Biotinylation of bacterial lipopolysaccharide and its applications to electron microscopy.

Odeyale, Charles O.; Kang, Yong-Cheol

doi:10.1177/36.9.3136207

Cited by 19 publications

(9 citation statements)

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“…Similarly complex as the mechanism of LPS-uptake are the pathways of its intracellular movement. It has been detected in endosomes, phagosomes, lysosomes cytoplasm, and in the golgi-complex (Odeyale and Kang, 1988;Kang et al, 1990;Hampton et al, 1991;Kriegsmann et al, 1993;Gegner et al, 1995;Thieblemont and Wright, 1999). It seems that distinct routes of LPS trafficking determine the biological consequences.…”

Section: Discussionmentioning

confidence: 99%

Intracellular LPS inhibits the activity of potassium channels and fails to activate NFκB in human macrophages

Gerth

Grosche

Nieber

et al. 2004

Journal Cellular Physiology

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Although much has been learned about signal transduction mechanisms and binding proteins involved in lipopolysaccharides (LPS)-induced activation of monocytes/macrophages, little is known about the ability of internalized LPS to activate cells. To approach this question we either exposed macrophages to LPS or microinjected the cells with LPS before studying early cellular events associated with LPS-mediated macrophage activation. We measured membrane currents and translocation of NFkappaB to the nucleus. Using the whole-cell patch clamp technique ion channels were analyzed and characterized as K+ sensitive inward and outward currents. Exogenous LPS was shown to increase the voltage-dependent outward current whereas the voltage-dependent inward current was unaffected. However when cells were microinjected with LPS both inward and outward current were completely abolished. The presence of LPS within the cells did not prevent them to perform phagocytosis or to respond to fMLP with an appropriate increase in [Ca2+]i. The immunocytological detection of NFkappaB p65 translocation revealed that exogenous LPS led to the nuclear localization of the p65 subunit of NFkappaB, whereas only the cytoplasmic localization of p65 was observed following microinjection of LPS. These data show that one major process in macrophage activation, the NFkappaB dependent transcription of a number of genes encoding for many inflammatory mediators cannot be induced by intracellular LPS but requires the interaction of LPS with external membrane components. However intracellular LPS causes a drastic decrease in potassium currents which by keeping the cell membrane at a depolarized potential may result in changed biological answers of these cells.

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Section: Discussionmentioning

confidence: 99%

Intracellular LPS inhibits the activity of potassium channels and fails to activate NFκB in human macrophages

Gerth

Grosche

Nieber

et al. 2004

Journal Cellular Physiology

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“…This analysis of "where the LPS goes" has revealed the cellular primary targets of endotoxins (the cells that receive and accumulate LPS) after their release in the bloodstream. Another approach that has been less developed is the study of LPS movement within the cell, usually performed on cells treated with bacteria or LPS in vitro (Dacosta et al, 1990;Kang et al, 1990;Lucas et al, 1985;Odeyale and Kang, 1988;Risco et al, 1991). These studies can help to define potential primary targets of endotoxins at the intracellular level (Risco et al, 1993b), and to design experimental models for the study of molecular interactions that play a physiological role in endotoxemias (Risco and Pinto da Silva, 1993).…”

Section: Introductionmentioning

confidence: 99%

Cellular functions during activation and damage by pathogens: Immunogold studies of the interaction of bacterial endotoxins with target cells

Risco

Silva

1995

Microscopy Res & Technique

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Bacterial endotoxins (lipopolysaccharides or LPS) are active components of Gram-negative bacteria that act on numerous cellular functions through the processes of cell activation and damage. The molecular mechanisms involved in the "endotoxic phenomenon" are not defined yet, although extensive studies have been carried out. Immunogold and electron microscopy (EM) have contributed to identify the primary target cells of endotoxins and the subcellular systems that receive the direct action of these bacterial agents. Here, we review our studies on immunogold detection of endotoxins in cellular and subcellular systems. The analysis of the interaction between endotoxins and cells was focussed on the following aspects: (1) morphological characteristics of the LPS aqueous suspensions used in experimental work; (2) binding of endotoxins to the plasma membrane of type II pneumocytes and alveolar macrophages (two of their cellular targets), and influence of the state of aggregation of the LPS; (3) movement and distribution of endotoxins inside the cell, from the plasma membrane to the nucleoplasm; and (4) interaction of LPS with microtubules and its effects on the integrity of the microtubular network. These approaches provide information at the molecular level as well as data for the establishment of physiological models of endotoxicity.

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“…Highly purified CP was prepared as described previously (6). CPs from strains of serotypes 1 and 7 were conjugated to biotin following oxidation with sodium meta-periodate to generate aldehyde groups (16) and reacted with biotin-LC-hydrazide according to the manufacturer's instructions (Pierce Chemical Co., Rockford, Ill.). Biotin-LChydrazide was coupled directly to the carboxyl groups of 2-keto-3-deoxy-Dmanno-2-octulosonic acid in the serotype 5 CP with ethyl-dimethylaminopropylcarbodiamide (Bio-Rad Laboratories, Richmond, Calif.) according to the manufacturer's instructions (Pierce Chemical Co.).…”

Section: Bacterialmentioning

confidence: 99%

Serologic Detection of Actinobacillus pleuropneumoniae in Swine by Capsular Polysaccharide-Biotin-Streptavidin Enzyme-Linked Immunosorbent Assay

Inzana

Fenwick

2001

J Clin Microbiol

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Serologic detection of Actinobacillus pleuropneumoniae infections in swine have been problematic due to antigenic cross-reactivity of Apx toxins, lipopolysaccharide, and outer membrane proteins between A. pleuropneumoniae serotypes and other bacterial species. To maximize serologic specificity and sensitivity, we developed an assay that uses highly purified A. pleuropneumoniae capsular polysaccharide (CP) conjugated to biotin, which is then bound to streptavidin-coated enzyme-linked immunosorbent assay (CP-BS-ELISA) plates. This assay was used to test a panel of 240 serum samples from pigs prior to challenge, after challenge with bacterial species other than A. pleuropneumoniae, or after challenge with A. pleuropneumoniae serotype 1, 5, or 7. Overall assay results for the individual sera tested were reproducible on the same day and on separate days. The sensitivity of the assay was 100% by ELISAs with biotin-CPs of serotypes 1 and 7 and 87.5% by ELISAs with biotin-CP of serotype 5. Specificity was 100% by ELISAs with biotin-CPs of serotypes 1 and 5 and 94.5% by ELISAs with biotin-CP of serotype 7. The biotin-CPs of at least three A. pleuropneumoniae serotypes could be combined for use in a screening assay to detect antibodies to CPs from strains of different serotypes. In conclusion, the CP-BS-ELISA proved to be a serotype-specific and species-specific assay with high sensitivity for the identification of pigs exposed to A. pleuropneumoniae.

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Biotinylation of bacterial lipopolysaccharide and its applications to electron microscopy.

Cited by 19 publications

References 0 publications

Intracellular LPS inhibits the activity of potassium channels and fails to activate NFκB in human macrophages

Intracellular LPS inhibits the activity of potassium channels and fails to activate NFκB in human macrophages

Cellular functions during activation and damage by pathogens: Immunogold studies of the interaction of bacterial endotoxins with target cells

Serologic Detection of Actinobacillus pleuropneumoniae in Swine by Capsular Polysaccharide-Biotin-Streptavidin Enzyme-Linked Immunosorbent Assay

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