Biochemical Studies on Lipopolysaccharides of <i>Salmonella</i> R Mutants

Gmeiner, Jobst; Lüderitz, Otto; Westphal, Otto

doi:10.1111/j.1432-1033.1969.tb19618.x

Cited by 166 publications

(59 citation statements)

References 41 publications

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“…The low macrophage content of intradermal L5178Y lymphomata (personal observation) compared to the 4000 present in FS6 fibrosarcomata (Evans, 1972) The finding that the polysaccharidefree endotoxin derivative lipid A has the structure shown in Fig. 5 (Gmeiner et al, 1969), allows some speculation concerning a possible molecular basis for the similarity in action of dsRNA and endotoxin. It is not inconceivable that both may bind to a common receptor on cells which relies for its specificity on a combination of recurring phosphate groups, present in both macromolecules, and of a hydroxyl group provided by the myristic acid in lipid A and by ribose in dsRNA.…”

Section: Discussionmentioning

confidence: 97%

“…The tumour vasculature without prior exposure to endotoxin seems to respond to endotoxin like the blood vessels in skin after initial sensitization. Since the local Shwartzman reaction can be inhibited by administration of heparin (Good and Thomas, 1953), the effect of full heparinization on the antitumour action of endotoxin was studied with both L5178Y lymphoma and the FS6 sarcoma. Twenty mice were heparinized on Day 7 after inoculation i.d.…”

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confidence: 99%

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Similarities of the Anti-tumour Actions of Endotoxin, Lipid A and Double-stranded RNA

Parr¹,

Wheeler²,

Alexander³

1973

Br J Cancer

120

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Summary.-Double stranded RNA (dsRNA) whether isolated from a fungal virus or prepared synthetically (i.e., Poly I Poly C) and endotoxin were found to exert very similar effects on syngeneic murine lymphomata and fibrosarcomata. They cause complete regressions of some established subcutaneous (s.c.) or intradermal (i.d.) tumours but not of intraperitoneal (i.p.) tumours when administered either systemically or directly into the tumour. To achieve this effect the tumours must be fully established and the best results were obtained when treatment was started 7 days after transplant. If treatment is started within the first 3 days following the transplantation of the tumour then only a slight inhibition of growth rate was observed. These agents can also act prophylactically and protect mice against a subsequent challenge but only if this is given i.p. and not if given s.c. or i.d. The prophylactic action is explained by the action of dsRNA and endotoxin on peritoneal macrophages which cause them to become cytotoxic to tumour cells (i.e., to become activated).The therapeutic effect of systemically administered endotoxin and dsRNA on established tumours is not the result of a direct action on the tumour cells themselves but is a complex process requiring the co-operation of several host factors. Haemorrhagic necrosis involving coagulation is essential (i.e., heparinization reduces the regression of tumours) but is not itself sufficient. Immunosuppression by whole body irradiation or by antilymphocyte serum also interferes with the antitumour action of dsRNA and endotoxin in spite of the fact that haemorrhagic necrosis still occurs. Also, the magnitude of the antitumour action correlated in a series of different tumours with their antigenicity. The observed tumour regressions are probably brought about by (1) vascular damage in the tumour which permits immune defence mechanisms of the host to gain access to the tumour and (2) activation of macrophages present within the tumour. The relative contribution of these two mechanisms may depend on the nature of the tumour and the route of administration of the active agents.Dibenyline, which protects against the lethal action of endotoxin by preventing the action of the catecholamines on the a-adrenergic receptors, makes it possible to increase the effectiveness of endotoxin in tumours by allowing a large dose to be given. Lipid A, a derivative of endotoxin which does not contain polysaccharide, has similar antitumour action to dsRNA and endotoxin. Some common features of the chemical structure of lipid A and dsRNA are discussed.Over a hundred years ago (Busch (Coley's toxin), which contained endotoxins 1866), a Viennese physician noted that amongst many other components, and regressions of some malignant tumours achieved some very striking results (cf. appeared to coincide with the occurrence review by Nauts, Fowler and Bogatko, of erysipelas. Coley, having made a 1953). The subject of the antitumour similar observation, deliberately treated action of endotoxins has attracte...

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Section: Discussionmentioning

confidence: 97%

mentioning

confidence: 99%

Similarities of the Anti-tumour Actions of Endotoxin, Lipid A and Double-stranded RNA

Parr¹,

Wheeler²,

Alexander³

1973

Br J Cancer

120

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“…I n contrast to the stable glucosamine glycosides, the phosphodiester linkages are very labile to mild acid hydrolysis [31-331. As a result of mild acid hydrolysis of phosphodiester linkages between or within polyglucosamine chains, the phosphate groups should remain linked to glucosamine especially as very stable monoesters in positions C, and C, [29,34]. Mild acid hydrolysis used for the isolation of lipid A may cleave the phosphodiester linkages and result in breakdown products of different sizes.…”

Section: Toxicity Studiesmentioning

confidence: 99%

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

Wober

Alaupovic

1971

European Journal of Biochemistry

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Conjugated protein isolated from the endotoxin complex of Xerratia marcescens 08 by lo/, acetic acid hydrolysis was characterized by analytical ultracentrifugation, electrophoretic and immunological properties and chemical analysis. The chemical composition of conjugated protein and other degraded fragments of endotoxin showed that all characteristic constituents of lipid A were present exclusively in conjugated protein. Successive treatment of conjugated protein with trypsin and pronase resulted in the isolation of corresponding tryptic and pronase cores which were characterized by an increased content of lipid A constituents. Lipid A was separated as an entity from the conjugated protein only after hydrolysis with 0.1 N hydrochloric acid. This result confirms our previous proposal that lipid A is covalently linked to the protein moiety in whole endotoxin of S. marcescens 08. "Conjugated" protein isolated by acetic acid hydrolysis and "simple" protein prepared by hot aqueous phenol treatment of whole endotoxin differ from one another not by the presence or absence of lipid A but rather by the presence of the entire lipid A moiety in the conjugated protein and only a fragment of lipid A in the simple protein.Conjugated protein and its tryptic and pronase cores were immunogenic and possessed a common antigenic determinant with simple protein and its corresponding cores. Toxicity studies revealed that both conjugated and simple proteins retained to different degrees the toxic properties of whole endotoxin. Sodium dodecyl sulfate had an enhancing rather than diminishing effect on the toxicity of endotoxin and its various fragments. The importance of lipid A as the toxic factor of endotoxins was established not only indirectly by determining the toxicity of each fragment containing lipid A, but also directly by demonstrating that lipid A dispersed in a Tris-dodecyl sulfate buffer displayed toxic properties similar to those of the lipopolysaccharide preparation.Morgan and Partridge [l --31 demonstrated that hydrolysis of whole endotoxin of Shigella dysenteriae by lo/, acetic acid resulted in the isolation of the so-called conjugated protein which may be converted into simple protein by treatment with either goo/,, phenol or 0.1 N sodium hydroxide. Because conjugated protein from Shigella pradysenteriae retained the typical toxic properties, Goebel and his coworkers [4,5] have referred to protein preparations isolated by lo/, acetic acid hydrolysis as toxic proteins. Since tryptic digestion of the intact endotoxin and of toxic protein did not destroy the toxic component, authors concluded that the toxin is either not a protein or that it represents a portion of the protein resistant to the action of proteolytic enzymes. The suggested existence of a toxic component other A preliminary report of this study was presented at the 158th National Meeting of the American Chemical Society, New York, September, 1969. than protein and polysaccharide marked the beginning not only of an extensive search for but also of a lastin...

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“…The bacteria were grown as described previously [6] and the lipopolysaccharides extracted with phenol-water [7].…”

Section: Abe-oacmentioning

confidence: 99%

Structural Investigations on the Core Polysaccharide of Salmonella typhimurium and the Mode of Attachment of the O-Specific Chains

1970

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Two lipopolysaccharides were analyzed comparatively : the lipopolysaccharide derived from a Salmonella typhimurium SR mutant, containing single repeating units linked to the core polysaccharide, and a lipopolysaccharide derived from a corresponding Ra mutant containing core polysaccharide only. Partial hydrolysis of these lipopolysaccharides led to the formation of a group of oligosaccharides occurring in both lipopolysaccharides. Their structural analysis provided information for the reconstruction of the hexose region of the core. From the SR lipopolysaccharide, four oligosaccharides were identified which were absent from the Ra lipopolysaccharide: (a) Man-al,4-rhamnose (derived from the repeating units) ; (b) Gal-j31,4-Glc ; (c) Gal-B1,4-(GlcNAc-a1,Z-)Glc ; (d) Gal-f?l,4-Glc-ccl,2-Gal. Oligosaccharides (b), (c), and (d) are derived from the linkage region between the repeating unit and the core. Methylation studies which were performed on the SR and Ra lipopolysaccharides led to the identification of the sugar linkages. On the basis of the present investigation, the partial structure of the SR lipopolysaccharide can be drawn as shown below, which confirms and extends previous results. The findings are discussed in regard to the biosynthesis and immunochemistry of SR and S form lipopolysaccharides. Abe-OAcGlcNAc (Glc) GalMan-ccl,4-Rha-~l,3-Gal-~l,4-Glc-al,2-Gal-ccl,3-Glc ------ Fig. 1). Glucose 11, therefore, represents a branching point ; it is substituted a t positions 2 and 4 [2], carrying N-acetylglucosamine as a small side chain (Fig. 1). The work described in this paper was performed with the aim of obtaining more information on the structure of Salmonella lipopolysaccharides, and, especially, on the linkage between 0-specific chains and the core. For this purpose, the lipopolysaccharides of two S. typhimurium mutants were compared :(a) The Ra mutant his 386, which is a deletion mutant defective in the rfb locus and which, therefore, produces a lipopolysaccharide containing, besides lipid A, only the complete core polysaccharide [3]. (b) The SR mutant SH777, which is defective in the rfc locus, and which produces a lipopolysaccharide containing the complete core, to which is attached one single 0-specific repeating unit (in contrast to the wild form lipopolysaccharide, which contains a sequence of repeating units linked to the core) [4].The analysis of oligosacchafide fragments, obtained by partial hydrolysis of the two lipopolysaccharides, as well as the results of methylation studies, confirmed the previously proposed structures of the core [5] and of the linkage region between the repeating unit and the core [2]. Furthermore, the results led to certain new details regarding sugar linkages in these lipopolysaccharides. It was found that in the SR lipopolysaccharide, the glucose I1 residue carries the N-acetylglucosamine residue a t position 2, and the terminal reducing galactose residue of the repeating unit a t position 4. Further-

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Biochemical Studies on Lipopolysaccharides of Salmonella R Mutants

Cited by 166 publications

References 41 publications

Similarities of the Anti-tumour Actions of Endotoxin, Lipid A and Double-stranded RNA

Similarities of the Anti-tumour Actions of Endotoxin, Lipid A and Double-stranded RNA

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

Structural Investigations on the Core Polysaccharide of Salmonella typhimurium and the Mode of Attachment of the O-Specific Chains

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