A mechanism of glycine and alanine cytoprotective action: Stimulation of stress-induced HSP70 mRNA

A mechanism of glycine and alanine cytoprotective action: Stimulation of stress-induced HSP70mRNA. Studies done both in vitro and in vivo have shown that glycine and alanine protect kidney cells from stress injury. However, the mechanism(s) of this cytoprotection is unknown. Our aim was to test the hypothesis that the cytoprotective action is in part due to stimulation of gene(s) expression encoding stress protein synthesis. Experiments were carried out using heat shock as a model for stress in the opossum kidney cell line (OK cells). The induction of HSP70 mRNA was evaluated in cell monolayers exposed to 45°C for 15 minutes followed by a recovery period at 37°C for either 0.5, 1, 2, 3, 4, 6 or 24 hours. The results demonstrate that the maximum level of HSP70mRNA occurred at ≈ three hours after heat treatment. Although the mRNA levels declined thereafter, appreciable amounts were still seen even 24 hours after heat-shock. To examine the effect of glycine or alanine on HSP70mRNA levels and on the synthesis of stress protein, cultures were preincubated for 30 minutes with Krebs-Henseleit buffer, pH 7.4, supplemented with either 1, 2, 5 or 10mM glycine or alanine, or with no added amino acids. Comparative studies were performed with 10mM glutamate, aspartate, arginine or leucine. Following preincubation, cultures were heat-shocked (45°C for 15min) and then reincubated at 37°C for three hours. Both glycine and alanine enhanced the level of HSP70mRNA and the synthesis of 72,73kDa stress proteins, but neither amino acid induced HSP70mRNA without concomitant heat treatment. Glutamate, aspartate, leucine and arginine had no enhancing effect, however, their inclusion in the incubation medium induced heat-shock-like response without heat treatment. The increased level of HSP70 mRNA and the synthesis of stress protein in the presence of glycine or alanine were associated with decreased cellular LDH release, suggesting greater thermotolerance of the cultured cells. Intracellular ATP levels declined following heat shock in all experiments. Supplementation of the medium with glycine or alanine did not alter this stress-induced reduction of intracellular ATP, supporting a previous suggestion that the cytoprotective action of glycine and alanine is independent of cellular ATP levels. The current data suggest a functional role for glycine and alanine in the stimulation of gene(s) expression encoding for stress protein(s) synthesis, and in protecting cells against stress damage. This characteristic is not shared by other amino acids, such as glutamate, asparate, arginine or leucine

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies

Metabolic networking in Brunfelsia calycina petals after flower opening

Brunfelsia calycina flowers change colour from purple to white due to anthocyanin degradation, parallel to an increase in fragrance and petal size. Here it was tested whether the production of the fragrant benzenoids is dependent on induction of the shikimate pathway, or if they are formed from the anthocyanin degradation products. An extensive characterization of the events taking place in Brunfelsia flowers is presented. Anthocyanin characterization was performed using ultraperfomance liquid chromatography–quadrupole time of flight–tandem mass specrometry (UPLC-QTOF-MS/MS). Volatiles emitted were identified by headspace solid phase microextraction–gas chromatography–mass spectrometry (HS-SPME-GC-MS). Accumulated proteins were identified by 2D gel electrophoresis. Transcription profiles were characterized by cross-species hybridization of Brunfelsia cDNAs to potato cDNA microarrays. Identification of accumulated metabolites was performed by UPLC-QTOF-MS non-targeted metabolite analysis. The results include characterization of the nine main anthocyanins in Brunfelsia flowers. In addition, 146 up-regulated genes, 19 volatiles, seven proteins, and 17 metabolites that increased during anthocyanin degradation were identified. A multilevel analysis suggests induction of the shikimate pathway. This pathway is the most probable source of the phenolic acids, which in turn are precursors of both the benzenoid and lignin production pathways. The knowledge obtained is valuable for future studies on degradation of anthocyanins, formation of volatiles, and the network of secondary metabolism in Brunfelsia and related species

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Investigação do ensaio transparências de lar, seu desdobramento em exposição ao público e sua composição e impressão em um livro de imagem, tendo como base a pesquisa e o estudo do arquivo gerado desde 2009. Neste ensaio, o olhar é lançado sobre o ritmo da vida humana num cotidiano real que é misturado com o imaginário, formando um mosaico de afetos. Provoca a reflexão sobre beleza, vida, intimidade, semelhança e diferença. Neste percurso, surge um novo ensaio poético: camponesa, que nasceu a partir de reflexões sobre o fazer artístico, pesquisa de arquivo pessoal e registros cotidianos.Investigation of the transparências de lar essay, its unfolding in public exposure and its composition and printing in an image book, based on the research and study of the archive generated since 2009. In this essay, the look is cast on the rhythm of human life in a real daily life that is mixed with the imaginary, forming a mosaic of affections. It provokes reflection on beauty, life, intimacy, similarity and difference. Along this path, a new poetic essay appears: camponesa, which was born from reflections on artistic practice, research of personal archives and daily records

Role of the glutamate dehydrogenase reaction in furnishing aspartate nitrogen for urea synthesis: studies in perfused rat liver with 15N.

The present study was designed to determine: (i) the role of the reductive amination of alpha-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of alpha-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N -acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate

3-Isobutylmethylxanthine Inhibits Hepatic Urea Synthesis: PROTECTION BY AGMATINE*

We previously showed that agmatine stimulated hepatic ureagenesis. In this study, we sought to determine whether the action of agmatine is mediated via cAMP signaling. A pilot experiment demonstrated that the phosphodiesterase inhibitor, 3-isobutylmethylxanthine (IBMX), inhibited urea synthesis albeit increased [cAMP]. Thus, we hypothesized that IBMX inhibits hepatic urea synthesis independent of [cAMP]. We further theorized that agmatine would negate the IBMX action and improve ureagenesis. Experiments were carried out with isolated mitochondria and 15NH4Cl to trace [15N]citrulline production or [5-15N]glutamine and a rat liver perfusion system to trace ureagenesis. The results demonstrate that IBMX induced the following: (i) inhibition of the mitochondrial respiratory chain and diminished O2 consumption during liver perfusion; (ii) depletion of the phosphorylation potential and overall hepatic energetic capacity; (iii) inhibition of [15N]citrulline synthesis; and (iv) inhibition of urea output in liver perfusion with little effect on [N-acetylglutamate]. The results indicate that IBMX directly and specifically inhibited complex I of the respiratory chain and carbamoyl-phosphate synthase-I (CPS-I), with an EC50 about 0.6 mm despite a significant elevation of hepatic [cAMP]. Perfusion of agmatine with IBMX stimulated O2 consumption, restored hepatic phosphorylation potential, and significantly stimulated ureagenesis. The action of agmatine may signify a cascade effect initiated by increased oxidative phosphorylation and greater ATP synthesis. In addition, agmatine may prevent IBMX from binding to one or more active site(s) of CPS-I and thus protect against inhibition of CPS-I. Together, the data may suggest a new experimental application of IBMX in studies of CPS-I malfunction and the use of agmatine as intervention therapy