Synthesis of alpha-galactooligosaccharides with alpha-galactosidase from Lactobacillus reuteri of canine origin

Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities. When raffinose was used as the carbon source, alpha-galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase. A 64 kDa enzyme was purified by ultra- and gel filtration, and characterized for its hydrolytic and synthetic activity. Highest hydrolytic activity was found at pH 5.0 at 50 degreesC (K-M 0.55 mM, V-max 0.80 mumol min(-1) mg(-1) of protein). The crude cell-free extract was further used in glycosyl transfer reactions to synthesize oligosaccharides from melibiose and raffinose. At a substrate concentration of 23% (w/v) oligosaccharide mixtures were formed with main products being a trisaccharide at 26% (w/w) yield from melibiose after 8 h and a tetrasaccharide at 18% (w/w) yield from raffinose after 7 h. Methylation analysis revealed the trisaccharide to be 6' alpha-galactosyl melibiose and the tetrasaccharide to be stachyose. In both cases synthesis ceased when hydrolysis of the substrate reached 50%.

The reaction of flavonoid metabolites with peroxynitrite

There is much interest in the bioactivity of in vivo flavonoid metabolites. We report for the first time the hierarchy of reactivity of flavonoid metabolites with peroxynitrite and characterise novel reaction products. O-Methylation of the B-ring catechol containing flavonoids epicatechin and quercetin, and O-glucuronidation of all flavonoids reduced their reactivity with peroxynitrite. The reaction of the flavanones hesperetin and naringenin and their glucuronides resulted in the formation of multiple mono-nitrated and nitrosated products. In contrast, the catechol-containing flavonoids epicatechin and quercetin yielded oxidation products which when trapped with glutathione led to the production of glutathionyl-conjugates. However, the O-methylated metabolites of epicatechin yielded both mono-and di-nitrated products and nitrosated metabolites. The 3'-O-methyl metabolite of quercetin also yielded a nitrosated species, although its counterpart 4'-O-methyl quercetin yielded only oxidation products. Such products may represent novel metabolic products in vivo and may also express cellular activity. (c) 2006 Elsevier Inc. All rights reserved.

Pectins and pectic-oligosaccharides inhibit Escherichia coli O157 : H7 Shiga toxin as directed towards the human colonic cell line HT29

Pectins and pectic-oligosaccharides, as derived by controlled enzymatic hydrolysis, were evaluated for their ability to interfere with the toxicity of Shiga-like toxins from Escherichia coli O157:H7. Both types of material resulted in some degree of protection but this was significantly higher (P > 0.01) with the oligosaccharide fractions (giving 90-100% cell survival, compared to 70-80% with the polymer). An effect of methylation on the protective effect was detected with lower degrees being more active. The pectic-oligosaccharides and galabiose, the minimum toxin receptor analogue, were shown to inhibit toxicity and were both protective at 10 mg ml(-1), but not at lower concentrations. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Synthesis of galacto-oligosaccharide from lactose using beta-galactosidase from Kluyveromyces lactis: studies on batch and continuous UF membrane-fitted bioreactors

A study of galacto-oligosaccharides (GOS) synthesis from lactose with beta-galactosidase from Kluyveromyces lactis (Maxilact(R) L2000) was carried out. The synthesis was performed using various initial lactose concentrations ranging from 220 to 400 mg/mL and enzyme concentrations ranging from 3 to 9 U/mL, and was investigated at 40degreesC and pH 7, in a stirred-tank reactor. In the experimental range examined, the results showed the amount of GOS formed depended on lactose concentration but not on enzyme concentration. Galactose was a competitive inhibitor, while glucose was a non-competitive inhibitor. In a further study, a laboratory-scale reactor system, fitted with a 10-kDa NMWCO composite regenerated cellulose membrane, was used in a continuous process. The reactor was operated in cross-flow mode. The effect of operating pressures on flux and productivity was investigated by applying different transmembrane pressures to the system. The continuous process showed better production performance compared to the batch synthesis with the same lactose and enzyme concentrations at 40degreesC, pH 7. Comparison of product structures from batch and continuous processes, analyzed by HPAEPAD and methylation analysis, showed similarities but differed from the structures found in a commercial GOS product (Vivinal(R)GOS). (C) 2004 Wiley Periodicals, Inc.

Regioselective synthesis of mannobiose and mannotriose by reverse hydrolysis using a novel 1,6-alpha-D-mannosidase from Aspergillus phoenicis

A novel 1,6-alpha-D-mannosidase was produced by Aspergillus phoenicis grown on a commercial manno-oligosaccharide preparation in liquid culture. The enzyme hydrolysed only alpha-D-Manp-(1 --> 6)-D-Manp and did not act on alpha-D-Manp-(1 --> 2)-D-Manp, or alpha-D-Manp-(1 --> 3)-D-Manp. The 1,6-alpha-D-mannosidase was used for synthesis of manno-oligosaccharides by reverse hydrolysis reaction. The highest yields, expressed as percentages (w/w) of total sugar, were similar to21% mannobiose and similar to5% mannotriose, and they were obtained with 45% (w/w) initial mannose concentration at pH 4.5 after 12 days incubation at 55 degreesC. The disaccharide and trisaccharide products were separated and their structures determined by methylation analysis. Only 1-6 linkages were found in both of them. (C) 2003 Elsevier B.V. All rights reserved.

Asymmetric synthesis using catalysts containing multiple stereogenic centres and a trans-1,2-diaminocyclohexane core; reversal of predominant enantioselectivity upon N-alkylation

N-Methylation of ligands containing a trans-1,2-diaminocyclohexane core and multiple stereogenic centres is shown to provide the product of the opposite configuration in significant enantiomeric excess, in the addition of diethylzinc to aldehydes. Some of the ligands were effective in an asymmetric Michael addition. (C) 2004 Elsevier Ltd. All rights reserved.

Analysis of chain and blood group type and branching pattern of sialylated oligosaccharides by negative ion electrospray tandem mass spectrometry

We previously reported sequence determination of neutral oligosaccharides by negative ion electrospray tandem mass spectrometry on a quadrupole-orthogonal time-of-flight instrument with high sensitivity and without the need of derivatization. In the present report, we extend our strategies to sialylated oligosaccharides for analysis of chain and blood group types together with branching patterns. A main feature in the negative ion mass spectrometry approach is the unique double glycosidic cleavage induced by 3-glycosidic substitution, producing characteristic D-type fragments which can be used to distinguish the type 1 and type 2 chains, the blood group related Lewis determinants, 3,6-disubstituted core branching patterns, and to assign the structural details of each of the branches. Twenty mono- and disialylated linear and branched oligosaccharides were used for the investigation, and the sensitivity achieved is in the femtomole range. To demonstrate the efficacy of the strategy, we have determined a novel complex disialylated and monofucosylated tridecasaccharide that is based on the lacto-N-decaose core. The structure and sequence assignment was corroborated by :methylation analysis and H-1 NMR spectroscopy.

Expressions and activities of cell cycle regulatory molecules during the transition from myocyte hyperplasia to hypertrophy

The role of cell cycle dependent molecules in controlling the switch from cardiac myocyte hyperplasia to hypertrophy remains unclear, although in the rat this process occurs between day 3 and 4 after birth. In this study we have determined (1) cell cycle profiles by fluorescence activated cell sorting (FACS); and (2) expressions, co-expressions and activities of a number of cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting andin vitrokinase assays in freshly isolated rat cardiac myocytes obtained from 2, 3, 4 and 5-day-old animals. The percentage of myocytes found in the S phase of the cell cycle decreased significantly during the transition from hyperplasia to hypertrophy (5.5, 3.5, 2.3 and 1.9% of cells in 2-, 3-, 4- and 5-day-old myocytes, respectively,P<0.05), concomitant with a significant increase in the percentage of G0/G1phase cells. At the molecular level, the expressions and activities of G1/S and G2/M phase acting cyclins and CDKs were downregulated significantly during the transition from hyperplasia to hypertrophy, whereas the expressions and activities of G1phase acting cyclins and CDKs were upregulated significantly during this transition. In addition, p21CIP1- and p27KIP1- associated CDK kinase activities remained relatively constant when histone H1 was used as a substrate, whereas phosphorylation of the retinoblastoma protein was upregulated significantly during the transition from hyperplasia to hypertrophy. Thus, there is a progressive and significant G0/G1phase blockade during the transition from myocyte hyperplasia to hypertrophy. Whilst CDK2 and cdc2 may be pivotal in the withdrawal of cardiac myocytes from the cell cycle, CDK4 and CDK6 may be critical for maintaining hypertrophic growth of the myocyte during development.

Progressive erosion of genetic and epigenetic variation in callus-derived cocoa (Theobroma cacao) plants

Relatively little is known about the timing of genetic and epigenetic forms of somaclonal variation arising from callus growth. We surveyed for both types of change in cocoa (Theobroma cacao) plants regenerated from calli of various ages, and also between tissues from the source trees. For genetic change, we used 15 single sequence repeat (SSR) markers from four source trees and from 233 regenerated plants. For epigenetic change, we used 386 methylation-sensitive amplified polymorphism (MSAP) markers on leaf and explant (staminode) DNA from two source trees and on leaf DNA from 114 regenerants. Genetic variation within source trees was limited to one slippage mutation in one leaf. Regenerants were far more variable, with 35% exhibiting at least one mutation. Genetic variation initially accumulated with culture age but subsequently declined. MSAP (epigenetic) profiles diverged between leaf and staminode samples from source trees. Multivariate analysis revealed that leaves from regenerants occupied intermediate eigenspace between leaves and staminodes of source plants but became progressively more similar to source tree leaves with culture age. Statistical analysis confirmed this rather counterintuitive finding that leaves of ‘late regenerants’ exhibited significantly less genetic and epigenetic divergence from source leaves than those exposed to short periods of callus growth.

Metabolism of tea flavonoids in the gastrointestinal tract

There is considerable interest in the bioavailability of flavan-3-ols such as tea catechins and their bioactivity in vivo. Although flavanols such as catechin and epicatechin have long been characterized as powerful antioxidants in vitro, evidence suggests that these compounds undergo significant metabolism and conjugation during absorption in the small intestine and in the colon. In the small intestine these modifications lead primarily to the formation of glucuronide conjugates that are more polar than the parent flavanol and are marked for renal excretion. Other phase II processes lead to the production of O-methylated forms that have reduced antioxidant potential via the methylation of the B-ring catechol. Significant modification of flavanols also occurs in the colon where the resident microflora degrade them to smaller phenolic acids, some of which may be absorbed. Cell, animal and human studies have confirmed such metabolism by the detection of flavanol metabolites in the circulation and tissues. This review will highlight the major sites of flavanol metabolism in the gastrointestinal tract and the processes that give rise to potential bioactive forms of flavan-3-ols in vivo.

N-nitroso compound exposure-associated transcriptomic profiles are indicative of an increased risk for colorectal cancer

Endogenous formation of N-nitroso compounds (NOCs), which are known animal carcinogens, could contribute to human carcinogenesis but definitive evidence is still lacking. To investigate the relevance of NOCs in human colorectal cancer (CRC) development, we analyzed whole genome gene expression modifications in human colon biopsies in relation to fecal NOC exposure. We had a particular interest in patients suffering from intestinal inflammation as this may stimulate endogenous NOC formation, and consequently predispose to CRC risk. Inflammatory bowel disease (IBD) patients diagnosed with ulcerative colitis and irritable bowel syndrome patients without inflammation, serving as controls, were therefore recruited. Fecal NOC were demonstrated in the majority of subjects. By associating gene expression levels of all subjects to fecal NOC levels, we identified a NOC exposure-associated transcriptomic response that suggests that physiological NOC concentrations may potentially induce genotoxic responses and chromatin modifications in human colon tissue, both of which are linked to carcinogenicity. In a network analysis, chromatin modifications were linked to 11 significantly modulated histone genes, pointing towards a possible epigenetic mechanism that may be relevant in comprehending NOC-induced carcinogenesis. In addition, pro-inflammatory transcriptomic modifications were identified in visually non-inflamed regions of the IBD colon. However, fecal NOC levels were slightly but not significantly increased in IBD patients, suggesting that inflammation did not strongly stimulate NOC formation. We conclude that NOC exposure is associated with gene expression modifications in the human colon that may suggest a potential role of these compounds in CRC development.

The impact of catechol-O-methyltransferase genotype on the acute responsiveness of vascular reactivity to a green tea extract

The beneficial effects of green tea catechins, such as the proposed improvement in endothelial function, may be influenced by phase II metabolism during and after absorption. The methylation enzyme, catechol-O-methyltransferase (COMT), has a missense mutation rs4680 (G to A), proposed to result in a 40 % reduction in enzyme activity. In the present pilot study, twenty subjects (ten of each homozygous COMT genotype) were recruited. Green tea extract capsules (836 mg green tea catechins) were given in a fasted state, and a high-carbohydrate breakfast was given after 60 min. Blood samples and vascular function measurements were taken at regular intervals. The change in digital volume pulse stiffness index (SI) from baseline was shown to be different between genotype groups at 120 and 240 min, with a lower SI in the GG individuals (P ≤ 0·044). The change in blood pressure from baseline also differed between genotype groups, with a greater increase in systolic (P = 0·023) and diastolic (P = 0·034) blood pressure at 120 min in the GG group. The AA group was shown to have a greater increase in insulin concentrations at 120 min (P = 0·019) and 180 min (P = 0·008) compared with baseline, despite similar glucose profiles. No genotypic differences were found in vascular reactivity measured using laser Doppler iontophoresis, total nitrite, lipids, plasma total antioxidant capacity or inflammatory markers after ingestion of the green tea extract. In conclusion, SI and insulin response to the glucose load differed between the COMT genotype groups, and this may be suggestive of a green tea extract and genotype interaction.

A preliminary investigation of the impact of catechol-O-methyltransferase genotype on the absorption and metabolism of green tea catechins

Purpose Green tea is thought to possess many beneficial effects on human health. However, the extent of green tea polyphenol biotransformation may affect its proposed therapeutic effects. Catechol-O-methyltransferase (COMT), the enzyme responsible for polyphenolic methylation, has a common polymorphism in the genetic code at position 158 reported to result in a 40% reduction in enzyme activity in in vitro studies. The current preliminary study was designed to investigate the impact of COMT genotype on green tea catechin absorption and metabolism in humans. Methods Twenty participants (10 of each homozygous COMT genotype) were recruited, and plasma concentration profiles were produced for epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) and 4′-O-methyl EGCG after 1.1 g of Sunphenon decaffeinated green tea extract (836 mg green tea catechins), with a meal given after 60 min. Results For the entire group, EGCG, EGC, EC, ECG and 4′-O-methyl EGCG reached maximum concentrations of 1.09, 0.41, 0.33, 0.16 and 0.08 μM at 81.5, 98.5, 99.0, 85.5 and 96.5 min, respectively. Bimodal curves were observed for the non-gallated green tea catechins EGC and EC as opposed to single-peaked curves for the gallated green tea catechins EGCG and ECG. No significant parametric differences between COMT genotype groups were found. Conclusions In conclusion, the COMT Val(158/108)Met does not appear to have a dramatic influence on EGCG absorption and elimination. However, further pharmacokinetic research is needed to substantiate these findings.

Fluorescence lifetime imaging microscopy (FLIM) to demonstrate the nuclear binding of flavanols and (--epigallocatechin gallate

The use of light microscopy and DMACA staining strongly suggested that plant and animal cell nuclei act as sinks for flavanols [1, 2]. Detailed uv-vis spectroscopic titration experiments indicated that histone proteins are the likely binding sites in the nucleus [2]. Here we report the development of a multi-photon excitation microscopy technique combined with fluorescent lifetime measurements of flavanols. Using this technique, (+) catechin, (-) epicatechin and (-) epigallocatechin gallate (EGCG) showed strikingly different excited state lifetimes in solution. Interaction of histone proteins with flavanols was indicated by the appearance of a significant τ2-component of 1.7 to 4.0ns. Tryptophan interference could be circumvented in the in vivo fluorescence lifetime imaging microscopy (FLIM) experiments with 2-photon excitation at 630nm. This enabled visualisation and semi-quantitative measurements that demonstrated unequivocally the absorption of (+)catechin, (-)epicatechin and EGCG by nuclei of onion cells. 3D FLIM revealed for the first time that externally added EGCG penetrated the whole nucleus in onion cells. The relative proportions of EGCG in cytoplasm: nucleus: nucleoli were ca. 1:10:100. FLIM experiments may therefore facilitate probing the health effects of EGCG, which is the major constituent of green tea.

Analysis of time-related metabolic fluctuations induced by ethionine in the rat

The time-course of metabolic events following response to a model hepatotoxin ethionine (800 mg/kg) was investigated over a 7 day period in rats using high-resolution (1)H NMR spectroscopic analysis of urine and multivariate statistics. Complementary information was obtained by multivariate analysis of (1)H MAS NMR spectra of intact liver and by conventional histopathology and clinical chemistry of blood plasma. (1)H MAS NMR spectra of liver showed toxin-induced lipidosis 24 h postdose consistent with the steatosis observed by histopathology, while hypertaurinuria was suggestive of liver injury. Early biochemical changes in urine included elevation of guanidinoacetate, suggesting impaired methylation reactions. Urinary increases in 5-oxoproline and glycine suggested disruption of the gamma-glutamyl cycle. Signs of ATP depletion together with impairment of the energy metabolism were given from the decreased levels in tricarboxylic acid cycle intermediates, the appearance of ketone bodies in urine, the depletion of hepatic glucose and glycogen, and also hypoglycemia. The observed increase in nicotinuric acid in urine could be an indication of an increase in NAD catabolism, a possible consequence of ATP depletion. Effects on the gut microbiota were suggested by the observed urinary reductions in the microbial metabolites 3-/4-hydroxyphenyl propionic acid, dimethylamine, and tryptamine. At later stages of toxicity, there was evidence of kidney damage, as indicated by the tubular damage observed by histopathology, supported by increased urinary excretion of lactic acid, amino acids, and glucose. These studies have given new insights into mechanisms of ethionine-induced toxicity and show the value of multisystem level data integration in the understanding of experimental models of toxicity or disease.