Endotoxic Glycolipids Research Articles

Radioprotective Properties of Detoxified Lipid A from Salmonella minnesota R595

In the past, the toxicity of bacterial lipopolysaccharide (LPS) or its principal bioactive component, lipid A, has detracted from their potential use as radioprotectants. Recently, a relatively nontoxic monophosphoryl Lipid A (LAM) that retains many of the immunobiologic properties of LPS has been isolated from a polysaccharide deficient Re mutant strain of Salmonella minnesota (R595). The ability of the native endotoxic glycolipid (GL) from S. minnesota (R595) as well as diphosphoryl lipid A (LAD) and nontoxic monophosphoryl lipid A (LAM) derived from GL to protect LPS responsive (CD2F1 or C3H/HeN) and nonresponsive (C3H/HeJ) mice from 60Co gamma irradiation has been studied. Administration of GL, LAD, or LAM to CD2F1 or C3H/HeN mice (400 micrograms/kg) 24 h prior to exposure provided significant radioprotection. No protection was afforded to C3H/HeJ mice. Experiments were also conducted to determine the relative abilities of GL, LAD, and LAM to stimulate hematopoiesis as reflected by the endogenous spleen colony (E-CFU) assay. Protection was not correlated with the ability of these substances to increase E-CFUs or to induce colony-stimulating activity (CSA).

Structural requirements of endotoxic glycolipid for antitumor and toxic activity.

Endotoxic glycolipids extracted from the polysaccharide heptose-less Re mutant of Salmonella typhimurium were hydrolyzed with alkaline and acid reagents. Treatment with hydroxylamine caused the liberation of all O-ester linked fatty acids and resulted in abrogation of the toxicity (lethality to chick embryos) and ability to regress tumors (line-10 tumors in strain 2 guinea pigs). Treatment with dilute sodium hydroxide caused partial removal of O-ester linked fatty acids without loss of these activities. Toxicity and tumor-regressive potency were retained after removal of 2-keto-3-deoxyoctonate (KDO) by exposing the glycolipids to sodium acetate solution at pH 4.5. The majority of the glycolipids of the endotoxic extracts were rendered non-toxic but retained antitumor activity when hydrolyzed with boiling 0.1 N hydrochloric acid, which split KDO and glycosidic phosphate from the glycolipid molecules. Non-toxic glycolipid fractions possessing antitumor activity were separated from the acid hydrolysate by means of preparative thin layer chromatography. It was concluded that glycosidic bound phosphate and at least a portion of the fatty acids of the lipid A moiety are essential for toxicity, but that this phosphate is not an essential structural feature for tumor-regression activity.

Different structural requirements of endotoxic glycolipid for tumor regression and endotoxic activity

Endotoxic glycolipid extracted from the heptose-less mutant of Salmonella typhimurium was treated with alkali and acid reagents. The glycolipid freed of all O-ester linked fatty acids by hydroxylamine had lost tumor regression activity and toxicity, whereas a partial removal of O-ester linked fatty acids by mild alkali did not impair with these activities. The glycolipid retained both activities after removal of 2-keto-3-deoxyotonate by sodium acetate (pH 4.5) but was rendered nontoxic while retaining antitumor activity when hydrolyzed by 0.1N HCl whereby 2-keto-3-deoxyoctonate and glycosidic phosphate was split off the glycolipid molecule. Nontoxic and tumor regressive fractions were separated by means of preparative thin layer chromatography of glycolipid hydrolyzed by mild acid. Thus, it was concluded that glycosidic bound phosphate and at least a portion of fatty acids of the lipid A moiety were essential for toxicity, but that this phosphate is not essential for tumor regression activity.

Preparation and antitumor activity of nontoxic lipid A

Highly refined, disaggregated endotoxic glycolipids (B5) from heptose-less (Re) mutant Salmonella typhimurium quantitatively converted to nontoxic (lethality for chick embryos) and nonpyrogenic (fever in rabbits) lipid A by treatment with boiling 0.1 N HCl (B5-HC1). Nontoxic B5-HCl, like toxic B5, caused regression of line-10 tumors and elimination of lymph node metastasis in 27 of 32 (84%) syngeneic strain 2 guinea pigs at a dosage of 150 μg. At this dosage, toxic B5 led to a cure in 54 of 67 (81%) tumor-bearing animals. All cured animals rejected a second line-10 tumor cell transplant. This activity depended on combining the toxic or nontoxic endotoxins with mycobacterial trehalose mycolate (P3) and an essentially nontoxic peptide-containing side-fraction (ACP) recovered during the isolation of B5. In contrast to toxic B5 or endotoxins in general, nontoxic B5-HCl did not cause endotoxic shock when combined with adjuvant dipeptide (MDP) and injected IV into guinea pigs. Chemical analysis showed that the phosphate content of nontoxic B5-HCl was about one-half that observed in toxic B5 or in toxic KDO-free lipid A, which was obtained by treating toxic B5 with sodium acetate at pH 4.5 at 100° C (B5-pH 4.5). The molar ratio of glucosamine: phosphorus: fatty acids was 2:1:4 for nontoxic B5-HCl and was 2:2:4 for toxic B5-pH 4.5. These results demonstrate that endotoxic extracts could be selectively detoxified while retaining antitumor properties. Thus, nontoxic B5-HCl may be a potential candidate for immunotherapy of human cancer.

Endotoxic glycolipid as a potent depressor of the hepatic drug-metabolizing enzyme systems in mice.

Inhibitory effects of the endotoxic glycolipid from Salmonella minnesota R595 on hepatic drug-metabolizing enzyme activities in mice were investigated, and the depressor activity of the glycolipid in the enzyme systems was confirmed. Among degradation products of lipopolysaccharides tested, lipid A preparations derived from the mild acetic acid hydrolysates of lipopolysaccharides were the most active, but the lipid A fractions prepared from the hydrolysates with 1 N-HCl were almost inactive. A degraded polysaccharide fraction from E. coli lipopolysaccharide was inactive. The activities of the glycolipid and the lipid A preparation were markedly reduced by treatment with alkaline-hydroxylamine, mild alkali or hydrazine. The data showed that the lipid A moiety of the glycolipid may be responsible for the inhibitory activity on the hepatic drug-metabolizing enzyme systems.

Relationship of structure to function in bacterial endotoxins: serologically cross-reactive components and their effect on protection of mice against some gram-negative infections.

Rabbit antisera were prepared against the heptoseless Re mutants, Salmonella minnesota R595 and S. typhimurium SLI102, as well as against purified R595 glycolipid coated on autologous erythrocytes. The antisera cross-reacted with the endotoxic glycolipids extracted from Re mutants of various bacterial strains, including S. minnesota R595, S. typhimurium SLI102, Escherichia coli D3Im4, E. coli D2If2 and E. coli F515, as shown by passive haemagglutination and gel diffusion tests. The anti-Re sera also cross-reacted with the RESI preparations (a purified 'lipid A' fraction) from the endotoxic lipopolysaccharides of various heterologous smooth Gram-negative bacteria including Serratia marcescens. Psuedomonas fluorescens and E. coli 0127. However, the same antisera failed to protect mice against infection by Gram-negative bacteria such as Klebsiella pneumoniae type II, S. typhi 0901, P. aeruginosa 119 and E. coli. The results suggest that although the lipid moieties of the lipopolysaccharides in the cell wall of Gram-negative bacteria share cross-reactive immunodeterminant groups, these groups may not be accessible to antibody against them.

Relationship of structure to function in bacterial endotoxins. IX. Differences in the lipid moiety of endotoxic glycolipids

Chemical, immunochemical, chromatographic, and endotoxic properties of five chromatographically pure glycolipids were compared. The preparations were extracted by chloroform-methanol from three Escherichia coli, one Salmonella minnesota, and one S. typhimurium Re heptoseless mutant strains. The local Shwartzman skin assay, the nonspecific resistance-enhancing effect, and the Limulus assays could not distinguish among the five glycolipids, all five being active in all three assays. Significant differences could be seen when the tumor resistance-enhancing effect of the glycolipids in mice was compared with the nonspecific TA3-Ha murine mammary adenocarcinoma growing in ascites form. Even greater variation was observed in the capacity of the preparations to enhance the nonspecific resistance of mice to virulent S. typhi 0901 infections. The data show that the five glycolipids are quite dissimilar in their biological effects. Similarly, thin-layer chromatography and molecular ratio determinations showed that differences exist in the chemical structure of the glycolipids. Accordingly, we claim that not only the polysaccharide but the lipid moiety as well may vary in various gram-negative endotoxin preparations.

Relation of structure to function in bacterial O-antigens—VII. Endotoxicity of ‘lipid A’

The ‘lipid A’ precipitate obtained from endotoxic lipopolysaccharide (LPS) by acidic hydrolysis is highly heterogenous, it consists of more than 30 components as shown by chromatography. This preparation represents approximately 1 10 of the original endotoxic activity of the parent endotoxin. The crude ‘lipid A’ was separated into three major fractions: (1) ethyl acetate-soluble (ETAC); (2) chloroform-soluble (CHLO); and (3) insoluble residue (RESI). Investigating the biological activity of these and comparing them to the activity of the parent endotoxin as well as to the crude, nonfractionated ‘lipid A’ preparation, it was found that almost all the biological activity residing in the crude ‘lipid A’ preparation can be found in the RESI. Chemical analysis as well as biological studies of this fraction strongly suggested that it contains incompletely degraded endotoxin derivatives. The insolubility of RESI in dilute acids or in water is due to partial degradation of the hydrophilic polysaccharide moiety of the endotoxic lipopolysaccharide molecule. While this degredation rendered this endotoxin derivative less soluble in water, it was apparently not strong enough to completely abolish its biological activity. Further separation of the fraction RESI on preparative thin-layer chromatography provided a chromatographically homogeneous component which is apparently equally or more active than the parent endotoxin. Biological activities and molecular ratios of this subtance reveal some similarities between endotoxic glycolipids and this material. This substance represents a relatively acid resistance active fragment of the parent endotoxin. This study also compared various preparations, all called ‘lipid A’, and established chemical as well as biological differences among them. The more limited use of the term ‘lipid A’ and more accurate description of the various lipid-rich endotoxin derivatives is therefore recommended.

Direct determination of molar ratios of various chemical constituents in endotoxic glycolipids in silicic acid scrapings from thin-layer chromatographic plates

Analytical procedures are described for the determination of the ratios of nitrogen, phosphorus, hexosamine, carbohydrates, fatty acids and 2-keto-3-deoxyoctonate (KDO) in bacterial endotoxic glycolipids separated by thin-layer chromatography (TLC). The methods make it possible to carry out analyses in the presence of silicic acid to obtain the molar ratios of the chemical constituents of natural products. For some micro-determinations, such as nitrogen, phosphorus or KDO, scrapings from a single TLC plate are sufficient for carrying out the quantitative analyses.

Heterogeneity and biological activity of endotoxic glycolipid from Salmonella minnesota R595.

The endotoxic glycolipid of Salmonella minnesota R595 was extracted either by the phenol-water method (GL) or directly with chloroform-methanol (GL-DE). Both glycolipids were soluble in chloroform-methanol (4:1). The yield of GL-DE was higher than that of GL. Examination of both materials by thin-layer chromatography (TLC) and column chromatography demonstrated that GL and GL-DE are chromatographically similar and that both are heterogeneous. Four fractions isolated from GL-DE with preparative TLC showed equal endotoxic activities in Shwartzman skin reaction, Limulus lysate gelation activity, and chick-embryo lethality. Chemical analyses indicate that all four are glycolipid in nature. The chromatographic behavior and the similar molar ratios of chemical constituents in these four components suggest that they may be chromatographically different due to polymerism. More detailed studies of some biological parameters have been done with GL and GL-DE by comparison with an endotoxin obtained from a smooth strain of Serratia marcescens. It was found that glycolipid showed activities similar to or higher than those observed for endotoxin.

Site of action of lipid A on mitochondria.

Lipopolysaccharide (LPS) of a number of gram-negative bacteria affected mitochondrial respiration and phosphorylation when it was preincubated with the mitochondrial suspension. The structural part responsible for this activity of LPS is the lipid moiety (lipid A), because the lipid A prepared from either the LPS of Escherichia coli or the endotoxic glycolipid of a heptose-less mutant (R595) of Salmonella minnesota affected mitochondrial oxidative phosphorylation as did LPS, whereas the polysaccharide moiety was inactive. Preincubation of the mitochondrial suspension with lipid A resulted in (i) inhibition of respiration and accompanying phosphorylation in the presence of either succinate or a number of reduced nicotinamide adenine dinucleotide-linked substrates, (ii) decrease of respiratory control, (iii) inhibition of the transfer of electrons at coupling site II without decrease of efficiency of phosphorylation, and the uncoupling at coupling site III, and (iv) stimulation of adenosine triphosphatase and the inhibition of 2,4-dinitrophenol-induced adenosine triphosphatase.

Fate and effect of endotoxin derivtives in tumor-bearing mice.

Summary1. A toxic endotoxin, its nontoxic chemical derivative called endotoxoid, and endotoxic glycolipids were tested in tumor-hemorrhage and lethality assays in tumor-bearing mice. All three materials were active in the tumor-hemorrhage assay. This indicates that neither toxicity nor the presence of O-antigenic polysaccharides is necessary to induce tumor hemorrhage.2. No evidence of a direct action of endotoxin on tumors or of altered reticuloendothelial function could be obtained by the fluorescein antibody technique, which was used to detect the presence of endotoxin preparations in the different tissues.3. Basic amines such as putrescine, spermine, and cadaverine, found in incompletely purified endotoxin preparations, and vasoactive agents such as histamine and serotonin could not duplicate the hemorrhagic activity of the endotoxic materials.4. The tumor-bearing state increases the susceptibility of mice to the lethal action of all three materials.5. Various inbred mouse strains, with or without tum...

Adjuvancy of endotoxin from heptoseless Salmonella mutants.

Japanese Journal of MicrobiologyVolume 15, Issue 1 p. 102-104 NoteFree Access Adjuvancy of Endotoxin from Heptoseless Salmonella Mutants Masayasu Nakano, Masayasu Nakano Department of Microbiology, Keio University School of Medicine, TokyoSearch for more papers by this authorTadakatsu Shimamura, Tadakatsu Shimamura Department of Microbiology, Keio University School of Medicine, TokyoSearch for more papers by this authorKazuhisa Saito, Kazuhisa Saito Department of Microbiology, Keio University School of Medicine, TokyoSearch for more papers by this author Masayasu Nakano, Masayasu Nakano Department of Microbiology, Keio University School of Medicine, TokyoSearch for more papers by this authorTadakatsu Shimamura, Tadakatsu Shimamura Department of Microbiology, Keio University School of Medicine, TokyoSearch for more papers by this authorKazuhisa Saito, Kazuhisa Saito Department of Microbiology, Keio University School of Medicine, TokyoSearch for more papers by this author First published: January 1971 https://doi.org/10.1111/j.1348-0421.1971.tb00557.xCitations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1Kasai, N., and Nowotny, A. 1967. Endotoxic glycolipid from a heptoseless mutant of Salmonella minnesota. J. Bacteriol. 94: 1824– 1836. 2Kim, Y. B., and Watson, D. W. 1967. Biologically active endotoxins from Salmonella mutants deficient in O- and R-polysaccharides and heptose. J. Bacteriol. 94: 1320– 1326. 3Lüderitz, O., Staub, A. M., and Westphal, O. 1966. Immunochemistry of O and R antigens of Salmonella and related Enterobacteriaceae. Bacteriol. Rev. 30: 192– 255. 4Nakano, M. 1962. Mutants of Salmonella with unusually low toxicity for mice. Nature 196: 1118– 1119. 5Nakano, M., and Saito, K. 1969. Chemical components in the cell wall of Salmonella typhimurium affecting its virulence and immunogenicity in mice. Nature 222: 1085– 1086. 6Nakano, M., and Saito, K. 1970. Antibody formation in mice infected with Salmonella typhimurium by primary immunization with sheep erythrocytes or bacterial cells. Japan. J. Microbiol. 14: 73– 81. 7Neter, E. 1969. Endotoxins and the immune response. Current Topics Microbiol. Immunol. 47: 82– 124. 8Nikaido, H. 1967. Bacterial cell wall–Deep layers, p. 3– 30. In B. D. Davis, and L. Warren (eds), The specificity of cell surfaces, Prentice Hall Inc., Englewood Cliffs, New Jersey. 9Ushiba, D., Kitasato, B., Saito, K., Nakano, M., and Nomura, T. 1962. Selective inbreeding of mouse strains uniformly susceptible to mouse typhoid and experimental infections of those strains. I. Trial of establishing inbred strains with different time-to-death after infection with Salmonella enteritidis. Japan. J. Exp. Med. 32: 505– 517. 10Westphal, O., and Lüderitz, O. 1954. Chemische Erforschung von Lipopolysaccha-riden Gramnegativer Bakterien. Angew. Chem. 66: 407– 417. Citing Literature Volume15, Issue1January 1971Pages 102-104 ReferencesRelatedInformation