Prebiotic evaluation of a novel galactooligosaccharide mixture produced by the enzymatic activity of Bifidobacterium bifidum NCIMB 41171, in healthy humans: a randomized, double-blind, crossover, placebo-controlled intervention study

Background: Galactooligosaccharides are selectively fermented by the beneficial member of the colonic microflora contributing to the health of the host. Objective: We assessed the prebiotic potential of a novel galactooligosaccharide produced through the action of beta-galactosidases, originating from a probiotic Bifidobacterium bifidum strain, against a galactooligosaccharide produced through the action of an industrial P-galactosidase and a placebo. Design: Fifty-nine healthy human volunteers participated in this study. Initially, the effect of the matrix on the prebiotic properties of a commercially available galactooligosaccharide (7 g/d) was assessed during 7-d treatment periods with a 7-d washout period in between. During the second phase, 30 volunteers were assigned to a sequence of treatments (7 d) differing in the amount of the novel galactooligosaccharide (0, 3.6, or 7 g/d). Stools were recovered before and after each intervention, and bacteria numbers were determined by fluorescent in situ hybridization. Results: Addition of the novel galactooligosaccharide mixture significantly increased the bifidobacterial population ratio compared with the placebo (P < 0.05), whereas 7 g/d of the novel galactooligosaccharide significantly increased the bifidobacterial ratio compared with the commercial galactooligosaccharide (P < 0.05). Moreover, a significant relation (P < 0.001) between the bifidobacteria proportion and the novel galactooligosaccharide dose (0, 3.6, and 7 g/d) was observed. This relation was similar to the effect of the novel galactooligosaccharide on the prebiotic index of each dose. Conclusions: This study showed that galactooligosaccharide mixtures produced with different beta-galactosidases show different prebiotic properties and that, by using enzymes originating from bifidobacterial species, an increase in the bifidogenic properties of the prebiotic product is achievable.

PEGylated amyloid peptide nanocontainer delivery and release system

A micellar nanocontainer delivery and release system is designed on the basis of a peptide-polymer conjugate. The hybrid molecules self-assemble into micelles comprising a modified amyloid peptide core surrounded by a PEG corona. The modified amyloid peptide previously studied in our group forms helical ribbons based on a beta-sheet motif and contains beta-amino acids that are excluded from the beta-sheet structure, thus being potentially useful as fibrillization inhibitors. In the model peptide-PEG hybrid system studied, enzymatic degradation using alpha-chymotrypsin leads to selective cleavage close to the PEG-peptide linkage, break up of the micelles, and release of peptides in unassociated form. The release of monomeric peptide is useful because aggregation of the released peptide into beta-sheet amyloid fibrils is not observed. This concept has considerable potential in the targeted delivery of peptides for therapeutic applications.

Multistep electrochemical reduction path of clusters [Os3(CO)10(α-diimine)]: comparison of electrochemical and photochemical Os-Os(α-diimine) bond cleavage

Electrochemical reduction of the triangular clusters [Os-3(CO)(10)(alpha-dimine)] (alpha-dimine = 2,2'-bipyridine (bpy), 2,2'-bipyrimidine (bpym)) and [Os-3(CO)(10)(mu-bpym) ReBr(CO)(3)] produces primarily the corresponding radical anions. Their stability is strongly determined by the pi acceptor ability of the reducible alpha-dimine ligand, which decreases in the order mu-bpym > bpym >> bpy. Along this series, increasing delocalisation of the odd electron density in the radical anion over the Os(alpha-dimine) chelate ring causes weakening of the axial (CO)(4)Os-Os(CO)(2)(alpha-dimine) bond and its facile cleavage for alpha-diimine = bpy. In contrast, the cluster radical anion is inherently stable for the bridging bpym ligand, the strongest pi-acceptor in the studied series. In the absence of the partial delocalisation of the unpaired electron over the Re( bpym) chelate bond, the Os-3-core of the radical anion remains intact only at low temperatures. Subsequent one-electron reduction of [Os-3(CO)(10)(bpym)](center dot-) at T = 223 K gives the open-triosmium core (= Os-3*) dianion, [Os-3*(CO)(10)(bpym)](2-). Its oxidation leads to the recovery of parent [Os-3(CO)(10)( bpym)]. At room temperature, [Os-3*( CO)(10)(bpym)](2-) is formed along a two-electron (ECE) reduction path. The chemical step (C) results in the formation of an open- core radical anion that is directly reducible at the cathodic potential of the parent cluster in the second electrochemical (E) step. In weakly coordinating tetrahydrofuran, [Os-3*(CO)(10)( bpym)](2-) rapidly attacks yet non- reduced parent cluster molecules, producing the relatively stable open- core dimer [Os-3*(CO)(10)(bpym)](2)(2-) featuring two open- triangle cluster moieties connected with an ( bpym) Os - Os( bpym) bond. In butyronitrile, [Os-3*( CO)(10)(bpym)](2-) is stabilised by the solvent and the dimer [Os-3*(CO)(10)(bpym)](2)(2-) is then mainly formed by reoxidation of the dianion on reverse potential scan. The more reactive cluster [Os-3(CO)(10)(bpy)] follows the same reduction path, as supported by spectroelectrochemical results and additional valuable evidence obtained from cyclic voltammetric scans. The ultimate process in the reduction mechanism is fragmentation of the cluster core triggered by the reduction of the dimer [Os-3*(CO)(10)(alpha- diimine)](2)(2-). The products formed are [Os-2(CO)(8)](2-) and {Os(CO)(2)(alpha- diimine)}(2). The latter dinuclear fragments constitute a linear polymeric chain [Os( CO)(2)(alpha-dimine)] n that is further reducible at the alpha-dimine ligands. For alpha-dimine = bpy, the charged polymer is capable of reducing carbon dioxide. The electrochemical opening of the triosmium core in the [Os-3( CO)(10)(alpha-dimine)] clusters exhibits several common features with their photochemistry. The same Os-alpha-dimine bond dissociates in both cases but the intimate mechanisms are different.

Coordination behavior of pyridylmethylthioether system with cupric chloride and cupric bromide: CS bond cleavage and crystal structures

The coordination behavior of pyridylmethylthioether type of organic moieties having N2S2 donor set [L-1=1,2-bis(2-pyridylmethylthio)ethane, L-2 = 1,3-bis(2-pyridylmethyl-thio)propane and L-3 = 1,4-bis(2-pyridylmethylthio)butane] with copper(II) chloride and copper(II) bromide have been studied in different chemical environments. Copper(II) chloride assisted C-S bond cleavage of the organic moieties leading to the formation of copper(II) picolinate derivatives, whereas, under similar experimental conditions, no C-S bond cleavage was observed in the reaction with copper(II) bromide. The resulted copper(II) complexes isolated from the different mediums have been characterized by spectroscopic and X-ray crystallographic tools.

Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process

The overall aim of this work was to characterize the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilization of β-lactoglobulin and casein derived peptides (CDP) from whey. The adsorbed proteins were hydrolyzed in-situ which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC50 =287μg/mL) and (ii) from β-lactoglobulin (IC50=128μg/mL). IC50 is the concentration of inhibitor needed to inhibit ACE by half. The well known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterized in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC50).

AID enzymatic activity is inversely proportional to the size of cytosine C5 orbital cloud

Activation induced deaminase (AID) deaminates cytosine to uracil, which is required for a functional humoral immune system. Previous work demonstrated, that AID also deaminates 5-methylcytosine (5 mC). Recently, a novel vertebrate modification (5-hydroxymethylcytosine - 5 hmC) has been implicated in functioning in epigenetic reprogramming, yet no molecular pathway explaining the removal of 5 hmC has been identified. AID has been suggested to deaminate 5 hmC, with the 5 hmU product being repaired by base excision repair pathways back to cytosine. Here we demonstrate that AID’s enzymatic activity is inversely proportional to the electron cloud size of C5-cytosine - H . F . methyl .. hydroxymethyl. This makes AID an unlikely candidate to be part of 5 hmC removal.

A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells

The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde.

Design, synthesis and in vitro degradation of a novel co-drug for the treatment of psoriasis

Psoriasis is a common, chronic and relapsing inflammatory skin disease. It affects approximately 2% of the western population and has no cure. Combination therapy for psoriasis often proves more efficacious and better tolerated than monotherapy with a single drug. Combination therapy could be administered in the form of a co-drug, where two or more therapeutic compounds active against the same condition are linked by a cleavable covalent bond. Similar to the pro-drug approach, the liberation of parent moieties post-administration, by enzymatic and/or chemical mechanisms, is a pre-requisite for effective treatment. In this study, a series of co-drugs incorporating dithranol in combination with one of several non-steroidal anti-inflammatory drugs, both useful for the treatment of psoriasis, were designed, synthesized and evaluated. An ester co-drug comprising dithranol and naproxen in a 1:1 stoichiometric ratio was determined to possess the optimal physicochemical properties for topical delivery. The co-drug was fully hydrolyzed in vitro by porcine liver esterase within four hours. When incubated with homogenized porcine skin, 9.5% of the parent compounds were liberated after 24 h, suggesting in situ esterase-mediated cleavage of the co-drug would occur within the skin. The kinetics of the reaction revealed first order kinetics, Vmax = 10.3 μM/min and Km = 65.1 μM. The co-drug contains a modified dithranol chromophore that was just 37% of the absorbance of dithranol at 375 nm and suggests reduced skin/clothes staining. Overall, these findings suggest that the dithranol-naproxen co-drug offers an attractive, novel approach for the treatment of psoriasis.

Zinc chelatase in human lymphocytes: detection of the enzymatic defect in erythropoietic protoporphyria

We describe a fluorometric assay for heme synthetase, the enzyme that is genetically deficient in erythropoietic protoporphyria. The method, which can readily detect activity in 1 microliter of packed human lymphocytes, is based on the formation of zinc protoheme from protoporphyrin IX. That zinc chelatase and ferrochelatase activities reside in the same enzyme was shown by the competitive action of ferrous ions and the inhibitory effects of N-methyl protoporphyrin (a specific inhibitor of heme synthetase) on zinc chelatase. The Km for zinc was 11 micrograms and that for protoporphyrin IX was 6 microM. The Ki fro ferrous ions was 14 microM. Zinc chelatase was reduced to 15.3% of the mean control activity in lymphocytes obtained from patients with protoporphyria, thus confirming the defect of heme biosynthesis in this disorder. The assay should prove to be useful for determining heme synthetase in tissues with low specific activity and to investigate further the enzymatic defect in protoporphyria.

Genetic heterogeneity in erythropoietic protoporphyria: a study of the enzymatic defect in nine affected families

Erythropoietic protoporphyria (EPP) is associated with a deficiency of protohaem ferrolyase. We have used a novel assay for this enzyme based on its ability to utilize zinc as a substrate to investigate the inheritance of EPP in nine affected families. Zinc chelatase activity was markedly reduced in peripheral blood mononuclear cells from 14 EPP patients (mean, 3.3 nmol Zn protohaem/h/mg protein; range, 0.3-8.0) when compared with 41 controls (16.8 +/- 3.6) p less than 0.01. In three families with parent-to-child transmission of disease, the asymptomatic parent had an enzymatic activity within the normal range. In three pedigrees where the parents were asymptomatic, enzymatic activities were below the 95% confidence limits in both. Zinc chelatase activity was below the mean control value in 17 of the 18 parents in nine affected pedigrees, and six of seven asymptomatic offspring of patients with protoporphyria. The findings suggest that EPP is not transmitted as a simple dominant trait and that inheritance of more than one gene may be required for disease expression.

Enhancing the recovery of tiger nut (Cyperus esculentus) oil by mechanical pressing: moisture content, particle size, high pressure and enzymatic pre-treatment effects

Tiger nut (Cyperus esculentus) tuber contains oil that is high in monounsaturated fatty acids, and this oil makes up about 23% of the tuber. The study aimed at evaluating the impact of several factors and enzymatic pre-treatment on the recovery of pressed tiger nut oil. Smaller particles were more favourable for pressing. High pressure pre-treatment did not increase oil recovery but enzymatic treatment did. The highest yield obtained by enzymatic treatment prior to mechanical extraction was 33 % on a dry defatted basis, which represents a recovery of 90 % of the oil. Tiger nut oil consists mainly of oleic acid; its acid and peroxide values reflect the high stability of the oil.

Edible oil from Tiger nut (Cyperus esculentus L.): mechanical pressing and aqueous enzymatic extraction methods

The tiger nut tuber of the Cyperus esculentus L. plant is an unusual storage system with similar amounts of starch and lipid. The extraction of its oil employing both mechanical pressing and aqueous enzymatic extraction (AEE) methods was investigated and an examination of the resulting products was carried out. The effects of particle size and moisture content of the tuber on the yield of tiger nut oil with pressing were initially studied. Smaller particles were found to enhance oil yields while a range of moisture content was observed to favour higher oil yields. When samples were first subjected to high pressures up to 700 MPa before pressing at 38 MPa there was no increase in the oil yields. Ground samples incubated with a mixture of α- Amylase, Alcalase, and Viscozyme (a mixture of cell wall degrading enzyme) as a pre-treatment, increased oil yield by pressing and 90% of oil was recovered as a result. When aqueous enzymatic extraction was carried out on ground samples, the use of α- Amylase, Alcalase, and Celluclast independently improved extraction oil yields compared to oil extraction without enzymes by 34.5, 23.4 and 14.7% respectively. A mixture of the three enzymes further augmented the oil yield and different operational factors were individually studied for their effects on the process. These include time, total mixed enzyme concentration, linear agitation speed, and solid-liquid ratio. The largest oil yields were obtained with a solid-liquid ratio of 1:6, mixed enzyme concentration of 1% (w/w) and 6 h incubation time although the longer time allowed for the formation of an emulsion. Using stationary samples during incubation surprisingly gave the highest oil yields, and this was observed to be as a result of gravity separation occurring during agitation. Furthermore, the use of high pressure processing up to 300 MPa as a pre-treatment enhanced oil yields but additional pressure increments had a detrimental effect. The quality of oils recovered from both mechanical and aqueous enzymatic extraction based on the percentage free fatty acid (% FFA) and peroxide values (PV) all reflected the good stabilities of the oils with the highest % FFA of 1.8 and PV of 1.7. The fatty acid profiles of all oils also remained unchanged. The level of tocopherols in oils were enhanced with both enzyme aided pressing (EAP) and high pressure processing before AEE. Analysis on the residual meals revealed DP 3 and DP 4 oligosaccharides present in EAP samples but these would require further assessment on their identity and quality.

Effect of enzymatic pretreatment and increasing the organic loading rate of lipid-rich wastewater treated in a hybrid UASB reactor

This study aimed at evaluating the effect of increasing organic loading rates and of enzyme pretreatment on the stability and efficiency of a hybrid upflow anaerobic sludge blanket reactor (UASBh) treating dairy effluent. The UASBh was submitted to the following average organic loading rates (OLR) 0.98 Kg.m(-3).d(-1), 4.58 Kg.m(-3).d(-1), 8.89 Kg.m(-3).d(-1) and 15.73 Kg.m(-3).d(-1), and with the higher value, the reactor was fed with effluent with and without an enzymatic pretreatment to hydrolyze fats. The hydraulic detention time was 24 h, and the temperature was 30 +/- 2 degrees C. The reactor was equipped with a superior foam bed and showed good efficiency and stability until an OLR of 8.89 Kg.m(-3).d(-1). The foam bed was efficient for solid retention and residual volatile acid concentration consumption. The enzymatic pretreatment did not contribute to the process stability, propitiating loss in both biomass and system efficiency. Specific methanogenic activity tests indicated the presence of inhibition after the sludge had been submitted to the pretreated effluent It was concluded that continuous exposure to the hydrolysis products or to the enzyme caused a dramatic drop in the efficiency and stability of the process, and the single exposure of the biomass to this condition did not inhibit methane formation. (C) 2011 Elsevier B.V. All rights reserved.

Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos

Objective: To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. Design: Comparison between oocyte groups and between early embryo stages. Setting: Laboratories of embryo manipulation and molecular biology from Departamento de Genetica (FMRP) and Departamento de Ciencias Basicas (FZEA) - University of Sao Paulo. Sample(s): Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. Main Outcome Measure(s): Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). Result(s): Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good-and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. Conclusion(s): Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development. (Fertil Steril (R) 2010; 93: 2507-12. (C) 2010 by American Society for Reproductive Medicine.)

Role of glutaredoxin 2 and cytosolic thioredoxins in cysteinyl-based redox modification of the 20S proteasome

The yeast 20S proteasome is subject to sulfhydryl redox alterations, such as the oxidation of cysteine residues (Cys-SH) into cysteine sulfenic acid (Cys-SOH), followed by S-glutathionylation (Cys-S-SG). Proteasome S-glutathionylation promotes partial loss of chymotrypsin-like activity and post-acidic cleavage without alteration of the trypsin-like proteasomal activity. Here we show that the 20S proteasome purified from stationary-phase cells was natively S-glutathionylated. Moreover, recombinant glutaredoxin 2 removes glutathione from natively or in vitro S-glutathionylated 20S proteasome, allowing the recovery of chymotrypsin-like activity and post-acidic cleavage. Glutaredoxin 2 deglutathionylase activity was dependent on its entry into the core particle, as demonstrated by stimulating S-glutathionylated proteasome opening. Under these conditions, deglutathionylation of the 20S proteasome and glutaredoxin 2 degradation were increased when compared to non-stimulated samples. Glutaredoxin 2 fragmentation by the 20S proteasome was evaluated by SDS-PAGE and mass spectrometry, and S-glutathionylation was evaluated by either western blot analyses with anti-glutathione IgG or by spectrophotometry with the thiol reactant 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. It was also observed in vivo that glutaredoxin 2 was ubiquitinated in cellular extracts of yeast cells grown in glucose-containing medium. Other cytoplasmic oxido-reductases, namely thioredoxins 1 and 2, were also active in 20S proteasome deglutathionylation by a similar mechanism. These results indicate for the first time that 20S proteasome cysteinyl redox modification is a regulated mechanism coupled to enzymatic deglutathionylase activity.