Determinación de ácido quinolínico en corteza renal de rata

Se describe una modificación al método descrito por McDaniel y col. (8) para la determinación de ácido quinolínico en diferentes tejidos animales. El método consiste en la extracción con ácido perclórico, adsorción selectiva sobre carbón activado y descarboxilación hasta ácido nicotínico, que se mide colorimétricamente. Con este método se han medido las concentraciones de ácido qUinolínico en corteza renal tras la administración de triptófano a las ratas, encontrándose que ninguno de los valores es suficiente para producir una inhibición de la fosfoenolpiruvato carboxicinasa y por tanto de la capacidad gluconeogénica renal.We have described a method based on McDaniel's (8) for the determination of quinolinic acid in several animal tissues. The method consist in the extraction of quinolinic acid, selective adsortion on activated charco al and decarboxylation to nicotinic acid which have been measured by colorimetric reaction. With this method we have measured the concentrations of quinolinic acid in rat renal cortex after the tryptophan injection to rato We have found that anyone of values is enough to make an inhibition of phosphoenol pyruvate carboxykinase activity and t he renal gluconeogenic capacity

Desarrollo de las capacidades gluconeogénica y glucolítica de corteza renal durante ciclos de ayuno y alimentación

Se han estudiado los efectos de diferentes ciclos alternativos de alimentación y ayuno sobre las capacidades gluconeogénica y glucolítica de corteza renal, así como sobre los principales enzimas implicados en ambos procesos. Durante el período de alimentación, caracterizado por someter a los animales a una dieta rica en carbohidratos, la capacidad gluconeogénica de corteza renal disminuyó hasta un máximo del 60 %, mientras que el flujo glucolítico se incrementó significativamente a lo largo del tiempo llegando a alcanzar un valor superior al 100 %. La situación cambia durante el período de ayuno, dando lugar a un importante y significativo (70 %) incremento de la síntesis de glucosa con una disminución (31 %) de la capacidad glucolítica. Los resultados presentados en este trabajo permiten concluir que la corteza renal es capaz de poner en marcha los mecanismos adecuados de adaptación frente a diferentes situaciones nutricionales, indicando el importante papel que juega este tejido en el mantenimiento del estado glucostático del animal.The effects of different starve-feed cycles on gluconeogenic and glycolytic capacities of kidney cortex have been studied. Opposite effects were found during these nutritional states (feeding, characterized by a high carbohydrate diet, and starvation) and both metabolic processes. During the feeding state, a decrease in the gluconeogenic flux was parallel to an increase in the glycolytic one. The behaviour of these metabolic processes was opposed during starvation. The results in this work show that kidney cortex, is able to adapt itself to different availability of exogenous macronutrients or nutritional situations, pointing out the important role that it plays in the maintenance of the glucostatic state of the animal

Unveiling the Differential Antioxidant Activity of Maslinic Acid in Murine Melanoma Cells and in Rat Embryonic Healthy Cells Following Treatment with Hydrogen Peroxide

Maslinic acid (MA) is a natural triterpene from Olea europaea L. with multiple biological properties. The aim of the present study was to examine MA’s effect on cell viability (by the MTT assay), reactive oxygen species (ROS levels, by flow cytometry) and key antioxidant enzyme activities (by spectrophotometry) in murine skin melanoma (B16F10) cells compared to those on healthy cells (A10). MA induced cytotoxic effects in cancer cells (IC50 42 M), whereas no effect was found in A10 cells treated withMA(up to 210 M). In order to produce a stress situation in cells, 0.15mMH2O2 was added. Under stressful conditions, MA protected both cell lines against oxidative damage, decreasing intracellular ROS, which were higher in B16F10 than in A10 cells. The treatment with H2O2 and without MA produced different responses in antioxidant enzyme activities depending on the cell line. In A10 cells, all the enzymes were up-regulated, but in B16F10 cells, only superoxide dismutase, glutathione S-transferase and glutathione peroxidase increased their activities. MA restored the enzyme activities to levels similar to those in the control group in both cell lines, highlighting that in A10 cells, the highest MA doses induced values lower than control. Overall, these findings demonstrate the great antioxidant capacity of MA.General Secretariat of Universities, Research and Technology of the Ministry of Economy, Innovation, Science and Employment Government of the Junta de Andalucia (Spain) BIO-157program FEDER-INNTERCONECTA from the Spanish Government C-3650-0

Influencia de ciclos alternativos alimentación-ayuno sobre los principales enzimas del metabolismo renal de carbohidratos

Con objeto de explicar molecularmente los cambios descritos en el metabolismo renal de carbohidratos ( 1) se han estudiado los efectos que diferentes ciclos de alimentación y ayuno presentan sobre los principales enzimas implicados en los procesos glucolítico y gluconeogénico. En general durante el período de alimentación se activan los enzimas glucolíticos y se inhiben los gluconeogénicos .. Efectos opuestos se observan durante los períodos de ayuno. Estos resultados permiten concluir que la corteza renal es capaz de adaptar continuamente los mecanismos enzimáticos necesarios para ajustar el destino de macronutrientes tanto de procedencia exógena como endógena.In order to explain the changes previously described by us in the renal carbohydrate metabolism during different starved-feed cycles ( 1), the effects of this nutritional situation on the most important enzymes in volved in the glycolytic and gluconeogenic processes have been studied. In general, during the feed-state all the glycolytic enzymes increased its activities whereas a decrease was found in the gluconeogenic ones. Opposite effects were obtanined during starvation. Several mechanism of enzymatic regulation are shown throughout the development of these nutritional conditions

Nutraceutical Role of Polyphenols and Triterpenes Present in the Extracts of Fruits and Leaves of Olea europaea as Antioxidants, Anti-Infectives and Anticancer Agents on Healthy Growth

This research has been funded by the Junta de Andalucia (Andalusian Research Plan, Junta de Andalucia, Spain) by the grant from the research group BIO-157 "Drugs, Environmental Toxics and Cellular Metabolism".There is currently a worldwide consensus and recognition of the undoubted health benefits of the so-called Mediterranean diet, with its intake being associated with a lower risk of mortality. The most important characteristics of this type of diet are based on the consumption of significant amounts of fruit, vegetables, legumes, and nuts, which provide, in addition to some active ingredients, fiber and a proportion of vegetable protein, together with extra virgin olive oil (EVOO) as the main sources of vegetable fat. Fish and meat from poultry and other small farm animals are the main sources of protein. One of the main components, as already mentioned, is EVOO, which is rich in monounsaturated fatty acids and to a lesser extent in polyunsaturated fatty acids. The intake of this type of nutrient also provides an important set of phytochemicals whose health potential is widely spread and agreed upon. These phytochemicals include significant amounts of anthocyanins, stilbenes, flavonoids, phenolic acids, and terpenes of varying complexities. Therefore, the inclusion in the diet of this type of molecules, with a proven healthy effect, provides an unquestionable preventive and/or curative activity on an important group of pathologies related to cardiovascular, infectious, and cancerous diseases, as well as those related to the metabolic syndrome. The aim of this review is therefore to shed light on the nutraceutical role of two of the main phytochemicals present in Olea europaea fruit and leaf extracts, polyphenols, and triterpenes, on healthy animal growth. Their immunomodulatory, anti-infective, antioxidant, anti-aging, and anti-carcinogenic capabilities show them to be potential nutraceuticals, providing healthy growth.Junta de Andalucia BIO-15

Effects of Erythrodiol on the Antioxidant Response and Proteome of HepG2 Cells

This research was funded by the University of Jaén (Plan Propio de Investigación, grant number UJA2014/07/13) and by Junta de Andalucía (Plan Andaluz de Investigación, Junta de Andalucía, Spain), grant BIO-341 “Enzymes and Metabolism”.Erythrodiol (EO) is a pentacyclic triterpenic alcohol found in olive tree leaves and olive oil, and it has important effects on the health properties and quality of olive oil. In this study, we characterized the cytotoxic effects of EO on human hepatocarcinoma (HepG2) cells by studying changes in cell viability, reactive oxygen species (ROS) production, antioxidant defense systems, and the proteome. The results reveal that EO markedly decreased HepG2 cell viability without changing ROS levels. The concentrations of glutathione and NADPH were significantly reduced, with selective changes in the activity of several antioxidant enzymes: glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. Proteomic data reveal that EO led to the complete elimination or decreased abundance of 41 and 3 proteins, respectively, and the abundance of 29 proteins increased. The results of functional enrichment analysis show that important metabolic processes and the nuclear transport of mature mRNA were impaired, whereas AMP biosynthesis and cell cycle G2/M phase transition were induced. The transcription factors and miRNAs involved in this response were also identified. These potent antiproliferative effects make EO a good candidate for the further analysis of its hepatic antitumor effects in in vivo studies.University of Jaén (Plan Propio de Investigación, grant number UJA2014/07/13)Junta de Andalucía (Plan Andaluz de Investigación, Junta de Andalucía, Spain), grant BIO-34

The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism

[Background] Maslinic acid, a pentacyclic triterpene found in the protective wax-like coating of the leaves and fruit of Olea europaea L., is a promising agent for the prevention of colon cancer. We have shown elsewhere that maslinic acid inhibits cell proliferation to a significant extent and activates mitochondrial apoptosis in colon cancer cells. In our latest work we have investigated further this compound's apoptotic molecular mechanism. [Methods] We used HT29 adenocarcinoma cells. Changes genotoxicity were analyzed by single-cell gel electrophoresis (comet assay). The cell cycle was determined by flow cytometry. Finally, changes in protein expression were examined by western blotting. Student's t-test was used for statistical comparison. [Results] HT29 cells treated with maslinic acid showed significant increases in genotoxicity and cell-cycle arrest during the G0/G1 phase after 72 hours' treatment and an apoptotic sub-G0/G1 peak after 96 hours. Nevertheless, the molecular mechanism for this cytotoxic effect of maslinic acid has never been properly explored. We show here that the anti-tumoral activity of maslinic acid might proceed via p53-mediated apoptosis by acting upon the main signaling components that lead to an increase in p53 activity and the induction of the rest of the factors that participate in the apoptotic pathway. We found that in HT29 cells maslinic acid activated the expression of c-Jun NH2-terminal kinase (JNK), thus inducing p53. Treatment of tumor cells with maslinic acid also resulted in an increase in the expression of Bid and Bax, repression of Bcl-2, release of cytochrome-c and an increase in the expression of caspases -9, -3, and -7. Moreover, maslinic acid produced belated caspase-8 activity, thus amplifying the initial mitochondrial apoptotic signaling. [Conclusion] All these results suggest that maslinic acid induces apoptosis in human HT29 colon-cancer cells through the JNK-Bid-mediated mitochondrial apoptotic pathway via the activation of p53. Thus we propose a plausible sequential molecular mechanism for the expression of the different proteins responsible for the intrinsic mitochondrial apoptotic pathway. Further studies with other cell lines will be needed to confirm the general nature of these findings.This study was supported by grants BIO157 from the Andalucian regional government; SAF2008-00164 and ISCIII-RTICC (RD06/0020/0046) grants from the Spanish national government and & European Regional Development Fund (ERDF) "Una manera de hacer Europa" and by AGAUR-Generalitat de Catalunya grant 2009SGR1308, 2009 CTP 00026 and Icrea Academia award 2010 granted to M. Cascante)

Diclofenac N-Derivatives as Therapeutic Agents with Anti-Inflammatory and Anti-Cancer Effect

A series of diclofenac N-derivatives (2, 4, 6, 8c, 9c, 10a-c) were synthesized in order to test their anti-cancer and anti-inflammatory effects. The anticarcinogen activity has been assayed against three cancer cell lines: HT29, human colon cancer cells; Hep-G2, human hepatic cells; and B16-F10, murine melanoma cells. First, we determined the cytotoxicity of the different compounds, finding that the most effective compound was compound 8c against all cell lines and both compounds 4 and 6 in human Hep-G2 and HT29 cell lines. Compounds 4 and 8c were selected for the percentage of apoptosis determination, cell cycle distribution, and mitochondrial membrane potential measure because these products presented the lowest IC50 values in two of the three cancer cell lines assayed (B16-F10 and HepG2), and were two of the three products with lowest IC50 in HT29 cell line. Moreover, the percentages of apoptosis induction were determined for compounds 4 and 8c, showing that the highest values were between 30 to 60%. Next, the effects of these two compounds were observed on the cellular cycle, resulting in an increase in the cell population in G2/M cell cycle phase after treatment with product 8c, whereas compound 4 increased the cells in phase G0/G1, by possible differentiation process induction. Finally, to determine the possible apoptosis mechanism triggered by these compounds, mitochondrial potential was evaluated, indicating the possible activation of extrinsic apoptotic mechanism. On the other hand, we studied the anti-inflammatory effects of these diclofenac (DCF) derivatives on lipopolysaccharide (LPS) activated RAW 264.7 macrophagesmonocytes murine cells by inhibition of nitric oxide (NO) production. As a first step, we determined the cytotoxicity of the synthesized compounds, as well as DCF, against these cells. Then, sub-cytotoxic concentrations were used to determine NO release at different incubation times. The greatest antiinflammatory effect was observed for products 2, 4, 8c, 10a, 10b, and 9c at 20 µg·mL−1 concentration after 48 h of treatment, with inhibition of produced NO between 60 to 75%, and a concentration that reduces to the 50% the production of NO (IC50 NO) between 2.5 to 25 times lower than that of DCF. In this work, we synthesized and determined for the first time the anti-cancer and anti-inflammatory potential of eight diclofenac N-derivatives. In agreement with the recent evidences suggesting that inflammation may contribute to all states of tumorigenesis, the development of these new derivatives capable of inducing apoptosis and anti-inflammatory effects at very low concentrations represent new effective therapeutic strategies against these diseases.MINISTERIO DE ECONOMÍA Y COMPETITIVIDAD, PID2019-106222RB-C32/SRA (State Research Agency, 10.13039/501100011033)“Consejería de Economía, Conocimiento, Empresas y Universidad. Junta de Andalucía”, grant number B1-BIO-281-UGR1

Synthesis and Biological Evaluation of Cassane Diterpene (5 alpha)-Vuacapane-8(14), 9(11)-Diene and of Some Related Compounds

A set of thirteen cassane-type diterpenes was synthesized and an expedient synthetic route was used to evaluate 14-desmethyl analogs of the most active tested cassane. The anti-inflammatory activities of these 13 compounds were evaluated on a lipopolysaccharide (LPS)-activated RAW 264.7 cell line by inhibition of nitric oxide (NO) production, some of them reaching 100% NO inhibition after 72 h of treatment. The greatest anti-inflammatory effect was observed for compounds 16 and 20 with an IC50 NO of 2.98 +/- 0.04 mu g/mL and 5.71 +/- 0.14 mu g/mL, respectively. Flow-cytometry analysis was used to determine the cell cycle distribution and showed that the inhibition in NO release was accompanied by a reversion of the differentiation processes. Moreover, the anti-cancer potential of these 13 compounds were evaluated in three tumor cell lines (B16-F10, HT29, and Hep G2). The strongest cytotoxic effect was achieved by salicylaldehyde 20, and pterolobirin G (6), with IC50 values around 3 mu g/mL in HT29 cells, with total apoptosis rates 80% at IC80 concentrations, producing a significant cell-cycle arrest in the G0/G1 phase, and a possible activation of the extrinsic apoptotic pathway. Additionally, initial SAR data analysis showed that the methyl group at the C-14 positions of cassane diterpenoids is not always important for their cytotoxic and anti-inflammatory activities.Junta de Andalucia BFQM-278-UGR20 B-FQM-650-UGR2

Semisynthesis and Evaluation of Anti-Inflammatory Activity of the Cassane-Type Diterpenoid Taepeenin F and of Some Synthetic Intermediates

A new strategy for the semisynthesis of the aromatic cassane-type diterpene taepeenin F (6) is reported. The introduction of the methyl group at C-14, characteristic of the target compound, was achieved via dienone 13, easily prepared from abietic acid (10), the major compound in renewable rosin. Biological assays of selected compounds are reported. The antiproliferative activity against HT29, B16-F10, and HepG2 tumor cell lines has been investigated. Salicylaldehyde 21 was the most active compound (IC50 = 7.72 μM). Products 16 and 21 displayed apoptotic effects in B16-F10 cells, with total apoptosis rates of 46 and 38.4%, respectively. This apoptotic process involves a significant arrest of the B16-F10 cell cycle, blocking the G0/G1 phase. Dienone 16 did not cause any loss of the mitochondrial membrane potential (MMP), while salicylaldehyde 21 caused a partial loss of the MMP. The anti-inflammatory activity of the selected compounds was investigated with the LPS-stimulated RAW 264.7 macrophages. All compounds showed potent NO inhibition, with percentages between 80 and 99% at subcytotoxic concentrations. Dienone 16 inhibited LPS-induced differentiation of RAW 264.7 cells, by increasing the proportion of cells in the S phase. In addition, salicylaldehyde 21 had effects on the cell cycle, recovering the cells from the G0/G1 full arrest produced in response to LPS action.Junta de Andalucia B-FQM-278-UGR20 B-FQM-650-UGR20 FQM-348 BIO-157Universidad de Granada/CBU