rRNA probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli

Objectives: Certain milk factors may help to promote the growth of a host-friendly colonic microflora (e.g. bifidobacteria, lactobacilli) and explain why breast-fed infants experience fewer and milder intestinal infections than those who are formula-fed. The effects of supplementation of formula with two such milk factors was investigated in this study. Materials and Methods: Infant rhesus macaques were breastfed, fed control formula, or formula supplemented with glycomacropeptide (GMP) or alpha-lactalburnin (alpha-LA) from birth to 5 months of age. Blood was drawn monthly and rectal swabs were collected weekly. At 4.5 months of age, 10(8) colonyforming units of enteropathogenic E.coli O127, strain 2349/68 (EPEC) was given orally and the response to infection assessed. The bacteriology of rectal swabs pre- and post-infection was determined by culture independent fluorescence in situ hybridization. Results: Post-challenge, breast-fed infants and infants fed alpha-LA-supplemented formula had no diarrhea, whilst those infants fed GMP-supplemented formula had intermittent diarrhea. In infants fed control formula the diarrhea was acute. Conclusions: Supplementation of infant formula with appropriate milk proteins may be useful for improving the infant's ability to resist acute infection caused by E.coli.

Interaction of flavonoids with bovine serum albumin: a fluorescence quenching study

The interaction between four flavonoids (catechin, epicatechin, rutin and quercetin) and bovine serum albumin (BSA) was investigated using tryptophan fluorescence quenching. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between the flavonoids and BSA. The binding affinity was found to be strongest for quercetin, and ranked in the order quercetin>rutin>epicatechin=catechin. The pH in the range of 5 to 7.4 does not affect significantly (p<0.05) the association of rutin, epicatechin and catechin with BSA, but quercetin exhibited a stronger affinity at pH 7.4 than at lower pH (p<0.05). Quercetin has a total quenching effect on BSA tryptophan fluorescence at a molar ratio of 10:1 and rutin at approximately 25:1. However, epicatechin and catechin did not fully quench tryptophan fluorescence over the concentration range studied. Furthermore, the data suggested that the association between flavonoids and BSA did not change molecular conformation of BSA and that hydrogen bonding, ionic and hydrophobic interaction are equally important driving forces for protein-flavonoid association.

Chlorophyll fluorescence technique as a rapid diagnostic test of the effects of the photosynthetic inhibitor chlorotoluron on two winter wheat cultivars

A modified chlorophyll fluorescence technique was evaluated as a rapid diagnostic test of the susceptibility of wheat cultivars to chlorotoluron. Two winter wheat cultivars (Maris Huntsman and Mercia) exhibited differential response to the herbicide. All of the parameters of chlorophyll fluorescence examined were strongly influenced by herbicide concentration. Additionally, the procedure adopted here for the examination of winter wheat cultivar sensitivity to herbicide indicated that the area above the fluorescence induction curve and the ratio F-V/F-M are appropriate chlorophyll fluorescence parameters for detection of differential herbicide response between wheat cultivars. The potential use of this technique as an alternative to traditional methods of screening new winter wheat cultivars for their response to photosynthetic inhibitor herbicide is demonstrated here.

The effects of temperature and light integral on early vegetative growth and chlorophyll fluorescence of four contrasting genotypes of cacao (Theobroma cacao)

The effect of temperature on early vegetative growth, leaf chlorophyll fluorescence and chlorophyll content was examined on four genotypes of cacao (Amelonado, AMAZ 15–15, SCA 6 and SPEC 54/1). A controlled environment glasshouse was used to simulate the temperature conditions of three cacao-growing regions (Bahia, Brazil; Tafo, Ghana and Lower Perak, Malaysia) over the course of a year. Base temperatures calculated from increments in main stem growth varied from 18.6°C for AMAZ 15/15 to 20.8°C for SPEC 54/1. Temporal variation in Fv/Fm observed for two of the clones (SCA 6 and SPEC 54/1) in two of the compartments were correlated with temperature differences over time. Significant differences were also recorded between genotypes in leaf chlorophyll content. It was shown that variation over time in leaf chlorophyll content could be quantified accurately as a function of temperature and light integral. The results imply that genetic variability exists in cacao in response to temperature stress.

Malacosporean-like spores in urine of rainbow trout react with antibody and DNA probes to Tetracapsuloides bryosalmonae

Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD) of salmonid fishes in Europe and North America. The complete life cycle of the parasite remains unknown despite recent discoveries that the stages infectious for fish develop in freshwater bryozoans. During the course of examinations of the urine of rainbow trout (Oncorhynchus mykiss) with or recovering from PKD we identified spores with features similar to those of T. bryosalmonae found in the bryozoan host. Spores found in the urine were subspherical, with a width of 16 mum and height of 14 mum, and possessed two soft valves surrounding two spherical polar capsules (2 mum in diameter) and a single sporoplasm. The absence of hardened valves is a distinguishing characteristic of the newly established class Malacosporea that includes T. bryosalmonae as found in the bryozoan host. The parasite in the urine of rainbow trout possessed only two polar capsules and two valve cells compared to the four polar capsules and four valves observed in the spherical spores of 19 mum in diameter from T. bryosalmonae from the bryozoan host. Despite morphological differences, a relationship between the spores in the urine of rainbow trout and T. bryosalmonae was demonstrated by binding of monoclonal and polyclonal antibodies and DNA probes specific to T. bryosalmonae.

Constitutive oligomerization of human D-2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells - Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer

Human D-2Long (D-2L) and D-2Short (D-2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [H-3] Spiperone labelled D-2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D-2L or D-2S receptors, with a pK(d) value of approximate to 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D-2L and D-2S receptors in Sf9 cells. When the FLAG-tagged D-2S and HIV-tagged D-2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D-2 receptors. In both cases, constitutive homo-oligomers were revealed for D-2L and D-2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D-2S receptors. The D-2 receptor ligands dopamine, R-(-) propylnorapomorphine, and raclopride did not affect oligomerization of D-2L and D-2S in Sf9 and HEK293 cells. Human D-2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D-2 oligomerization in these cells is not regulated by ligands.

Chlorophyll fluorescence technique as a rapid diagnostic test of the effects of the photosynthetic inhibitor chlorotoluron on two winter wheat cultivars

A modified chlorophyll fluorescence technique was evaluated as a rapid diagnostic test of the susceptibility of wheat cultivars to chlorotoluron. Two winter wheat cultivars (Maris Huntsman and Mercia) exhibited differential response to the herbicide. All of the parameters of chlorophyll fluorescence examined were strongly influenced by herbicide concentration. Additionally, the procedure adopted here for the examination of winter wheat cultivar sensitivity to herbicide indicated that the area above the fluorescence induction curve and the ratio F-V/F-M are appropriate chlorophyll fluorescence parameters for detection of differential herbicide response between wheat cultivars. The potential use of this technique as an alternative to traditional methods of screening new winter wheat cultivars for their response to photosynthetic inhibitor herbicide is demonstrated here.

Use of 16S rRNA-targeted oligonucleotide probes to investigate function and phylogeny of sulphate-reducing bacteria and methanogenic archaea in a UK estuary

Sulphate-reducing bacteria (SRB) and methanogenic archaea (MA) are important anaerobic terminal oxidisers of organic matter. However, we have little knowledge about the distribution and types of SRB and MA in the environment or the functional role they play in situ. Here we have utilised sediment slurry microcosms amended with ecologically significant substrates, including acetate and hydrogen, and specific functional inhibitors, to identify the important SRB and MA groups in two contrasting sites on a UK estuary. Substrate and inhibitor additions had significant effects on methane production and on acetate and sulphate consumption in the slurries. By using specific 16S-targeted oligonucleotide probes we were able to link specific SRB and MA groups to the use of the added substrates. Acetate consumption in the freshwater-dominated sediments was mediated by Methanosarcinales under low-sulphate conditions and Desulfobacter under the high-sulphate conditions that simulated a tidal incursion. In the marine-dominated sediments, acetate consumption was linked to Desulfobacter. Addition of trimethylamine, a non-competitive substrate for methanogenesis, led to a large increase in Methanosarcinales signal in marine slurries. Desulfobulbus was linked to non-sulphate-dependent H-2 consumption in the freshwater sediments. The addition of sulphate to freshwater sediments inhibited methane production and reduced signal from probes targeted to Methanosarcinales and Methanomicrobiales, while the addition of molybdate to marine sediments inhibited Desulfobulbus and Desulfobacterium. These data complement our understanding of the ecophysiology of the organisms detected and make a firm connection between the capabilities of species, as observed in the laboratory, to their roles in the environment. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children

Children with autistic spectrum disorders (ASDs) tend to suffer from severe gastrointestinal problems. Such symptoms may be due to a disruption of the indigenous gut flora promoting the overgrowth of potentially pathogenic micro-organisms. The faecal flora of patients with ASDs was studied and compared with those of two control groups (healthy siblings and unrelated healthy children). Faecal bacterial populations were assessed through the use of a culture-independent technique, fluorescence in situ hybridization, using oligonucleotide probes targeting predominant components of the gut flora. The faecal flora of ASD patients contained a higher incidence of the Clostridium histolyticum group (Clostridium clusters I and 11) of bacteria than that of healthy children. However, the non-autistic sibling group had an intermediate level of the C. histolyticum group, which was not significantly different from either of the other subject groups. Members of the C. histolyticum group are recognized toxin-producers and may contribute towards gut dysfunction, with their metabolic products also exerting systemic effects. Strategies to reduce clostridial population levels harboured by ASD patients or to improve their gut microflora profile through dietary modulation may help to alleviate gut disorders common in such patients.

Activity and location of olive oil phenolic antioxidants in liposomes

The antioxidant activity of hydroxytyrosol, hydroxytyrosol acetate, oleuropein, 3,4-dihydroxyphenylelenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenyielenolic acid dialdehyde (3,4-DHPEA-EDA) towards oxidation initiated by 2,2'-azobis (2-amidinopropane) hydrochloride in a soybean phospholipid liposome system was studied. The antioxidant activity of these olive oil phenols was similar and the duration of the lag phase was almost twice that of alpha-tocopherol. Trolox(R), a water-soluble analogue of alpha-tocopherol, showed the worst antioxidant activity. However, oxidation before the end of the lag phase was inhibited less effectively by the olive oil phenols than by alpha-tocopherol and Trolox(R). Synergistic effects (11-20% increase in lag phase) were observed in the antioxidant activity of combinations of alpha-tocopherol with olive oil phenols both with and without ascorbic acid. Fluorescence anisotropy of probes and fluorescence quenching studies showed that the olive oil phenols did not penetrate into the membrane, but their effectiveness as antioxidants showed they were associated with the surface of the phospholipid bilayer. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Molecular characterisation of the gut microflora of healthy and inflammatory bowel disease cats using fluorescence in situ hybridisation with special reference to Desulfovibrio spp

Inflammatory bowel disease (IBD) is a common cause of chronic large bowel diarrhoea in cats. Although the aetiology of IBD is unknown, an immune-mediated response to a luminal antigen is thought to be involved. As knowledge concerning the colonic microflora of cats is limited and requires further investigation, the purpose of this study was to determine the presence of specific bacterial groups in normal and IBD cats, and the potential role they play in the health of the host. Total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum subgp., Lactobacillus-Enterococcus subgp. and Desulfovibrio spp. were enumerated in 34 healthy cats and 11 IBD cats using fluorescence in situ hybridisation. The study is one of the first to show the presence of Desulfovibrio in cats. Total bacteria, Bifidobacterium spp. and Bacteroides spp. counts were all significantly higher in healthy cats when compared with IBD cats, whereas Desulfovibrio spp. (producers of toxic sulphides) numbers were found to be significantly higher in colitic cats. The information obtained from this study suggests that modulation of bacterial flora by increasing bifidobacteria and decreasing Desulfovibrio spp. may be beneficial to cats with IBD. Dietary intervention may be an important aspect of their treatment.

Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions

Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewis(x) trisaccharide to antibodies to Lewisx, (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core.

Longitudinal investigation of the faecal microbiota of healthy full-term infants using fluorescence in situ hybridization and denaturing gradient gel electrophoresis.

From birth onwards, the gastrointestinal (GI) tract of infants progressively acquires a complex range of micro-organisms. It is thought that by 2 years of age the GI microbial population has stabilized. Within the developmental period of the infant GI microbiota, weaning is considered to be most critical, as the infant switches from a milk-based diet (breast and/or formula) to a variety of food components. Longitudinal analysis of the biological succession of the infant GI/faecal microbiota is lacking. In this study, faecal samples were obtained regularly from 14 infants from 1 month to 18 months of age. Seven of the infants (including a set of twins) were exclusively breast-fed and seven were exclusively formula-fed prior to weaning, with 175 and 154 faecal samples, respectively, obtained from each group. Diversity and dynamics of the infant faecal microbiota were analysed by using fluorescence in situ hybridization and denaturing gradient gel electrophoresis. Overall, the data demonstrated large inter- and intra-individual differences in the faecal microbiological profiles during the study period. However, the infant faecal microbiota merged with time towards a climax community within and between feeding groups. Data from the twins showed the highest degree of similarity both quantitatively and qualitatively. Inter-individual variation was evident within the infant faecal microbiota and its development, even within exclusively formula-fed infants receiving the same diet. These data can be of help to future clinical trials (e.g. targeted weaning products) to organize protocols and obtain a more accurate outline of the changes and dynamics of the infant GI microbiota.