Deborah L. Chance

Gas-liquid chromatography-mass spectrometry of hydroxy fatty acids as their methyl esters tert.-butyldimethylsilyl ethers.

tert.-Butyldimethylsilyl ethers of secondary hydroxy fatty acid methyl esters (tBDMS-O-FAMEs) produce stable derivatives amenable to gas-liquid chromatography (GLC) and mass spectrometry (MS). Derivatives produce prominent molecular mass minus 57 [M-57]+ fragment ions and unique marker fragment ions indicating the location of the secondary hydroxyl groups along the aliphatic chain from the omega-2 carbon to carbon numbers 5 from the carboxylic terminus, in addition to yielding information regarding carbon chain length, and degree of unsaturation. The tBDMS-derivatives of C-2, C-3 hydroxy fatty acids and the unique GLC-MS data of gamma- and delta-lactones are also presented. Though several tBDMS-O-FAMEs with centrally located hydroxyl groups were not chromatographically resolved, the combination of GLC retention times and monitoring of key diagnostic fragment ions of each tBDMS-derivative, when applied to mixtures containing all hydroxy isomers of palmitic through arachidic acid methyl esters, and to several monounsaturated, monohydroxylated fatty acid methyl esters, allowed for their unambiguous identification. Coupled with derivative stability, permitting their purification and concentration, this method was applied to the identification of trace lipids isolated from bovine skim milk which contained a complex mixture of hydroxy fatty acids of which 19 were newly identified.

Carbohydrate sulfation effects on growth of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a key player in the pathology and morbidity of cystic fibrosis. Chronic obstructive pulmonary disease, which results from the most common and severe mutations in this genetic disorder, typically includes chronic infection with P. aeruginosa which, even with rugged antibiotic and physical therapy regimens, is rarely eradicated. It is not known whether the increased oligosaccharide sulfation characteristic of cystic fibrosis tracheobronchial mucins plays a role in the survival of P. aeruginosa in the airway. In this study, sulfated monosaccharides were synthesized and tested for their effects on the growth of clinical isolates and laboratory strains of this organism when supplied as the sole carbon source in vitro. Carbohydrate sulfation was observed to reduce, but not prohibit, growth of P. aeruginosa on carbohydrates normally utilized in their nonsulfated form. The various sulfated sugars employed as the sole carbon source gave characteristic and consistent growth profiles and maximum growth values across the strains tested. P. aeruginosa isolates from patients with cystic fibrosis often express a mucoid phenotype, which is thought to contribute to their ability to survive in harsh conditions. Carbohydrate sulfation effects on growth did not differ significantly between mucoid and nonmucoid strains. These results suggest that the additional sulfation of tracheobronchial mucin documented in cystic fibrosis may in fact contribute to the mucins resistance to utilization by P. aeruginosa and potentially other pathogens, providing an additional level of host protection, and limiting the available nutrient pool and thereby bacterial growth.

RAFT-based tri-component fluorescent glycopolymers: synthesis, characterization and application in lectin-mediated bacterial binding study

A group of fluorescent statistical glycopolymers, prepared via reversible addition–fragmentation chain-transfer (RAFT)-based polymerizations, were successfully employed in lectin-mediated bacterial binding studies. The resultant glycopolymers contained three different monomers: N-(2-hydroxyethyl) acrylamide (HEAA), N-(2-aminoethyl) methacrylamide (AEMA) and N-(2-glyconamidoethyl)-methacrylamides possessing different pendant sugars. Low dispersities (≤1.32) and predictable degrees of polymerization were observed among the products. After the polymerization, the glycopolymers were further modified by different succinimidyl ester fluorophores targeting the primary amine groups on AEMA. With their binding specificities being confirmed by testing with lectin coated agarose beads, the glycopolymers were employed in bacterial binding studies, where polymers containing α-galactose or β-galactose as the pendant sugar were specifically bound by two clinically important pathogens Pseudomonas aeruginosa and Staphylococcus aureus, respectively. This is the first report of using RAFT-based glycopolymers in bacterial binding studies, and the ready access to tri-component statistical glycopolymers also warrants further exploration of their utility in other glycobiological applications.

Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens

ABSTRACT Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was ∼31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3′ and 5′ nucleoside monophosphates at pH 6. Calculated Kms ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 μmol of Pi/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

Stabilization of the acyclic tautomer in reducing carbohydrates.

Since the advent of carbohydrate chemistry, it has been established that numerous reactions leading to chemical transformations at the carbonyl/anomeric group, such as oxidation, reduction, isomerization, addition, condensation, degradation, etc., involve the acyclic saccharide as an intermediate. Enzymatic carbohydrate isomerization or redox reactions have been of considerable interest for years, due to their significant theoretical and practical importance for biological, medicinal, and food chemistries and biofuel technologies. The acyclic intermediates of these transformations are difficult to characterize accurately, due to their low to trace populations in anomeric equilibria (Scheme 1) and their inability to crystallize. We have previously demonstrated, however, that 1-amino-1-deoxypentulose derivatives can provide crystallographic data about acyclic keto tautomers. An X-ray diffraction study of the acyclic 1-amino-1-deoxy-d-xylulose tautomer revealed similarities between the conformations of the carbohydrate part of this compound and the xylo-pentose substrate in the active site of d-xylose isomerase, as well as the keto tautomer of xylulose. Encouraged by this initial success, we have obtained, for the first time, the crystal structures of several acyclic dfructose keto tautomers, specifically d-fructosamine derivatives 6 and 8. Initially, we sought to verify that crystalline N-methyl-Np-tolyl-d-fructosamine (6) may contain the keto isomer, as was suggested on the basis of the IR spectrum. In the solidstate C NMR spectrum of 6, only one set of carbon resonances is present, with an unambiguous carbonyl signal at d = 209 ppm and no anomeric carbon peaks in the interval of d = 95–110 ppm, which indicates that crystalline 6 exists solely in the keto tautomeric form (Figure 1A). To further

Structural Elucidation by Fast Atom Bombardment Mass Spectrometry of Multisulfated Oligosaccharides Isolated from Human Respiratory Mucous Glycoproteins

Abstract Underivatized mono- and multisulfated oligosaccharides obtained by the alkalineborohydride treatment of human respiratory mucous glycoproteins were analyzed by positive ion fast atom bombardment mass spectrometry (FAB MS). Employing three unique and structurally homologous groups, the FAB mass spectra of a mono- and a disulfated tri- and tetrasaccharide and a mono-, di- and trisulfated branched hexasaccharide were compared. Each produced mass spectra displaying molecular weight-related and structurally significant fragment ions including fragments differing in mass by multiples of 102 amu reflecting the loss of one or more sulfate esters. From these data, combined with monosaccharide composition, the carbohydrate sequence and number and location of sulfate esters can readily be determined. These findings, with other chemical and enzymatic analyses, make FAB MS a valuable tool applicable to the unambiguous structural elucidation of underivatized reduced, linear and branched, mucous glycoprotein ol...

Acid phosphatase activity as a measure of Haemophilus influenzae adherence to mucin

Haemophilus influenzae is an important respiratory tract pathogen. Toward understanding the progression of H. influenzae from commensal to pathogen, we need to understand the steps of colonization and infection, processes which must involve overcoming the normal host mucociliary clearance mechanism. A reliable method for the screening and quantitation of mucin-H. influenzae binding to allow for the assessment of the physiological variables significant to H. influenzae-mucin interactions in the normal and diseased conditions, will provide insight on how to intervene to prevent, inhibit, or treat infection. The current methods for enumeration of mucin-bound H. influenzae are labor intensive and rely on viable organisms. In this report, we present a new detection method, which reduces the number of variables, processing steps, and time involved, providing an economical, rapid, and reliable means to screen for and quantitate mucin-bound H. influenzae. Organisms are applied to mucin-coated microtiter wells for a set time; nonadherent organisms are removed with gentle rinses; wells are incubated with the phosphomonoesterase substrate p-nitrophenyl phosphate; and the absorbance, reflecting phosphatase activity of the mucin-bound organisms, is read at 410 nm in a microtiter plate reader against enzymatic activity calibration curves. All nonencapsulated and encapsulated H. influenzae tested exhibited significant acid phosphate activity within 20 min, which provided linear relationships with the numbers of organisms present. H. influenzae mucin binding characteristics obtained by this method were generally comparable to published data, and ranged from 10(3) to 10(6) organisms per well, depending on both strain of organism and type of mucin employed. This convenient, rapid and economical mucin adherence assay, will enable more extensive and comprehensive studies of the interactions of H. influenzae adhesins and specific ligands on mucin macromolecules, as well as the nonspecific means by which mucins function in preventing bacterial infection.

Gas-liquid chromatography-mass spectrometry of primary and secondary fatty alcohols and diols as their tert.-butyldimethylsilyl derivatives

Abstract The use of gas-liquid chromatography (GLC) and mass spectrometry (MS) with derivatizing agents which give stable derivatives and informative fragmentation patterns allows for accurate identification of a variety of compounds. In this comprehensive study, the tert.-butyldimethylsilyl (tBDMS) derivatives of saturated and unsaturated primary alcohols, iso-and anteiso-primary alcohols, branched alcohols, secondary alcohols and primary diols were produced and analyzed by GLC-MS. Most derivatized alcohols produced prominent mass minus 57 [M-57]+ fragment ions, which, combined with their respective retention data and other key fragment ions, allows for their identification and analysis.

Quality of Intrathecal Baclofen From Different Sources

To compare the quality of intrathecal baclofen obtained from a national compounding pharmacy (AnazaoHealth) with the manufactured product (Lioresal) with regard to accuracy and precision of baclofen concentration, and the content of the baclofen degradation product, 4‐(4‐chlorophenyl)‐2‐pyrrolidinone (PYR).

Transient Proteotoxicity of Bacterial Virulence Factor Pyocyanin in Renal Tubular Epithelial Cells Induces ER-Related Vacuolation and Can Be Efficiently Modulated by Iron Chelators

Persistent infections of biofilm forming bacteria, such as Pseudomonas aeruginosa, are common among human populations, due to the bacterial resistance to antibiotics and other adaptation strategies, including release of cytotoxic virulent factors such as pigment pyocyanin (PCN). Urinary tract infections harbor P. aeruginosa strains characterized by the highest PCN-producing capacity, yet no information is available on PCN cytotoxicity mechanism in kidney. We report here that renal tubular epithelial cell (RTEC) line NRK-52E responds to PCN treatments with paraptosis-like activity features. Specifically, PCN-treated cells experienced dilation of endoplasmic reticulum (ER) and an extensive development of ER-derived vacuoles after about 8 h. This process was accompanied with hyper-activation of proteotoxic stress-inducible transcription factors Nrf2, ATF6, and HSF-1. The cells could be rescued by withdrawal of PCN from the culture media before the vacuoles burst and cells die of non-programmed necrosis after about 24–30 h. The paraptosis-like activity was abrogated by co-treatment of the cells with metal-chelating antioxidants. A microscopic examination of cells co-treated with PCN and agents aiming at a variety of the cellular stress mediators and pathways have identified iron as a single most significant co-factor of the PCN cytotoxicity in the RTECs. Among biologically relevant metal ions, low micromolar Fe2+ specifically mediated anaerobic oxidation of glutathione by PCN, but catechol derivatives and other strong iron complexing agents could inhibit the reaction. Our data suggest that iron chelation could be considered as a supplementary treatment in the PCN-positive infections.