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Beginning at 16 weeks of age and continuing for 44 weeks, male C57BL/6J were fed either a control (CON) diet; a high-fat (HF) diet (60% unsaturated); or the HF diet containing an extract of unripe avocados (AvX) enriched in the 7-carbon sugar mannoheptulose (MH), designed to act as a glycolytic inhibitor (HF + MH). Compared to the CON diet, mice on the HF diet exhibited higher body weights; body fat; blood lipids; and leptin with reduced adiponectin levels, insulin sensitivity, VO2max, and falls from a rotarod. Mice on the HF + MH diet were completely protected against these changes in the absence of significant diet effects on food intake. Compared to the CON diet, oxidative stress was also increased by the HF diet indicated by higher levels of total reactive oxygen species, superoxide, and peroxynitrite measured in liver samples by electron paramagnetic resonance spectroscopy, whereas the HF + MH diet attenuated these changes. Compared to the CON, the HF diet increased signaling in the mechanistic target of the rapamycin (mTOR) pathway, and the addition of the MH-enriched AvX to this diet attenuated these changes. Beyond generating further interest in the health benefits of avocados, these results draw further new attention to the effects of this rare sugar, MH, as a botanical intervention for preventing obesity.

Plant origin, physicochemical parameters and composition were analysed to characterize the avocado honey (Persea americana Mill.) from Andalusia (Southern, Spain). Ashes content, total polyphenol, and electrical conductivity corresponded to these of a typical dark honey (>80 mm scale Pfund). Regarding mineral elements, K was predominant, followed by P and Mg. Antioxidant and invertase activities presented some desirable values. In the 20 analysed samples, 48 pollen types corresponding to 33 families were identified. Avocado pollen was found in high variability (13-58%). At least a 20% was suggested to guarantee the authentic avocado honey. Perseitol, sugar-alcohol identified only in avocado honey, fundamentally contributes to distinguish this kind of honey. The content varied between 0.31 and 1.56 g/100 g. The correlation between perseitol and avocado pollen was found to be significant. A minimum concentration of 0.30 g/100 g of perseitol is suggested to characterize the proposed monofloral avocado honey., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, we describe a reinvestigation of the lipopolysaccharide (LPS) structure of Helicobacter pylori strain Sydney (SS1) based on the NMR analysis of oligosaccharides obtained through the use of various degradations of the LPS as well as capillary electrophoresis-MS data. The results of the analysis indicated that the core region of a major H. pylori SS1 LPS glycoform consists of a backbone core oligosaccharide substituted at the D-glycero-D-manno-heptose (DD-Hep) residue by a linear chain composed of a trisaccharide fragment α-ddHep-3-α-L-Fuc-3-β-GlcNAc, as previously demonstrated for H. pylori strain 26695, further elongated by consecutively added α-Glc and β-Gal residues, and terminating in a novel linear chain consisting of 1,2-linked β-ribofuranosyl residues, where the last β-ribofuranosyl residue provides a point of attachment for the O-chain polysaccharide: [Formula: see text] where [2-β-Ribf-](n) is a short (three to five residues) oligomer of 1,2-linked β-ribofuranose (riban), and PS is a polysaccharide chain consisting of N-acetyllactosamine, substituted with α-Fuc to form Lewis (Le)-type structures. In addition to the previously identified LacNAc, Le(y) and Le(x) components, the O-chain polysaccharide of H. pylori SS1 LPS was found to contain a novel LacNAc unit carrying a phosphoethanolamine substituent at the O-6 position of β-GlcNAc residues., (© 2011 National Research Council Canada. Journal compilation © 2011 FEBS.)

The detection of antibodies specific to meningococcal lipo-oligosaccharides (LOSs; outer-core-->inner-core-->lipid A) in sera of patients convalescent from meningococcal infection suggests the potential use of LOS as a vaccine to combat pathogenic Neisseria spp. Removal of the outer-core region, which expresses glycans homologous to human blood-group antigens, is a required first-step in order to avoid undesirable immunological reactions following vaccination. To this end, we describe here the structural makeup of the LOS produced by serogroup B N. meningitidis NMB isogenic phosphoglucomutase (Pgm) mutant (NMB-R6). The dominant LOS types produced by NMB-R6 expressed a deep-truncated inner-core region, GlcNAc-(1-->2)-LDHepII-(1-->3)-LDHepI-(1-->5)-[Kdo-2-->4]-Kdo-->lipid A, with one PEA unit attached at either O-6 or O-7 of LDHepII, or with two simultaneously PEA moieties attached at O-3 and O-6 or O-3 and O-7 of the same unit. Unexpectedly, this mutation did not completely deactivate the production of Glc, as some LOS molecules were observed to carry Glc at O-4 of LDHepI and at O-3 of LDHepII. A glycoconjugate vaccine comprised of NMB-R6 LOSs is currently being evaluated in our laboratory.

The lipopolysaccharide of Pseudomonas aeruginosa O-12 was studied by strong alkaline and mild acid degradations and dephosphorylation followed by fractionation of the products by GPC and high-performance anion-exchange chromatography and analyses by ESI FT-MS and NMR spectroscopy. The structures of the lipopolysaccharide core and the O-polysaccharide repeating unit were elucidated and the site and the configuration of the linkage between the O-polysaccharide and the core established. The core was found to be randomly O-acetylated, most O-acetyl groups being located on the terminal rhamnose residue of the outer core region.

Structural analysis of the lipopolysaccharide (LPS) from nontypeable Haemophilus influenzae strain 981 has been achieved using NMR spectroscopy and ESI-MS on O-deacylated LPS and core oligosaccharide (OS) material as well as by ESI-MSn on permethylated dephosphorylated OS. A heterogeneous glycoform population was identified, resulting from the variable length of the OS branches attached to the glucose residue in the common structural element of H. influenzae LPS, l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-Glcxp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A. Notably, the O-6 position of the beta-d-Glcp residue was either substituted by PCho or the disaccharide branch beta-d-Galp-(1-->4)-d-alpha-d-Hepp, while the O-4 position was substituted by the globotetraose unit, beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp, or sequentially truncated versions thereof. This is the first time a branching sugar residue has been reported in the outer-core region of H. influenzae LPS. Additionally, a PEtn group was identified at O-3 of the distal heptose residue in the inner-core.

Structural elucidation of the lipopolysaccharide (LPS) from three nontypeable Haemophilus influenzae clinical isolates, 1209, 1207 and 1233 was achieved using NMR spectroscopy and ESI-MS on O-deacylated LPS and core oligosaccharide (OS) material as well as ESI-MS(n) on permethylated dephosphorylated OS. It was found that the organisms expressed a tremendous heterogeneous glycoform mixture resulting from the variable length of the OS chains attached to the common structural element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A. Notably, the O-6 position of the beta-D-Glcp residue could either be occupied by PCho or L-glycero-D-manno-heptose (L,D-Hep), which is a location for L,D-Hep that has not been seen previously in H. influenzae LPS. The outer-core L,D-Hep residue was further chain elongated at the O-6 position by the structural element beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp, or sequentially truncated versions thereof. The distal heptose residue in the inner-core was found to be chain elongated at O-2 by the globotetraose unit, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp, or sequentially truncated versions thereof. Investigation of LPS from an lpsA mutant of isolate 1233 and a lic1 mutant of isolate 1209 was also performed, which aside from confirming the functions of the gene products, simplified elucidation of the OS extending from the proximal heptose (the lpsA mutant), and showed that the organism exclusively expresses LPS glycoforms comprising the outer-core l,d-Hep residue when PCho is not expressed (the lic1 mutant).

Lipopolysaccharides (LPSs) from four strains of Ralstonia solanacearum belonging to biovar I (ICMP 6524, 8115, 5712, and 8169) were isolated and investigated. The structural components of the LPS molecule, such as lipid A, the core oligosaccharide, and O-specific polysaccharide (O-PS), were obtained after mild acid hydrolysis of the LPS preparations. In lipid A from all the LPS samples studied, 3-hydroxyhexadecanoic, 2-hydroxyhexadecanoic, tetradecanoic, and hexadecanoic fatty acids prevailed. The dominant monosaccharides of the core oligosaccharides of all of the strains studied were rhamnose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, and heptose. However, individual strains varied in the content of galactose, ribose, xylose, and arabinose. Three types of the O-PS structure were established, which differed in their configuration (alpha or beta), as well as in the type of the bond between glucosamine and rhamnose residues (1-->2 or 1-->3).

This paper reports the application of near-infrared (NIR) reflectance spectroscopy to determine the concentration in honey of perseitol, a sugar that is specific to avocado honey. Reference values for perseitol were obtained by high-performance liquid chromatography analysis in 109 honey samples. Although the average concentration of perseitol in honey samples was only 0.48%, accurate prediction equations were successfully developed. The regression model of modified partial least squares was superior to that of principal component regressions. Calibrations based on the first or second derivative of Log(1/R) were equally good (R(2) > 0.95). Using half of the samples for calibration and the second half for validation, the correlation between actual and predicted values of the second half was satisfactory (R(2) = 0.87), the slope did not differ from 1, bias was low (0.005%), and the standard error of prediction was relatively low (0.13%). It was concluded that NIRS analysis may be used to detect to what extent honeybees have harvested avocado nectar but not to authenticate avocado honey as unifloral.

Synthesis of the title trisaccharide was accomplished by sugar chain extension starting from the non-reducing terminus: coupling of Glcp NAc with LD-Hepp, then adding Glcp-OAll. An alternative route started from the reducing end: coupling of LD-Hepp with Glcp-OAll, then addition of Glcp NAc. In the synthesis of the title disaccharide a modification of the first approach was employed. The allyl glycosides were coupled with cysteamine, activated with thiophosgene and conjugated to bovine serum albumin (BSA). The neoglycoconjugates obtained were used in immunochemical studies of monoclonal and polyclonal antibodies directed against Escherichia coli K-12 lipopolysaccharide.

The O-specific polysaccharide was isolated from Yersinia mollaretii strain WS 42/89 and characterized. Studies of the partial hydrolysis and methylation products and the 1H and 13C NMR spectroscopy data enabled the following structure to be proposed for the repeating unit of the polysaccharide: -->2)-beta-D-Galp-(1-->3)-alpha-D-6dGulp-(1--> The same structure was ascribed to the O-specific polysaccharide from Yersinia enterocolitica serovar O:6.31. The difference between these two O-antigens from various Yersinia species is in the absence of D-glycero-D-manno-heptose residues in the structure of the lipopolysaccharide from Y. mollaretii.

Syntheses are described of the tetrasaccharide 2-(4-trifluoroacetamidophenyl)ethyl O-(beta-D-galactopyranosyl)-(1-->2)-O- (L-glycero-alpha-D-manno-heptopyranosyl)-(1-->2)-O-(L-glycero-alpha-D- manno-heptopyranosyl)-(1-->3)-L-glycero-alpha-D-manno-heptopyranoside (20) and the three trisaccharides 2-(4-trifluoroacetamidophenyl)ethyl O-(L-glycero-alpha-D-manno-heptopyranosyl)-(1-->2)-O-(L-glycero-alpha-D- manno-heptopyranosyl)-(1-->3)-L-glycero-alpha-D-manno-heptopyranoside (17), 2-(4-trifluoroacetamidophenyl)ethyl O-(beta-D-glucopyranosyl)-(1-->4)- O-(beta-D-glucopyranosyl)-(1-->4)-L-glycero-alpha-D-manno-heptopyrano side (5), and 2-(4-trifluoro-acetamidophenyl)ethyl O-(beta-D-galactopyranosyl)-(1-->4)-O-(beta-D-glucopyranosyl)-(1-->4)- L-glycero-alpha-D-manno-heptopyranoside (8), corresponding to structures found in the lipooligosaccharides of Haemophilus influenzae.

Chemical and serological studies of LPS from Vibrio cholerae O76 (O76) were performed. The LPS of O76 contained D-glucose, D-galactose, L-glycero-D-manno-heptose, D-fructose, D-glucosamine, D-quinovosamine (2-amino-2,6-dideoxy-D-glucose) and L-perosamine (4-amino-4,6-dideoxy-L-mannopyranose). The sugar composition of the LPS from O76 was quite similar to that of LPS from V. cholerae O1 with the exception of the presence of a small amount of D-galactose in the LPS of O76. However, perosamine, a major sugar component of the LPS from O76, was in the L configuration in contrast to the D configuration of the perosamine in the LPS of V. cholerae O1. The L-perosamine was N-acylated with an (S)-(+)-2-hydroxypropionyl group in the LPS from O76. Structural analysis by NMR spectroscopy, as well as GC/MS, revealed that the O polysaccharide chain of the LPS from O76 was an alpha(1-->2)-linked homopolymer of N-[(S)-(+)-2-hydroxypropionyl]-L-perosamine. The serological cross-reactivity between the LPS of O76 and the LPS from other strains, such as V. cholerae O1 (Ogawa and Inaba O forms), Vibrio bio-serogroup 1875 (Original and Variant strains), V. cholerae O140 (Hakata) and Yersinia enterocolitica O9, was examined in passive haemolysis tests with sheep red blood cells that had been sensitized with LPS and antisera raised against whole cells of these bacteria. The latter six strains have in common the O antigen that includes Inaba antigen factor C, in addition to their own O-antigenic factors. Thus, they crossreact serologically. The O polysaccharide chains of the LPS of these six trains are known to consist exclusively of alpha(1-->2)-linked D-perosamine homopolymers and differences are found only among the N-acyl substituents. In passive haemolysis tests, the LPS of O76 did not cross-react serologically with any of the other LPS examined. Thus, the results obtained in this study support the hypothesis that Inaba antigen factor C, associated with the O antigens of these six strains, which include V. cholerae O1, is related substantially and exclusively to their alpha(1-->2)-linked homopolymers of N-acylated D-perosamine, and not to such homopolymers of N-acylated L-perosamine.

A lipopolysaccharide isolated from Coxiella burnetti strain Nine Mile in avirulent phase II contains in the lipid A proximal region D-mannose, D-glycero-D-manno-heptose, and 3-deoxy-D- manno-oct-2-ulosonic acid (Kdo) in the molar ratio 2:2:3. The primary structure 1 of the heptasaccharide was determined by glycose analysis, methylation analysis, ESI-MS, and FABMS. [sequence: see text]

Lipo-oligosaccharide from phenol-water extraction of cells of Campylobacter lari strain PC 637 was separated as a water-insoluble gel of low relative molecular mass (M(r)) from a water-soluble extracellular polysaccharide of high M(r). Structural investigations were performed on the lipo-oligosaccharide and the extracellular polysaccharide, variously using 1H, 13C, and 31P NMR spectroscopy, linkage analysis, and fast atom bombardment-mass spectrometry of permethylated derivatives of the glycans and their products of chemical and enzymic degradation. The following structures are proposed for the highly branched oligosaccharide region: [formula: see text] and for the tetraglycosyl phosphate repeating unit of the extracellular polysaccharide: [formula; see text]

Lipo-oligosaccharide (LOS) from phenol-water extraction of cells of the Campylobacter lari type strain (ATCC 35221) was separated as a water-insoluble gel of low relative molecular mass (M(r)) from a water-soluble extracellular polysaccharide of high M(r). Structural investigations were performed on the liberated oligosaccharide and the extracellular polysaccharide, variously using 1H, 13C, and 31P NMR spectroscopy, linkage analysis, and fast atom bombardment-mass spectrometry of permethylated derivatives of the glycans and their products of chemical and enzymic degradation. The following structures are proposed for the highly branched region of the LOS: [formula: see text] and for the tetraglycosyl phosphate repeating unit of the extracellular polysaccharide: [-(PO3-)-->3)-beta-D-GlcpNAc-(1-->2)-6-d-alpha-L-gul-Hepp -(1-->2)-3-d-beta-D-threo-Penp-(1-->3)-6-d-alpha-L-gul-He pp-]n

A branched-chain hexasaccharide containing 3-deoxy-D-manno-oct-2-ulosonic acid was released by detergent-promoted hydrolysis from Bordetella pertussis endotoxin preparations that were first dephosphorylated with aqueous HF and then treated with nitrous acid. Its structure (2) [Formula: See text] was determined by chemical and physical methods. This hexasaccharide is present in all four lipopolysaccharides that make up the B. pertussis strain 1414 (phase 1) endotoxin preparations analysed, and is situated near to the hydrophobic domains. An analogous structure reported previously (ref 7) is erroneous and should be disregarded.

The structure of the saccharide part of the lipopolysaccharide from Haemophilus influenzae strain AH1-3 (lic3+) has been investigated. The saccharide was obtained from the lipopolysaccharide by mild acid hydrolysis followed by high-performance anion-exchange chromatography, and isolated fractions were studied by methylation analysis, NMR spectroscopy, and FAB mass spectrometry. The major saccharide is a heptasaccharide with the following structure, [formula: see text] in which Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid and PEA is 2-aminoethyl phosphate. Hep is identified as L-glycero-D-manno-heptose. The absolute configuration of the phosphorylated heptose is tentative only.

A compositional sugar analysis was carried out on lipopolysaccharide (LPS) from Shigella sonnei form II in which a plasmid with cloned form I antigen genes had been introduced. The recipient form II strains contained galactose, glucose, heptose, glucosamine, and 2-keto-3-deoxyoctonic acid (KDO) (2: 3: 1: 2: 2) in its LPS, while the transformant form I LPS contained, besides these sugars, N-acetyl-L-altrosaminouronic acid as an additional sugar constituent, which is known to be one of the antigenic determinants of form I antigen.

The lipopolysaccharide (LPS) of the outer membrane of Caulobacter crescentus was purified and analyzed. Two distinct strains of the species, NA 1000 and CB2A, were examined; despite differences in other membrane-related polysaccharides, the two gave similar LPS composition profiles. The LPS was the equivalent of the rough LPS described for other bacteria in that it lacked the ladder of polysaccharide-containing species that results from addition of variable amounts of a repeated sequence of sugars, as detected by gel electrophoresis in smooth LPS strains. The purified LPS contained two definable regions: (i) an oligosaccharide region, consisting of an inner core of three residues of 2-keto-3-deoxyoctonate, two residues of alpha-L-glycero-D-mannoheptose, and one alpha-D-glycero-D-mannoheptose unit and an outer core region containing one residue each of alpha-D-mannose, alpha-D-galactose, and alpha-D-glucose, with the glucose likely phosphorylated and (ii) a region equivalent to the lipid A region of the archetype, consisting primarily of an esterified fatty acid, 3-OH-dodecanoate. The lipid A-like region was resistant to conclusive analysis; in particular, although a variety of analytical methods were used, no amino sugars were detected, as is found in the lipid A of the LPS of most bacteria.

An antigenic surface polysaccharide produced by Eubacterium saburreum strain T18, isolated from human dental plaque, was purified from formamide extract of whole cells. Methylation analysis, Smith degradation, optical rotation data and nuclear magnetic resonance spectra demonstrated that the purified antigen was a homopolysaccharide composed of D-glycero-D-galacto-heptose (Hep.) residues. The structure of the repeating unit in the polysaccharide was: -[----6)-[alpha-Hep.furanosyl-(1----4)]-beta-Hep.pyranosyl- (1----6)-[alpha-Hep.furanosyl-(1----2), alpha-Hep.furanosyl-(1----4)]-beta- Hep.pyranosyl-(1-)4----6)-beta-Hep.pyranosyl-(1----. No heptose residues were acetylated. Immunodiffusion reactions in agar gel suggested that the immunodeterminant of the antigenic polysaccharide was D-glycero-D-galacto-heptofuranosyl residues as branched nonreducing terminals.

The lipopolysaccharides of Actinobacillus actinomycetemcomitans strain Y4 and a human clinical isolate PO 1021-7 were examined by SDS/PAGE, deoxycholate/PAGE and mass spectrometry. PAGE analysis revealed an electrophoretic pattern similar to the SR-type lipopolysaccharide (LPS) of Salmonella. Deoxycholate/PAGE indicated the LPS of A. actinomycetemcomitans to consist of short sugar chains. Chemical analysis revealed the presence of thiobarbituric-acid-positive material (3-deoxy-D-manno-octulosonic acid equivalents) and four neutral sugars: glucose, galactose, D-glycero-D-manno-heptose and L-glycero-D-manno-heptose. Phosphate, glucosamine, glycine, and the fatty acids, 3-hydroxymyristic acid, myristic acid and palmitic acid, comprised the remainder of the molecule. The structure of the free lipid A revealed it to consist of a 1,6-glucosamine disaccharide esterified at C4' by a phosphomonoester. The hydroxyl group at C3 and the amide group of the non-reducing glucosamine were both acylated by 3-myristoylmyristic acid; analogous sites on the reducing glucosamine were acylated by 3-hydroxymyristic acid. Hydroxyl groups at C4 and C6' in the free lipid A were unsubstituted, with C6 being the proposed attachment site of the polysaccharide moiety. Chemical analysis revealed the presence of glycine in the intact LPS; its exact location in the A. actinomycetemcomitans LPS is still to be determined. Both intact LPS and free lipid A were highly lethal to galactosamine-sensitized mice, comparable to that of Salmonella.

The disaccharide L-glycero-D-manno-heptosyl-D-glucose was isolated from the lipopolysaccharide (LPS) of Escherichia coli K-12 strain W3100 after partial hydrolysis with acid, and the structure was determined by methylation analysis, n.m.r. spectroscopy, and comparison with a synthetic standard. In addition, the oligosaccharides L,D-Hep-D-Glc-D-Glc and L,D-Hep-D-Glc-D-Glc-D-Glc were isolated, and their structures were established by g.l.c.-m.s. and methylation analysis. The results indicated that L-glycero-D-manno-heptose, a characteristic constituent of the inner core region, may also occur in the outer core region which, in E. coli, is generally composed of hexoses. A revised structure of the carbohydrate backbone of the hexose/heptose region of the LPS is given.

A heptose-deficient lipopolysaccharide strain of Escherichia coli O8, strain F515, was found to release portions of its outer membrane when cells were exposed to 10 mM citrate buffer (pH 2.75) for 30 min and subsequently exposed to 100 mM tris(hydroxymethyl)aminomethane buffer (pH 8.00). The outer membrane component release was found to be composed of protein, lipopolysaccharide, phospholipid (cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol), and alkaline phosphatase. The outer membrane component was released from the cell envelope in the absence of cell lysis, as no glucose-6-phosphate dehydrogenase activity or succinic dehydrogenase activity was detected. Morphologically, the outer membrane component appeared to consist of laminar fragments and vesicles which had an associated alkaline phosphatase activity.

1. A singly branched heptaose produced as a limit dextrin in the digest of beta-limit dextrin with liquefying alpha-amylase [EC 3.2.1.1] of Bacillus amyloliquefaciens was isolated in a paper chromatographically pure state. 2. Analysis using several enzymes revealed that the isolated branched dextrin was a mixture of six singly branched heptaoses with different ramifying points. 3. All the branched heptaoses contained a 62-alpha-maltosylmaltotriose moiety in their molecules, differing only in the mode of attachment of one maltose or two glucose residues by alpha-1,4-glucosidic bonds from this core dextrin. 4. The formation of various singly branched heptaoses (the present paper) and hexaoses (the previous paper) is discussed regarding the attack site specificity of the enzyme on beta-limit dextrin.

Studies of the lipopolysaccharide of Pseudomonas alcaligenes strain BR 1/2 were extended to the polysaccharide moiety. The crude polysaccharide, obtained by mild acid hydrolysis of the lipopolysaccharide, was fractionated by gel filtration. The major fraction was the phosphorylated polysaccharide, for which the approximate proportions of residues were; glucose (2), rhamnose (0.7), heptose (2-3), galactosamine (1), alanine (1), 3-deoxy-2-octulonic acid (1), phosphorus (5-6). The heptose was l-glycero-d-manno-heptose. The minor fractions from gel filtration contained free 3-deoxy-2-octulonic acid, P(i) and PP(i). The purified polysaccharide was studied by periodate oxidation, methylation analysis, partial hydrolysis, and dephosphorylation. All the rhamnose and part of the glucose and heptose occur as non-reducing terminal residues. Other glucose residues are 3-substituted, and most heptose residues are esterified with condensed phosphate residues, possibly in the C-4 position. Free heptose and a heptosylglucose were isolated from a partial hydrolysate of the polysaccharide. The location of galactosamine in the polysaccharide was not established, but either the C-3 or C-4 position appears to be substituted and a linkage to alanine was indicated. In its composition, the polysaccharide from Ps. alcaligenes resembles core polysaccharides from other pseudomonads: no possible side-chain polysaccharide was detected.

Lipopolysaccharide prepared from cells of Yersinia (Pasteurella) pseudotuberculosis of serogroups I, II, III, IV, and V is known to contain the 3,6-dideoxyhexose (DDH) paratose, abequose, paratose, tyvelose, and ascarylose in its respective O-specific side chains. Lipopolysaccharides or lipid-free polysaccharides of all of the 10 known serogroups and subgroups were subjected to methylation analysis and determined as alditol acetates by gas-liquid chromatography and mass spectrometry. The results indicated that the O-specific side chains of nine serotypes are composed of oligosaccharide repeating units in the form of four alternative general structures in which a terminal DDH may vary. These structures are DDH [Formula: see text] 6-deoxy-d-manno-heptose [Formula: see text] d-galactose (serogroups IA, IIA, and IVB), DDH [Formula: see text] d-mannose [Formula: see text] l-fucose (serogroups IB and IIB), and two configurations similar to the latter except that the 4-position of l-fucose was either linked to the d-mannose residue (serogroups VA and VB) or to the DDH residue (serogroups III and IVA). In contrast, O-groups in lipopolysaccharide of the newly discovered serogroup VI contained the DDH colitose and 2-acetamido-2-deoxy-d-galactose. Accordingly, all five known types of DDH have now been detected in lipopolysaccharides of Y. pseudotuberculosis. The sugar 6-deoxy-d-manno-heptose, present in O-specific side chains of serogroups IA, IIA, and IVB, has not yet been reported to occur elsewhere in nature.

A cellular phenol-water extract of Acetobacter xylinum NRC 17007 was fractionated on Sepharose 4 B. The fraction eluting with the void volume consisted to about 95% of glycogen-like material. The lipopolysaccharide fraction was of lower molecular weight and had the following composition (%, w/w): Mannose, 42; glucose, 7; galactose, 3.8; heptose, 2; 2-keto-3-deoxy-octonate, 1.2; glucosamine, 3.3; phosphate, 4.5; total fatty acids, 3.9. Among the fatty acids, 3-hydroxy-tetradecanoic acid was present, and 2-hydroxy-hexadecanoic acid predominated.

With a single exception among 11 strains, each strain of Y. enterocolitica representing one of the six O-groups possesses, as judged by the sugar composition of its lipopolysaccharides, a particular chemotype. All 14 of the strains examined possessed glucosamine, KDO, two heptoses and glucose. In the most complicated chemotype 10 sugars were demonstrable. Of three Y. enterocolitica strains not included in the 6 O-groups, two had the same chemotype as the representatives of the O-groups IV and VI. Details can be seen in the two tables.

The protein composition of the outer membrane of Salmonella typhimurium has been analyzed by electrophoresis on slabs of sodium dodecyl sulfate-acrylamide gel. This powerful technique allows very high resolution of protein mixtures and has permitted the identification of multiple major protein components of the outer membrane; no evidence for a single major component of molecular weight 44,000 was obtained. These proteins were shown to be decreased in amount in mutants which have defective lipopolysaccharides. Mutants of an apparently new type were also found which contain decreased amounts of the proteins and the parent-like lipopolysaccharide, yet are resistant to a lipopolysaccharide-specific phage, C21. Several outer membrane proteins are insoluble in sodium dodecyl sulfate unless heated at high temperature (above 70 C). A purification procedure based on this property is tentatively suggested.

In the preceding paper we presented a formula for the composition of lipopolysaccharides (LPS) from Escherichia coli K-12. This formula contains four regions defined from analyses of LPS from four key strains, the parent and mutants which had lost one, two, or three regions of their carbohydrates. Support for the formula was derived from the susceptibility of the key mutants to several bacteriophages. One of these, phage phi W, was found specific for strains which had lost region 4. In this paper we described inactivation in vitro of phage phi W and its host-range mutant phi 5, using LPS devoid of regions 2 to 4. The blocking of phi W was found to require about 0.15 M concentrations of monovalent cations and to be inhibited by low concentrations of calcium and magnesium ions. One particle of phage phi W required 2 times 10-16 g of LPS devoid of region 4 for stoichiometric inactivation. Phage phi 5 was blocked by both heptose-less LPS (devoid of regions 2 to 4) and glucose-less LPS (devoid of regions 3 to 4) but was unaffected by LPS devoid of region 4. LPS from a heptose-less mutant of Salmonella minnesota showed the same inactivation ability as did LPS from heptose-less strains of E. coli K-12. Lipid A was prepared from LPS containing all four regions. Such lipid A was found to inactivate phi 5, whereas both the polysaccharide moiety as well as the intact LPS were without effect. It is suggested that lipid A is part of the receptor site for phage phi 5.

Methylation analysis of the core oligosaccharide of the Proteus mirabilis mutant R4 (derived from serotype 028 was carried out in order to obtain information on the internal (glucose-heptose) region of the P. mirabilis R core. The isolated core oligosaccharide was composed of glucose, L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid (dOclA) and phosphorus in a molar ratio of about 1:2:1:1.4. It was methylated either directly or after dephosphorylation. To localize the position of the phosphate substituents, the permethylated product was dephosphorylated with hydrogen fluoride and the oligosaccharide obtained was remethylated using C2H3I. Location of phosphate at C-7 of the terminal heptose was shown by isolation of the sugar phosphate from partial hydrolysates and gas-liquid chromatography/mass spectrometry of the permethylated product. Combining the data of the methylation analysis with the data of an NMR study allows one to formulate the structure of the core oligosaccharide as folllows: (formula: see text).

Endotoxic lipopolysachharide (LPS) was obtained from phenol-water extraction of cell walls prepared from mass-cultivated Fusobacterium necrophorum. The LPS was relatively free of nucleic acids and low in protein, and constituted about 4% of the cell walls. Upon acid hydrolysis, some of the components detected were hexosamines (7.0%), neutral and reducing sugars (50.5%), heptose (6.4%), 2-keto-3-deoxyoctonate (0.8%), lipid A (21.0%), and phosphorus (1.7%). Under electron microscopy the LPS appeared mainly as ribbon-like trilaminar structures, and upon chemical treatment it displayed a behavior resembling that reported in certain enterobacterial LPS. The LPS was lethal to mice, 11-day-old chicken embryos, and rabbits. Endotoxicity in mice was enhanced at least 1,380-fold by the addition of 12.5 mug of actinomycin D. Induced tolerance to lethal effect of the endotoxin and rapidly acquired resistance to infection by F. necrophrum viable cells were also demonstrated in mice. The endotoxin produced both localized and generalized Shwartzman reactions as well as biphasic pyrogenic responses in rabbits. These results firmly establish the presence of a classical endotoxin in F. necrophorum, thus providing strong support to our recent suggestion that cell wall-associated components may contribute significantly to the pathogenicity of F. necrophorum.

Factors that may determine the variable resistance of urinary strains of Escherichia coli to the bactericidal activity of normal human serum have been analysed. No statistically significant difference was found in the amount of lipopolysaccharide (LPS) that could be extracted from serum-sensitive and serum-resistant strains by either the phenol-water or warm-saline techniques. The ratio of LPS O-side-chain sugars to core sugars was not found to be significantly greater in serum-resistant than in serum-sensitive strains. A sugar resembling D-glycero-D-mannoheptose was found in LPS from some of the strains; in one case the sugar was shown to be associated with the O-side chain moiety. Lipopolysaccharides from all but two of the strains contained the E. coli R1 core structure. No consistent difference was observed between serum-sensitive and serum-resistant strains in either the amount of acidic polysaccharide extracted or its red-cell agglutination-inhibiting activity; nor was a clear relationship found between sensitivity to serum and sensitivity to R-specific bacteriophages. It is concluded that no one mechanism of serum resistance explains the response to serum of the E. coli strains examined in this study.

The F6R rough mutants isolated from Shigella flexneri F6S, serotype 5b, and the FH rough mutants, derived from other serotypes of S. flexneri, were chemotyped according to the chemical analysis of their lipopolysaccharides. Further, the following stages of lipopolysaccharide core biosynthesis in S. flexneri have been established: --(KDO)3--heptose--heptose--glucose--galactose; the last three stages are: either --glucose--glucosamine--glucose, or --glucosamine--glucose--glucose. The results of the chemical study of the R lipopolysaccharides are compatible with the assumption of the existence of a similar core in all considered S. flexneri serotypes.

The composition of the cell envelope of a heptose-deficient lipopolysaccharide mutant of Escherichia coli, GR467, was studied after fractionation into its outer and cytoplasmic membrane components by means of sucrose density gradient centrifugation. The outer membrane of GR467 had a lower density than that of its parent strain, CR34. Analysis of the fractionated membranes of GR467 indicated that the phospholipid-to-protein ratio had increased 2.4-fold in the outer membrane. The ratio in the mutant cytoplasmic membrane was also increased, although to a lesser extent. By employing a third parameter, the lipid A content of the outer membrane, it was found that the observed phospholipid-to-protein change in the outer membrane was due predominantly to a decrease in the relative amount of protein. This decrease in protein was particularly significant, since it was concomitant with a 68% decrease in the lipid A recovered in the outer membrane of GR467 relative to the lipid A recovered in the outer membrane of CR34. Similar findings were observed in a second heptose-deficient mutant of E. coli, RC-59. The apparent protein deficiency in GR467 was further studied by subjecting solubilized envelope proteins to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that major envelope proteins which were localized in the outer membrane were greatly diminished in GR467. Two revertants of GR467 with the wild-type amounts of heptose had wild-type relative levels of protein in their outer membranes. A partial heptose revertant had a relative level of protein in its outer membrane between those of the mutant and wild type.

There is a common structure (core region) in the lipopolysaccharides of Rhodospirillum tenue. It is composed of a branched trisaccharide of L-glycero-D-mannoheptose (and of 2-keto-3-deoxyoctonate), as revealed by methylation analyses of degraded polysaccharides of four different R. tenue strains. The structure is similar or might even be identical to the inner core of enterobacterial O antigens. In addition, each of the four R. tenue lipopolysaccharides contains a strain-specific region that consists of heptose(s) (L-glycero-D-mannoheptose or D-glycero-D-mannoheptose or both) or hexoses. There is a partial substitution of the core region and the strain-specific region by phosphorus, showing microheterogeneity.

Extraction of mycelium or walls of Micropolyspora faeni with cold or hot aqueous phenol yielded a lipopolysaccharide consisting of lipid A, phosphate, galactose, arabinose, glucose, glucosamine, and a dideoxy sugar. Extraction with trichloroacetic acid (TCA) yielded an incomplete molecule lacking lipid A. Part of an O-chain was secreted into the culture medium. Phenol and TCA extracts gave three lines of precipitation with human serum from cases of farmer's lung disease, and one of these was given by the culture medium polysaccharide. Serologically-reactive sugars were arabinose, galactose and glucose. The lipopolysaccharide fixed on to red cells which agglutinated in the presence of specific antibody and lysed on the addition of complement. The lipopolysaccharide appeared to elicit mainly IgM antibodies in animals, but IgM and IgG antibodies in humans.

Lipopolysaccharides (LPS, O-antigens) of 12 strains of the photosynthetic bacterium Rhodopseudomonas gelatinosa were obtained by the phenol/chloroform/petroleum ether method, recommended for extracting lipophilic glycolipids of enterobacterial R-mutants. All R. gelatinosa LPS have essentially the same chemical composition. Similar to LPS of Salmonella R-mutants of chemotypes Rd1 and Rd2, the sole neutral sugar constituent is an aldoheptose. The heptose of R. gelatinosa LPS has the D-glycero-D-manno- configuration, in contrast to the L-glycero-D-mannoheptose of enterobacterial LPS. 2-Keto-3-deoxyoctonate forms the acid-labile linkage between the lipid moiety (lipid A) and the oligosaccharide moiety of R. gelatinosa LPS. Like enterobacterial lipid A, lipid A of this species contains phosphate and D-glucosamine as the sole amino sugar. The fatty acid spectrum conprises beta-hydroxycapric, lauric, and myristic acids. Beta-Hydroxymyristic acid, the typical fatty acid of enterobacterial LPS, is lacking. The R. gelatinosa LPS show O-antigenic acitivity; passive hemagglutinations with untreated or heat-treated (not well alkali-treated) LPS and antisera prepared against heat-killed cells yield high titers. According to the serological cross-reactions observed, the LPS of the 12 strains could be arranged into two different serotypes: serotype I comprising strains 29/1, 29/2, 25/2, and serotype II comprising strains 44/K/6, 3/1, IS/10, 39/2, Dr2, 2150, P8P9, K32, P18f3.1. No serological cross-reactions were observed between LPS of these two different serotypes in passive hemagglutinations.

The oligosaccharide moiety of the lipooligosaccharide of Haemophilus influenzae type b strain Eag was isolated from the lipid component by mild acid hydrolysis and purified by gel filtration. Fast atom bombardment-mass spectrometry indicated that the lipid-free oligosaccharide had a basic molecular weight of 1,768; polysaccharides comparable to high-molecular-weight O side chains were not found. Glucose, galactose, galactosamine, heptose, 3-deoxy-D-manno-2-octulosonic acid (KDO), ethanolamine, and phosphate were identified in the lipid-free oligosaccharide by colorimetric assays, gas chromatography-mass spectrometry, or an amino acid analyzer. The presence of KDO was not clearly established by a thiobarbituric acid assay or by growth inhibition by a diazaborine derivative thought to block KDO synthesis. However, the semicarbizide assay and gas chromatography-mass spectrometry confirmed the presence of KDO. Lectin precipitation by Eag lipooligosaccharide in gels indicated that beta-D-galactose was present and that some of this monosaccharide was a terminal, nonreducing residue linked to N-acetyl-D-galactosamine. The lipid-free oligosaccharide was antigenic and completely inhibited lipooligosaccharide antibody (predominantly immunoglobulin G [IgG] and IgM) in an enzyme-linked immunosorbent assay, whereas the solubilized lipid A moiety did not. H. influenzae type b lipid-free oligosaccharide differed from core oligosaccharide of Salmonella lipooligosaccharide by the presence of galactosamine and a smaller percentage of heptose and KDO.

The individual polar lipids produced by Psuedomonas vesicularis NCTC 10 900 during surface culture have been isolated. The major lipids are phosphatidylglycerol, a phosphatidyl-alpha-D-glucopyranosyldiacylglycerol, 1,2-di-O-acyl-3-O-alpha-D-glucopyranosylglycerol. 1,2-di-O-acyl-3-O-alpha-D-glucopyranuronosylglycerol, and 1,2-di-O-acyl-3-O[beta-D-glucopyranosyl-(1 yields 4)-alpha-D-glucopyranuronosyl]glycerol. These are also the major polar lipids of Pseudomonas diminuta. Additional lipids present in P. vesicularis are unidentified carotenoids and a novel lipid characterised as 1,2-di-O-acyl-3-O-alpha-D-glycero-D-glucoheptopyranosylglycerol. A cis-octadecenoic acid and hexadecanoic acid are the major fatty acids: C15 and C17 acids are significant minor components. The fatty methyl ester fractions derived from three of the lipids (most notably the glucosyldiacylglycerol) contained substantial amounts of a compound with chromatographic properties resembling those of an octadecenoic ester: the identity and origin or this compound remained uncertain.