Cell density dependent restriction of N2O reductase in denitrifying Pseudomonas spp., a potential driver of N2O emissions in natural systems.

Denitrification is a major biological source and sink for the ozone-depleting greenhouse gas N2. Thus, the respiratory physiology of denitrifiers and the mechanisms determining their propensity for accumulation of N-oxides are of fundamental interest. Here, we report a pervasive positive correlation between cell density and N2O accumulation in Pseudomonas aeruginosa and P. fluorescens F113. We show that this was a result of quorum sensing by comparing the P. aeruginosa PAO1-UW wild type to a rhlI/lasI knockout mutant able to sense, but not synthesize the N-acyl-homoserine lactones (AHL) of the Rhl and Las circuits. Neither the transcription of nosZ (encoding N2O reductase, N2OR) nor the abundance of peptides of known relevance to denitrification could explain the restriction of N2O reduction in AHL-affected cultures. However, a protein shown to be involved in synthesis and repair of iron-sulphur (Fe-S) centers under NO stress, CyaY, was significantly downregulated in the AHL producing wild type. This hints to a possible route of N2OR-suppression via compromised Fe-S centers in the ancillary protein NosR. While the exact mechanism remains obscure, it appears that quorum sensing driven restriction of N2OR activity is common. Thus, given its ubiquity among prokaryotes, and the potential for cross-species and -strain effects, quorum sensing is plausibly a driver of N2O emissions in a range of systems.

A bet-hedging strategy for denitrifying bacteria curtails their release of N2O.

When oxygen becomes limiting, denitrifying bacteria must prepare for anaerobic respiration by synthesizing the reductases NAR (NO3- → NO2-), NIR (NO2- → NO), NOR (2NO → N2O), and NOS (N2O → N2), either en bloc or sequentially, to avoid entrapment in anoxia without energy. Minimizing the metabolic burden of this precaution is a plausible fitness trait, and we show that the model denitrifier Paracoccus denitrificans achieves this by synthesizing NOS in all cells, while only a minority synthesize NIR. Phenotypic diversification with regards to NIR is ascribed to stochastic initiation of gene transcription, which becomes autocatalytic via NO production. Observed gas kinetics suggest that such bet hedging is widespread among denitrifying bacteria. Moreover, in response to oxygenation, P. denitrificans preserves NIR in the poles of nongrowing persister cells, ready to switch to anaerobic respiration in response to sudden anoxia. Our findings add dimensions to the regulatory biology of denitrification and identify regulatory traits that decrease N2O emissions.

Investigation of the essentiality of glutamate racemase in Mycobacterium smegmatis.

The mycobacterial cell wall frequently has been used as a target for drug development, and d-glutamate, synthesized by glutamate racemase (MurI), is an important component of peptidoglycan. While the essentiality of the murI gene has been shown in several bacterial species, including Escherichia coli, Bacillus anthracis, and Streptococcus pneumoniae, studies in mycobacteria have not yet provided definitive results. This study aimed to determine whether murI is indeed essential and can serve as a possible target for structure-aided drug design. We have achieved this goal by creating a ΔmurI strain of Mycobacterium smegmatis, a close relative of Mycobacterium tuberculosis. The deletion of the murI gene in M. smegmatis could be achieved only in minimal medium supplemented with D-glutamate, demonstrating that MurI is essential for growth and that glutamate racemase is the only source of D-glutamate for peptidoglycan synthesis in M. smegmatis.

Headspace analysis of Italian and New Zealand parmesan cheeses.

UNLABELLED: New Zealand is a leader in the global dairy industry. Milk powder is the principal export product, but there is also a prominent cheese manufacturing industry, catering more for the domestic market. The Selected Ion Flow Tube-Mass Spectrometric (SIFT-MS) technique was used to compare 4 New Zealand cheeses marketed as "parmesan" with 4 Italian Parmigiano Reggiano and Grana Padano cheeses. The cheese headspace was analyzed in real time without any sample preconcentration. Total of 38 volatile compounds in the cheese headspace were monitored with headspace concentrations varying between single digit parts per billion (ppb) to tens of parts per million (ppm). When the results were subjected to multivariate statistical analysis, a clear discrimination was found between the New Zealand "parmesan" and Italian cheeses based solely on the measured concentrations of these volatile compounds. If the volatile compounds used in the analyses were restricted to known odor-active compounds in Parmigiano Reggiano cheese, the ability to discriminate between the cheeses was maintained. The analyses also showed that it was possible to clearly differentiate between the different processing plants in individual countries. Important discriminatory volatiles in the samples tested were butanoic acid and phenylacetaldehyde for discriminating between Italian cheeses and ethyl butyrate, acetaldehyde and methylbutanals between New Zealand cheeses. We conclude that the New Zealand "parmesans" do not provide a good representation of the aroma of Italian "parmesans." PRACTICAL APPLICATION: SIFT-MS has been shown to clearly differentiate both country of origin and the manufacturer of "parmesan" cheeses made in Italy and New Zealand based on differences in volatile organic compounds. Thus this method will have benefit for use in the quality control of "parmesan" and other cheese varieties.

Rate of gas phase association of hydroxyl radical and nitrogen dioxide.

The reaction of OH and NO(2) to form gaseous nitric acid (HONO(2)) is among the most influential in atmospheric chemistry. Despite its importance, the rate coefficient remains poorly determined under tropospheric conditions because of difficulties in making laboratory rate measurements in air at 760 torr and uncertainties about a secondary channel producing peroxynitrous acid (HOONO). We combined two sensitive laser spectroscopy techniques to measure the overall rate of both channels and the partitioning between them at 25°C and 760 torr. The result is a significantly more precise value of the rate constant for the HONO(2) formation channel, 9.2 (±0.4) × 10(-12) cm(3) molecule(-1) s(-1) (1 SD) at 760 torr of air, which lies toward the lower end of the previously established range. We demonstrate the impact of the revised value on photochemical model predictions of ozone concentrations in the Los Angeles airshed.

Monitoring chloramines and bromamines in a humid environment using selected ion flow tube mass spectrometry.

The selectivity and sensitivity of selected ion flow tube mass spectrometry (SIFT-MS) for individual breath analysis of haloamines has been improved by heating the flow tube in a commercial instrument to around 106 degrees C. Data is presented showing the marked reduction in the number density of water clusters of product ions of common breath metabolites that are isobaric with the product ions from monochloramine and monobromamine that are used to monitor the haloamine concentrations. These results have direct relevance to the real-time monitoring of chloramines in drinking water, swimming pools and food processing plants. However, once the isobaric overlaps from water cluster ions are reduced at the higher temperatures, there is no conclusive evidence showing the presence of haloamines on single breath exhalations in the mid parts per trillion range from examination of the breaths of volunteers.

Application of selected ion flow tube mass spectrometry to real-time atmospheric monitoring.

Data are presented for real-time atmospheric monitoring of volatile organic chemicals (VOCs) in air using selected ion flow tube mass spectrometry (SIFT-MS) technology. These measurements were made by one of the new generation of SIFT-MS instruments. Results are shown for five VOCs that were continually monitored from a stationary sampling point over a 4-day period: ethene, ethanol, 1,3-butadiene, benzene and toluene. All analytes except ethene in the study have at least two simultaneous and independent measures of concentration. These results demonstrate the great advances in SIFT-MS that have been made in recent years. 1,3-Butadiene is measured at a concentration of 9 pptv with a precision of 44%. For a 1-s integration time, a detection limit of 50 pptv is achieved. Instrument sensitivities are reported for all five analytes.

Real-time measurement of peroxyacetyl nitrate using selected ion flow tube mass spectrometry.

The on-line detection of gaseous peroxyacetyl nitrate (PAN) using selected ion flow tube mass spectrometry (SIFT-MS) has been investigated using a synthetic sample of PAN in air at a humidity of approximately 30%. Using the H(3)O(+) reagent ion, signals due to PAN at m/z 122, 77 and 95 have been identified. These correspond to protonated PAN, protonated peractetic acid and its water cluster, respectively. These products and their energetics have been probed through quantum mechanical calculations. The rate coefficient of H(3)O(+) has been estimated to be 4.5 x 10(-9) cm(3) s(-1), leading to a PAN sensitivity of 138 cps/ppbv. This gives a limit of detection of 20 pptv in 10 s using the [M+H](+) ion of PAN at m/z 122.

Detection and quantification of chemical warfare agent precursors and surrogates by selected ion flow tube mass spectrometry.

The rate coefficients and branching ratios of 15 chemical warfare agent precursor and surrogate compounds reacting with H(3)O(+), NO(+), and O(2)(+) have been measured in the laboratory using the selected ion flow tube (SIFT) technique. Measurement of the relevant kinetic parameters for these agents has enabled quantitative monitoring using the SIFT-MS analytical technique. Thirteen of the 15 compounds studied were found to have real-time detection limits in the parts-per-trillion-by-volume concentration range when measured on a standard commercial Voice100 instrument, with specific compounds having detection limits below 100 parts-per-trillion-by-volume.

Real-time monitoring of hazardous air pollutants.

Selected ion flow tube mass spectrometry (SIFT-MS) is a technique that offers real-time alternatives to existing methods for monitoring hazardous air pollutants (HAPs) in the environment using chemical ionization. The use of this technique requires knowledge of the kinetic parameters of the reagent ions H(3)O(+), NO(+), and O(2)(+) that are most commonly used. We report here measurements with these reagent ions of kinetic parameters for 17 HAP molecules ranging from 1,1-dichloroethene to nitrobenzene. From these data, limits of quantitation are established for all 17 compounds on a commercial SIFT-MS instrument and are found to be well below the time-weighted averages required by legislating bodies for workplace conditions.

Arylphthalazines as potent, and orally bioavailable inhibitors of VEGFR-2.

A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting materials, was found to be a potent inhibitor of VEGFR-2 in enzymatic, cellular and mitogenic assays (comparable activity to ZD-6474). Additionally, 57 inhibited the related receptor, VEGF receptor I (VEGFR-1), and showed excellent exposure when dosed orally to female CD-1 mice.

The alanine racemase of Mycobacterium smegmatis is essential for growth in the absence of D-alanine.

Alanine racemase, encoded by the gene alr, is an important enzyme in the synthesis of d-alanine for peptidoglycan biosynthesis. Strains of Mycobacterium smegmatis with a deletion mutation of the alr gene were found to require d-alanine for growth in both rich and minimal media. This indicates that alanine racemase is the only source of d-alanine for cell wall biosynthesis in M. smegmatis and confirms alanine racemase as a viable target gene for antimycobacterial drug development.

Gas-phase reactions and rearrangements of alkyl esters with H3O+, NO+, and O2*+: a selected ion flow tube study.

Selected ion flow tube mass spectrometry (SIFT-MS) has been employed to study the ion-molecule reactions of 17 alkyl esters reacting with the common SIFT-MS reagent ions, H3O+, H3O+.nH2O (n = 1, 2, 3), NO+, and O2+. The majority of reactions were observed to proceed at or near collision rate, with the exception of H3O+.3H2O, which was found to be slow for 8 of 17 alkyl esters. Unexpected product ions in the form of the parent carboxylic acid cation were observed to arise from the H3O+ and NO+ reactions of some alkyl esters. The observed reactions have been probed by the ab initio CBS-4M and G2(MP2,SVP) methods. The postulated reaction pathway involves a 1,5 H atom migration from a beta-carbon onto the carbonyl oxygen.

1H-1,2,4-triazol-3-yl-anilines: novel potent inhibitors of vascular endothelial growth factor receptors 1 and 2.

Novel derivatives of 1,2,4-triazoles are described as potent ATP-competitive inhibitors of vascular endothelial growth factor receptors I and II (VEGFR-1/2). A number of compounds display VEGFR-2 inhibitory activity comparable to that of Vatalanib and Vandetanib in both homogenous time-resolved fluorescence enzymatic and cellular assays. Several active molecules feature high intrinsic permeability (>30 x 10(-5) cm/min) across Caco-2 cell monolayer.

Novel inhibitors of VEGF receptors-1 and -2 based on azole-5-carboxamide templates.

We have developed a series of novel potent 1-(2-(pyridin-4-yl)ethyl)-1H-azole-5-carboxamides active against kinases VEGFR-2 and -1. Both specific and dual ATP-competitive inhibitors of VEGFR-2 were identified. Kinase selectivity could be controlled by varying the 5-carboxamide substituent at the azole ring. The most specific molecules displayed >10-fold selectivity for VEGFR-2 over VEGFR-1. Compound activities in vitro and in cell-based assays (IC(50)<100 nM) were similar to those of reported clinical and development candidates, including PTK787 (Vatalanib(trade)) and ZD6474 (Vandetanib(trade mark)). High permeability of active compounds across the Caco-2 cell monolayer (>40 x 10(-5)cm/min) is indicative of their potential for intestinal absorption upon oral administration.

Demonstration of selected ion flow tube MS detection in the parts per trillion range.

The rate coefficients of the ion-molecule reactions between H3O+, NO+, O2+, and phosphine were determined using a selected ion flow tube. Using these data, the selected ion flow tube mass spectrometry (SIFT-MS) method was applied to the real-time measurement of phosphine in nitrogen without sample preparation down to concentrations in the mid parts per trillion range. This is the first reported measurement using SIFT-MS in the parts per trillion range. Linear dependencies on concentration were found from 190 ppt to the ppm range, and the limit of detection for a 10-s scan was 190 ppt (0.27 pg/mL).

4-(Azolylphenyl)-phthalazin-1-amines: Novel inhibitors of VEGF receptors I and II.

Novel potent derivatives of phthalazine are described as ATP-competitive inhibitors of vascular endothelial growth factor receptors I and II (VEGFR-1/2). A number of compounds display VEGFR-2 inhibitory activity reaching that of Vatalanib 3 (IC50 < 100 nm) in an HTRF enzymatic assay. Several derivatives also show good potential for the development as VEGFR-2 specific inhibitors showing 15-20-fold selectivity over VEGFR-1.

Novel tricyclic azepine derivatives: Biological evaluation of pyrimido[4,5-b]-1,4-benzoxazepines, thiazepines, and diazepines as inhibitors of the epidermal growth factor receptor tyrosine kinase.

Novel tricyclic derivatives containing an oxazepine, thiazepine, or diazepine ring were studied for their EGFR tyrosine kinase inhibitory activity. While the oxazepines were in general more potent than thiazepines, the diazepines displayed somewhat different structure-activity relationships. Moreover, the diazepines, in contrast to the oxazepines, showed appreciable inhibitory activity against the KDR tyrosine kinase. Furthermore, both oxazepines and diazepines demonstrated significant ability to inhibit autophosphorylation of EGFR in DiFi cells (generally, IC(50) values in the single-digit micromolar to submicromolar range).

Inhibitors of VEGF receptors-1 and -2 based on the 2-((pyridin-4-yl)ethyl)pyridine template.

We have developed a series of novel potent ((pyridin-4-yl)ethyl)pyridine derivatives active against kinases VEGFR-1 and -2. Both specific and dual ATP-competitive inhibitors of VEGFR-2 were identified. Kinase selectivity could be controlled by varying the arylamino substituent at the 1,3,4-oxadiazole ring. The most specific molecules displayed >10-fold selectivity for VEGFR-2 over VEGFR-1. Compound activities in vitro and in cell-based assays (IC(50)<100nM) were similar to those of reported clinical and development candidates, including PTK787 (Vatalanibtrade mark). High permeability of active compounds across the Caco-2 cell monolayer (>30x10(-5)cm/min) is indicative of their potential for intestinal absorption upon oral administration.

Arylphthalazines. Part 2: 1-(Isoquinolin-5-yl)-4-arylamino phthalazines as potent inhibitors of VEGF receptors I and II.

A novel class of 1-(isoquinolin-5-yl)-4-arylamino-phthalazines is described as inhibitors of vascular endothelial growth factor receptor II (VEGFR-2). Many compounds display VEGFR-2 inhibitory activity with an IC(50) as low as 0.017 microM in an HTRF enzymatic assay. The compounds also inhibit VEGFR-1, a related tyrosine kinase.