AMPK inhibits voltage-gated calcium channel-current in rat chromaffin cells.

Metabolic changes are critical in the regulation of Ca2+ influx in central and peripheral neuroendocrine cells. To study the regulation of L-type Ca2+ channels by AMPK we used biochemical reagents and ATP/glucose-concentration manipulations in rat chromaffin cells. AICAR and Compound-C, at low concentration, significantly induce changes in L-type Ca2+ channel-current amplitude and voltage dependence. Remarkably, an overlasting decrease in the channel-current density can be induced by lowering the intracellular level of ATP. Accordingly, Ca2+ channel-current density gradually diminishes by decreasing the extracellular glucose concentration. By using immunofluorescence, a decrease in the expression of CaV1.2 is observed while decreasing extracellular glucose, suggesting that AMPK reduces the number of functional Ca2+ channels into the plasma membrane. Together, these results support for the first time the dependence of metabolic changes in the maintenance of Ca2+ channel-current by AMPK. They reveal a key step in Ca2+ influx in secretory cells.

The plant organellar primase-helicase directs template recognition and primosome assembly via its zinc finger domain.

BACKGROUND: The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD). RESULTS: Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase. CONCLUSIONS: We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.

Alarmones as Vestiges of a Bygone RNA World.

All known alarmones are ribonucleotides or ribonucleotide derivatives that are synthesized when cells are under stress conditions, triggering a stringent response that affects major processes such as replication, gene expression, and metabolism. The ample phylogenetic distribution of alarmones (e.g., cAMP, Ap(n)A, cGMP, AICAR, and ZTP) suggests that they are very ancient molecules that may have already been present in cellular systems prior to the evolutionary divergence of the Archaea, Bacteria, and Eukarya domains. Their chemical structure, wide biological distribution, and functional role in highly conserved cellular processes support the possibility that these modified nucleotides are molecular fossils of an epoch in the evolution of chemical signaling and metabolite sensing during which RNA molecules played a much more conspicuous role in biological catalysis and genetic information.

Influenza virus RNA polymerase may be activated inside the virion.

Influenza A and B virions are packaged with their polymerases to catalyse RNA-dependent RNA polymerase activity. Since there is no evidence to rule in or out the permissiveness of influenza virions to triphosphate ribonucleotides, we functionally evaluated this. We found the means to stimulate influenza A and B RNA polymerase activity inside the virion, called natural endogenous RNA polymerase (NERP) activity. Stimulation of NERP activity increased up to 3 log10 viral RNA content, allowing the detection of influenza virus in otherwise undetectable clinical samples. NERP activation also improved our capacity to sequence misidentified regions of the influenza genome from clinical samples. By treating the samples with the ribavirin triphosphate we inhibited NERP activity, which confirms our hypothesis and highlights that this assay could be used to screen antiviral drugs. Altogether, our data show that NERP activity could be explored to increase molecular diagnostic sensitivity and/or to develop antiviral screening assays.

AMPK activation promotes gastroprotection through mutual interaction with the gaseous mediators H2S, NO, and CO.

Activation of 5' adenosine monophosphate-activated protein kinase (AMPK) stimulates production of the gaseous mediators nitric oxide (NO) and carbon monoxide (CO), which are involved in mucosal defense and gastroprotection. As AMPK itself has gastroprotective effects against several gastric ulcer etiologies, in the present study, we aimed to elucidate whether AMPK may also prevent ethanol-induced injury and play a key role in the associated gastroprotection mediated by hydrogen sulfide (H2S), NO, and CO. Mice were pretreated with AICAR (20 mg/kg, an AMPK activator) alone or with 50% ethanol. Other groups were pretreated with respective gaseous mediator inhibitors PAG, l-NAME, or ZnPP IX 30 min prior to AICAR, or with gaseous mediator donors NaHS, Lawesson's reagent and l-cysteine (H2S), SNP, l-Arginine (NO), Hemin, or CORM-2 (CO) 30 min prior to ethanol with or without compound C (10 mg/kg, a non-selective AMPK inhibitor). H2S, nitrate/nitrite (NO3-/NO2-), bilirubin levels, GSH and MDA concentration were evaluated in the gastric mucosa. The gastric mucosa was also collected for histopathological analysis and AMPK expression assessment by immunohistochemistry. Pretreatment with AICAR attenuated the ethanol-induced injury and increased H2S and bilirubin levels but not NO3-/NO2- levels in the gastric mucosa. In addition, inhibition of H2S, NO, or CO synthesis exacerbated the ethanol-induced gastric damage and inhibited the gastroprotection by AICAR. Pretreatment with compound C reversed the gastroprotective effect of NaHS, Lawesson's reagent, l-cysteine, SNP, l-Arginine, CORM-2, or Hemin. Compound C also reversed the effect of NaHS on H2S production, SNP on NO3-/NO2- levels, and Hemin on bilirubin levels. Immunohistochemistry revealed that AMPK is present at basal levels mainly in the gastric mucosa cells, and was increased by pretreatment with NaHS, SNP, and CORM-2. In conclusion, our findings indicate that AMPK activation exerts gastroprotection against ethanol-induced gastric damage and mutually interacts with H2S, NO, or CO to facilitate this process.

5'-AMP-Activated Protein Kinase Regulates Papillary (TPC-1 and BCPAP) Thyroid Cancer Cell Survival, Migration, Invasion, and Epithelial-to-Mesenchymal Transition.

BACKGROUND: Differentiated thyroid carcinomas (DTC) are associated with a good prognosis and a high survival rate. However, tumor recurrence occurs in approximately 20-30% of DTC patients, reinforcing the importance of identifying new molecular targets for cancer management. It has been shown that the 5'-AMP-activated protein kinase (AMPK) is over-activated in papillary thyroid cancer (PTC). This study aimed to investigate the effects of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR), an AMPK activator, on various aspects of thyroid cancer cell behavior, including cell survival, apoptosis, migration, invasion, and epithelial-to-mesenchymal transition (EMT), in the human thyroid cancer cell lines BCPAP and TPC-1. METHODS: BCPAP and TPC-1 cells were cultivated in Dulbecco's modified Eagle's medium, and the non-tumor-derived cell line Nthy-ORI was grown in RPMI. Cells were treated or not with AICAR for different periods of time. The cell growth rate, cell cycle phase, apoptosis, cell migration, and invasion were analyzed using transwell inserts, and EMT was quantified by the expression of mesenchymal and epithelial markers. RESULTS: AMPK is activated in thyroid cancer cell lines, and AICAR treatment further increased AMPK phosphorylation. After 48 hours of AICAR treatment, the percentage of cells in the G2/M phase decreased, and a G0/G1-phase arrest was induced in both cell lines. AMPK activation effectively induced apoptosis in the BCPAP and TPC-1 cancer cell lines, while no apoptosis induction was observed in Nthy-ORI cells. AICAR also reduced the migration of Nthy-ORI and BCPAP cells by 30% and approximately 60% in TPC-1 cells. AICAR had no effect on cell invasion in Nthy-ORI and TPC-1 cells, but a significant reduction of cell invasion was observed in BCPAP cells. AICAR induced a significant reduction of N-cadherin and no changes in the expression of vimentin or TCF/Zeb1 protein in BCPAP cells. No differences in the expression of EMT markers were found in the AICAR-treated Nthy-ORI cells. A remarkable reduction of vimentin, TCF/Zeb1, and N-cadherin protein expression was detected in the TPC-1 cells. CONCLUSIONS: Increased activation of AMPK in PTC cell lines leads to a strong antitumor response, as measured by the inhibition of cell proliferation, cell migration, and induction of cell death. AMPK activation also reverses EMT in TPC-1 cells.

Effects of alpha-lipoic acid on chemerin secretion in 3T3-L1 and human adipocytes.

Chemerin is a novel adipokine associated with obesity and insulin resistance. α-Lipoic acid (α-LA) has shown beneficial properties on diabetes and obesity. The aim of this study was to examine the effects of α-LA on chemerin production in adipocytes in absence or presence of TNF-α, insulin and AICAR. The potential signaling pathways involved in α-LA effects on chemerin were also analyzed. α-LA actions on chemerin were tested in differentiated 3T3-L1 adipocytes and in some cases in human subcutaneous and omental adipocytes. Chemerin mRNA levels were measured by RT-PCR and the amount of chemerin secreted to culture media was determined by ELISA. α-LA induced a concentration-dependent inhibition on both chemerin secretion and mRNA levels in 3T3-L1 adipocytes. The AMPK activator AICAR and the PI3K inhibitor LY294002 dramatically abrogated both chemerin secretion and gene expression, and further potentiated the inhibitory effect of α-LA on chemerin secretion. Insulin was able to partially reverse the inhibitory action of α-LA on chemerin secretion. α-LA also reduced basal chemerin secretion in both subcutaneous and omental adipocytes from overweight/obese subjects. Moreover, α-LA was able to abolish the stimulatory effects of the pro-inflammatory cytokine TNF-α on chemerin secretion. Our data demonstrated the ability of α-LA to inhibit chemerin production, an adipokine associated to obesity and metabolic syndrome, suggesting that the reduction of chemerin could contribute to the antiobesity/antidiabetic properties described for α-LA.

AMP-Activated Protein Kinase Regulates Oxidative Metabolism in Caenorhabditis elegans through the NHR-49 and MDT-15 Transcriptional Regulators.

Cellular energy regulation relies on complex signaling pathways that respond to fuel availability and metabolic demands. Dysregulation of these networks is implicated in the development of human metabolic diseases such as obesity and metabolic syndrome. In Caenorhabditis elegans the AMP-activated protein kinase, AAK, has been associated with longevity and stress resistance; nevertheless its precise role in energy metabolism remains elusive. In the present study, we find an evolutionary conserved role of AAK in oxidative metabolism. Similar to mammals, AAK is activated by AICAR and metformin and leads to increased glycolytic and oxidative metabolic fluxes evidenced by an increase in lactate levels and mitochondrial oxygen consumption and a decrease in total fatty acids and lipid storage, whereas augmented glucose availability has the opposite effects. We found that these changes were largely dependent on the catalytic subunit AAK-2, since the aak-2 null strain lost the observed metabolic actions. Further results demonstrate that the effects due to AAK activation are associated to SBP-1 and NHR-49 transcriptional factors and MDT-15 transcriptional co-activator, suggesting a regulatory pathway that controls oxidative metabolism. Our findings establish C. elegans as a tractable model system to dissect the relationship between distinct molecules that play a critical role in the regulation of energy metabolism in human metabolic diseases.

Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5' AMP-activated protein kinase (AMPK).

This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 µM compound C AMPK inhibitor or 2 mM AMP+40 µM compound C. Our results showed that the utilization of a hypometabolic TRIS extender supplemented or not with AMP or metformin significantly improves stallion sperm freezability when compared with a commercial extender. Additionally, high cooling rates do not affect stallion sperm quality after cooling and post-thawing. Finally, stallion spermatozoa present several putative AMPK sperm isoforms that do not seem to respond to classical activators, but do respond to the Compound C inhibitor.

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

AMP-activated protein kinase upregulates glucose uptake in thyroid PCCL3 cells independent of thyrotropin.

BACKGROUND: Glucose is transported into cells by specific glucose transporter proteins (GLUTs) that are widely expressed in a tissue-specific manner. The mechanisms that regulate glucose uptake and metabolism in thyroid cells are poorly defined. Recently, our group showed that AMP-activated protein kinase (AMPK) plays a pivotal role in the rat thyroid gland, downregulating iodide uptake by thyroid cells even in the presence of its main stimulator thyrotropin (TSH). Since AMPK increases glucose uptake in different tissues, and taken into consideration that in pathophysiological conditions such as thyroid cancer a negative correlation between iodide and glucose uptake occurs, we hypothesized that AMPK might modulate glucose uptake in thyroid cells. METHODS: Rat follicular thyroid PCCL3 cells cultivated in Ham's F-12 supplemented with 5% calf serum and hormones were exposed to the AMPK pharmacological activator 5-aminoimidazole-4 carboxamide ribonucleoside (AICAR) or AMPK antagonist compound C for 24 hours either in the presence or absence of TSH. Glucose uptake was assessed in vitro using 2-deoxy-D-[(3)H]glucose. RESULTS: AMPK activation by AICAR induced a significant increase in glucose uptake by PCCL3 cells, an effect that was completely reversed by the AMPK inhibitor compound C. Also, the AICAR mediated increase in glucose uptake was detected either in the presence or absence of TSH. The mechanism by which AICAR increases glucose uptake is related to higher levels of GLUT 1 protein content and hexokinase (HK) activity in thyroid cells. CONCLUSION: Our results show that AMPK activation significantly upregulates GLUT 1 content and glucose uptake, and it also stimulates hexokinase activity, the first step of glycolysis.

The Arabidopsis F-box protein AtFBS1 interacts with 14-3-3 proteins.

AtFBS1 is an F-box protein whose transcript accumulates in response to biotic and abiotic stresses. Previous evidence suggests that a postranscriptional event regulates AtFBS1 expression [1]. We now found that AtFBS1 interacts with 14-3-3 proteins through its amino-terminus and the F-box motif. Deletion of any of these regions abolishes the interaction between AtFBS1 and 14-3-3 proteins. On the other hand, the treatment with the proteasome inhibitor MG132 or the deletion of the F-box from AtFBS1 increases ß-glucuronidase (GUS) activity in plants containing a translational fusion of AtFBS1 with the GUS reporter gene, indicating that AtFBS1 is degraded by the 26S proteasome. MG132 treatment of seedlings containing a gene fusion between AtFBS1 and the TAP (Tandem Affinity Purification) cassette causes an increase in the half-life of the protein. In an attempt to understand the role of 14-3-3 interactions, we treated Arabidopsis seedlings with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl 5'-monophosphate (AICAR), an inhibitor of 14-3-3 client interactions. We observed an increase in AtFBS1-TAP stability as a consequence of AICAR treatment. Based on these data we propose that 14-3-3 proteins promote the dimerization of SCF(AtFBS1). This also may enhance the AtFBS1 autoubiquitination activity and its degradation by the 26S proteasome. AICAR also affects Cullin1 (CUL1) modification by RUB1, which would provide an additional element to the effect of this compound on AtFBS1 stability.

Biotechnological utilization of biodiesel-derived glycerol for the production of ribonucleotides and microbial biomass.

Ten yeast strains were evaluated concerning their capabilities to assimilate biodiesel-derived glycerol in batch cultivation. The influence of glycerol concentration, temperature, pH and yeast extract concentration on biomass production was studied for the yeast selected. Further, the effect of agitation on glycerol utilization by the yeast Hansenula anomala was also studied. The yeast H. anomala CCT 2648 showed the highest biomass yield (0.30 g g(-1)) and productivity (0.19 g L(-1) h(-1)). Citric acid, succinic acid, acetic acid and ethanol were found as the main metabolites produced. The increase of yeast extract concentration from 1 to 3 g L(-1) resulted in high biomass production. The highest biomass concentration (21 g L(-1)), yield (0.45 g g(-1)) and productivity (0.31 g L(-1) h(-1)), as well as ribonucleotide production (13.13 mg g(-1)), were observed at 700 rpm and 0.5 vvm. These results demonstrated that glycerol from biodiesel production process showed to be a feasible substrate for producing biomass and ribonucleotides by yeast species.

A novel role for AMP-kinase in the regulation of the Na+/I--symporter and iodide uptake in the rat thyroid gland.

The aim of this study was to investigate the role of AMP-kinase (AMPK) in the regulation of iodide uptake by the thyroid gland. Iodide uptake was assessed in PCCL3 follicular thyroid cells exposed to the AMPK agonist 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR), and also in rat thyroid glands 24 h after a single intraperitoneal injection of AICAR. In PCCL3 cells, AICAR-induced AMPK and acetyl-CoA carboxylase (ACC) phosphorylation decreased iodide uptake in a concentration-dependent manner, while the AMPK inhibitor compound C prevented this effect. In the thyroid gland of rats injected with AICAR, AMPK and ACC phosphorylation was increased and iodide uptake was reduced by ~35%. Under conditions of increased AMPK phosphorylation/activation such as TSH deprivation or AICAR treatment, significant reductions in cellular Na(+)/I(-)-symporter (NIS) protein (~41%) and mRNA content (~65%) were observed. The transcriptional (actinomycin D) and translational (cycloheximide) inhibitors, as well as the AMPK inhibitor compound C prevented AICAR-induced reduction of NIS protein content in PCCL3 cells. The presence of TSH in the culture medium reduced AMPK phosphorylation in PCCL3 cells, while inhibition of protein kinase A (PKA) with H89 prevented this effect. Conversely, the adenylyl cyclase activator forskolin abolished the AMPK phosphorylation response induced by TSH withdrawal in PCCL3 cells. These findings demonstrate that TSH suppresses AMPK phosphorylation/activation in a cAMP-PKA-dependent manner. In summary, we provide novel evidence that AMPK is involved in the physiological regulation of iodide uptake, which is an essential step for the formation of thyroid hormones as well as for the regulation of thyroid function.

Application of lysine, taurine, disodium inosinate and disodium guanylate in fermented cooked sausages with 50% replacement of NaCl by KCl.

The effects of 50% replacement of NaCl by KCl and addition of the amino acids lysine and taurine and the 5'-ribonucleotide disodium inosinate and disodium guanylate on some sensory and physicochemical parameters of fermented cooked sausages were evaluated. The partial replacement of NaCl by KCl did not alter the manufacturing process; however, defects in the sensory quality were detected. Lysine at a concentration of 0.313% and a mixture of taurine (750 mg/kg) with disodium inosinate (300 mg/kg) and disodium guanylate (300 mg/kg) reduced the sensory defects caused by KCl, allowing the fermented cooked sausages to be elaborated with reduced sodium content and high sensory quality.

Adenosine regulates Sertoli cell function by activating AMPK.

This work evaluates adenosine effects on Sertoli cell functions, which are different to those resulting from occupancy of purinergic receptors. The effects of adenosine and N(6)-cyclohexyladenosine (CHA) - an A(1) receptor agonist resistant to cellular uptake - on Sertoli cell physiology were compared. Adenosine but not CHA increased lactate production, glucose uptake, GLUT1, LDHA and MCT4 mRNA levels, and stabilized ZO-1 protein at the cell membrane. These differential effects suggested a mechanism of action of adenosine that cannot be solely explained by occupancy of type A(1) purinergic receptors. Activation by adenosine but not by CHA of AMPK was observed. AMPK participation in lactate production and ZO-1 stabilization was confirmed by utilizing specific inhibitors. Altogether, these results suggest that activation of AMPK by adenosine promotes lactate offer to germ cells and cooperates in the maintenance of junctional complex integrity, thus contributing to the preservation of an optimum microenvironment for a successful spermatogenesis.

Aceptabilidad de sopas deshidratadas de leguminosas adicionadas de realzadores del sabor (UMAMI) / Acceptability of dehydrated legume soups with the addition of flavor enhancers (UMAMI)

The enhancer effect of glutamate monosodium (MSG) flavor was evaluated and its synergistic action with 5'-ribonucleotides: ionone rib nucleotides 5'-monophosphate (IMP) and guano sine monophosphate (GMP) in dehydrated soups consisting of lentils and peas. Four formulations were developed for both soups: the first was the target with the original level of MSG, the following had different concentrations and mixtures of these enhancers (6 percent MSG; 6 percent MSG and 0.26 percent IMP; 0.6 MSG and 0.12 percent IMP-GMP). A five-.point Graphic Hedonic Scale test was used, where 1 represented the most upset face and 5 represented the happiest face. The most accepted soup was selected by thirty elderly adults. The lentils soup with 0,6 MSG and 0J2 percent IMP-GMP and the pea's soup with 6 percent MSG and 0.26 percent IMP obtained the greatest level of acceptance. So, the effectiveness of the synergistic action between the MSG and 5'-ribonucleotides was demonstrated, because they can improve the acceptance of the evaluated formulation.

Se evaluó el efecto realzador del sabor del glutamato monosódico (GMS) y su acción sinergista con 5'-ribonucleótidos: inosinato monofosfato (IMP) y guanilato monofosfato (GMP), cuando se adicionaron a sopas deshidratadas de lentejas y arvejas. Se elaboraron 4 formulaciones para cada sopa, la primera formulación correspondió al control con su nivel de GMS original, las siguientes formulaciones contaron con distintas concentraciones y mezclas de estos realzadores (6 por ciento GMS; 6 por ciento GMS mas 0,26 por ciento IMP y 0,6 GMS mas 0,12 por ciento IMP-GMP). Se utilizó la evaluación sensorial de Escala Hedónica Gráfica, con una escala de 1 al 5, donde 1: representa "la carita más disgustada" y 5: "la más feliz". Treinta adultos mayores determinaron la formulación más aceptada. La sopa de lentejas con 6 por ciento de GMS mas 0,12 por ciento de IMP-GMP fue la que tuvo mayor aceptación, mientras que para la sopa de arvejas fue aquella que contenía 6 por ciento de GMS más 0,26 por ciento de IMP. Por tanto, se pudo demostrar la efectividad de la acción sinergista entre el GMS y los 5'-ribonucleótidos, al mejorar las aceptación de las formulaciones evaluadas.

DNAzymes and ribozymes carrying 2'-C-methyl nucleotides.

DNAzymes and Ribozymes find applications as inhibitors of gene expression and in detection systems such as biosensors, among others. In particular, we are interested in the properties of hammerhead ribozymes carrying 2'-C-methylnucleotides and 10-23 DNAzymes containing (2'R) or (2'S)- 2'-C-methyl 2'-deoxynucleotides. In this work a new synthesis of 2'-C-Methyluridine phosphoramidite is presented. Special emphasis is dedicated to the improvement of the protection of the tertiary 2'-hydroxyl group. Comparison to previous protecting strategies as well as the stability under oligonucleotide synthesis conditions is discussed.2'-C-methyl-2'-deoxynucleosides show differential preferred conformations depending on the configuration of the 2'-carbon. The influence of these modifications on the activity of 10-23 DNAzymes is also assessed.

Immune response to nucleotide-supplemented infant formulae: systematic review and meta-analysis.

Human milk is recommended as the only alimentary source for the first six months of life. Additionally there is a medical and social need for safe and effective alternative forms of nutrition for infants who cannot be fed with breast milk. Recently the safety and efficacy of some ingredients in infant formulae, such as nucleotides have been discussed. This systematic review analyzed the available evidence to establish the efficacy, safety and dose-response effect of ribonucleotide-supplemented infant formulae (RSIFs). Randomised controlled clinical trials (RCTs) comparing RSIFs to formulae without nucleotides or breast milk were considered in this review. Outcome measures were: antibody titres to common paediatric vaccinations, total lymphocytes, lymphocyte subclasses and NK-cells, episodes of diarrhoea and acute respiratory infection. Publication quality was determined using Jadad and CONSORT guidelines. Results were combined using a random effects model and reported through standardised mean differences (WMD) or risk ratio (RR). Systematic review and meta-analysis showed that RSIFs were associated with a better antibody response to immunisation with Haemopillus influenzae vaccine [SMD 1.74 (99%CI 1.43-2.05), P=0.001], diphtheria toxoid [SMD 0.94 (0.75-1.12), P=0.001], oral polio vaccine [SMD 0.73 (0.51-0.95), P=0.001], and fewer episodes of diarrhoea [RR 0.67 (0.58-0.76), P=0.02]. We did not find a major risk of upper respiratory infections [RR 1.11 (0.90-1.36), P=0.50]. Available evidence suggests a positive benefit of RSFIs on infant health without any risk. These benefits begin with nucleotide addition of 1.9 mg/418.4 kJ and are maintained or increased with 10.78 mg/418.4 kJ.