Insight into the Mechanism of Reduced IgG/IgE Binding Capacity in Ovalbumin as Induced by Glycation with Monose Epimers through Liquid Chromatography and High-Resolution Mass Spectrometry.

Ovalbumin (OVA) is one of the major food allergens in hen eggs. In this work, it was demonstrated that glycation with d-glucose and its epimers, including d-mannose, d-allose, d-galactose, and l-idose, could effectively attenuate the IgG/IgE binding of OVA, which was attributed to the covalent masking by sugars and to its structural changes. The glycation sites were determined, and their average degree of substitution was found using liquid chromatography coupled with high-resolution mass spectrometry. Fluctuations in OVA conformation were monitored by conventional spectrometry. Compared to those of OVA-Man and OVA-Glu, OVA-All, OVA-Gal, and OVA-Ido showed a higher glycation extent, and the alterations on their steric layouts were more drastic, suggesting that the configuration of hydroxyl groups at positions C-3, C-4, and C-5 in sugars might be important for the glycation reactivity; as such, their capabilities in binding with IgG/IgE decreased more significantly. Attempts were made to provide valuable information for in-depth understanding of the differences in biochemical functionality among epimeric sugars. These insights would be helpful for designing sweetened food products with a desirable level of safety.

Molecular characterization of a transmembrane C-type lectin receptor gene from ayu (Plecoglossus altivelis) and its effect on the recognition of different bacteria by monocytes/macrophages.

C-type lectin receptors (CTLRs) play vital roles in immune responses as pattern-recognition receptors (PRRs). In this study, we identified a novel C-type lectin receptor (PaCTLRC) gene from ayu, Plecoglossus altivelis. Predicted PaCTLRC is a single transmembrane receptor with a typical carbohydrate recognition domain (CRD) at its C-terminus. Sequence comparison and phylogenetic tree analysis showed that PaCTLRC was most closely related to Atlantic salmon (Salmo salar) CLRC, but was significantly different from two other ayu CTLRs, aCLR and PaCD209L. PaCTLRC transcript was detected in all tested tissues and cells, with high levels in the liver; and its expression was significantly altered upon Vibrio anguillarum infection. Refolded recombinant PaCTLRC (rPaCTLRC) agglutinated three types of Gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus and Streptococcus iniae) and four types of Gram-negative bacteria (Aeromonas hydrophila, Escherichia coli, V. anguillarum and Vibrio parahaemolyticus) in a Ca(2+)-dependent manner in vitro, and Gram-positive bacteria were shown to be biologically relevant ligands for PaCTLRC. rPaCTLRC bound to d-mannose, d-galactose, l-fucose, N-acetyl-d-glucosamine (GlcNAc), lipopolysaccharide (LPS) and peptidoglycan (PGN), exhibiting a relative binding strength to d-mannose and PGN. d-Mannose, l-fucose, GlcNAc, LPS and PGN could inhibit the agglutinating activity of rPaCTLRC, while d-galactose did not functioned. PaCTLRC neutralization using anti-PaCTLRC IgG resulted in the inhibition of phagocytosis by ayu monocytes/macrophages (MO/MΦ) of S. aureus but not of E. coli, and produced a consistently higher survival rate of S. aureus than that of E. coli. d-Mannose, LPS and PGN treatment had no significant influence on the phagocytosis of ayu MO/MΦ. These results suggest that PaCTLRC may serve as a Gram-positive bacteria-preferred PRR which is involved in pathogen recognition and signal transduction in ayu MO/MΦ.

A novel emulsion-type adjuvant containing CpG oligodeoxynucleotides enhances CD8+ T-cell-mediated anti-tumor immunity.

PELC is a novel emulsion-type adjuvant that contains the bioresorbable polymer poly (ethylene glycol)-block-poly (lactide-co-ε-caprolactone) (PEG-b-PLACL), Span®85 and squalene. To investigate whether PELC is able to enhance CTL responses of antigens for treating tumor, peptides or protein antigens derived from HPV16 E7 were formulated with PELC nanoparticles and CpG oligodeoxynucleotide. We identified that PELC formulation could delay the release of antigens in vitro and in vivo. We assessed the immunogenicity of an H-2D(b)-restricted CTL epitope RAHYNIVTF (RAH) formulated with PELC or PELC/CpG and investigated the ability of these formulations to promote tumor regression. Following a single-dose subcutaneous injection in mice, we found that the RAH peptide formulated with PELC/CpG (RAH/PELC/CpG) resulted in increased numbers of IFN-γ-secreting cells and RAH-specific CD8(+) T cells and an enhanced cytotoxic T cell response compared with RAH formulated with PELC or CpG alone. The tumor-bearing mice received a single-dose injection of RAH/PELC/CpG, which induced complete tumor regression. These results demonstrated that peptide antigen formulated with PELC/CpG nanoparticles is feasible for cancer immunotherapy.

Contact allergy to sorbitans: a follow-up study.

BACKGROUND: Sorbitan sesquioleate (SSO), an emulsifier in many corticosteroids, was previously found positive in 8.9% of 112 dermatitis patients. OBJECTIVE: The objective of this study was to present data on 24 of 591 dermatitis patients with reactions to SSO and/or sorbitan monooleate (SMO) on patch testing. METHODS: A retrospective chart review was conducted on 591 consecutive dermatitis patients patch tested from November 2008 to May 2010. In addition to being tested to a modified North American Contact Dermatitis Group standard series, all patients were tested to a cosmetic series. RESULTS: Of the 591 patients tested, 24 reacted to SSO and/or SMO (4.1%), 19 (3.2%) reacted to SSO alone, 1 (0.17%) to SMO alone, and 4 (0.68%) reacted to both. Of the 24 sorbitan-allergic patients, 2 (8.3%) reacted to any of 4 corticosteroid screening chemicals tested. CONCLUSIONS: In this follow-up study, 4.1% of 591 dermatitis patients reacted to SSO and/or SMO. Given the presence of SSO in many popular topical corticosteroid formulations, clinicians should consider allergy to sorbitans when patients do not improve with topical corticosteroid therapy.

Unique interplay between sugar and lipid in determining the antigenic potency of bacterial antigens for NKT cells.

Invariant natural killer T (iNKT) cells are an evolutionary conserved T cell population characterized by features of both the innate and adaptive immune response. Studies have shown that iNKT cells are required for protective responses to Gram-positive pathogens such as Streptococcus pneumoniae, and that these cells recognize bacterial diacylglycerol antigens presented by CD1d, a non-classical antigen-presenting molecule. The combination of a lipid backbone containing an unusual fatty acid, vaccenic acid, as well as a glucose sugar that is weaker or not stimulatory when linked to other lipids, is required for iNKT cell stimulation by these antigens. Here we have carried out structural and biophysical studies that illuminate the reasons for the stringent requirement for this unique combination. The data indicate that vaccenic acid bound to the CD1d groove orients the protruding glucose sugar for TCR recognition, and it allows for an additional hydrogen bond of the glucose with CD1d when in complex with the TCR. Furthermore, TCR binding causes an induced fit in both the sugar and CD1d, and we have identified the CD1d amino acids important for iNKT TCR recognition and the stability of the ternary complex. The studies show also how hydrogen bonds formed by the glucose sugar can account for the distinct binding kinetics of the TCR for this CD1d-glycolipid complex. Therefore, our studies illuminate the mechanism of glycolipid recognition for antigens from important pathogens.

Mycobacterium avium glycopeptidolipids require specific acetylation and methylation patterns for signaling through toll-like receptor 2.

Toll-like receptors (TLRs) recognize pathogen-associated molecules and play a vital role in promoting an immune response against invading microbes. TLR2, one of the key members of the TLR family, recognizes a wide variety of microbial products, including lipoproteins and lipopeptides, from a number of pathogens. Recent studies from our laboratory indicate that glycopeptidolipids (GPLs), a major surface component of Mycobacterium avium and other non-tuberculosis mycobacteria, are ligands for TLR2. However, the molecular requirements necessary for the GPL-TLR2 interaction were not defined in this report. In the present study we isolated different GPL species from M. avium, and using mass spectrometry and NMR analyses, characterized the molecular requirements of the GPL-TLR2 interaction. Interestingly, the extent of the respective acetylation and methylation of the 6-deoxytalose and rhamnose contained within the core GPL structure dictated whether the GPL signaled through TLR2. These experiments illustrate how subtle changes in a complex TLR2 ligand can alter its affinity for this important receptor, and suggest that M. avium could potentially modify its GPL structure to limit its interaction with TLR2.

Comparison of two antigens for demonstration of Trichinella spp. antibodies in blood and muscle fluid of foxes, pigs and wild boars.

For the surveillance of trichinellosis, the digestion method is reliable but also labour intensive. The serological methods for the detection of Trichinella-specific antibodies using ELISA offer a sensitive and relatively specific alternative. For serological studies, sera or plasma from blood samples are the most common source of antibodies, but although the concentration of antibodies is approximately 10-fold lower, muscle fluid can be a good alternative particularly for testing of wildlife samples. In the present study, an indirect ELISA technique was evaluated on both sera and muscle fluids from experimentally infected foxes, pigs, and wild boars using both excretory/secretory (E/S) antigens and a synthetic glycan antigen, beta-tyvelose. Although the synthetic antigen appears to be less sensitive than the E/S antigens, Trichinella-specific IgG antibodies were detected in both serum samples and muscle fluid samples from pigs, wild boars and foxes infected at levels which would be important for food safety or represent a significant reservoir for further transmission.

Comparison of synthetic tyvelose antigen with excretory-secretory antigen for the detection of trichinellosis in swine using enzyme-linked immunosorbent assay.

Two enzyme-linked immunosorbent assay (ELISA) systems, one using natural excretory-secretory (ES) antigens and the other a synthetic glycan antigen (3,6-dideoxy-D-arabinohexose [tyvelose, TY]), were evaluated for the serological diagnosis of trichinellosis in swine. Sensitivity was estimated using samples (n = 113) collected 3-21 wk PI from 15 experimentally infected pigs, and specificity was estimated using samples (n = 397) from a population of Trichinella spp.-free pigs. Results were analyzed using 2 cutoff values recommended in international guidelines (Office Internationale des Epizooties [OIE]) and by the optimal cutoff level as determined by receiver-operator characteristic (ROC) analysis. The ROC-optimized TY-ELISA consistently performed better than all other combinations. None of the combinations of test and cut-off detected infected pigs sooner than 35 days; however, the ROC-optimized TY-ELISA identified 8 of 15 pigs earlier than the ES-ELISA and detected 2 pigs missed by all other tests. At 49 days PI the sensitivity and specificity of the ROC-optimized TY-ELISA were 94.3 and 96.7%, respectively, as compared with the ROC-optimized ES-ELISA at 84.9 and 96.0%, respectively. The ROC-optimized TY-ELISA was 100% specific at OIE-recommended cut-offs. This study indicates that the TY-ELISA is as good or better than the ES-ELISA for the detection of trichinellosis in swine.

Heterogeneity and immunogenicity of the Trichinella TSL-1 antigen gp53.

This study investigates the heterogeneity and immunogenicity of the Trichinella TSL-1 antigen gp53. Western blotting analysis of several Trichinella isolates with the gp53-specific monoclonal antibodies (mAbs) US5 and US9, produced in Btkxid mice, revealed that gp53 from the species T. britovi, T. murrelli and genotype T8 had higher MW (60 kDa) than gp53 from T. spiralis, T. nelsoni and genotype T6 (53 kDa) and from T. nativa (55 kDa). mAb US5 reacted only with gp53 from T. spiralis. Experiments including immunoassays of gp53 binding by sera from T. spiralis-infected mice, in the presence of different potential inhibitors (recombinant gp53, US5, T. britovi-crude larval extract (CLE), and CLE N- and O-glycans), indicate (i) that gp53 from T. spiralis bears specific epitopes that induce antibody formation during infection; (ii) that the protein epitopes of gp53 are much more important (76 or 68% of total antibody reactivity in BALB/c and Swiss CD-1 mice, respectively) than the corresponding glycan epitopes including tyvelose (11 or 32% of total reactivity) for the induction of anti-gp53 circulating antibodies; and (iii) that the species-specific epitopes present on gp53 are differentially recognized in different mouse strains. Whereas in BALB/c mice US5- and non-US5-recognized species-specific epitopes on gp53 bind about 84% of circulating antibodies on day 80 post-infection, this percentage was only 38% in Swiss CD-1 mice. These data on the antigenicity of gp53 contrast with data for Trichinella CLE antigens, in that most circulating antibodies reactive with CLE antigens recognized tyvelose-containing epitopes (57% and 58% of circulating antibodies in BALB/c and Swiss CD-1 mice, respectively). Together these results demonstrate that gp53 is recognized during infection but is antigenically different from other Trichinella TSL-1 antigens.

A putative serine protease among the excretory-secretory glycoproteins of L1 Trichinella spiralis.

Trichinella spiralis first-stage larvae infect susceptible hosts by invading epithelial cells that line the small intestine. During this process the larva disgorges several glycoproteins that bear an unusual, highly antigenic sugar moiety, tyvelose (3,6-dideoxy arabinohexose). Monoclonal antibodies specific for tyvelose protect the intestine against infection, implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment in the intestinal epithelium. In order to investigate these glycoproteins at the molecular level, we first prepared monoclonal anti-peptide antibodies. The antibodies bind a family of glycoproteins that are present in excretory-secretory products of first-stage larvae and are delivered to epithelial cells during invasion by T. spiralis. The major species present in an affinity purified fraction of crude T. spiralis antigens were subjected to tryptic peptide digestion. De novo amino acid sequencing of the peptides using Q-TOF tandem mass spectrometry, in combination with database searches and antibody screening of an L1 cDNA library, showed that the glycoproteins are variably glycosylated homologues of the serine protease family.

Tyvelose and protective responses to the intestinal stages of Trichinella spiralis.

The unusual sugar tyvelose is the immunodominant portion of the major larval glycoprotein antigens of Trichinella spiralis, which play an important role in generating immunity against the intestinal stages of infection. The possibility that the tyvelose component itself may have a host- or parasite-protective role in the intestine was tested by following the outcome of challenge infections in mice primed and boosted with tyvelose-BSA, or in mice primed with tyvelose-BSA before boosting with larval antigen. Although antibody responses were raised against tyvelose there was no evidence of protective immunity against the intestinal stages, as assessed by total adult worm recovery or by size and fecundity of female worms in immunized mice. Equally, priming with tyvelose-BSA before boosting with larval antigen had no effect on the expression of immunity against a challenge infection. The predominant antibody isotype recorded in all immunized mice was IgG1, suggesting the induction of type 2 T cell responses, and this was confirmed by cytokine analysis, mesenteric node lymphocytes of all mice showing production of IL-5 but not IFN-gamma. Clearly immunization with tyvelose had no significant effect on T cell polarization. The data show that, with the experimental design employed, there was no evidence for a functional role of tyvelose in either host- or parasite-protection during the intestinal phase of infection.

The use of a synthetic antigen for the serological diagnosis of human trichinellosis.

Hosts infected with Trichinella produce antibodies specific for an epitope common to the TSL-1 family antigens. This epitope contained uncommon terminal 3, 6-dideoxy-D-arabinohexose (so called tyvelose) residues. The disaccharide moiety was synthesized and an immunodiagnostic assay was developed, which was specific and sensitive in swine trichinellosis. We aimed to verify the specificity and sensitivity of this immunodiagnostic test in human trichinellosis. 15 sera from normal subjects, 12 from patients with other parasitic diseases and 50 from trichinellosis patients were tested. Indirect enzyme linked immunosorbent assay (ELISA) for specific IgG and an amplified ELISA for specific IgE were performed using beta-tyvelose-GalNAc-bovine serum albumin (BSA) disaccharide conjugate or T. spiralis muscle larvae excretory/secretory (E/S) products, as antigens. Neither control sera nor other parasitic infection sera resulted positive both for IgG and IgE when synthetic or E/S antigens were used. In trichinellosis patient sera, specific IgG were present in 100% of cases, irrespective of the antigen used, but whereas specific IgE were detected in 78% using E/S antigens, a 100% positivity rate was obtained, using the beta-tyvelose-BSA conjugate.

Sensitive and rapid immunoassays for Salmonella enteritidis.

In order to develop more sensitive, specific, and rapid immunoassays to detect Salmonella enteritidis in food supplies, we have applied various approaches by using several different antibody preparations. Utilizing ELISA in both a plate and immunodot assay, we employed (i) a polycolonal rabbit antiserum to a boiled suspension of the organism; (ii) a monoclonal antibody to the cell surface of the bacterium; and (iii) mouse antisera to two oligosaccharides each containing the rare sugar tyvelose, and exhibited by S. enteritidis, a member of the group D salmonellae. We showed that the polyclonal antiserum and monoclonal antibody IG-10 to the cell surface could specifically detect from 10(2) to 10(3) organisms in a 10-microl sample in the plate and immunodot assay. Both assays are read in 4-5 hr. Further, in the mice immunized to the trisaccharide, (alpha-D-galactose-alpha-tyvelose-alpha-D-mannose), as well as those mice immunized to the tetrasaccharide, (alpha-D-galactose-alpha-tyvelose-alpha-D-mannose-alpha-L-rhamnose), specificity to tyvelose was determined by inhibition studies. The inhibitors of the antisera to the trisaccharide included the single sugar tyvelose conjugated to bovine serum albumin (BSA), a tetrasaccharide in which tyvelose is excluded, but contains alpha-D-galactose, alpha-ascarose, alpha-D-mannose, and alpha-L-rhamnose (conjugated to BSA), and others. The inhibition studies suggest that the mouse antisera are specific for tyvelose and also contain antibodies for mannose and rhamnose. The antibodies that have been made to the unique sugar tyvelose should improve the specificity in assays for S. enteritidis.

Antibodies to tyvelose exhibit multiple modes of interference with the epithelial niche of Trichinella spiralis.

Infection with the parasitic nematode Trichinella spiralis is initiated when the L1 larva invades host intestinal epithelial cells. Monoclonal antibodies specific for glycans on the larval surface and secreted glycoproteins protect the intestine against infection. Protective antibodies recognize tyvelose which caps the target glycan. In this study, we used an in vitro model of invasion to further examine the mechanism(s) by which tyvelose-specific antibodies protect epithelial cells against T. spiralis. Using cell lines that vary in susceptibility to invasion, we confirmed and clarified the results of our in vivo studies by documenting three modes of interference: exclusion of larvae from cells, encumbrance of larvae as they migrated within epithelial monolayers, and inhibition of parasite development. Excluded larvae bear cephalic caps (C. S. McVay et al., Infect. Immun. 66:1941-1945, 1998) of immune complexes that may physically block invasion or may interfere with sensory reception. Monovalent Fab fragments prepared from a tyvelose-specific antibody also excluded larvae from cells, demonstrating that antibody binding can inhibit the parasite in the absence of antigen aggregation and cap formation. In contrast, encumbered larvae caused extensive damage to the monolayer yet were not successful in establishing a niche, as reflected by their failure to molt. These results show that antibodies to tyvelose exhibit multiple modes of inhibitory activity, further implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment by T. spiralis.

Nuclear antigens in Trichinella spiralis infected muscle cells: nuclear extraction, compartmentalization and complex formation.

Infection of mammalian skeletal muscle cells by Trichinella spiralis induces a series of changes that include: reentry of the terminally differentiated host cell into the cell cycle; suspension of infected cells in apparent G2/M; and transcriptional inactivation of the differentiated skeletal muscle gene program. Cell cycle repositioning and genetic reprogramming are chronic characteristics of host cells that can remain infected for years. Nuclear antigens (NA, 79, 86 and 97 kDa) that localize to host cell nuclei have been detected with antibodies against T. spiralis proteins. Since NA may play a role in regulating the infected cell phenotype, their origin, nuclear compartmentalization, and biochemical properties were investigated. We show that a monoclonal antibody to a defined epitope of T. spiralis glycans binds these NA, which indicates the parasite origin of these proteins. NA were not extracted under conditions that solubilized chromatin from infected cell nuclei. In contrast, NA were coextracted with B lamins (nuclear envelope) by 4 M urea. Urea extraction was pH dependent (8.0), suggesting ionic interaction of NA in protein complexes. Nevertheless, confocal microscopy demonstrated colocalization of NA with host chromatin, and not B lamins. Nuclear protein complexes containing NA were observed under non-reducing conditions, and NA were readily cross-linked in isolated nuclei by succinimidyl protein conjugating reagents. The results establish methods to extract NA from infected cell nuclei for further biochemical analysis, establish the existence of nuclear protein complexes containing NA and demonstrate colocalization of NA with host chromatin. Collectively, the results provide a foundation from which to investigate the role of NA in regulating the T. spiralis infected skeletal muscle cell phenotype.

Terminal beta-linked tyvelose creates unique epitopes in Trichinella spiralis glycan antigens.

Indirect evidence that the immunodominant N-glycans of the parasite, Trichinella spiralis are capped by novel beta-linked 3,6-dideoxy-D-arabinohexopyranosyl residues (tyvelose, Tyv) was obtained from immunochemical assays employing monoclonal antibodies and synthetic oligosaccharides. Three of four previously characterized monoclonal antibodies generated from the lymphocytes of T.spiralis infected rats bind BSA glycoconjugates bearing the synthetic epitope beta-D-Tyvp(1-->3)-beta-D-GalNAcp but not to the corresponding alpha-D-Tyvp(1-->3)-beta-D-GalNAcp-BSA glycoconjugate. Monosaccharide and disaccharide glycoside inhibition data mirrors the results of the direct binding experiments. The branched tetrasaccharide beta-D-Tyv(1-->3)-beta-D-GalNAcp(1-->4)[alpha-L-Fucp(1 -->3)] beta-D-GalNAcp is the most active synthetic oligosaccharide inhibitor for all four monoclonal antibodies studied, while the corresponding alpha-D-Tyv containing tetrasaccharide and the core trisaccharide beta-D-GalNAcp(1-->4)[alpha-L-Fucp(1-->3)]beta-D-GlcNAcp+ ++ are inactive. The exceptional inhibitory activity of the disaccharide beta-D-Tyvp(1-->3)-beta-D-GalNAcp with one mAb (18H) compared to that of the branched tetrasaccharide beta-D-Tyvp(1-->3)-beta-D-GalNAcp(1-->4)[alpha-L-Fucp( 1-->3)]-beta-D-GlcNAcp is indicative of the presence of linear, nonfucosylated glycan epitopes (beta-D-Tyvp(1-->3)-beta-D-GalNAcp(1-->4) beta-D-GlcNAcp) that lack a fucose residue in one arm of the antigenic, tetra-antennary N-glycan. This observation supports earlier FAB-mass spectrometry evidence for the existence of tetra-antennary, core fucosylated glycans that lack a fucose residue on one of their antennae.

Testing with fragrance mix. Is the addition of sorbitan sesquioleate to the constituents useful?

In a multicentre study, the value of adding sorbitan sesquioleate (SSO) to the constituents of the 8% fragrance mix (FM) was investigated. In 7 centres, 709 consecutive patients were tested with 2 types of FM from different sources, its 8 constituents with 1% SSO, its 8 constituents without SSO, and 20% SSO. 5 patients (0.71%) reacted to the emulsifier SSO itself, read as definitely allergic on day 3/4. 53 patients reacted to either one of the mixes with an allergic type of reaction. When tested with the constituents without SSO, 41.5% showed an allergic reaction versus 54.7% with SSO. If both types of reactions were considered (allergic and irritant) 38.3% of 73 patients showed a positive "breakdown" result without SSO, versus 54.8% with SSO. The differences were statistically significant. Reactivity to FM constituents was changed in a specific pattern by addition of SSO--irritant reactions increased, particularly for cinnamic alcohol, eugenol, geraniol, oak moss and hydroxycitronellal, whereas others showed only a slight change. Allergic reactions were also increased by SSO, but the rank order of the top 3 sensitizers (isoeugenol, oak moss and eugenol) did not change. Cinnamic alcohol was the only constituent with decreased reactivity after addition of SSO. A positive history of fragrance sensitivity (HFS) was clearly associated with a positive allergic reaction to either the mix or 1 of its constituents (51% versus 28.6% with a negative HFS). Irritant reactions were linked to a negative HFS in a high proportion (64.3%).(ABSTRACT TRUNCATED AT 250 WORDS)

Hemagglutinins in fungus extracts and their blood group specificity.

A total of 833 fungi harvested from 1977 to 1994 were tested and 422 extracts (47.8%) produced hemagglutination of human red cells. The lectins in fungus extracts which showed blood-group-specific or related reactions were partially purified by ammonium sulfate precipitation and gel filtration on a Sephadex G-100 column. Anti-H-like agglutinins were found in extracts of Pleurocybella porrigens, Naematoloma sublateritium and Pholiota squarrosa. These extracts agglutinated strongly with human group O red cells and rather weakly with A and B cells. Anti-A agglutinins were found in extracts of Hohenbuehelia serotina, Paxillus panuoides, Melanoleuca melaleuca and Hygrophorus capreolarius. The extract of Clavulinopsis fusiformis contained anti-B agglutinin. The ABH reactivities of the extracts were cofirmed by an agglutination inhibition test with ABH secretor saliva and blood group substances from human gastric linings and by the destruction of inhibiting activity using blood-group-specific decomposing enzymes. L-Fucose was the most active inhibiting monosacharide of anti-H-like agglutinins. The reaction of anti-A agglutinins was strongly inhibited by N-acetyl-D-galactosamine. D-Galactose and raffinose and melibiose which contain alpha-galactosyl residues were potent inhibitors of C. fusiformis agglutinin.

Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP-L-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems.

The Yersinia enterocolitica O:3 lipopolysaccharide O-antigen is a homopolymer of 6-deoxy-L-altrose. The cloned rfb region was sequenced, and 10 open reading frames were identified. Transposon mutagenesis, deletion analysis and transcomplementation experiments showed that eight of the genes, organized into two operons, rfbABC and rfbDEFGH, are essential for O-antigen synthesis. Functional tandem promoters were identified upstream of both operons. Of the deduced polypeptides RfbA, RfbF and RfbG were similar to Salmonella proteins involved in the dTDP-L-rhamnose biosynthesis. Rhamnose and 6-deoxy-L-altrose are C3-epimers suggesting that analogous pathways function in their biosynthesis. RfbD and RfbE were similar to capsular polysaccharide export proteins, e.g. KpsM and KpsT of Escherichia coli. This and transposon mutagenesis showed that RfbD and RfbE function as O-antigen exporters.