Exogenous Ammonia Research Articles

A fluorescent probe with large Stokes for rapid detection of ammonia based on BINOL

Ammonia is a common toxic gas in industrial and agricultural production, which will cause harm to the environment and human health in certain extent. Here, a novel fluorescent probe (R)-1 based on 1,1′-binaphthol (BINOL) was synthesized for real-time, on-site and quantitative detection of ammonia. The mechanism study shows that the functional groups of –CHO and –OH on (R)-1 interact with ammonia, which changes the electronic state of the probe molecule and excites strong fluorescence. A test strip for rapid detection of ammonia was developed, which changed from light yellow to reddish brown under natural light after interacting with ammonia, thus realizing the rapid detection and easy visualization of ammonia. Meanwhile, bioimaging experiments shows that probe (R)-1 can detect exogenous ammonia in cells.

Biochar facilitates methanogens evolution by enhancing extracellular electron transfer to boost anaerobic digestion of swine manure under ammonia stress

This study explored the mechanisms by which biochar mitigates ammonia inhibition in anaerobic digestion (AD) of swine manure. Findings show 2–8 g/L exogenous ammonia dosages gradually inhibited AD, leading to decreases in the efficiencies of hydrolysis, acidogenesis and methanogenesis by 3.4–70.8%, 6.0–82.0%, and 4.9–93.8%, respectively. However, biochar addition mitigated this inhibition and facilitated methane production. Biochar enhanced microbial activities related to electron transport and extracellular electron transfer. Moreover, biochar primarily enriched Methanosarcina, which, consequently, upregulated the genes encoding formylmethanofuran dehydrogenase and methenyltetrahydromethanopterin cyclohydrolase for the CO2-reducing methanogenesis pathway by 26.9–40.8%. It is believed that biochar mediated direct interspecies electron transfer between syntrophic partners, thereby enhancing methane production under ammonia stress. Interestingly, biochar removal did not significantly impact the AD performance of the acclimated microbial community. This indicated the pivotal role of biochar in triggering methanogen evolution to mitigate ammonia stress rather than the indispensable function after the enrichment of ammonia-resistance methanogen.

A nitrifier‐enriched microbial community contributes to the degradation of environmental DNA

AbstractEnvironmental DNA (eDNA) surveys are a promising alternative to traditional monitoring of invasive species, rare species, and biodiversity. Detecting organism‐specific eDNA reduces the need to collect physical specimens for population estimates, and the high sensitivity of eDNA assays may improve detection of rare or cryptic species. However, correlating estimated concentrations of eDNA with species abundance can be difficult due to the many abiotic and biotic factors that influence eDNA persistence and degradation. Here, we assessed the impact of a nitrifier‐enriched microbial (NEM) community on the persistence and degradation of Hypophthalmichthys molitrix (silver carp) milt eDNA using experimental aquatic mesocosms and a quantitative PCR approach. The NEM community was cultured from combined sediment and water samples collected from a golf course pond in Columbia, Missouri (USA), and experiments were conducted in the dark at 22°C. We found that the NEM community transformed organic nitrogen from silver carp milt to measurable amounts of nitrate, both in the presence and absence of ammonia nitrogen. Additionally, regardless of ammonia availability, milt eDNA followed a one‐phase exponential decay pattern after an initial 24‐h plateau in the presence of the NEM community. However, milt eDNA had a shorter half‐life (12.5 h) in the absence of exogenous ammonia compared to when ammonia was present (15 h). In sterile mesocosms, eDNA was stable during the 72‐h experiment. Together, these results suggest that the presence of microorganisms is necessary for short‐term degradation of eDNA. Furthermore, nitrifying microbial communities, which are ubiquitous in most soil and water environments, could limit eDNA persistence in the environment. Understanding the contributions of environmental microbial communities will allow more confidence in sampling design and eDNA result interpretations for biodiversity management applications.

In Streptococcus thermophilus, Ammonia from Urea Hydrolysis Paradoxically Boosts Acidification and Reveals a New Regulatory Mechanism of Glycolysis.

ABSTRACTStreptococcus thermophilus is widely used in the dairy industry for the manufacturing of fermented milk and cheeses and probiotic formulations. S. thermophilus evolved from closely phylogenetically related pathogenic streptococci through loss-of-function events counterbalanced by the acquisition of relevant traits, such as lactose and urea utilization for the adaptation to the milk environment. In the context of regressive evolution, the urease gene cluster accounts for 0.9% of the total coding sequence belonging to known functional categories. The fate of ammonia and carbon dioxide derived by urea hydrolysis in several biosynthetic pathways have been depicted, and the positive effect of urease activity on S. thermophilus growth fitness and lactic acid fermentation in milk has been already addressed by several authors. However, the mechanistic effect of urea hydrolysis on the energetic metabolisms of S. thermophilus is still unclear. This study aimed to assess the effect of urease activity on the growth and energy metabolism of Streptococcus thermophilus in milk. In milk, 13C-urea was completely hydrolyzed in the first 150 min of S. thermophilus growth, and urea hydrolysis was accompanied by an increase in cell density and a reduction in the generation time. By using energetically discharged cells with gene transcription and translation blocked, we showed that in the presence of fermentable carbon sources, urease activity, specifically the production of ammonia, could dramatically boost glycolysis and, in cascade, homolactic fermentation. Furthermore, we showed that ammonia, specifically ammonium ions, were potent effectors of phosphofructokinase, a key glycolytic enzyme.IMPORTANCE Finding that ammonia-generating enzymes, such as urease, and exogenous ammonia act on phosphofructokinase activity shed new light on the regulatory mechanisms that govern glycolysis. Phosphofructokinase is the key enzyme known to exert a regulatory role on glycolytic flux and, therefore, ammonia as an effector of phosphofructokinase acts, in cascade, modulating the glycolytic pathway. Apart from S. thermophilus, due to the high conservation of glycolytic enzymes in all branches of the tree of life and being aware of the role of ammonia as an effector of phosphofructokinase, we propose to reevaluate the physiological role of the ammonia production pathways in all organisms whose energy metabolism is supported by glycolysis.

Symbiotic Dinoflagellates of the Giant Clam, Tridacna squamosa, Express Ammonium Transporter 2 at the Plasma Membrane and Increase Its Expression Levels During Illumination

Giant clams harbor dinoflagellates generally of the three genera (Symbiodinium, Cladocopium, and Durusdinium) of phototrophic Symbiodiniaceae. Coccoid dinoflagellates (alias zooxanthellae) are found mainly inside zooxanthellal tubules located in the colorful outer mantle. The symbionts need to obtain carbon, nitrogen and phosphorus from the host for growth and metabolism. The host can absorb exogenous ammonia through the ctenidium and assimilate it into glutamine. Although the host does not normally excrete ammonia, its hemolymph contains only low concentrations of ammonia, indicating that the symbionts can absorb and recycle the ammonia produced metabolically by the host. In this study, we had obtained from the outer mantle of the giant clam, Tridacna squamosa, three major ammonium transporter 2 (AMT2) sequences, one each for Symbiodinium spp. (Symb-AMT2), Cladocopium spp. (Clad-AMT2), and Durusdinium spp. (Duru-AMT2), which comprised 1341 bp, 1308 bp, and 1296 bp, respectively. The respective deduced amino acid sequences contained 447 (~ 46.5 kDa), 436 (~ 45.5 kDa), and 432 (~ 45.0 kDa) residues. Phenogramic and sequence similarity analyses confirmed that these sequences were derived from dinoflagellates. Zooxanthellae-AMT2 (Zoox-AMT2), which represented comprehensively AMT2 of Symbiodinium spp., Cladocopium spp., and Durusdinium spp. was localized at the dinoflagellates’ plasma membranes, indicating that it could partake in the absorption of ammonia from the luminal fluid of the zooxanthellal tubules. Zoox-AMT2 expression was detected in the outer mantle, inner mantle, foot muscle, hepatopancreas and ctenidium of T. squamosa, indicating that the coccoid dinoflagellates residing in all five organs had the potential of ammonia absorption. The outer mantle had the highest transcript level of Zoox-AMT2, and illumination upregulated the protein abundance of Zoox-AMT2 therein. Therefore, it can be deduced that the coccoid dinoflagellates residing in the outer mantle could augment the potential of ammonia absorption in alignment with photosynthesis as the assimilation of ammonia required an increased supply of carbon chains.

Ammonia exposure causes the imbalance of the gut-brain axis by altering gene networks associated with oxidative metabolism, inflammation and apoptosis

Ammonia is an acknowledged environment pollutant in atmosphere with irritating smell. Previous studies have shown that excessive ammonia has toxic effects on farm animals and humans. However, the detail toxicity mechanism of ammonia to pigs is still unknown so far. In order to clarify the mechanism of ammonia toxicity, we established a porcine exogenous ammonia poisoning model and assessed the effects of ammonia on the gut-brain axis by transcriptome sequencing, histological observation and chemical analysis. Our results showed that after 30 d of ammonia exposure, 578 differentially expressed genes (DEGs) and 407 DEGs were obtained in the hypothalamus and jejunum, respectively. These DEGs were enriched into Gene Ontology terms associated with inflammation, oxidative metabolism, apoptosis, and the highly expressed genes among these DEGs were verified by real-time quantitative PCR. The content of glutathione and the activities of glutathione peroxidase and superoxide dismutase were significantly decreased, while malondialdehyde content was increased after ammonia exposure. Corticotropin releasing factor, substance P, 5-hydroxytryptamine and ghrelin contents in serum elevated significantly. Furthermore, pathologic observation in the ammonia group revealed infiltration of lymphocytes in the hypothalamus and significant decrease of jejunal epithelial cells. Our results indicated that ammonia exposure mediated changes in transcriptional profiles, pathological damage, oxidative stress and brain-gut peptide of the pig jejunum and hypothalamus, and induced the imbalance of the brain-gut axis through the “oxidative stress-inflammation-apoptosis” interaction network. Our study not only provides a new perspective for the toxicity assessment of ammonia, but also enriches the toxicology mechanism of ammonia.

Ammonia induces Treg/Th1 imbalance with triggered NF-κB pathway leading to chicken respiratory inflammation response

Ammonia (NH3) is a severe air pollutant and a component of haze (PM2.5). The respiratory tract is the first route of exogenous ammonia's entry into the human and animal body. Many studies have suggested that exposure to NH3 is associated with a higher risk of respiratory tract tracheitis; however, the underlying mechanism remains unclear. In this study, chicken tracheas were used as a model to investigate toxic effects and genetic changes induced by NH3 exposure, as evaluated by scanning electron microscopy (SEM) and bioinformatic analyses. The transcript analysis illustrated that NH3 exposure caused immune disorders, which play key roles in regulating inflammatory responses from NH3 exposure. Therefore, we carried out Real-time quantitative PCR (RT-PCR) and Western Blot analyses to detect the immune response genes; Treg/Th1, Th2 and Th17 secretions were found that led to inflammatory responses. Next, we also detected the NF-κB pathway and downstream genes, accompanied by cytochromes P450 (CYPs), antioxidative genes, and heat shock proteins (HSPs). Our results are consistent with transcriptome detection, indicating that ammonia has a negative effect on immune responses and causes inflammatory injury of the trachea. This study provided baseline information for exploration of the molecular mechanism of NH3-PM2.5 induced respiratory diseases.

The ctenidium of the giant clam, Tridacna squamosa, expresses an ammonium transporter 1 that displays light-suppressed gene and protein expression and may be involved in ammonia excretion

Ammonium transporters (AMTs) can participate in ammonia uptake or excretion across the plasma membrane of prokaryotic, plant and invertebrate cells. The giant clam, Tridacna squamosa, harbors nitrogen-deficient symbiotic zooxanthellae, and normally conducts light-enhanced ammonia absorption to benefit the symbionts. Nonetheless, it can excrete ammonia when there is a supply of exogenous nitrogen or exposed to continuous darkness. This study aimed to elucidate the role of AMT1 in the ctenidium of T. squamosa by cloning and characterizing the AMT1/AMT1, determining its subcellular localization, and examining changes in its transcript and protein expression levels in response to light exposure. The cDNA coding sequence of AMT1 from T. squamosa consisted of 1527bp and encoded 508 amino acids of 54.6kDa. AMT1-immunofluorescence was detected mainly at the apical epithelium of ctenidial filaments, and it decreased significantly after 12h of exposure to light. By contrast, the epithelial cells surrounding the tertiary water channels in the ctentidium, which are known to exhibit light-enhanced glutamine synthetase expression and take part in the assimilation of exogenous ammonia in light, did not display any AMT1-immunolabelling. Furthermore, the transcript level and protein abundance of ctenidial AMT1/AMT1 decreased significantly at the 6th and 12th h of light exposure. Taken together, these results indicate that AMT1 might participate in ammonia excretion instead of ammonia absorption and assimilation in T. squamosa. It is probable that the expression levels of AMT1/AMT1 need to be down-regulated during light exposure to achieve light-enhanced ammonia uptake.

Ammonia modifies enteric neuromuscular transmission through glial γ-aminobutyric acid signaling.

Impaired gut motility may contribute, at least in part, to the development of systemic hyperammonemia and systemic neurological disorders in inherited metabolic disorders, or in severe liver and renal disease. It is not known whether enteric neurotransmission regulates intestinal luminal and hence systemic ammonia levels by induced changes in motility. Here, we propose and test the hypothesis that ammonia acts through specific enteric circuits to influence gut motility. We tested our hypothesis by recording the effects of ammonia on neuromuscular transmission in tissue samples from mice, pigs, and humans and investigated specific mechanisms using novel mutant mice, selective drugs, cellular imaging, and enzyme-linked immunosorbent assays. Exogenous ammonia increased neurogenic contractions and decreased neurogenic relaxations in segments of mouse, pig, and human intestine. Enteric glial cells responded to ammonia with intracellular Ca2+ responses. Inhibition of glutamine synthetase and the deletion of glial connexin-43 channels in hGFAP::CreERT2+/-/connexin43f/f mice potentiated the effects of ammonia on neuromuscular transmission. The effects of ammonia on neuromuscular transmission were blocked by GABAA receptor antagonists, and ammonia drove substantive GABA release as did the selective pharmacological activation of enteric glia in GFAP::hM3Dq transgenic mice. We propose a novel mechanism whereby local ammonia is operational through GABAergic glial signaling to influence enteric neuromuscular circuits that regulate intestinal motility. Therapeutic manipulation of these mechanisms may benefit a number of neurological, hepatic, and renal disorders manifesting hyperammonemia.NEW & NOTEWORTHY We propose that local circuits in the enteric nervous system sense and regulate intestinal ammonia. We show that ammonia modifies enteric neuromuscular transmission to increase motility in human, pig, and mouse intestine model systems. The mechanisms underlying the effects of ammonia on enteric neurotransmission include GABAergic pathways that are regulated by enteric glial cells. Our new data suggest that myenteric glial cells sense local ammonia and directly modify neurotransmission by releasing GABA.

Design, synthesis, and evaluation of substituted nicotinamide adenine dinucleotide (NAD+) synthetase inhibitors as potential antitubercular agents

Nicotinamide adenine dinucleotide (NAD+) synthetase catalyzes the last step in NAD+ biosynthesis. Depletion of NAD+ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD+ synthetase (NadE) from Mtb, we expect to eliminate NAD+ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure–activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC50=1000µM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC50 value of 90µM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19–100µg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.

Reversible Gas-Solid Ammonia N-H Bond Activation Mediated by an Organopalladium Complex.

N-H bond activation of gaseous ammonia is achieved at room temperature in a reversible solvent-free reaction using a solid dicyclopalladated azobenzene complex. Monitoring of the gas-solid reaction in real-time by in situ solid-state Raman spectroscopy enabled a detailed insight into the stepwise activation pathway proceeding to the final amido complex via a stable diammine intermediate. Gas-solid synthesis allowed for isolation and subsequent structural characterization of the intermediate and the final amido product, which presents the first dipalladated complex with the PdII-(μ-NH2)-PdII bridge. Gas-solid reaction is readily followed via color changes associated with conformational switching of the palladated azobenzene backbone. The reaction proceeds analogously in solution and was characterized by UV-vis and NMR spectroscopies showing the same stepwise route to the amido complex. Combining the experimental data with density functional theory calculations we propose a stepwise mechanism of this heterolytic N-H bond activation assisted by exogenous ammonia.

A light-dependent ammonia-assimilating mechanism in the ctenidia of a giant clam

Giant clams harbor symbiotic zooxanthellae and manifest light-enhanced calcification. In light, they also increase the absorption and assimilation of exogenous ammonia, but the roles of the host and symbionts are unclear and the reason for light dependency remains enigmatic. Here, we report the complete coding cDNA sequence of a glutamine synthetase (GS), from the ctenidia (gill) of Tridacna squamosa. The deduced GS amino acid sequence was closely related to other animal GSs, indicating a host origin. The GS/GS transcript level and protein abundance increased significantly in the ctenidia after 12 h of light exposure. These results denote the ctenidia as the site of light-enhanced ammonia absorption with the absorbed ammonia being assimilated to glutamine by the host GS. Immunofluorescence microscopy confirmed that GS was expressed predominantly in the cytoplasm of the epithelial cells separating the tertiary water channels from the hemolymph inside the ctenidia. Hence, unlike symbiotic cnidarians, the host clam probably supplies exogenous nitrogen as glutamine from the ctenidia, through the hemolymph and tubular fluid, to the nitrogen-deficient zooxanthellae which reside extracellularly in the extensible outer mantle. This is the first report on light-dependent gene and protein expression of a host enzyme involved in nitrogen metabolism in an alga–invertebrate association.

Differential Activation of Ammonium Transporters During the Accumulation of Ammonia by Colletotrichum gloeosporioides and Its Effect on Appressoria Formation and Pathogenicity

Ammonium secreted by the post-harvest pathogen Colletotrichum gloeosporioides during host colonization accumulates in the host environment due to enhanced fungal nitrogen metabolism. Two types of ammonium transporter-encoding genes, AMET and MEP, are expressed during pathogenicity. Gene disruption of AMET, a gene modulating ammonia secretion, showed twofold reduced ammonia secretion and 45% less colonization on avocado fruit, suggesting a contribution to pathogenicity. MEPB, a gene modulating ammonium transport, is expressed by C. gloeosporioides during pathogenicity and starvation conditions in culture. Gene disruption of MEPB, the most highly expressed gene of the MEP family, resulted in twofold overexpression of MEPA and MEPC but reduced colonization, suggesting MEPB expression's contribution to pathogenicity. Analysis of internal and external ammonia accumulation by ΔmepB strains in mycelia and germinated spores showed rapid uptake and accumulation, and reduced secretion of ammonia in the mutant versus wild-type (WT) strains. Ammonia uptake by the WT germinating spores but not by the ΔmepB strain with compromised ammonium transport activated cAMP and transcription of PKA subunits PKAR and PKA2. ΔmepB mutants showed 75% less appressorium formation and colonization than the WT, which was partially restored by 10 mM exogenous ammonia. Thus, whereas both AMET and MEPB genes modulate ammonia secretion, only MEPB contributes to ammonia accumulation by mycelia and germinating spores that activate the cAMP pathways, inducing the morphogenetic processes contributing to C. gloeosporioides pathogenicity.

Effect of Different Ammonia Concentrations on Community Succession of Ammonia-oxidizing Microorganisms in a Simulated Paddy Soil Column

Ammonia oxidation is performed by both ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). To explore the effect of ammonia concentration on the population dynamic changes of ammonia-oxidizing microorganisms, we examined changes in the abundance and community composition of AOA and AOB in different layers. Most of the archaeal amoA sequences were Nitrosotalea-related and the proportion that Nitrosotalea cluster occupied decreased in the surface layer and increased in the deep layer during the cultivation process. Nitrosopumilus-related sequences were only detected in the deep layer in the first stage and disappeared later. Both phylogenetic and quantitative analysis showed that there were increased Nitrosomonas-related sequences appeared in the surface layer where the ammonia concentration was the highest. Both AOA and AOB OTU numbers in different layers decreased under selective pressure and then recovered. The potential nitrification rates were 25.06 µg·N·L−1·g−1 dry soil·h−1 in the mid layer which was higher than the other two layers. In general, obvious population dynamic changes were found for both AOA and AOB under the selective pressure of exogenous ammonia and the changes were different in three layers of the soil column.

Procurement of exogenous ammonia by the swallowtail butterfly, Papilio polytes, for protein biosynthesis and sperm production

How to acquire sufficient quantity of nitrogen is a pivotal issue for herbivores, particularly for lepidopterans (butterflies and moths) of which diet quality greatly differs among their life stages. Male Lepidoptera often feed from mud puddles, dung, and carrion, a behavior known as puddling, which is thought to be supplementary feeding targeted chiefly at sodium. During copulation, males transfer a spermatophore to females that contains, besides sperm, nutrients (nuptial gifts) rich in sodium, proteins, and amino acids. However, it is still poorly understood how adults, mostly nectarivores, extract nitrogen from the environment. We examined the availability of two ubiquitous inorganic nitrogenous ions in nature, viz. ammonium (or ammonia) and nitrate ions, as nutrients in a butterfly, and show that exogenous ammonia ingested by adult males of the swallowtail, Papilio polytes, can serve as a resource for protein biosynthesis. Feeding experiments with (15)N-labeled ammonium chloride revealed that nitrogen was incorporated into eupyrene spermatozoa, seminal protein, and thoracic muscle. Ammonia uptake by males significantly increased the number of eupyrene sperms in the reproductive tract tissues. The females also had the capacity to assimilate ammonia into egg protein. Consequently, it is evident that acquired ammonia is utilized for the replenishment of proteins allocable for reproduction and somatic maintenance. The active exploitation of exogenous ammonia as a nutrient by a butterfly would foster better understanding of the foraging and reproductive strategies in insects.

Effects of elevated ammonia concentrations on survival, metabolic rates, and glutamine synthetase activity in the Antarctic pteropod mollusk Clione limacina antarctica

Information on the effects of elevated ammonia on invertebrates in general, and polar Mollusks in particular, is scant. Questions of ammonia sensitivity are interesting for several reasons, particularly since predicted global change scenarios include increasing anthropogenic nitrogen and toxic ammonia. Furthermore, polar zooplankton species are often lipid-rich, and authors have speculated that there is a linkage between elevated levels of lipids/trimethylamine oxide and enhanced ammonia tolerance. In the present study, we sought to examine ammonia tolerance and effects of elevated exogenous ammonia on several key aspects of the physiology and biochemistry of the pteropod mollusk, Clione limacina antarctica. We determined that the 96-h LC50 value for this species is 7.465 mM total ammonia (Upper 95% CL = 8.498 mM and Lower 95% CL = 6.557 mM) or 0.51 mg/L as unionized ammonia (NH3) (at a pH of 7.756). While comparative data for mollusks are limited, this value is at the lower end of reported values for other species. When the effects of lower ammonia concentrations (0.07 mM total ammonia) on oxygen consumption and ammonia excretion rates were examined, no effects were noted. However, total ammonia levels as low as 0.1 mM (or 0.007 mg/l NH3) elevated the activity of the ammonia detoxification enzyme glutamine synthetase by approximately 1.5-fold. The values for LC50 and observable effects on biochemistry for this one species are very close to permissible marine ammonia concentrations, indicating a need to more broadly determine the sensitivity of zooplankton to potential elevated ammonia levels in polar regions.

Revisiting the effects of crowding and feeding in the gulf toadfish, Opsanus beta: the role of Rhesus glycoproteins in nitrogen metabolism and excretion

Models of branchial transport in teleosts have been reshaped by the recent discovery of Rhesus (Rh) glycoproteins, a family of proteins that facilitate the movement of NH(3) across cell membranes. This study examines the effects of crowding and feeding on ammonia excretion in gulf toadfish (Opsanus beta) within the context of Rh glycoproteins and the ammonia-fixing enzyme, glutamine synthetase (GS). Four Rh isoforms (Rhag, Rhbg, Rhcg1 and Rhcg2) were isolated from toadfish. Tissue distributions showed higher levels of mRNA expression in the gills and liver, moderate levels in the intestine and lower levels in the stomach. Crowding significantly lowered branchial Rh expression and ammonia excretion rates in fasted toadfish. A comparison of Rh expression in the digestive tract revealed relatively low levels of Rhcg1 and Rhcg2 in the stomach and high mRNA abundance of Rhbg, Rhcg1 and Rhcg2 in the intestine of fasted, crowded toadfish. We speculate that these trends may reduce secretion and enhance absorption, respectively, to minimize the amount of ammonia that is lost through gastrointestinal routes. By contrast, these patterns of expression were modified in response to an exogenous ammonia load via feeding. Post-prandial ammonia excretion rates were elevated twofold, paralleled by similar increases in branchial Rhcg1 mRNA, gastric Rhcg1 mRNA and mRNA of all intestinal Rh isoforms. These changes were interpreted as an attempt to increase post-prandial ammonia excretion rates into the environment owing to a gradient created by elevated circulating ammonia concentrations and acidification of the digestive tract. Overall, we provide evidence that toadfish modulate both the expression of Rh isoforms and urea synthesis pathways to tightly control and regulate nitrogen excretion.

Effects of acute exposure to exogenous ammonia on cerebral monoaminergic neurotransmitters in juvenile Solea senegalensis

The present study explored the potential role of brain catecholaminergic and serotoninergic systems as neuronal targets for the toxicological effects of acute ammonia exposure (0.28 mg l(-1) of un-ionized ammonia for 12 and 24 h) in juvenile sole (Solea senegalensis). In addition, plasma cortisol levels were measured. The results showed significant increases in their concentrations that were similar after 12 and 24 h of exposure. These data indicate that acute exposure (12 and 24 h) to ammonia initiates a typical stress response in the Senegalese sole, with stimulation of the hypothalamus-pituitary-interrenal axis. The concentrations of dopamine (DA), serotonin (5-hydroxytryptamine; 5-HT) and noradrenaline (NA), and their metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxy-3-indoleacetic acid (5HIAA), were measured in the hypothalamus, telencephalon and optic tectum. The main changes induced by acute exposure to ammonia were decreases in the concentrations of 5-HT and DA, which were significant in most of the brain regions studied. The ratios of 5-HIAA/5-HT and DOPAC/DA increased in all regions and at all times studied, although in the case of the DOPAC/DA ratio, the increases were only significant in the hypothalamus (24 h exposure) and in the optic tectum (12 and 24 h exposure). These changes indicated that exposure to ammonia elicited time-dependent increases in serotoninergic and dopaminergic activity in the hypothalamus, telencephalon and optic tectum.

Variceal Recurrence, Rebleed Rates, and Alterations in Clinical and Laboratory Parameters Following Post Variceal Obliteration in Cirrhotic Patients

© 2011, INASL 21 Ex-vivo Treatment of Neutrophils with a p38-MAPK Agonist in Patients with Acute and Chronic Liver Failure and Elevated Arterial Ammonia Improves Their Bacteriocidal Capacity A Nishtala, NJ Taylor, RD Abeles, JA Wendon, DL Shawcross Institute of Liver Studies and Transplantation, King’s College London School of Medicine at King’s College Hospital, Denmark Hill, London, UK Background: Ammonia reduces neutrophil phagocytic and bacteriocidal capacity. Neutrophils swell in response to ammonia exposure and activation of the p38-MAPK pathway has been implicated in protecting the neutrophil against osmotic shock and normalizing neutrophil phagocytic dysfunction. In patients with acute (ALF) and chronic liver failure (CLF) with elevated arterial ammonia, we hypothesized that treating neutrophils ex vivo with isoproterenol, a β non-specific adrenergic receptor and p38-MAPK agonist would improve neutrophil bacteriocidal capacity. Aims and Methods: Out of 20 patients (10 ALF and 10 CLF) enrolled in study of neutrophil function encompassing 125 patients, we investigated the role of ex vivo incubation of neutrophils with an exogenous ammonia load, a p38-MAPK agonist (Isoproterenol) and the specific p38-MAPK antagonist SB203580 (Calbiochem) on neutrophil function. Phagocytic capacity in heparinized whole blood was quantitatively determined by flow cytometry using FITC-labelled opsonized Escherichia coli at baseline, and following incubation for 90 minutes with NH4Cl (200 μmol/L), isoproterenol (2 μmol/L) or SB203580 (40 μmol/L) at 37°C. Results: ALF patients had a median SOFA score of 16 and APACHE II score of 22. CLF patients had median MELD score 27. The median arterial ammonia level in ALF group was 88 and CLF group was 62 μmol/L. Baseline bactericidal capacity was 67% in the ALF group and 72% in the CLF group compared to > 85% in healthy controls. The exogenous ammonia load further reduced neutrophil phagocytic capacity by median of 7.3%. The p38-MAPK agonist isoproterenol significantly abrogated the ammonia-induced phagocytic impairment and improved bacteriocidal capacity (p < 0.007). The p38-MAPK antagonist SB203580 however, exacerbated the ammonia-induced reduction in neutrophil phagocytic capacity by median of 5.2%; p = 0.003. Conclusion: Data shows that in patients with liver failure and elevated arterial ammonia that neutrophil bacteriocidal capacity is impaired. Activation of the p38-MAPK pathway serves as an important cellular protective mechanism against ammonia-induced impairment. Conflict of Interest: None Variceal Recurrence, Rebleed Rates, and Alterations in Clinical and Laboratory Parameters Following Post Variceal Obliteration in Cirrhotic Patients K Arun Kumar, B Siva Subramaniam, V Jayanthi Department of Gastroenterology and Hepatology, Stanley Medical College and Hospital, Chennai Background: Band ligation is considered as an effective method for obliteration of esophageal varices. Injection sclerotherapy is an alternative method. Aim: To determine the natural history of esophageal varices after its obliteration following sclerotherapy. Material and Method: Consecutive patients with endoscopic proven varices between June 2006 and May 2007 were registered. All the patients had an elective or an emergency variceal endoscopic injection with 0.5% sodium tetradecyl sulphate at an interval of 3 weeks. The patients were followed-up in the liver clinic for recurrences of varices, variceal rebleed, changes in clinical, laboratory parameters and hemodynamic changes in portal venous system at 3 monthly intervals for the first year and thereafter at 6 monthly intervals until May 2010. Patients who had an EVL or a devascularization procedure were excluded. Results: A total of 104 patients with cirrhosis were registered. Eradication of varices required a mean of 5 sessions. Eighteen patients with cirrhosis liver had recurrence of varices at the end of 3 years. Twenty patients had Gr I varices and the remaining Gr II. The varices appeared after a mean period of 11.4 months. Only 4 patients rebled following reappearance of varices. A significant improvement in laboratory findings was noted in hemoglobin, platelet count, prothrombin time (PT), and INR. There was a significant improvement in ascites and regression of jaundice following endotherapy in GpI patients (P < 0.05). A significant (P < 0.05) number of patients in Gp I reverted from Child’s C to B or A. Conclusion: The rebleed rates and bleed-related death was significantly low in cirrhotic patients with portal hypertension after sclerotherapy. There was a significant improvement in clinical and laboratory findings after obliteration of esophageal varices. A significant number of patients reverted from Child’s C to B or A. Conflict of Interest: None 03_JCEH-Abstract.indd 21 3/18/2011 11:13:04 AM

P40 Ex vivo treatment of neutrophils with a p38-MAPK agonist in patients with liver failure improves their bacteriocidal capacity

IntroductionAmmonia reduces neutrophil phagocytic and bacteriocidal capacity. Neutrophils swell in response to ammonia exposure and activation of the p38-MAPK pathway has been implicated in protecting the neutrophil against osmotic shock...