Flavonoids and the gastrointestinal tract: Local and systemic effects.

The gastrointestinal (GI) tract plays a central role in the absorption, distribution, metabolism, and excretion of flavonoids, which ultimately define the health effects of these bioactives. These aspects are modulated by the interactions of flavonoids with other dietary components, environmental factors, the host, and the GI microbiota. Flavonoid can target molecules in the luminal content, the different GI tract cell types, and the microbiota. Importantly, flavonoid actions at the GI tract can have an impact systemically, e.g. on glucose homeostasis, lipid and energy metabolism, or cardiovascular risk factors. The beneficial actions of flavonoids at the GI include their capacity to: i) protect the intestinal epithelium against pharmacological insults and food toxins; ii) modulate the activity of enzymes involved in lipid and carbohydrate absorption; iii) maintain the intestinal barrier integrity; iv) modulate the secretion of gut hormones; v) modulate the GI tract immune system; vi) exert potential anti-colorectal cancer activity; and vii) shape microbiota composition and function. Further understanding of the mechanisms mediating the effects of flavonoids on the intestine (and its microbiota) is of critical importance given the relevance of the GI tract on sustaining overall health and of the widespread recommendations of increasing the intake of plant bioactives.

Characterization of porcine milk oligosaccharides during early lactation and their relation to the fecal microbiome.

The composition of porcine milk oligosaccharides (PMO) was analyzed during early lactation and their relation to piglet gut microbiome was investigated. Pigs are considered ideal intestinal models to simulate humans because of the striking similarity in intestinal physiopathology to humans. The evolution of PMO was investigated in the milk from 3 healthy sows at prefarrowing, farrowing, and d 7 and 14 postpartum by Nano-LC Chip Quadrupole-Time-of-Flight mass spectrometer (Agilent Technologies, Santa Clara, CA). Previously sequenced metagenome libraries were reanalyzed to examine changes with specific gut bacterial populations. Over 30 oligosaccharides (OS) were identified in the milk, with 3'-sialyllactose, lacto-N-tetraose, α1-3,ß1-4-d-galactotriose, 2'-fucosyllactose, and 6'-sialyllactose being the most abundant species (accounting for ~70% of the total OS). Porcine milk had lower OS diversity (number of unique structures) than human milk, and appeared closer to bovine and caprine milk. In agreement with previous studies, only 3 fucosylated OS were identified. Surprisingly, their contribution to total OS abundance was greater than in bovine milk (9 vs. 1%). Indeed, fucosylated PMO increased during lactation, mirroring a similar trend observed for neutral and type I OS content during early lactation. Taken together, these results suggest that, in terms of abundance, PMO are closer to human milk than other domestic species, such as bovine and caprine milks. Metagenomic sequencing revealed that fucose-consuming bacterial taxa in the gut microbiota of piglets were qualitatively but not quantitatively different between nursing and weaning stages, suggesting that both the composition and structure of dietary glycans may play a critical role in shaping the distal gut microbiome. The similarity of both intestinal physiopathology and milk OS composition in human and porcine species suggests similar effects on gastrointestinal development of early nutrition, reinforcing the use of the pig intestinal model to simulate human intestinal models in the clinical setting.

Buccal administration of human colostrum: impact on the oral microbiota of premature infants.

OBJECTIVE: To determine whether the administration of mother's colostrum into the buccal pouch in the first days of life alters the oral microbiota compared with control infants. STUDY DESIGN: In this pilot study, 12 very low birth weight (VLBW) infants were randomly assigned to receive either colostrum from their mothers directly into the buccal pouch every 2 h for 46 h or standard care. We analyzed the oral microbiota at initiation and 48 and 96 h later using next-generation sequencing. RESULT: The oral microbiota changed markedly over the 96 h period in all babies. Patterns of colonization differed between groups with Planococcaceae, the dominant family at 48 and 96 h in the colostrum group, and Moraxellaceae and Staphylococcaceae, the dominant families at 48 and 96 h, respectively, in the control group. CONCLUSION: Buccal administration of mother's colostrum to VLBW infants influenced the colonization of the oral cavity with differences persisting 48 h after completion of the intervention.

Potential of Lactobacillus curvatus LFC1 to produce slits in Cheddar cheese.

Defects in Cheddar cheese resulting from undesired gas production are a sporadic problem that results in significant financial losses in the cheese industry. In this study, we evaluate the potential of a facultatively heterofermentative lactobacilli, Lactobacillus curvatus LFC1, to produce slits, a gas related defect in Cheddar cheese. The addition of Lb. curvatus LFC1 to cheese milk at log 3 CFU/ml resulted in the development of small slits during the first month of ripening. Chemical analyses indicated that the LFC1 containing cheeses had less galactose and higher levels of lactate and acetate than the control cheeses. The composition the cheese microbiota was examined through a combination of two culture independent approaches, 16S rRNA marker gene sequencing and automated ribosomal intergenic spacer analysis; the results indicated that no known gas producers were present and that high levels of LFC1 was the only significant difference between the cheese microbiotas. A ripening cheese model system was utilized to examine the metabolism of LFC1 under conditions similar to those present in cheeses that exhibited the slit defect. The combined cheese and model system results indicate that when Lb. curvatus LFC1 was added to the cheese milk at log 3 CFU/ml it metabolized galactose to lactate, acetate, and CO2. For production of sufficient CO2 to result in the formation of slits there needs to be sufficient galactose and Lb. curvatus LFC1 present in the cheese matrix. To our knowledge, facultatively heterofermentative lactobacilli have not previously been demonstrated to result in gas-related cheese defects.

Probiotic administration in congenital heart disease: a pilot study.

OBJECTIVE: To investigate the impact of probiotic Bifidobacterium longum ssp. infantis on the fecal microbiota and plasma cytokines in neonates with congenital heart disease. STUDY DESIGN: Sixteen infants with congenital heart disease were randomly assigned to receive either B. infantis (4.2 × 10(9) colony-forming units two times daily) or placebo for 8 weeks. Stool specimens from enrolled infants and from six term infants without heart disease were analyzed for microbial composition. Plasma cytokines were analyzed weekly in the infants with heart disease. RESULTS: Healthy control infants had increased total bacteria, total Bacteroidetes and total bifidobacteria compared to the infants with heart disease, but there were no significant differences between the placebo and probiotic groups. Plasma interleukin (IL)10, interferon (IFN)γ and IL1ß levels were transiently higher in the probiotic group. CONCLUSION: Congenital heart disease in infants is associated with dysbiosis. Probiotic B. infantis did not significantly alter the fecal microbiota. Alterations in plasma cytokines were found to be inconsistent.

The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome.

Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp cluster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteomic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.

Modeling Uhthoff's phenomenon in MS patients with internuclear ophthalmoparesis.

OBJECTIVE: The goal of this investigation was to demonstrate that internuclear ophthalmoparesis (INO) can be utilized to model the effects of body temperature-induced changes on the fidelity of axonal conduction in multiple sclerosis (Uhthoff's phenomenon). METHODS: Ocular motor function was measured using infrared oculography at 10-minute intervals in patients with multiple sclerosis (MS) with INO (MS-INO; n = 8), patients with MS without INO (MS-CON; n = 8), and matched healthy controls (CON; n = 8) at normothermic baseline, during whole-body heating (increase in core temperature 0.8 degrees C as measured by an ingestible temperature probe and transabdominal telemetry), and after whole-body cooling. The versional disconjugacy index (velocity-VDI), the ratio of abducting/adducting eye movements for velocity, was calculated to assess changes in interocular disconjugacy. The first pass amplitude (FPA), the position of the adducting eye when the abducting eye achieves a centrifugal fixation target, was also computed. RESULTS: Velocity-VDI and FPA in MS-INO patients was elevated (p < 0.001) following whole body heating with respect to baseline measures, confirming a compromise in axonal electrical impulse transmission properties. Velocity-VDI and FPA in MS-INO patients was then restored to baseline values following whole-body cooling, confirming the reversible and stereotyped nature of this characteristic feature of demyelination. CONCLUSIONS: We have developed a neurophysiologic model for objectively understanding temperature-related reversible changes in axonal conduction in multiple sclerosis. Our observations corroborate the hypothesis that changes in core body temperature (heating and cooling) are associated with stereotypic decay and restoration in axonal conduction mechanisms.

Use of human lysozyme transgenic goat milk in cheese making: effects on lactic acid bacteria performance.

Genetically engineered goats expressing elevated levels of the antimicrobial enzyme lysozyme in their milk were developed to improve udder health, product shelf life, and consumer well-being. The purpose of this study was to evaluate the effect of lysozyme on the development of lactic acid bacteria (LAB) throughout the cheese-making process. Raw and pasteurized milk from 7 lysozyme transgenic goats and 7 breed-, age-, and parity-matched nontransgenic controls was transformed into cheeses by using industry methods, and their microbiological load was evaluated. The numbers of colony-forming units of LAB were determined for raw and pasteurized goat milk, whey, and curd at d 2 and at d 6 or 7 of production. Selective plating media were used to enumerate lactococcal species separately from total LAB. Although differences in the mean number of colony-forming units between transgenic and control samples in raw milk, whey, and cheese curd were non-significant for both total LAB and lactococcal species from d 2 of production, a significant decrease was observed in both types of LAB among d 6 transgenic raw milk cheese samples. In pasteurized milk trials, a significant decrease in LAB was observed only in the raw milk of transgenic animals. These results indicate that lysozyme transgenic goat milk is not detrimental to LAB growth during the cheese-making process.

Topographical memory impairments after unilateral lesions of the anterior thalamus and contralateral inferotemporal cortex.

Monkeys with crossed unilateral excitotoxic lesions of the anterior thalamus and unilateral inferotemporal cortex ablation were severely impaired at learning two tasks which required the integration of information about the appearance of objects and their positions in space. The lesioned monkeys were also impaired at learning a spatial task and a task which required the integration of information about the appearance of objects and the background on which the objects were situated. Monkeys with only one of the unilateral lesions were not impaired and previous work has shown that monkeys with bilateral lesions of the anterior thalamus were not impaired on these tasks. These results indicate that the whole of the inferotemporal cortex-anterior thalamic circuit, which passes via the hippocampus, fornix, mamillary bodies and mamillothalamic tract, is essential for the topographical analysis of information about specific objects in different positions in space. Together with previous work, the results show that a unilateral lesion may affect cognition in the presence of other brain damage when an equivalent bilateral lesion alone does not. The tasks required the slow acquisition of information into long term memory and therefore assessed semantic knowledge although other research has shown impairment on topographical processing within working or episodic memory following lesions of the hippocampal-diencephalic circuit. It is argued that the hippocampal-diencephalic circuit does not have a role in a specific form of memory such as episodic memory but rather is involved in topographical analysis of the environment in perception and across all types of declarative memory.

Evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of fungal communities in compost.

AIMS: Three previously published fungal specific PCR primer sets, referred to as the NS, EF and NL primer sets, were evaluated for use in compost microbial community analysis by PCR and denaturing gradient gel electrophoresis (DGGE). METHODS AND RESULTS: Primers were first evaluated based on their tolerance to PCR inhibitors. Due to its sensitivity to inhibitors, the NS primer set was determined to require a 10-fold smaller volume addition of compost DNA to PCR than the EF and NL primer sets, based on a logistic regression model for a 75% PCR success rate. Further evaluation of the EF and NL primer sets involved testing the resolution of PCR products from pure fungal cultures on DGGE. The NL primer set, which targets the more variable 28S rDNA, resulted in multiple bands for each pure culture. Thus, the EF primer set was used to monitor the microbial community during compost colonization studies, where three fungi were inoculated onto autoclaved grape pomace and rice straw compost. CONCLUSIONS: Of the three primer sets evaluated, the EF primer set was determined to be the best for PCR-DGGE of compost fungal populations; however, concerns with the EF primer set included the lack of sequence divergence in the targeted region of 18S rDNA and PCR artifacts which interfered with detection of inoculated fungi in the colonization studies. SIGNIFICANCE AND IMPACT OF THE STUDY: There are many factors related to PCR primers that need to be assessed prior to applying PCR-DGGE to fungal communities in complex environments such as compost.

Complementary therapy involvement of physicians: implications for practice and learning.

OBJECTIVE: We studied physicians' current and desired clinical role functions within the complementary health paradigm and their perceptions of the necessary educational programs to support them. DESIGN AND SETTING: A questionnaire to determine clinical activities within different complementary therapies was developed and mailed-out to 837 eligible physicians in Hamilton, Ontario, Canada, using a modified Dillman approach. RESULTS: The overall response rate to the mailed survey was 49.8% (417/837), with response rates of 50.2% (115/229) for family physicians and 49.7% (302/608) for specialists. The amount of interactions around complementary therapies between physicians, their patients and complementary therapists appears to be low. At the same time, there is a growing interest among physicians about complementary therapies, particularly with respect to developing their knowledge about efficacy and enhancing their skills in assessment and counselling. CONCLUSIONS: The differential levels of acceptance of different therapies by physicians will influence integration of complementary therapies in mainstream medicine.

Mutagenesis in the post genomics era: tools for generating insertional mutations in the lactic acid bacteria.

The increasing availability of whole genome sequences has increased the demand for effective tools to generate insertional mutations in the lactic acid bacteria (LAB). Several novel approaches, such as shuttle-, transposome- and intron-based mutagenesis methods, are possible additions to the existing repertoire of transposon- and recombination-based tools available for mutagenesis of LAB.

Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes.

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II reveals the native processing site, shows involvement of the C-terminus in cytochrome c binding, and improves the assay for proton pumping.

To enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-terminal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Characterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values for cytochrome c that are 6-14-fold higher for the truncated and his-tagged forms than for the wildtype. This lowered ability to bind cytochrome c indicates a previously undetected role for the C-terminus in cytochrome c binding and is mimicked by reduced affinity for an FPLC anion exchange column. The elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Paracoccus denitrificans oxidase, does not produce the same enzyme as the native processing reaction. MALDI-TOF MS data show the true C-terminus of subunit II is at serine 290, three amino acids longer than expected. When the his-tagged form is reconstituted into lipid vesicles and further purified by metal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results are attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.

Redesign of the proton-pumping machinery of cytochrome c oxidase: proton pumping does not require Glu(I-286).

One of the putative proton-transfer pathways leading from solution toward the binuclear center in many cytochrome c oxidases is the D-pathway, so-called because it starts with a highly conserved aspartate [D(I-132)] residue. Another highly conserved amino acid residue in this pathway, glutamate(I-286), has been indicated to play a central role in the proton-pumping machinery of mitochondrial-type enzymes, a role that requires a movement of the side chain between two distinct positions. In the present work we have relocated the glutamate to the opposite side of the proton-transfer pathway by constructing the double mutant EA(I-286)/IE(I-112). This places the side chain in about the same position in space as in the original enzyme, but does not allow for the same type of movement. The results show that the introduction of the second-site mutation, IE(I-112), in the EA(I-286) mutant enzyme results in an increase of the enzyme activity by a factor of >10. In addition, the double mutant enzyme pumps approximately 0.4 proton per electron. This observation restricts the number of possible mechanisms for the operation of the redox-driven proton pump. The proton-pumping machinery evidently does require the presence of a protonatable/polar residue at a specific location in space, presumably to stabilize an intact water chain. However, this residue does not necessarily have to be at a strictly conserved location in the amino acid sequence. In addition, the results indicate that E(I-286) is not the "proton gate" of cytochrome c oxidase controlling the flow of pumped protons from one to the other side of the membrane.

Characterization of a phosphate binding domain on the alpha-subunit of chloroplast ATP synthase using the photoaffinity phosphate analogue 4-azido-2-nitrophenyl phosphate.

The photoaffinity phosphate analogue 4-azido-2 nitrophenyl phosphate (ANPP) was shown previously (Pougeois, R., Lauquin, G. J.-M., and Vignais, P. V. (1983) Biochemistry 22, 1241-1245) to bind covalently and specifically to a single catalytic site on one of the three beta-subunits of the isolated chloroplast coupling factor 1 (CF(1)). Modification by ANPP strongly inhibited ATP hydrolysis activity. In this study, we examined labeling of membrane-bound CF(1) by ANPP by exposing thylakoid membranes to increasing concentrations of the reagent. ANPP exhibited saturable binding to two sites on CF(1), one on the beta-subunit and one on the alpha-subunit. Labeling by ANPP resulted in the complete inhibition of both ATP synthesis and ATP hydrolysis by the membrane-bound enzyme. Labeling of both sites by ANPP was reduced by more than 80% in the presence of P(i) (> or = 10 mM) and ATP (> or = 0.5 mM). ADP was less effective in competing with ANPP for binding, giving a maximum of approximately 35% inhibition at concentrations > or = 2 mM. ANPP-labeled tryptic peptides of the alpha-subunit were isolated and sequenced. The majority of the probe was contained in three peptides corresponding to residues Gln(173) to Arg(216), Gly(217) to Arg(253), and His(256) to Arg(272) of the alpha-subunit. In the mitochondrial F(1) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), all three analogous peptides are located within the nucleotide binding pocket and within close proximity to the gamma-phosphate binding site. The data indicate, however, that the azidophenyl group of bound ANPP is oriented at approximately 180 degrees in the opposite direction to the adenine binding site with reference to the phosphate binding site on the alpha-subunit. The study has confirmed that ANPP is a bona fide phosphate analogue and suggests that it specifically targets the gamma-phosphate binding site within the nucleotide binding pockets on the alpha- and beta-subunits of CF(1). The study also indicates that in the resting state of the chloroplast F(1)-F(0) complex both the alpha- and beta-subunits are structurally asymmetric.

Direct profiling of the yeast dynamics in wine fermentations.

We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml(-1). PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine.

Where is 'outside' in cytochrome c oxidase and how and when do protons get there?

Cytochrome c oxidase moves both electrons and protons in its dual role as a terminal electron acceptor and a contributor to the proton motive force which drives the formation of ATP. Although the sequence of electron transfer events is well-defined, the correlated mechanism and routes by which protons are translocated across the membrane are not. A recent model [Michel, Proc. Natl. Acad. Sci. USA 95 (1998) 12819] offers a detailed molecular description of when and how protons are translocated through the protein to the outside, which contrasts with previous models in several respects. This article reviews the behavior of site-directed mutants of Rhodobacter sphaeroides cytochrome c oxidase in the context of these different models. Studies of the internally located lysine 362 on the K channel and aspartate 132 on the D channel, indicate that D132, but not K362, is connected to the exterior region. Analysis of the externally located arginine pair, 481 and 482, and the Mg/Mn ligands, histidine 411 and aspartate 412, which are part of the hydrogen-bonded network that includes the heme propionates, indicates that alterations in this region do not strongly compromise proton pumping, but do influence the pH dependence of overall activity and the control of activity by the pH gradient. The results are suggestive of a region of 'sequestered' protons: beyond a major energetic gate, but selectively responsive to the external environment.

Proton uptake and release in cytochrome c oxidase: separate pathways in time and space?

Analysis of mutant forms of cytochrome c oxidase in conjunction with knowledge from high resolution crystal structures is providing important clues as to the location and specificity of proton channels and the timing of proton movements with respect to electron transfer events. Mutant forms of Rhodobacter sphaeroides cytochrome c oxidase at the highly conserved aspartate 132, in the 'D-channel' and at lysine 362, in the 'K-channel', are compared with respect to the nature of their residual activity and their reactions with H2O2. The results argue for physical separation and specificity in these two proton input routes, due to their distinctive kinetics with peroxide and the apparent connection of the D-channel, but not the K-channel, to the proton exit pathway. The reversible nature and possible location of the exit pathway are discussed in the context of direct and indirect mechanisms of energy coupling.