In der Pathogenese der hepatischen Enzephalopathie nimmt die systemische Hyperam-moniämie eine zentrale Stellung ein. Es konnte kürzlich gezeigt werden, dass der Dünndarm in der postprandialen Hyperammoniämie bei Patienten mit Leberzirrhose eine wichtige Rolle spielt. Somit wird das über die Pfortader der Leber zugeführte Ammonium nicht ausschließlich von Bakterien im Kolon produziert, sondern entstammt in relevanter Menge, beim Versuchstier zu etwa 50%, dem Glutaminstoffwechsel der Dünndarmmukosa . Unter pathologischen Verhältnissen wie bei einer Leberzirrhose und dem Vorliegen portosystemischer Umgehungskreisläufe führt die intestinale Ammoniogenese zur systemischen Hyperammoniämie und hepatischen Enzephalopathie. Hauptziele dieser Studie waren zu untersuchen, welche Stickstoffgruppe des Glutaminmoleküls der intestinalen Ammoniogenese dient, welche Rolle die zweite Stickstoffgruppe des Glutamins spielt, welche anderen Metabolite als Produkte des intestinalen Glutaminmetabolismus entstehen. Wir untersuchten 2 Gruppen à 4 Patienten mit Leberzirrhose, bei denen zuvor ein transju-gulärer intrahepatischer portosystemischer Stent-Shunt (TIPS) angelegt worden war. Die Patienten erhielten während 240 min über eine nasoduodenale Sonde eine Nährlösung bestehend aus markiertem Glutamin, Kohlenhydraten und Fetten. Entweder die Amino- oder Amid-Gruppe des Glutamins war durch das stabile Stickstoff-Isotope (15 N) markiert. Je drei Blutentnahmen wurden aus einem arteriellen, einem Mesenterialvenen- und einem Hepatalvenenkatheter vor (-10, -5 und 0 min) und während der Nährlösungsapplikation (150, 195 und 240 min) entnommen. Die Messung der quantitativen Ammoniumkonzentration erfolgte enzymatisch. Die Aminosäurenkonzentrationen wurde mittels HPLC bestimmt. Die Anreicherung des markierten Stickstoffs im Ammonium und in den Aminosäuren erfolgte mittels GC-MS. Der Transfer des markierten Stickstoffs aus der Amid-Gruppe des Glutamins auf das Ammonium war signifikant höher als aus der Amino-Gruppe (1.66 +/- 0.15 MPE vs 0.05 +/- 0.15 MPE). Dagegen war der Transfer auf die Amino-Gruppen anderer Aminosäuren wie Ala-nin, Serin, Glycin und Prolin nach der Applikation von Amino-markiertem Glutamin signifikant höher als der nach der Applikation von Amid-markiertem Glutamin. Aus diesen Daten kann geschlossen werden, dass das durch den Dünndarm produzierte Ammonium überwiegend aus der Deamidierung des Glutamins über die Gluaminase-Reaktion entstammt. / Systemic hyperammonemia plays a central role in the pathogenesis of the hepatic encephalopathy. Recently it could be demonstrated, that the small intestine is an important source of postfeeding hyperammonemia in patients with cirrhosis. The portal venous ammonia load forms partly, in animals up to 50 %, from glutamine metabolism in small intestine. Under pathologic conditions as in liver cirrhosis with portal-systemic shunting, however, this ammonia load causes systemic hyperammonemia. The Main goals of our study were to investigate, which nitrogen group of glutamine molecule serves the intestinal am-moniogenesis, what role the secound nitrogen group of glutamine plays, and which metabolites are produced within the intestinal glutamine metabolism. We investigate 2 Groups of 4 patients each with liver cirrhosis who had received a transjugular intrahepatic porto-systemic stent-shunt (TIPS) before. The patients got a nutrient solution containing labelled glutamine, carbohydrate and fat over 240 min via a nasoduodenal tube. The glutamine molecule was labelled on its amid or amino group respectively with the stable 15 N-isotope. Blood was taken from an arterial, a mesenterial venous and a hepatic venous catheter before (-10, -5 and 0 min) and during (150, 195 and 240 min) application of the nutrient solution. Ammonia concentrations were measured enzymatically, the concentration of the amino acids by HPLC. The enrichment of the labelled nitrogen in ammonia and amino acids was measured by GC-MS. The amount of labelled nitrogen of the amid group which was transferred to ammonia was significantly higher than the labelled nitrogen transferred from amino group (1.66 +/- 0.15 MPE vs 0.05 +/- 0.15 MPE). In contrast the amino nitrogen was significantly transferred to the amino acids such as alanine, serine, glycine and proline. We conclude that the ammonia produced by small intestine predominantly originates from The amid nitrogen of glutamine by deamidation via glutaminase pathway.

The guanosine monophosphate synthetase (GMPS) is a class I glutamine amidotransferase, involved in the de-novo purine nucleotide biosynthesis. The enzyme catalyzes the biochemical transformation of xantosine (XMP) into guanosine monophosphate (GMP) in presence of ATP, Mg2+ and glutamine. All GMPSs consist of two catalytic sites 1) for GATase activity 2) for the ATPPase activity. The two catalytic sites may be housed in the same polypeptide (two-domain-type) or in separate polypeptides (two-subunit-type). Most of the studies have been performed on two-domain-type GMPSs, while only one study has been reported from two-subunit-type GMPS (Maruoka et al. 2009). The two-subunit-type GMPS presents an example where the component reactions of a single enzymatic reaction are carried out by two distinct subunits. In order to get better understanding of structural aspects and mechanistic principle that governs the GMPS activity in two-subunit-type GMPSs, we initiated the study by taking GMPS of Methanocaldococcus jannaschii as a model system. The GMPS of M. jannaschii (Mj) is a two-subunit-type protein. The GATase subunit catalyzes the hydrolysis of glutamine to produce glutamate and ammonia. The ATPPase subunit catalyses the amination of XMP to produce GMP using the ammonia generated in GATase subunit. Since the two component reactions are catalysed by two separate subunits and are coupled in the way that product of one reaction (ammonia) acts as a nucleophile in the second reaction. The cross-talk between these two subunits in order to maximise the efficiency of overall GMPS warrants investigation. The GATase activity is tightly regulated by the interaction with ATPPase domain/subunit, in all GMPS except in the case of P. falciparum. This interaction is facilitated by substrate binding to the ATPPase domain/subunit. Though, the conditions for the interaction between two subunits is known in a two-subunit-type GMP synthetase from P. horikoshii, the structural basis of substrate dependent interaction is not known. As a first step to understand the structural basis of interaction between the Mj GATase and Mj ATPPase subunits, we have determined the structure of Mj GATase (21 kDa) subunit using high resolution, multinuclear, multidimensional NMR spectroscopy. Sequence specific resonance assignments were obtained through analysis of various 2D and 3D hetero-nuclear multidimensional NMR experiments. NMR based distance restraints were obtained from assignment of correlations observed in NOE based experiments. Data were acquired on isotopically enriched samples of Mj GATase. The structure of Mj GATase (2lxn) was solved by using cyana-3.0 using NMR based restraints as input for the structure calculation. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35±0.06 Å for backbone atoms and 0.8±0.06 Å for all heavy atoms. Attempts were also made to obtain assignments for the 69.6 kDa dimeric ATPPase subunit. Partial assignments have been obtained for this subunit. The GATase subunit is catalytically inactive. So far, there has been only one published report on a two-subunit-type GMPS from P. horikashii. The study has shown that the catalytic activity of GATase is regulated by the GATase-ATPPase interaction which is facilitated by the substrate binding to the ATPPase subunit. For the first time, we have provided the structural basis of interaction between GATase-ATPPase (112 kDa) in a two-subunit-type GMPS. Observed line width changes were used to identify residues in GATase residues that are involved in the Mj GATase-ATPPase interaction. Our data provides a possible explanation for conformational changes observed in the Mj GATase subunit upon GATase-ATPPase interaction that lead to GATase activation. Ammonia is generated in GATase subunit and is very reactive and labile. Thus, the faithful transportation of ammonia from GATase to ATPPase subunit is very crucial for optimal GMPS activity. Till date, a PDB query for GMPS retrieves only one structure which belongs to two-subunit-type GMPS, where authors have determined the structures of GATase and ATPPase subunits separately. However, the structure of holo-GMPS is not determined yet. Using interface information from experimental data and HADDOCK, we have constructed a model for the holo-GMPS from M. jannaschii. A possible ammonia channel has been deduced using the programs MOLE 2.0 and CAVER 2.0. This ammonia channel has a length of 46 Å, which is well within the range of the lengths calculated for similar channels in other glutamine amidotransferase. It had been suggested earlier that in addition to the magnesium required for charge stabilization of ATP, additional binding sites were present on GMPS. The effect of excess Mg2+ requirement on the GMPS activity has been studied in two-domain-type GMPS. However, the interaction between GATase and Mg2+ has been not investigated in any GMPS. This prompted us to investigate the effect of MgCl2 on Mj GATase subunit. For the first time, using chemical shift perturbation, we have established interaction between Mj GATase and Mg2+. The dissociation constant (Kd) of the Mj GATase-Mg2+ interaction was determined. The Kd value was found to be 1 mM, which indicates a very weak interaction. The substrate of the GATase subunit is glutamine. The condition of the hydrolysis of the glutamine is known in GMPS. However, the binding of the glutamine and associated conformational changes in GATase have been not studied in GMPS. Furthermore, till date there is no structure available for the glutamine bound GMPS/GATase. Using isotope edited one dimensional and two-dimensional NMR spectroscopy; we have shown that the Mj GATase catalytic residues are not in a compatible conformation to bind with glutamine. Thus, a conformational change in Mj GATase subunit is a pre-requisite condition for the binding of glutamine. These conformational changes are brought by the Mj GATase-ATPPase interaction.

Ruminant animals consuming low N-diet have been reported to have increased urea reabsorption with the Nguni being categorized as N-recycling ruminant. The enzymes associated with N-cycling are hypothesized to contribute to survival of the Nguni in harsh conditions. Enzymes responsible for such a function needed to be characterized in order to determine their effect in the functioning of the Nguni as opposed to Hereford breed. Crude enzymes from both breeds were separated from most or some contaminants by sephadex G-25, DEAE sephacel, and different affinity column chromatography. CPS and GDH were successfully purified and characterized by LC-MS/MS and further analysed by ProteinPilot™, blasted and matched >95% with those of Bos Taurus. Comparison of characterized enzymes and those which failed to ionise such as ARG, GS and GA was done using kinetics and graphs annotating specific activities. Partial purification and characterization was in part achieved. / Life & Consumer Sciences / M. Sc. (Life Sciences)

No description available.

INTRODUÇÃO: Relatos conflitantes têm dificultado para se estabelecer o potencial benefício da glutamina (GLN) no tratamento de condições inflamatórias agudas. Nós avaliamos o efeito da infusão parenteral de GLN, prévia à pancreatite aguda (PA) experimental, nos mediadores inflamatórios, morbidade e mortalidade. MÉTODOS: Ratos Lewis (n = 131) receberam glutamina parenteral (grupo GG), solução salina (grupo SS ou controle), ou permaneceram sem infusão parenteral (grupo Sham) por 48h. Após este período, foi induzida PA por meio da injecção retrógrada de taurocolato de sódio no ducto pancreático. Sangue, amostras de pulmão, fígado, pâncreas e líquido ascítico foram colhidos a partir de 2, 12 e 24 horas após PA para avaliação das variáveis propostas (citocinas, hsp, histologia, amilase). Sessenta animais permaneceram vivos após PA para a análise da mortalidade em sete dias. RESULTADOS: A análise entre grupos não mostrou diferenças significativas nos níveis de citocinas (p > 0,05). Análise cinética dentro de cada grupo ao longo do tempo mostrou maior INF-y no grupo Sham e SS às 2h do que em 12h e 24h, maior IL-2 e inferior IL-10 no Sham, às 24h do que em 2h e 12h, e menor IL-10 no SS e GG em 24 h do que no tempo de 2h (p <= 0.05). O grupo GG exibiu maior expressão de HSP 90 no pulmão e no fígado do que no grupo Sham nos tempos de 2h e 12h, respectivamente; e maior expressão no fígado de HSP90 e HSP70 no grupo SS no tempo 12 horas (p < 0,01). O grupo Sham apresentou maior expressão de HSP 70 no pulmão e HSP 90 no fígado do que os outros grupos no tempo de 24h. Não ocorreram alterações na taxa de mortalidade. CONCLUSÕES: Em modelo de PA experimental induzida por taurocolato de sódio, o pré-tratamento com GLN parenteral melhorou o perfil dos mediadores inflamatórios, sem afetar a mortalidade / INTRODUCTION: Conflicting reports have hindered establish the potential glutamine (GLN) benefit in treating acute inflammatory conditions. We evaluated the effect of parenteral GLN infusion before experimental acute pancreatitis (AP), as systemic inflammation-reproducing model, on inflammatory mediators and mortality. METHODS: Lewis rats (n=131) received parenteral glutamine (GG group), saline (SS or Control group), or remained without parenteral infusion ( Sham group) for 48h. Thereafter, AP was induced by retrograde injection of sodium taurocholate into pancreatic duct. Blood, lung, liver and pancreas samples were collected from 2, 12 and 24h post-AP to assess serum cytokines levels, tissue HSP expression, histology and amylase. Sixty animals remained alive post-PA for seven-day mortality analysis. RESULTS: Punctual between-groups analysis did not show differences in cytokine levels (p > 0.05). Intragroup analysis over time showed higher INF-y in Sham and SS at 2h than at 12h and 24h, higher IL-2 and lower IL-10 in Sham at 24h than at 2h and 12h, and lower IL-10 in SS and GG at 24h than at 2h timepoint (p <= 0.05). GG group exhibited higher lung and liver HSP90 than Sham at 2h and 12h timepoints, respectively; and higher liver HSP90 and HSP70 than SS at 12h timepoint (p < 0.01). Sham group presented higher lung HSP70 and liver HSP90 than the others at 24h timepoint (p < 0.02). No changes occurred on mortality rate. CONCLUSIONS: In sodium taurocholate-induced PA model, pretreatment with parenteral GLN improved inflammatory mediator\'s profile, without affecting mortality

O melhor entendimento sobre os aspectos metabólicos, nutricionais e produtivos das gramíneas forrageiras quanto ao uso do nitrogênio é necessário para aumentar a eficiência de uso do nutriente e para a sustentabilidade da produção pecuária. Com isso, objetivou-se analisar alguns cultivares de gramíneas forrageiras utilizados em pastagens no Brasil, quanto a: I) modificações morfológicas e produtivas da parte aérea e das raízes; II) formas de partição do nitrogênio absorvido para melhorar a eficiência de uso do nitrogênio e III) forma de assimilação do nitrogênio absorvido, sendo avaliados pelas variáveis: produção de massa seca, números de folhas e perfilhos, área foliar, concentração e conteúdos de nitrogênio total, nitrato e amônio, eficiência de uso do nitrogênio, atividades das enzimas nitrato redutase e glutamina sintetase e concentração de aminoácidos livres totais na parte aérea e nas raízes dessas gramíneas, quando submetidas a alta e baixa dose de nitrogênio. Na baixa dose de nitrogênio os cultivares Marandu, Xaraés, Piatã, Basilisk e Mombaça mantiveram o número de perfilhos constante e diminuíram o número de folhas e a área foliar, enquanto o cultivar Tanzânia manteve o número de perfilhos e área foliar constantes e reduziu o número de folhas e os cultivares Aruana e Massai aumentaram o número de perfilhos e de folhas e reduziram a área foliar. A baixa dose de nitrogênio resultou em proporções da massa seca da parte aérea e das raízes distintas entre os cultivares quando comparada à alta dose de nitrogênio, favorecendo o crescimento das raízes. Os cultivares estudados modificaram a partição de nitrogênio, nitrato e amônio da parte aérea e raízes, quando em alto e baixo suprimento de nitrogênio. Essas diferenças refletiram na mais alta eficiência de uso do nitrogênio pelos cultivares Mombaça e Tanzânia. A assimilação do nitrato ocorreu principalmente na parte aérea desses capins. Os cultivares Mombaça e Aruana apresentaram elevadas atividades da enzima nitrato redutase nas folhas diagnósticas. A atividade da enzima glutamina sintetase nas folhas diagnósticas foi mais elevada nos cultivares de Panicum. A concentração de amônio nas raízes foi mais alta nos cultivares Piatã e Xaraés, na baixa dose de nitrogênio, e nos capins Aruana e Mombaça, na dose alta de nitrogênio. A concentração de aminoácidos totais nas folhas diagnósticas foi mais elevada nos cultivares de Panicum, quando submetidos à alta dose de nitrogênio, enquanto nas raízes foi maior nos cultivares de Brachiaria, nas duas doses de nitrogênio. / A better understanding of the metabolic, nutritional and production aspects of nitrogen use by grasses are needed to increase nutrient use efficiency and livestock production sustainability. Thus, this study aimed to analyze some forage grasses cultivars used in Brazilian pastures, for: i) morphological and production changes in shoots and roots; II) absorbed nitrogen partition to improve nitrogen use efficiency and III) nitrogen assimilation, evaluated by the variables: dry matter production, number of leaves and tillers, leaf area, concentration and content of total nitrogen, nitrate and ammonium, nitrogen use efficiency, nitrate reductase and glutamine synthetase activities and free amino acids concentration in shoots and roots of these grasses, when supplied with high and low nitrogen rates. At low nitrogen rate, Marandu, Xaraés, Piatã, Basilisk and Mombaça cultivars kept the number of tillers constant and decreased the number of leaves and leaf area, while Tanzânia cultivar kept the number of tillers and leaf area constant and reduced the number of leaves, and Aruana and Massai cultivars increased the number of tillers and leaves and reduced leaf area. The low nitrogen rate resulted in different shoots to roots proportions among cultivars when compared to the higher nitrogen rate, which favored roots growth. The cultivars changed the partition of nitrogen, nitrate and ammonium between roots and shoots, in both nitrogen rates. These differences reflect the higher nitrogen use efficiency by Mombaça and Tanzânia cultivars. The nitrate assimilation occurred mainly in the shoots of these grasses. Mombaça and Aruana cultivars showed high nitrate reductase activity in diagnostic leaves. The glutamine synthetase activity in diagnostic leaves was higher in Panicum cultivars. Ammonium concentration in the roots was high in Piatã and Xaraés, at low nitrogen rate, and Aruana and Mombaça grasses, at high nitrogen rate. The total amino acids concentration in diagnostic leaves was higher in Panicum cultivars than in Brachiaria cultivars, when high nitrogen rate was supplied, whereas Brachiaria cultivars had this concentration high in the roots, at the two nitrogen rates.

A Glutamina (Gln) melhora a regeneração hepática em ratos nutridos. Para avaliar o efeito molecular da Gln endovenosa no remanescente hepático, 52 ratos foram classificados em: 1. Nutridos com hepatectomia parcial (HP) e Gln (N-Gln=10) ou prolina (N-Pro=10); 2. Desnutridos com HP: (D-Gln=10) ou (D-Pro=10); 3. Nutridos e Desnutridos sem HP (N-Controle=6) e (D-Controle=6). Após 96 horas da HP os resultados foram: DNA = (D-Gln, D-Pro e D-Controle < N-Gln, N-Pro e N-controle), (N-Gln, N-Pro, D-Gln e D-Pro < N e D-Controle), (N-Gln > D-Gln); RNA= (N-Gln, N-Pro, D-Gln e D-Pro < N e D-Controle), (N-Gln > D-Gln e N-Pro). Não houve diferença nos genes transcritos (GT) para HGF, TGF-a e timidina kinase. Concluímos que: A desnutrição e HP reduzem DNA e RNA; A Gln não altera os GT estudados em ratos desnutridos 96 horas após HP / Glutamine dipeptide (Gln) improve hepatic regeneration of nourished rats. To evaluate molecular effect of Gln on liver remnant 52 rats was classified into: 1. Nourished with partial hepatectomy (PH) and Gln (N-Gln=10) or proline (N-Pro=10); 2. Malnourished (MN) with PH: (MN-Gln=10) or (MN-Pro=10); 3. Nourished and Malnourished rat without PH (N-Control=6) and (MN-Control=6). Results: DNA = (MN-Gln, MN-Pro and MN-Control < N-Gln, N-Pro and N-control), (N-Gln, N-Pro, MN-Gln and MN-Pro < N and MN-Control), (N-Gln > MN-Gln); RNA= (N-Gln, N-Pro, MN-Gln and MN-Pro < N and MN-Control), (N-Gln > MN-Gln and N-Pro). There was no difference on transcript genes (TG) for HGF, TGF-a and thymidine kinase. Conclusions: Malnutrition and PH decrease hepatic DNA and RNA; Gln does not modify TG studied 96 hours after PH in MN rats

O metabolismo da glutamina (Gln) é alvo de atenções recentes para a compreensão da reprogramação metabólica para o suprimento energético das células tumorais em proliferação e para o desenvolvimento de novas estratégias terapêuticas em câncer. Tanto a absorção de glutamina quanto a taxa de glutaminólise, o catabolismo da Gln para gerar adenosina trifosfato (ATP) e lactato na mitocôndria estão aumentados em diferentes tumores. A Gln e glicose participam do processo da proliferação de células tumorais tanto na produção de (ATP) como no fornecimento de produtos intermediários utilizados na síntese de macromoléculas e Gln é utilizado para anaplerose do ciclo do ácido tricarboxílico. Nesse estudo, nosso objetivo foi analisar a expressão dos genes envolvidos na glutaminólise: ASCT2, LAT1, GLS, GLSISO1, GLSISO2, GLS2, GOT1, GOT2, GLUD1 e GPT2 em astrocitomas de diferentes graus de malignidade (AGI-AGIV), classificados de acordo com a Organização Mundial de Saúde (OMS), em relação às expressões em tecidos cerebrais não neoplásicos e correlacionar os níveis de expressão destes genes aos dados clínicos. PCR quantitativo em tempo real (qRT-PCR) foi realizado em 175 amostras, sendo 22 dentre estes de tecidos não neoplásicos. Observou-se tempo de sobrevida menor entre os pacientes com hiperexpressão de LAT1, na presença de hipoexpressão de ASCT2. A expressão de GLS foi comparativamente maior que a expressão de GLS2 entre os astrocitomas de diferentes graus de malignidade, corroborando descrições prévias de que GLS relaciona-se à proliferação tumoral e GLS2 à supressão do crescimento tumoral. Observou-se, adicionalmente, o aumento da associação das expressões destes genes conforme o aumento do grau de malignidade, culminando em GBM, onde estas correlações foram estatisticamente significativas. Apesar da demonstração da ativação gradativa desta via da glutaminólise com o aumento da malignidade, a hiperexpressão dos genes relacionados a esta via mostrou-se hiperexpressa em apenas um subgrupo de pacientes com GBM. Esta observação ressalta a heterogeneidade observada em GBM e a elegibilidade restrita deste subgrupo a eventuais estratégias terapêuticas que forem desenvolvidas com alvos nesta via / The metabolism of glutamine (Gln) is the target of recent attentions to understand the metabolic reprogramming of cancer cell for the energetic needs for cell proliferation, and to develop new cancer therapeutic strategies. Glutamine absortion and glutamine conversion to ATP and lactate in the mitochondria through glutaminolysis are both increased in different cancer types. Gln and glucose participate in metabolic pathways which provide ATP and intermediate substrats for synthesis of macromolecules, and Gln is used for anaplesoris of tricyclic acid cycle. The aims of the present study were to analyze the differential mRNA expressions of genes involved in the glutaminolysis pathways: ASCT2, LAT1, GLS, GLSISO1, GLSISO2, GLS2, GOT1, GOT2, GLUD1 e GPT2 in astrocytomas of different grades of malignancy (WHO grades I to IV) compared to non-neoplastic brain tissues, and to correlate these expression data to clinical outcome. Shorter overall survival time was observed among a subset of GBM patients presenting hyperexpression of LAT1 while ASCT2 was hypoexpressed. GLS expression was comparatively higher than GLS2 expression among astrocytomas of different grades of malignancy, which corrobates previous reports relating GLS to tumor proliferation and GLS2 to suppression of tumor growth. Additionally, increased gene expression correlation was observed in parallel to the increase of malignancy, and these associated expressions were significant among GBM. Although a stepwise increase of the glutaminolysis pathway was demonstrated with the increase of malignancy in astrocytomas, the hyperexpression of genes involved in this pathway were detected only in a subset of GBM patients. This finding confirm the heterogeneity observed among GBM, and highlights the fact that any therapeutic strategy aiming this pathway will be restricted to a subset of GBM patients

INTRODUÇÃO: Metionina (Met), cisteína (Cys), homocisteína (Hcy) e taurina (Tau) são os quatro aminoácidos sulfurados (AAS), mas apenas a Met e Cys são incorporadas em proteínas. Os três principais produtos doS AAS, glutationa, (GSH), Hcy e Tau influenciam, principalmente, as respostas inflamatória e imune. A Tau e GSH diminuem a inflamação, enquanto que a Hcy apresenta efeito oposto. Os pacientes HIV+ apresentam baixos níveis de GSH e outros nutrientes antioxidantes, mostrando relação direta entre Cys (e GSH) com células CD4+. Não se conhece o mecanismo pelo qual as mudanças na ingestão dos AAS influenciam este fenômeno. Paralelamente, as relações entre Hcy, doenças inflamatórias e alterações in vitro no comportamento das células imunes levantou ressalvas sobre a suplementação de dietas com AAS. OBJETIVOS : investigar as vias dos AAS em pacientes HIV+ nas condições de jejum e pós-sobrecarga de Met frente à dieta habitual (OH) isolada ou acompanhada da suplementação de Cys (NAC) ou glutamina (Gln). MÉTODOS : 12 pacientes HIV+ (6 M e 6 F, de 25 a 36 anos), sob tratamento anti-retroviral pelo esquema tríplice, sem infecções secundárias e 20 controles saudáveis (10M e 10F, 23-28 anos) foram randomicamente distribuídos para suplementação com NAC (N-acetilcisteína, 1g/d) ou Gln (20 g/d) em estudo cruzado com 7 dias de dieta separados por uma semana de washout (Wo com DH). Amostras de sangue após jejum noturno de 10 a 12 horas foram coletadas antes (MO) e após (M1) cada regime dietético. A seguir, os indivíduos ingeriram metionina (100 mg/kg), com coletas de sangue após 2 e 4 horas para a determinação da área abaixo da curva (AAC). No MO, ambos os grupos foram avaliados quanto à antropometria (IMC, kg/m2), funções glomerular (uréia, creatinina) e hepatocelular (&#947;-GT), estados nutricional (albumina, cálcio, ácido fólico e vitamina 812) e antioxidante (ácido úrico, GSH, GSSG, Hcy), glicose, lipídios (triacilgliceróis e frações de colesterol) e AAS, serina (Ser), glicina (Gly), glutamato (Glu) e Gln. O grupo HIV também foi caracterizado pela carga viral e contagem de CD4+ e CD8+. As comparações estatísticas entre os grupos e entre as dietas mostraram homogeneidade para IMC, albumina, cálcio, vitamina 812, Hcy, HDL-colesterol, uréia e creatinina. Os pacientes apresentaram valores maiores de glicose, triacilgliceróis, &#947;-GT, LDL-colesterol e GSSG paralelalemente às menores concentrações de ácido úrico, GSH e todos os AAS, exceto Hcy. A sobrecarga de metionina igualou (pelos valores de delta) os grupos para Met, Hcy, Tau e Gln. As suplementações de NAC e Gln levaram o grupo HIV+ a concentrações maiores de GSH (NAC > Gln), atuando diferentemente em seus precursores: G/y (Gln > NAC) e Cys (NAC > Gln) e resultando em consumo similar de Ser e produção de Tau. Ambas as dietas reduziram GSSG/GSH (NAC > Gln) e apenas NAC aumentou (6 x) a Hcy. Esta última foi piorada pela sobrecarga de Mel. Assim, HIV+ resulta em deficiências múltiplas de vitaminas e aminoácidos levando a menores níveis de GSH e GSSG/GSH mais elevada. Os principais problemas de menor formação de Cys e menor incorporação de Cys em GSH foram resolvidos dando-se Met, NAC e Gln aos pacientes, ainda permanecendo a desvantagem do aumento da Hcy com Met ou suplementação de NAC. / BACKGROUNO: Methionine (Met), cysteine (Cys), homocysteine (Hcy), and, taurine (Tau) are the 4 sulfur-containing amino acids (SAA), but only Met and Cys are incorporated into proteins. The 3 major products of SAA, glutathione (GSH), Hcy and Tau influence, mainly, inflammatory and of immune responses. Tau and GSH ameliorate inflammation whereas Hcy has the opposite effect. HIV+ patients present low levelis of GSH and other antioxidants nutrients, showing a direct relationship between Cys (and GSH) with CD4+/ cells. How changes in SAA intake influence this phenomenon is unknown and the relationships among Hcy, inflammatory diseases, and in vitro alterations in immune cell behavior create a cautionary note about supplementation of diets with SAA. OBJECTIVE: To investigate SAA pathways in HIV+ patients on fast and Met-overload (Met-DL) states after taken diet habitual without (HD) or with supplements of Cys (NAC) or glutamine (Gln). METHOOS: 12 HIV+ (6M and 6F, 25-36 yrs old) patients under HAART without secondary infections and 20 healthy (10M and 10F, 23-28 yrs old) controls were randomly assigned to either NAC (N-acetylcysteine, 1g/d) or Gln (20g/d) diets, in a 7-day diet crossover design, separated by a 7-day washout (with HD) period. Blood samples were drawn after overnight fast before (MO) and after each dietary treatments (M1) for the resting measurements. Immediately after blood sampling ali subjects started the Met-DL by ingesting at once 100 mg Met/kg BW and having the blood draw after 2 and 4 hours for the area under the curve (AUC) determination. At MO both groups were assessed for anthropometry (BMI, kg/m2), glomerular (plasma urea and creatinina) and hepatocellular (plasma &#947;GT activity) funetions, nutritional (albumin, calcium, folic acid and vitamin B12) and antioxidant (uric acid, GSH, GSSG, Hey) states, glucose, lipids (triglycerides and cholesterol fractions) and SAA, serine (Ser), glyeine (Gly), glutamate (Glu) and Gln. The HIV+ group was characterized also by viral load, CD4+ and CD8+ counts. The statistical comparisons between groups and among diets showed group homogeneity for 8MI, albumin, calcium, vitamin B12, Hey, HDL-cholesterol, urea and creatinine. The patients presented higher values of glucose, triglycerides, &#947;-GT, LDL-cholesterol, and GSSG along with lower concentrations of uric acid, GSH and all but Hcy amino acids. The Met-OL equalized (&#916; values) the groups for Met, Hcy, Tau and Gln. NAC and Gln diets led the HIV+ group to a higher concentrations of GSH (NAC > Gln) by acting differently on its precursors: Gly (Gln > NAC) and Cys (NAC > Gln), resulting similar consumption of Ser and production of Tau. Both diets reduced GSSG/GSH (NAC > Gln) and only NAC increased (6 x) Hey. The later was worsened by Met-OL. Thus HIV+ results in multiple deficiencies of vitamins and amino acids leading to lower levels of GSH and higher GSSG/GSH ration. The main problems of lower formation of Cys and low ineorporation of Cys and Gly into GSH were greatly solved by giving Met, NAC and Gln to the patients, hence remaining the drawback of increasing Hcy with Met or NAC supplements.

O melhor entendimento sobre os aspectos metabólicos, nutricionais e produtivos das gramíneas forrageiras quanto ao uso do nitrogênio é necessário para aumentar a eficiência de uso do nutriente e para a sustentabilidade da produção pecuária. Com isso, objetivou-se analisar alguns cultivares de gramíneas forrageiras utilizados em pastagens no Brasil, quanto a: I) modificações morfológicas e produtivas da parte aérea e das raízes; II) formas de partição do nitrogênio absorvido para melhorar a eficiência de uso do nitrogênio e III) forma de assimilação do nitrogênio absorvido, sendo avaliados pelas variáveis: produção de massa seca, números de folhas e perfilhos, área foliar, concentração e conteúdos de nitrogênio total, nitrato e amônio, eficiência de uso do nitrogênio, atividades das enzimas nitrato redutase e glutamina sintetase e concentração de aminoácidos livres totais na parte aérea e nas raízes dessas gramíneas, quando submetidas a alta e baixa dose de nitrogênio. Na baixa dose de nitrogênio os cultivares Marandu, Xaraés, Piatã, Basilisk e Mombaça mantiveram o número de perfilhos constante e diminuíram o número de folhas e a área foliar, enquanto o cultivar Tanzânia manteve o número de perfilhos e área foliar constantes e reduziu o número de folhas e os cultivares Aruana e Massai aumentaram o número de perfilhos e de folhas e reduziram a área foliar. A baixa dose de nitrogênio resultou em proporções da massa seca da parte aérea e das raízes distintas entre os cultivares quando comparada à alta dose de nitrogênio, favorecendo o crescimento das raízes. Os cultivares estudados modificaram a partição de nitrogênio, nitrato e amônio da parte aérea e raízes, quando em alto e baixo suprimento de nitrogênio. Essas diferenças refletiram na mais alta eficiência de uso do nitrogênio pelos cultivares Mombaça e Tanzânia. A assimilação do nitrato ocorreu principalmente na parte aérea desses capins. Os cultivares Mombaça e Aruana apresentaram elevadas atividades da enzima nitrato redutase nas folhas diagnósticas. A atividade da enzima glutamina sintetase nas folhas diagnósticas foi mais elevada nos cultivares de Panicum. A concentração de amônio nas raízes foi mais alta nos cultivares Piatã e Xaraés, na baixa dose de nitrogênio, e nos capins Aruana e Mombaça, na dose alta de nitrogênio. A concentração de aminoácidos totais nas folhas diagnósticas foi mais elevada nos cultivares de Panicum, quando submetidos à alta dose de nitrogênio, enquanto nas raízes foi maior nos cultivares de Brachiaria, nas duas doses de nitrogênio. / A better understanding of the metabolic, nutritional and production aspects of nitrogen use by grasses are needed to increase nutrient use efficiency and livestock production sustainability. Thus, this study aimed to analyze some forage grasses cultivars used in Brazilian pastures, for: i) morphological and production changes in shoots and roots; II) absorbed nitrogen partition to improve nitrogen use efficiency and III) nitrogen assimilation, evaluated by the variables: dry matter production, number of leaves and tillers, leaf area, concentration and content of total nitrogen, nitrate and ammonium, nitrogen use efficiency, nitrate reductase and glutamine synthetase activities and free amino acids concentration in shoots and roots of these grasses, when supplied with high and low nitrogen rates. At low nitrogen rate, Marandu, Xaraés, Piatã, Basilisk and Mombaça cultivars kept the number of tillers constant and decreased the number of leaves and leaf area, while Tanzânia cultivar kept the number of tillers and leaf area constant and reduced the number of leaves, and Aruana and Massai cultivars increased the number of tillers and leaves and reduced leaf area. The low nitrogen rate resulted in different shoots to roots proportions among cultivars when compared to the higher nitrogen rate, which favored roots growth. The cultivars changed the partition of nitrogen, nitrate and ammonium between roots and shoots, in both nitrogen rates. These differences reflect the higher nitrogen use efficiency by Mombaça and Tanzânia cultivars. The nitrate assimilation occurred mainly in the shoots of these grasses. Mombaça and Aruana cultivars showed high nitrate reductase activity in diagnostic leaves. The glutamine synthetase activity in diagnostic leaves was higher in Panicum cultivars. Ammonium concentration in the roots was high in Piatã and Xaraés, at low nitrogen rate, and Aruana and Mombaça grasses, at high nitrogen rate. The total amino acids concentration in diagnostic leaves was higher in Panicum cultivars than in Brachiaria cultivars, when high nitrogen rate was supplied, whereas Brachiaria cultivars had this concentration high in the roots, at the two nitrogen rates.