Development of the false-memory illusion.

The counterintuitive developmental trend in the Deese-Roediger-McDermott (DRM) illusion (that false-memory responses increase with age) was investigated in learning-disabled and nondisabled children from the 6- to 14-year-old age range. Fuzzy-trace theory predicts that because there are qualitative differences in how younger versus older children and disabled versus nondisabled children connect meaning information across the words on DRM lists, certain key effects that are observed in adult studies will be absent in young children and in learning-disabled children. Data on 6 such adult effects (list strength, recall inflation, delayed inflation, delayed stability, thematic intrusion, and true-false dissociation) were used to investigate this hypothesis, and the resulting data were consistent with prediction.

Structural variability and originality of the Bordetella endotoxins.

Structural studies of Bordetella endotoxins (LPSs) have revealed remarkable differences: (i) between their LPSs and those of other bacterial pathogens; (ii) among the LPSs of the seven identified Bordetella species; and (iii) among the LPSs of some Bordetella strains. The lipid As have the "classical" bisphosphorylated diglucosamine backbone but tend to have fewer and species-specific fatty acid components compared to those of other genera. Nevertheless, three strains of B. bronchiseptica have at least three different fatty acid distributions; however, the recently identified B. hinzii and B. trematum LPSs had identical lipid A structures. The B. pertussis core is a dodecasaccharide multi-branched structure bearing amino and carboxylic groups. Another unusual feature is the presence of free amino sugars in the central core region and a complex distal trisaccharide unit containing five amino groups of which four are acetylated and one is methylated. The B. pertussis LPS does not have O-chains and that of B. trematum had only a single O-unit, unlike the LPSs of all the other species of the smooth-type. The O-chain-free cores of non-B. pertussis LPSs were always built on the B. pertussis core model but most were species-specifically incomplete. The LPS structures of three B. bronchiseptica strains were found to be different from each other. The O-chains of B. bronchiseptica and B. parapertussis were almost identical and had some features in common with B. hinzii O-chain. Serological analyses are consistent with the determined LPS structures.

Structure of the Bordetella pertussis 1414 endotoxin.

The endotoxin (lipopolysaccharide) of Bordetella pertussis, the agent of whooping cough, consists of a lipid A linked to a highly branched dodecasaccharide containing several acid and amino sugars. The elucidation of the polysaccharide structure was accomplished by first analyzing the structures of fragments obtained by hydrolysis and nitrous deamination and then piecing the fragments together. The fine structure of the antigenic distal pentasaccharide, presented here, was determined by chemical analyses as well as by high-resolution nuclear magnetic resonance and mass spectrometry. The complete structure was reconstituted and confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The following structure was derived from the combined experimental data:The detailed structure combined with previously reported serological data now allows the synthesis of its epitopes for potential vaccines.

Novel variation of lipid A structures in strains of different Yersinia species.

The Yersinia genus includes human and animal pathogens (plague, enterocolitis). The fine structures of the endotoxin lipids A of seven strains of Yersinia enterocolitica, Yersinia ruckeri and Yersinia pestis were determined and compared using mass spectrometry. These lipids differed in secondary acylation at C-2': this was dodecanoic acid (C(12)) for two strains of Y. enterocolitica and Y. ruckeri, tetradecanoic acid (C(14)) in two other Y. enterocolitica and hexadecenoic acid (C(16:1)) in Y. pestis. The enterocolitica lipids having a mass identical to that of Escherichia coli were found to be structurally different. The results supported the idea of a relation between membrane fluidity and environmental adaptability in Yersinia.

Characterization of the common antigenic lipopolysaccharide O-chains produced by Bordetella bronchiseptica and Bordetella parapertussis.

Representative strains of Bordetella bronchiseptica and B. parapertussis were found to produce smooth lipopolysaccharides (LPS) having identical antigenic O-polysaccharide components composed of linear unbranched polymers of 1,4-linked 2,3-diacetamido-2,3-dideoxy-alpha-L-galacto-pyranosyluronic acid residues. These LPSs differed from the LPS of B. pertussis which produces only rough-type LPS, devoid of O-polysaccharide. While B. bronchiseptica and B. parapertussis had chemically and immunologically identical O-polysaccharide structures, their core oligosaccharide components differed. The core oligosaccharide of B. parapertussis was chemically distinct from the core of B. bronchiseptica which appeared to be structurally and immunologically similar to a core oligosaccharide of B. pertussis.

Several uses for isobutyric Acid-ammonium hydroxide solvent in endotoxin analysis.

Many steps in the analysis of rough and semirough endotoxins were found to be facilitated by the use of isobutyric acid-ammonium hydroxide solvent.

252Cf plasma desorption mass spectrometry applied to the analysis of underivatized rough-type endotoxin preparations.

Plasma desorption mass spectrometry has recently been used with success to characterize native, underivatized Re- to Rc-type endotoxins in terms of their constituent lipopolysaccharides. The spectra give masses for the major molecular species of lipopolysaccharide present from which their probable compositions could be inferred using the overall composition determined by chemical analyses. Moreover, the relative intensities of the signals are roughly proportional to the abundance of their corresponding molecular species. Native Rc-, Rb-, and Ra-type enterobacterial endotoxins with 5-10 core sugar units have been rendered amenable to plasma-desorption mass spectrometry analysis by improvement in their solubility and the use of cellobiose as an additive. The spectra of four Salmonella and Escherichia endotoxin preparations demonstrated heterogeneity in acylation and phosphorylation. Since these sources of heterogeneity are critical for many biological activities, the spectra underline the need to define the composition of each preparation of endotoxin used in structure-function studies.

Comparison of lipids A of several Salmonella and Escherichia strains by 252Cf plasma desorption mass spectrometry.

Plasma desorption mass spectrometry has recently been used with success to characterize underivatized lipid A preparations: the major molecular species present give signals indicating their masses, from which probable compositions could be inferred by using the overall composition determined by chemical analyses. In the present study, plasma desorption mass spectrometry was used to compare structures in lipid A preparations isolated from several smooth and rough strains of Escherichia and Salmonella species. Preparations isolated from strains of both genera revealed considerable variation in degree of heterogeneity (number of fatty acids and presence or absence of hexadecanoic acid, phosphorylethanolamine, and aminoarabinose). Molecular species usually associated with Salmonella lipid A were found in preparations from Escherichia sp. In addition, preparations from three different batches of lipid A from one strain of Salmonella minnesota showed significant differences in composition. These results demonstrate that preparations used for biological and structural analyses should be defined in terms of their particular molecular constituents and that no generalizations based on analysis of a single preparation should be made.

Autolysis of a division mutant of Escherichia coli.

The pleiotropic character of the envC chain-forming mutant of Escherichia coli was found to include leakage of periplasmic enzymes and an abnormal tendency to autolyse. Washed suspensions of envC cells released murein fragments into the supernatant, and cell extracts from the mutant were richer than those of wild type in exo-beta-N-acetylglucosaminidase (187% of the wild-type value) and in soluble endopeptidase (256%) activities, but n-acetylmuramoylamidase, D,D-carboxypeptidase, L,Dj-carboxypeptidase and transglycosylase were not markedly different. When envC cells were grown in medium containing 0.58 M-sucrose, the chains broke up into rods, the L,D-carboxypeptidase activity increased about sixfold and D,Dj-carboxypeptidase 1B about twofold. It is suggested that L,D-carboxypeptidase is involved in septum splitting. The results suggest that the triggering of autolysis in E. coli envC depends on the alteration of envelope constituents rather than on an enhanced activity of murein hydrolases.

Is there a correlation between membrane phospholipid metabolism and cell division?

The specificity of lipid/protein interactions in bacterial membranes is based on the diversity and variability of whole phospholipid molecules and of their polar and non-polar moieties. Of particular interest is the synthesis and turnover of anionic phospholipids facilitating variations in the phosphatidylglycerol (PG)/diphosphatidylglycerol (DPG) ratio in correlation with cell growth and division. Accumulation of DPG increases the anionic character of the cell membrane which is known to hinder the septation process. Consequently, the decrease in PG/DPG observed in stationary and in penicillin- or UV-inhibited non-dividing bacteria can be explained in this manner. On the other hand, the presence of lysophosphatidylethanolamine observed in the chain-forming mutant envC PM61 of Escherichia coli, and resulting from an anomaly in the deacylation-reacylation cycle, appears to be an indirect consequence rather than the cause of septation deficiency. Variations in the ratio of saturated/unsaturated fatty acids in phospholipids are also correlated with the physiological state of the cell. Hence, the levels of fluid and non-fluid phospholipids, their polymorphism and interaction with membrane proteins appear to be implicated in cell wall modelling. Future work will show how all these correlations function on the genetic and molecular levels.

Analysis of unmodified endotoxin preparations by 252Cf plasma desorption mass spectrometry. Determination of molecular masses of the constituent native lipopolysaccharides.

Nine unmodified endotoxin preparations constituted of Re-, Rd-, and Rc-type lipopolysaccharides (2 to 5 glycoses), representing four species of enterobacteria were analyzed by 252Cf plasma desorption mass spectrometry. The constituent lipopolysaccharides were characterized by the ion pair: (M-H)- and its corresponding lipid fragment ion. The lipid fragment ion is produced by cleavage of the glycosidic bond of the 3-deoxy-D-manno-oct-2-ulosonic acid unit that substitutes O-6' of the glucosamin beta 1'-6glucosamine ("lipid A backbone") disaccharide of the lipid A moiety. These lipid fragment ions were identical to the (M-H)- ions seen in the spectra of homologous isolated lipid A preparations that were obtained by hydrolysis (pH 4.5, 100 degrees C) promoted by sodium dodecyl sulfate. Since the molecular components present in the endotoxin preparations analyzed are known, the ion pair (M-H)(-)-lipid fragment ion defines the molecular compositions of each individual lipopolysaccharide. Heterogeneity of the R-type endotoxin preparations analyzed was due almost exclusively to differing lipid A moieties. In three Salmonella minnesota 595 Re endotoxin preparations 10 different lipopolysaccharides were identified, only two of which were common to all three preparations. Of the nine lipopolysaccharides identified in two S. minnesota R7 endotoxin preparations, only two were present in both.

Distribution of lipid A species between long and short chain lipopolysaccharides isolated from Salmonella, Yersinia, and Escherichia as seen by 252Cf plasma desorption mass spectrometry.

Smooth type endotoxins of Salmonella, Yersinia, and Escherichia were fractionated into long and short chain lipopolysaccharides by silica gel chromatography. Lipid A was prepared from the fractions and analyzed by plasma desorption mass spectrometry. Both Yersinia and Salmonella endotoxins had a large proportion of aminoarabinose-containing lipopolysaccharide molecular species that were found to be concentrated in the long chain fraction. In the Escherichia endotoxin, hypoacylated lipopolysaccharides (lacking the tetradecanoate and one of the four hydroxytetradecanoates) were found mostly in the short chain fraction. Possible implications of these results for the lipopolysaccharide biosynthetic pathway and for studies on the influence of sugar chain length on the biological effects of endotoxins are discussed.

Structural characterization of the lipid A of Bordetella pertussis 1414 endotoxin.

The structure of Bordetella pertussis 1414 lipid A was investigated by classical methods of chemical analysis as well as plasma desorption mass spectrometry and fast atom bombardment mass spectrometry. Previous analysis showed that it contained a bisphosphorylated beta-(1-->6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkage. The presence of two main molecular species as seen by thin-layer chromatography was confirmed by plasma desorption mass spectrometry, in which the larger signal was attributable to a molecular ion containing two glucosamine, two phosphate, one tetradecanoic acid, one hydroxydecanoic acid, and three hydroxytetradecanoic acid residues. The ion of the smaller signal was lighter by the mass of one hydroxytetradecanoic acid residue (226 Da). The fatty acids in ester linkage were localized by chemical and fast atom bombardment mass spectrometry analysis. C-4 and C-6' hydroxyl groups of the backbone disaccharide were unsubstituted, the latter being the proposed attachment site for Kdo (3-deoxy-D-manno-octulosonic acid).

252Cf-plasma desorption mass spectrometry of unmodified lipid A: fragmentation patterns and localization of fatty acids.

The fragmentation patterns of synthetic Escherichia coli-type lipid A in plasma desorption mass spectrometry (PDMS) in both negative- and positive-ion modes were determined. Negative-ion spectra gave signals for the main diphosphorylated (intact) molecular species in their native proportions. Intact and alkaline-treated lipid A in this mode gave, for the glucosamine I moiety, easily identified signals that have not been previously reported in PDMS. These spectra gave enough information to localize the fatty acids. The procedure was verified with relatively homogeneous lipids A prepared from Salmonella minnesota R595 and Neisseria meningitidis lipopolysaccharides, and then applied to the previously unstudied Yersinia entercolitica O:11,24 lipid A to obtain the localization of its fatty acids. The possibility of obtaining this much information from two negative-ion spectra was attributed to the method, described earlier, of preparing the samples. In the positive-ion mode, about half of the E. coli ions containing diglucosamine appeared as monodephosphorylated species and/or as Na adducts. The intact glucosamine II moiety and its fragment ions gave signals none of which were Na adducts. With lipids A prepared from S. minnesota, N. meningitidis, and Y. enterocolitica, similar fragmentation patterns were observed. For lipid A structure determination, the positive-ion mode could play a confirmatory role. The above results and some of those reported by others were compared.

Variations in the carbohydrate regions of Bordetella pertussis lipopolysaccharides: electrophoretic, serological, and structural features.

Structural and immunological differences between the two components that are usually present in unequal quantities in Bordetella pertussis endotoxin preparations and are visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been studied by using strains 1414, A100, and 134, all in phase I. According to analyses by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer chromatography, the minor (8%) component of the endotoxin of strain 1414 (endotoxin 1414) appeared to be the predominating component of endotoxins A100 and 134. The masses of the carbohydrate chains isolated from endotoxin A100 and from the major component of endotoxin 1414 were 1,649 and 2,311 atomic mass units, respectively, as determined by 252Cf plasma desorption mass spectrometry. Comparison of the 1H nuclear magnetic resonance spectra of these chains established that four N-acetyl groups, an N-methyl group, and a 6-deoxy function, which characterize the nonreducing, distal trisaccharide of the glycose chain of strain 1414, were absent from that of strain A100. The antigenicity of endotoxin 1414, as measured by enzyme-linked immunosorbent assay, was higher than that of endotoxin A100, but fell below it when the glycose chain of endotoxin 1414 was deprived of seven sugars by treatment with nitrous acid. This observation suggests that at least three (distal, proximal, and intermediate) regions of the glycose chain of endotoxin 1414 carry antigenic determinants. One of these, located in the distal trisaccharide, is absent from both endotoxins A100 and 134.

252Cf-plasma desorption mass spectrometry analysis of lipids A obtained by an elimination reaction under mild conditions.

Lipids A are the hydrophobic domains of bacterial endotoxic lipopolysaccharides. Since they are responsible for most of the biological activities (both pathogenic and beneficial) of endotoxins, the characterization of their structure is crucial to the understanding of their mode of action. However, the inadequacy of existing methods for preparing certain lipids A has prompted us to devise a new, mild procedure which gives intact products. Use was made of the special features of 252Cf-plasma desorption mass spectrometry for forming molecular ions from these species and giving qualitative and quantitative information from the primary mass spectrum.

Structural characterization of the lipids A of three Bordetella bronchiseptica strains: variability of fatty acid substitution.

The structures of lipids A isolated from the lipopolysaccharides (LPSs; endotoxins) of three different pathogenic Bordetella bronchiseptica strains were investigated by chemical composition and methylation analysis, gas chromatography-mass spectrometry, nuclear magnetic resonance, and plasma desorption mass spectrometry (PDMS). The analyses revealed that the LPSs contain the classical lipid A bisphosphorylated beta-(1-->6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkages. Their structures differ from that of the lipid A of Bordetella pertussis endotoxin by the replacement of hydroxydecanoic acid on the C-3 position with hydroxydodecanoic acid or dodecanoic acid and the presence of variable amounts of hexadecanoic acid. The dodecanoic acid is the first nonhydroxylated fatty acid to be found directly linked to a lipid A glucosamine. The lipids A were heterogeneous and composed of one to three major and several minor molecular species. The fatty acids in ester linkage were localized by PDMS of chemically modified lipids A. B. pertussis lipids A are usually hypoacylated with respect to those of enterobacterial lipids A. However, one of the three B. bronchiseptica strains had a major hexaacylated molecular species. C-4 and C-6' hydroxyl groups of the backbone disaccharide were unsubstituted, the latter being the proposed attachment site of the polysaccharide. The structural variability seen in these three lipids A was unusual for a single species and may have consequences for the pathogenicity of this Bordetella species.