Presence Of MSX Research Articles

Survey of Bivalve Molluscs forBonamiaspp. and Other Parasitic Pathogens in Florida East Coast Lagoons

A 2007 study of Suminoe (Crassostrea ariakensis) and eastern (Crassostrea virginica) oysters exposed to Indian River Lagoon (IRL), Florida waters revealed the presence of Bonamia spp. in exposed oysters. In 2010, a survey of oysters and other bivalves (n = 721, 18 species) was conducted in the southern IRL (10 sites) and in the more southerly Lake Worth Lagoon (LWL, 3 sites). Polymerase chain reaction (PCR) was used to assess the prevalence of three oyster parasites, Bonamia spp., Perkinsus marinus, and Haplosporidium nelsoni, in collected samples. Histology, fluorescent in situ hybridization (FISH) and standard H&E staining, was used to confirm PCR-positive Bonamia results. Bonamia spp. was detected in eight species (Brachidontes exustus, Crassostrea virginica, Geukensia demissa, Ischadium recurvum, Isognomon alatus, Isognomon bicolor, Mercenaria campechiensis, and Ostrea stentina). The prevalence was 17.3% in the IRL and 9.9% in LWL with 66% of PCR-positive samples subsequently identified as either Bonamia exitiosa or Bonamia perspora. The presence of Bonamia spp. was confirmed by FISH in C. virginica, O. stentina, I. alatus, I. bicolor, and G. demissa, although only C. virginica and O. stentina showed evidence of infection. The presence of P. marinus was detected in eight species (Atrina rigida, B. exustus, C. virginica, G. demissa, I. recurvum, I. alatus, I. bicolor, and O. stentina). The prevalence was 12.4% in the IRL and 7.7% in LWL. The presence of MSX was not detected in any of the collected bivalves. This study confirms the presence of Bonamia spp. and P. marinus in Florida east coast waters and widens the described geographic range for Bonamia spp., and provides further evidence that MSX is unlikely to be found in Florida waters

Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1.

Understanding how nutrients affect gene expression will help us to understand the mechanisms controlling plant growth and development as a function of nutrient availability. Nitrate has been shown to serve as a signal for the control of gene expression in Arabidopsis. There is also evidence, on a gene-by-gene basis, that downstream products of nitrogen (N) assimilation such as glutamate (Glu) or glutamine (Gln) might serve as signals of organic N status that in turn regulate gene expression. To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. Phase response curve analysis shows that distinct N-metabolites can advance or delay the CCA1 phase. Regulation of CCA1 by organic N signals may represent a novel input mechanism for N-nutrients to affect plant circadian clock function.

Evaluating the Contribution of Glutamate Dehydrogenase and the Glutamate Synthase Cycle to Ammonia Assimilation by Four Ectomycorrhizal Fungal Isolates

Combined gas chromatography-mass spectrometry were used to evaluate the contributions of glutamate dehydrogenase (GDH) and the glutamate synthase cycle in 15N-labelled ammonium assimilation by four ectomycorrhizal fungal isolates. In all four species (Elaphomyces, Amanita, Pisolithus and Gautieria), glutamine was the major product accumulated following transfer of 14-day-old nitrogen-limited cultures to fresh medium. Label was rapidly assimilated into fungal tissue, with rates of 733 nmol g-1 FW h-1 in Pisolithus, 972 nmol g-1 FW h-1 in Amanita, 2760 nmol g-1 FW h-1 in Gautieria and 6756 nmol g-1 FW h-1 in Elaphomyces sp in the first 4 h of incubation. Incorporation of [15N]ammonium was sensitive to the inhibitory effects of both methionine sulfoximine (MSX, an inhibitor of glutamine synthetase (GS)) and albizziin (an inhibitor of glutamate synthase (GOGAT)) in three species (Amanita, Gautieria and Pisolithus) and labelling patterns were consistent with the action of the glutamate synthase cycle in ammonium assimilation. In all three species glutamine synthesis was almost totally blocked by MSX and there was no continued incorporation of 15N into glutamate. Elaphomyces displayed high levels of total incorporation of labelled ammonium in mycelium even in the presence of MSX, although incorporation into glutamine was reduced by 88%. This inhibition of GS by MSX, in addition to its partial inhibition by albizziin suggests strongly the action of glutamate synthase cycle in ammonium assimilation. The reduction in label entering glutamate under the influence of albizziin is direct evidence for the inhibition of GOGAT activity. However, MSX treatment had the effect of increasing significantly the quantity of label recovered in both glutamate and alanine. In the absence of GS inhibition there is clearly competition for ammonium which under normal physiological conditions results in assimilation through the glutamate synthase cycle. However, when GS is blocked by MSX label is able to cycle through the GDH pathway. Extra keywords: ectomycorrhiza, ammonium assimilation, glutamate synthase cycle, glutamate dehydrogenase, amino acid metabolism.

Role of glutamine synthetase activity in the urea regulation of heterocyst and nitrogenase formation in the cyanobacterium Anabaena cycadeae

AbstractGrowth and urea regulation of heterocyst and nitrogenase formation have been studied in the diazotrophic cyanobacterium Anabaena cycadeae and its glutamine auxotrophic mutant strain lacking glutamine synthetase (GS) activity. The parent strain having normal GS activity utilized urea as sole nitrogen source without producing N2‐fixing heterocysts. In contrast, the mutant strain lacking GS activity could not utilize urea as sole nitrogen source although similar to the parent strain it lacked N2‐fixing heterocysts in urea‐medium. Both parent and mutant strain showed high levels of urea uptake and urease activity in the presence and absence of a GS inhibitor, l‐methionine‐dl‐sulphoximine (MSX). Urea‐dependent NH production by MSX‐untreated cells was only confined to the mutant strain lacking GS activity whereas the parent strain having normal GS activity produced NH only in the presence of MSX. These results suggest that (i) GS is the sole NH assimilating as well as glutamine‐forming route in heterocystous N2‐fixing cyanobacteria; (ii) while GS activity is necessarily required for the assimilation of urea as sole nitrogen source, it is however, not required for the urea inhibition of heterocysts and nitrogenase formation; (iii) that NH resulting from the hydrolysis of urea and not GS‐mediated pathway of NH assimilation appears to be the initial repressor signal for heterocysts and nitrogenase formation; and (iv) GS does not control the formation of N2‐fixing heterocysts, urea uptake and urease activity systems.

Regulation of assimilatory nitrate reductase activity in soil by microbial assimilation of ammonium.

It is well established that assimilatory nitrate reductase (ANR) activity in soil is inhibited by ammonium (NH4+). To elucidate the mechanism of this inhibition, we studied the effect of L-methionine sulfoximine (MSX), an inhibitor of NH4+ assimilation by microorganisms, on assimilatory reduction of nitrate (NO3-) in aerated soil slurries treated with NH4+. We found that NH4+ strongly inhibited ANR activity in these slurries and that MSX eliminated this inhibition. We also found that MSX induced dissimilatory reduction of NO3- to NH4+ in soil and that the NH4+ thus formed had no effect on the rate of NO-3 reduction. We concluded from these observations that the inhibition of ANR activity by NH4+ is due not to NH4+ per se but to products formed by microbial assimilation of NH4+. This conclusion was supported by a study of the effects of early products of NH4+ assimilation (L amino acids) on ANR activity in soil, because this study showed that the biologically active, L isomers of glutamine and asparagine strongly inhibited ANR activity, whereas the D isomers of these amino acids had little effect on ANR activity. Evidence that ANR activity is regulated by the glutamine formed by NH4+ assimilation was provided by studies showing that inhibitors of glutamine metabolism (azaserine, albizziin, and aminooxyacetate) inhibited ANR activity in soil treated with NO3- but did not do so in the presence of MSX.

Interactions between nitrogen metabolism and photosynthesis in nitrogen-limited Monoraphidium falcatus

Nitrogen and carbon metabolism of Monoraphidium falcatus were shown to compete for carbon skeletons under high PPFD and atmospheric levels of CO2. The uptake of was dependent upon the presence of a Ci-source and was inhibited in the presence of MSX with no detectable efflux of N H 4 + from the cells. These results indicate that either Ci or an intermediate of carbon metabolism was essential for the maintenance and stabilization of the NO 3 − uptake system. The addition of inorganic-N ( NO 3 − or N H 4 + ) to the N-limited cells resulted in a suppression in the rates of nett photosynthetic CO2 fixation and O2 evolution. The inhibition of CO2 fixation and O2 evolution by NO 3 − and N H 4 + was partially alleviated in the presence of MSX. The carbon skeleton demand of amino acid biosynthesis may be met by anaplerotic reactions which would drain off triose-phosphates from the RPP pathway via the TCA cycle resulting in reduced rates of RuBP regeneration and increased mitochondrial respiratory activity.

Effect of Methionine Sulfoximine on Asparaginase Activity and Ammonium Levels in Pea Leaves

In developing leaves of Pisum sativum the levels of ammonium did not change during the light-dark photoperiod even though asparaginase (EC 3.5.1.1) did; asparaginase activity in detached leaves doubled during the first 2.5 hours in the light. When these leaves were supplied with 1 millimolar methionine sulfoximine (MSX, an inhibitor of glutamine synthetase, GS, activity) at the beginning of the photoperiod, levels of ammonium increased 8-to 10-fold, GS activity was inhibited 95%, and the light-stimulated increase in asparaginase activity was completely prevented, and declined to less than initial levels. When high concentrations of ammonium were supplied to leaves, the light-stimulated increase of asparaginase was partially prevented. However, it was also possible to prevent asparaginase increase, in the absence of ammonium accumulation, by the addition of MSX together with aminooxyacetate (AOA, which inhibits transamination and some other reactions of photorespiratory nitrogen cycling). AOA alone did not prevent light-stimulated asparaginase increase; neither MSX, AOA, or elevated ammonium levels inhibited the activity of asparaginase in vitro. These results suggest that the effect of MSX on asparaginase increase is not due solely to interference with photorespiratory cycling (since AOA also prevents cycling, but has no effect alone), nor to the production of high ammonium concentration or its subsequent effect on photosynthetic mechanisms. MSX must have further inhibitory effects on metabolism. It is concluded that accumulation of ammonium in the presence of MSX may underestimate rates of ammonium turnover, since liberation of ammonium from systems such as asparaginase is reduced by the effects of MSX.

Nitrogen‐stimulated potassium influx into maize roots: differential response of components resistant and sensitive to ambient ammonium1

Abstract Potassium (86Rb) influx from 200 mmol m −3 KCl into dark grown, decapitated maize seedlings 6 d old) was stimulated by nitrate pretreatment. The stimulus was clearly evident by 6h exposure to nitrate and required 12–24 h for maximal expression. Decay of the nitrate‐stimulated potassium influx was more than 50% complete within 3 h after transfer to nitrogen‐free solutions. The stimulation of potassium influx was entirely accounted for by an increase in the influx component that was resistant to inhibition by presence of 200 mmol m−3 ambient ammonium. In contrast, the component of potassium influx that was sensitive to inhibition by ambient ammonium was unaffected by nitrate pretreatment. Exposure to the glutamine synthetase inhibitor L‐methionine‐dl‐sulphoximine (MSX) during nitrate pretreatment stimulated the resistant component but the sensitive component was nearly eliminated. Pretreatment with ammonium increased the resistant component of potassium influx within 3 h, i.e. before it was increased by nitrate pretreatment, but the sensitive component was concomitantly restricted. The latter recovered partially during extended pretreatment with ammonium. The data indicate that the resistant component responded positively to increases in tissue ammonium concentrations whereas the sensitive component was unaffected by tissue ammonium except at concentrations in excess of 10μmol g−1. Ammonium influx was also stimulated by nitrate pretreatment and to a greater extent than potassium influx. Presence of MSX with nitrate during pretreatment resulted in a further stimulation in ammonium influx. The parallel increases in root ammonium concentrations with the two pretreatments imply that part of the increase in ammonium influx was a consequnce of increased counter‐transport with endogenous ammonium.

Effect of methionine-sulfoximine on nitrate uptake and photosynthesis in the cyanobacteriumAnabaena doliolum Bharadwaja

l-Methionine-dl-sulfoximine (MSX) stimulated nitrate uptake but inhibited14CO2 fixation and O2 evolution inAnabaena doliolum. Nitrate uptake was inhibited by ammonium (NH4+) in the absence of MSX, but not in the presence of MSX. Glutamine or a derivative of it appears to be the actual negative effector of nitrate utilization. In presence of nitrate, MSX-treated cells ofA. doliolum evolve more O2 than do untreated cells. Our results suggest a close relation between photoassimilation of carbon and utilization of nitrogen.

Effect of L‐methionine‐DL‐sulphoximine, a specific inhibitor of glutamine synthetase, on ammonium and nitrate metabolism in the unicellular alga Cyanidium caldarium

In the unicellular alga Cyanidium caldarium nitrate utilization is strongly inhibited by ammonium and it is resumed when ammonium has been depleted. In the presence of L‐methionine‐DL‐sulphoximine (MSX), which prevents ammonium assimilation through a specific irreversible inhibition of glutamine synthetase, nitrate reduction is no longer inhibited by ammonium, and most of the ammonium derived from nitrate reduction is excreted into the external medium. However, in the presence of MSX, nitrate reduction to ammonium proceeds at a reduced rate (45 to 70% of the control); this is particularly marked at low nitrate concentration. It is hypothesized that either MSX or accumulating ammonium bring about decrease in the rate of nitrate entry into the cell.