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Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis  [PDF]
Ryoma Nakao, Madeleine Ramstedt, Sun Nyunt Wai, Bernt Eric Uhlin
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0051241
Abstract: Lipopolysaccharide (LPS) is the major component of the surface of Gram-negative bacteria and its polysaccharide portion is situated at the outermost region. We investigated the relationship between the polysaccharide portion of LPS and biofilm formation using a series of Escherichia coli mutants defective in genes earlier shown to affect the LPS sugar compositions. Biofilm formation by a deep rough LPS mutant, the hldE strain, was strongly enhanced in comparison with the parental strain and other LPS mutants. The hldE strain also showed a phenotype of increased auto-aggregation and stronger cell surface hydrophobicity compared to the wild-type. Similar results were obtained with another deep rough LPS mutant, the waaC strain whose LPS showed same molecular mass as that of the hldE strain. Confocal laser scanning microscopy (CLSM) analysis and biofilm formation assay using DNase I revealed that biofilm formation by the hldE strain was dependent on extracellular DNA. Furthermore, a loss of flagella and an increase in amount of outer membrane vesicles in case of the hldE strain were also observed by transmission electron microscopy and atomic force microscopy, respectively. In addition, we demonstrated that a mutation in the hldE locus, which alters the LPS structure, caused changes in both expression and properties of several surface bacterial factors involved in biofilm formation and virulence. We suggest that the implication of these results should be considered in the context of biofilm formation on abiotic surfaces, which is frequently associated with nosocominal infections such as the catheter-associated infections.
A role for lymphocytes and cytokines on the eosinophil migration induced by LPS
Castro-Faria-Neto, Hugo C;Penido, Carmen M;Larangeira, Andréa P;Silva, Adriana R;Bozza, Patrícia T;
Memórias do Instituto Oswaldo Cruz , 1997, DOI: 10.1590/S0074-02761997000800026
Abstract: in the present work we review the existing evidence for a lps-induced cytokine-mediated eosinophil accumulation in a model of acute inflammation. intrathoracic administration of lps into rodents (mice, rats or guinea pigs) induces a significant increase in the number of eosinophils recovered from the pleural fluid 24 hr later. this phenomenon is preceded by a neutrophil influx and accompanied by lymphocyte and monocyte accumulation. the eosinophil accumulation induced by lps is not affected by inhibitors of cyclo or lipoxygenase nor by paf antagonists but can be blocked by dexamethasone or the protein synthesis inhibitor cycloheximide. transfer of cell-free pleural wash from lps injected rats (lps-pw) to naive recipient animals induces a selective eosinophil accumulation within 24 hr. the eosinophilotactic activity present on the lps-pw has a molecular weight ranging between 10 and 50 kda and its effect is abolished by trypsin digestion of the pleural wash indicating the proteic nature of this activity. the production of the eosinophilotactic activity depends on the interaction between macrophages and t-lymphocytes and its effect can not be blocked by anti-il-5 monoclonal antibodies. accumulated evidence suggest that the eosinophil accumulation induced by lps is a consequence of a eosinophilotactic cytokine produced through macrophage and t-cell interactions in the site of a lps-induced inflammatory reaction.
A role for lymphocytes and cytokines on the eosinophil migration induced by LPS
Castro-Faria-Neto Hugo C,Penido Carmen M,Larangeira Andréa P,Silva Adriana R
Memórias do Instituto Oswaldo Cruz , 1997,
Abstract: In the present work we review the existing evidence for a LPS-induced cytokine-mediated eosinophil accumulation in a model of acute inflammation. Intrathoracic administration of LPS into rodents (mice, rats or guinea pigs) induces a significant increase in the number of eosinophils recovered from the pleural fluid 24 hr later. This phenomenon is preceded by a neutrophil influx and accompanied by lymphocyte and monocyte accumulation. The eosinophil accumulation induced by LPS is not affected by inhibitors of cyclo or lipoxygenase nor by PAF antagonists but can be blocked by dexamethasone or the protein synthesis inhibitor cycloheximide. Transfer of cell-free pleural wash from LPS injected rats (LPS-PW) to naive recipient animals induces a selective eosinophil accumulation within 24 hr. The eosinophilotactic activity present on the LPS-PW has a molecular weight ranging between 10 and 50 kDa and its effect is abolished by trypsin digestion of the pleural wash indicating the proteic nature of this activity. The production of the eosinophilotactic activity depends on the interaction between macrophages and T-lymphocytes and its effect can not be blocked by anti-IL-5 monoclonal antibodies. Accumulated evidence suggest that the eosinophil accumulation induced by LPS is a consequence of a eosinophilotactic cytokine produced through macrophage and T-cell interactions in the site of a LPS-induced inflammatory reaction.
The Transcription Factor C/EBP-β Mediates Constitutive and LPS-Inducible Transcription of Murine SerpinB2  [PDF]
Ekemini A. Udofa, Brett W. Stringer, Padmaja Gade, Donna Mahony, Marguerite S. Buzza, Dhananjaya V. Kalvakolanu, Toni M. Antalis
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0057855
Abstract: SerpinB2 or plasminogen activator inhibitor type 2 (PAI-2) is highly induced in macrophages in response to inflammatory stimuli and is linked to the modulation of innate immunity, macrophage survival, and inhibition of plasminogen activators. Lipopolysaccharide (LPS), a potent bacterial endotoxin, can induce SerpinB2 expression via the toll-like receptor 4 (TLR4) by ~1000-fold over a period of 24 hrs in murine macrophages. To map the LPS-regulated SerpinB2 promoter regions, we transfected reporter constructs driven by the ~5 kb 5'-flanking region of the murine SerpinB2 gene and several deletion mutants into murine macrophages. In addition, we compared the DNA sequence of the murine 5′ flanking sequence with the sequence of the human gene for homologous functional regulatory elements and identified several regulatory cis-acting elements in the human SERPINB2 promoter conserved in the mouse. Mutation analyses revealed that a CCAAT enhancer binding (C/EBP) element, a cyclic AMP response element (CRE) and two activator protein 1 (AP-1) response elements in the murine SerpinB2 proximal promoter are essential for optimal LPS-inducibility. Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated that LPS induces the formation of C/EBP-β containing complexes with the SerpinB2 promoter. Importantly, both constitutive and LPS-induced SerpinB2 expression was severely abrogated in C/EBP-β-null mouse embryonic fibroblasts (MEFs) and primary C/EBP-β-deficient peritoneal macrophages. Together, these data provide new insight into C/EBP-β-dependent regulation of inflammation-associated SerpinB2 expression.
On the effects of cycloheximide on cell motility and polarisation in Dictyostelium discoideum
Margaret Clotworthy, David Traynor
BMC Cell Biology , 2006, DOI: 10.1186/1471-2121-7-5
Abstract: We found that treatment with cycloheximide also causes the amoebae to retract their pseudopodia, round up and cease movement. Furthermore, fluid phase endocytosis, phagocytosis and capping cease in the presence of 2 mM cycloheximide, although membrane uptake, as measured using FM1-43, is unaffected. In the presence of cycloheximide, aggregation-competent amoebae sensitive to cAMP, although round, can still localise CRAC, ABP120, PI3K and actin polymerisation in response to a micropipette filled with cAMP. The behaviour of wild-type amoebae in the presence of cycloheximide is surprisingly similar to that of amoebae having a temperature-sensitive version of NSF at the restrictive temperature.Our results may suggest that, upon cycloheximide treatment, either a labile protein required for polarised membrane recycling is lost, or a control mechanism linking protein synthesis to membrane recycling is activated.Using Dictyostelium discoideum amoebae, Gonzalez and Satre [1] investigated the effect of protein synthesis inhibition on fluid phase uptake. They found that cycloheximide, which acts specifically on the 60S subunit of eukaryotic ribosomes [2], was the most potent inhibitor. Treatment of amoebae with cycloheximide for 30 minutes essentially eliminated fluid phase uptake, as measured using FITC-dextran. This, coupled with their observation that several other inhibitors of protein synthesis have a similar effect, suggested that a labile protein is essential for endocytosis.We examined Dictyostelium amoebae after treatment with cycloheximide and confirmed the observations of Gonzalez and Satre [1]. Furthermore, we noticed that under these conditions the cells withdrew their pseudopodia and rounded up. The loss of NSF (N-ethyl maleimide-Sensitive Factor) also leads to Dictyostelium amoebae rounding up and ceasing to move or endocytose fluid phase [3]. NSF is an essential ATPase required for SNARE complex dissociation after membrane fusion events [4-6]. As SNARE proteins
Construction of Monophosphoryl Lipid A Producing Escherichia coli Mutants and Comparison of Immuno-Stimulatory Activities of Their Lipopolysaccharides  [PDF]
Yaning Han,Ye Li,Jiuzhou Chen,Yanzhen Tan,Feng Guan,Xiaoyuan Wang
Marine Drugs , 2013, DOI: 10.3390/md11020363
Abstract: The lipid A moiety of Escherichia coli lipopolysaccharide is a hexaacylated disaccharide of glucosamine phosphorylated at the 1- and 4′-positions. It can be recognized by the TLR4/MD-2 complex of mammalian immune cells, leading to release of proinflammatory cytokines. The toxicity of lipid A depends on its structure. In this study, two E. coli mutants, HW001 and HW002, were constructed by deleting or integrating key genes related to lipid A biosynthesis in the chromosome of E. coli W3110. HW001 was constructed by deleting lacI and replacing lacZ with the Francisella novicida lpxE gene in the chromosome and only synthesizes monophosphoryl lipid A. HW002 was constructed by deleting lpxM in HW001 and synthesizes only the pentaacylated monophosphoryl lipid A. The structures of lipid A made in HW001 and HW002 were confirmed by thin layer chromatography and electrospray ionization mass spectrometry. HW001 and HW002 grew as well as the wild-type W3110. LPS purified from HW001 or HW002 was used to stimulate murine macrophage RAW264.7 cells, and less TNF-α were released. This study provides a feasible way to produce interesting lipid A species in E. coli.
Abl Kinase Inhibits the Engulfment of Apopotic Cells in Caenorhabditis elegans  [PDF]
Michael E. Hurwitz,Pamela J. Vanderzalm,Laird Bloom,Julia Goldman,Gian Garriga,H. Robert Horvitz
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1000099
Abstract: The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway includes the adaptor protein CED-2 CrkII and the small GTPase CED-10 Rac, and acts to rearrange the cytoskeleton of the engulfing cell. The other pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Although many components required for engulfment have been identified, little is known about inhibition of engulfment. The tyrosine kinase Abl regulates the actin cytoskeleton in mammals and Drosophila in multiple ways. For example, Abl inhibits cell migration via phosphorylation of CrkII. We tested whether ABL-1, the C. elegans ortholog of Abl, inhibits the CED-2 CrkII-dependent engulfment of apoptotic cells. Our genetic studies indicate that ABL-1 inhibits apoptotic cell engulfment, but not through CED-2 CrkII, and instead acts in parallel to the two known engulfment pathways. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. The loss of ABL-1 function partially restores normal DTC migration in the CED-10 Rac pathway mutants. We found that ABI-1 the C. elegans homolog of mammalian Abi (Abl interactor) proteins, is required for engulfment of apoptotic cells and proper DTC migration. Like Abl, Abi proteins are cytoskeletal regulators. ABI-1 acts in parallel to the two known engulfment pathways, likely downstream of ABL-1. ABL-1 and ABI-1 interact physically in vitro. We propose that ABL-1 opposes the engulfment of apoptotic cells by inhibiting ABI-1 via a pathway that is distinct from the two known engulfment pathways.
Abl Kinase Inhibits the Engulfment of Apopotic Cells in Caenorhabditis elegans  [PDF]
Michael E Hurwitz,Pamela J Vanderzalm,Laird Bloom,Julia Goldman,Gian Garriga,H. Robert Horvitz
PLOS Biology , 2009, DOI: 10.1371/journal.pbio.1000099
Abstract: The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway includes the adaptor protein CED-2 CrkII and the small GTPase CED-10 Rac, and acts to rearrange the cytoskeleton of the engulfing cell. The other pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Although many components required for engulfment have been identified, little is known about inhibition of engulfment. The tyrosine kinase Abl regulates the actin cytoskeleton in mammals and Drosophila in multiple ways. For example, Abl inhibits cell migration via phosphorylation of CrkII. We tested whether ABL-1, the C. elegans ortholog of Abl, inhibits the CED-2 CrkII-dependent engulfment of apoptotic cells. Our genetic studies indicate that ABL-1 inhibits apoptotic cell engulfment, but not through CED-2 CrkII, and instead acts in parallel to the two known engulfment pathways. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. The loss of ABL-1 function partially restores normal DTC migration in the CED-10 Rac pathway mutants. We found that ABI-1 the C. elegans homolog of mammalian Abi (Abl interactor) proteins, is required for engulfment of apoptotic cells and proper DTC migration. Like Abl, Abi proteins are cytoskeletal regulators. ABI-1 acts in parallel to the two known engulfment pathways, likely downstream of ABL-1. ABL-1 and ABI-1 interact physically in vitro. We propose that ABL-1 opposes the engulfment of apoptotic cells by inhibiting ABI-1 via a pathway that is distinct from the two known engulfment pathways.
Senescence and Postharvest Performance of Cut Nerine sarniensis Flowers: Effects of Cycloheximide  [PDF]
Fahima Gul,Inayatullah Tahir,Inam Ul Rasool
International Journal of Botany , 2012,
Abstract: A study was conducted to determine the effects of pretreatment at varying concentrations of Cycloheximide (CHI) on senescence and postharvest performance in Nerine sarniensis. At a particular threshold concentration CHI delays senescence and above which it prevents flower opening and promotes senescence. The fact that cycloheximide delays tepal senescence demonstrates the synthesis of particular proteins probably enzymes, responsible for degradation of cellular constituents, executes the cell death programme in flower tepals. Pretreatment of scapes with CHI at 0.01 and 0.05 mM concentrations was found to delay visible signs of senescence, maintain a sustained rate of flower blooms and increase fresh and dry mass, besides lowering electrical conductivity of ion leachates and solution absorption. An increase in total sugar, phenolic and soluble protein content was observed with a concomitant decrease in a-amino acid content. Pretreatment of scapes with 0.05 or 0.01 CHI for 1 h can be used as an effective treatment to improve the post harvest longevity in this flower system.
Modulatory effects of salmonella lap-lps on murine macrophages  [cached]
Rishi P,Batra N,Sood S,Tiwari R
Indian Journal of Medical Microbiology , 2002,
Abstract: PURPOSE: To study the modulatory effects of Salmonella lipid associated protein - lipopolysaccharides (LAP-LPS) on murine macrophages as the intracellular survival within the host macrophages is an important feature for a number of gram-negative pathogens like S.typhi. METHODS: Macrophage functions were studied in two groups of mice immunized with either LPS or LAP-LPS. RESULTS: Comparison of protective efficacy of mice preimmunized with LPS based preparations, against challenge infectious doses, showed higher protection in LAP-LPS complex immunized mice group as compared to the mice group immunized with LPS alone. Aggregation of S.typhi cells was lesser with intestinal mucus extracted from LAP-LPS immunized mice as compared to LPS immunized challenged group. A significant increase in the number of macrophages in LAP-LPS immunized mice was also observed in comparison to control and LPS immunized mice groups. Nitric oxide (NO) and superoxide dismutase (SOD) production were also more in macrophages derived from LAP-LPS immunized mice group. Phagocytic uptake studies showed that there was enhanced uptake of bacteria in the LAP-LPS immunized animals in comparison to LPS immunized and controls. Similar trend was observed in intracellular killing of bacteria by the macrophages. CONCLUSIONS: The results indicated the involvement of protein moiety in LAP on modulation of effects of LPS on macrophages.
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