siRNA high throughput screening identifies regulators of chloropicrin and hydrogen fluoride injury in human corneal epithelial cell models.

Corneal injuries induced by various toxicants result in similar clinical presentations such as corneal opacity and neovascularization. Many studies suggest that several weeks post-exposure a convergence of the molecular mechanisms drives these progressive pathologies. However, chemical agents vary in toxicological properties, and early molecular responses are anticipated to be somewhat dissimilar for different toxicants. We chose 3120 targets from the Dharmacon Human Druggable genome to screen for chloropicrin (CP) and hydrogen fluoride (HF) corneal injury as we hypothesized that targets identified in vitro may be effective as therapeutic targets in future studies. Human immortalized corneal epithelial cells (SV40-HCEC) were used for screening. Cell viability and IL-8 were analyzed to down-select hits into validation studies, where multiplex cytokine analysis and high content analysis were performed to understand toxicant effect and target function. Some endpoints were also evaluated in a second human immortalized corneal epithelial cell line, TCEpi. Over 20 targets entered validation studies for CP and HF; of these, only three targets were shared: NR3C1, RELA, and KMT5A. These findings suggest that early molecular responses to different toxicants may be somewhat distinctive and present dissimilar targets for possible early intervention.

Metabolome and transcriptome analyses of plants grown in naturally attenuated soil after hydrogen fluoride exposure.

Accidental chemical leaks and illegal chemical discharges are a global environmental issue. In 2012, a hydrogen fluoride leak in Gumi, South Korea, killed several people and contaminated the environment. This leak also led to a significant decline in crop yield, even after the soil concentration of hydrogen fluoride decreased to below the standard level following natural attenuation. To determine the cause of this decreased plant productivity, we designed direct and indirect exposure tests by evaluating the metabolome, transcriptome, and phenome of the plants. In an indirect exposure test, soil metabolomics revealed downregulation of metabolites in vitamin B6, lipopolysaccharide, osmolyte, and exopolysaccharide metabolism. Next-generation sequencing of the plants showed that ABR1 and DREB1A were overexpressed in response to stress. Plant metabolomics demonstrated upregulation of folate biosynthesis and nicotinate and nicotinamide metabolism associated with detoxification of reactive oxygen species. These results demonstrate impaired metabolism of soil microbes and plants even after natural attenuation of hydrogen fluoride in soil. The novel chemical exposure testing used in this study can be applied to identify hidden damage to organisms after natural attenuation of chemicals in soil, as well as biomarkers for explaining the decline in yield of plants grown in soil near pollutant-emitting industrial facilities.

Toxicological response of Sprague Dawley rats from inhalation exposure to perfluorooctane sulfonyl fluoride (POSF).

Perfluorooctane sulfonyl fluoride (POSF) was a volatile starting material in the production of perfluorooctane sulfonate (PFOS), a stable surfactant that has been extensively studied due to its ubiquitous environmental distribution and slow clearance in humans. Because the inhalation toxicity of POSF on repeated exposure has not been previously reported, the current study evaluated the inhalation toxicity of POSF at 30, 100, and 300ppm (v/v) in rats for up to 13 weeks with a four-week recovery period. The extent of PFOS formation was also measured because POSF hydrolyzed to form PFOS. In addition, detailed urinalysis and examination of the urinary bladder were included to determine if factors associated with the development of bladder cancer were present. Exposure to POSF at 300ppm was associated with reduction in body weight-gain, necrosis of laryngeal cartilage, increased lung and bronchi weight with septal thickening, and changes in alveolar macrophages. The microscopic observations in larynx and lung are consistent with likely hydrolysis of POSF to form hydrogen fluoride (HF). Exposure to POSF at 100 and 300ppm was associated with increased relative liver weight, hepatocellular hypertrophy (except for females exposed to 100ppm POSF), and lowering of serum cholesterol (male only). After 13 weeks of exposure to 30, 100, or 300ppm POSF, serum PFOS concentration approximated 7, 35, or 100µg/mL, respectively. Approximately 0.1% of inhaled POSF was converted to PFOS. No changes indicative of bladder effects were observed in these rats exposed to POSF at any dose.

ATP-Dependent C-F Bond Cleavage Allows the Complete Degradation of 4-Fluoroaromatics without Oxygen.

UNLABELLED: Complete biodegradation of the abundant and persistent fluoroaromatics requires enzymatic cleavage of an arylic C-F bond, probably the most stable single bond of a biodegradable organic molecule. While in aerobic microorganisms defluorination of fluoroaromatics is initiated by oxygenases, arylic C-F bond cleavage has never been observed in the absence of oxygen. Here, an oxygen-independent enzymatic aryl fluoride bond cleavage is described during the complete degradation of 4-fluorobenzoate or 4-fluorotoluene to CO2 and HF in the denitrifying Thauera aromatica: the ATP-dependent defluorination of 4-fluorobenzoyl-coenzyme A (4-F-BzCoA) to benzoyl-coenzyme A (BzCoA) and HF, catalyzed by class I BzCoA reductase (BCR). Adaptation to growth with the fluoroaromatics was accomplished by the downregulation of a promiscuous benzoate-CoA ligase and the concomitant upregulation of 4-F-BzCoA-defluorinating/dearomatizing BCR on the transcriptional level. We propose an unprecedented mechanism for reductive arylic C-F bond cleavage via a Birch reduction-like mechanism resulting in a formal nucleophilic aromatic substitution. In the proposed anionic 4-fluorodienoyl-CoA transition state, fluoride elimination to BzCoA is favored over protonation to a fluorinated cyclic dienoyl-CoA. IMPORTANCE: Organofluorides are produced as pesticides, pharmaceuticals, and other chemicals and comprise approximately one quarter of all organic compounds in the pharmaceutical and agricultural sectors; they are considered a growing class of environmentally relevant persistent pollutants. Especially in the case of fluoroaromatics, biodegradation is hampered by the extreme stability of the arylic C-F bond. In aerobic microorganisms, degradation proceeds via oxygenase-dependent C-F bond cleavage reactions, whereas the enzymes involved in the degradation of fluoroaromatics at anoxic sites are unknown. Here we report a strategy for the complete biodegradation of a fluoroaromatic to CO2 and HF in a denitrifying bacterium via activation to a CoA ester, followed by oxygen-independent arylic C-F bond cleavage catalyzed by an ATP-dependent enzyme. This reaction, in conjunction with a transcriptional adaptation to fluorinated growth substrates, is essential for the anoxic biodegradation of 4-fluorobenzoate/4-F-toluene and probably other fluoroaromatics.

More Than Just Oligomannose: An N-glycomic Comparison of Penicillium Species.

N-glycosylation is an essential set of post-translational modifications of proteins; in the case of filamentous fungi, N-glycans are present on a range of secreted and cell wall proteins. In this study, we have compared the glycans released by peptide/N-glycosidase F from proteolysed cell pellets of three Penicillium species (P. dierckxii, P. nordicum and P. verrucosum that all belong to the Eurotiomycetes). Although the major structures are all within the range Hex(5-11)HexNAc(2) as shown by mass spectrometry, variations in reversed-phase chromatograms and MS/MS fragmentation patterns are indicative of differences in the actual structure. Hydrofluoric acid and mannosidase treatments revealed that the oligomannosidic glycans were not only in part modified with phosphoethanolamine residues and outer chain och1-dependent mannosylation, but that bisecting galactofuranose was present in a species-dependent manner. These data are the first to specifically show the modification of N-glycans in fungi with zwitterionic moieties. Furthermore, our results indicate that mere mass spectrometric screening is insufficient to reveal the subtly complex nature of N-glycosylation even within a single fungal genus.

Evaluation of the effect of skin cleaning procedures on the dermal absorption of chemicals.

To reduce the internal exposure, skin decontamination is the most important measure after dermal contact to chemicals. However, no harmonized skin cleaning procedure for experimental ex vivo studies is published. In our study, the impact of two skin cleaning techniques on dermal penetration kinetics and intradermal deposition of 1,4-dioxane, 5% hydrofluoric acid (HF, detected in terms of fluoride ions), and anisole was evaluated to develop a reliable ex vivo skin cleaning method using the diffusion cell technique. After exposure (duration: 3 min (HF); 1h (1,4-dioxane and anisole)) of excised human skin (n=6-8) decontamination was performed by (I) water-soaked cotton swabs or (II) direct application of water on the exposure area. The effect of skin cleaning was investigated by analysing the concentration time course of chemicals in the receptor fluid of diffusion cells and by determining the deposition in skin. Both skin cleaning procedures reduced the amount of fluoride in the skin compartments (p<0.05) and the receptor fluid (p<0.1). However, the effect of cleaning on the dermal absorption of the organic test compounds was not significant. The results demonstrate the suitability of the applied ex vivo protocol for investigating the effectiveness of skin cleaning measures following dermal exposure. In addition, data reveal that the determination of test compounds in both, skin compartments as well as receptor fluid as equivalent for the systemic uptake needs to be considered in studies assessing the effectiveness of skin decontamination procedures.

Theoretical study on HF elimination and aromatization mechanisms: a case of pyridoxal 5' phosphate-dependent enzyme.

Pyridoxal 5-phosphate (PLP), the phosphorylated and the oxidized form of vitamin B6 is an organic cofactor. PLP forms a Schiff base with the ϵ-amino group of a lysine residue of PLP-dependent enzymes. γ-Aminobutyric acid (GABA) aminotransferase is a PLP-dependent enzyme that degrades GABA to succinic semialdehyde, while reduction of GABA concentration in the brain causes convolution besides several neurological diseases. The fluorine-containing substrate analogues for the inactivation of the GABA-AT are synthesized extensively in cases where the inactivation mechanisms involve HF elimination. Although two proposed mechanisms are present for the HF elimination, the details of the base-induced HF elimination are not well identified. In this density functional theory (DFT) study, fluorine-containing substrate analogue, 5-amino-2-fluorocyclohex-3-enecarboxylic acid, is particularly chosen in order to explain the details of the HF elimination reactions. On the other hand, the experimental studies revealed that aromatization competes with Michael addition mechanism in the presence of 5-amino-2-fluorocyclohex-3-enecarboxylic acid. The results allowed us to draw a conclusion for the nature of HF elimination, besides the elucidation of the mechanism preference for the inactivation mechanism. Furthermore, the solvent phase calculations carried out in this study ensure that the proton transfer steps should be assisted either by a water molecule or a base for lower activation energy barriers.

Fluoride metabolism.

Knowledge of all aspects of fluoride metabolism is essential for comprehending the biological effects of this ion in humans as well as to drive the prevention (and treatment) of fluoride toxicity. Several aspects of fluoride metabolism - including gastric absorption, distribution and renal excretion - are pH-dependent because the coefficient of permeability of lipid bilayer membranes to hydrogen fluoride (HF) is 1 million times higher than that of F(-). This means that fluoride readily crosses cell membranes as HF, in response to a pH gradient between adjacent body fluid compartments. After ingestion, plasma fluoride levels increase rapidly due to the rapid absorption from the stomach, an event that is pH-dependent and distinguishes fluoride from other halogens and most other substances. The majority of fluoride not absorbed from the stomach will be absorbed from the small intestine. In this case, absorption is not pH-dependent. Fluoride not absorbed will be excreted in feces. Peak plasma fluoride concentrations are reached within 20-60 min following ingestion. The levels start declining thereafter due to two main reasons: uptake in calcified tissues and excretion in urine. Plasma fluoride levels are not homeostatically regulated and vary according to the levels of intake, deposition in hard tissues and excretion of fluoride. Many factors can modify the metabolism and effects of fluoride in the organism, such as chronic and acute acid-base disturbances, hematocrit, altitude, physical activity, circadian rhythm and hormones, nutritional status, diet, and genetic predisposition. These will be discussed in detail in this review.

Biosilicification of loricate choanoflagellate: organic composition of the nanotubular siliceous costal strips of Stephanoeca diplocostata.

Loricate choanoflagellates (unicellular, eukaryotic flagellates; phylum Choanozoa) synthesize a basket-like siliceous lorica reinforced by costal strips (diameter of approximately 100 nm and length of 3 µm). In the present study, the composition of these siliceous costal strips is described, using Stephanoeca diplocostata as a model. Analyses by energy-dispersive X-ray spectroscopy (EDX), coupled with transmission electron microscopy (TEM), indicate that the costal strips comprise inorganic and organic components. The organic, proteinaceous scaffold contained one major polypeptide of mass 14 kDa that reacted with wheat germ agglutinin. Polyclonal antibodies were raised that allowed mapping of the proteinaceous scaffold, the (glyco)proteins, within the costal strips. Subsequent in vitro studies revealed that the organic scaffold of the costal strips stimulates polycondensation of ortho-silicic acid in a concentration- and pH-dependent way. Taken together, the data gathered indicate that the siliceous costal strips are formed around a proteinaceous scaffold that supports and maintains biosilicification. A scheme is given that outlines that the organic template guides both the axial and the lateral growth of the strips.

Hydrogen bonding interactions in PN...HX complexes: DFT and ab initio studies of structure, properties and topology.

Spin-restricted DFT (X3LYP and B3LYP) and ab initio (MP2(fc) and CCSD(fc)) calculations in conjunction with the Aug-CC-pVDZ and Aug-CC-pVTZ basis sets were performed on a series of hydrogen bonded complexes PN...HX (X = F, Cl, Br) to examine the variations of their equilibrium gas phase structures, energetic stabilities, electronic properties, and vibrational characteristics in their electronic ground states. In all cases the complexes were predicted to be stable with respect to the constituent monomers. The interaction energy (Delta E) calculated using a super-molecular model is found to be in this order: PN...HF > PN...HCl > PN...HBr in the series examined. Analysis of various physically meaningful contributions arising from the Kitaura-Morokuma (KM) and reduced variational space self-consistent-field (RVS-SCF) energy decomposition procedures shows that the electrostatic energy has significant contribution to the over-all interaction energy. Dipole moment enhancement (Delta mu) was observed in these complexes expected of predominant dipole-dipole electrostatic interaction and was found to follow the trend PN...HF > PN...HCl > PN...HBr at the CCSD level. However, the DFT (X3LYP and B3LYP) and MP2 levels less accurately determined these values (in this order HF < HCl < HBr). Examination of the harmonic vibrational modes reveals that the PN and HX bands exhibit characteristic blue- and red shifts with concomitant bond contraction and elongation, respectively, on hydrogen bond formation. The topological or critical point (CP) analysis using the static quantum theory of atoms in molecules (QTAIM) of Bader was considered to classify and to gain further insight into the nature of interaction existing in the monomers PN and HX, and between them on H-bond formation. It is found from the analysis of the electron density rho ( c ), the Laplacian of electron charge density nabla(2)rho(c), and the total energy density (H ( c )) at the critical points between the interatomic regions that the interaction N...H is indeed electrostatic in origin (rho(c) > 0, nabla(2)rho(c) > 0 and H(c) > 0 at the BCP) whilst the bonds in PN (rho(c) > 0, nabla(2)rho(c) > 0 and H(c) < 0) and HX ((rho(c) > 0, nabla(2)rho(c) < 0 and H(c) < 0)) are predominantly covalent. A natural bond orbital (NBO) analysis of the second order perturbation energy lowering, E((2)), caused by charge transfer mechanism shows that the interaction N...H is n(N) --> BD*(HX) delocalization.

Lipoprotein lipase and hydrofluoric acid deactivate both bacterial lipoproteins and lipoteichoic acids, but platelet-activating factor-acetylhydrolase degrades only lipoteichoic acids.

To identify the Toll-like receptor 2 ligand critically involved in infections with gram-positive bacteria, lipoprotein lipase (LPL) or hydrogen peroxide (H(2)O(2)) is often used to selectively inactivate lipoproteins, and hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) is used to selectively inactivate lipoteichoic acid (LTA). However, the specificities of these chemical reactions are unknown. We investigated the reaction specificities by using two synthetic lipoproteins (Pam(3)CSK(4) and FSL-1) and LTAs from pneumococci and staphylococci. Changes in the structures of the two synthetic proteins and the LTAs were monitored by mass spectrometry, and biological activity changes were evaluated by measuring tumor necrosis factor alpha production by mouse macrophage cells (RAW 264.7) following stimulation. PAF-AH inactivated LTA without reducing the biological activities of Pam(3)CSK(4) and FSL-1. Mass spectroscopy confirmed that PAF-AH monodeacylated pneumococcal LTA but did not alter the structure of either Pam(3)CSK(4) or FSL-1. As expected, HF treatment reduced the biological activity of LTA by more than 80% and degraded LTA. HF treatment not only deacylated Pam(3)CSK(4) and FSL-1 but also reduced the activities of the lipoproteins by more than 60%. Treatment with LPL decreased the biological activities by more than 80%. LPL also removed an acyl chain from the LTA and reduced its activity. Our results indicate that treatment with 1% H(2)O(2) for 6 h at 37 degrees C inactivates Pam(3)CSK(4), FSL-1, and LTA by more than 80%. Although HF, LPL, and H(2)O(2) treatments degrade and inactivate both lipopeptides and LTA, PAF-AH selectively inactivated LTA with no effect on the biological and structural properties of the two lipopeptides. Also, the ability of PAF-AH to reduce the inflammatory activities of cell wall extracts from gram-positive bacteria suggests LTA to be essential in inflammatory responses to gram-positive bacteria.

Glycosylation pattern of brush border-associated glycoproteins in enterocyte-like cells: involvement of complex-type N-glycans in apical trafficking.

We have previously reported that galectin-4, a tandem repeat-type galectin, regulates the raft-dependent delivery of glycoproteins to the apical brush border membrane of enterocyte-like HT-29 cells. N-Acetyllactos-amine-containing glycans, known as galectin ligands, were found enriched in detergent-resistant membranes. Here, we analyzed the potential contribution of N- and/or O-glycans in this mechanism. Structural studies were carried out on the brush border membrane-enriched fraction using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and nano-ESI-QTOF-MS/MS. The pattern of N-glycans was very heterogeneous, with the presence of high mannose- and hybrid-type glycans as well as a multitude of complex-type glycans. In contrast, the pattern of O-glycans was very simple with the presence of two major core type 1 O-glycans, sialylated and bisialylated T-antigen structures [Neu5Acalpha2-3Galbeta1-3GalNAc-ol and Neu5Acalpha2- 3Galbeta1-3(Neu5Acalpha2-6)GalNAc-ol]. Thus, N-glycans rather than O-glycans contain the N-acetyllactosamine recognition signals for the lipid raft-based galectin-4-dependent apical delivery. In the presence of 1-deoxymannojirimycin, a drug which inhibits the generation of hybrid-type or complex type N-glycans, the extensively O-glycosylated mucin-like MUC1 glycoprotein was not delivered to the apical brush border but accumulated inside the cells. Altogether, our data demonstrate the crucial role of complex N-glycans in the galectin-4-dependent delivery of glycoproteins to the apical brush border membrane of enterocytic HT-29 cells.

Discriminative detection and enumeration of microbial life in marine subsurface sediments.

Detection and enumeration of microbial life in natural environments provide fundamental information about the extent of the biosphere on Earth. However, it has long been difficult to evaluate the abundance of microbial cells in sedimentary habitats because non-specific binding of fluorescent dye and/or auto-fluorescence from sediment particles strongly hampers the recognition of cell-derived signals. Here, we show a highly efficient and discriminative detection and enumeration technique for microbial cells in sediments using hydrofluoric acid (HF) treatment and automated fluorescent image analysis. Washing of sediment slurries with HF significantly reduced non-biological fluorescent signals such as amorphous silica and enhanced the efficiency of cell detachment from the particles. We found that cell-derived SYBR Green I signals can be distinguished from non-biological backgrounds by dividing green fluorescence (band-pass filter: 528/38 nm (center-wavelength/bandwidth)) by red (617/73 nm) per image. A newly developed automated microscope system could take a wide range of high-resolution image in a short time, and subsequently enumerate the accurate number of cell-derived signals by the calculation of green to red fluorescence signals per image. Using our technique, we evaluated the microbial population in deep marine sediments offshore Peru and Japan down to 365 m below the seafloor, which provided objective digital images as evidence for the quantification of the prevailing microbial life. Our method is hence useful to explore the extent of sub-seafloor life in the future scientific drilling, and moreover widely applicable in the study of microbial ecology.

N-glycans of the porcine nematode parasite Ascaris suum are modified with phosphorylcholine and core fucose residues.

In recent years, the glycoconjugates of many parasitic nematodes have attracted interest due to their immunogenic and immunomodulatory nature. Previous studies with the porcine roundworm parasite Ascaris suum have focused on its glycosphingolipids, which were found, in part, to be modified by phosphorylcholine. Using mass spectrometry and western blotting, we have now analyzed the peptide N-glycosidase A-released N-glycans of adults of this species. The presence of hybrid bi- and triantennary N-glycans, some modified by core alpha1,6-fucose and peripheral phosphorylcholine, was demonstrated by LC/electrospray ionization (ESI)-Q-TOF-MS/MS, as was the presence of paucimannosidic N-glycans, some of which carry core alpha1,3-fucose, and oligomannosidic oligosaccharides. Western blotting verified the presence of protein-bound phosphorylcholine and core alpha1,3-fucose, whereas glycosyltransferase assays showed the presence of core alpha1,6-fucosyltransferase and Lewis-type alpha1,3-fucosyltransferase activities. Although, the unusual tri- and tetrafucosylated glycans found in the model nematode Caenorhabditis elegans were not found, the vast majority of the N-glycans found in A. suum represent a subset of those found in C. elegans; thus, our data demonstrate that the latter is an interesting glycobiological model for parasitic nematodes.

Comparison of the acid combinations in microwave-assisted digestion of marine sediments for heavy metal analyses.

Sediments as a tool for monitoring contamination by heavy metals in the environment has long been considered. It is therefore a necessity to produce reliable data for such purposes. Microwave-assisted acid dissolution has proved to be a suitable method for digesting complex matrices, such as sediments. However, due to the infancy of the technique, the procedures are numerous and varied in both the reagents used and microwave conditions. In this study, the efficiency of two recommended acid mixtures, a HNO3-HF mixture and an aqua regia-HF mixture, under the same microwave digestion conditions were compared using certified reference materials. It was observed that the HNO3-HF mixture showed better efficiency than the aqua regia-HF mixture in most of the heavy metals analyzed in all certified reference materials used.

Active-site-mediated elimination of hydrogen fluoride from a fluorinated substrate analogue by isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is a non-haem iron oxidase that catalyses the formation of bicyclic isopenicillin N from delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV). In this study we report a novel activity for the iron of the IPNS active site, which behaves as a Lewis acid to catalyse the elimination of HF from the fluorinated substrate analogue, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-beta-fluorovaline (ACbetaFV). X-Ray crystallographic studies of IPNS crystals grown anaerobically with ACbetaFV reveal that the valinyl beta-fluorine is missing from the active site region, and suggest the presence of the unsaturated tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-isodehydrovaline in place of substrate ACbetaFV. (19)F NMR studies confirm the release of fluoride from ACbetaFV in the presence of the active IPNS enzyme. These results suggest a new mode of reactivity for the IPNS iron centre, a mechanism of action that has not previously been reported for any of the iron oxidase enzymes.

Study on fluoride emission from soils at high temperature related to brick-making process.

Characteristics of fluoride emission from 12 soils at temperatures of 400-1,100 degrees C related to the brick-making process were studied. The results obtained in this study indicate that fluoride emission as gaseous HF and SiF4 was related to the firing temperature, soil total fluoride content, soil composition and calcium compounds added to soils. Soils began to release fluoride at temperatures between 500 and 700 degrees C. Marked increases of the average fluoride mission rate from 57.2% to 85.4% of soil total fluoride were noticed as the heating temperature was increased from 700 to 1,100 degrees C. It was found that the major proportion (over 50%) of the soil total fluoride was emitted from soils at approximate 800 degrees C. The amount of fluoride released into the atmosphere when heated depended on the total fluoride contents in the soils. Correlation analysis showed that the soil composition, such as cation exchange capacity, exchangeable calcium and CaCO3, had some influence on fluoride emission below 900 degrees C, but had no influence at temperatures above 900 degrees C. Addition of four calcium compounds (CaO, CaCO3, Ca(OH)2, and CaSO4) at 1.5% by weight raised the temperature at which fluoride began to be released to 700 degrees C. The greatest decrease in fluoride emission among the four calcium compound treatments was found with CaCO3.

Structural analysis of Francisella tularensis lipopolysaccharide.

The structure of the lipid A and core region of the lipopolysaccharide (LPS) from Francisella tularensis (ATCC 29684) was analysed using NMR, mass spectrometry and chemical methods. The LPS contains a beta-GlcN-(1-6)-GlcN lipid A backbone, but has a number of unusual structural features; it apparently has no substituent at O-1 of the reducing end GlcN residue in the lipid part in the major part of the population, no substituents at O-3 and O-4 of beta-GlcN, and no substituent at O-4 of the Kdo residue. The largest oligosaccharide, isolated after strong alkaline deacylation of NaBH4 reduced LPS had the following structure: where Delta-GalNA-(1-3)-beta-QuiNAc represents a modified fragment of the O-chain repeating unit. Two shorter oligosaccharides lacking the O-chain fragment were also identified. A minor amount of the disaccharide beta-GlcN-(1-6)-alpha-GlcN-1-P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end. The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2-N-(3-O-acyl4-acyl2)-beta-GlcN-(1-6)-2-N-acyl1-3-O-acyl3-GlcN where acyl1, acyl2 and acyl3 are 3-hydroxyhexadecanoic or 3-hydroxyoctadecanoic acids, acyl4 is tetradecanoic or (minor) hexadecanoic acids. No phosphate substituents were found in this compound. OH-1 of the reducing end glucosamine, and OH-3 and OH-4 of the nonreducing end glucosamine residues were not substituted. LPS of F. tularensis exhibits unusual biological properties, including low endoxicity, which may be related to its unusual lipid A structure.