Altered nitrogen balance and decreased urea excretion in male rats fed cafeteria diet are related to arginine availability

Hyperlipidic diets limit glucose oxidation and favor amino acid preservation, hampering the elimination of excess dietary nitrogen and the catabolic utilization of amino acids.We analyzed whether reduced urea excretion was a consequence of higherNO ; (nitrite,nitrate, and other derivatives) availability caused by increased nitric oxide production in metabolic syndrome. Rats fed a cafeteria diet for 30 days had a higher intake and accumulation of amino acid nitrogen and lower urea excretion.There were no differences in plasma nitrate or nitrite. NO and creatinine excretion accounted for only a small part of total nitrogen excretion. Rats fed a cafeteria diet had higher plasma levels of glutamine, serine, threonine, glycine, and ornithinewhen comparedwith controls,whereas arginine was lower. Liver carbamoyl-phosphate synthetase I activity was higher in cafeteria diet-fed rats, but arginase I was lower. The high carbamoyl-phosphate synthetase activity and ornithine levels suggest activation of the urea cycle in cafeteria diet-fed rats, but low arginine levels point to a block in the urea cycle between ornithine and arginine, thereby preventing the elimination of excess nitrogen as urea. The ultimate consequence of this paradoxical block in the urea cycle seems to be the limitation of arginine production and/or availability.

Schizophrenia: glutathione deficit as a new vulnerability factor for disconnectivity syndrome

(from the journal abstract) Schizophrenia, a major psychiatric disease, affects individuals in the centre of their personality. Its aetiology is not clearly established. In this review, we will present evidence that patients suffering of schizophrenia present a brain deficit in glutathione, a major endogenous redox regulator and antioxidant. We will also show that, in experimental models, a decrease in glutathione, particularly during development, induces morphological, electrophysiological and behavioural anomalies consistent with those observed in the disease. In the cerebrospinal fluid of drug-naive schizophrenics, glutathione level was decreased by 27% and its direct metabolite of glutathione by 16%. Glutathione level in prefrontal cortex of patients, measured by magnetic resonance spectroscopy, was 52% lower than in controls. Patients' fibroblasts reveal a decrease in mRNA levels of the two glutathione synthesising enzymes, glutamatecysteine ligase modulatory subunit (GCLM) and glutathione synthetase. GCLM expression level in fibroblasts correlates negatively with symptoms severity. Glutathione is an important endogenous redox regulator and neuroactive substance. It is protecting cells from damage by reactive oxygen species generated, among others, by dopamine metabolism. A glutathione deficit-induced oxidative stress would lead to lipid peroxidation and micro-lesions at the level of dendritic spines, a synaptic damage responsible for abnormal nervous connections or structural disconnectivity. On the other hand, a glutathione deficit could also lead to a functional disconnectivity by depressing NMDA neurotransmission, in analogy to phencyclidine effects. Present experimental data are consistent with the proposed hypothesis: decreasing pharmacologically glutathione level in experimental models, with or without blocking dopamine (DA) uptake (GBR12909), induces morphological, electrophysiological and behavioural changes similar to those observed in patients. In summary, a deficit of glutathione and/or glutathione-related enzymes during early development would lead to both a functional and a structural disconnectivity, which could be at the basis of some perceptive, cognitive and behavioural troubles of the disease. It could constitute a major vulnerability factor for schizophrenia. Attempts to restore physiological glutathione functions could open new therapeutic avenues. This translational research, made possible by a close interaction between clinicians and neuroscientists, should also pave the way to the identification of biological markers for schizophrenia. In turn, they should allow early diagnostic and hopefully preventive intervention to this devastating disease. (PsycINFO Database Record (c) 2005 APA, all rights reserved)

Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation.

Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [(3)H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [(3)H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release.

BRAIN DAMAGE IN METHYLMALONIC ACIDURIA: 2-METHYLCITRATE LEADS TO AMMONIA INCREASE AND APOPTOSIS

A 3D in vitro model of rat organotypic brain cell cultures in aggregates was used to investigate neurotoxicity mechanisms in methylmalonic aciduria. 1 mM methylmalonate (MMA), 2-methylcitrate (2-MCA) or propionate (PA) were repeatedly added to the culture media at two different time points of the cultures. In cultures treated with 2-MCA, we observed a significant increase of lactate in the medium, consistent with a possible inhibition of Krebs cycle and respiratory chain, as described earlier in the literature. Interestingly, we further observed that 2-MCA induced an important increase in ammonia production with concomitant decrease of glutamine concentrations, which suggests an inhibition of the astrocytic enzyme glutamine synthetase. These previously unreported findings may uncover a pathogenic mechanism in this disease with deleterious effects on early stages of brain development. By immunohistochemistry we could show that 2-MCA substantially increased the number of apoptotic cells. On the cellular level, 2-MCA had a toxic effect (cell swelling and cell death) on glial cells, but not on neurons. Surprisingly, MMA seemed to have a growth stimulating effect on the cultures. We can conclude that 2-MCA was the most toxic metabolite in our model for methylmalonic aciduria inducing ammonia accumulation and massive apoptosis in brain cells.

En face optical coherence tomography of foveal microstructure in full-thickness macular hole: a model to study perifoveal müller cells.

PURPOSE: To characterize perifoveal intraretinal cavities observed around full-thickness macular holes (MH) using en face optical coherence tomography and to establish correlations with histology of human and primate maculae. DESIGN: Retrospective nonconsecutive observational case series. METHODS: Macular en face scans of 8 patients with MH were analyzed to quantify the areas of hyporeflective spaces, and were compared with macular flat mounts and sections from 1 normal human donor eye and 2 normal primate eyes (Macaca fascicularis). Immunohistochemistry was used to study the distribution of glutamine synthetase, expressed by Müller cells, and zonula occludens-1, a tight-junction protein. RESULTS: The mean area of hyporeflective spaces was lower in the inner nuclear layer (INL) than in the complex formed by the outer plexiform (OPL) and the Henle fiber layers (HFL): 5.0 × 10(-3) mm(2) vs 15.9 × 10(-3) mm(2), respectively (P < .0001, Kruskal-Wallis test). In the OPL and HFL, cavities were elongated with a stellate pattern, whereas in the INL they were rounded and formed vertical cylinders. Immunohistochemistry confirmed that Müller cells followed a radial distribution around the fovea in the frontal plane and a "Z-shaped" course in the axial plane, running obliquely in the OPL and HFL and vertically in the inner layers. In addition, zonula occludens-1 co-localized with Müller cells within the complex of OPL and HFL, indicating junctions in between Müller cells and cone axons. CONCLUSION: The dual profile of cavities around MHs correlates with Müller cell morphology and is consistent with the hypothesis of intra- or extracellular fluid accumulation along these cells.

Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies.

Inherited peripheral neuropathies are a genetically heterogeneous group of disorders characterized by distal muscle weakness and sensory loss. Mutations in genes encoding aminoacyl-tRNA synthetases have been implicated in peripheral neuropathies, suggesting that these tRNA charging enzymes are uniquely important for the peripheral nerve. Recently, a mutation in histidyl-tRNA synthetase (HARS) was identified in a single patient with a late-onset, sensory-predominant peripheral neuropathy; however, the genetic evidence was lacking, making the significance of the finding unclear. Here, we present clinical, genetic, and functional data that implicate HARS mutations in inherited peripheral neuropathies. The associated phenotypic spectrum is broad and encompasses axonal and demyelinating motor and sensory neuropathies, including four young patients presenting with pure motor axonal neuropathy. Genome-wide linkage studies in combination with whole-exome and conventional sequencing revealed four distinct and previously unreported heterozygous HARS mutations segregating with autosomal dominant peripheral neuropathy in four unrelated families (p.Thr132Ile, p.Pro134His, p.Asp175Glu and p.Asp364Tyr). All mutations cause a loss of function in yeast complementation assays, and p.Asp364Tyr is dominantly neurotoxic in a Caenorhabditis elegans model. This study demonstrates the role of HARS mutations in peripheral neuropathy and expands the genetic and clinical spectrum of aminoacyl-tRNA synthetase-related human disease.

Gene transfer engineering for astrocyte-specific silencing in the CNS.

Cell-type-specific gene silencing is critical to understand cell functions in normal and pathological conditions, in particular in the brain where strong cellular heterogeneity exists. Molecular engineering of lentiviral vectors has been widely used to express genes of interest specifically in neurons or astrocytes. However, we show that these strategies are not suitable for astrocyte-specific gene silencing due to the processing of small hairpin RNA (shRNA) in a cell. Here we develop an indirect method based on a tetracycline-regulated system to fully restrict shRNA expression to astrocytes. The combination of Mokola-G envelope pseudotyping, glutamine synthetase promoter and two distinct microRNA target sequences provides a powerful tool for efficient and cell-type-specific gene silencing in the central nervous system. We anticipate our vector will be a potent and versatile system to improve the targeting of cell populations for fundamental as well as therapeutic applications.

Síntese, avaliação biológica e modelagem molecular de arilfuranos como inibidores da enzima tripanotiona redutase

Trypanosoma cruzi is a protozoan parasite that causes a severe disease (Chagas'disease) in Central and South America. The currently available chemotherapeutic agents against this disease are still inadequate. The enzyme trypanothione reductase (TR) is considered a validated molecular target for the development of new drugs against this parasite. In this regard, a series of arylfurans based on 2,5-bis-(4-acetamidophenyl)furan was synthesized and tested for their in vitro inhibitory activity against TR. Molecular modeling studies of putative enzyme-inhibitor complexes revealed a possible mechanism of interaction. From synthesized compounds, a benzylaminofuran derivative was found to be more active than the lead compound.

Altered nitrogen balance and decreased urea excretion in male rats fed cafeteria diet are related to arginine availability

Hyperlipidic diets limit glucose oxidation and favor amino acid preservation, hampering the elimination of excess dietary nitrogen and the catabolic utilization of amino acids.We analyzed whether reduced urea excretion was a consequence of higherNO ; (nitrite,nitrate, and other derivatives) availability caused by increased nitric oxide production in metabolic syndrome. Rats fed a cafeteria diet for 30 days had a higher intake and accumulation of amino acid nitrogen and lower urea excretion.There were no differences in plasma nitrate or nitrite. NO and creatinine excretion accounted for only a small part of total nitrogen excretion. Rats fed a cafeteria diet had higher plasma levels of glutamine, serine, threonine, glycine, and ornithinewhen comparedwith controls,whereas arginine was lower. Liver carbamoyl-phosphate synthetase I activity was higher in cafeteria diet-fed rats, but arginase I was lower. The high carbamoyl-phosphate synthetase activity and ornithine levels suggest activation of the urea cycle in cafeteria diet-fed rats, but low arginine levels point to a block in the urea cycle between ornithine and arginine, thereby preventing the elimination of excess nitrogen as urea. The ultimate consequence of this paradoxical block in the urea cycle seems to be the limitation of arginine production and/or availability.

Metabolism and gene polymorphisms of the folate pathway in Brazilian women with history of recurrent abortion

PURPOSE: To investigate the association between polymorphisms in genes that encode enzymes involved in folate- and vitamin B12-dependent homocysteine metabolism and recurrent spontaneous abortion (RSA).METHODS: We investigated the C677T and A1298C polymorphisms of the methylenetetrahydrofalate reductase gene (MTHFR), the A2756G polymorphism of the methionine synthase gene (MS) and the 844ins68 insertion of the cystathionine beta synthetase gene (CBS). The PCR technique followed by RFLP was used to assess the polymorphisms; the serum levels of homocysteine, vitamin B12 and folate were investigated by chemiluminescence. The EPI Info Software version 6.04 was used for statistical analysis. Parametric variables were compared by Student's t-test and nonparametric variables by the Wilcoxon rank sum test.RESULTS: The frequencies of gene polymorphisms in 89 women with a history of idiopathic recurrent miscarriage and 150 controls were 19.1 and 19.6% for the C677T, insertion, 20.8 and 26% for the A1298C insertion, 14.2 and 21.9% for the A2756G insertion, and 16.4 and 18% for the 844ins68 insertion, respectively. There were no significant differences between case and control groups in any of the gene polymorphisms investigated. However, the frequency of the 844ins68 insertion in the CBS gene was higher among women with a history of loss during the third trimester of pregnancy (p=0.003). Serum homocysteine, vitamin B12 and folate levels id not differ between the polymorphisms studied in the case and control groups. However, linear regression analysis showed a dependence of serum folate levels on the maintenance of tHcy levels.CONCLUSION: The investigated gene polymorphisms and serum homocysteine, vitamin B12 and folate levels were not associated with idiopathic recurrent miscarriage in the present study. Further investigations are needed in order to confirm the role of the CBS 844ins68 insertion in recurrent miscarriage.

Effects of different ammoniacal nitrogen sources on N-metabolism of the atmospheric bromeliad Tillandsia pohliana Mez

Increasing levels of atmospheric ammonia from anthropogenic sources have become a serious problem for natural vegetation. Short-term effects of different ammoniacal sources on the N metabolism of Tillandsia pohliana, an atmospheric bromeliad, were investigated. One-year-old, aseptically grown plants were transferred to a modified Knudson medium lacking N for three weeks. Plants were subsequently transferred to Knudson media supplemented with 0.5, 1.0, or 1.5 mM of N in the forms of NH3 or NH4+ as the sole N source. The activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH-NADH) were determined after 40 h. The GS activity was stimulated significantly by increasing the levels of the gaseous form. The GDH-NADH activity increased significantly under increasing N concentrations with NH3, while no significant differences were observed with NH4+ as a N source. These results may reflect a faster NH3 absorption by T. pohliana compared to NH4+ uptake. The increased activity of GDH-NADH in NH3 treatment may play a role in protecting the cells from the toxic effects of increased endogenous level of free ammonium. A raise in the concentration of N, especially in the form of NH3, greatly increased the content of free amino acids and soluble proteins. A possible utilisation of T. pohliana to evaluate the changes of atmospheric gaseous ammonia is proposed.

Estudo da eficiência de uso do nitrogênio em variedades locais e melhoradas de milho

O uso de fertilizantes, além dos riscos de contaminação ambiental, onera o agricultor, chegando a representar 40% dos custos de produção na cultura do milho. O presente estudo visa identificar características fisiológicas relacionadas com o aumento da eficiência do uso do nitrogênio e assim subsidiar programas de melhoramento genético direcionados para obtenção de genótipos de milho produtivos em solos com baixa disponibilidade de nitrogênio. Foram estudadas as variedades de milho Pedra Dourada, Catetão, Carioca (variedades locais, não melhoradas), BR 106, BR 105 (variedades melhoradas em solos férteis), Nitroflint e Nitrodente (variedades melhoradas em solos pobres em N). Plântulas de milho receberam solução nutritiva de Hoagland modificada quanto às fontes de N, sendo utilizadas duas doses de N (1 mM e 15 mM), 75% na forma nítrica e 25% na forma amoniacal. O experimento, composto por um fatorial 2 × 7 (duas doses de N e sete variedades) foi conduzido em casa de vegetação em blocos completos casualizados com três repetições. A deficiência de N afetou de modo muito mais intenso o crescimento das partes aéreas em todos os genótipos. As características bioquímicas estudadas (atividades da nitrato redutase, glutamina sintetase e conteúdo de pigmentos fotossintéticos) foram sensíveis à disponibilidade de N mas não permitiram discriminar diferenças genotípicas. A massa seca das plantas deficientes em N apresentou elevada correlação positiva (0,86) com a massa seca acumulada nas raízes dos diferentes genótipos. Tais resultados sugerem a importância do estudo das características morfológicas e fisiológicas do sistema radicular na seleção de genótipos eficientes quanto ao uso do nitrogênio.

JAK/STAT-deficient cell lines

Mutant cell lines B3 and B10, which are unresponsive to both interferon (IFN)-alpha and IFN-gamma, and line B9, which does not respond to IFN-gamma stimulation, are described. The mutants were submitted to fluorescence-activated cell sorting (FACS) from a cellular pool, which was obtained from the parental cell line 2C4 after several rounds of mutagenesis. The unresponsiveness to IFN stimulation was observed both in terms of expression of cell surface markers (CD2, class I and II HLAs) and mRNA expression of IFN-stimulated genes (2'-5' oligoadenylate synthetase (OAS), 9-27, and guanylate binding protein (GBP)). Genetic crossing of B3, B9 and B10 with U3 (STAT1-), gamma2a (JAK2-) and U4 (JAK1-) mutants, respectively, did not restore IFN responsiveness to the hybrid cell lines. However, when these cell lines were crossed with the same mutants, but using the pairwise crosses B3 x U4, B9 x U3 and B10 x U3, the cell hybrids recovered full IFN responsiveness. The present genetic experiments permitted us to assign the mutant cell lines B3, B9 and B10 to the U3, gamma2 and U4 complementation groups, respectively. These conclusions were supported by the analysis of IFN-stimulated genes in the mutants.

Metabolite and light regulation of metabolism in plants: lessons from the study of a single biochemical pathway

We are using molecular, biochemical, and genetic approaches to study the structural and regulatory genes controlling the assimilation of inorganic nitrogen into the amino acids glutamine, glutamate, aspartate and asparagine. These amino acids serve as the principal nitrogen-transport amino acids in most crop and higher plants including Arabidopsis thaliana. We have begun to investigate the regulatory mechanisms controlling nitrogen assimilation into these amino acids in plants using molecular and genetic approaches in Arabidopsis. The synthesis of the amide amino acids glutamine and asparagine is subject to tight regulation in response to environmental factors such as light and to metabolic factors such as sucrose and amino acids. For instance, light induces the expression of glutamine synthetase (GLN2) and represses expression of asparagine synthetase (ASN1) genes. This reciprocal regulation of GLN2 and ASN1 genes by light is reflected at the level of transcription and at the level of glutamine and asparagine biosynthesis. Moreover, we have shown that the regulation of these genes is also reciprocally controlled by both organic nitrogen and carbon metabolites. We have recently used a reverse genetic approach to study putative components of such metabolic sensing mechanisms in plants that may be conserved in evolution. These components include an Arabidopsis homolog for a glutamate receptor gene originally found in animal systems and a plant PII gene, which is a homolog of a component of the bacterial Ntr system. Based on our observations on the biology of both structural and regulatory genes of the nitrogen assimilatory pathway, we have developed a model for metabolic control of the genes involved in the nitrogen assimilatory pathway in plants.

The decrease in uroporphyrinogen decarboxylase activity induced by ethanol predisposes rats to the development of porphyria and accelerates xenobiotic-triggered porphyria, regardless of hepatic damage

We evaluated the porphyrinogenic ability of ethanol (20% in drinking water) per se, its effect on the development of sporadic porphyria cutanea tarda induced by hexachlorobenzene in female Wistar rats (170-190 g, N = 8/group), and the relationship with hepatic damage. Twenty-five percent of the animals receiving ethanol increased up to 14-, 25-, and 4.5-fold the urinary excretion of delta-aminolevulinate, porphobilinogen, and porphyrins, respectively. Ethanol exacerbated the precursor excretions elicited by hexachlorobenzene. Hepatic porphyrin levels increased by hexachlorobenzene treatment, while this parameter only increased (up to 90-fold) in some of the animals that received ethanol alone. Ethanol reduced the activities of uroporphyrinogen decarboxylase, delta-aminolevulinate dehydrase and ferrochelatase. In the ethanol group, many of the animals showed a 30% decrease in uroporphyrinogen activity; in the ethanol + hexachlorobenzene group, this decrease occurred before the one caused by hexachlorobenzene alone. Ethanol exacerbated the effects of hexachlorobenzene, among others, on the rate-limiting enzyme delta-aminolevulinate synthetase. The plasma activities of enzymes that are markers of hepatic damage were similar in all drug-treated groups. These results indicate that 1) ethanol exacerbates the biochemical manifestation of sporadic hexachlorobenzene-induced porphyria cutanea tarda; 2) ethanol per se affects several enzymatic and excretion parameters of the heme metabolic pathway; 3) since not all the animals were affected to the same extent, ethanol seems to be a porphyrinogenic agent only when there is a predisposition, and 4) hepatic damage showed no correlation with the development of porphyria cutanea tarda.