Effects of the naturally occurring food mycotoxin ochratoxin A on brain cells in culture.

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.

Les neutrophiles ne sont pas résistants aux glucocorticoides

Les neutrophiles sont généralement considérés résistants aux glucocorticoïdes. Cependant, peu d’études comparant l’effet de ces drogues sur les neutrophiles et les autres leucocytes sanguins (monocytes, lymphocytes et éosinophiles) ont été rapportées. Dans notre étude, nous avons évalué la réponse aux glucocorticoïdes de ces deux populations cellulaires chez le cheval et l’homme. Les cellules, préalablement isolées du sang de 6 chevaux et 4 sujets humains sains, ont été incubées pendant 5 h en présence de lipopolysaccharide (LPS; 100 ng/mL) seul ou combiné avec de l’hydrocortisone, de la prednisolone ou de la dexaméthasone (10-8M et 10-6M). L’expression d’ARNm pour l’IL-1β, le TNF-α, l’IL-8, la glutamine synthétase et le récepteur α des glucocorticoïdes (GR-α) a été quantifiée par qPCR. Les neutrophiles équins ont également été incubés pendant 20 h en présence de ces 3 glucocorticoïdes et la survie cellulaire a été évaluée par cytométrie de flux et microscopie optique. Nous avons démontré que les glucocorticoïdes inhibaient l’expression des gènes pro-inflammatoires induite par le LPS pour les deux populations cellulaires chez les deux espèces étudiées. L’expression de la glutamine synthétase était également significativement augmentée par les glucocorticoïdes chez les neutrophiles et les autres leucocytes sanguins équins. De manière générale, l’intensité de la réponse aux glucocorticoïdes s’est avérée similaire dans les 2 populations leucocytaires et chez les deux espèces. Les glucocorticoïdes augmentaient également la survie des neutrophiles équins, phénomène également rapporté dans d’autres espèces. Ainsi, les glucococorticoïdes exercent des effets d’intensité comparable sur les neutrophiles et les autres leucocytes sanguins. Nous spéculons que la faible réponse à la corticothérapie observée lors de maladies inflammatoires chroniques neutrophiliques comme l’asthme sévère ou la Maladie Pulmonaire Obstructive Chronique (MPOC) ne s’explique pas par une corticorésistance intrinsèque des neutrophiles.

Limited capacity for ammonia removal by brain in chronic liver failure: potential role of nitric oxide.

Chronic liver failure leads to hyperammonemia and consequently increased brain ammonia concentrations, resulting in hepatic encephalopathy. When the liver fails to regulate ammonia concentrations, the brain, devoid of a urea cycle, relies solely on the amidation of glutamate to glutamine through glutamine synthetase, to efficiently clear ammonia. Surprisingly, under hyperammonemic conditions, the brain is not capable of increasing its capacity to remove ammonia, which even decreases in some regions of the brain. This non-induction of glutamine synthetase in astrocytes could result from possible limiting substrates or cofactors for the enzyme, or an indirect effect of ammonia on glutamine synthetase expression. In addition, there is evidence that nitration of the enzyme resulting from exposure to nitric oxide could also be implicated. The present review summarizes these possible factors involved in limiting the increase in capacity of glutamine synthetase in brain, in chronic liver failure.

Direct molecular and spectroscopic evidence for increased ammonia removal capacity of skeletal muscle in acute liver failure.

BACKGROUND/AIMS: It has been proposed that, in acute liver failure, skeletal muscle adapts to become the principle organ responsible for removal of blood-borne ammonia by increasing glutamine synthesis, a reaction that is catalyzed by the cytosolic ATP-dependent enzyme glutamine synthetase. To address this issue, glutamine synthetase expression and activities were measured in skeletal muscle of rats with acute liver failure resulting from hepatic devascularization. METHODS: Glutamine synthetase protein and gene expression were investigated using immunoblotting and semi-quantitative RT-PCR analysis. Glutamine synthetase activity and glutamine de novo synthesis were measured using, respectively, a standard enzymatic assay and [13C]-nuclear magnetic resonance spectroscopy. RESULTS: Glutamine synthetase protein (but not gene) expression and enzyme activities were significantly up-regulated leading to increased de novo synthesis of glutamine and increased skeletal muscle capacity for ammonia removal in acute liver failure. In contrast to skeletal muscle, expression and activities of glutamine synthetase in the brain were significantly decreased. CONCLUSIONS: These findings demonstrate that skeletal muscle adapts, through a rapid induction of glutamine synthetase, to increase its capacity for removal of blood-borne ammonia in acute liver failure. Maintenance of muscle mass together with the development of agents with the capacity to stimulate muscle glutamine synthetase could provide effective ammonia-lowering strategies in this disorder.

L-ornithine phenylacetate attenuates increased arterial and extracellular brain ammonia and prevents intracranial hypertension in pigs with acute liver failure.

Hyperammonemia is a feature of acute liver failure (ALF), which is associated with increased intracranial pressure (ICP) and brain herniation. We hypothesized that a combination of L-ornithine and phenylacetate (OP) would synergistically reduce toxic levels of ammonia by (1) L-ornithine increasing glutamine production (ammonia removal) through muscle glutamine synthetase and (2) phenylacetate conjugating with the ornithine-derived glutamine to form phenylacetylglutamine, which is excreted into the urine. The aims of this study were to determine the effect of OP on arterial and extracellular brain ammonia concentrations as well as ICP in pigs with ALF (induced by liver devascularization). ALF pigs were treated with OP (L-ornithine 0.07 g/kg/hour intravenously; phenylbutyrate, prodrug for phenylacetate; 0.05 g/kg/hour intraduodenally) for 8 hours following ALF induction. ICP was monitored throughout, and arterial and extracellular brain ammonia were measured along with phenylacetylglutamine in the urine. Compared with ALF + saline pigs, treatment with OP significantly attenuated concentrations of arterial ammonia (589.6 +/- 56.7 versus 365.2 +/- 60.4 mumol/L [mean +/- SEM], P= 0.002) and extracellular brain ammonia (P= 0.01). The ALF-induced increase in ICP was prevented in ALF + OP-treated pigs (18.3 +/- 1.3 mmHg in ALF + saline versus 10.3 +/- 1.1 mmHg in ALF + OP-treated pigs;P= 0.001). The value of ICP significantly correlated with the concentration of extracellular brain ammonia (r(2) = 0.36,P< 0.001). Urine phenylacetylglutamine levels increased to 4.9 +/- 0.6 micromol/L in ALF + OP-treated pigs versus 0.5 +/- 0.04 micromol/L in ALF + saline-treated pigs (P< 0.001).Conclusion:L-Ornithine and phenylacetate act synergistically to successfully attenuate increases in arterial ammonia, which is accompanied by a significant decrease in extracellular brain ammonia and prevention of intracranial hypertension in pigs with ALF.

Nitrogen Metabolism in leaves of a tank epiphytic bromeliad: Characterization of a spatial and functional division

The leaf is considered the most important vegetative organ of tank epiphytic bromeliads due to its ability to absorb and assimilate nutrients. However, little is known about the physiological characteristics of nutrient uptake and assimilation. In order to better understand the mechanisms utilized by some tank epiphytic bromeliads to optimize the nitrogen acquisition and assimilation, a study was proposed to verify the existence of a differential capacity to assimilate nitrogen in different leaf portions. The experiments were conducted using young plants of Vriesea gigantea. A nutrient solution containing NO(3)(-)/NH(4)(+) or urea as the sole nitrogen source was supplied to the tank of these plants and the activities of urease, nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (NADH-GDH) were quantified in apical and basal leaf portions after 1, 3, 6, 9, 12, 24 and 48 h. The endogenous ammonium and urea contents were also analyzed. Independent of the nitrogen sources utilized, NR and urease activities were higher in the basal portions of leaves in all the period analyzed. On the contrary. GS and GDH activities were higher in apical part. It was also observed that the endogenous ammonium and urea had the highest contents detected in the basal region. These results suggest that the basal portion was preferentially involved in nitrate reduction and urea hydrolysis, while the apical region could be the main area responsible for ammonium assimilation through the action of GS and GDH activities. Moreover, it was possible to infer that ammonium may be transported from the base, to the apex of the leaves. In conclusion, it was suggested that a spatial and functional division in nitrogen absorption and NH(4)(+) assimilation between basal and apical leaf areas exists, ensuring that the majority of nitrogen available inside the tank is quickly used by bromeliad`s leaves. (C) 2011 Elsevier GmbH. All rights reserved.

Antígenos da forma amastigota de Leishmania chagasi identificados por dupla varredura da biblioteca de cDNA

Control of human visceral leishmaniasis in endemic regions is hampered in part by the lack of knowledge with respect of the role reservoirs and vector. In addition, there is not yet an understanding of how non-symptomatic subclinical infection might influence the maintenance of infection in a particular locality. Of worrisome is the limited accessibility to medical care in places with emerging drug resistance. There is still no available protective vaccine either for humans or other reservoirs. Leishmania species are protozoa that express multiple antigens which are recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the causative agent of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T and T-dependent B cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second step screen for their ability to cause proliferation and IFN-γ responses of T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The clones encoded part of the coding sequence of glutamine synthetase, transitional endoplasmic reticulum ATPase, elongation factor 1γ, kinesin K-39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these protein Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines against Leishmania

Nitrogen assimilation in Citrus based on CitEST data mining

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Velocidade de absorção do glufosinate e seus efeitos em plantas daninhas e algodão

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Sombreamento e aplicação de nitrogênio em plantas jovens de Jatropha curcas L

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metodologia para determinação de efeitos fisiológicos e metabólicos do glufosinate em soja

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Qualidade de sementes, teor de nitrogênio e expressão gênica em genótipos de milho inoculados com Azospirillum brasilense

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Nitrogen metabolism in coffee plants in response to nitrogen supply by fertigation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Manejo de azevém (Lolium multiflorum Lam.) resistente ao glyphosate com o uso de diferentes herbicidas

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon

Abstract Background Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter. Conclusions Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon.