The chemical topology of a bacterial swarm.

Microbes respond to antibiotics by initiating a suite of defense mechanisms, including the production of small-molecule effectors. However, it is not well-known how these defenses vary according to the particular effector or antibiotic and bacterial state, due in part to the challenges of monitoring small molecules in complex environments. A new study uses state-of-the-art imaging techniques to track the location of secreted small molecules produced by a bacterial swarm in response to different antibiotics, providing unexpected insights into the spatial heterogeneity of bacterial stress responses.

Determination of metoserpate, buquinolate, and diclofenac in pork, eggs, and milk using liquid chromatography-tandem mass spectrometry.

In this work, a method was developed for the simultaneous determination of residual metoserpate, buquinolate and diclofenac in pork, milk, and eggs. Samples were extracted with 0.1% formic acid in acetonitrile, defatted with n-hexane, and filtered prior to analysis using liquid chromatography-tandem mass spectrometry. The analytes were separated on a C18 column using 0.1% acetic acid and methanol as the mobile phase. The matrix-matched calibration curves showed good linearity over a concentration range of 5-50 ng/g with coefficients of determination (R2 ) ≥0.991. The intra- and inter-day accuracies (expressed as recovery percentage values) calculated using three spiking levels (5, 10, and 20 µg/kg) were 80-108.65 and 74.06-107.15%, respectively, and the precisions (expressed as relative standard deviation) were 2.86-13.67 and 0.05-11.74%, respectively, for the tested drugs determined in various matrices. The limits of quantification (1 and 2 µg/kg) were below the uniform residual level (0.01 mg/kg) set for compounds that have no specific maximum residue limit (MRL). The developed method was tested using market samples and none of the target analytes was detected in any of the samples. The validated method proved to be practicable for detection of the tested analytes in pork, milk, and eggs.

Elucidation of Biosynthetic Pathways of Natural Products.

During the last decade, we have revealed biosynthetic pathways responsible for the formation of important and chemically complex natural products isolated from various organisms through genetic manipulation. Detailed in vivo and in vitro characterizations enabled elucidation of unexpected mechanisms of secondary metabolite biosynthesis. This personal account focuses on our recent efforts in identifying the genes responsible for the biosynthesis of spirotryprostatin, aspoquinolone, Sch 210972, pyranonigrin, fumagillin and pseurotin. We exploit heterologous reconstitution of biosynthetic pathways of interest in our study. In particular, extensive involvement of oxidation reactions is discussed. Heterologous hosts employed here are Saccharomyces cerevisiae, Aspergillus nidulans and A. niger that can also be used to prepare biosynthetic intermediates and product analogs by engineering the biosynthetic pathways using the knowledge obtained by detailed characterizations of the enzymes. (998 char.).

Quantitative determination of a synthetic amide derivative of gallic acid, SG-HQ2, using liquid chromatography tandem mass spectrometry, and its pharmacokinetics in rats.

An amide derivative of gallic acid (GA), 3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide) (SG-HQ2) was recently synthesized, and its inhibitory actions were previously shown on histamine release and pro-inflammatory cytokine expression. In this study, a simultaneous quantification method was developed for the determination of SG-HQ2 and its possible metabolite, GA, in rat plasma using liquid chromatography with a tandem mass spectrometry (LC-MS/MS). After simple protein precipitation with acetonitrile including diclofenac (internal standard, IS), the analytes were chromatographed on a reversed phased column with a mobile phase of acetonitrile and water (60:40, v/v, including 0.1% formic acid). The ion transitions of the precursor to the product ion were principally protonated ion [M+H]+ at m/z 313.2→160.6 for SG-HQ2, and deprotonated ions [M-H]- at m/z 168.7→124.9 for GA and 296.0→251.6 for the IS. The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This method was successfully applied to a pharmacokinetic study of SG-HQ2 after intravenous administration in rats.

[Determination of nequinate and buquinolate in livestock products using liquid chromatography-tandem mass spectrometry].

We studied the simultaneous determination of nequinate and buquinolate, which are used as feed additives to prevent coccidiosis, by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The sample was extracted with acetonitrile, then loaded onto an HLB mini-column with 20% methanol. After clean-up with 20% methanol, the analytes were eluted with acetonitrile-methanol (1 : 1). The coccidiostats in the purified samples were determined using ESI-MRM mode LC-MS/MS with a sample matrix calibration curve. Mean recoveries of nequinate and buquinolate from 8 kinds of livestocks samples (chicken muscle, chicken liver, chicken heart, swine muscle, swine heart, cattle muscle, sheep muscle, egg) were in the range of 89.5% to 108.6%, and the relative standard deviation values were <20% (n=10) at the levels of 0.01 µg/g and 0.05 µg/g, respectively. The limits of quantification of these compounds were 0.001 µg/g in each sample.

Rapid determination of quinoline and 2-hydroxyquinoline in quinoline biodegradation process by tri-wavelength UV/Vis spectroscopy.

For the present study, a tri-wavelength UV/Vis spectrophotometric method for rapid determination of quinoline (Q) and 2-hydroxyquinoline (HQ) during Q biodegradation was developed. Based on the spectral measurements at 289 nm (the isosbestic point of Q and HQ), 326 and 380 nm, the spectral interference of extracellular polymeric substances (EPS) in the process samples could be minimized, and the amounts of Q and HQ could be simultaneously quantified. Our results indicated that the relative standard deviations in the repeatability tests were 2.7 and 1.7% for Q and HQ, respectively. The method validation was conducted by comparing the data obtained using the present method with those generated from high performance liquid chromatography (HPLC). The same set of samples from Q biodegradation process was used. The relative differences between the two methods were within 10%. In conclusion, the present method is simple, rapid, and suitable for the investigation in Q biodegradation processes.

Quinoline biodegradation and its nitrogen transformation pathway by a Pseudomonas sp. strain.

A Pseudomonas sp. strain, which can utilize quinoline as its sole carbon, nitrogen and energy source, was isolated from activated sludge in a coking wastewater treatment plant. Quinoline can be degraded via the 8-hydroxycoumarin pathway. We quantified the first two organic intermediates of the biodegradation, 2-hydroxyquinoline and 2,8-dihydroxyquinoline. We tracked the transformation of the nitrogen in quinoline in two media containing different C/N ratios. At least 40.4% of the nitrogen was finally transformed into ammonium when quinoline was the sole C and N source. But addition of an external carbon source like glucose promoted the transformation of N from NH3 into NO3(-), NO2(-), and then to N2. The product analysis and gene characteristics indicated that the isolate accomplished heterotrophic nitrification and aerobic denitrification simultaneously. The study also demonstrated that quinoline and its metabolic products can be eliminated if the C/N ratio is properly controlled in the treatment of quinoline-containing wastewater.

Characterization of new metabolites from in vivo biotransformation of 2-amino-3-methylimidazo[4,5-f]quinoline in mouse by mass spectrometry.

In studying the metabolic pathways underlying the mechanism of carcinogenesis of the heterocyclic amine of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), we recently found a new metabolite which gave an [M + H](+) ion of m/z 217 when subjected to electrospray ionization (ESI) in positive-ion mode. Following i.p. injection of this metabolite of m/z 217 (designated as m/z 217) to beta-naphthoflavone-treated mice, 57% of the total radioactivity was recovered in a 24-h mouse urine sample. HPLC separation followed by MS analysis indicates that the urine sample contained m/z 217 (36 +/- 3% of total recovered radioactivity) and two other peaks that gave rise to the [M + H](+) ions of m/z 393 (31 +/- 4%, designated as m/z 393) and m/z 233 (14 +/- 1%, designated as m/z 233). Beta-glucuronidase treatment of m/z 393 resulted in a radioactive peak corresponding to m/z 217. ESI in combination with various mass spectrometry techniques, including multiple-stage mass spectrometry, exact mass measurements and H/D exchange followed by tandem mass spectrometry, was used for structural characterization. The urinary metabolites of m/z 217, 393 and 233 were identified as 1,2-dihydro-2-amino-5-hydroxy-3-methylimidazo[4,5-f]quinoline, 1,2-dihydro-2-amino-5-O-glucuronide-3-methylimidazo[4,5-f]quinoline and 1,2-dihydro-2-amino-5,7-dihydroxy-3-methylimidazo[4,5-f]quinoline, respectively. Our results demonstrated that m/z 217 is biotransformed in vivo to m/z 393 by O-glucuronidation and to m/z 233 by oxidation. The observation of these more polar metabolites relative to IQ suggests that they may arise from a previously undescribed detoxification pathway.

Modified ferron assay for speciation characterization of hydrolyzed Al(III): a precise k value based judgment.

The speciation of Al-OH complexes in terms of Al(a), Al(b) and Al(c) could be achieved by traditional ferron assay and Al(b) is generally considered as Al(13), however, the inherent correlation between them remains an enigma. This paper presents a modified ferron assay to get precise determination of Al(13) using nonlinear least squares analysis, and to clarify the correlation between Al(b) and Al(13). Two parallel reactions conforming to pseudo-first-order kinetics can simulate the complicate reactions between polynuclear complexes and ferron successfully. Four types of experimental kinetic constant (k value) of Al-OH complexes can be observed by this method when investigating three typical aluminium solutions. Comparing with the results of (27)Al NMR, the species with moderate kinetics around 0.001 s(-1) can be confirmed to resemble to Al(13) polycation. The other types of kinetics are also well-regulated in partially neutralized aluminium solutions with various OH/Al ratios (b values) in the range 0 approximately 2.5. It would provide potential means to trace the in-situ formation of Al(13) in dilute solutions such as coagulation with Al-based coagulants.

Preconcentration of Zr, Hf, Nb, Ta and W in seawater using solid-phase extraction on TSK-8-hydroxyquinoline resin and determination by inductively coupled plasma-mass spectrometry.

Here, we present the first simultaneous preconcentration and determination of ultratrace (pmol kg(-1) level) Zr, Hf, Nb, Ta and W in seawater, both in the form of dissolved and acid-dissolvable species. 8-Hydroxyquinoline (8HQ) bonded covalently to a vinyl polymer resin, TSK-8HQ, was used in a chelating adsorbent column to concentrate the metals. The greatest advantage of this resin is its endurance to 5M HF, since this is an effective eluent for all five metals. The analytes were successfully concentrated from 250 mL seawater with a 50-fold concentration factor through the column extraction and evaporation. The detection limit was 0.009-0.15 pmol kg(-1). The procedure blank determined using ultra pure water as a sample was 0.005-0.37 pmol kg(-1). The five metals were quantitatively recovered from seawater with good precision (2-4%). The effect of sample pH, sample flow rate, eluent composition and sample pretreatment were carefully studied. This method was applied to seawater.

Supramolecular structures and stereochemical versatility of azoquinoline containing novel rare earth metal complexes.

Rare earth complexes of 5-(phenylazo)-8-hydroxyquinoline (HL) of composition [M(L)(2)X.H(2)O] [where M=La, Ce, Pr, Nd and X=NO(3)(-) or NCS(-)] have been prepared and characterized on the basis of their chemical analyses, (1)H NMR, magnetic measurements, conductance, and visible and IR spectral data. Composition, conductance and IR spectral data of the complexes show that the HL acts as a bidentate monobasic ligand. The visible spectra of Pr(3+) and Nd(3+) show characteristic f-f transitions, and the nephelauxetic effect (1-beta) of these transitions has been evaluated. These data indicate the weak involvement of the 4f orbitals in complex formation.

CYP3A4 activity in four different animal species liver microsomes using 7-benzyloxyquinoline and HPLC/spectrofluorometric determination.

Some microplate-based direct assays with different fluorometric substrates have been developed, among which 7-benzyloxyquinoline (BOQ) has demonstrated the highest degree of selectivity for CYP3A subfamily. In our study, we firstly developed and validated an efficient, fast and cheap HPLC/spectrofluorometric analytical method to quantify 7-hydroxyquinoline (BOQ metabolite). Secondly, BOQ oxidation rate (1.95 +/- 0.24 microM/mg protein/min) was compared to that of midazolam (MDZ) (1.4 +/- 0.21 microM/mg protein/min), an other specific CYP3A probe. However, the difference did not reach statistically significance (test of Sign; p = 0.125, two tailed). Thirdly, the potential use of BOQ in other species than the rat (mouse, dog and monkey) was studied. The highest BOQ activity was observed in rat microsomes (3.75 micromol/mg protein/min) with lower P450 content (0.3 nmol/mg protein) compared to other species. Finally, the effect of CYP3A enzymes-selective inhibitor ketoconazole on the dealkylation of BOQ in control and dexamethasone (DM)-treated rat microsomes was studied. Ketoconazole inhibition potency was greater in control (IC(50) approximately 21.6 microM) compared to DM induced (IC(50) approximately 32.3 microM) microsomes. At concentrations greater than that considered to be enzyme-selective (e.g., 10-30 microM), ketoconazole inhibitory activity did not rise significantly, and at the maximal concentration tested (1,000 microM) a nearly similar inhibition (76%) was observed than that at 50 microM concentration (68.2%).

[Assay of three kinds of aluminum fractions (Al(a), Al(b) and Al(c)) in polynuclear aluminum solutions by Al-Ferron timed spectrophotometry and demarcation of their time limits].

Al-Ferron timed spectrophotometry assay is a basic method in the study on the formation of polynuclear hydroxyl aluminum species and their transformation laws in aqueous systems. In actual working process, this methodology has some dogmatism and arbitrariness in the time limits demarcation of the three kinds of aluminum fractions (Al(a), Al(b) and Al(c)) in polynuclear aluminum solutions, which makes this kind of classification rougher, and the experimental results non-reproducible. The reason for this difference is that the specific species within Al(a), Al(b) and Al(c) have different reaction mechanism and dynamics, and that specific species of Al(b) having different OH/Al ratios have different reaction rates with ferron. In this paper, the ExpAssoc distribution was applied to quantitatively fit the Al-Ferron reaction dynamics curve, and the extrapolation method was used to survey the 1 min measured value [Al(a)] of monomeric Al, which is hard to obtain in manual manipulation. The time demarcation between Al(b) and Al(c) should reach the point of the experimental data curve up to horizontal platform. The microwave-radiated technology was used to fast assay the total aluminum concentration [Al(T)]. With these methods, the contents of monomeric Al(a), polynuclear Al(b) and gel Al(c) can be conveniently and quantitatively measured. It offers a novel method for surmounting the arbitrariness in the measurement of the three kinds of aluminum fractions and the repetitive calculation of Al(a) and Al(b).

[Study on the fluorescence spectra of 7-hydroxyquinoline in dimethyl sulfoxide solution].

7-hydroxyquinoline (7-HQ) is a kind of organic molecule with excited-state proton transfer (ESPT) effect. 7-HQ in ethanol solution causes ESPT reaction under the excitation of ultraviolet light. The fluorescence spectrum of the sample exhibits two bands. In contrast, 7-HQ in dimethyl sulfoxide (DMS) solution does not cause ESPT reaction. The fluorescence spectrum of the sample exhibits a single band. But after the sample was irradiated with a strong UV light, its fluorescence spectrum also exhibits two bands. This phenomenon is reported for the first time in the present paper, and its cause is investigated through the study on the absorption spectra and fluorescence spectra of 7-HQ in ethanol, dimethyl sulfoxide and N, N-dimethyl formamide solution. The conclusion is that the change in the fluorescence spectrum of 7-HQ in DMS solution is due to the fact that 7-HQ causes ESPT reaction which results from the photodecomposition of DMS and the product of water after the solution was irradiated with strong UV light.

Carbostyril derivatives as antenna molecules for luminescent lanthanide chelates.

Luminescent lanthanide complexes consisting of a lanthanide-binding chelate and organic-based antenna molecule have unusual emission properties, including millisecond excited state lifetimes and sharply spiked spectra, compared to standard organic fluorophores. We have previously used carbostyril (cs124, 7-amino-4-methyl-2(1H)-quinolinone) as an antenna molecule (Li and Selvin, J. Am. Chem. Soc., 1995) attached to a polyaminocarboxylate chelate such as DTPA. Here, we report the chelate syntheses of DTPA conjugated with cs124 derivatives substituted on the 1-, 3-, 4-, 5-, 6-, and 8-position. Among them, the DTPA chelate of cs124-6-SO(3)H has similar lifetime and brightness for both Tb(3+) and Eu(3+) compared to the corresponding DTPA-cs124 complexes, yet it is significantly more soluble in water. The Tb(3+) complex of DTPA-cs124-8-CH(3) has significantly longer lifetime compared to DTPA-cs124 (1.74 vs 1.5 ms), indicating higher lanthanide quantum yield resulting from the elimination of back emission energy transfer from Tb(3+) to the antenna molecule. Thiol-reactive forms of chelates were made for coupling to proteins. These lanthanide complexes are anticipated to be useful in a variety of fluorescence-based bioassays.

A high-throughput monoamine oxidase inhibition assay using liquid chromatography with tandem mass spectrometry.

A highly efficient method utilizing liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed and employed for high-throughput screening of compounds for monoamine oxidase (MAO) inhibition. The method used kynuramine as a common substrate for both MAO-A and MAO-B in incubations, and the 4-hydroxyquinoline (4-HQ) resulting from deamination of kynuramine followed by intramolecular condensation was analyzed using LC/MS/MS; formation of 4-HQ was used as the marker of MAO activity to evaluate the effects of test compounds. Isocratic liquid chromatography was applied to reduce the run time to 2 min. Because of the high specificity and sensitivity of detection of 4-HQ by LC/MS/MS, this method was able to use MAO enzymes at very low concentrations and to perform short incubations; as a result, consumable cost was minimized, and sample preparations were completely avoided. The inhibition data are highly reproducible, and the IC(50) values determined by the method are in good agreement with literature values. The results suggest that this method is very robust and can be used as a generic approach to screen for MAO inhibitors in drug discovery.

Differential pulse polarographic determination of poly(8-hydroxyquinoline) in the presence and absence of an insulating poly(vinyl alcohol) matrix.

The electrochemical activity of poly(8-hydroxyquinoline) (PHQ) in acid and alkaline media has been investigated by use of differential pulse polarography (DPP). The reduction peak height (I(p)) of PHQ in universal buffer solutions is not useful as an analytical signal, because it is highly affected by hydrogen evolution in acid media and appears as a small peak located at more negative potential values in alkaline media. A new and highly sensitive reduction peak (E(p)=-0.45, pH 9.25) appears, however, after addition of trace amounts of PHQ to Cu(II), or vice versa. This reduction peak is a result of the reduction of Cu(II) chelates in the PHQ-Cu(II) complex and is highly promising for the trace determination of PHQ at nanomolar and submicromolar levels. The response current (I(p)/mu A) for the reduction peak of Cu(II) chelates in a PHQ-Cu(II) matrix results in sensitivity to the concentration of PHQ at least three orders of magnitude higher than that for the reduction peak of PHQ alone under the same conditions. The limit of detection is as low as 5.264 ppb (microg L(-1)). The effect of a variety of anions and cations and of an insulating poly(vinyl alcohol) (PVA) matrix has been investigated. Electroactive PHQ-Cu(II) at a level of 0.685% could induce a current of approximately 240 nA in an insulating PVA matrix, suggesting possible application for the preparation of a PHQ-Cu(II)-PVA electroactive composite.

A rapid spectrophotometric method for the determination of molybdenum in industrial, environmental, biological and soil samples using 5,7-dibromo-8-hydroxyquinoline.

A very simple, ultra-sensitive and highly selective non-extractive spectrophotometric method for the determination of trace amount of molybdenum(VI) using 5,7-dibromo-8-hydroxyquinoline (DBHQ) has been developed. 5,7-Dibromo-8-hydroxyquinoline reacts in a slightly acidic solution (0.05 - 1.0 M H2SO4) with molybdenum(VI) to give a deep greenish-yellow chelate which has an absorption maximum at 401 nm. The reaction is instantaneous and the absorbance remains stable for over 24 h. The average molar absorption coefficient and Sandell's sensitivity were found to be 4.13 x 10(3) L mol(-1) cm(-1) and 7 ng cm(-2) of molybdenum(VI), respectively. Linear calibration graphs were obtained for 0.1 - 50 microg mL(-1) of molybdenum(VI). The stoichiometric composition of the chelate is 1:3 (Mo:DBHQ). A large excess of over 50 cations, anions and some common complexing agents (e.g. EDTA, oxalate, citrate, phosphate, thiourea, SCN-) do not interfere with the determination. The method was successfully used in the determination of molybdenum in several Standard Reference Materials (alloys, steels and waters) as well as in some environmental waters (inland and surface), biological samples (human blood and urine), soil samples, solution containing both molybdenum(V) and molybdenum(VI) and complex synthetic mixtures. The method has high precision and accuracy (S = +/-0.01 for 0.5 microg mL(-1)).

[The biosynthesis of low-molecular-weight nitrogen-containing secondary metabolite-alkaloids by the resident strains of Penicillium chrysogenum and Penicillium expansum isolated on the board of the Mir space station ]. / Biosintez nizkomolekuliarnykh azotsoderzhashchikh vtorichnykh metabolitov-alkaloidov shtammami-rezidentami gribov vidov Penicillium chrysogenum i Penicillium expansum, vydelennykh na orbital'noi stantsii "Mir".

The analysis of the absorption spectra of the low-molecular-weight nitrogen-containing secondary metabolites--alkaloids--of 4 Penicillium chrysogenum strains and 6 Penicillium expansum strains isolated on board the Mir space station showed that all these strains synthesize metabolites of alkaloid origin (roquefortine, 3,12-dihydroroquefortine, meleagrin, viridicatin, viridicatol, isorugulosuvin, rugulosuvin B, N-acetyl-tryptamine, and a "yellow metabolite" containing the benzoquinone chromophore).

Determination of viridicatin in Penicillium cyclopium by capillary gas chromatography.

A sensitive gas chromatographic method for the determination of the quinoline alkaloid viridicatin, a fungal metabolite isolated from several Penicillium species and presumably a biogenetic precursor to naturally occurring diazepam-like 1,4-benzo-diazepines, has been developed. Prior to conversion of viridicatin into a volatile trimethylsilyl derivative, a rapid sample clean-up was accomplished by solid-phase extraction from mould mycelium on a C18 cartridge. Using synthetical 6-bromo-viridicatin as an internal standard, the amount of viridicatin produced de novo in emerged grown cultures of Penicillium cyclopium strain SM 72 was calculated in correlation to the duration of mould cultivation.