Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite.

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

Characterization of O-antigen delivered by Generalized Modules for Membrane Antigens (GMMA) vaccine candidates against nontyphoidal Salmonella.

Invasive nontyphoidal Salmonella disease (iNTS) is a leading cause of death and morbidity in Africa. The most common pathogens are Salmonella enterica serovars Typhimurium and Enteritidis. The O-antigen portion of their lipopolysaccharide is a target of protective immunity and vaccines targeting O-antigen are currently in development. Here we investigate the use of Generalized Modules for Membrane Antigens (GMMA) as delivery system for S. Typhimurium and S. Enteritidis O-antigen. Gram-negative bacteria naturally shed outer membrane in a blebbing process. By deletion of the tolR gene, the level of shedding was greatly enhanced. Further genetic modifications were introduced into the GMMA-producing strains in order to reduce reactogenicity, by detoxifying the lipid A moiety of lipopolysaccharide. We found that genetic mutations can impact on expression of O-antigen chains. All S. Enteritidis GMMA characterized had an O-antigen to protein w/w ratio higher than 0.6, while the ratio was 0.7 for S. Typhimurium ΔtolR GMMA, but decreased to less than 0.1 when further mutations for lipid A detoxification were introduced. Changes were also observed in O-antigen chain length and level and/or position of O-acetylation. When tested in mice, the GMMA induced high levels of anti-O-antigen-specific IgG functional antibodies, despite variation in density and O-antigen structural modifications. In conclusion, simplicity of manufacturing process and low costs of production, coupled with encouraging immunogenicity data, make GMMA an attractive strategy to further investigate for the development of a vaccine against iNTS.

Structural analysis of the O-acetylated O-polysaccharide isolated from Salmonella paratyphi A and used for vaccine preparation.

Salmonella paratyphi A is increasingly recognized as a common cause of enteric fever cases and there are no licensed vaccines against this infection. Antibodies directed against the O-polysaccharide of the lipopolysaccharide of Salmonella are protective and conjugation of the O-polysaccharide to a carrier protein represents a promising strategy for vaccine development. O-Acetylation of S. paratyphi A O-polysaccharide is considered important for the immunogenicity of S. paratyphi A conjugate vaccines. Here, as part of a programme to produce a bivalent conjugate vaccine against both S. typhi and S. paratyphi A diseases, we have fully elucidated the O-polysaccharide structure of S. paratyphi A by use of HPLC-SEC, HPAEC-PAD/CD, GLC, GLC-MS, 1D and 2D-NMR spectroscopy. In particular, chemical and NMR studies identified the presence of O-acetyl groups on C-2 and C-3 of rhamnose in the lipopolysaccharide repeating unit, at variance with previous reports of O-acetylation at a single position. Moreover HR-MAS NMR analysis performed directly on bacterial pellets from several strains of S. paratyphi A also showed O-acetylation on C-2 and C-3 of rhamnose, thus this pattern is common and not an artefact from O-polysaccharide purification. Conjugation of the O-polysaccharide to the carrier protein had little impact on O-acetylation and therefore should not adversely affect the immunogenicity of the vaccine.

Activity of antimicrobial peptides in the presence of polysaccharides produced by pulmonary pathogens.

Antimicrobial peptides (AMPs) are secreted in the airway and contribute to initial defence against inhaled pathogens. Infections of the respiratory tract are a major cause of morbidity and mortality in preterm newborns and in patients with cystic fibrosis (CF). In this latter group, the state of chronic lung infection is due to the ability of bacteria to grow as mucoid biofilm, a condition characterised by overproduction and release of polysaccharides (PSs). In this study, we investigate the effect of PSs produced by lung pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae and members of the Burkholderia cepacia complex on the antibacterial activity of structurally different peptides. The AMPs tested in this study include the cathelicidin LL-37 and the beta-defensin hBD-3 from humans, both released at the alveolar level, as well as peptides from other mammals, i.e. SMAP-29, PG-1 and Bac7(1-35). Susceptibility assays, time killing and membrane permeabilization kinetics experiments were carried out to establish whether PSs produced by lung pathogens may be involved in the poor defence reaction of infected lungs and thus explain infection persistence. All the PSs investigated inhibited, albeit to a different extent, the antibacterial activity of the peptides tested, suggesting that their presence in the lungs of patients with CF may contribute to the decreased defence response of this district upon infection by PS-producing microorganisms. The results also show that inhibition of the antibacterial activity is not simply due to ionic interaction between the negatively charged PSs and the cationic AMPs, but it also involves other structural features of both interactors.

Divalent cation interactions with oligogalacturonides.

The conformational properties of high and low molecular weight galacturonides were investigated in relation to the ability of oligomers with degree of polymerization >10 to act as elicitors of plant defense mechanisms. Oligomers from polygalacturonate were obtained by means of enzymatic hydrolysis. Two fractions exhibiting high and low average degrees of polymerization were isolated by solvent fractionation and characterized by means of electrospray mass spectrometry. The conformational behaviors of the two fractions were investigated in the presence of different divalent cations using circular dichroism. Calcium, copper, and zinc ions were able to induce a conformational transition in both fractions. When in the presence of the high molecular weight fraction, copper and zinc ions were much more effective than calcium ions, whereas the efficiency was much reduced with low molecular weight oligomers.

Host-guest complex formation in cyclotrikis-(1-->6).

The possibility that cyclotrikis-(1-->6)-[alpha-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl] (CGM6) forms inclusion complexes, like cycloamyloses (cyclodextrins), was investigated by means of electrospray mass spectrometry and fluorescence spectroscopy. The complexing ability of both 1-anilinonaphthalene-8-sulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS), which were already used with cyclodextrins, was investigated. The former showed very little or no tendency to be complexed by CGM6, while the latter produced detectable adducts with CGM6. Fixed 90 degree angle light scattering experiments supported the findings obtained by molecular modelling calculations, which indicated a polar character for the CGM6 internal cavity. CGM6-TNS complexes were probably formed throughout interaction of the polar regions of the two molecules.

Structural study of the exopolysaccharide produced by a clinical isolate of Burkholderia cepacia.

The primary structure of the exopolysaccharide produced by a clinical isolate of the bacterium Burkholderia cepacia was studied by means of methylation analysis, selective degradation, NMR spectroscopy, and electrospray mass spectrometry. The resulting data showed that the parent repeating unit of the exopolysaccharide is a highly branched heptasaccharide with the following structure: Two acetyl groups are present per repeating unit, as noncarbohydrate substituents.

Studies on the primary structure of short polysaccharides using SEC MALDI mass spectroscopy.

The introduction of size-exclusion chromatography (SEC) analysis of polysaccharides prior to MALDI mass spectroscopy accounts for the determination of the molecular mass of the repeating unit when neutral homopolymers are investigated. In the case of natural polysaccharides characterised by more complicated structural features (presence of non-carbohydrate substituents, charged groups, etc.), this mass value usually is in agreement with more than one sugar composition. Therefore, it is not sufficient to give the correct monosaccharidic composition of the polysaccharide investigated. To solve this problem, MALDI spectra were recorded on the permethylated sample and post-source decay experiments were performed on precursor ions. In this way, the composition (in terms of Hex, HexNAc, etc.), size and sequence of the repeating unit were determined.

Molecular typing and exopolysaccharide biosynthesis of Burkholderia cepacia isolates from a Portuguese cystic fibrosis center.

This work describes the first epidemiological survey of Burkholderia cepacia involved in pulmonary infections among the Portuguese population with cystic fibrosis (CF) who attended the major CF treatment Center in Lisbon at Sta. Maria Hospital from 1995 to the end of 1997. The characterization of the genomic relatedness of the isolates was based on the analysis of their ribopatterns (with EcoRI) followed by construction of a ribotype-based phylogenetic tree. This study was complemented with macrorestriction fragment analysis by pulsed-field gel electrophoresis. After optimization of the solid growth medium, we found that exopolysaccharide (EPS) production by B. cepacia CF isolates is not as rare a phenomenon as was thought before; indeed, 70% of the isolates examined were EPS producers.

Exopolysaccharide-producing lactic acid bacteria strains from traditional Thai fermented foods: isolation, identification and exopolysaccharide characterization.

Lactic Acid Bacteria (LAB) isolated from various traditional Thai fermented foods were screened for exopolysaccharides (EPS) production. From 104 isolates, two rod-shaped and five coccal-shaped LAB were able to produce EPS from sucrose on solid media. However, only the cocci were capable of producing EPS in liquid media and these were identified as Pediococcus pentosaceus. Pediococcus pentosaceus strains AP-1 and AP-3 produced EPS in high yield. In liquid media containing sucrose as carbon source, the amount of EPS produced by AP-1 and AP-3 strains was 6.0 and 2.5 g/L, respectively. The isolated and purified EPSs were chemically characterized. On the basis of sugar composition, methylation analysis and nuclear magnetic resonance spectroscopy, both the EPSs were shown to belong to the same dextran class. In particular, both EPSs differed from linear dextran by branching through 3,6-di-Osubstituted alpha-D-glucopyranosyl residues. The EPS from P. pentosaceus AP-3 was characterized by a relatively higher degree of branching and by a higher molecular weight than that from P. pentosaceus AP-1.

Influence of substituents on the solution conformation of the exopolysaccharide produced by Pseudomonas 'gingeri' strain Pf9.

The influence of pyruvate ketals and acetyl groups on the conformational behaviour of the exopolysaccharide produced by Pseudomonas 'gingeri' strain Pf9 has been investigated experimentally through studies of intrinsic viscosity and circular dichroism experiments. A conformational variation was detected as a function of the ionic strength. Measurements carried out on the native polymer, as well as on both de-pyruvated and de-acetylated samples, suggested a critical role for the acetyl group on the solution conformation of the polysaccharide. Molecular mechanics calculations indicated the possibility of intramolecular hydrogen bonding between acetyl substituents on the mannose and the C(2)OH group of the preceding saccharidic unit. NMR linewidth measurements, carried out as a function of temperature, on the low molecular weight de-pyruvated sample indicated different polymeric backbone dynamics in aqueous solutions with respect to that observed in 0.3 M NaCl solutions.

Size determination of bacterial capsular oligosaccharides used to prepare conjugate vaccines.

We recently described the use of ion exchange chromatography for analysis and the industrial scale preparation of pools of oligosaccharides of intermediate chain length from polysaccharides of Haemophilus influenzae type b (Hib) and Neisseria meningitidis groups A and C. These negatively charged "sized" oligosaccharides are activated and conjugated to the carrier protein (CRM197) to prepare the corresponding glycoconjugate vaccines. Characterization and accurate determination of the degree of polymerization (DP) of the pool of oligosaccharides is essential for the consistent production of these conjugate vaccines. This paper describes the colorimetric assays used for determination of the average DP of the Hib and meningococcal oligosaccharides, and the qualification of these assays achieved by size characterization of the respective oligosaccharides by use of physicochemical methods, including liquid chromatography, mass spectrometry (ionspray) and NMR spectroscopy.

Structural determination of the acidic exopolysaccharide produced by a Pseudomonas sp. strain 1.15.

Pseudomonas strain 1.15 was isolated from a freshwater biofilm and shown to produce considerable amounts of an acidic polysaccharide which was investigated by methylation analysis, NMR spectroscopy and ionspray mass spectrometry (ISMS). The polysaccharide was depolymerised by a bacteriophage-associated endoglucosidase and by autohydrolysis, and the resulting oligosaccharides were investigated by NMR spectroscopy and mass spectrometry. The resulting data showed that the parent repeating unit of the 1.15 exopolysaccharide (EPS) is a branched hexasaccharide. The main chain is constituted of the trisaccharide -->4)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-beta-D-Glcp- (1--> and the side chain alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->is linked to O-3 of the first Fuc residue. The terminal non-reducing Gal carries a 1-carboxyethylidene acetal in the R configuration at the positions 4 and 6. Of the four different O-acetyl groups present in non-stoichiometric amounts, two were established to be on O-2 of the 3-linked Gal and on O-2 of the 4-linked Fuc.

Structural investigation of the exopolysaccharide produced by Pseudomonas flavescens strain B62--degradation by a fungal cellulase and isolation of the oligosaccharide repeating unit.

Pseudomonas flavescens strain B62 (NCPPB 3063) is a recently described bacterium isolated from walnut blight cankers. This strain has been designated as the type strain of a Pseudomonas rRNA group-I species. Strain B62 produced a mixture of two exopolysaccharides, differing in weight average relative molecular mass and composition. Only the most abundant exopolysaccharide (90% by mass), corresponding to the one with the lower molecular mass, was investigated by use of methylation analysis, partial acid hydrolysis, and NMR spectroscopy. The polysaccharide was depolymerised by the action of the cellulase produced by Penicillum funiculosum and the oligosaccharide obtained, corresponding to the repeating unit, was characterised by NMR spectroscopy and ion-spray mass spectrometry. The repeating unit of the B62 exopolysaccharide is [structure in text] where X is glucose (75%) or mannose (25%), and Lac is lactate. The O-acetyl groups are present only on 75% of the repeating units, and they are linked to the C6 of the hexose residues in non-stoichiometric amounts.

All-transglycolytic synthesis and characterization of sialyl(alpha2-3)galactosyl(beta1-4)xylosyl-p-nitrophenyl(beta1-), an oligosaccharide derivative related to glycosaminoglycan biosynthesis.

Beta-D-Xylopyranosides, such as p-nitrophenyl-beta-D-xylopyranoside (Xyl-Np) or 4-methylumbelliferyl-beta-D-xylopyranoside (Xyl-MeUmb), when added to the culture medium of human skin fibroblasts have previously been shown to produce some Np- or MeUmb-oligosaccharides related to the regulation of glycosaminoglycan biosynthesis. Among these oligosaccharide derivatives, we synthesized the trisaccharide derivative NeuAc(alpha2-3)Gal(beta1-4)Xyl-Np(beta1- as a potential inhibitor of human skin fibroblast glycosaminoglycan biosynthesis. This synthesis was achieved by sequential use of transglycosylating activities of Escherichia coli beta-galactosidase and Trypanosoma cruzi trans-sialidase. The structure of the oligosaccharide obtained was determined by HPLC, ion-spray mass spectrometry, and NMR.

The structure of the exopolysaccharide of Pseudomonas fluorescens strain H13.

An acidic exopolysaccharide was isolated from P. fluorescens strain H13. The structure of the polysaccharide repeating unit was determined using chemical methods and 1D and 2D NMR techniques. The repeating unit was characterized as a trisaccharide composed of D-glucose, 2-acetamido-2-deoxy-D-glucose and 4-O-acetyl-2-acetamido-2-deoxy-D-mannuronic acid.

Determination of the size and degree of acetyl substitution of oligosaccharides from Neisseria meningitidis group A by ionspray mass spectrometry.

The capsular polysaccharide produced by Neisseria meningitidis group A has the following structure: [formula: see text] [formula: see text] This polysaccharide was partially hydrolysed with acetic acid, and the oligomers obtained were separated by fast performance liquid chromatography. Six fractions were collected and characterised by ionspray mass spectrometry in the positive ion mode. This soft ionisation technique established the size of the obtained oligosaccharides and the degree of O-acetyl substitution for each fraction.

The structure of the acidic exopolysaccharide produced by Pseudomonas "gingeri" strain Pf9.

The structure of the acidic exopolysaccharide produced by the mushroom pathogen Pseudomonas "gingeri" strain Pf9, a bacterium which causes ginger blotch, was investigated by chemical analysis, mass spectrometry and 1D and 2D NMR spectroscopy. The polysaccharide consists of the linear trisaccharide repeating unit [formula: see text] where the cyclic pyruvic acetal groups at O-4 and O-6 of the mannopyranosyl residues have the S-configuration. Methylation analysis under neutral conditions and NMR data showed that the mannose residues are acetylated at O-2. This exopolysaccharide has the same structure as the E. coli K55 capsular polysaccharide and differs from the Klebsiella K5 capsular polysaccharide only in the position of acetylation (C-2 of the glucopyranose residue).

Identification of exopolysaccharides produced by fluorescent pseudomonads associated with commercial mushroom (Agaricus bisporus) production.

The acidic exopolysaccharides (EPSs) from 63 strains of mushroom production-associated fluorescent pseudomonads which were mucoid on Pseudomonas agar F medium (PAF) were isolated, partially purified, and characterized. The strains were originally isolated from discolored lesion which developed postharvest on mushroom (Agaricus bisporus) caps or from commercial lots of mushroom casing medium. An acidic galactoglucan, previously named marginalan, was produced by mucoid strains of the saprophyte Pseudomonas putida and the majority of mucoid strains of saprophytic P. fluorescens (biovars III and V) isolated from casing medium. One biovar II strain (J1) of P. fluorescens produced alginate, a copolymer of mannuronic and guluronic acids, and one strain (H13) produced an apparently unique EPS containing neutral and amino sugars. Of 10 strains of the pathogen "P. gingeri," the causal agent of mushroom ginger blotch, 8 gave mucoid growth on PAF. The "P. gingeri" EPS also was unique in containing both neutral sugar and glucuronic acid. Mucoid, weakly virulent strains of "P. reactans" produced either alginate or marginalan. All 10 strains of the pathogen P. tolaasii, the causal agent of brown blotch of mushrooms were nonnmucoid on PAF. Production of EPS by these 10 strains plus the 2 nonmucoid strains of "P. gingeri" also was negative on several additional solid media as well as in two broth media tested. The results support our previous studies indicating that fluorescent pseudomonads are a rich source of novel EPSs.