Expression and localization of an agmatinase-like protein in the rat brain

Agmatinase catalyzes the hydrolysis of agmatine into putrescine and urea, and agmatine (decarboxylated l-arginine) plays several roles in mammalian tissues, including neurotransmitter/neuromodulatory actions in the brain. Injection of agmatine in animals produces anticonvulsant, antineurotoxic and antidepressant-like actions. Information regarding the enzymatic aspects of agmatine metabolism in mammals, especially related to its degradation, is relatively scarce. The explanation for this is the lack of enzymatically active preparations of mammalian agmatinase. Recently, we have cloned a protein from a cDNA rat brain library having agmatinase activity although its amino acid sequence greatly differs from all known agmatinases, we called agmatinase-like protein. In this work, we analyzed the expression of this enzyme in the rat brain by means of RT-PCR and immunohistochemical analysis using a polyclonal antibody generated against the recombinant agmatinase-like protein. The agmatinase-like protein was detected in the hypothalamus in glial cells and arcuate nucleus neurons, and in hippocampus astrocytes and neurons, but not in brain cortex. In general, detected localization of agmatinase-like protein coincides with that described for its substrate agmatine and our results help to explain several reported effects of agmatine in the brain. Concretely, a role in the regulation of intracellular concentrations of the neurotransmitter/neuromodulator agmatine is suggested for the brain agmatinase-like protein.     

Role of obesity-associated dysfunctional adipose tissue in cancer: a molecular nutrition approach

Obesity is a complex disease caused by the interaction of a myriad of genetic, dietary, lifestyle and environmental factors, which favors a chronic positive energy balance, leading to increased body fat mass. There is emerging evidence of a strong association between obesity and an increased risk of cancer. However, the mechanisms linking both diseases are not fully understood. Here, we analyze the current knowledge about the potential contribution that expanding adipose tissue in obesity could make to the development of cancer via dysregulated secretion of pro-inflammatory cytokines, chemokines and adipokines such as TNF-α, IL-6, leptin, adiponectin, visfatin and PAI-1. Dietary factors play an important role in the risk of suffering obesity and cancer. The identification of bioactive dietary factors or substances that affect some of the components of energy balance to prevent/reduce weight gain as well as cancer is a promising avenue of research. This article reviews the beneficial effects of some bioactive food molecules (n-3 PUFA, CLA, resveratrol and lipoic acid) in energy metabolism and cancer, focusing on the molecular mechanisms involved, which may provide new therapeutic targets in obesity and cancer.     

Efflux of bile acids in Lactobacillus reuteri is mediated by ATP

Purpose of work  

To study whether an active bile acid (BA) efflux occurs in Lactobacillus reuteri CRL 1098 as well as the nature (ATP or proton motive force [PMF] mediated primary transport) of the BA extrusion mechanism.     

Application of fluorescent techniques to evaluate the survival of probiotic lactobacilli to bile acid

Purpose of work  

To apply a fluorescent dye as an alternative technique to evaluate the survival of potentially probiotic lactobacilli to bile acids (BA) as first step in the design of probiotic functional foods.     

Determination of procyanidins and their metabolites in plasma samples by improved liquid chromatography–tandem mass spectrometry

An off-line solid-phase extraction (SPE) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determining procyanidins, catechin, epicatechin, dimer, and trimer in plasma samples. In the validation procedure of the analytical method, linearity, precision, accuracy, detection limits (LODs), quantification limits (LOQs), and the matrix effect were studied. Recoveries of the procyanidins were higher than 84%, except for the trimer, which was 65%. The LODs and LOQs were lower than 0.003 and 0.01 μM, respectively, for all the procyanidins studied, except for the trimers, which were 0.8 and 0.98 μM, respectively. This methodology was then applied for the analysis of rat plasma obtained 2 h after ingestion of grape seed phenolic extract. Monomers (catechin and epicatechin), dimer and trimer in their native form were detected and quantified in plasma samples, and their concentration ranged from 0.85 to 8.55 μM. Moreover, several metabolites, such as catechin and epicatechin glucuronide, catechin and epicatechin methyl glucuronide, and catechin and epicatechin methyl-sulphate were identified. These conjugated forms were quantified, in reference to the respective unconjugated form, showing concentrations between 0.06 and 23.90 μM.     

α-SNAP prevents docking of the acrosome during sperm exocytosis because it sequesters monomeric syntaxin

α-SNAP has an essential role in membrane fusion that consists of bridging cis SNARE complexes to NSF. α-SNAP stimulates NSF, which releases itself, α-SNAP, and individual SNAREs that subsequently re-engage in the trans arrays indispensable for fusion. α-SNAP also binds monomeric syntaxin and NSF disengages the α-SNAP/syntaxin dimer. Here, we examine why recombinant α-SNAP blocks secretion in permeabilized human sperm despite the fact that the endogenous protein is essential for membrane fusion. The only mammalian organism with a genetically modified α-SNAP is the hyh mouse strain, which bears a M105I point mutation; males are subfertile due to defective sperm exocytosis. We report here that recombinant α-SNAP-M105I has greater affinity for the cytosolic portion of immunoprecipitated syntaxin than the wild type protein and in consequence NSF is less efficient in releasing the mutant. α-SNAP-M105I is a more potent sperm exocytosis blocker than the wild type and requires higher concentrations of NSF to rescue its effect. Unlike other fusion scenarios where SNAREs are subjected to an assembly/disassembly cycle, the fusion machinery in sperm is tuned so that SNAREs progress uni-directionally from a cis configuration in resting cells to monomeric and subsequently trans arrays in cells challenged with exocytosis inducers. By means of functional and indirect immunofluorescense assays, we show that recombinant α-SNAPs--wild type and M105I--inhibit exocytosis because they bind monomeric syntaxin and prevent this SNARE from assembling with its cognates in trans. Sequestration of free syntaxin impedes docking of the acrosome to the plasma membrane assessed by transmission electron microscopy. The N-terminal deletion mutant α-SNAP-(160-295), unable to bind syntaxin, affects neither docking nor secretion. The implications of this study are twofold: our findings explain the fertility defect of hyh mice and indicate that assembly of SNAREs in trans complexes is essential for docking.     

Elemental and molecular detection for Quantum Dots-based immunoassays: a critical appraisal

A critical comparison between Elemental Mass Spectrometry (ICP-MS) and molecular fluorescence, as detection techniques for CdSe/ZnS Quantum Dots (QDs)-based immunoassays is presented here. Using a QDs-based progesterone immunoassay as "model" analytical system the features of both detection modes has been investigated. Minimal changes, compared to the previously developed fluorescent approach, were necessary to build the corresponding inhibition curve for the progesterone immunoassay using ICP-MS detection of cadmium (contained in the QDs core). Adequate agreement between results obtained using both elemental and molecular techniques for the determination of progesterone in cow milk has been obtained. Moreover, results from the comparison showed that fluorescence detection of the QDs is simpler, less time consuming and less expensive, but ICP-MS detection affords alternative and useful information unattainable using luminescence detection. First of all, ICP-MS allowed mass balances to be carried out (all along the sample preparation) providing an internal validation of the immunoassay procedure. Secondly, matrix-independent quantification as provided by ICP-MS enabled a direct determination of progesterone in raw milk without any further sample preparation (dilution) step. As a matter of fact, ICP-MS results showed that the quenching matrix effect suffered on bioconjugated QDs fluorescence emission (e.g. when the immunoassay was carried out directly in whole milk without any dilution) could be unequivocally attributed to nonspecific interactions between the matrix of the whole milk and the QDs surface. Finally, better sensitivity could be obtained with ICP-MS detection, IC(10)=0.028 ng/mL, versus 0.11 ng/mL using conventional fluorimetric detection, just by using lower reagents concentrations.