vasculatures. In this study, we characterized stromal vascular cells obtained through the explant culture method from three different vessel walls in humans: arterial wall (ART; >500 μm in diameter), venous wall (VN; >500 μm in diameter), and small vessels in adipose tissue (SV; arterioles and venules, adipose-derived stem/stromal cells (ASCs). All stromal vascular cells of different origins presented fibroblast-like morphology and we could not visually discriminate one population from another. Flow cytometry showed that the cultured population heterogeneously expressed a variety of surface antigens associated with stem/progenitor cells, but CD105 was expressed by most cells in all groups, suggesting that the cells generally shared the characteristics of mesenchymal stem cells. Our histological and flow cytometric data suggested that the main population of vessel wall-derived stromal vascular cells were CD34(+)/CD31(-) and came from the tunica adventitia and areola tissue surrounding the adventitia. CD271 (p75NTR) was expressed by the vasa vasorum in the VN adventitia and by a limited population in the adventitia of SV. All three populations differentiated into multiple lineages as did ASCs. ART cells induced the largest quantity of calcium formation in the osteogenic medium, whereas ASCs showed the greatest adipogenic differentiation. SV and VN stromal cells had greater potency for network formation than did ART stromal cells. In conclusion, the three stromal vascular populations exhibited differential functional properties. Our results have clinical implications for vascular diseases such as arterial wall calcification and possible applications to regenerative therapies

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

    DEFF Research Database (Denmark)

    Mikkelsen, Maria Dalgaard; Harholt, Jesper; Ulvskov, Peter

    2014-01-01

    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...... 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...

  6. A DDB2 mutant protein unable to interact with PCNA promotes cell cycle progression of human transformed embryonic kidney cells.

    Science.gov (United States)

    Perucca, Paola; Sommatis, Sabrina; Mocchi, Roberto; Prosperi, Ennio; Stivala, Lucia Anna; Cazzalini, Ornella

    2015-01-01

    DNA damage binding protein 2 (DDB2) is a protein involved in the early step of DNA damage recognition of the nucleotide excision repair (NER) process. Recently, it has been suggested that DDB2 may play a role in DNA replication, based on its ability to promote cell proliferation. We have previously shown that DDB2 binds PCNA during NER, but also in the absence of DNA damage; however, whether and how this interaction influences cell proliferation is not known. In this study, we have addressed this question by using HEK293 cell clones stably expressing DDB2(Wt) protein, or a mutant form (DDB2(Mut)) unable to interact with PCNA. We report that overexpression of the DDB2(Mut) protein provides a proliferative advantage over the wild type form, by influencing cell cycle progression. In particular, an increase in the number of S-phase cells, together with a reduction in p21(CDKN1A) protein level, and a shorter cell cycle length, has been observed in the DDB2(Mut) cells. These results suggest that DDB2 influences cell cycle progression thanks to its interaction with PCNA.

  7. Effect of commercial enzymes on berry cell wall deconstruction in the context of intravineyard ripeness variation under winemaking conditions

    DEFF Research Database (Denmark)

    Gao, Yu; Fangel, Jonatan Ulrik; Willats, William George Tycho;

    2016-01-01

    at the berry cell wall polymer level and occurred within the experimental vineyard block. Furthemore, all enzyme treatments reduced cell wall variation via depectination. Interestingly, cell wall esterification levels were unaffected by enzyme treatments. This study provides clear evidence that enzymes can...

  8. Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation.

    Science.gov (United States)

    Bottai, Daria; Di Luca, Mariagrazia; Majlessi, Laleh; Frigui, Wafa; Simeone, Roxane; Sayes, Fadel; Bitter, Wilbert; Brennan, Michael J; Leclerc, Claude; Batoni, Giovanna; Campa, Mario; Brosch, Roland; Esin, Semih

    2012-03-01

    The chromosome of Mycobacterium tuberculosis encodes five type VII secretion systems (ESX-1-ESX-5). While the role of the ESX-1 and ESX-3 systems in M. tuberculosis has been elucidated, predictions for the function of the ESX-5 system came from data obtained in Mycobacterium marinum, where it transports PPE and PE_PGRS proteins and modulates innate immune responses. To define the role of the ESX-5 system in M. tuberculosis, in this study, we have constructed five M. tuberculosis H37Rv ESX-5 knockout/deletion mutants, inactivating eccA(5), eccD(5), rv1794 and esxM genes or the ppe25-pe19 region. Whereas the Mtbrv1794ko displayed no obvious phenotype, the other four mutants showed defects in secretion of the ESX-5-encoded EsxN and PPE41, a representative member of the large PPE protein family. Strikingly, the MtbeccD(5) ko mutant also showed enhanced sensitivity to detergents and hydrophilic antibiotics. When the virulence of the five mutants was evaluated, the MtbeccD(5) ko and MtbΔppe25-pe19 mutants were found attenuated both in macrophages and in the severe combined immune-deficient mouse infection model. Altogether these findings indicate an essential role of ESX-5 for transport of PPE proteins, cell wall integrity and full virulence of M. tuberculosis, thereby opening interesting new perspectives for the study of this human pathogen.

  9. Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant.

    Science.gov (United States)

    Ginart, Paul; Kalish, Jennifer M; Jiang, Connie L; Yu, Alice C; Bartolomei, Marisa S; Raj, Arjun

    2016-03-01

    Imprinting is a classic mammalian epigenetic phenomenon that results in expression from a single parental allele. Imprinting defects can lead to inappropriate expression from the normally silenced allele, but it remains unclear whether every cell in a mutant organism follows the population average, which would have profound implications for human imprinting disorders. Here, we apply a new fluorescence in situ hybridization method that measures allele-specific expression in single cells to address this question in mutants exhibiting aberrant H19/Igf2 (insulin-like growth factor 2) imprinting. We show that mutant primary embryonic mouse fibroblasts are comprised of two subpopulations: one expressing both H19 alleles and another expressing only the maternal copy. Only in the latter cell population is Igf2 expression detected. Furthermore, the two subpopulations are stable in that cells do not interconvert between the two expression patterns. Combined small input methylation analysis and transcriptional imaging revealed that these two mutant subpopulations exhibit distinct methylation patterns at their imprinting control regions. Consistently, pharmacological inhibition of DNA methylation reduced the proportion of monoallelic cells. Importantly, we observed that the same two subpopulations are also present in vivo within murine cardiac tissue. Our results establish that imprinting disorders can display striking single-cell heterogeneity in their molecular phenotypes and suggest that such heterogeneity may underlie epigenetic mosaicism in human imprinting disorders.

  10. Mouse mutants for the nicotinic acetylcholine receptor ß2 subunit display changes in cell adhesion and neurodegeneration response genes.

    Directory of Open Access Journals (Sweden)

    Carol M Rubin

    Full Text Available Mice lacking expression of the ß2 subunit of the neuronal nicotinic acetylcholine receptor (CHRNB2 display abnormal retinal waves and a dispersed projection of retinal ganglion cell (RGC axons to their dorsal lateral geniculate nuclei (dLGNs. Transcriptomes of LGN tissue from two independently generated Chrnb2-/- mutants and from wildtype mice were obtained at postnatal day 4 (P4, during the normal period of segregation of eye-specific afferents to the LGN. Microarray analysis reveals reduced expression of genes located on the cell membrane or in extracellular space, and of genes active in cell adhesion and calcium signaling. In particular, mRNA for cadherin 1 (Cdh1, a known axon growth regulator, is reduced to nearly undetectable levels in the LGN of P4 mutant mice and Lypd2 mRNA is similarly suppressed. Similar analysis of retinal tissue shows increased expression of crumbs 1 (Crb1 and chemokine (C-C motif ligand 21 (Ccl21 mRNAs in Chrnb2-/- mutant animals. Mutations in these genes are associated with retinal neuronal degeneration. The retinas of Chrnb2-/- mutants are normal in appearance, but the increased expression of these genes may also be involved in the abnormal projection patterns of RGC to the LGN. These data may provide the tools to distinguish the interplay between neural activity and molecular expression. Finally, comparison of the transcriptomes of the two different Chrnb2-/- mutant strains reveals the effects of genetic background upon gene expression.

  11. Expression of S-adenosylmethionine Hydrolase in Tissues Synthesizing Secondary Cell Walls Alters Specific Methylated Cell Wall Fractions and Improves Biomass Digestibility

    Directory of Open Access Journals (Sweden)

    Aymerick Eudes

    2016-07-01

    Full Text Available Plant biomass is a large source of fermentable sugars for the synthesis of bioproducts using engineered microbes. These sugars are stored as cell wall polymers, mainly cellulose and hemicellulose, and are embedded with lignin, which makes their enzymatic hydrolysis challenging. One of the strategies to reduce cell wall recalcitrance is the modification of lignin content and composition. Lignin is a phenolic polymer of methylated aromatic alcohols and its synthesis in tissues developing secondary cell walls is a significant sink for the consumption of the methyl donor S-adenosylmethionine (AdoMet. In this study, we demonstrate in Arabidopsis stems that targeted expression of S-adenosylmethionine hydrolase (AdoMetase, E.C. 3.3.1.2 in secondary cell-wall synthesizing tissues reduces the AdoMet pool and impacts lignin content and composition. In particular, both NMR analysis and pyrolysis gas chromatography mass spectrometry of lignin in engineered biomass showed relative enrichment of non-methylated p-hydroxycinnamyl (H units and a reduction of dimethylated syringyl (S units. This indicates a lower degree of methylation compared to that in wild-type lignin. Quantification of cell wall-bound hydroxycinnamates revealed a reduction of ferulate in AdoMetase transgenic lines. Biomass from transgenic lines, in contrast to that in control plants, exhibits an enrichment of glucose content and a reduction in the degree of hemicellulose glucuronoxylan methylation. We also show that these modifications resulted in a reduction of cell wall recalcitrance, because sugar yield generated by enzymatic biomass saccharification was greater than that of wild type plants. Considering that transgenic plants show no important diminution of biomass yields, and that heterologous expression of AdoMetase protein can be spatiotemporally optimized, this novel approach provides a valuable option for the improvement of lignocellulosic biomass feedstock.

  12. Nonsense mediated decay resistant mutations are a source of expressed mutant proteins in colon cancer cell lines with microsatellite instability.

    Directory of Open Access Journals (Sweden)

    David S Williams

    Full Text Available BACKGROUND: Frameshift mutations in microsatellite instability high (MSI-High colorectal cancers are a potential source of targetable neo-antigens. Many nonsense transcripts are subject to rapid degradation due to nonsense-mediated decay (NMD, but nonsense transcripts with a cMS in the last exon or near the last exon-exon junction have intrinsic resistance to nonsense-mediated decay (NMD. NMD-resistant transcripts are therefore a likely source of expressed mutant proteins in MSI-High tumours. METHODS: Using antibodies to the conserved N-termini of predicted mutant proteins, we analysed MSI-High colorectal cancer cell lines for examples of naturally expressed mutant proteins arising from frameshift mutations in coding microsatellites (cMS by immunoprecipitation and Western Blot experiments. Detected mutant protein bands from NMD-resistant transcripts were further validated by gene-specific short-interfering RNA (siRNA knockdown. A genome-wide search was performed to identify cMS-containing genes likely to generate NMD-resistant transcripts that could encode for antigenic expressed mutant proteins in MSI-High colon cancers. These genes were screened for cMS mutations in the MSI-High colon cancer cell lines. RESULTS: Mutant protein bands of expected molecular weight were detected in mutated MSI-High cell lines for NMD-resistant transcripts (CREBBP, EP300, TTK, but not NMD-sensitive transcripts (BAX, CASP5, MSH3. Expression of the mutant CREBBP and EP300 proteins was confirmed by siRNA knockdown. Five cMS-bearing genes identified from the genome-wide search and without existing mutation data (SFRS12IP1, MED8, ASXL1, FBXL3 and RGS12 were found to be mutated in at least 5 of 11 (45% of the MSI-High cell lines tested. CONCLUSION: NMD-resistant transcripts can give rise to expressed mutant proteins in MSI-High colon cancer cells. If commonly expressed in primary MSI-High colon cancers, MSI-derived mutant proteins could be useful as cancer specific

  13. Interplay between calcineurin and the Slt2 MAP-kinase in mediating cell wall integrity, conidiation and virulence in the insect fungal pathogen Beauveria bassiana.

    Science.gov (United States)

    Huang, Shuaishuai; He, Zhangjiang; Zhang, Shiwei; Keyhani, Nemat O; Song, Yulin; Yang, Zhi; Jiang, Yahui; Zhang, Wenli; Pei, Yan; Zhang, Yongjun

    2015-10-01

    The entomopathogenic fungus, Beauveria bassiana, is of environmental and economic importance as an insect pathogen, currently used for the biological control of a number of pests. Cell wall integrity and conidiation are critical parameters for the ability of the fungus to infect insects and for production of the infectious propagules. The contribution of calcineurin and the Slt2 MAP kinase to cell wall integrity and development in B. bassiana was investigated. Gene knockouts of either the calcineurin CNA1 subunit or the Slt2 MAP kinase resulted in decreased tolerance to calcofluor white and high temperature. In contrast, the Δcna1 strain was more tolerant to Congo red but more sensitive to osmotic stress (NaCl, sorbitol) than the wild type, whereas the Δslt2 strain had the opposite phenotype. Changes in cell wall structure and composition were seen in the Δslt2 and Δcna1 strains during growth under cell wall stress as compared to the wild type. Both Δslt2 and Δcna1 strains showed significant alterations in growth, conidiation, and viability. Elevation of intracellular ROS levels, and decreased conidial hydrophobicity and adhesion to hydrophobic surfaces, were also seen for both mutants, as well as decreased virulence. Under cell wall stress conditions, inactivation of Slt2 significantly repressed CN-mediated phosphatase activity suggesting some level of cross talk between the two pathways. Comparative transcriptome profiling of the Δslt2 and Δcna1 strains revealed alterations in the expression of distinct gene sets, with overlap in transcripts involved in cell wall integrity, stress response, conidiation and virulence. These data illustrate convergent and divergent phenotypes and targets of the calcineurin and Slt2 pathways in B. bassiana.

  14. BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses

    Directory of Open Access Journals (Sweden)

    Deborah ePetrik

    2016-02-01

    Full Text Available Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames (ORFs into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels

  15. FvBck1, a Component of Cell Wall Integrity MAP Kinase Pathway, is Required for Virulence and Oxidative Stress Response in Sugarcane Pokkah Boeng Pathogen

    Directory of Open Access Journals (Sweden)

    Chengkang eZhang

    2015-10-01

    Full Text Available Fusarium verticillioides (formerly F. moniliforme is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only required for growth, micro- and macro-conidia production, and cell wall integrity but also for response to osmotic and oxidative stresses. The deletion of FvBCK1 caused a significant reduction in virulence and FB1 production, a carcinogenic mycotoxin produced by the fungus. Moreover, we found the expression levels of three genes, which are known to be involved in superoxide scavenging, were down regulated in the mutant. We hypothesized that the loss of superoxide scavenging capacity was one of the reasons for reduced virulence, but overexpression of catalase or peroxidase gene failed to restore the virulence defect in the deletion mutant. When we introduced Magnaporthe oryzae MCK1 into the FvBck1 deletion mutant, while certain phenotypes were restored, the complemented strain failed to gain full virulence. In summary, FvBck1 plays a diverse role in F. verticillioides, and detailed investigation of downstream signaling pathways will lead to a better understanding of how this MAPK pathway regulates Pokkah Boeng on sugarcane.

  16. Mutant huntingtin regulates EGF receptor fate in non-neuronal cells lacking wild-type protein.

    Science.gov (United States)

    Melone, Mariarosa A B; Calarco, Anna; Petillo, Orsolina; Margarucci, Sabrina; Colucci-D'Amato, Luca; Galderisi, Umberto; Koverech, Guido; Peluso, Gianfranco

    2013-01-01

    Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild-type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.

  17. Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea.

    Science.gov (United States)

    May-Simera, Helen

    2016-02-21

    In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

  18. Molecular characterization of two Arabidopsis thaliana glycosyltransferase mutants, rra1 and rra2, which have a reduced residual arabinose content in a polymer tightly associated with the cellulosic wall residue

    DEFF Research Database (Denmark)

    Egelund, Jack; Obel, Nicolai; Ulvskov, Peter

    2007-01-01

    Two putative glycosyltransferases in Arabidopsis thaliana, designated reduced residual arabinose-1 and -2 (RRA1 and RRA2), are characterized at the molecular level. Both genes are classified in CAZy GT-family-77 and are phylogenetically related to putative glycosyltranferases of Chlamydomonas...... identified and characterized at the molecular and biochemical level. Monosaccharide compositional analyses of cell wall material isolated from the meristematic region showed a ca. 20% reduction in the arabinose content in the insoluble/undigested cell wall residue after enzymatic removal of xyloglucan...... and pectic polysaccharides. These data indicate that both RRA-1 and -2 play a role in the arabinosylation of cell wall component(s)....

  19. Investigation on Adsorption of Lithospermum erythrorhizon onto Fungal Cell Wall Polysaccharides

    Institute of Scientific and Technical Information of China (English)

    孟琴; 薛莲

    2003-01-01

    A culture of Lithosperrnum erythrorhizon adsorbed on fungal cell wall polysaccharides, a novel bioadsorbent made from fungal cell wall, has been established in this paper. Three steps were involved in this immobilization. The first step was preparation of suspended plant cells from tightly aggregated plant cell clumps. The disassembled ratio of 0.715g·g-1 (the disassembled cells over total cells) was obtained under optimum condition for the enzymatic reaction. Then, the adsorption of plant cells onto fungal cell wall polysaccharides was conducted and the saturated capacity of 12g cell per gram of carrier was obtained in adsorption immobilization. Finally, the culture of cells adsorbed on fungal cell wall polysaccharides was compared with that of cells entrapped in alginate or suspension cell culture. While exposed to in situ liquid paraffin extraction coupled with cell culture, the shikonin productivity of immobilized cells by adsorption was 10.67g·L-1, which was 1.8 times of that in suspension culture and 1.5 times of that entrapped in alginate.

  20. A type 2C protein phosphatase FgPtc3 is involved in cell wall integrity, lipid metabolism, and virulence in Fusarium graminearum.

    Directory of Open Access Journals (Sweden)

    Jinhua Jiang

    Full Text Available Type 2C protein phosphatases (PP2Cs play important roles in regulating many biological processes in eukaryotes. Currently, little is known about functions of PP2Cs in filamentous fungi. The causal agent of wheat head blight, Fusarium graminearum, contains seven putative PP2C genes, FgPTC1, -3, -5, -5R, -6, -7 and -7R. In order to investigate roles of these PP2Cs, we constructed deletion mutants for all seven PP2C genes in this study. The FgPTC3 deletion mutant (ΔFgPtc3-8 exhibited reduced aerial hyphae formation and deoxynivalenol (DON production, but increased production of conidia. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents on potato dextrose agar plates. Pathogencity assays showed that ΔFgPtc3-8 is unable to infect flowering wheat head. All of the defects were restored when ΔFgPtc3-8 was complemented with the wild-type FgPTC3 gene. Additionally, the FgPTC3 partially rescued growth defect of a yeast PTC1 deletion mutant under various stress conditions. Ultrastructural and histochemical analyses showed that conidia of ΔFgPtc3-8 contained an unusually high number of large lipid droplets. Furthermore, the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Quantitative real-time PCR assays showed that basal expression of FgOS2, FgSLT2 and FgMKK1 in the mutant was significantly higher than that in the wild-type strain. Serial analysis of gene expression in ΔFgPtc3-8 revealed that FgPTC3 is associated with various metabolic pathways. In contrast to the FgPTC3 mutant, the deletion mutants of FgPTC1, FgPTC5, FgPTC5R, FgPTC6, FgPTC7 or FgPTC7R did not show aberrant phenotypic features when grown on PDA medium or inoculated on wheat head. These results indicate FgPtc3 is the key PP2C that plays a critical role in a variety of cellular and biological functions, including cell wall integrity, lipid and secondary metabolisms, and virulence in F. graminearum.

  1. Inhibition of Cell Proliferation in an NRAS Mutant Melanoma Cell Line by Combining Sorafenib and α-Mangostin.

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    Yun Xia

    Full Text Available α-Mangostin is a natural product commonly used in Asia for cosmetic and medicinal applications including topical treatment of acne and skin cancer. Towards finding new pharmacological strategies that overcome NRAS mutant melanoma, we performed a cell proliferation-based combination screen using a collection of well-characterized small molecule kinase inhibitors and α-Mangostin. We found that α-Mangostin significantly enhances Sorafenib pharmacological efficacy against an NRAS mutant melanoma cell line. The synergistic effects of α-Mangostin and Sorafenib were associated with enhanced inhibition of activated AKT and ERK, induced ER stress, and reduced autophagy, eventually leading to apoptosis. The structure of α-Mangostin resembles several inhibitors of the Retinoid X receptor (RXR. MITF expression, which is regulated by RXR, was modulated by α-Mangostin. Molecular docking revealed that α-Mangostin can be accommodated by the ligand binding pocket of RXR and may thereby compete with RXR-mediated control of MITF expression. In summary, these data demonstrate an unanticipated synergy between α-Mangostin and sorafenib, with mechanistic actions that convert a known safe natural product to a candidate combinatorial therapeutic agent.

  2. Nanostructured carbon electrocatalyst supports for intermediate-temperature fuel cells: Single-walled versus multi-walled structures

    Science.gov (United States)

    Papandrew, Alexander B.; Elgammal, Ramez A.; Tian, Mengkun; Tennyson, Wesley D.; Rouleau, Christopher M.; Puretzky, Alexander A.; Veith, Gabriel M.; Geohegan, David B.; Zawodzinski, Thomas A.

    2017-01-01

    It is unknown if nanostructured carbons possess the requisite electrochemical stability to be used as catalyst supports in the cathode of intermediate-temperature solid acid fuel cells (SAFCs) based on the CsH2PO4 electrolyte. To investigate this application, single-walled carbon nanohorns (SWNHs) and multi-walled carbon nanotubes (MWNTs) were used as supports for Pt catalysts in SAFCs operating at 250 °C. SWNH-based cathodes display greater maximum activity than their MWNT-based counterparts at a cell voltage of 0.8 V, but are unstable in the SAFC cathode as a consequence of electrochemical carbon corrosion. MWNT-based cells are resistant to this effect and capable of operation for at least 160 h at 0.6 V and 250 °C. Cells fabricated with nanostructured carbon supports are more active (52 mA cm-1vs. 28 mA cm-1 at 0.8 V) than state-of-the-art carbon-free formulations while simultaneously displaying enhanced Pt utilization (40 mA mgPt-1vs. 16 mA mgPt-1 at 0.8 V). These results suggest that MWNTs are a viable support material for developing stable, high-performance, low-cost air electrodes for solid-state electrochemical devices operating above 230 °C.

  3. N-acetylglucosamine deacetylases modulate the anchoring of the gamma-glutamyl capsule to the cell wall of Bacillus anthracis.

    Science.gov (United States)

    Candela, Thomas; Balomenou, Stavroula; Aucher, Willy; Bouriotis, Vassilis; Simore, Jean-Pierre; Fouet, Agnes; Boneca, Ivo G

    2014-06-01

    Bacillus anthracis has a complex cell wall structure composed of a peptidoglycan (PG) layer to which major structures are anchored such as a neutral polysaccharide, an S-layer, and a poly-γ-D-glutamate (PDGA) capsule. Many of these structures have central roles in the biology of B. anthracis, particularly, in virulence. However, little attention has been devoted to structurally study the PG and how it is modified in the presence of these secondary cell wall components. We present here the fine structure of the PG of the encapsulated RPG1 strain harboring both pXO1 and pXO2 virulence plasmids. We show that B. anthracis has a high degree of cross-linking and its GlcNAc residues are highly modified by N-deacetylation. The PG composition is not dependent on the presence of either LPXTG proteins or the capsule. Using NMR analysis of the PG-PDGA complex, we provide evidence for the anchoring of the PDGA to the glucosamine residues. We show that anchoring of the PDGA capsule is impaired in two PG N-deacetylase mutants, Ba1961 and Ba3679. Thus, these multiple N-deactylase activities would constitute excellent drug targets in B. anthracis by simultaneously affecting its resistance to lysozyme and to phagocytosis impairing B. anthracis survival in the host.

  4. Size, Shape, and Arrangement of Cellulose Microfibril in Higher Plant Cell Walls

    Energy Technology Data Exchange (ETDEWEB)

    Ding, S. Y.

    2013-01-01

    Plant cell walls from maize (Zea mays L.) are imaged using atomic force microscopy (AFM) at the sub-nanometer resolution. We found that the size and shape of fundamental cellulose elementary fibril (CEF) is essentially identical in different cell wall types, i.e., primary wall (PW), parenchyma secondary wall (pSW), and sclerenchyma secondary wall (sSW), which is consistent with previously proposed 36-chain model (Ding et al., 2006, J. Agric. Food Chem.). The arrangement of individual CEFs in these wall types exhibits two orientations. In PW, CEFs are horizontally associated through their hydrophilic faces, and the planar faces are exposed, forming ribbon-like macrofibrils. In pSW and sSW, CEFs are vertically oriented, forming layers, in which hemicelluloses are interacted with the hydrophobic faces of the CEF and serve as spacers between CEFs. Lignification occurs between CEF-hemicelluloses layers in secondary walls. Furthermore, we demonstrated quantitative analysis of plant cell wall accessibility to and digestibility by different cellulase systems at real-time using chemical imaging (e.g., stimulated Raman scattering) and fluorescence microscopy of labeled cellulases (Ding et al., 2012, Science, in press).

  5. The Basal Level Ethylene Response is Important to the Wall and Endomembrane Structure in the Hypocotyl Cells of Etiolated Arabidopsis Seedlings

    Institute of Scientific and Technical Information of China (English)

    Chan Xu; Xiaoyan Gao; Xiaobin Sun; Chi-Kuang Wen

    2012-01-01

    The sub-cellular events that occur during the ethylene-modulated cell elongation were characterized by examining the ultra-structure of etiolated Arabidopsis seedling hypocotyl cells.Preventing the basal level ethylene response facilitated cell elongation,and the cells exhibited wall loosening and separation phenotype.Nearby the wall separation sites were frequently associated with an increase in the cortical rough endoplasmic reticulum (rER) membranes,the presence of paramural bodies,and the circular Golgi formation.The cortical rER proliferation and circular Golgi phenotype were reverted by the protein biosynthesis inhibitor cycloheximide.The cortical rER membranes were longer when the ethylene response was prevented and shortened with elevated ethylene responses.Proteomic changes between wild type and the ethylene-insensitive mutant ethylene insensitive2 (ein2) seedling hypocotyls indicated that distinct subsets of proteins involving endomembrane trafficking,remodeling,and wall modifications were differentially expressed.FM4-64 staining supported the proteomic changes,which indicated reduced endocytosis activity with alleviation of the ethylene response.The basal level ethylene response has an important role in endomembrane trafficking,biological materials transport and maintenance of the endomembrane organization.It is possible that endomembrane alterations may partly associate with the wall modifications,though the biological significance of the alterations should be addressed in future studies.

  6. [Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase].

    Science.gov (United States)

    Pokrovskaya, M V; Zhdanov, D D; Eldarov, M A; Aleksandrova, S S; Veselovskiy, A V; Pokrovskiy, V S; Grishin, D V; Gladilina, Ju A; Sokolov, N N

    2017-01-01

    The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.

  7. The plant cell wall integrity maintenance mechanism-concepts for organization and mode of action.

    Science.gov (United States)

    Hamann, Thorsten

    2015-02-01

    One of the main differences between plant and animal cells are the walls surrounding plant cells providing structural support during development and protection like an adaptive armor against biotic and abiotic stress. During recent years it has become widely accepted that plant cells use a dedicated system to monitor and maintain the functional integrity of their walls. Maintenance of integrity is achieved by modifying the cell wall and cellular metabolism in order to permit tightly controlled changes in wall composition and structure. While a substantial amount of evidence supporting the existence of the mechanism has been reported, knowledge regarding its precise mode of action is still limited. The currently available evidence suggests similarities of the plant mechanism with respect to both design principles and molecular components involved to the very well characterized system active in the model organism Saccharomyces cerevisiae. There the system has been implicated in cell morphogenesis as well as response to abiotic stresses such as osmotic challenges. Here the currently available knowledge on the yeast system will be reviewed initially to provide a framework for the subsequent discussion of the plant cell wall integrity maintenance mechanism. The review will then end with a discussion on possible design principles for the cell wall integrity maintenance mechanism and the function of the plant turgor pressure in this context.

  8. Eukaryotic-type Ser/Thr protein kinase mediated phosphorylation of mycobacterial phosphodiesterase affects its localization to the cell wall

    Directory of Open Access Journals (Sweden)

    Neha eMalhotra

    2016-02-01

    Full Text Available Phosphodiesterase enzymes, involved in cAMP hydrolysis reaction, are present throughout phylogeny and their phosphorylation mediated regulation remains elusive in prokaryotes. In this context, we focused on this enzyme from Mycobacterium tuberculosis. The gene encoded by Rv0805 was PCR amplified and expressed as a histidine-tagged protein (mPDE utilizing Escherichia coli based expression system. In kinase assays, upon incubation with mycobacterial Clade I eukaryotic-type Ser/Thr kinases (PknA, PknB and PknL, Ni-NTA purified mPDE protein exhibited transphosphorylation ability albeit with varying degree. When mPDE was co-expressed one at a time with these kinases in E. coli, it was also recognized by an anti-phosphothreonine antibody, which further indicates its phosphorylating ability. Mass spectrometric analysis identified Thr-309 of mPDE as a phosphosite. In concordance with this observation, anti-phosphothreonine antibody m