Sensitive Reverse Staining of Bacterial Lipopolysaccharides on Polyacrylamide Gels by Using Zinc and Imidazole Salts

Hardy, Eugenio; Pupo, Elder; Castellanos‐Serra, Lila; Reyes, J.; Fernández-Patrón, Carlos

doi:10.1006/abio.1996.9719

Cited by 36 publications

(44 citation statements)

References 1 publication

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“…Fimbriae from the P. gingivalis 381 strain were isolated by the method of Arai et al (4), while the gingipains HRgpA, RgpB, and Kgp were purified from P. gingivalis strain HG66 as previously described (62,65). Lipopolysaccharide (LPS) was obtained from the P. gingivalis W83 strain using phenol-water extraction according to the published procedure (83), and after dialysis against deionized water, the purity of the LPS preparation was checked by SDS-PAGE and zinc-imidazole staining (22).…”

Section: Methodsmentioning

confidence: 99%

Adsorption of Components of the Plasma Kinin-Forming System on the Surface ofPorphyromonas gingivalisInvolves Gingipains as the Major Docking Platforms

et al. 2011

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Enhanced production of proinflammatory bradykinin-related peptides, the kinins, has been suggested to contribute to the pathogenesis of periodontitis, a common inflammatory disease of human gingival tissues. In this report, we describe a plausible mechanism of activation of the kinin-generating system, also known as the contact system or kininogen-kallikrein-kinin system, by the adsorption of its plasma-derived components such as high-molecular-mass kininogen (HK), prekallikrein (PK), and Hageman factor (FXII) to the cell surface of periodontal pathogen Porphyromonas gingivalis. The adsorption characteristics of mutant strains deficient in selected proteins of the cell envelope suggested that the surface-associated cysteine proteinases, gingipains, bearing hemagglutinin/adhesin domains (RgpA and Kgp) serve as the major platforms for HK and FXII adhesion. These interactions were confirmed by direct binding tests using microplate-immobilized gingipains and biotinylated contact factors. Other bacterial cell surface components such as fimbriae and lipopolysaccharide were also found to contribute to the binding of contact factors, particularly PK. Analysis of kinin release in plasma upon contact with P. gingivalis showed that the bacterial surface-dependent mechanism is complementary to the previously described kinin generation system dependent on HK and PK proteolytic activation by the gingipains. We also found that several P. gingivalis clinical isolates differed in the relative significance of these two mechanisms of kinin production. Taken together, these data show the importance of this specific type of bacterial surface-host homeostatic system interaction in periodontal infections.

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Section: Methodsmentioning

confidence: 99%

Adsorption of Components of the Plasma Kinin-Forming System on the Surface ofPorphyromonas gingivalisInvolves Gingipains as the Major Docking Platforms

et al. 2011

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“…Cell envelopes were isolated by the procedure of Lugtenberg et al (45) and treated with pronase overnight at 37°C to obtain the LPS samples. The isolated LPS was resolved by Tricine polyacrylamide gel electrophoresis (PAGE) (42,60) and analyzed by silver staining (69), zinc staining (35), and Western blotting (47). C. jejuni 81116 antiserum diluted 1:1,000 in TST (10 mM Tris-HCl [pH 7.4], 100 mM NaCl, 0.1% Tween 20) containing 5% skimmed milk was used in immunoblotting.…”

Section: Methodsmentioning

confidence: 99%

The galE Gene of Campylobacter jejuni Is Involved in Lipopolysaccharide Synthesis and Virulence

et al. 2000

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Lipopolysaccharide (LPS) is one of the main virulence factors of gram-negative bacteria. The LPS fromCampylobacter spp. has endotoxic properties and has been shown to play a role in adhesion. We previously cloned a gene cluster (wla) which is involved in the synthesis of the Campylobacter jejuni 81116 LPS molecule. Sequence alignment of the first gene in this cluster indicated similarity with galE genes. These genes encode a UDP-glucose 4-epimerase, which catalyzes the interconversion of UDP-galactose and UDP-glucose. A Salmonella galE mutant was transformed with the galE gene from C. jejuni. The LPS analysis of wild-type, galE, and complemented galE Salmonella strains showed that the C. jejuni galE gene could restore the smooth wild-type Salmonella LPS. A UDP-glucose 4-epimerase assay was used to demonstrate that the galE gene from C. jejuni encoded this epimerase. We constructed a C. jejuni galE mutant which expressed a lipid A-core molecule of reduced molecular weight that did not react with antiserum raised against the parental strain. These results show an essential role for the galE gene in the synthesis of C. jejuni LPS. The galE mutant also showed a reduction in its ability to adhere to and invade INT407 cells. However, it was still able to colonize chickens to the same level as the wild-type strain. The serum resistance and hemolytic activity of this mutant were not changed compared to the parent strain. The ability of the mutant to take up DNA and integrate it in its genome was reduced 20-fold. These results show that LPS of C. jejuni is an important virulence factor.

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“…Also, zinc staining is not specific to protein and can also effectively detect nucleic acids and lipopolysaccharides [112,113]. Since this stain has no definitive endpoint, its use also involves the risk of overdevelopment.…”

Section: Negative Stainsmentioning

confidence: 99%

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

2010

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Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

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Sensitive Reverse Staining of Bacterial Lipopolysaccharides on Polyacrylamide Gels by Using Zinc and Imidazole Salts

Cited by 36 publications

References 1 publication

Adsorption of Components of the Plasma Kinin-Forming System on the Surface ofPorphyromonas gingivalisInvolves Gingipains as the Major Docking Platforms

Adsorption of Components of the Plasma Kinin-Forming System on the Surface ofPorphyromonas gingivalisInvolves Gingipains as the Major Docking Platforms

The galE Gene of Campylobacter jejuni Is Involved in Lipopolysaccharide Synthesis and Virulence

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

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