Desenvolvimento de bebidas lácteas probióticas adicionadas de polpa de morango e suplementadas com L-triptofano elaboradas a partir de leite bubalino e bovino / Development of probiotic dairy beverages with strawberry pulp and supplemented with L-tryptophan from buffalo and bovine milk

O objetivo deste estudo foi desenvolver uma formulação de bebida láctea bubalina probiótica adicionada de polpa de morango, comparando os efeitos do uso do leite de búfala e de vaca na elaboração dos produtos e verificando a possibilidade de suplementação com triptofano nos produtos lácteos probióticos. Como primeira etapa do trabalho, bebidas lácteas probióticas foram elaboradas a partir de leite bubalino e bovino, fermentadas com Streptococcus thermophilus TA040, Lactobacillus bulgaricus LB340 e Lactobacillus acidophilus La5, e formuladas com 0, 25 e 50% de soro em sua formulação. As bebidas foram avaliadas quanto à cinética de fermentação das culturas láticas utilizadas, ao teor de proteína, gordura e sólidos totais não gordurosos, pós-acidificação, viabilidade das culturas fermentadoras e sua capacidade de sobrevivência ao estresse gastrointestinal in vitro. As bebidas lácteas bubalinas apresentaram resultados superiores as bebidas bovinas. O uso do leite de búfala na elaboração das bebidas lácteas promoveu benefícios quanto as culturas láticas presentes nos produtos, exercendo efeito protetivo e influindo na preservação da viabilidade das bactérias ao longo do armazenamento refrigerado e durante a simulação do estresse gastrointestinal in vitro. As bebidas lácteas elaboradas com 25% apresentaram os resultados mais próximos aos obtidos pelos produtos controle, sem adição de soro, sendo selecionadas para a segunda parte do estudo. Nesta etapa, as formulações de bebida láctea com 25% de soro, foram acrescidas de um preparado com polpa de morango e bebidas sem adição da fruta, utilizadas como controle. As bebidas lácteas bubalinas frutadas, apresentaram menor teor de gordura e melhores características reológicas, com maior viscosidade e consistência do que os produtos controle, sem afetar a pós-acidificação, o perfil de ácido graxo, assim como, a viabilidade e a resistência às condições de estresse gastrointestinal in vitro das culturas fermentadoras. A avaliação da possibilidade de suplementar lácteos probióticos com triptofano foi realizada em conjunto com a Universidade de Milão. Para isso, iogurtes probióticos receberam adição de triptofano antes ou após a fermentação, sendo avaliados com relação ao perfil de pós-acidificação, quantidade de triptofano nos produtos, número de células viáveis por plaqueamento e citometria de fluxo ao longo do armazenamento a 25° e 4°C. Complementarmente, a influência da presença do triptofano no crescimento e produção de compostos antimicrobianos pelas culturas láticas, também foi avaliada. A adição de triptofano após a fermentação dos iogurtes, que foram armazenados sob refrigeração (4°C), além de não afetar a pós-acidificação dos produtos, apresentou benefícios quanto a viabilidade L. acidophilus, redução do dano e aumento do número de células vivas, promovendo teor maior do aminoácido nos iogurtes. A presença do triptofano nos meios de cultivo, também influenciou de forma positiva o crescimento de S. thermophilus e L. acidophilus, melhorando o desenvolvimento das bactérias durante a fermentação e influindo em uma maior atividade antilistérica por parte do S. thermophilus. Diante da influência positiva da aplicação do leite de búfala na elaboração das bebidas lácteas, assim como, a adição do triptofano em iogurtes probióticos, a suplementação do aminoácido em bebidas lácteas bubalinas frutadas permitiria a obtenção de um produto funcional, onde seus benefícios estariam relacionados tanto ao consumo do probiótico presente no produto quanto a complementação de triptofano na dieta do consumidor

The aim of this study was to develop a formulation of probiotic buffalo dairy beverage added with strawberry pulp, comparing the effects of using buffalo and cow's milk in the preparation of products and verifying the possibility of tryptophan supplementation in probiotic dairy products. As a first stage of the work, probiotic dairy beverages were made from buffalo and bovine milk, fermented with Streptococcus thermophiles TA040, Lactobacillus bulgaricus LB340 and Lactobacillus acidophilus La5, and formulated with 0, 25 and 50% whey in their formulation. The beverages were evaluated for the fermentation kinetics of the used lactic cultures, the levels of protein, fat and total no fat solids, post-acidification, fermenting cultures viability and their ability to survive gastrointestinal stress in vitro. Buffalo milk use in dairy beverages production promoted benefits regarding the lactic cultures present in the products, exerting a protective effect and influencing the viability preservation of bacteria during the cold storage and simulation of gastrointestinal stress in vitro. Dairy beverages made with 25% whey addition showed results similar to those obtained by the control products, without whey addition, being selected for the second part of the study. In this part, the dairy beverages formulations with 25% whey, were added with a preparation were added with a strawberry pulp preparation and dairy beverages without added fruit, used as a control. Fruity bubaline dairy beverages had lower fat content and better rheological characteristics, with higher viscosity and consistency than control products, without affecting post-acidification, fatty acid profile, as well as viability and resistance to in vitro gastrointestinal condition of fermented cultures. The possibility of supplementing probiotic dairy products with tryptophan was evaluated in partnership with the University of Milan. For this, probiotic yogurts received the addition of tryptophan before or after fermentation, being evaluated in relation to the post-acidification profile, tryptophan amount in the products, viable cell number per plating and flow cytometry during storage at 25°C and 4°C. In addition, the influence of the tryptophan presence on the growth and production of antimicrobial compounds by lactic cultures was also evaluated. The addition of tryptophan after the yogurt fermentation, which were stored under refrigeration (4°C), in addition to not affecting the post-acidification of the products, showed benefits to the viability of L. acidophilus, reduced the damage and increased the number of cells promoting higher amino acid content in yogurts. Tryptophan presence in the culture media also positively influenced the growth of S. thermophiles and L. acidophilus, improving the development of bacteria during fermentation and influencing better antilisteric activity in the part of S. thermophiles. In view of the buffalo milk positive influence observed after the application in dairy beverage preparation, as well as the addition of tryptophan in probiotic yoghurts, amino acid supplementation in fruity buffalo dairy beverages would allow to obtain a functional product, where its benefits would be related both to the consumption of the probiotic present in the product as to the supplementation of tryptophan in the consumer's diet

Characterization of modeled inhibitory binding sites on isoform one of the Na+/H+ exchanger.

Mammalian Na+/H+ exchanger isoform one (NHE1) is a plasma membrane protein responsible for pH regulation in mammalian cells. Excess activity of the protein promotes heart disease and is a trigger of metastasis in cancer. Inhibitors of the protein exist but problems in specificity have delayed their clinical application. Here we examined amino acids involved in two modeled inhibitor binding sites (A, B) in human NHE1. Twelve mutations (Asp159, Phe348, Ser351, Tyr381, Phe413, Leu465, Gly466, Tyr467, Leu468, His473, Met476, Leu481) were made and characterized. Mutants S351A, F413A, Y467A, L468A, M476A and L481A had 40-70% of wild type expression levels, while G466A and H473A expressed 22% ~ 30% of the wild type levels. Most mutants, were targeted to the cell surface at levels similar to wild type NHE1, approximately 50-70%, except for F413A and G466A, which had very low surface targeting. Most of the mutants had measurable activity except for D159A, F413A and G466A. Resistance to inhibition by EMD87580 was elevated in mutants F438A, L465A and L468A and reduced in mutants S351A, Y381A, H473A, M476A and L481A. All mutants with large alterations in inhibitory properties showed reduced Na+ affinity. The greatest changes in activity and inhibitor sensitivity were in mutants present in binding site B which is more closely associated with TM4 and C terminal of extracellular loop 5, and is situated between the putative scaffolding domain and transport domain. The results help define the inhibitor binding domain of the NHE1 protein and identify new amino acids involved in inhibitor binding.

Dibenzodiazepinone-type muscarinic receptor antagonists conjugated to basic peptides: Impact of the linker moiety and unnatural amino acids on M2R selectivity.

The family of human muscarinic acetylcholine receptors (MRs) is characterized by a high sequence homology among the five subtypes (M1R-M5R), being the reason for a lack of subtype selective MR ligands. In continuation of our work on dualsteric dibenzodiazepinone-type M2R antagonists, a series of M2R ligands containing a dibenzodiazepinone pharmacophore linked to small basic peptides was synthesized (64 compounds). The linker moiety was varied with respect to length, number of basic nitrogens (0-2) and flexibility. Besides proteinogenic basic amino acids (Lys, Arg), shorter homologues of Lys and Arg, containing three and two methylene groups, respectively, as well as D-configured amino acids were incorporated. The type of linker had a marked impact on M2R affinity and also effected M2R selectivity. In contrast, the structure of the basic peptide rather determined M2R selectivity than M2R affinity. For example, the most M2R selective compound (UR-CG188, 89) with picomolar M2R affinity (pKi 9.60), exhibited a higher M2R selectivity (ratio of Ki M1R/M2R/M3R/M4R/M5R: 110:1:5200:55:2300) compared to the vast majority of reported M2R preferring MR ligands. For selected ligands, M2R antagonism was confirmed in a M2R miniG protein recruitment assay.

Discovery of Potential Chemical Probe as Inhibitors of CXCL12 Using Ligand-Based Virtual Screening and Molecular Dynamic Simulation.

CXCL12 are small pro-inflammatory chemo-attractant cytokines that bind to a specific receptor CXCR4 with a role in angiogenesis, tumor progression, metastasis, and cell survival. Globally, cancer metastasis is a major cause of morbidity and mortality. In this study, we targeted CXCL12 rather than the chemokine receptor (CXCR4) because most of the drugs failed in clinical trials due to unmanageable toxicities. Until now, no FDA approved medication has been available against CXCL12. Therefore, we aimed to find new inhibitors for CXCL12 through virtual screening followed by molecular dynamics simulation. For virtual screening, active compounds against CXCL12 were taken as potent inhibitors and utilized in the generation of a pharmacophore model, followed by validation against different datasets. Ligand based virtual screening was performed on the ChEMBL and in-house databases, which resulted in successive elimination through the steps of pharmacophore-based and score-based screenings, and finally, sixteen compounds of various interactions with significant crucial amino acid residues were selected as virtual hits. Furthermore, the binding mode of these compounds were refined through molecular dynamic simulations. Moreover, the stability of protein complexes, Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), and radius of gyration were analyzed, which led to the identification of three potent inhibitors of CXCL12 that may be pursued in the drug discovery process against cancer metastasis.

Oncology Therapeutics Targeting the Metabolism of Amino Acids.

Amino acid metabolism promotes cancer cell proliferation and survival by supporting building block synthesis, producing reducing agents to mitigate oxidative stress, and generating immunosuppressive metabolites for immune evasion. Malignant cells rewire amino acid metabolism to maximize their access to nutrients. Amino acid transporter expression is upregulated to acquire amino acids from the extracellular environment. Under nutrient depleted conditions, macropinocytosis can be activated where proteins from the extracellular environment are engulfed and degraded into the constituent amino acids. The demand for non-essential amino acids (NEAAs) can be met through de novo synthesis pathways. Cancer cells can alter various signaling pathways to boost amino acid usage for the generation of nucleotides, reactive oxygen species (ROS) scavenging molecules, and oncometabolites. The importance of amino acid metabolism in cancer proliferation makes it a potential target for therapeutic intervention, including via small molecules and antibodies. In this review, we will delineate the targets related to amino acid metabolism and promising therapeutic approaches.

Small molecules-Giant leaps for immuno-oncology.

Immuno-oncology therapies are revolutionizing the oncology landscape with checkpoint blockade becoming the treatment backbone for many indications. While inspiring, much work remains to increase the number of cancer patients that can benefit from these treatments. Thus, a new era of immuno-oncology research has begun which is focused on identifying novel combination regimes that lead to improved response rates. This review highlights the significance of small molecules in this approach and illustrates the huge progress that has been made to date.

Protonation states of central amino acids in a zinc metalloprotease complexed with inhibitor: Molecular mechanics optimizations and ab initio molecular orbital calculations.

The zinc-metalloprotease pseudolysin (PLN) secreted from bacteria degrades extracellular proteins to produce bacterial nutrition. Since PLN has a Zn ion at the inhibitor-binding site, the interactions between Zn and PLN residues as well as inhibitor can be significantly changed depending on the protonation states of PLN residues at the inhibitor-binding site. To determine stable protonation states of these residues, we here considered different protonation states for Glu and His residues located around Zn and investigated the electronic states of the PLN + inhibitor complex, using ab initio molecular simulations. The protonation state of His223 was found to significantly affect the specific interactions between PLN and the inhibitor.

Biological activity of synthesized 5-{1-[(4-chlorophenyl)sulfonyl]piperidin-4-yl}-2-mercapto-1, 3, 4-oxadiazole derivatives demonstrated by in silico and BSA binding studies

We synthesized a series of compounds bearing pharmacologically important 1,3,4-oxadiazole and piperidine moieties. Spectral data analysis by 1H-NMR, 13C-NMR, IR and EI-MS was used to elucidate the structures of the synthesized molecules. Docking studies explained the different types of interaction of the compounds with amino acids, while bovine serum albumin (BSA) binding interactions showed their pharmacological effectiveness. Antibacterial screening of these compounds demonstrated moderate to strong activity against Salmonella typhi and Bacillus subtilis but only weak to moderate activity against the other three bacterial strains tested. Seven compounds were the most active members as acetyl cholinesterase inhibitors. All the compounds presented displayed strong inhibitory activity against urease. Compounds 7l, 7m, 7n, 7o, 7p, 7r, 7u, 7v, 7x and 7v were highly active, with respective IC50 values of 2.14±0.003, 0.63±0.001, 2.17±0.006, 1.13±0.003, 1.21±0.005, 6.28±0.003, 2.39±0.005, 2.15±0.002, 2.26±0.003 and 2.14±0.002 µM, compared to thiourea, used as the reference standard (IC50 = 21.25±0.15 µM). These new urease inhibitors could replace existing drugs after their evaluation in comprehensive in vivo studies.

(2R,6R)-hydroxynorketamine exerts mGlu2 receptor-dependent antidepressant actions.

Currently approved antidepressant drugs often take months to take full effect, and ∼30% of depressed patients remain treatment resistant. In contrast, ketamine, when administered as a single subanesthetic dose, exerts rapid and sustained antidepressant actions. Preclinical studies indicate that the ketamine metabolite (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] is a rapid-acting antidepressant drug candidate with limited dissociation properties and abuse potential. We assessed the role of group II metabotropic glutamate receptor subtypes 2 (mGlu2) and 3 (mGlu3) in the antidepressant-relevant actions of (2R,6R)-HNK using behavioral, genetic, and pharmacological approaches as well as cortical quantitative EEG (qEEG) measurements in mice. Both ketamine and (2R,6R)-HNK prevented mGlu2/3 receptor agonist (LY379268)-induced body temperature increases in mice lacking the Grm3, but not Grm2, gene. This action was not replicated by NMDA receptor antagonists or a chemical variant of ketamine that limits metabolism to (2R,6R)-HNK. The antidepressant-relevant behavioral effects and 30- to 80-Hz qEEG oscillation (gamma-range) increases resultant from (2R,6R)-HNK administration were prevented by pretreatment with an mGlu2/3 receptor agonist and absent in mice lacking the Grm2, but not Grm3-/-, gene. Combined subeffective doses of the mGlu2/3 receptor antagonist LY341495 and (2R,6R)-HNK exerted synergistic increases on gamma oscillations and antidepressant-relevant behavioral actions. These findings highlight that (2R,6R)-HNK exerts antidepressant-relevant actions via a mechanism converging with mGlu2 receptor signaling and suggest enhanced cortical gamma oscillations as a marker of target engagement relevant to antidepressant efficacy. Moreover, these results support the use of (2R,6R)-HNK and inhibitors of mGlu2 receptor function in clinical trials for treatment-resistant depression either alone or in combination.

A label-free MALDI TOF MS-based method for studying the kinetics and inhibitor screening of the Alzheimer's disease drug target ß-secretase.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities including sensitivity, minute sample consumption, and label-free detection, all of which are highly desired in enzyme assays. On the other hand, the application of MALDI TOF MS is usually limited by high concentrations of MS-incompatible compounds in the reaction mixture such as salts or organic solvents. Here, we introduce kinetic and inhibition studies of ß-secretase (BACE1), a key enzyme of the progression of Alzheimer's disease. Compatibility of the enzyme assay with MALDI TOF MS was achieved, providing both a complex protocol including a desalting step designed for rigorous kinetic studies and a simple mix-and-measure protocol designed for high-throughput inhibitor screening. In comparison with fluorescent or colorimetric assays, MALDI TOF MS represents a sensitive, fast, and label-free technique with minimal sample preparation. In contrast to other MS-based methodological approaches typically used in drug discovery processes, such as a direct injection MS or MS-coupled liquid chromatography or capillary electrophoresis, MALDI TOF MS enables direct analysis and is a highly suitable approach for high-throughput screening. The method's applicability is strongly supported by the high correlation of the acquired kinetic and inhibition parameters with data from the literature as well as from our previous research. Graphical abstract ᅟ.

fabH deletion increases DHA production in Escherichia coli expressing Pfa genes.

BACKGROUND: Some marine bacteria, such as Moritella marina, produce the nutraceutical docosahexaenoic acid (DHA) thanks to a specific enzymatic complex called Pfa synthase. Escherichia coli heterologously expressing the pfa gene cluster from M. marina also produces DHA. The aim of this study was to find genetic or metabolic conditions to increase DHA production in E. coli. RESULTS: First, we analysed the effect of the antibiotic cerulenin, showing that DHA production increased twofold. Then, we tested a series of single gene knockout mutations affecting fatty acid biosynthesis, in order to optimize the synthesis of DHA. The most effective mutant, fabH, showed a threefold increase compared to wild type strain. The combination of cerulenin inhibition and fabH deletion rendered a 6.5-fold improvement compared to control strain. Both strategies seem to have the same mechanism of action, in which fatty acid synthesis via the canonical pathway (fab pathway) is affected in its first catalytic step, which allows the substrates to be used by the heterologous pathway to synthesize DHA. CONCLUSIONS: DHA-producing E. coli strain that carries a fabH gene deletion boosts DHA production by tuning down the competing canonical biosynthesis pathway. Our approach can be used for optimization of DHA production in different organisms.

Cerebrolysin and Aquaporin 4 Inhibition Improve Pathological and Motor Recovery after Ischemic Stroke.

BACKGROUND: Edema represents one of the earliest negative markers of survival and consecutive neurological deficit following stroke. The mixture of cellular and vasogenic edema makes treating this condition complicated, and to date, there is no pathogenically oriented drug treatment for edema, which leaves parenteral administration of a hypertonic solution as the only non-surgical alternative. OBJECTIVE: New insights into water metabolism in the brain have opened the way for molecular targeted treatment, with aquaporin 4 channels (AQP4) taking center stage. We aimed here to assess the effect of inhibiting AQP4 together with the administration of a neurotropic factor (Cerebrolysin) in ischemic stroke. METHODS: Using a permanent medial cerebral artery occlusion rat model, we administrated a single dose of the AQP4 inhibitor TGN-020 (100 mg/kg) at 15 minutes after ischemia followed by daily Cerebrolysin dosing (5ml/kg) for seven days. Rotarod motor testing and neuropathology examinations were next performed. RESULTS: We showed first that the combination treatment animals have a better motor function preservation at seven days after permanent ischemia. We have also identified distinct cellular contributions that represent the bases of behavior testing, such as less astrocyte scarring and a larger neuronalsurvival phenotype rate in animals treated with both compounds than in animals treated with Cerebrolysin alone or untreated animals. CONCLUSION: Our data show that water diffusion inhibition and Cerebrolysin administration after focal ischemic stroke reduces infarct size, leading to a higher neuronal survival in the peri-core glial scar region.

Competitive inhibition of amino acid transport in human preovulatory ovarian follicles.

To date we have yet to examine whether amino acid (AA) transport in human ovarian follicles is affected by competitive inhibition. In contrast, transplacental transfer of AAs in late-gestation sheep is characterized by reciprocal competition. This phenomenon has been described by algebraic equations of umbilical uptake of AAs based on maternal arterial concentrations. In the present translational study at a university teaching hospital, we verified whether these equations apply to the transport of AAs from blood to follicular fluid (FF) in human preovulatory follicles. For this purpose we used our data on AA concentrations in blood and FF measured earlier by high-performance liquid chromatography in specimens from 14 patients undergoing oocyte retrieval for in vitro fertilization after controlled ovarian stimulation. The main outcome measure was statistical significance of Spearman correlation coefficients for measured versus calculated concentrations of 8 AAs: isoleucine, leucine, valine, phenylalanine, methionine, threonine, lysine, and arginine. Equations for umbilical uptake provided a highly accurate description of blood-to-FF transport for 7 AAs with the exception of lysine: R ≥ 0.899 (p < 0.0001) for the branched-chain AAs, R = 0.829 (p = 0.0003) for threonine, R = 0.754 (p = 0.0019) for arginine, and R = 0.631 (p = 0.0156) for phenylalanine and methionine. We conclude that these equations indicate competitive inhibition between the AAs studied. Our study strongly suggests that many AA transport systems operating in the placenta should also be active in the cells of the preovulatory follicle. Future studies on AA fluxes in human ovarian follicles must consider possible competitive inhibition. ABBREVIATIONS: AA: amino acid; FF: follicular fluid; HPLC: high-performance liquid chromatography.

Anti-ulcer effect and potential mechanism of licoflavone by regulating inflammation mediators and amino acid metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhiza is the dry root and rhizome of the leguminous plant, Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L., which was firstly cited in Shennong's Herbal Classic in Han dynasty and was officially listed in the Chinese Pharmacopoeia, has been widely used in China during the past millennia. Licoflavone is the major component of Glycyrrhiza with anti-ulcer activity. The present study is based on clarifying the anti-ulcer effect of licoflavone, aiming at elucidating the possible molecule mechanisms of its action for treating gastric ulcer rats induced by acetic acid. MATERIALS AND METHODS: Rats were divided into 7 groups, and drugs were administered from on the day after the onset of gastric ulcer (day 3) until day 11 of the experiment once daily continuously. The plasma were analyzed by high-performance liquid chromatography combined with time-of-flight mass spectrometry (HPLC/ESI-TOF-MS), significant different metabolites were investigated to explain its therapeutic mechanism. Furthermore, quantitative real time polymerase chain reaction (RT-PCR) analysis was performed to detect the expression of RNA in stomach tissue for verifying the above results. RESULTS: Licoflavone can effectively cure the gastric ulcer, particularly the middle dose group. According to the statistical analysis of the plasma different metabolites from each groups and the expression of genes in tissues, sixteen significant different metabolites, including histamine, tryptophan, arachidonic acid, phingosine-1-phosphate etc., contributing to the treatment of gastric ulcer were discovered and identified. In RT-PCR analysis, the results of the expression of RNA were corresponded with what we discovered. CONCLUSIONS: Our study indicated licoflavone plays the role of treating gastric ulcer by regulating inflammation mediators and amino acid metabolism. We demonstrated that metabolomics technology combined with gene technology is a useful tool to search different metabolites and to dissect the potential mechanisms of traditional Chinese medicine (TCM) in treating gastric ulcer.

Understanding Amino Acid Mutations in Hepatitis B Virus Proteins for Rational Design of Vaccines and Drugs.

The hepatitis B virus (HBV) genome encodes four proteins, i.e., DNA polymerase, surface protein, X, and core proteins. HBV undergoes different selective pressures for drug resistance and immune/vaccine escape and mutations are common for the HBV proteins. We here collected all the reported amino acid mutations happened in these four HBV proteins and studied their patterns. The relationship between the mutations and epitopic functions are investigated with bioinformatics tools, based on their sequence information. Some interesting results are observed for the mutation patterns, such as we found the serine and threonine are both for frequently mutated residues and mutant residues, while the tryptophan and methionine have low mutability. The results provide important information for the understanding of the molecular mechanism of virus evolution and therefore will facilitate the future rational design of HBV vaccines or drugs.

Glycine release is regulated by metabotropic glutamate receptors sensitive to mGluR2/3 ligands and activated by N-acetylaspartylglutamate (NAAG).

The presence of metabotropic glutamate receptors (mGluRs) of group II modulating glycine exocytosis from glycinergic nerve endings of mouse spinal cord was investigated. Purified synaptosomes were selectively prelabeled with [(3)H]glycine through the neuronal transporter GlyT2 and subsequently depolarized by superfusion with 12 mM KCl. The selective mGluR2/3 agonist LY379268 inhibited the K(+)-evoked overflow of [(3)H]glycine in a concentration-dependent manner (EC(50) about 0.2 nM). The effect of LY379268 was prevented by the selective mGluR2/3 antagonist LY341495 (IC(50) about 1 nM). N-acetylaspartylglutamate (NAAG) inhibited [(3)H]glycine overflow with extraordinary potency (EC(50) about 50 fmol). In contrast, glutamate was ineffective up to 0.1 nM, excluding that glutamate contamination of commercial NAAG samples is responsible for the reported activity of NAAG at mGluR3. LY341495 antagonized the NAAG inhibition of [(3)H]glycine release. The effect of a combination of maximally effective concentrations of LY379268 and NAAG exhibited no additivity. The non-hydrolysable NAAG analogue N-acetylaspartyl-ß-linked glutamate (ß-NAAG) antagonized NAAG and LY379268. In conclusion, our results show that glycinergic nerve endings in spinal cord are endowed with group II mGluRs mediating inhibition of glycine exocytosis. NAAG can activate these presynaptic receptors with extremely high affinity and with characteristics compatible with the reported mGluR3 pharmacology. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Sensing of amino acids by the gut-expressed taste receptor T1R1-T1R3 stimulates CCK secretion.

CCK is secreted by endocrine cells of the proximal intestine in response to dietary components, including amino acids. CCK plays a variety of roles in digestive processes, including inhibition of food intake, consistent with a role in satiety. In the lingual epithelium, the sensing of a broad spectrum of L-amino acids is accomplished by the heteromeric amino acid (umami) taste receptor (T1R1-T1R3). T1R1 and T1R3 subunits are also expressed in the intestine. A defining characteristic of umami sensing by T1R1-T1R3 is its potentiation by IMP or GMP. Furthermore, T1R1-T1R3 is not activated by Trp. We show here that, in response to L-amino acids (Phe, Leu, Glu, and Trp), but not D-amino acids, STC-1 enteroendocrine cells and mouse proximal small intestinal tissue explants secrete CCK and that IMP enhances Phe-, Leu-, and Glu-induced, but not Trp-induced, CCK secretion. Furthermore, small interfering RNA inhibition of T1R1 expression in STC-1 cells results in significant diminution of Phe-, Leu-, and Glu-stimulated, but not Trp-stimulated, CCK release. In STC-1 cells and mouse intestine, gurmarin inhibits Phe-, Leu-, and Glu-induced, but not Trp-stimulated, CCK secretion. In contrast, the Ca(2+)-sensing receptor antagonist NPS2143 inhibits Phe-stimulated CCK release partially and Trp-induced CCK secretion totally in mouse intestine. However, NPS2143 has no effect on Leu- or Glu-induced CCK secretion. Collectively, our data demonstrate that functional characteristics and cellular location of the gut-expressed T1R1-T1R3 support its role as a luminal sensor for Phe-, Leu-, and Glu-induced CCK secretion.

Impact of gestational and lactational phthalate exposure on hypothalamic content of amino acid neurotransmitters and FSH secretion in peripubertal male rats.

This study investigated the effect of the pre- and perinatal exposure to di-(2-ethylhexyl) phthalate (DEHP) on the neuroendocrine parameters that regulate reproduction in peripubertal male rats. DEHP at dose of 3 and 30mg/kg bw/day was administered orally to female rat since pregnancy onset until weaning. The male litters were sacrificed at 30 days of age to determine gonadotropin serum level and the hypothalamic contents of the amino acids aspartate and gamma-aminobutyric acid. No changes in gonadotropin, aspartate and gamma-aminobutyric acid levels were detected at the low dose. DEHP 30mg/kg bw/day reduced testis weight and serum FSH, in correlation with a significant increase in the inhibitory GABAergic tone and a reduction in the stimulatory effect of aspartate on gonadotropin level. This study provides unknown data regarding changes in the hypothalamic contents of the amino acid neurotransmitters, which are involved in the neuroendocrine regulation of reproductive axis, in peripubertal male rat offspring from dams exposed to DEHP during gestational and lactational periods. This could be related with the gonadotropin modifications also here described.

Determinación de acrilamida y furosina en harinas y papillas infantiles / Determination of acrylamide and furosine in flour and infant formulas

Una de las modificaciones más importantes inducidas en el alimento durante el calentamiento es la reacción de Maillard, donde participan aminoácidos y azúcares reductores, pudiendo producirse mejora de las características organolépticas del alimento pero también pérdida del valor nutritivo y aparición de compuestos tóxicos. Los cereales infantiles son hidrolizados durante el procesado, aumentando los niveles de azúcares reductores, por lo que en el tostado o desecado de estas muestras puede favorecerse el desarrollo de la reacción de Maillard. Los niños son especialmente susceptibles ala disminución del valor nutritivo y al consumo de compuestos potencialmente tóxicos, de ahí la importancia de conocer el grado de desarrollo de esta reacción en dichos productos. En este estudio se determinó el contenido de acrilamida y furosina en harinas crudas y tostadas y en papillas infantiles, con el fin de evaluar la extensión de la reacción de Maillard. El contenido de furosina osciló entre11,5-34,6 mg/100 g de proteínas en las muestras de harinas y entre 122-1193 mg/100 g de proteínas en las muestras de papillas. No se detectó acrilamida en las muestras de harinas y osciló entre 0,22 y 9,6Cg/kg en las papillas. Las pérdidas de lisina pueden considerarse altas en algunas muestras, pero no suponen riesgo de déficit nutricional al consumirse estos productos con leche. La acrilamida sólo se encontró en niveles cuantificables en cuatro muestras y en concentraciones muy bajas; éstas se encuentran lejos de la dosis máxima permitida y por lo tanto no deberían representar problemas adversos tras su consumo(AU)

One of the most important modifications originated in foods during thermal treatment is Maillard reaction, in which amino acids and reducing sugars are participants. This reaction can improve the organoleptic characteristics of foods but, moreover, can lead to nutritional value losses and apparition of toxicological compounds. Infant cereals are hydrolyzed during processing, increasing reducing sugar levels thereby Maillard reaction can be favored in toasted or dried samples. Infants are especially susceptible to the decreased nutritional value and to the toxicological compounds consumption. Thus it is important to know the development rate of this reaction in these products. In this study acrylamide and furosine content in raw and toasted flour and in infant formulas were determined, in order to evaluate Maillard reaction evolution. Furosine content ranged from 11.5 to34.6 mg/100 g of proteins in toasted flour samples and from 122 to 1193 mg/100 g of proteins in formula samples. Acrylamide was not detected in flour samples ranging from 0.22 and 9.6 Cg/kg in formula samples. Lysine losses can be considered to be high in several samples, but it does not suppose a nutritional deficiency risk because these products are consumed together with milk. Acrylamide only was found in quantified levels in four samples and in very low concentrations. These values are lower than the maximum dose allowed and, therefore, they should not represent adverse problems after their consumption(AU)

The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity.

Excess copper is poisonous to all forms of life, and copper overloading is responsible for several human pathologic processes. The primary mechanisms of toxicity are unknown. In this study, mutants of Escherichia coli that lack copper homeostatic systems (copA cueO cus) were used to identify intracellular targets and to test the hypothesis that toxicity involves the action of reactive oxygen species. Low micromolar levels of copper were sufficient to inhibit the growth of both WT and mutant strains. The addition of branched-chain amino acids restored growth, indicating that copper blocks their biosynthesis. Indeed, copper treatment rapidly inactivated isopropylmalate dehydratase, an iron-sulfur cluster enzyme in this pathway. Other enzymes in this iron-sulfur dehydratase family were similarly affected. Inactivation did not require oxygen, in vivo or with purified enzyme. Damage occurred concomitant with the displacement of iron atoms from the solvent-exposed cluster, suggesting that Cu(I) damages these proteins by liganding to the coordinating sulfur atoms. Copper efflux by dedicated export systems, chelation by glutathione, and cluster repair by assembly systems all enhance the resistance of cells to this metal.