A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of gram-negative bacteria

Karkhanis, Yashwant D.; Zeltner, Johanna Y.; Jackson, Jesse J.; Carlo, Dennis J.

doi:10.1016/0003-2697(78)90260-9

Cited by 691 publications

(334 citation statements)

References 9 publications

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“…4. KDO was detected in the control (0.011 ± 0.003 mg/g VSS) because some dead microorganisms (including G − bacteria) in sludge had released KDO to water (Karkhanis et al, 1978;Kumada et al, 1993). The KDO in the water of sludge after formaldehyde extraction, formaldehyde-NaOH extraction and cation exchange resin extraction were 0.010 ± 0.004, 0.013 ± 0.006 and 0.012 ± 0.007 mg/g VSS, respectively (Fig.…”

Section: Damage Of Cell Walls In Eps Extractionmentioning

confidence: 99%

“…The DNA content of extracted EPS was measured by the diphenylamine colorimetric method using fish sperm DNA and sodium salt as the standard (Sun et al, 1999). KDO content was determined according to Karkhanis et al (1978), the activity of G6PD according to the method of Lessie and Vander Wyk (1972) and N-acetylglucosamine content by the Morgan-Elson colorimetric method with glucosamine as a standard (Morgan and Elson, 1934). Divided into two parts: one pa rt for ce ntrif ugation and the othe r part for anal ysis 20000 g Ce ntri fugatio n, 4° C, 20 min …”

Section: Chemical Analysismentioning

confidence: 99%

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Evaluation of the damage of cell wall and cell membrane for various extracellular polymeric substance extractions of activated sludge

Guo

Liu

Xiao

2014

Journal of Biotechnology

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a b s t r a c t Extracellular polymeric substances (EPS) are susceptible to contamination by intracellular substances released during the extraction of EPS owing to the damage caused to microbial cell structures. The damage to cell walls and cell membranes in nine EPS extraction processes of activated sludge was evaluated in this study. The extraction of EPS (including proteins, carbohydrates and DNA) was the highest using the NaOH extraction method and the lowest using formaldehyde extraction. All nine EPS extraction methods in this study resulted in cell wall and membrane damage. The damage to cell walls, evaluated by 2-keto-3-deoxyoctonate (KDO) and N-acetylglucosamine content changes in extracted EPS, was the most significant in the NaOH extraction process. Formaldehyde extraction showed a similar extent of damage to cell walls to those detected in the control method (centrifugation), while those in the formaldehydeNaOH and cation exchange resin extractions were slightly higher than those detected in the control. N-acetylglucosamine was more suitable than KDO for the evaluation of cell wall damage in the EPS extraction of activated sludge. The damage to cell membranes was characterized by two fluorochromes (propidium iodide and FITC Annexin V) with flow cytometry (FCM) measurement. The highest proportion of membrane-damaged cells was detected in NaOH extraction (26.54% of total cells) while membranedamaged cells comprised 8.19% of total cells in the control.

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Section: Damage Of Cell Walls In Eps Extractionmentioning

confidence: 99%

Section: Chemical Analysismentioning

confidence: 99%

Evaluation of the damage of cell wall and cell membrane for various extracellular polymeric substance extractions of activated sludge

Guo

Liu

Xiao

2014

Journal of Biotechnology

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“…LPSs were solubilized in water by sonication at the appropriate concentration and autoclaved before use. Determination of 2-keto-3-deoxyoctonate (KDO) was performed as described by Karkhanis et al (23). The Limulus lysate gelation activity (24), which estimates the state of aggregation of endotoxins, was 2 ng for B. abortus LPS and 1 ng for S. flexneri LPS.…”

Section: Lipopolysaccharidementioning

confidence: 99%

Brucella abortus Lipopolysaccharide in Murine Peritoneal Macrophages Acts as a Down-Regulator of T Cell Activation

Forestier

Deleuil

Lapaque

et al. 2000

The Journal of Immunology

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Macrophages play a central role in host immune responses against pathogens by acting as both professional phagocytic cells and as fully competent APCs. We report here that the LPS from the facultative intracellular Gram-negative bacteria Brucella abortus interferes with the MHC class II Ag presentation pathway. LPS inhibits the capacity of macrophages to present hen egg lysozyme (HEL) antigenic peptides to specific CD4+ T cells but not those of OVA to specific CD8+ T cells. This defect was neither related to a decrease of MHC class II surface expression nor to a deficient uptake or processing of HEL. In addition, B. abortus LPS did not prevent the formation of SDS-resistant MHC class II complexes induced by HEL peptides. At the cell surface of macrophages, we observed the presence of LPS macrodomains highly enriched in MHC class II molecules, which may be responsible for the significant down-regulation of CD4+ T cell activation. This phenomenon may account for the avoidance of the immune system by certain bacterial pathogens and may explain the immunosuppression observed in individuals with chronic brucellosis.

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“…The product(s) obtained at various times from the polysaccharide with coliphage K5 (100-l portions of the incubation mixture) was treated with the periodate-thiobarbituric acid reagent (15,25). This treatment converted the ⌬ 4,5 GlcA residue at the nonreducing end to formyl pyruvate, which in turn reacted to form a violet product.…”

mentioning

confidence: 99%

Analysis of the enzymatic cleavage (beta elimination) of the capsular K5 polysaccharide of Escherichia coli by the K5-specific coliphage: reexamination

et al. 1996

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The capsular K5 polysaccharide of Escherichia coli is the receptor of the capsule-specific coliphage K5, which harbors an enzyme that degrades the capsular K5 polysaccharide to a number of oligosaccharides. Analysis of the degradation products using gel permeation chromatography, the periodate-thiobarbituric acid and bicinchoninic acid reactions, and nuclear magnetic resonance spectroscopy showed that the major reaction products are hexa-, octa-, and decasaccharides with 4,5-unsaturated glucuronic acid (⌬ 4,5 GlcA) at their nonreducing end. Thus, the bacteriophage enzyme is a K5 polysaccharide lyase and not, as we had reported previously, an endo-N-acetylglucosaminidase.

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A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of gram-negative bacteria

Cited by 691 publications

References 9 publications

Evaluation of the damage of cell wall and cell membrane for various extracellular polymeric substance extractions of activated sludge

Evaluation of the damage of cell wall and cell membrane for various extracellular polymeric substance extractions of activated sludge

Brucella abortus Lipopolysaccharide in Murine Peritoneal Macrophages Acts as a Down-Regulator of T Cell Activation

Analysis of the enzymatic cleavage (beta elimination) of the capsular K5 polysaccharide of Escherichia coli by the K5-specific coliphage: reexamination

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