Combined application of asparagine and thiourea improves tolerance to lead stress in wheat by modulating AsA-GSH cycle, lead detoxification and nitrogen metabolism.

Lead (Pb), like other heavy metals, is not essentially required for optimal plant growth; however, plants uptake it from the soil, which poses an adverse effect on growth and yield. Asparagine (Asp) and thiourea (Thi) are known to assuage the negative impacts of heavy metal pollution on plant growth; however, combined application of Asp and Thi has rarely been tested to discern if it could improve wheat yield under Pb stress. Thus, this experimentation tested the role of individual and combined applications of Asp (40 mM) and Thi (400 mg/L) in improving wheat growth under lead (Pb as PbCl2, 0.1 mM) stress. Lead stress significantly reduced plant growth, chlorophyll contents and photosystem system II (PSII) efficiency, whereas it increased Pb accumulation in the leaves and roots, leaf proline contents, phytochelatins, and oxidative stress related attributes. The sole or combined application of Asp and Thi increased the vital antioxidant biomolecules/enzymes, including reduced glutathione (GSH), ascorbic acid (AsA), ascorbate peroxsidase (APX), catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), dehydroascorbate reductase (DHAR), and glutathione reductase (GR). Furthermore, the sole or the combined application of Asp and Thi modulated nitrogen metabolism by stimulating the activities of nitrate and nitrite reductase, glutamate synthase (GOGAT) and glutamine synthetase (GS). Asp and Thi together led to improve plant growth and vital physiological processes, but lowered down Pb accumulation compared to those by their sole application. The results suggest that Asp and Thi synergistically can improve wheat growth under Pb-toxicity.

The adaptive mechanism of halophilic Brachybacterium muris in response to salt stress and its mitigation of copper toxicity in hydroponic plants.

Serious environmental pollution of heavy metals has attracted people's attention in recent years and halophiles seem to be potential bioremediation in the controlling of heavy metals contamination. In this study, the adaptive mechanism of halophilic Brachybacterium muris (B. muris) in response to salt stress and its mitigation of copper (Cu) toxicity in hydroponic plants were investigated. The cell morphology was observed using transmission electron microscopy. The cell membrane composition and fluidity were examined by the combination of gas chromatography, gas chromatography-mass spectrometry, ultra-high performance liquid chromatography-mass spectrometry, and fluorescence spectrophotometry. Moreover, the metabolic pathways of B. muris in response to salt stress were analyzed using the prokaryotic transcriptomics approach. A hydroponic co-culture model was further conducted to explore the effects of B. muris on wheat seedlings subjected to Cu toxicity. It was found that B. muris can respond to high osmotic pressure by improving the cell membrane fluidity, altering the cell morphology and cell membrane compositions. The proportion of unsaturated fatty acids, phosphatidylethanolamine, and phosphatidylinositol in B. muris cell membranes increased significantly, while zymosterol, fecosterol, and ergosterol contents decreased under a high salinity situation. Further transcriptomic analysis showed that genes encoding L-glutamate synthase, glutamate ABC transporter ATP-binding protein, and sodium cotransporter were up-regulated, indicating that both the synthesis and transport of glutamate were significantly enhanced under high osmotic pressure. Additionally, B. muris alleviated the inhibitory effect of Cu2+ on wheat seedlings' growth, causing a 30.14% decrease in H2O2 content and a significant increase of 83.86% and 45.96% in POD activity and GSH content in wheat roots, respectively. The findings of this study suggested that the salt-tolerant B. muris may serve as a promising strategy for improving the bioremediation of metal-contaminated saline water and soils.

New Insights into the Transcriptional Regulation of Genes Involved in the Nitrogen Use Efficiency under Potassium Chlorate in Rice (Oryza sativa L.).

Potassium chlorate (KClO3) has been widely used to evaluate the divergence in nitrogen use efficiency (NUE) between indica and japonica rice subspecies. This study investigated the transcriptional regulation of major genes involved in the NUE in rice treated with KClO3, which acts as an inhibitor of the reducing activity of nitrate reductase (NR) in higher plants. A set of two KClO3 sensitive nitrate reductase (NR) and two nitrate transporter (NRT) introgression rice lines (BC2F7), carrying the indica alleles of NR or NRT, derived from a cross between Saeilmi (japonica, P1) and Milyang23 (indica, P2), were exposed to KClO3 at the seedling stage. The phenotypic responses were recorded 7 days after treatment, and samples for gene expression, physiological, and biochemical analyses were collected at 0 h (control) and 3 h after KClO3 application. The results revealed that Saeilmi (P1, japonica) and Milyang23 (P2, indica) showed distinctive phenotypic responses. In addition, the expression of OsNR2 was differentially regulated between the roots, stem, and leaf tissues, and between introgression lines. When expressed in the roots, OsNR2 was downregulated in all introgression lines. However, in the stem and leaves, OsNR2 was upregulated in the NR introgression lines, but downregulation in the NRT introgression lines. In the same way, the expression patterns of OsNIA1 and OsNIA2 in the roots, stem, and leaves indicated a differential transcriptional regulation by KClO3, with OsNIA2 prevailing over OsNIA1 in the roots. Under the same conditions, the activity of NR was inhibited in the roots and differentially regulated in the stem and leaf tissues. Furthermore, the transcriptional divergence of OsAMT1.3 and OsAMT2.3, OsGLU1 and OsGLU2, between NR and NRT, coupled with the NR activity pattern in the roots, would indicate the prevalence of nitrate (NO3¯) transport over ammonium (NH4+) transport. Moreover, the induction of catalase (CAT) and polyphenol oxidase (PPO) enzyme activities in Saeilmi (P1, KClO3 resistant), and the decrease in Milyang23 (P2, KClO3 sensitive), coupled with the malondialdehyde (MDA) content, indicated the extent of the oxidative stress, and the induction of the adaptive response mechanism, tending to maintain a balanced reduction-oxidation state in response to KClO3. The changes in the chloroplast pigments and proline content propose these compounds as emerging biomarkers for assessing the overall plant health status. These results suggest that the inhibitory potential of KClO3 on the reduction activity of the nitrate reductase (NR), as well as that of the genes encoding the nitrate and ammonium transporters, and glutamate synthase are tissue-specific, which may differentially affect the transport and assimilation of nitrate or ammonium in rice.

Glutamate synthase 1 is involved in iron-deficiency response and long-distance transportation in Arabidopsis.

Iron is an essential microelement for plant growth. After uptake from the soil, iron is chelated by ligands and translocated from roots to shoots for subsequent utilization. However, the number of ligands involved in iron chelation is unclear. In this study, we identified and demonstrated that GLU1, which encodes a ferredoxin-dependent glutamate synthase, was involved in iron homeostasis. First, the expression of GLU1 was strongly induced by iron deficiency condition. Second, lesion of GLU1 results in reduced transcription of many iron-deficiency-responsive genes in roots and shoots. The mutant plants revealed a decreased iron concentration in the shoots, and displayed severe leaf chlorosis under the condition of Fe limitation, compared to wild-type. Third, the product of GLU1, glutamate, could chelate iron in vivo and promote iron transportation. Last, we also found that supplementation of glutamate in the medium can alleviate cadmium toxicity in plants. Overall, our results provide evidence that GLU1 is involved in iron homeostasis through affecting glutamate synthesis under iron deficiency conditions in Arabidopsis.

The effects of magnetic treatment on nitrogen absorption and distribution in seedlings of Populus × euramericana 'Neva' under NaCl stress.

A potted experiment with Populus × euramericana 'Neva' was carried out to assess whether there are positive effects of magnetic treatment of saline water (MTSW) on nitrogen metabolism under controlled conditions in a greenhouse. Growth properties, nitrogen contents, enzyme activities and metabolite concentrations were determined based on field experiments and laboratory analysis after a 30-day treatment. The results were as follows: (1) Biomass accumulation, root morphological properties and total nitrogen content were improved by MTSW. (2) Magnetization led to a greater increase in nitrate-nitrogen (NO3--N) content in roots than in leaves, accompanied by greater NO3- efflux and activated nitrate reductase. (3) MTSW led to a higher ammonium-nitrogen (NH4+-N) content and greater uptake of net NH4+ in the leaves than that in the roots. (4) Magnetization stimulated glutamine synthase, glutamate dehydrogenase and glutamate synthase activities, whereas the concentrations of glutathione and oxidized glutathione were increased in leaves but decreased in roots, and the total glutathione content was increased. Overall, these results indicated some beneficial impacts of MTSW on nitrogen translocation under field conditions, especially for equilibrating the distribution of NO3--N and NH4+-N. Moreover, these findings confirmed the potential of using low-quality water for agriculture.

Polyaspartic acid (PASP)-urea and optimised nitrogen management increase the grain nitrogen concentration of rice.

Increase in grain nitrogen concentration (GNC), which is directly affected by nitrogen (N) application, can help overcome the issues of malnutrition. Here, the effects of urea type (polyaspartic acid (PASP) urea and conventional urea) and N management method (two splits and four splits) on GNC and N concentration of head rice were investigated in field experiments conducted in Sichuan, China, in 2014 and 2015. N concentration of grain and head rice were significantly (P < 0.05) increased by N redistribution from the leaf lamina, activities of glutamine synthetase (GS), and glutamate synthase (GOGAT) at the heading stage, and N concentration and GOGAT activity in the leaf lamina at the maturity stage. Compared to conventional urea, PASP-urea significantly improved N concentration of grain and head rice by improving the activities of GS and GOGAT, thereby increasing N distribution in the leaf lamina. The four splits method, unlike the two splits method, enhanced N concentration and activities of key N metabolism enzymes of leaf lamina, leading to increased GNC and N concentration in head rice too. Overall, four splits is a feasible method for using PASP-urea and improving GNC.

[Impairments of placental amino acid metabolism in fetal growth restriction]. / Narushenie platsentarnogo obmena aminokislot pri zaderzhke rosta ploda.

The content of the amino acids in the placenta during physiological pregnancy and fetal growth restriction (FGR) has been investigated my means of the method of ion-exchange chromatography. It has been found that in FGR the placental amino acid pool is characterized by a decreased content of arginine, proline, alanine, serine, cysteine, methionine, tryptophan, leucine, threonine, tyrosine, phenylalanine, glutamine and an increased content of dicarboxylic amino acids, lysine, histidine and glycine. These changes are accompanied by altered activity of some enzymes of amino acid metabolism, and the degree of these changes correlates with the level of corresponding amino acids.

PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate.

The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can't be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein PII from Synechococcus elongatus PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine in situ glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in Synechococcus elongatus PCC 7942 cells and measured 2-OG concentrations in cell extracts from Escherichia coli in vitro. Finally, we could show the sensors' functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.

The role of 24-epibrassinolide in the regulation of photosynthetic characteristics and nitrogen metabolism of tomato seedlings under a combined low temperature and weak light stress.

In the present study, we aimed to investigate the effects of exogenous 24-epibrassinolide (EBR) on growth, photosynthetic characteristics, chlorophyll fluorescence imaging, and nitrogen metabolism of tomato leaves under low temperature and weak light conditions. The results showed that foliar application of EBR significantly alleviated the inhibition of plant growth, and increased the fresh and dry weights of tomato plants under a combined low temperature and weak light stress. Moreover, EBR also increased the net photosynthetic rate (Pn), light saturation point (LSP), maximal quantum yield of PSII photochemistry (Fv/Fm), actual photochemical efficiency of PSII (ФPSII), and photochemical quenching coefficient (qP), but decreased the intercellular CO2 concentration (Ci), light compensation point (LCP) and apparent quantum efficiency (AQE) under low temperature and weak light conditions. In addition, application of EBR to tomato leaves significantly enhanced the activities of nitrate reductase (NR), glutamate dehydrogenase (GDH), glutamine synthetase (GS), and glutamate synthase (GOGAT), but decreased the ammonium content and nitrite reductase (NiR) activity. We observed that EBR remarkably increased the contents of aspartic acid, threonine, serine, glycine, and phenylalanine, while decreasing the accumulation of cysteine, methionine, arginine, and proline under a combined low temperature and light stress. These results suggest that EBR could alleviate the combined stress-induced harmful effects on photosynthesis and nitrogen metabolism, thus leading to improved plant growth.

Bacillithiol has a role in Fe-S cluster biogenesis in Staphylococcus aureus.

Staphylococcus aureus does not produce the low-molecular-weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism, we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH-deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activities of the iron-sulfur (Fe-S) cluster-dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH-deficient cells also had decreased aconitase and glutamate synthase activities, suggesting a general defect in Fe-S cluster biogenesis. The phenotypes of the BSH-deficient strains were exacerbated in strains lacking the Fe-S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu, suggesting functional overlap between BSH and Fe-S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe-S clusters to apo-aconitase, verifying that it serves as an Fe-S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe-S clusters to apo-proteins in S. aureus.

Ammonium nitrate and iron nutrition effects on some nitrogen assimilation enzymes and metabolites in Spirulina platensis.

The effect of various concentrations of ammonium nitrate (5-60 mM), an economical nitrogen source, on the growth, nitrate-ammonium uptake rates, production of some pigments and metabolites, and some nitrogen assimilation enzymes such as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) in Spirulina platensis (Gamont) Geitler was investigated. Ten millimolars of ammonium nitrate stimulated the growth, production of pigments and the other metabolites, and enzyme activities, whereas 30 and 60 mM ammonium nitrate caused inhibition. In the presence of 10 mM ammonium nitrate, different concentrations of iron were tried in the growth media of S. platensis. After achieving the best growth, levels of metabolite and pigment production, and enzyme activities in the presence of 10 mM ammonium nitrate as a nitrogen source, different iron concentrations (10-100 µM) were tried in the growth medium of S. platensis. The highest growth, pigment and metabolite levels, and enzyme activities were determined in the medium containing 50 µM iron and 10 mM ammonium nitrate. In this optimum condition, the highest dry biomass level, chlorophyll a, and pyruvate contents were obtained as 55.42 ± 3.8 mg mL(-1) , 93.114 ± 7.9 µg g(-1) , and 212.5 ± 18.7 µg g(-1) , respectively. The highest NR, NiR, GS, and GOGAT activities were 67.16 ± 5.1, 777.92 ± 52, 0.141 ± 0.01, and 44.45 ± 3.6, respectively. Additionally, 10 mM ammonium nitrate is an economical and efficient nitrogen source for nitrogen assimilation of S. platensis, and 50 µM iron is optimum for the growth of S. platensis.

Lanthanum (III) regulates the nitrogen assimilation in soybean seedlings under ultraviolet-B radiation.

Ultraviolet-B (UV-B, 280-320 nm) radiation has seriously affected the growth of plants. Finding the technology/method to alleviate the damage of UV-B radiation has become a frontal topic in the field of environmental science. The pretreatment with rare earth elements (REEs) is an effective method, but the regulation mechanism of REEs is unknown. Here, the regulation effects of lanthanum (La(III)) on nitrogen assimilation in soybean seedlings (Glycine max L.) under ultraviolet-B radiation were investigated to elucidate the regulation mechanism of REEs on plants under UV-B radiation. UV-B radiation led to the inhibition in the activities of the key enzymes (nitrate reductase, glutamine synthetase, glutamate synthase) in the nitrogen assimilation, the decrease in the contents of nitrate and soluble proteins, as well as the increase in the content of amino acid in soybean seedlings. The change degree of UV-B radiation at the high level (0.45 W m(-2)) was higher than that of UV-B radiation at the low level (0.15 W m(-2)). The pretreatment with 20 mg L(-1) La(III) could alleviate the effects of UV-B radiation on the activities of nitrate reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase, promoting amino acid conversion and protein synthesis in soybean seedlings. The regulation effect of La(III) under UV-B radiation at the low level was better than that of UV-B radiation at the high level. The results indicated that the pretreatment with 20 mg L(-1) La(III) could alleviate the inhibition of UV-B radiation on nitrogen assimilation in soybean seedlings.

Modulation of glutamine metabolism by the PI(3)K-PKB-FOXO network regulates autophagy.

The PI(3)K-PKB-FOXO signalling network provides a major intracellular hub for the regulation of cell proliferation, survival and stress resistance. Here we report an unexpected role for FOXO transcription factors in regulating autophagy by modulating intracellular glutamine levels. To identify transcriptional targets of this network, we performed global transcriptional analyses after conditional activation of the key components PI(3)K, PKB/Akt, FOXO3 and FOXO4. Using this pathway approach, we identified glutamine synthetase as being transcriptionally regulated by PI(3)K-PKB-FOXO signalling. Conditional activation of FOXO also led to an increased level of glutamine production. FOXO activation resulted in mTOR inhibition by preventing the translocation of mTOR to lysosomal membranes in a glutamine-synthetase-dependent manner. This resulted in an increased level of autophagy as measured by LC3 lipidation, p62 degradation and fluorescent imaging of multiple autophagosomal markers. Inhibition of FOXO3-mediated autophagy increased the level of apoptosis, suggesting that the induction of autophagy by FOXO3-mediated glutamine synthetase expression is important for cellular survival. These findings reveal a growth-factor-responsive network that can directly modulate autophagy through the regulation of glutamine metabolism.

Ammonium formation and assimilation in P(SARK)∷IPT tobacco transgenic plants under low N.

Wild Type (WT) and transgenic tobacco plants expressing isopentenyltransferase (IPT), a gene encoding the enzyme regulating the rate-limiting step in cytokinins (CKs) synthesis, were grown under limited nitrogen (N) conditions. We analyzed nitrogen forms, nitrogen metabolism related-enzymes, amino acids and photorespiration related-enzymes in WT and P(SARK)∷IPT tobacco plants. Our results indicate that the WT plants subjected to N deficiency displayed reduced nitrate (NO3⁻) assimilation. However, an increase in the production of ammonium (NH4⁺), by the degradation of proteins and photorespiration led to an increase in the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle in WT plants. In these plants, the amounts of amino acids decreased with N deficiency, although the relative amounts of glutamate and glutamine increased with N deficiency. Although the transgenic plants expressing P(SARK)∷IPT and growing under suboptimal N conditions displayed a significant decline in the N forms in the leaf, they maintained the GS/GOGAT cycle at control levels. Our results suggest that, under N deficiency, CKs prevented the generation and assimilation of NH4⁺ by increasing such processes as photorespiration, protein degradation, the GS/GOGAT cycle, and the formation of glutamine.

Exogenous calcium affects nitrogen metabolism in root-zone hypoxia-stressed muskmelon roots and enhances short-term hypoxia tolerance.

We investigated the effects of short-term root-zone hypoxic stress and exogenous calcium application or deficiency in an anoxic nutrient solution on nitrogen metabolism in the roots of the muskmelon cultivar Xiyu No. 1. Seedlings grown in the nutrient solution under hypoxic stress for 6d displayed significantly reduced plant growth and soluble protein concentrations. However, NO3⁻ uptake rate and activities of nitrate reductase and glutamate synthase were significantly increased. We also found higher amounts of nitrate, ammonium, amino acids, heat-stable proteins, polyamines, H2O2, as well as higher polyamine oxidase activity in the roots. In comparison to the reactions seen under hypoxic stress, exogenous calcium application led to a marked increase in plant weights, photosynthesis parameters, NO3⁻ uptake rate and contents of nitrate, ammonium, amino acids (e.g., glutamic acid, proline, glycine, cystine, γ-aminobutyric acid), soluble and heat-stable proteins, free spermine, and insoluble bound polyamines. Meanwhile, exogenous calcium application resulted in significantly increased activities for nitrate reductase, glutamine synthetase, and glutamate synthase but decreased activities for diamine and polyamine oxidase, as well as lower H2O2 content in roots during exposure to hypoxia. However, calcium deficiency in the nutrient solution decreased plant weight, photosynthesis parameters, NO3⁻ reduction, amino acids (e.g., alanine, aspartic acid, glutamic acid, γ-aminobutyric acid), protein, all polyamines except for free putrescine, and the activities of glutamate synthase and glutamine synthetase. Additionally, there was an increase in the NO3⁻ uptake rate, polyamine oxidase activity and H2O2 contents under hypoxia-Ca. Simultaneously, exogenous calcium had little effect on nitrate absorption and transformation, photosynthetic parameters, and plant growth under normoxic conditions. These results suggest that calcium confers short-term hypoxia tolerance in muskmelon, most likely by promoting nitrate uptake and accelerating its transformation into amino acids, heat-stable proteins or polyamines, as well as by decreasing polyamine degradation in muskmelon seedlings.

Adenylylation: renaissance of a forgotten post-translational modification.

The stable post-translational modification of proteins by adenylylation or uridylylation was discovered more than four decades ago as a mechanism to regulate the activity of enzymes. Although many other processes involving the covalent transfer of an AMP residue to an amino acid side chain have been identified since then, these are transient adenylylation events that essentially use the free energy of ATP hydrolysis to activate specific processes. Recently, new examples of stable adenylylation of small GTPases involved in signal transduction and regulation of cellular events were discovered, which appear to modulate downstream processes such as cytoskeletal rearrangement and vesicular trafficking. We present a survey of the historical and modern phases of research in this area, focusing on the common and differing aspects of protein adenylylation.

Evidence for an operative glutamine translocator in chloroplasts from maritime pine (Pinus pinaster Ait.) cotyledons.

In higher plants, ammonium is assimilated into amino acids through the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle. This metabolic cycle is distributed in different cellular compartments in conifer seedlings: glutamine synthesis occurs in the cytosol and glutamate synthesis within the chloroplast. A method for preparing intact chloroplasts of pine cotyledons is presented with the aim of identifying a glutamine-glutamate translocator. Glutamine-glutamate exchange has been studied using the double silicone layer system, suggesting the existence of a translocator that imports glutamine into the chloroplast and exports glutamate to the cytoplasm. The translocator identified is specific for glutamine and glutamate, and the kinetic constants for both substrates indicate that it is unsaturated at intracellular concentrations. Thus, the experimental evidence obtained supports the model of the GS/GOGAT cycle in developing pine seedlings that accounts for the stoichiometric balance of metabolites. As a result, the efficient assimilation of free ammonia produced by photorespiration, nitrate reduction, storage protein mobilisation, phenylpropanoid pathway or S-adenosylmethionine synthesis is guaranteed.

Genetics of the glutamate-mediated methylamine utilization pathway in the facultative methylotrophic beta-proteobacterium Methyloversatilis universalis FAM5.

The ability of some microbial species to oxidize monomethylamine via glutamate-mediated pathways was proposed in the 1960s; however, genetic determinants of the pathways have never been described. In the present study we describe a gene cluster essential for operation of the N-methylglutamate pathway in the methylotrophic beta-proteobacterium Methyloversatilis universalis FAM5. Four major polypeptides from protein fractions displaying high activities of N-methylglutamate synthetase, N-methylglutamate dehydrogenase and gamma-glutamylmethylamide synthetase were selected for mass spectrometry-based identification. The activities of enzymes were associated with the presence of peptides identified as ferredoxin-dependent glutamate synthase (GltB2), large subunit of putative heterotetrameric sarcosine oxidase (SoxA) and glutamine synthetase type III (GSIII) respectively. A gene cluster (8.3 kb) harbouring gltB2, soxA and gsIII-like genes was amplified from M. universalis FAM5, sequenced and assembled. Two partial and six complete open reading frames arranged in the order soxBDAG-gsIII-gltB132 were identified and subjected to mutational analysis, functional and metabolic profiling. We demonstrated that gltB-like and sox-like genes play a key role in methylamine utilization and encode N-methylglutamate synthetase and N-methylglutamate dehydrogenase respectively. Metabolic, enzymatic and mutational analyses showed that the gsIII-like gene encodes gamma-glutamylmethylamide synthetase; however, this enzyme is not essential for oxidation of methylamine.

Differential CRX and OTX2 expression in human retina and retinoblastoma.

The histogenesis of retinoblastoma tumors remains controversial, with the cell-of-origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone-rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner-Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell-of-origin of retinoblastoma tumors and cells expressing CRX and OTX2.