L-ornithine-L-aspartate in experimental portal-systemic encephalopathy: therapeutic efficacy and mechanism of action.

Strategies aimed at the lowering of blood ammonia remain the treatment of choice in portal-systemic encephalopathy (PSE). L-ornithine-L-aspartate (OA) has recently been shown to be effective in the prevention of ammonia-precipitated coma in humans with PSE. These findings prompted the study of mechanisms of the protective effect of OA in portacaval-shunted rats in which reversible coma was precipitated by ammonium acetate administration (3.85 mmol/kg i.p.). OA infusions (300 mg/kg/h, i.v) offered complete protection in 12/12 animals compared to 0/12 saline-infused controls. This protective effect was accompanied by significant reductions of blood ammonia, concomitant increases of urea production and significant increases in blood and cerebrospinal fluid (CSF) glutamate and glutamine. Increased CSF concentrations of leucine and alanine also accompanied the protective effect of OA. These findings demonstrate the therapeutic efficacy of OA in the prevention of ammonia-precipitated coma in portacaval-shunted rats and suggest that this protective effect is both peripherally-mediated (increased urea and glutamine synthesis) and centrally-mediated (increased glutamine synthesis).

Oxidative DNA cleavage, cytotoxicity and antimicrobial studies of L-ornithine copper (II) complexes

New ternary copper (II) complexes, Cu(L-orn)(B)(Cl)](Cl center dot 2H(2)O) (1-2) where L-orn is L-ornithine, B is an N,N-donor heterocyclic base, viz. 2,2'-bipyridine (bpy, 1) and 1,10-phenanthroline (phen, 2), were synthesized and characterized by various spectroscopic techniques. Complex 2 is characterized by the X-ray single crystallographic method. The complex shows a distorted square-pyramidal (4 + 1) CuN3OCl coordination sphere. Binding interactions of the complexes with calf thymus DNA (CT-DNA) were investigated by UV-Vis absorption titration, ethidium bromide displacement assay, viscometric titration experiment and DNA melting studies. Complex 2 shows appreciable chemical nuclease activity in the presence of 3-mercaptopropionic acid (MPA). The complexes were subjected to in vitro cytotoxicity studies against carcinomic human alveolar basal epithelial cells (A-549) and human epithelial (HEp-2) cells. The IC50 values of 1 and 2 are less than that of cisplatin against HEp-2 cell lines. MIC values for 1 against the bacterial strains Streptococcus mutans and Pseudomonas aeruginosa are 0.5 mM. (C) 2012 Elsevier Ltd. All rights reserved.

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.

Putrescine-Sensitive (Artifactual) and Insensitive (Biosynthetic) S-Adenosyl-L-Methionine Decarboxylase Activities of Lathyrus sativus Seedlings

The crude extracts of 3-day-old etiolated seedlings of Lathyrus sativus contained two S-adenosyl-L-methionine decarboxylase activities. The artifactual putrescine-dependent activity was due to the H2O2 generated by diamine oxidase (EC 1.4.3.6) of this plant system and was inhibited by catalase. This observation was confirmed by using an electrophoretically and immunologically homogeneous preparation of L. sativus diamine oxidase. In the presence of putrescine, diamine oxidase, in addition to S-adenosylmethionine, decarboxylated L-lysine, L-arginine, L-ornithine, L-methionine and L-glutamic acid to varying degrees. The decarboxylation was not metal-ion dependent. The biosynthetic S-adenosylmethionine decarboxylase (EC 4.1.1.21) was detected after removing diamine oxidase specifically from the crude extracts by employing an immunoaffinity column. This Mg2+ -dependent decarboxylase was not stimulated by putrescine or inhibited by catalase. The enzyme activity was inhibited by semicarbazide, 4-bromo-3-hydroxybenzoylamine dihydrogen phosphate and methylglyoxal-bis (guanylhydrazone). It was largely localized in the shoots of the etiolated seedlings and was purified 40-fold by employing a p-hydroxymercuribenzoate/AH-Sepharose affinity column, which also separated the decarboxylase activity from spermidine synthase.

Substrate-induced conformational change in a trimeric ornithine transcarbamoylase

The crystal structure of Escherichia coli ornithine transcarbamoylase (OTCase, EC 2.1.3.3) complexed with the bisubstrate analog N-(phosphonacetyl)-l-ornithine (PALO) has been determined at 2.8-Å resolution. This research on the structure of a transcarbamoylase catalytic trimer with a substrate analog bound provides new insights into the linkages between substrate binding, protein–protein interactions, and conformational change. The structure was solved by molecular replacement with the Pseudomonas aeruginosa catabolic OTCase catalytic trimer (Villeret, V., Tricot, C., Stalon, V. & Dideberg, O. (1995) Proc. Natl. Acad. Sci. USA 92, 10762–10766; Protein Data Bank reference pdb 1otc) as the model and refined to a crystallographic R value of 21.3%. Each polypeptide chain folds into two domains, a carbamoyl phosphate binding domain and an l-ornithine binding domain. The bound inhibitor interacts with the side chains and/or backbone atoms of Lys-53, Ser-55, Thr-56, Arg-57, Thr-58, Arg-106, His-133, Asn-167, Asp-231, Met-236, Leu-274, Arg-319 as well as Gln-82 and Lys-86 from an adjacent chain. Comparison with the unligated P. aeruginosa catabolic OTCase structure indicates that binding of the substrate analog results in closure of the two domains of each chain. As in E. coli aspartate transcarbamoylase, the 240s loop undergoes the largest conformational change upon substrate binding. The clinical implications for human OTCase deficiency are discussed.

Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family.

The crystal structure of the Glu-105-->Gly mutant of catabolic ornithine transcarbamoylase (OTCase; carbamoyl phosphate + L-ornithine = orthophosphate + L-citrulline, EC 2.1.3.3) from Pseudomonas aeruginosa has been determined at 3.0-A resolution. This mutant is blocked in the active R (relaxed) state. The structure was solved by the molecular replacement method, starting from a crude molecular model built from a trimer of the catalytic subunit of another transcarbamoylase, the extensively studied aspartate transcarbamoylase (ATCase) from Escherichia coli. This model was used to generate initial low-resolution phases at 8-A resolution, which were extended to 3-A by noncrystallographic symmetry averaging. Four phase extensions were required to obtain an electron density map of very high quality from which the final model was built. The structure, including 4020 residues, has been refined to 3-A, and the current crystallographic R value is 0.216. No solvent molecules have been added to the model. The catabolic OTCase is a dodecamer composed of four trimers organized in a tetrahedral manner. Each monomer is composed of two domains. The carbamoyl phosphate binding domain shows a strong structural homology with the equivalent ATCase part. In contrast, the other domain, mainly implicated in the binding of the second substrate (ornithine for OTCase and aspartate for ATCase) is poorly conserved. The quaternary structures of these two allosteric transcarbamoylases are quite divergent: the E. coli ATCase has pseudo-32 point-group symmetry, with six catalytic and six regulatory chains; the catabolic OTCase has 23 point-group symmetry and only catalytic chains. However, both enzymes display homotropic and heterotropic cooperativity.

Water stress induced alterations in ornithine aminotransferase of ragi (Eleusine coracana): Protection by proline against heat inactivation and denaturation by urea and guanidinium chloride

Water stress resulted in a specific response leading to a large and significant increase (80-fold) in free proline content of ragi (Eleusine coracana) leaves and seedlings. L-Proline protected ornithine aminotransferase, an enzyme in the pathway for proline biosynthesis, isolated from normal and stressed ragi leaves against heat inactivation and denaturation by urea and guanidinium chloride. The protection of the stressed enzyme by L-proline was much more complete than that of the enzyme isolated from normal leaves. While L-ornithine, one of the substrates, protected the stressed enzyme against inactivation, it enhanced the rate of inactivation of the normal enzyme. α-Ketoglutarate protected both the normal and stressed enzyme against inactivation and denaturation. These results support the suggestion that ornithine aminotransferase has undergone a structural alteration during water stress. In view of the causal relationship between elevated temperature and water stress of plants under natural conditions, the protection afforded by proline against inactivation and denaturation of the enzyme from stressed leaves assumes significance. These results provide an explanation for a possible functional importance of proline accumulation during water stress.

Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-L-lysine, poly-L-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-L-lysine and poly-L-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.

Arginine Decarboxylase from Lathyrus sativus Seedlings Purification and Properties

Arginine decarboxylase which makes its appearance in Lathyrus sativus seedlings after 24 h of seed germination reaches its highest level around 5–7 days, the cotyledons containing about 60% of the total activity in the seedlings at day 5. The cytosol enzyme was purified 977-fold from whole seedlings by steps involving manganese chloride treatment, ammonium sulphate and acetone fractionations, positive adsorption on alumina C-γ gel, DEAE-Sephadex chromatography followed by preparative disc gel electrophoresis. The enzyme was shown to be homogeneous by electrophoretic and immunological criteria, had a molecular weight of 220000 and appears to be a hexamer with identical subunits. The optimal pH and temperature for the enzyme activity were 8.5 and 45 °C respectively. The enzyme follows typical Michaelis-Menten kinetics with a Km value of 1.73 mM for arginine. Though Mn2+ at lower concentrations stimulated the enzyme activity, there was no dependence of the enzyme on any metal for the activity. The arginine decarboxylase of L. sativus is a sulfhydryl enzyme. The data on co-factor requirement, inhibition by carbonyl reagents, reducing agents and pyridoxal phosphate inhibitors, and a partial reversal by pyridoxal phosphate of inhibition by pyridoxal · HCl suggests that pyridoxal 5'-phosphate is involved as a co-factor for the enzyme. The enzyme activity was inhibited competitively by various amines including the product agmatine. Highest inhibition was obtained with spermine and arcain. The substrate analogue, l-canavanine, homologue l-homoarginine and other basic amino acids like l-lysine and l-ornithine inhibited the enzyme activity competitively, homoarginine being the most effective in this respect.

Doenças cardiovasculares em pacientes bipolares: uma investigação da biodisponibilidade do óxido nítrico

Os transtornos psiquiátricos são um problema de saúde pública, ocupando cinco das dez principais causas de incapacitação no mundo. O transtorno bipolar (TB) é um transtorno de humor, segundo o DSM-IV (manual diagnóstico e estatístico de doenças mentais), o qual afeta cerca de 1% da população mundial. O TB do tipo I (TBI) é o mais frequente entre os TBs e é caracterizado pela presença de episódios maníacos ou mistos acompanhados por episódios depressivos. Assim como outros transtornos psiquiátricos, como a depressão, ansiedade e esquizofrenia, o TB representa um importante fator de risco cardiovascular, e pacientes com este transtorno apresentam mortalidade cardiovascular duas vezes maior que a população em geral. No entanto, os exatos mecanismos envolvidos nesta relação permanecem desconhecidos. Estudos sugerem o envolvimento da via L-arginina-óxido nitrico (NO) na patofisiologia do TB. O NO é responsável por diversas funções fisiológicas, incluindo a inibição da função plaquetária. A L-arginina, sua precursora, é transportada em plaquetas pelo carreador y+L, ativando a enzima NO sintase (NOS), a qual produz NO e e L-citrulina. Uma vez produzido, o NO ativa a enzima guanilato ciclase (GC), levando ao aumento dos níveis de guanosina monofosfato cíclica (GMPc). Adicionalmente, a L-arginina não é exclusivamente utilizada pela NOS, ela também pode ser metabolizada pela arginase e produzir L-ornitina e uréia. A biodisponibilidade do NO depende tanto de sua síntese como de sua degradação pelo estresse oxidativo ou pela inflamação. O objetivo deste estudo foi investigar detalhadamente a via L-arginina-NO-GMPc em plaquetas de pacientes com TBI, a expressão da arginase e outros marcadores de estresse oxidativo e inflamação. Vinte e oito pacientes com TB e dez indivíduos saudáveis foram incluídos no estudo. Nossos estudos mostraram uma redução da atividade da NOS em todos os grupos de pacientes bipolares (fases de eutimia, depressão e mania), quando comparados aos controles. Isto ocorreu na presença de concentrações normais do substrato e de seu transporte, e da expressão inalterada das isoformas eNOS e iNOS. A expressão da arginase II não diferiu entre os grupos estudados, indicando que a disponibilidade da L-arginina não está sendo desviada para o ciclo de uréia em plaquetas. A produção reduzida de GMPc foi observada mesmo com a expressão inalterada da GC. A atividade e marcadores de estresse oxidativo, avaliada através da quantificação da oxidação de proteínas e atividade da catalase, não foram modificadas em plaquetas de pacientes com TB, enquanto que a atividade da SOD estava aumentada em todas as fases. Os níveis séricos da proteína C-reativa (PCR), um marcador inflamatório, estão aumentados em pacientes maníacos, comparados aos controles. A reduzida produção de NO observada em plaquetas de pacientes bipolares pode ser um elo entre esta complexa associação entre TB e a doença cardiovascular.

The outer membrane of Brucella ovis shows increased permeability to hydrophobic probes and is more susceptible to cationic peptides than are the outer membranes of mutant rough Brucella abortus strains

The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-L-lysine, and poly-L-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells.

Outer membrane differences between pathogenic and environmental Yersinia enterocolitica biogroups probed with hydrophobic permeants and polycationic peptides

Sensitivities to polycationic peptides and EDTA were compared in Yersinia enterocolitica pathogenic and environmental biogroups. As shown by changes in permeability to the fluorescent hydrophobic probe N-phenylnaphthylamine (NPN), the outer membranes (OMs) of pathogenic and environmental strains grown at 26 degrees C in standard broth were more resistant to poly-L-lysine, poly-L-ornithine, melittin, cecropin P1, polymyxin B, and EDTA than Escherichia coli OMs. At 37 degrees C, OMs of pathogenic biogroups were resistant to EDTA and polycations and OMs of environmental strains were resistant to EDTA whereas E. coli OMs were sensitive to both EDTA and polycations. Similar results were found when testing deoxycholate sensitivity after polycation exposure or when isogenic pairs with or without virulence plasmid pYV were compared. With bacteria grown without Ca++ available, OM permeability to NPN was drastically increased in pathogenic but not in environmental strains or E. coli. Under these conditions, OMs of pYV+ and pYV- cells showed small differences in NPN permeability but differences in polycation sensitivity could not be detected by fluorimetry. O:1,6 (environmental type) lipopolysaccharide (LPS), but not O:3 or O:8 LPS, was markedly rough at 37 degrees C, and this could explain the differences in polycation sensitivity. LPSs from serotypes O:3 and O:8 grown at 37 degrees C were more permeable to NPN than O:1,6 LPS, and O:8 LPS was resistant to polycation-induced permeabilization. These data suggest that LPSs relate to some but not all the OM differences described. It is hypothesized that the different OM properties of environmental and pathogenic biogroups reflect the adaptation of the latter biogroups to pathogenicity.

Nerve Growth Factor Sensitisation of TRPA1 on Sensory Neurones

Background: Sensory neurones from the trigeminal nerve innervate the oro-facial region and teeth. Transient receptor potential channels (TRPs) expressed by these neurones are responsible for relaying sensory information such as changes in ambient temperature, mechanical sensations and pain. Study of TRP channel expression and regulation in human sensory neurones therefore merits investigation to improve our understanding of allodynia and hyperalgesia. Objective: The objective of this study was to differentiate human dental pulp stem cells (hDPSCs) towards a neuronal phenotype (peripheral neuronal equivalents; PNEs) and employ this model to study TRP channel sensitisation. Method: hDPSCs were enriched by preferential adhesion to fibronectin, plated on coverslips (thickness 0) coated with poly-l-ornithine and laminin and then differentiated for 7 days in neurobasal A medium with additional supplementation. A whole cell patch clamp technique was used to investigate whether TRP channels on PNE membranes were modulated in the presence of nerve growth factor (NGF). PNEs were treated with NGF for 20 minutes immediately before experimentation and then stimulated for TRPA1 activity using cinnamaldehyde. Peak currents were read at 80 mV and -80 mV and compared to peak currents recorded in untreated PNEs. Data were analysed and plotted using Clampfit9 software (Molecular Devices, Sunnyvale, California, USA). Result: Results showed for the first time that pre-treatment of PNEs by NGF produced significantly larger inward and outward currents demonstrating that TRPA1 channels on PNE membranes were capable of becoming sensitised following treatment with NGF. Conclusion: Sensitisation of TRPA1 by NGF provides evidence of a mechanism for rapid neuronal sensitisation that is independent of TRPA1 gene expression

Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis Of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K-M and V-max. values of 23.9(+/- 0.96) mM and 192.3 mu mol/min mg protein (+/- 14.3), respectively, for the native enzyme. For the recombinant counterpart, K-M was 21.5(+/- 0.90) mM and V-max was 144.9(+/- 8.9) mu mol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy. (c) 2008 Elsevier B.V. All rights reserved.

Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity

In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg(-) L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg(-) mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol-targeted ARG lacking the glycosomal SKL targeting sequence (arg Delta SKL) restored growth but failed to restore infectivity. Further study showed that the ARG Delta SKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration.