Exercise and the Kynurenine pathway: Current state of knowledge and results from a randomized cross-over study comparing acute effects of endurance and resistance training.

INTRODUCTION: The essential amino acid tryptophan (TRP) is primarily degraded through the kynurenine (KYN) pathway, which is dysregulated in several chronic diseases. KYN pathway metabolites have immune- and neuro-modulatory properties and are involved in th de novo synthesis of nicotinamide adenine dinucleotide (NAD+). Currently, little evidence exists demonstrating that physical exercise may influence this pathway. However, differences between acute and chronic stimuli as well as the influence of exercise modalities remain to be investigated. Here, we provide an overview of existing studies and present results of a randomized cross-over trial on acute effects of a single-bout of resistance and endurance exercise. METHODS: 24 healthy male adults conducted both an acute endurance exercise (EE) and resistance exercise (RE) session. Blood samples were collected before, immediately after and one hour after cessation of each exercise session. Outcomes comprised serum levels of TRP, KYN, kynurenic acid (KA), quinolinic acid (QA) and calculated ratios. Gene expression of the enzymes indoleamine 2,3 dioxygenase (IDO) 1 and kynurenine aminotransferase (KAT) 4 was measured in peripheral blood mononuclear cells (PBMCs). Moreover, serum concentrations of the potential KYN pathway mediators interleukin (IL)-6 and cortisol were determined. Finally, we investigated baseline correlations between immune cell subsets, potential mediators and initial KYN pathway activation outcomes. RESULTS: The KYN/TRP ratio correlated positively with IL-6 and CD56bright NK-cells and negatively with CD56dim NKcells. Expression of IDO1 in PBMCs correlated positively with IL-6, regulatory T-cells and CD56bright NK-cells, whereas negative correlations to cytotoxic T-cells and CD56dim NKcells were revealed. A significant time effect on KYN/TRP ratio was detected for RE. Regarding KA and KA/KYN ratio, an increase after exercise followed by a decrease at the follow- up measurement was revealed in EE. KAT4 expression also increased after exercise in EE. Moreover, elevated QA levels were observed after the EE session. CONCLUSIONS: In contrast to chronic exercise interventions, single-bouts of endurance exercise provoke acute alterations on KYN pathway outcomes in humans. Our results indicate that EE induces stronger alterations than RE. Enhanced conversion of KYN to both, KA and QA suggest a peripheral KYN clearance, thereby preventing pathological accumulation within the CNS. Future acute and chronic exercise studies are needed to examine the role of NAD+ synthesis starting with TRP and the interplay between KYN pathway activation and mid- to long-term immunological modulations.

Molecular and biochemical characterization of key enzymes in the cysteine and serine metabolic pathways of Acanthamoeba castellanii.

BACKGROUND: Acanthamoeba spp. can cause serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis and cutaneous acanthamoebiasis. Cysteine biosynthesis and the L-serine metabolic pathway play important roles in the energy metabolism of Acanthamoeba spp. However, no study has confirmed the functions of cysteine synthase (AcCS) in the cysteine pathway and phosphoglycerate dehydrogenase (AcGDH) or phosphoserine aminotransferase (AcSPAT) in the non-phosphorylation serine metabolic pathway of Acanthamoeba. METHODS: The AcCS, AcGDH and AcSPAT genes were amplified by PCR, and their recombinant proteins were expressed in Escherichia coli. Polyclonal antibodies against the recombinant proteins were prepared in mice and used to determine the subcellular localisation of each native protein by confocal laser scanning microscopy. The enzymatic activity of each recombinant protein was also analysed. Furthermore, each gene expression level was analysed by quantitative PCR after treatment with different concentrations of cysteine or L-serine. RESULTS: The AcCS gene encodes a 382-amino acid protein with a predicted molecular mass of 43.1 kDa and an isoelectric point (pI) of 8.11. The AcGDH gene encodes a 350-amino acid protein with a predicted molecular mass of 39.1 kDa and a pI of 5.51. The AcSPAT gene encodes a 354-amino acid protein with a predicted molecular mass of 38.3 kDa and a pI of 6.26. Recombinant AcCS exhibited a high cysteine synthesis activity using O-acetylserine and Na2S as substrates. Both GDH and SPAT catalysed degradation, rather than synthesis, of serine. Exogenous L-serine or cysteine inhibited the expression of all three enzymes in a time- and dose-dependent manner. CONCLUSIONS: This study demonstrated that AcCS participates in cysteine biosynthesis and serine degradation via the non-phosphorylation serine metabolic pathway, providing a molecular basis for the discovery of novel anti-Acanthamoeba drugs.

Biochemical Principles and Functional Aspects of Pipecolic Acid Biosynthesis in Plant Immunity.

The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/µ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction.

AGY, a Novel Egg Yolk-Derived Anti-gliadin Antibody, Is Safe for Patients with Celiac Disease.

BACKGROUND: Celiac disease (CD) is a gluten-triggered autoimmune disorder of the small intestine. A lifelong gluten-free diet (GFD) is the only approved treatment; however, strict adherence is difficult and many suffer from inadvertent gluten exposure. Oral egg yolk anti-gliadin antibody (AGY) is a novel treatment to neutralize gluten and may improve the efficacy of the GFD. AIMS: To determine the safety, tolerability, and potential efficacy of AGY in patients with CD. METHODS: This 6-week, open-label, single-arm study was conducted in adults with biopsy-proven CD on a GFD. Safety measures included adverse events, physical examination, and clinical laboratory tests. Additional measures included a daily Celiac Symptom Index, Health-Related Quality of life, anti-tissue transglutaminase and anti-gliadin IgA/IgG, and lactulose/mannitol excretion ratio (LMER). A 2-week run-in period to assess questionnaire compliance and acceptability of baseline safety laboratory results was followed by a 4-week treatment period with two AGY capsules taken before meals. RESULTS: Ten patients completed the study (mean age 43.4 years, nine female). All followed a GFD for at least 6 months (mean 5 years). No safety concerns were identified. Most patients had fewer celiac symptoms (especially tiredness, headache, and bloating), improved quality of life, lowered antibodies, and lowered LMER when taking AGY compared to the run-in period. CONCLUSION: In our cohort, AGY was safe and potentially associated with improved CD-related outcome measures in patients on a GFD. A larger study powered for further safety and efficacy evaluation is planned.

Transient hypertransaminasemia related to cow's milk protein antigen induced proctocolitis in infants

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ALD1 Regulates Basal Immune Components and Early Inducible Defense Responses in Arabidopsis.

Robust immunity requires basal defense machinery to mediate timely responses and feedback cycles to amplify defenses against potentially spreading infections. AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1) is needed for the accumulation of the plant defense signal salicylic acid (SA) during the first hours after infection with the pathogen Pseudomonas syringae and is also upregulated by infection and SA. ALD1 is an aminotransferase with multiple substrates and products in vitro. Pipecolic acid (Pip) is an ALD1-dependent bioactive product induced by P. syringae. Here, we addressed roles of ALD1 in mediating defense amplification as well as the levels and responses of basal defense machinery. ALD1 needs immune components PAD4 and ICS1 (an SA synthesis enzyme) to confer disease resistance, possibly through a transcriptional amplification loop between them. Furthermore, ALD1 affects basal defense by controlling microbial-associated molecular pattern (MAMP) receptor levels and responsiveness. Vascular exudates from uninfected ALD1-overexpressing plants confer local immunity to the wild type and ald1 mutants yet are not enriched for Pip. We infer that, in addition to affecting Pip accumulation, ALD1 produces non-Pip metabolites that play roles in immunity. Thus, distinct metabolite signals controlled by the same enzyme affect basal and early defenses versus later defense responses, respectively.

Molecular insights into primary hyperoxaluria type 1 pathogenesis.

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder of glyoxylate metabolism caused by the deficiency of liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme. The PH1 pathogenesis is mostly due to single point mutations (more than 150 so far identified) on the AGXT gene, and is characterized by a marked heterogeneity in terms of genotype, enzymatic and clinical phenotypes. This article presents an up to date review of selected aspects of the biochemical properties of the two allelic forms of AGT and of some PH1-causing variants. These recent discoveries highlight the effects at the protein level of the pathogenic mutations, and, together with previous cell biology and clinical data, (i) improve the understanding of the molecular basis of PH1 pathogenesis, and (ii) help to delineate perspectives for predicting the response to pyridoxine treatment or for suggesting new strategies for PH1 patients bearing the analyzed mutations.

Autoimmune liver diseases in a paediatric population with coeliac disease - a 10-year single-centre experience.

BACKGROUND: Coeliac disease (CD) can be associated with liver disease. Gluten-free diet (GFD) normalizes cryptogenic forms, but most likely not autoimmune hepatitis (AIH). For this condition, immunosuppressants represent the treatment. However, when these are stopped, AIH generally relapses. AIM: To determine in CD children liver test abnormality frequency, the effect of GFD alone, or plus prolonged immunosuppressants on AIH course. METHODS: Coeliac disease patients with abnormal transaminases were selected; if transaminases <5 x UNL (upper normal limits), GFD alone was administered; if >5 x UNL, liver examinations and biopsy were performed. In AIH, immunosuppressants were administered (5 years). Treatment was stopped only if patients remained in remission during the entire maintenance period and normalized liver histology. RESULTS: A total of 140 out of 350 CD children had hypertransaminaemia: 133 cryptogenic disease, 7 AIH. GFD normalized only cryptogenic hepatitis. During treatment, all AIH persistently normalized clinical and biochemical parameters; after withdrawal, six patients maintained a sustained remission (follow-up range: 12-63 months), while one relapsed. CONCLUSIONS: In CD children with AIH, only GFD plus immunosuppressants determines a high remission rate. When clinical remission is reached, a prolonged immunosuppressive regimen induces a high sustained remission rate after treatment withdrawal, indicating that this regimen may prevent early relapse.

Colestasis gravidica: a propósito de un caso / Cholestasis gravidarum: a purpose of a case

La Colestasis Gravídica en un síndrome que habitualmente se produce en el tercer trimestre del embarazo y se resuelve en el embarazo. Se caracteriza clínicamente por la presencia de prurito, ictericia o ambos sin otra disfunción hepática importante. En el siguiente trabajo se reporta caso de paciente femenino de 24 años III gestas II para FUR: 12/8/06 quien refiere de 6 días de evolución orinas coluricas, prurito generalizado predominio de manos y tórax, exámenes para clínicos con aumento de un transaminasas, fosfatasas alcalinas, se diagnóstica colestasis gravídica, patología de baja incidencia de 1/300 y 1/2000 embarazos, se considera importante realizar una revisión académica del mismo.

Do we need to measure total serum IgA to exclude IgA deficiency in coeliac disease?

BACKGROUND: Screening for IgA deficiency in patients with coeliac disease is essential because of the increased incidence of IgA deficiency associated with the disease, which usually relies on the estimation of IgA levels in each case. AIM: To devise a method of excluding IgA deficiency without measuring total serum IgA in each case. MATERIALS AND METHODS: The optical density readings on enzyme-linked immunosorbent assay (ELISA) of 608 routine samples received for tissue transglutaminase (TTG) antibody testing for coeliac disease were compared with their total IgA concentrations. Dilution experiments were also carried out to ensure linear relationships between optical density on ELISA and IgA concentrations and to compare the sensitivities for TTG and endomysium antibodies in TTG-positive samples. RESULTS AND DISCUSSION: A clear relationship was shown between total IgA concentration and TTG optical density readings by ELISA. To ensure a positive TTG result if antibodies are present, it was possible to recommend an optical density level above which all samples have sufficient IgA. Samples with optical density <0.05 should be investigated further by estimating total IgA and, if low, samples should be subjected to immunofluorescence microscopy testing for IgA and IgG endomysium antibodies. CONCLUSIONS: An easier, more cost-effective and practical way of excluding IgA deficiency in the investigation on coeliac disease is reported.

Intestinal involvement is not sufficient to explain hypertransaminasemia in celiac disease?

Chronic unexplained hypertransaminasemia is an isolated clinical manifestation of celiac disease (CD) and lacks of a clear physiopathological explanation. Since CD and tropical sprue (TS) have similar intestinal functional and histological pattern of injury and that an increased inflammatory response has been reported to occur in patients with irritable bowel syndrome (IBS), liver involvement might be expected to occur either in TS or IBS. However, according to author's prior observations, the frequency of hypertransaminasemia is significantly higher in CD than in TS and IBS-diarrhea predominant patients (IBS-D). Thus, based on current knowledge, intestinal mucosal damage, increased intestinal permeability and/or an active intestinal inflammatory response do not completely explain liver damage in CD. We hypothesize that other factors, unique to CD not present in TS or IBS-D, like gluten toxicity and the presence of tissular transglutaminase (tTG) an auto-antigen with pro-inflammatory and remodeling properties, act in addition to intestinal mucosal injury and account to hypertransaminasemia in CD. Further research focusing on the mechanisms of gluten and tTG hepatic toxicity, and/or the characterization of the expression, secretion and enteral-hepatic transport of certain pro-inflammatory cytokines is needed, to understand the possible links between intestinal and liver disorders seen in CD.

Demonstration of kynurenine aminotransferases I and II and characterization of kynurenic acid synthesis in cultured cerebral cortical neurons.

The present study characterizes the synthesis of kynurenic acid (KYNA) from exogenously added kynurenine and its regulation by extrinsic factors, in cultured cerebral cortical neurons and, for comparison, in astrocytes incubated under identical conditions. The neuronal culture showed positive immunostaining for both kynurenic acid aminotransferase (KAT) isoforms I and II. Neurons synthesized KYNA at a rate about 2.3 times higher than astrocytes. Neuronal, but not astrocytic, KYNA synthesis was lowered approximately 30% by ionotropic glutamate receptor agonists [(R,S)-3-hydroxy-5-methoxyloxasole-4-propionic acid (AMPA; 100 microM) and N-methyl-D-aspartic acid (NMDA; 100 microM)] and depolarizing agents [KCl (50 mM) and 4-aminopyridine (4-AP; 10 microM)]. Neuronal and astrocytic synthesis alike were vulnerable to inhibition exerted by the aminotransferase inhibitor aminooxyacetic acid (AOAA), glutamate (IC50: 31 and 85 microM, respectively), substrates of the L-amino transport system [leucine (Leu); IC50: 19 and 42 microM, respectively] and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH; IC50: 19 and 28 microM, respectively). Glutamine (Gln), which is a metabolic precursor of glutamate in astrocytes and L-system substrate in both cell types, inhibited KYNA synthesis both in neurons and in astrocytes (IC50: 268 and 318 microM, respectively). alpha-Ketoisocaproic acid (KIC), a Leu transamination product that is produced mainly in astrocytes and shuttled to neurons to modulate intraneuronal concentration of glutamate, stimulated KYNA synthesis in neurons but did not affect the synthesis in astrocytes. In conclusion, this study is the first to demonstrate active, regulation-prone KYNA synthesis in neurons.

Estudo clinico, laboratorial e da histologia hepática de candidatos a doadores de sangue HBsAg positivos em Belo Horizonte / Volunters blood donnors HBsAg positives in Belo Horizonte, State of Minas Gerais, Brasil : Clinical, laboratorial and histological study

Duzentos e cinquenta e dois candidatos a doadores de sangue HBsAg positivos (media de idade= 32,6 anos, 97,7masculino) foram investigados num estudo transversal para determinar suas caracteristicas clinicas, laboratoriais e histologicas. Foi tambem comparada a positividade e a negatividade dos marcadores sorologicos HBeAg, anti-HBe e IgM anti-HBc com os valores das aminotransferases sericas...

Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2.

Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase, key enzymes for the assimilation of one-carbon compounds in methylotrophs, was performed using antibodies raised against these enzymes purified from Hyphomicrobium methylovorum GM2. Immunodiffusion studies indicated that serine-glyoxylate aminotransferase and hydroxypyruvate reductase of all seven Hyphomicrobium strains tested were immunochemically similar. In immunotitration experiments and Western blot analyses of both enzymes in the genera Hyphomicrobium and Methylobacterium, the serine-glyoxylate aminotransferase of the genus Methylobacterium exhibited low similarity to that of the genus Hyphomicrobium. For hydroxypyruvate reductase, no immunological relationship was observed between the genera Hyphomicrobium and Methylobacterium, which was in agreement with the differences in primary structure and enzymological properties.

Strategies for the prenatal diagnosis of primary hyperoxaluria type 1.

Primary hyperoxaluria type 1 (PH1) is a potentially lethal autosomal recessive disorder of glyoxylate metabolism caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). Over the past 13 years, various strategies have been adopted for its prenatal diagnosis, including (1) glyoxylate metabolite analysis of amniotic fluid in the second trimester; (2) AGT enzyme assay, immunoassay, and immuno-electron microscopy of fetal liver biopsies also in the second trimester; and (3) linkage and mutation analysis of DNA isolated from chorionic villus samples in the first trimester. These methods have evolved in parallel with our increased understanding of the molecular aetiology and pathogenesis of the disease. Although the usefulness of metabolite analysis remains unproven, all the other methods have been successfully applied to the prenatal diagnosis of PH1. In this review, examples of the use of the available methodologies are provided, and their pros and cons are discussed with reference to specific cases.

A rapid, high-yield purification of L-alanine:4,5-dioxovalerate transaminase from rat kidney mitochondria using an improved enzyme assay method.

The present report documents an improved enzyme assay method for the mammalian L-alanine:4,5-dioxovalerate transaminase which is of significant utility in work with crude tissue homogenates, cell cultures, or purified enzyme preparations. We also describe a new and rapid purification procedure for this enzyme from rat kidney mitochondria. The three-step procedure involves the use of digitonin and lubrol for mitochondrial matrix preparation and L-alanine-Sepharose 4B column chromatography followed by gel filtration on Sepharose 6B. By this procedure it is possible to obtain a highly purified enzyme preparation in a relatively short time with a 37.5% yield.

Purification and characterization of kynurenine aminotransferase I from human brain.

Two kynurenine aminotransferases (KATs), arbitrarily termed KAT I and KAT II, are capable of producing the neuroinhibitory brain metabolite kynurenic acid from L-kynurenine in human brain tissue. Here we describe the purification of KAT I to homogeneity and the subsequent characterization of the enzyme using physicochemical, biochemical, and immunological methods. KAT I was purified from human brain approximately 2,000-fold with a yield of 2%. Assessed by polyacrylamide gel electrophoresis, KAT I migrated toward the anode as a single protein with a mobility of 0.5. The pure enzyme was found to be a dimer consisting of two identical subunits of approximately 60 kDa. Among several oxo acids tested, KAT I showed highest activity with 2-oxoisocaproate. Kinetic analyses of the pure enzyme revealed an absolute Km of 2.0 mM and 10.0 mM for L-kynurenine and pyruvate, respectively. KAT I activity was substantially inhibited by L-glutamine, L-phenylalanine, and L-tryptophan, using either pyruvate (1 mM) or 2-oxoisocaproate (1 mM) as a cosubstrate. L-Tryptophan inhibited enzyme activity noncompetitively with regard to pyruvate (Ki = 480 microM) and competitively with regard to L-kynurenine (Ki = 200 microM). Anti-KAT I antibodies were produced against pure KAT I and were partially purified by conventional techniques. Immunotitration and immunoblotting analyses confirmed that KAT I is clearly distinct from both human KAT II and rat kynurenine-pyruvate aminotransferase. Pure human KAT I and its antibody will serve as valuable tools in future studies of kynurenic acid production in the human brain under physiological and pathological conditions.

In vitro translocation of L-alanine:4,5-dioxovalerate transaminase into rat kidney mitochondria.

A specific rabbit antibody was prepared against rat kidney mitochondrial L-alanine: 4,5-dioxovalerate transaminase, which is one of the two enzymes catalyzing the synthesis of delta-aminolevulinic acid in the heme biosynthetic pathway. Total polyadenylated RNA isolated from rat kidney was translated in vitro using rabbit reticulocyte cell-free translation system, and L-alanine:4,5-dioxovalerate transaminase was estimated by indirect immunoprecipitation to represent 0.85% of the total translation product. When the total in vitro translated product was incubated with homologous kidney mitochondria, 59% of the [35S]methionine labeled enzyme was translocated into the mitochondria where it was no longer accessible to externally added protease. In relation to total protein translocation, the translocation of L-alanine:4,5-dioxovalerate transaminase remained unaltered by addition of hemin up to 50 microM. These results show that, unlike the other enzyme of the heme biosynthetic pathway (delta-aminolevulinic acid synthetase), this enzyme is not under tight control by heme, but nonetheless, functions as an important source to maintain a housekeeping level of delta-aminolevulinic acid.

Cysteine conjugate beta-lyase of rat kidney cytosol: characterization, immunocytochemical localization, and correlation with hexachlorobutadiene nephrotoxicity.

Cysteine conjugate beta-lyase (beta-lyase) was purified to electrophoretic homogeneity from the kidney cytosol of male Wistar rats. The highly purified enzyme exhibited a monomeric molecular weight of 50,000 Da and was active in the alpha-beta elimination of cysteine conjugates including S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), and S-(2-benzothiazolyl)-L-cysteine, particularly toward DCVC and TFEC. The purified enzyme also exhibited glutamine transaminase K activity with phenylalanine and alpha-keto-gamma-methiolbutyrate as substrates. An antibody was raised to the purified rat protein in sheep and the crude immune serum affinity purified, yielding a specific antibody that recognized only the beta-lyase protein in whole kidney homogenates. Immunocytochemical studies on rat kidney sections stained with the purified antibody revealed that the cytosolic beta-lyase enzyme was mainly localized in the pars recta of the proximal tubule in untreated rats. This localization is coincident with the site-specific kidney necrosis produced by hexachloro-1,3-butadiene (HCBD). These results indicate that the tissue localization of beta-lyase in the proximal tubule plays an important role in determining the specific nephrotoxicity produced by halogenated alkenes such as HCBD.