Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

Effect of Batch and Fed-Batch Growth Modes on Biofilm Formation by <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> at Different Temperatures

The influence of Listeria monocytogenes (L. monocytogenes) biofilm formation feeding conditions (batch and fed-batch) at different temperatures on biofilm biomass and activity was determined. Biofilm biomass and cellular metabolic activity were assessed by Crystal Violet (CV) staining and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) colorimetric method, respectively. Live/Dead staining was also performed in order to get microscopic visualization of the different biofilms. Results revealed that at refrigeration temperature (4°C) a higher amount of biofilm was produced when batch conditions were applied, while at higher temperatures the fed-batch feeding condition was the most effective on biofilm formation. Moreover, independently of the temperature used, biofilms formed under fed-batch conditions were metabolically more active than those formed in batch mode. In conclusion, this work shows that different growth modes significantly influence L. monocytogenes biofilm formation on abiotic surfaces as well as the metabolic activity of cells within biofilms.     

In vitro Antioxidant Activity of <Emphasis Type="Italic">Valeriana officinalis</Emphasis> Against Different Neurotoxic Agents

Valeriana officinalis L. (Valerian) is widely used as a traditional medicine to improve the quality of sleep. Although V. officinalis have been well documented as promising pharmacological agent; the exact mechanisms by which this plant act is still unknown. Limited literature data have indicated that V. officinalis extracts can exhibit antioxidant properties against iron in hippocampal neurons in vitro. However, there is no data available about the possible antioxidant effect of V. officinalis against other pro-oxidants in brain. In the present study, the protective effect of V. officinalis on lipid peroxidation (LPO) induced by different pro-oxidant agents with neuropathological importance was examined. Ethanolic extract of valerian (0–60 μg/ml) was tested against quinolinic acid (QA); 3-nitropropionic acid; sodium nitroprusside; iron sulfate (FeSO₄) and Fe²⁺/EDTA induced LPO in rat brain homogenates. The effect of V. officinalis in deoxyribose degradation and reactive oxygen species (ROS) production was also investigated. In brain homogenates, V. officinalis inhibited thiobarbituric acid reactive substances induced by all pro-oxidants tested in a concentration dependent manner. Similarly, V. officinalis caused a significant decrease on the LPO in cerebral cortex and in deoxyribose degradation. QA-induced ROS production in cortical slices was also significantly reduced by V. officinalis. Our results suggest that V. officinalis extract was effective in modulating LPO induced by different pro-oxidant agents. These data may imply that V. officinalis extract, functioning as antioxidant agent, can be beneficial for reducing insomnia complications linked to oxidative stress.     

Activation of the PI3K/Akt Pathway Early during Vaccinia and Cowpox Virus Infections Is Required for both Host Survival and Viral Replication

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to ≥90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.The family Poxviridae is a family of large, linear, double-stranded DNA viruses that carry out their entire life cycle within the cytoplasmic compartment of infected cells. Vaccinia virus (VACV) is a prototypical member of the genus Orthopoxvirus, which also includes the closely related cowpox virus (CPXV) (12, 52). The genomes of these viruses are approximately 200 kbp in length, with a coding capacity of approximately 200 genes. The genes involved in virus-host interactions are situated at both ends of the genome and are associated with the evasion of host immune defenses (1). These evasion mechanisms operate mainly extracellularly. For example, the secretion of soluble cytokine and chemokine receptor homologues blocks the receptor recognition by intercepting the cognate cytokine/chemokine in the extracellular environment. This mechanism facilitates viral attachment and entry into cells (1, 70). Therefore, decoy receptors for alpha interferon (IFN-α), IFN-β, IFN-γ, and tumor necrosis factor alpha play an important immunomodulatory role by affecting both the host antiviral and apoptotic responses.To counteract the host proapoptotic response, poxviruses have developed a number of antiapoptotic strategies, including the inhibition of apoptotic signals triggered by the extrinsic pathway (those mediated by death receptors such as tumor necrosis factor and Fas ligand) or the intrinsic pathway (mediated by the mitochondria and triggered upon viral infection) (1, 25, 70, 74). Many studies previously identified viral inhibitors that block specific steps of the intrinsic pathway. These include the VACV-encoded E3L, F1L, and N1L genes and the myxoma virus (MYXV)-encoded M11L gene, which block cytochrome c release (14, 20, 34, 39, 45, 75, 90), and the CPXV-encoded cytokine response modifier gene (CrmA) as well as the VACV-encoded SPI-2 gene, which inhibits both caspase-1 and caspase-8 (25, 58, 61, 74).An emerging body of evidence has also highlighted the pivotal role played by intracellular signaling pathways in Orthopoxvirus biology (18, 48, 92). We and others have shown that poxvirus manipulation of signaling pathways can be virus specific. For example, while both VACV and CPXV stimulate the MEK/extracellular signal-regulated kinase (ERK)/EGR-1 pathway during a substantial length of time of their infective cycle, the pathway is required only for VACV replication, whereas its role in CPXV biology has yet to be identified (71). MYXV, a rabbit-specific poxvirus, also activates the MEK/ERK pathway in a mouse model of poxvirus-host interactions. However, this stimulation led to the expression of IFN-β, which consequently blocked virus replication and possibly explains why MYXV has such a restricted host range (87).Another signaling molecule associated with viral replication is Akt kinase (also known as protein kinase B). The MYXV host range factor M-T5 is able to reprogram the intracellular environment, thereby increasing human tumor cell permissiveness to viral replication, which is directly associated with levels of phosphorylated Akt (88). In addition, M-T5 is functionally replaced by the host phosphatidylinositol 3-kinase (PI3K) enhancer A protein (92).The transmission of intracellular signals mediated by the serine/threonine kinase Akt to downstream molecules in response to diverse stimuli such as growth factors, insulin, and hormones is dependent upon the phosphorylation of serine 473 (S473-P) and threonine 308 (T308-P). This phosphorylation is mediated by mammalian target of rapamycin complex 2 (mTORC2) and phosphoinositide-dependent protein kinase 1 (PDK1), which act as downstream effectors of the PI3K/Akt/mTORC1 pathway (2, 66). PI3Ks are a family of enzymes (classes I to III) that generate lipid second messengers by the phosphorylation of plasma membrane phosphoinositides. Class IA PI3Ks consist of a catalytic subunit (p110, comprising the three isoforms α, β, and δ) and an adaptor/regulatory subunit (p85, comprising the two isoforms α and β) (for a detailed review, see reference 80).The Akt family of proteins is comprised of the three isoforms α, β, and γ, which are composed of an N-terminal pleckstrin homology domain, a central catalytic domain, and a C-terminal hydrophobic domain. Akt is recruited to the plasma membrane through the binding of its pleckstrin homology domain to the phosphatidylinositol 3,4,5-triphosphate (PIP3), which is a product of PI3K that is anchored to the plasma membrane. PDK1 is also recruited to the plasma membrane through interactions with PIP3. As both PDK1 and Akt interact with PIP3, PDK1 colocalizes with Akt and activates it by phosphorylating threonine 308 (T308-P) (2, 66). Following its activation, Akt phosphorylates a number of downstream substrates such as caspase-9, BAD, glycogen synthase kinase 3β (GSK-3β), and FKHR. This leads to the suppression of apoptosis, cell growth, survival, and proliferation (11, 16, 56).Another downstream target of PI3K/Akt is mTOR, a serine/threonine kinase that plays a central role in the regulation of cell growth, proliferation, survival, and protein synthesis (26). mTOR kinase has recently been found to be associated with two functionally distinct complexes in mammalian cells, known as mTORC1 and mTORC2 (63, 66). Although these multiprotein complexes share molecules in common, distinct adaptor proteins are recruited into each complex: regulatory-associated protein of TOR (raptor) is recruited into mTORC1, while rapamycin-insensitive companion of TOR (rictor) is recruited into mTORC2 (33, 64). While mTORC1 controls cell growth and protein translation and has proven to be rapamycin sensitive, mTORC2 regulates the actin cytoskeleton and is assumed to be rapamycin insensitive, even though under conditions of prolonged exposure to the drug, it appears to inhibit mTORC2 assembly (29, 64, 65). Additionally, it has been demonstrated that mTORC2 regulates the activity of Akt through the phosphorylation of S473 (S473-P). S473-P appears to be required for the full activation of Akt, since S473-P has been shown to enhance the subsequent phosphorylation of T308 by PDK1 (66, 67, 94). Moreover, the phosphorylation of both S473 and T308 results in a four- to fivefold increase in Akt activity compared to T308-P by PDK1 alone (66).The PI3K/PDK1/Akt(T308)/mTORC1 pathway regulates vital cellular processes that are important for viral replication and propagation, including cell growth, proliferation, and protein translation. This pathway is particularly important for the replication of DNA viruses, as their replication is cap dependent. However, the Akt signaling pathway can also negatively affect viral replication. The stress response downstream of Akt signaling, including hypoxia and energy and amino acid depletion, inhibits mTORC1 (5, 9, 69). Therefore, DNA viruses must overcome these constraints to translate their mRNAs.Pharmacological disruption of the PI3K/Akt pathway with the specific PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (2-morpholino-8-phenyl-4H-1-benzopyran-4-one) (82) has been reported to not only increase the cleavage of downstream molecules associated with proapoptotic activity [e.g., poly(ADP-ribose) polymerase (PARP) and the executioner caspase-3] (38, 41) but also promote microtubule stabilization, actin filament remodeling/cell migration, and bleb formation/viral infectivity (10, 35, 49, 54, 59).Because the PI3K/Akt and PI3K/Akt/mTOR pathways influence diverse cellular functions and possibly a healthy antiviral response, usurping these pathways could support an increase in viral replication. In support of this, a number of reports have demonstrated that either the PI3K/Akt or the PI3K/Akt/mTOR pathway plays a role in the replication of many viruses including flavivirus (38), hepatitis C virus (27), human immunodeficiency virus type 1 (93), human papillomavirus (44, 96), respiratory syncytial virus (77), coxsackievirus B3 (19), Epstein-Barr virus (17, 50, 73), human cytomegalovirus (36, 37, 72), herpes simplex virus type 1 (7, 83), varicella-zoster virus (60), Kaposi''s sarcoma-associated herpesvirus (89), adenovirus (55), and simian virus 40 (SV40) (95). With this in mind, we also investigated whether the PI3K/Akt pathway played a pivotal role in orthopoxvirus biology. In this study, we show that the VACV- and CPXV-stimulated PI3K/Akt pathway not only contributes to the prevention of host-cell death but also plays a beneficial role in the viral replication cycle.     

Production of β-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of β-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of β-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular β-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 °C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 °C. β-Fructofuranosidase activity was slightly activated by Cu²⁺, Mn²⁺, Mg²⁺, and Na⁺ at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K_d values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively.     

Partitioning of glycomacropeptide in aqueous two-phase systems

The partition behavior of glycomacropeptide (GMP) was determined in polyethylene glycol (PEG) and sodium citrate aqueous two-phase systems (ATPS). It was found that the partitioning of GMP depends on PEG molar mass, tie line length, pH, NaCl concentration and temperature. The obtained data indicates that GMP is preferentially partitioned into the PEG phase without addition of NaCl at pH 8.0. Larger tie line lengths and higher temperatures favor GMP partition to the PEG phase. Furthermore, it was verified that PEG molar mass and concentration have a slight effect on GMP partition. The increase in the molar mass of PEG induces a reduction of the protein solubility in the top PEG rich phase, being shown that the use of PEG1500 is beneficial for the extraction of GMP. A protein recovery higher than 85% was obtained in the top phase of these systems, clearly demonstrating its suitability as a starting point for the separation of GMP.     

Production of 3-hydroxy-γ-decalactone, the precursor of two decenolides with flavouring properties, by the yeast Yarrowia lipolytica

3-Hydroxy-γ-decalactone is the precursor of dec-2 and dec-3-en-4-olides which are valuable aroma compounds not yet produced. To promote the accumulation of this lactone, the yeast Yarrowia lipolytica was placed in different environmental conditions aiming at altering β-oxidation fluxes. The concentration of substrate, pH, aeration and dissolved oxygen level were modified. We observed an important accumulation at low aeration (0.40 molar yields) and, to a lesser extent, at lower pH (0.15). As oxygen played a key-role, we evaluated its effect at fixed dissolved oxygen and at the pH which was the most favourable to the biotransformation (pH 4.5). At 5% and 30% dissolved oxygen, yields reached 0.50. β-Oxidation fluxes are very dependent on the presence of oxygen and conditions of accumulation of 3-hydroxy-γ-decalactone with very high yields were identified. These results are an important step in the production of the two decenolides. Moreover, they show the high dependence of β-oxidation fluxes on environmental conditions and relate these conditions to the accumulation of intermediates, results that are of interest to all the processes using yeast on lipids or alkanes.     

Spatio-temporal expression patterns of anandamide-binding receptors in rat implantation sites: evidence for a role of the endocannabinoid system during the period of placental development

Background  

Although there is growing evidence that endocannabinoids play a critical role in early pregnancy, there are no studies describing the possible targets for this system after implantation. The endometrial stroma, which undergoes extensive proliferation and differentiation giving rise to the decidua and the trophoblast cells that invade after the initial stages of implantation, are potential targets. Since high anandamide (AEA) levels, the main endocannabinoid, are detrimental to implantation and in order to gain insight into the role of the endocannabinoid system in the development of the fetoplacental unit, the spatio-temporal pattern of expression of the anandamide-binding receptors, CB1, CB2 and the vanilloid receptor (TRPV1), were investigated by quantitative RT-PCR, western blot and immunohistochemistry.