The hydrolytic degradation of the antigenic complex of <i>Bact. typhosum</i> Ty 2

Freeman, G. G.; Anderson, Thomas

doi:10.1042/bj0350564

Cited by 29 publications

(4 citation statements)

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“…A similar, partial degradation of the protein moiety by tryptic digestion has also been reported for the endotoxin complex of Salmonella typhosa [44,45], Shigella paradysenteriae [6,7], E . coli [19] and Neisseria gonorrhoeae [46].…”

Section: Chemical Characterization Of the Tryptic And Pronase Cores Osupporting

confidence: 65%

Studies on the Protein Moiety of Endotoxin from Gram‐Negative Bacteria

Wober

Alaupovic

1971

European Journal of Biochemistry

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The so-called simple proteins isolated from the endotoxin of Serratia marcescens 08 and whole cells Escherichia coli 0 141 :K85(B) by aqueous phenol treatment were characterized by the determination of hydrodynamic properties, electrophoretic behavior, immunochemical specificity and chemical analysis. The chemical composition of both proteins revealed the presence of small amount of lipid A constituents such as glucosamine, phosphorus and fatty acids ; the p-hydroxymyristic acid regarded as the characteristic marker for lipid A was present in all protein preparations. Since the treatment of intact endotoxin or bacterial cells with aqueous phenol results in the formation of two fragments (a lipopolysaccharide and a lipid A-protein), both of which are characterized by the presence of lipid A constituents, this dissociation seems to be caused by cleavage of a phenolsensitive linkage within the molecule of lipid A rather than at its point of attachment to the protein moiety. Thus, the %imple'' protein preparations consist of the protein moiety and a small segment of lipid A. Contrary to previous views, such protein preparations are not simple proteins if this designation implies absence of constituents other than amino acids. However, for a historical reason we suggest that the term simple protein be retained for protein fragments isolated by aqueous phenol treatment of intact endotoxins.The simple protein from Serratia marcescens 0s was treated successively with trypsin and pronase. The resulting trypsin and pronase cores contained increasing amounts of lipid A constituents firmly bound to the residual protein moiety. Since lipid A was separated as an entity from the protein moiety only after acid hydrolysis of the pronase core, it is proposed that in intact endotoxin lipid A is covalently linked to the protein moiety. The absence of any detectable glucosamine and fatty acids in the mixture of peptides and amino acids released by trypsin and pronase, indicated a single point attachment between the lipid A and protein moieties.The protein moieties from Serratia marcemens and Escherichia coli were immunogenic and possessed a common antigenic determinant. Both trypsin and pronase cores retained the immunogenicity and revealed the presence ofa common antigen with the parent simple protein. This second set of antigenic determinant(s) different from the specific 0-antigens seems to be located in the protein moiety close to lipid A.It is generally accepted that the endotoxin or somatic 0-antigen complexes occurring in the cell walls of gram-negative bacteria represent macromolecular entities composed of lipid, polysaccharide and protein moieties [1,2]. Evidence for this structural complexity was presented in early studies by Morgan and Partridge [3-51 who showed that endotoxins isolated from Shigella dysenteria and Salmonella typhosa could be resolved by successive treatments with formamide and 900/, phenol into a

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Section: Chemical Characterization Of the Tryptic And Pronase Cores Osupporting

confidence: 65%

Studies on the Protein Moiety of Endotoxin from Gram‐Negative Bacteria

Wober

Alaupovic

1971

European Journal of Biochemistry

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“…L'amélioration des techniques d'étude y est pour beau coup : en particulier, la chromatographie sur plaques (8) et en phase gazeuse (5)(6)(7), qui permettent l'étude des constituants qui ne représentent souvent que 5 à 10% du poids des bactéries.…”

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Etude des lipides de la forme L stable de Protéus P 18: Identification des acides gras de la fraction acétonosoluble

Krembel

Pinson

1963

Pathobiology

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“…LPS (5 mg ml −1 ) was hydrolysed with 1% (v/v) acetic acid at 100°C for 90 min according to Leone et al (2006). This standard procedure, originally established by Freeman & Anderson (1941), cleaves the acid labile ketosidic linkage between the polysaccharide moiety and lipid A, leaving the polysaccharide chain intact. The insoluble lipid A was separated from the polysaccharide component of LPS by centrifugation (15,000 g, 10 min).…”

Section: Degradation Of Lpsmentioning

confidence: 99%

Inhibition of cardiac pacemaker channel hHCN2 depends on intercalation of lipopolysaccharide into channel‐containing membrane microdomains

Klöckner

Rueckschloss

Großmann

et al. 2014

The Journal of Physiology

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Key pointsr The regulation of cardiac function is seriously impaired in severe inflammatory diseases. One characteristic of this dysfunction is a strong reduction in heart rate variability (HRV) so that the cardiac cycle is more regular. This phenomenon is strongly correlated with an unfavourable prognosis in patients with systemic inflammation.r Although the depression in HRV can be partially explained by the interplay between cardiac pacemaker channels and lipopolysaccharide (LPS) liberated from the outer walls of Gram-negative bacteria, the underlying mechanism is still elusive.r Using HEK293 cells stably expressing a cardiac pacemaker channel, we demonstrate that only intact LPS molecules can intercalate into target cell membranes and then directly interact with extracellular parts of pacemaker channels. Intracellular signalling cascades do not contribute to LPS-dependent channel modulation.r The present results help to elucidate how LPS interacts with pacemaker channels to attenuate the regularity of the cardiac cycle.Abstract Depressed heart rate variability in severe inflammatory diseases can be partially explained by the lipopolysaccharide (LPS)-dependent modulation of cardiac pacemaker channels. Recently, we showed that LPS inhibits pacemaker current in sinoatrial node cells and in HEK293 cells expressing cloned pacemaker channels, respectively. The present study was designed to verify whether this inhibition involves LPS-dependent intracellular signalling and to identify structures of LPS responsible for pacemaker current modulation. We examined the effect of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) stably expressed in HEK293 cells. In whole-cell recordings, bath application of LPS decreased pacemaker current (I hHCN2 ) amplitude. The same protocol had no effect on channel activity in cell-attached patch recordings, in which channels are protected from the LPS-containing bath solution. This demonstrates that LPS must interact directly with or close to the channel protein. After cleavage of LPS into lipid A and the polysaccharide chain, neither of them alone impaired I hHCN2 , which suggests that modulation of channel activity critically depends on the integrity of the entire LPS molecule. We furthermore showed that β-cyclodextrin interfered with LPS-dependent channel modulation predominantly via scavenging of lipid A, thereby abrogating the capability of LPS to intercalate into target cell membranes. We conclude that LPS impairs I hHCN2 by a local mechanism that is restricted to the vicinity of the channels. Furthermore, intercalation of lipid A into target cell membranes is a prerequisite for the inhibition that is suggested to depend on the direct interaction of the LPS polysaccharide chain with cardiac pacemaker channels.

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The hydrolytic degradation of the antigenic complex of Bact. typhosum Ty 2

Cited by 29 publications

References 4 publications

Studies on the Protein Moiety of Endotoxin from Gram‐Negative Bacteria

Studies on the Protein Moiety of Endotoxin from Gram‐Negative Bacteria

Etude des lipides de la forme L stable de Protéus P 18: Identification des acides gras de la fraction acétonosoluble

Inhibition of cardiac pacemaker channel hHCN2 depends on intercalation of lipopolysaccharide into channel‐containing membrane microdomains

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