Free-amino acid profiles of thua nao, a Thai fermented soybean

Thua nao, a rich source of free-amino acids, is a fermented soybean, usually used as seasoning or flavouring enhancer in northern Thailand. Free-amino acids (FAA) of unfermented/cooked soybeans, thua nao, fermented by pure Bacillus subtilis TN51 (TNB51), and a naturally fermented product (TNMX), were investigated by pre-column derivatisation with 9-fluorenylmethyl chloroformate, followed by reversed-phase HPLC. Total FAA and essential amino acids were found at significantly higher concentrations in TNB51 thua nao than in TNMX thua nao (naturally fermented). Both fermented thua nao had much higher concentrations of FAA than had their unfermented counterparts. With respect to taste-enhancing FAA, typical bitter attributes of thua nao came mainly from hydrophobic and basic FAA, whereas an umami attribute came predominantly from acidic FAA.

Soya phytoestrogens change cortical and hippocampal expression of BDNF mRNA in male rats

Adult male hooded Lister rats were either fed a diet containing 150 microg/g soya phytoestrogens or a soya-free diet for 18 days. This concentration of phytoestrogens should have been sufficient to occupy the oestrogen-beta, but not the oestrogen-alpha, receptors. Using in situ hybridisation, significant reductions were found in brain-derived neurotrophic factor (BDNF) mRNA expression in the CA3 and CA4 region of the hippocampus and in the cerebral cortex in the rats fed the diet containing phytoestrogens, compared with those on the soya-free diet. No changes in glutamic acid decarboxylase-67 or glial fibrillary acidic protein mRNA were found. This suggests a role for oestrogen-beta receptors in regulating BDNF mRNA expression.

Electronic properties, redox behavior, and interactions with H2O2 of pH-sensitive hydroxyphenyl-1,2,4-triazole-based oxovanadium(V)complexes

The syntheses and spectroscopic characterization of two 1,2,4-triazole-based oxovanadium(V) complexes are reported: 1(-)[VO(2)L1](-) and 2 [(VOL2)(2)(OMe)(2)] (where H(2)L1 = 3-(2'-hydroxyphenyl)-5-(pyridin-2"-yl)-H-1-1,2,4-triazole, H3L2 = bis-3,5-(2'-hydroxyphenyl)-1H-1,2,4-triazole). The ligand environment (N,N,O vs O,N,O) is found to have a profound influence on the properties and reactivity of the complexes formed. The presence of the triazolato ligand allows for pH tuning of the spectroscopic and electrochemical properties, as well as the interaction and stability of the complexes in the presence of hydrogen peroxide. The vanadium(IV) oxidation states were generated electrochemically and characterized by UV-vis and EPR spectroscopies, For 2, under acidic conditions, rapid exchange of the methoxide ligands with solvent [in particular, in the vanadium(IV) redox state] was observed.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin- was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans

Dietary nitrate is metabolized to nitrite by bacterial flora on the posterior surface of the tongue leading to increased salivary nitrite concentrations. In the acidic environment of the stomach, nitrite forms nitrous acid, a potent nitrating/nitrosating agent. The aim of this study was to examine the pharmacokinetics of dietary nitrate in relation to the formation of salivary, plasma, and urinary nitrite and nitrate in healthy subjects. A secondary aim was to determine whether dietary nitrate increases the formation of protein-bound 3-nitrotyrosine in plasma, and if dietary nitrate improves platelet function. The pharmacokinetic profile of urinary nitrate excretion indicates total clearance of consumed nitrate in a 24 h period. While urinary, salivary, and plasma nitrate concentrations increased between 4- and 7-fold, a significant increase in nitrite was only detected in saliva (7-fold). High dietary nitrate consumption does not cause a significant acute change in plasma concentrations of 3-nitrotyrosine or in platelet function.

Hydrogen-bonding-driven self-assembly of PEGylated organosilica nanoparticles with poly(acrylic acid) in aqueous solutions and in layer-by-layer deposition at solid surfaces

PEGylated organosilica nanoparticles have been synthesized through self-condensation of (3-mercaptopropyl)trimethoxysilane in dimethyl sulfoxide into thiolated nanoparticles with their subsequent reaction with methoxypoly(ethylene glycol) maleimide. The PEGylated nanoparticles showed excellent colloidal stability over a wide range of pH in contrast to the parent thiolated nanoparticles, which have a tendency to aggregate irreversibly under acidic conditions (pH < 3.0). Due to the presence of a poly(ethylene glycol)-based corona, the PEGylated nanoparticles are capable of forming hydrogen-bonded interpolymer complexes with poly(acrylic acid) in aqueous solutions under acidic conditions, resulting in larger aggregates. The use of hydrogen-bonding interactions allows more efficient attachment of the nanoparticles to surfaces. The alternating deposition of PEGylated nanoparticles and poly(acrylic acid) on silicon wafer surfaces in a layer-by-layer fashion leads to multilayered coatings. The self-assembly of PEGylated nanoparticles with poly(acrylic acid) in aqueous solutions and at solid surfaces was compared to the behavior of linear poly(ethylene glycol). The nanoparticle system creates thicker layers than the poly(ethylene glycol), and a thicker layer is obtained on a poly(acrylic acid) surface than on a silica surface, because of the effects of hydrogen bonding. Some implications of these hydrogen-bonding-driven interactions between PEGylated nanoparticles and poly(acrylic acid) for pharmaceutical formulations are discussed.

NAADP regulates human platelet function.

Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid-adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.

Riluzole neuroprotection in a parkinson’s disease model involves suppression of reactive astrocytosis but not GLT-1 regulation

Background Riluzole is a neuroprotective drug used in the treatment of motor neurone disease. Recent evidence suggests that riluzole can up-regulate the expression and activity of the astrocyte glutamate transporter, GLT-1. Given that regulation of glutamate transport is predicted to be neuroprotective in Parkinson's disease, we tested the effect of riluzole in parkinsonian rats which had received a unilateral 6-hydroxydopamine injection into the median forebrain bundle. Results Rats were treated with intraperitoneal riluzole (4 mg/kg or 8 mg/kg), 1 hour before the lesion then once daily for seven days. Riluzole produced a modest but significant attenuation of dopamine neurone degeneration, assessed by suppression of amphetamine-induced rotations, preservation of tyrosine hydroxylase positive neuronal cell bodies in the substantia nigra pars compacta and attenuation of striatal tyrosine hydroxylase protein loss. Seven days after 6-hydroxydopamine lesion, reactive astrocytosis was observed in the striatum, as determined by increases in expression of glial fibrillary acidic protein, however the glutamate transporter, GLT-1, which is also expressed in astrocytes was not regulated by the lesion. Conclusions The results confirm that riluzole is a neuroprotective agent in a rodent model of parkinson’s disease. Riluzole administration did not regulate GLT-1 levels but significantly reduced GFAP levels, in the lesioned striatum. Riluzole suppression of reactive astrocytosis is an intriguing finding which might contribute to the neuroprotective effects of this drug.

Purified galactooligosaccharide, derived from a mixture produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium adhesion and invasion in vitro and in vivo

The prebiotic Bimuno (R) is a mixture containing galactooligosaccharides (GOSs), produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 411 71 using lactose as the substrate Previous in vivo and in vitro studies demonstrating the efficacy of Bimuno (R) in reducing Salmonella enterica serovar Typhimurium (S Typhimurium) colonization did not ascertain whether or not the protective effects could be attributed to the prebiotic component GOS Here we wished to test the hypothesis that GOS, derived from Bimuno (R) may confer the direct anti-invasive and protective effects of Bimuno (R) In this study the efficacy of Bimuno (R), a basal solution of Bimuno (R) without GOS [which contained glucose, galactose, lactose, maltodextrin and gum arabic in the same relative proportions (w/w) as they are found in Bimuno (R)] and purified GOS to reduce S Typhimurium adhesion and invasion was assessed using a series of in vitro and in vivo models The novel use of three dimensionally cultured HT-29-16E cells to study prebiotics in vitro demonstrated that the presence of similar to 5 mg Bimuno (R) ml(-1) or similar to 2 5 mg GOS ml(-1) significantly reduced the invasion of S Typhimurium (SL1344nal(r)) (P<0 0001) Furthermore, similar to 2 5 mg GOS ml(-1) significantly reduced the adherence of S Typhimurium (SU 344nal(r)) (P<0 0001) It was demonstrated that cells produced using this system formed multi-layered aggregates of cells that displayed excellent formation of brush borders and tight junctions In the murine ligated deal gut loops, the presence of Bimuno (R) or GOS prevented the adherence or invasion of S Typhimurium to enterocytes, and thus reduced its associated pathology This protection appeared to correlate with significant reductions in the neutral and acidic mucins detected in goblet cells, possibly as a consequence of stimulating the cells to secrete the mucin into the lumen In all assays, Bimuno (R) without GOS conferred no such protection, indicating that the basal solution confers no protective effects against S Typhimurium Collectively, the studies presented here clearly indicate that the protective effects conferred by Bimuno (R) can be attributed to GOS

Intracellular accumulation of high levels of gamma-aminobutyrate by Listeria monocytogenes 10403S in response to low pH: uncoupling of gamma-aminobutyrate synthesis from efflux in a chemically defined medium

It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular gamma-aminobutyrate (GABA(i)). The glutamate is then decarboxylated to GABA(i), a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA(e)) in response to acidification. In addition, high levels of GABA(i) (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA(i) in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA(i). These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.

Characterisation of the intracellular-glutamate decarboxylase system (GAD): analysis of its function, transcription and role in the acid resistance of various strains of Listeria monocytogenes

The glutamate decarboxylase (GAD) system is important for the acid resistance of Listeria monocytogenes. We previously showed that under acidic conditions, glutamate (Glt)/γ-aminobutyrate (GABA) antiport is impaired in minimal media but not in rich ones, like brain heart infusion. Here we demonstrate that this behavior is more complex and it is subject to strain and medium variation. Despite the impaired Glt/GABA antiport, cells accumulate intracellular GABA (GABA(i)) as a standard response against acid in any medium, and this occurs in all strains tested. Since these systems can occur independently of one another, we refer to them as the extracellular (GAD(e)) and intracellular (GAD(i)) systems. We show here that GAD(i) contributes to acid resistance since in a ΔgadD1D2 mutant, reduced GABA(i) accumulation coincided with a 3.2-log-unit reduction in survival at pH 3.0 compared to that of wild-type strain LO28. Among 20 different strains, the GAD(i) system was found to remove 23.11% ± 18.87% of the protons removed by the overall GAD system. Furthermore, the GAD(i) system is activated at milder pH values (4.5 to 5.0) than the GAD(e) system (pH 4.0 to 4.5), suggesting that GAD(i) is the more responsive of the two and the first line of defense against acid. Through functional genomics, we found a major role for GadD2 in the function of GAD(i), while that of GadD1 was minor. Furthermore, the transcription of the gad genes in three common reference strains (10403S, LO28, and EGD-e) during an acid challenge correlated well with their relative acid sensitivity. No transcriptional upregulation of the gadT2D2 operon, which is the most important component of the GAD system, was observed, while gadD3 transcription was the highest among all gad genes in all strains. In this study, we present a revised model for the function of the GAD system and highlight the important role of GAD(i) in the acid resistance of L. monocytogenes.

Survival of freeze dried Lactobacillus plantarum in instant fruit powders and reconstituted fruit juices

The aim of this study was to investigate the survival of freeze dried Lactobacillus plantarum cells mixed with several freeze dried instant fruit powders (strawberry, pomegranate, blackcurrant and cranberry) during storage for 12 months as well as after reconstitution with water each month. Inulin and gum arabic were also added to the instant fruit powders at two levels (10% and 20% w/w of dry weight) to improve the cell survival and functional properties of the product. The best cell survival over the 12 months of storage was observed for the blackcurrant powder (almost no decrease) followed by strawberry (~ 0.3 log decrease), pomegranate (~ 0.9 log decrease), whereas the worst survival was obtained in cranberry powder (~ 4.5 logs). To explain these results multiple regression analysis was conducted with the log decrease [log10N0 month − log10N12 months] as the dependent variable and water activity, pH, citric acid, dietary fibre and total phenol as the independent variables. The results indicated that among all the examined factors, the [log10N0 month − log10N12 months] depended only on the water activity (P < 0.05). Inulin and gum arabic demonstrated a substantial protective effect on cell survival (1–1.5 log) in the case of cranberry, which was likely due to a physical interaction between the cells and the carbohydrates. After reconstituting the dried fruit powders at room temperature and measuring cell viability for up to 4 h, it was shown that in the case of strawberry juice there was no decrease, and very little in the case of pomegranate and blackcurrant juices (< 0.5 log). On the other hand, a significant decrease was observed for cranberry juice (P < 0.05), which increased as the storage time of the dried cranberry powder increased, indicating that the cells became more susceptible with prolonged storage. Multiple regression analysis indicated that the main factors influencing cell survival were water activity and pH, while citric acid, dietary fibre and total phenol did not have an effect. Furthermore, inulin and gum arabic addition did not have a significant (P > 0.05) effect upon reconstitution of the dried fruit powder. This study showed that instant juice powders are very good carriers of probiotic cells and constitute good alternatives to highly acidic fruit juices.

Endothelin-converting enzyme-1 degrades internalized somatostatin-14

Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with beta-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized (125)I-Tyr(11)-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized (125)I-Tyr(1)-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A(1). ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

N-(Aryl)picolinamide complexes of rhodium : synthesis, structure and, spectral and electrochemical properties

Reaction of a series of N-(aryl)picolinamide ligands (HL-R, where II denotes the acidic proton and R (R = OCH3, CH3, H, Cl and NO2) is the para substituent in the aryl fragment) with RhCl3 center dot 3H(2)O in refluxing ethanal in the presence of a base (NEt3) affords two groups of yellow complexes of type [Rh(H-R)(L-R)Cl-2] and [Rh(L-R)(2)(H2O)Cl]. In [Rh(HL-R)(L-R)Cl-2], HL-R is coordinated as neutral N,O-donor and L-R as monoanionic N,N-donor, and the two chlorides are mutually trans. In [Rh(L-R)(2)(H2O)CI] both the amide ligands are coordinated as monoanionic N,N-donor, and the chloro and aquo ligands are mutually cis. Structures of the [Rh(HL-OCH3)(L-CH3)Cl-2] and [Rh(L-Cl)(2)(H2O)CI] complexes have been determined by X-ray crystallography. All the complexes show characteristic H-1 NMR signals and intense LLCT transitions in the ultraviolet region. Cyclic voltammetry on the complexes shows an oxidation of the coordinated amide ligand within 0.78-1.80 V vs SCE and a reductive response within -0.20 to -0.75 V vs SCE. DFT calculations have been done to explain the electronic spectral and electrochemical properties.

Syntheses, structures and efficient catalysis for C–C coupling of some benzaldehyde thiosemicarbazone complexes of palladium

Reaction of the 4-R-benzaldehyde thiosemicarbazones (denoted in general as L-R; R = OCH(3), CH(3), H, Cl and NO(2)) with trans-[Pd(PPh(3))(2)Cl(2)] afforded a group of mixed-ligand complexes (denoted in general as 1-R) incorporating a N,S-coordinated thiosemicarbazone. a triphenylphosphine and a chloride. Similar reaction with Na(2)[PdCl(4)] afforded a family of bis-thiosemicarbazone complexes (denoted in general as 2-R), where each ligand is N,S-coordinated. Crystal structures of 1-CH(3), 1-NO(2), 2-OCH(3), 2-NO(2) and L-NO(2) have been determined. In all the complexes the thiosemicarbazones are coordinated to the metal center, via dissociation of the acidic proton, as bidentate N,S-donors forming five-membered chelate rings. With reference to the structure of the uncoordinated thiosemicarbazone, this coordination mode is associated with a conformational change around the C=N bond. All the 1-R and 2-R complexes display intense absorptions in the visible region. Catalytic activity of the 1-R and 2-R complexes towards some C-C coupling reactions (e.g. Suzuki, Heck and Sonogashira) has been examined and while both are found to be efficient catalysts, 1-R is much better catalyst than 2-R.