Isolation of 'Candidatus Nitrosocosmicus franklandus', a novel ureolytic soil archaeal ammonia oxidiser with tolerance to high ammonia concentration

Acknowledgements. The authors would like to thank Mr Kevin Mackenzie and Mrs Gillian Milne (University of Aberdeen) for technical support with scanning electron microscopy, and Dr Robin Walker for access to the Woodlands Field experimental plots at the SRUC,Craibstone Estate, Aberdeen. This work was financially supported by Natural Environmental Research Council (standard grants NE/I027835/1 and NE/L006286/1 and fellowship NE/J019151/1), EC Marie Curie ITN NORA, Grant Agreement No. 316472, the AXA Research Fund and the Centre for Genome Enabled Biology and Medicine, University of Aberdeen.

A different approach to treatment of phenylketonuria: Phenylalanine degradation with recombinant phenylalanine ammonia lyase

Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N′-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)—both pharmacologic (with a clear dose–response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease.

Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis

We have spectroscopically determined breath ammonia levels in seven patients with end-stage renal disease while they were undergoing hemodialysis at the University of California, Los Angeles, dialysis center. We correlated these measurements against simultaneously taken blood samples that were analyzed for blood urea nitrogen (BUN) and creatinine, which are the accepted standards indicating the level of nitrogenous waste loading in a patient's bloodstream. Initial levels of breath ammonia, i.e., at the beginning of dialysis, are between 1,500 ppb and 2,000 ppb (parts per billion). These levels drop very sharply in the first 15–30 min as the dialysis proceeds. We found the reduction in breath ammonia concentration to be relatively slow from this point on to the end of dialysis treatment, at which point the levels tapered off at 150 to 200 ppb. For each breath ammonia measurement, taken at 15–30 min intervals during the dialysis, we also sampled the patient's blood for BUN and creatinine. The breath ammonia data were available in real time, whereas the BUN and creatinine data were available generally 24 h later from the laboratory. We found a good correlation between breath ammonia concentration and BUN and creatinine. For one of the patients, the correlation gave an R2 of 0.95 for breath ammonia and BUN correlation and an R2 of 0.83 for breath ammonia and creatinine correlation. These preliminary data indicate the possibility of using the real-time breath ammonia measurements for determining efficacy and endpoint of hemodialysis.

Ozone Sensitivity in Hybrid Poplar Is Correlated with a Lack of Defense-Gene Activation1

Ozone is a major gaseous pollutant thought to contribute to forest decline. Although the physiological and morphological responses of forest trees to ozone have been well characterized, little is known about the molecular basis for these responses. Our studies compared the response to ozone of ozone-sensitive and ozone-tolerant clones of hybrid poplar (Populus maximowizii × Populus trichocarpa) at the physiological and molecular levels. Gas-exchange analyses demonstrated clear differences between the ozone-sensitive clone 388 and the ozone-tolerant clone 245. Although ozone induced a decrease in photosynthetic rate and stomatal conductance in both clones, the magnitude of the decrease in stomatal conductance was significantly greater in the ozone-tolerant clone. RNA-blot analysis established that ozone-induced mRNA levels for phenylalanine ammonia-lyase, O-methyltransferase, a pathogenesis-related protein, and a wound-inducible gene were significantly higher in the ozone-tolerant than in the ozone-sensitive plants. Wound- and pathogen-induced levels of these mRNAs were also higher in the ozone-tolerant compared with the ozone-sensitive plants. The different physiological and molecular responses to ozone exposure exhibited by clones 245 and 388 suggest that ozone tolerance involves the activation of salicylic-acid- and jasmonic-acid-mediated signaling pathways, which may be important in triggering defense responses against oxidative stress.

Accumulation of Salicylic Acid and 4-Hydroxybenzoic Acid in Phloem Fluids of Cucumber during Systemic Acquired Resistance Is Preceded by a Transient Increase in Phenylalanine Ammonia-Lyase Activity in Petioles and Stems1

Cucumber (Cucumis sativa) leaves infiltrated with Pseudomonas syringae pv. syringae cells produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation. The production of a mobile signal by inoculated leaves was followed by a transient increase in phenylalanine ammonia-lyase (PAL) activity in the petioles of inoculated leaves and in stems above inoculated leaves; with peaks in activity at 9 and 12 h, respectively, after inoculation. In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h. No increases in PAL activity were detected in healthy leaves of inoculated plants. Two benzoic acid derivatives, salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation. The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation, and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA. These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf.

The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

Phenylalanine ammonia-lyase (EC 4.3.1.5) from parsley is posttranslationally modified by dehydrating its Ser-202 to the catalytically essential dehydroalanine prosthetic group. The codon of Ser-202 was changed to those of alanine and threonine by site-directed mutagenesis. These mutants and the recombinant wild-type enzyme, after treatment with sodium borohydride, were virtually inactive with L-phenylalanine as substrate but catalyzed the deamination of L-4-nitrophenylalanine, which is also a substrate for the wild-type enzyme. Although the mutants reacted about 20 times slower with L-4-nitrophenylalanine than the wild-type enzyme, their Vmax for L-4-nitrophenylalanine was two orders of magnitude higher than for L-phenylalanine. In contrast to L-tyrosine, which was a poor substrate, DL-3-hydroxyphenylalanine (DL-m-tyrosine) was converted by phenylalanine ammonia-lyase at a rate comparable to that of L-phenylalanine. These results suggest a mechanism in which the crucial step is an electrophilic attack of the prosthetic group at position 2 or 6 of the phenyl group. In the resulting carbenium ion, the beta-HSi atom is activated in a similar way as it is in the nitro analogue. Subsequent elimination of ammonia, concomitant with restoration of both the aromatic ring and the prosthetic group, completes the catalytic cycle.

Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley.

We describe a complete gene family encoding phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in one particular plant species. In parsley (Petroselinum crispum), the PAL gene family comprises two closely related members, PAL1 and PAL2, whose TATA-proximal promoter and coding regions are almost identical, and two additional members, PAL3 and PAL4, with less similarity to one another and to the PAL1 and PAL2 genes. Using gene-specific probes derived from the 5' untranslated regions of PAL1/2, PAL3, and PAL4, we determined the respective mRNA levels in parsley leaves and cell cultures treated with UV light or fungal elicitor and in wounded leaves and roots. For comparison, the functionally closely related cinnamate 4-hydroxylase (C4H) and 4-coumarate:CoA ligase (4CL) mRNAs were measured in parallel. The results indicate various degrees of differential responsiveness of PAL4 relative to the other PAL gene family members, in contrast to a high degree of coordination in the overall expression of the PAL, C4H, and 4CL genes. The only significant sequence similarities shared by all four PAL gene promoters are a TATA-proximal set of three putative cis-acting elements (boxes P, A, and L). None of these elements alone, or the promoter region containing all of them together, conferred elicitor or light responsiveness on a reporter gene in transient expression assays. The elements appear to be necessary but not sufficient for elicitor- or light-mediated PAL gene activation, similar to the situation previously reported for 4CL.

Ammonia observations of the nearby molecular cloud MBM 12

We present NH_3(1,1) and (2,2) observations of MBM 12, the closest known molecular cloud (65-pc distance), aimed at finding evidence for on-going star formation processes, No local temperature (with a T_rot upper limit of 12 K) or linewidth enhancement is found, which suggests that the area of the cloud that we have mapped (∼ 15-arcmin size) is not currently forming stars. Therefore this nearby 'starless' molecular gas region is an ideal laboratory to study the physical conditions preceding new star formation. A radio continuum source has been found in Very Large Array archive data, close to but outside the NH_3 emission. This source is likely to be a background object.

Ammonia removal using activated carbons: effect of the surface chemistry in dry and moist conditions

The effect of surface chemistry (nature and amount of oxygen groups) in the removal of ammonia was studied using a modified resin-based activated carbon. NH3 breakthrough column experiments show that the modification of the original activated carbon with nitric acid, that is, the incorporation of oxygen surface groups, highly improves the adsorption behavior at room temperature. Apparently, there is a linear relationship between the total adsorption capacity and the amount of the more acidic and less stable oxygen surface groups. Similar experiments using moist air clearly show that the effect of humidity highly depends on the surface chemistry of the carbon used. Moisture highly improves the adsorption behavior for samples with a low concentration of oxygen functionalities, probably due to the preferential adsorption of ammonia via dissolution into water. On the contrary, moisture exhibits a small effect on samples with a rich surface chemistry due to the preferential adsorption pathway via Brønsted and Lewis acid centers from the carbon surface. FTIR analyses of the exhausted oxidized samples confirm both the formation of NH4+ species interacting with the Brønsted acid sites, together with the presence of NH3 species coordinated, through the lone pair electron, to Lewis acid sites on the graphene layers.

Chemical characterization of emissions from a municipal solid waste treatment plant

Gaseous emissions are an important problem in municipal solid waste (MSW) treatment plants. The sources points of emissions considered in the present work are: fresh compost, mature compost, landfill leaks and leachate ponds. Hydrogen sulphide, ammonia and volatile organic compounds (VOCs) were analysed in the emissions from these sources. Hydrogen sulphide and ammonia were important contributors to the total emission volume. Landfill leaks are significant source points of emissions of H2S; the average concentration of H2S in biogas from the landfill leaks is around 1700 ppmv. The fresh composting site was also an important contributor of H2S to the total emission volume; its concentration varied between 3.2 and 1.7 ppmv and a decrease with time was observed. The mature composting site showed a reduction of H2S concentration (<0.1 ppmv). Leachate pond showed a low concentration of H2S (in order of ppbv). Regarding NH3, composting sites and landfill leaks are notable source points of emissions (composting sites varied around 30–600 ppmv; biogas from landfill leaks varied from 160 to 640 ppmv). Regarding VOCs, the main compounds were: limonene, p-cymene, pinene, cyclohexane, reaching concentrations around 0.2–4.3 ppmv. H2S/NH3, limonene/p-cymene, limonene/cyclohexane ratios can be useful for analysing and identifying the emission sources.