Cell surface receptor kinase FERONIA linked to nutrient sensor TORC signaling controls root hair growth at low temperature linked to low nitrate in Arabidopsis thaliana.

Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is determined both by intrinsic and environmental signals. Although nutrient availability and temperature are key factors for a sustained plant growth, the molecular mechanisms underlying their sensing and downstream signaling pathways remain unclear. We use genetics to address the roles of the cell surface receptor kinase FERONIA (FER) and the nutrient sensing TOR Complex 1 (TORC) in RH growth. We identified that low temperature (10°C) triggers a strong RH elongation response in Arabidopsis thaliana involving FER and TORC. We found that FER is required to perceive limited nutrient availability caused by low temperature. FERONIA interacts with and activates TORC-downstream components to trigger RH growth. In addition, the small GTPase Rho of plants 2 (ROP2) is also involved in this RH growth response linking FER and TOR. We also found that limited nitrogen nutrient availability can mimic the RH growth response at 10°C in a NRT1.1-dependent manner. These results uncover a molecular mechanism by which a central hub composed by FER-ROP2-TORC is involved in the control of RH elongation under low temperature and nitrogen deficiency.

Csk αC Helix: A Computational Analysis of an Essential Region for Conformational Transitions.

Conformational changes are an essential feature for the function of some dynamic proteins. Understanding the mechanism of such motions may allow us to identify important properties, which may be directly related to the regulatory function of a protein. Also, this knowledge may be employed for a rational design of drugs that can shift the balance between active and inactive conformations, as well as affect the kinetics of the activation process. Here, the conformational changes in carboxyl-terminal Src kinase, the major catalytic repressor to the Src family of kinases, was investigated, and it was proposed as a functionally related hypothesis. A Cα Structure-Based Model (Cα-SBM) was applied to provide a description of the overall conformational landscape and further analysis complemented by detailed molecular dynamics simulations. As a first approach to Cα-SBM simulations, reversible transitions between active (closed) and inactive (open) forms were modeled as fluctuations between these two energetic basins. It was found that, in addition to the interdomain Carboxyl-terminal SRC Kinase (Csk) correlated motions, a conformational change in the αC helix is required for a complete conformational transition. The result reveals this as an important region of transition control and domain coordination. Restrictions in the αC helix region of the Csk protein were performed, and the analyses showed a direct correlation with the global conformational changes, with this location being propitious for future studies of ligands. Also, the Src Homology 3 (SH3) and SH3 plus Src Homology 2 (SH2) domains were excluded for a direct comparison with experimental results previously published. Simulations where the SH3 was deleted presented a reduction of the transitions during the simulations, while the SH3-SH2 deletion vanishes the Csk transitions, corroborating the experimental results mentioned and linking the conformational changes with the catalytic functionality of Csk. The study was complemented by the introduction of a known kinase inhibitor close to the Csk αC helix region where its consequences for the kinetic behavior and domain displacement of Csk were verified through detailed molecular dynamics. The findings describe the mechanisms involving the Csk αC helix for the transitions and also support the dynamic correlation between SH3 and SH2 domains against the Csk lobes and how local energetic restrictions or interactions in the Csk αC helix can play an important role for long-range motions. The results also allow speculation if the Csk activity is restricted to one specific conformation or a consequence of a state transition, this point being a target for future studies. However, the αC helix is revealed as a potential region for rational drug design.

G-Protein Phosphorylation: Aspects of Binding Specificity and Function in the Plant Kingdom.

Plant survival depends on adaptive mechanisms that constantly rely on signal recognition and transduction. The predominant class of signal discriminators is receptor kinases, with a vast member composition in plants. The transduction of signals occurs in part by a simple repertoire of heterotrimeric G proteins, with a core composed of α-, ß-, and γ-subunits, together with a 7-transmembrane Regulator G Signaling (RGS) protein. With a small repertoire of G proteins in plants, phosphorylation by receptor kinases is critical in regulating the active state of the G-protein complex. This review describes the in vivo detected phosphosites in plant G proteins and conservation scores, and their in vitro corresponding kinases. Furthermore, recently described outcomes, including novel arrestin-like internalization of RGS and a non-canonical phosphorylation switching mechanism that drives G-protein plasticity, are discussed.

Trypanosomatid selenophosphate synthetase structure, function and interaction with selenocysteine lyase.

Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) complex and the tRNASec-specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T. brucei selenophosphate synthetase in the regulation of the parasite's ER stress response.

Discovery of a transcriptomic core of genes shared in 8 primary retinoblastoma with a novel detection score analysis.

PURPOSE: Expression microarrays are powerful technology that allows large-scale analysis of RNA profiles in a tissue; these platforms include underexploited detection scores outputs. We developed an algorithm using the detection score, to generate a detection profile of shared elements in retinoblastoma as well as to determine its transcriptomic size and structure. METHODS: We analyzed eight briefly cultured primary retinoblastomas with the Human transcriptome array 2.0 (HTA2.0). Transcripts and genes detection scores were determined using the Detection Above Background algorithm (DABG). We used unsupervised and supervised computational tools to analyze detected and undetected elements; WebGestalt was used to explore functions encoded by genes in relevant clusters and performed experimental validation. RESULTS: We found a core cluster with 7,513 genes detected and shared by all samples, 4,321 genes in a cluster that was commonly absent, and 7,681 genes variably detected across the samples accounting for tumor heterogeneity. Relevant pathways identified in the core cluster relate to cell cycle, RNA transport, and DNA replication. We performed a kinome analysis of the core cluster and found 4 potential therapeutic kinase targets. Through analysis of the variably detected genes, we discovered 123 differentially expressed transcripts between bilateral and unilateral cases. CONCLUSIONS: This novel analytical approach allowed determining the retinoblastoma transcriptomic size, a shared active transcriptomic core among the samples, potential therapeutic target kinases shared by all samples, transcripts related to inter tumor heterogeneity, and to determine transcriptomic profiles without the need of control tissues. This approach is useful to analyze other cancer or tissue types.

Impact of colibacillosis on production in laying hens associated with interference of the phosphotransfer network and oxidative stress.

The objective of this study was to evaluate the productive impact of colibacillosis on laying hens and to investigate whether energetic metabolism and oxidative stress were involved in the pathogenesis of the disease. An experimental shed containing 270 laying hens of the Hy-Line lineage (32 weeks old) presented approximately 40% daily laying, and many birds presented with diarrhea and apathy followed by death. Necropsy revealed macroscopic lesions compatible with colibacillosis and infectious agent Escherichia coli was isolated from fecal samples of all birds in the infected group, as well as from tissue (ovary, liver and peritoneum). Sixteen chickens were selected for this study, divided into two groups: Control (animals without clinical alterations) and infected (with diarrhea and apathetic). E. coli isolates were subjected to the antimicrobial susceptibility testing according to the methodology approved by CLSI, 2018. This testing showed sensitivity to gentamicin, amoxicillin, norfloxacin and colistin. It was then determined that laying hens would be treated with norfloxacin (15 mg/kg) diluted in water offered at will to the birds for three days. Blood collections were performed via brachial vein after the diagnosis of E. coli (before starting treatment) and seven days after treatment. Three debilitated chickens died on the second day after initiating therapy. Before treatment, birds with clinical signs had higher levels of lipoperoxidation (LPO) and activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) than in the control group (asymptomatic animals). After treatment, LPO levels remained higher in birds that had clinical disease (infected group), whereas the activity of SOD and GPx enzymes did not differ between groups. Activity levels of creatine kinase (CK) and pyruvate kinase (PK) were higher in the group of chickens with clinical disease before treatment. Post-treatment, no differences were observed between groups in terms of CK; however, PK activity remained high in these animals. In the hens that died, there were lesions characteristic of avian colibacillosis, with ovary involvement, explaining the low laying activity of the birds at their peak of production. For 10 days after starting treatment, the percentage of laying increased to 90%. Therefore, we conclude that colibacillosis interferes with the phosphotransfer network by stimulating ATP production, in addition to causing oxidative stress of the birds during laying, that negatively affects health and productive efficiency.

Heterologous expression and kinetic characterization of the α, ß and αß blend of the PPi-dependent phosphofructokinase from Citrus sinensis.

This work reports the molecular cloning and heterologous expression of the genes coding for α and ß subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg-1 for α and ß subunits, respectively). On the other hand, co-expression rendered a α3ß3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism.

Nitroxide Tempol down-regulates kinase activities associated with NADPH oxidase function in phagocytic cells and potentially decreases their fungicidal response.

AIMS: The identification of novel targets to control inflammation in humans is probably the primary challenge that impairs the development of new anti-inflammatory drugs. Therefore, the modulation of intracellular signaling pathways in phagocytes may be an interesting means of achieving this goal. However, this change to signaling can compromise the host's susceptibility to invading pathogens. We investigated whether the antioxidant nitroxide Tempol regulates the activity of kinases associated with the production of oxidants in neutrophils, which affects the fungicidal capability of these cells. MAIN METHODS: The effects of Tempol on PMA- or fMLP-activated neutrophils were examined by oxygen consumption as an index of the oxidative burst, a release of extracellular and total Reactive Oxygen Species (ROS) by chemiluminescence, kinase activities through analysis of ATP consumption during enzyme activities and the dot blot immunoassay and, finally, by neutrophil capacity of killing Candida albicans. KEY FINDINGS: Tempol significantly inhibited the neutrophil oxidative burst in a concentration-dependent manner and decreased oxygen consumption (IC50 = 45 µM) and extracellular/total ROS formation with an increase on the lag period response. In addition, Tempol inhibited neutrophil kinase activities (i.e., a decrease in protein phosphorylation) elicited through different biochemical pathways and consequently impaired the fungicidal activity of these cells. SIGNIFICANCE: Although Tempol has potential anti-inflammatory activity that acts on different intracellular pathways (such as those involving kinases), researchers should be cautious, since this nitroxide down-regulated oxidants production and the fungicidal response of neutrophils.

Biosynthesis of an antiviral compound using a stabilized phosphopentomutase by multipoint covalent immobilization.

Ribavirin is a synthetic guanosine analogue with a broad-spectrum of antiviral activity. It is clinically effective against several viruses, such as respiratory syncytial virus, several hemorrhagic fever viruses and HCV when combined with pegylated interferon-α. Phosphopentomutase (PPM) catalyzes the transfer of intramolecular phosphate (from C1 to C5) on ribose, and is involved in pentose phosphate pathway and in purine metabolism. Reactions catalyzed by this enzyme are useful for nucleoside analogues production. However, out of its natural environment PPM is unstable and its stability is affected by parameters such as pH and temperature. Therefore, to irreversibly immobilize this enzyme, it needs to be stabilized. In this work, PPM from Escherichia coli ATCC 4157 was overexpressed, purified, stabilized at alkaline pH and immobilized on several supports. The activity of different additives as stabilizing agents was evaluated, and the best result was found using 10% (v/v) glycerol. Under this condition, PPM maintained 86% of its initial activity at pH 10 after 18h incubation, which allowed further covalent immobilization of this enzyme on glyoxyl-agarose with a high yield. This is the first time that PPM has been immobilized by multipoint covalent attachment on glyoxyl support, this derivative being able to biosynthesize ribavirin from α-d-ribose-5-phosphate.

New insights into the interaction between the quorum-sensing receptor NprR and its DNA target, or the response regulator Spo0F.

The NprR protein and NprRB signaling peptide comprise a bifunctional quorum-sensing system from the Bacillus cereus group that is involved in transcriptional activation through DNA-binding and in sporulation initiation by binding to Spo0F. We characterized in vitro the direct interactions established by NprR that may be relevant for performing its two functions. Apo-NprR interacted with Spo0F, but not with the target DNA. The NprRB signaling peptide SSKPDIVG that binds strongly to Apo-NprR, failed to bind and disrupt the NprR-Spo0F complex. Finally, the NprR-NprRB complex bound both to Spo0F and the target DNA with similar affinity. Based on our findings, we propose that rather than a switch triggered by NprRB, the NprR/NprRB ratio and the availability of Spo0F binding sites define the function of NprR.

N-acetylcysteine inhibits lipid accumulation in mouse embryonic adipocytes.

Oxidative stress plays critical roles in the pathogenesis of diabetes, hypertension, and atherosclerosis; some authors reported that fat accumulation correlates to systemic oxidative stress in human and mice, but cellular redox environment effect on lipid accumulation is still unclear. In our laboratory we used mouse embryonic fibroblasts (undifferentiated cells: CC), which are capable of differentiating into mature adipocytes (differentiated cells: DC) and accumulate lipids, as obesity model. Here we analyzed the role of the well-known antioxidant and glutathione precursor N-acetylcysteine (NAC) in cellular MAPK modulation and lipid accumulation. We evaluated the effect of NAC on the adipogenic differentiation pathway using different doses: 0.01, 0.1, 1 and 5mM; no toxic doses in these cells. A dose of 5mM NAC [DCN-5] provoked a significant decrease in triglyceride accumulation (72±10 [DCN-5] vs 169±15 [DC], p<0.01), as well in Oil Red O stained neutral lipid content (120±2 [DCN-5] vs 139±12 [DC], p<0.01). Molecular mechanisms responsible for adipogenic differentiation involve increase of the expression of phosphoERK½ and phosphoJNK, 5mM NAC treatment inhibited both pERK½ and pJNK protein levels. We also evaluated the mitotic clonal expansion (MCE) which takes place during adipogenesis and observed an increase in DC at a rate of 1.5 cells number compared to CC at day 2, whereas the highest doses of NAC significantly inhibited MCE. Our results suggest that NAC inhibits lipid accumulation and the MAPK phosphorylation in mouse embryonic fibroblasts during adipogenic differentiation and further contribute to probe the importance of cellular redox environment in adipogenesis.

The receptor-like kinase SlSOBIR1 is differentially modulated by virus infection but its overexpression in tobacco has no significant impact on virus accumulation.

KEY MESSAGE: The role of the tomato receptor-like kinase SlSOBIR1 in antiviral defense was investigated. SlSOBIR1 was transcriptionally modulated by unrelated viruses but its ectopic expression had no effect on virus accumulation. Leucine-rich repeat receptor-like kinases (LRR-RLK) constitute a diverse group of proteins allowing the cell to recognize and respond to the extracellular environment. In the present study we focused on a gene encoding a tomato LRR-RLK (named SlSOBIR1) involved in the host defense against fungal pathogens. Curiously, SlSOBIR1 has been previously reported to be down-regulated by Pepper yellow mosaic virus (PepYMV) infection. Here, we show that SlSOBIR1 is responsive to wounding and differentially modulated by unrelated virus infection, i.e., down-regulated by PepYMV and up-regulated by Tomato chlorotic spot virus (TCSV). Despite these divergent expression profiles, SlSOBIR1 overexpression in transgenic tobacco plants had no evident effect on TCSV and PepYMV accumulation. On the other hand, overexpression of SlSOBIR1 significantly increased the expression of selected defense genes (PR-1a and PR-6) and exacerbated superoxide production in wounded leaves. Our data indicate that the observed modulation of SlSOBIR1 expression is probably triggered by secondary effects of the virus infection process and suggest that SlSOBIR1 is not directly involved in antiviral signaling response.

Whole-body magnetic resonance imaging in children: state of the art / Ressonância magnética de corpo inteiro em pediatria: estado da arte

Whole-body imaging in children was classically performed with radiography, positron-emission tomography, either combined or not with computed tomography, the latter with the disadvantage of exposure to ionizing radiation. Whole-body magnetic resonance imaging (MRI), in association with the recently developed metabolic and functional techniques such as diffusion-weighted imaging, has brought the advantage of a comprehensive evaluation of pediatric patients without the risks inherent to ionizing radiation usually present in other conventional imaging methods. It is a rapid and sensitive method, particularly in pediatrics, for detecting and monitoring multifocal lesions in the body as a whole. In pediatrics, it is utilized for both oncologic and non-oncologic indications such as screening and diagnosis of tumors in patients with genetic syndromes, evaluation of disease extent and staging, evaluation of therapeutic response and post-therapy follow-up, evaluation of non neoplastic diseases such as multifocal osteomyelitis, vascular malformations and syndromes affecting multiple regions of the body. The present review was aimed at describing the major indications of whole-body MRI in pediatrics added of technical considerations.

A avaliação de corpo inteiro em crianças era classicamente realizada com radiografias simples, cintilografia e tomografia por emissão de pósitrons combinada ou não à tomografia computadorizada, estes com a desvantagem de exposição à radiação ionizante. A ressonância magnética de corpo inteiro (RMCI), associada ao desenvolvimento de técnicas metabólicas e funcionais como difusão, trouxe a vantagem de uma avaliação global do paciente pediátrico sem os riscos da radiação ionizante habitualmente presente nos métodos radiológicos convencionais. A RMCI é um método rápido e sensível, com aplicação especial na área de pediatria na detecção e no monitoramento de lesões multifocais no corpo como um todo. Em pediatria, esta técnica é utilizada tanto em oncologia - no diagnóstico e rastreamento de tumores em pacientes portadores de síndromes genéticas, na avaliação da extensão de doenças e estadiamento oncológico, na avaliação da resposta terapêutica e no seguimento pós-terapêutico - como em lesões não neoplásicas - osteomielite multifocal, malformações vasculares e síndromes que comprometam múltiplas regiões do corpo. Esta revisão tem como objetivo mostrar as principais indicações do exame na população pediátrica e técnica de realização.

A comparative study on phosphotransferase activity of acid phosphatases from Raoultella planticola and Enterobacter aerogenes on nucleosides, sugars, and related compounds.

Natural and modified nucleoside-5'-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.

Silencing of the tomato sugar partitioning affecting protein (SPA) modifies sink strength through a shift in leaf sugar metabolism.

Limitations in our understanding about the mechanisms that underlie source-sink assimilate partitioning are increasingly becoming a major hurdle for crop yield enhancement via metabolic engineering. By means of a comprehensive approach, this work reports the functional characterization of a DnaJ chaperone related-protein (named as SPA; sugar partition-affecting) that is involved in assimilate partitioning in tomato plants. SPA protein was found to be targeted to the chloroplast thylakoid membranes. SPA-RNAi tomato plants produced more and heavier fruits compared with controls, thus resulting in a considerable increment in harvest index. The transgenic plants also displayed increased pigment levels and reduced sucrose, glucose and fructose contents in leaves. Detailed metabolic and enzymatic activities analyses showed that sugar phosphate intermediates were increased while the activity of phosphoglucomutase, sugar kinases and invertases was reduced in the photosynthetic organs of the silenced plants. These changes would be anticipated to promote carbon export from foliar tissues. The combined results suggested that the tomato SPA protein plays an important role in plastid metabolism and mediates the source-sink relationships by affecting the rate of carbon translocation to fruits.

Constitutive expression of selected genes from the pentose phosphate and aromatic pathways increases the shikimic acid yield in high-glucose batch cultures of an Escherichia coli strain lacking PTS and pykF.

BACKGROUND: During the last two decades many efforts have been directed towards obtaining efficient microbial processes for the production of shikimic acid (SA); however, feeding high amounts of substrate to increase the titer of this compound has invariably rendered low conversion yields, leaving room for improvement of the producing strains. In this work we report an alternative platform to overproduce SA in a laboratory-evolved Escherichia coli strain, based on plasmid-driven constitutive expression of six genes selected from the pentose phosphate and aromatic amino acid pathways, artificially arranged as an operon. Production strains also carried inactivated genes coding for phosphotransferase system components (ptsHIcrr), shikimate kinases I and II (aroK and aroL), pyruvate kinase I (pykF) and the lactose operon repressor (lacI). RESULTS: The strong and constitutive expression of the constructed operon permitted SA production from the beginning of the cultures, as evidenced in 1 L batch-mode fermentors starting with high concentrations of glucose and yeast extract. Inactivation of the pykF gene improved SA production under the evaluated conditions by increasing the titer, yield and productivity of this metabolite compared to the isogenic pykF+ strain. The best producing strain accumulated up to 43 g/L of SA in 30 h and relatively low concentrations of acetate and aromatic byproducts were detected, with SA accounting for 80% of the produced aromatic compounds. These results were consistent with high expression levels of the glycolytic pathway and synthetic operon genes from the beginning of fermentations, as revealed by transcriptomic analysis. Despite the consumption of 100 g/L of glucose, the yields on glucose of SA and of total aromatic compounds were about 50% and 60% of the theoretical maximum, respectively. The obtained yields and specific production and consumption rates proved to be constant with three different substrate concentrations. CONCLUSIONS: The developed production system allowed continuous SA accumulation until glucose exhaustion and eliminated the requirement for culture inducers. The obtained SA titers and yields represent the highest reported values for a high-substrate batch process, postulating the strategy described in this report as an interesting alternative to the traditionally employed fed-batch processes for SA production.

Effects of ß-alanine administration on selected parameters of oxidative stress and phosphoryltransfer network in cerebral cortex and cerebellum of rats.

ß-Alanine is a ß-amino acid derivative of the degradation of pyrimidine uracil and precursor of the oxidative substrate acetyl-coenzyme A (acetyl-CoA). The accumulation of ß-alanine occurs in ß-alaninemia, an inborn error of metabolism. Patients with ß-alaninemia may develop neurological abnormalities whose mechanisms are far from being understood. In this study we evaluated the effects of ß-alanine administration on some parameters of oxidative stress and on creatine kinase, pyruvate kinase, and adenylate kinase in cerebral cortex and cerebellum of 21-day-old rats. The animals received three peritoneal injections of ß-alanine (0.3 mg /g of body weight) and the controls received the same volume (10 µL/g of body weight) of saline solution (NaCl 0.85 %) at 3 h intervals. CSF levels of ß-alanine increased five times, achieving 80 µM in the rats receiving the amino acid. The results of ß-alanine administration in the parameters of oxidative stress were similar in both tissues studied: reduction of superoxide dismutase activity, increased oxidation of 2',7'-dihydrodichlorofluorescein, total content of sulfhydryl and catalase activity. However, the results of the phosphoryltransfer network enzymes were similar in all enzymes, but different in the tissues studied: the ß-alanine administration was able to inhibit the enzyme pyruvate kinase, cytosolic creatine kinase, and adenylate kinase activities in cerebral cortex, and increase in cerebellum. In case this also occurs in the patients, these results suggest that oxidative stress and alteration of the phosphoryltransfer network may be involved in the pathophysiology of ß-alaninemia. Moreover, the ingestion of ß-alanine to improve muscular performance deserves more attention in respect to possible side-effects.

Effect of leucine administration to female rats during pregnancy and lactation on oxidative stress and enzymes activities of phosphoryltransfer network in cerebral cortex and hippocampus of the offspring.

Maple Syrup Urine Disease is an inborn error of metabolism caused by severe deficiency in the activity of branched-chain α-keto acid dehydrogenase complex. Neurological disorder is common in patients with maple syrup urine disease. Although leucine is considered the main toxic metabolite, the mechanisms underlying the neuropathology of brain injury are poorly understood. In the present study, we evaluated the possible preventive effect of the co-administration of creatine plus pyruvate on the effects elicited by leucine administration to female Wistar rats during pregnancy and lactation on some oxidative stress parameters as well as the activities of some enzymes involved in the phosphoryltransfer network in the brain cortex and hippocampus of the offspring at 21 days of age. Leucine administration induced oxidative stress and altered the activities of pyruvate kinase, adenylate kinase, mitochondrial and cytosolic creatine kinase. Co-administration of creatine plus pyruvate was partially effective in the prevention of some alterations provoked by leucine administration on the oxidative stress but not in the enzymes of phosphoryltransfer network. These results suggest that non-treated maternal hyperleucinemia may be toxic to the brain of the offspring.

Comparative phytohormone profiles, lipid kinase and lipid phosphatase activities in barley aleurone, coleoptile, and root tissues.

We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone levels by liquid chromatography-tandem mass spectrometry in root and coleoptile tissues following germination of barley (Hordeum vulgare) seeds. The enzymes showing highest activity in aleurone cells were diacylglycerol kinase (DAG-k, EC 2.7.1.107) and phosphatidate kinase (PA-k). The ratio of gibberellins (GAs) to abscisic acid (ABA) was 2-fold higher in aleurone than in coleoptile or root tissues. In coleoptiles, phosphatidylinositol 4-kinase (PI4-k, EC 2.7.1.67) showed the highest enzyme activity, and jasmonic acid (JA) level was higher than in aleurone. In roots, activities of PI4-k, DAG-k, and PA-k were similar, and salicylic acid (SA) showed the highest concentration. In the assays to evaluate the hydrolysis of DGPP (diacylglycerol pyrophosphate) and PA (phosphatidic acid) we observed that PA hydrolysis by LPPs (lipid phosphate phosphatases) was not modified; however, the diacylglycerol pyrophosphate phosphatase (DGPPase) was strikingly higher in coleoptile and root tissues than to aleurone. Relevance of these findings in terms of signaling responses and seedling growth is discussed.