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The siderophore and virulence factor yersiniabactin is produced by Pseudomonas syringae. Yersiniabactin was originally detected by high-pressure liquid chromatography (HPLC); commonly used PCR tests proved ineffective. Yersiniabactin production in P. syringae correlated with the possession of irp1 located in a predicted yersiniabactin locus. Three similarly divergent yersiniabactin locus groups were determined: the Yersinia pestis group, the P. syringae group, and the Photorhabdus luminescens group; yersiniabactin locus organization is similar in P. syringae and P. luminescens. In P. syringae pv. tomato DC3000, the locus has a high GC content (63.4% compared with 58.4% for the chromosome and 60.1% and 60.7% for adjacent regions) but it lacks high-pathogenicity-island features, such as the insertion in a tRNA locus, the integrase, and insertion sequence elements. In P. syringae pv. tomato DC3000 and pv. phaseolicola 1448A, the locus lies between homologues of Psyr_2284 and Psyr_2285 of P. syringae pv. syringae B728a, which lacks the locus. Among tested pseudomonads, a PCR test specific to two yersiniabactin locus groups detected a locus in genospecies 3, 7, and 8 of P. syringae, and bNA hybridization within P. syringae also detected a locus in the pathovars phaseolicolla and glycinea. The PCR and HPLC methods enabled analysis of nonpathogenic Escherichia coli. HPLC-proven yersiniabactin-producing E. coli lacked modifications found in irp1 and irp2 in the human pathogen CF17073, and it is not clear whether CFT073 produces yersiniabactin. The study provides clues about the evolution and dispersion of yersiniabactin genes. It describes methods to detect and study yersiniabactin producers, even where genes have evolved.

Ethylsuccinylcarnitine, a previously undescribed acylcarnitine, was identified in urines obtained from 81% of adult volunteers. Its chemical structure was obtained by 1H nuclear magnetic resonance spectroscopy of the urinary purified compound and confirmed by its chemical synthesis. Its urinary excretion followed a circadian rhythm with a maximum occurring between 8 p.m. and 1 a.m. Excretion of this compound was enhanced after a load of L-carnitine and in this case, total 24-h urinary excretion may raise up to about 75 microM. It was observed that the day after adryamicine treatment, the compound was no more excreted. This molecule was absent or in trace amounts in urines obtained from few adults as well as in urines obtained from young subjects. In the positive urines, we detected an unknown organic acid whose excretion was almost parallel to that of ethylsuccinylcarnitine.

This work reports a detailed study of the fragmentations of aporphine alkaloids by electrospray ionization with multistage mass spectrometry (ESI-MSn) in positive mode. In a first step the loss of the amino group and its substituent is observed. Further steps display the loss of the peripheral groups. Losses of methanol and CO are observed if an OH is vicinal to an OCH3 on the aromatic ring. Otherwise the spectra show radical losses of CH3. or CH3O. as the main fragmentations. If a methylenedioxy group is present losses of formaldehyde followed by CO are observed. These fragmentations yield important information on the structures of aporphines.

The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopathogenic Pseudomonas species was investigated. A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins. Nonfluorescent strains were found in Pseudomonas amygdali, Pseudomonas meliae, Pseudomonas fuscovaginae, and P. syringae. Pseudomonas agarici and Pseudomonas marginalis produced typical pyoverdins. Among the arginine dihydrolase-negative fluorescent Pseudomonas species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species. Within this group, the oxidase-negative species Pseudomonas viridiflava and Pseudomonas ficuserectae and the pathovars of P. syringae produced the same atypical pyoverdin, whereas the oxidase-positive species Pseudomonas cichorii produced a similar atypical pyoverdin that contained a glycine instead of a serine. The more distantly related species Pseudomonas asplenii and Pseudomonas fuscovaginae both produced a less similar atypical pyoverdin. The spectral characteristics of Fe(Ill)-chelated atypical pyoverdins at pH 7.0 were related to the presence of two beta-hydroxyaspartic acids as iron ligands, whereas in typical pyoverdins one of the ligands is always ornithine based. The peptide chain influenced the chelation of iron more in atypical pyoverdins. Our results demonstrated that there is relative pyoverdin conservation in the amino acids involved in iron chelation and that there is faster evolution of the other amino acids, highlighting the usefulness of pyoverdins in systematics and in identification.

Purification of a cytotoxic crude alkaloid extract of Cassytha filiformis led to the isolation of four known aporphine alkaloids: neolitsine, dicentrine, cassythine ( = cassyfiline) and actino-daphnine. Their structures were determined by analysis of spectroscopic data. All isolated alkaloids were tested for their cytotoxic activities on cancer and non-cancer cell lines in vitro. Neolitsine was the most active against HeLa and 3T3 cells (IC50:21.6 muM, and 21.4 muM, respectively). Cassythine and actinodaphnine showed the highest activity against Mel-5 (IC50:24.3 muM and 25.7 muM, respectively) and HL-60 (IC50: 19.9 muM and 15.4 muM, respectively). This is the first report on the cytotoxic activity of C. filiformis extract and of neolitsine and cassythine. Furthermore, the complete NMR data of cassythine and actinodaphnine are given here for the first time.

The dichloromethane extract of leaves of Croton zambesicus (Euphorbiaceae) showing in vitro cytotoxicity against human cervix carcinoma cells was investigated in order to identify its active compounds. A bio-guided fractionation by HSCCC followed by MPLC led us to isolate a trachylobane diterpene, ent-trachyloban-3beta-ol, with cytotoxic properties (IC50 on HeLa cells = 7.3 mug/ml). This is the first report on the cytotoxicity of a trachylobane diterpene.

Eleven naturally occurring flavonoid aglycones, belonging to the representative flavone, flavonol, and flavanone types were separated by high performance liquid chromatography and analyzed on-line with negative ion electrospray ionization tandem mass spectrometry (ESI-MS/MS). In order to resolve the MS/MS spectra obtained, each compound was reinvestigated by direct loop injections using an ion trap mass spectrometer. The MSn spectra obtained allowed us to propose plausible schemes for their fragmentation supported by the analysis of five complementary synthetic flavonoid aglycones. The negative ion ESI-MS/MS behavior of the different aglycones investigated in this study revealed interesting differences when compared with the previously described patterns obtained using various ionization techniques in positive ion. Thus, concerning the retro Diels-Alder (RDA) fragmentation pathways, several structurally informative anions appeared highly specific of the negative ion mode. In addition, a new lactone-type structure, instead of a ketene, was proposed for a classic RDA diagnostic ion. We also observed unusual CO, CO2, and C3O2 losses which appear to be characteristic of the negative ion mode. All these results and these unusual neutral losses show that the negative ion mode was a powerful complementary tool of the positive ion mode for the structural characterization of flavonoid aglycones by ESI-MS/MS.

The nonvolatile radiolytic compounds produced by irradiation of cefotaxime were studied by a liquid chromatography-electrospray mass spectrometry method. Full scan LC-MS was first performed in order to obtain the m/z value of the protonated molecules of all detected peaks. LC-MS-MS was then carried out on the compounds of interest. A comparison between the MS-MS spectrum of cefotaxime and those of the radiolytic compounds showed that their fragmentation patterns were very similar suggesting that they were structural analogues of the main drug. The examination of the two main fragmentation pathways also permitted the location of the modified substructures. Moreover, it was shown that some stereoisomers appeared with the irradiation process. The complete fragmentation pattern of cefotaxime was studied by MSn and used to obtain information about the structure of the radiolytic compounds. A complete structure was proposed for four of these.

The goal of this study was to clarify the mechanism responsible for the catabolism of a-tocopherol. The vitamin, bound to albumin, was incubated with rat liver microsomes and appeared to be broken down. Optimal production of the metabolite was obtained when 1 mg of microsomal protein was incubated with 36 muM of alpha -tocopherol in the presence of 1.5 mM of NADPH. Chromatographic and mass spectrometric analyses of the metabolite led to the conclusion that it consists of an omega -acid with an opened chroman ring, although we could not perform nuclear magnetic resonance analysis to confirm this. Our data show that ol-tocopherol is omega -oxidized to a carboxylic acid and that this process can occur in rat liver microsomes in the presence of NADPH and O-2. The oxidation to the quinone structure appears to be a subsequent event that may be artifactual and/or catalyzed by a microsomal enzyme(s).

Effects of 2-beta -D-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-Glc) and DIMBOA-Glc N-O-methylated (HDMBOA-Glc), two compounds present in high concentration in maize, were rested on the aphid Metopolophium dirhodum reared on artificial diet. HDMBOA-Glc and DIMBOA-Glc decrease survival of adults with an LD50 of 1 mM and 5.6 mM, respectively, after 72 hr of feeding. These compounds also decrease the fecundity of the aphids at concentrations of 2 mM and 1 mM, respectively. At concentrations of 2 mM HDMBOA-Glc and 8 mM DIMBOA-Glc in the diet, the average lifetime fecundity of 10 females is near zero. Offspring mortality on diet with 2 mM DIMBOA-Glc is significantly higher than with the control diet. In contrast, HDMBOA-Glc has no effect on the survival of offspring. The possibility that these compounds protect Poaceae against aphids is discussed.

Nonfluorescent highly virulent strains of Pseudomonas syringae pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent P. syringae strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydro-pyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent P. syringae strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in beta -hydroxyaspartic acid residues of the peptide chains. These differences can he detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent Pseudomonas species and fluorescent P. syringae and P. viridiflava strains and to distinguish between the two siderovars in P. syringae pv. aptata.

Isomeric flavonoid O-diglycosides were analyzed by positive and negative nano-electrospray ionization (ESI) ion trap mass spectrometry (ITMS} in order to evaluate whether the two most common interglycosidic linkage Types, i.e. 1 --> 2 and 1 --> 6, found for glycosides containing a rhamnosylglucose glycan part can be differentiated. In the positive ion mode the degree of internal glucose residue loss was found to be strongly dependent on the aglycone type and was very pronounced for aglycones of the flavanone type. The relative abundance of the Y-type ions formed by fragmentation at glycosidic bonds only allows one to infer the interglycosidic linkage types in the case of flavone O-diglycosides. In contrast, the negative ion mode makes a clear differentiation between a rutinoside (1 --> 6) and a neohesperidoside (1 --> 2) glycan residue possible for all aglycone types. The neohesperidose-containing compounds could be characterized by additional product ions. When the compounds were dissolved in pure methanol a molecular radical ion tvas found to be the base peak in nano-ESI. Copyright (C) 2001 John Wiley & Sons, Ltd.

In urine of patients with propionyl-CoA carboxylase deficiency or with methylmalonic acidemia, carnitine esters of 2-methyl-branched fatty acids of all chain lengths between 4 and 9 atoms of carbon were identified during the acute phase of the diseases. The chemical structure of these compounds was obtained by gas chromatography-mass spectrometry analysis of their fatty acid moieties in their free and picolinyl ester forms. We suggest mechanisms for the biosynthesis of these branched fatty acids, and their accumulation in urine during episodes of caloric imbalance.

We report the variation of all 1,4-benzoxazin-3-one derivatives content detectable in maize with plant age in roots and aerial parts. Our results show that the concentration of hydroxamic acids, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc) and its 8-methoxylated analogue (DIM(2)BOA-Glc) is high after seed germination and then decreases with plant age. Nevertheless, these compounds continue to be biosynthesised during 6-10 days after germination. Variation in concentration of N-O-methylated DIMBOA-Glc (HDMBOA-Glc) is similar to the one of hydroxamic acids in aerial parts. On the contrary, in roots, its concentration remains relatively stable with plant age. After 10 days, HDMBOA-Glc becomes the main compound in roots. This compound is also present in higher concentration than hydroxamic acids in the oldest leaf of 20-day-old maize. The presence of four other DIMBOA related compounds in maize plants depends on variety, age and tissue. The role of these compounds in plant resistance to aphids is discussed. (C) 2000 Elsevier Science Ltd. All rights reserved.

A low percentage of cyclic oligomers can be found in poly(ethylene terephthalate) (PET) from its synthesis onward. In this article, a cyclic-oligomer-fr ee PET (COFP) obtained by solvent extraction was used to study the thermodynamics of the re-formation of cyclics from the melt. The cyclic-oligomer content re-increased into molten COFP, finally reaching an equilibrium. An analysis of the fraction of the re-formed cyclic oligomers showed that a majority of cyclic trimer (60-70%) was found at the equilibrium Before the establishment of the equilibrium, an unusual behavior was observed in the relative proportion of cyclic trimer and tetramer during the first steps of their formation that was probably due to a competition between kinetic and thermodynamic products. (C) 2000 John Wiley & Sons, Inc.

Previously undescribed medium-chain acylcarnitines were identified in a urine sample from a patient with medium-chain acyl-CoA dehydrogenase deficiency. These are the 4-methylvaleryl, 4- and 5-methylhexanoyl, 6-methyl-heptanoyl-, 6-methyloctanoyl-, 4,5-dimethylhexanoyl- and 4,7-decadienoylcarnitines. Their chemical structures were obtained by gas chromatography-mass spectrometry analysis of their fatty acid moieties as picolinyl esters.

A rapid, simple, and sensitive method is described for the determination of the anomeric configuration of sugar 1-phosphates, sugar nucleotides, and polyisoprenyl-phospho-sugars. Negative-ion electrospray ionization of picomole amounts of glycosyl 1-phosphate derivatives produces an intense signal of the [M-H]-deprotonated molecule which, by collision-induced dissociation, decomposes in a characteristic manner depending on cis/trans configuration of the 2-hydroxyl and phosphate groups of the glycosyl residue. A distinct feature of the product ion spectra of glycosyl 1-P and polyisoprenyl-P-sugars with cis configuration is the presence of abundant ions that correspond to the [M-H2O-H]- dehydration product and the [R-PO4-(C2H3O]- fragment arising from a cleavage across the sugar ring, where R is -H or -polyprenyl/dolichyl for glycosyl 1-P and polyisoprenyl-P-sugar, respectively. These two fragments, [M-H2O-H]- and [R-PO4-(C2H3O)]- are absent from the product ion spectra of sugar 1-P and polyisoprenyl-P-sugars with trans configuration. For sugar nucleotides, compounds with cis configuration produce, in tandem mass spectrometry, only one abundant fragment of nucleoside monophosphate, whereas those with trans configuration give nucleoside diphosphate as a major fragment ion. Accordingly, the anomeric configuration of a glycosyl 1-phosphate derivative can be easily determined by using electrospray-ionization tandem mass spectrometry provided that the glycosyl residue of known absolute configuration has a free 2-hydroxyl group and no other charge location.

The structures of chelirubine and chelilutine free bases have been examined by 2D NMR spectroscopy and mass spectrometry. Chelirubine chloride (la) upon treatment with Na,CO, yielded free base which possessed the constitution of bis(5,6-dihydrochelirubin-6-yl) ether (2a). The free base of chelilutine (Ib) was determined to be bis(5,6-dihydrochelilutin-6-yl) ether (2b). The aqueous NH3 treatment of chelilutine (Ib) produced bis(5,6-dihydrochelilutin-6-yl)amine (3b). 6-Hydroxy-5,6-dihydrochelirubine (4a) and 6-hydroxy-5,6-dihydrochelilutine (4b) were detected only in CDCl3 solution by NMR spectroscopy. In CDCl3, the compound 2b underwent hydrolysis to 4b that was immediately followed by the reverse condensation to a diastereomer of 2b. Pseudokinetics of this reaction was followed by NMR spectroscopy and quantum chemical calculations were carried out to support the suggested type of molecular symmetry alteration. The known alkaloid dihydrochelirubine (5) was isolated for the first time from Sanguinaria canadensis.

Sodium carbonate treatment of sanguilutine chloride yielded 6-hydroxy-5,6-dihydrosanguilutine. In a non-polar environment, 6-hydroxy-5,6-dihydrosanguilutine spontaneously transforms to bis[6-(5,6-dihydrosanguilutinyl)] ether, whose structure was confirmed by X-ray analysis. Treatment of sanguilutine chloride with aqueous ammonia gave bis[6-(5,6-dihydrosanguilutinyl)]amine. Reaction of sanguilutine chloride with potassium cyanide yielded 6-cyanodihpdrosanguilutine; reaction of bis[6-(5,6-dihydrosanguilutinyl)]amine with ethanol gave 6-ethoxydihydrosanguilutine. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.

In a chemical ionization source with CH4/N2O, carbon disulfide (CS2) yields its radical anion by electron capture and CHS2- by hydride attachment, By combining tandem mass spectrometric experiments and theoretical ab initio calculations, we demonstrate that hydride attachment can yield either thioformate anion or thiohydroxythiocarbonyl carbanion without interconversion. The thioformate anion reacts with neutral at CS2 carbon, whereas the syn thiohydroxythiocarbonyl carbanion attacks at the sulfur site to ultimately form HSCS2- and CS species.