Jose Maria Vieites - Profile on Academia.edu (original) (raw)

Papers by Jose Maria Vieites

Molecular Methods to Study Complex Microbial Communities

Methods in molecular biology, 2010

Microbes, which constitute a major fraction of the total biomass, are the main source of biodiver... more Microbes, which constitute a major fraction of the total biomass, are the main source of biodiversity on our Planet and play an essential role in maintaining global processes, which ultimately regulate the functioning of the Biosphere. Recent emergence of "metagenomics" allows for the analysis of microbial communities without tedious cultivation efforts. Metagenomics approach is analogous to the genomics with the difference that it does not deal with the single genome from a clone or microbe cultured or characterized in laboratory, but rather with that from the entire microbial community present in an environmental sample; it is the community genome. Global understanding by metagenomics depends essentially on the possibility of isolating the entire bulk DNA and identifying the genomes, genes, and proteins more relevant to each of the environmental sample under investigation. Following on this, in this chapter, we provide an analysis of methods available to isolate environmental DNA and to establish metagenomic libraries that can further be used for extensive activity screens.

Metagenomic Mining of Enzyme Diversity

Springer eBooks, 2010

ABSTRACT

Microbial biotechnology, Jul 17, 2009

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structura... more Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k cat/Km)]CE/ [(k cat/Km)]FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys 281 , Asp 282 , Asn 316 and Lys 317 ) situated close to the catalytic core (Ser 143 /Asp 273 /His 305 ) and a deletion of a 34-AA-long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k cat/Km values) and enzymes with inverted specificity ((k cat/Km)CE/(kcat/Km)FAE ratio of ~0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol -1 ), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol -1 ) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of 'hot spot' mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.

PLOS ONE, Jun 25, 2012

Microbial communities from cow rumen are known for their ability to degrade diverse plant polymer... more Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional bxylosidase, a-xylanase, a-L-arabinase and a-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit b-1,4 xylosidase, a-1,5 arabinofur(pyr)anosidase, b-1,4 lactase, a-1,6 raffinase, a-1,6 stachyase, b-galactosidase and a-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose-and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose-and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose-and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.

Isolation, identification and characterisation of three novel probiotic strains (Lactobacillus paracaseiCNCM I-4034,Bifidobacterium breveCNCM I-4035 andLactobacillus rhamnosusCNCM I-4036) from the faeces of exclusively breast-fed infants

British Journal of Nutrition, Jan 29, 2013

British Journal of Nutrition, May 1, 2012

Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce ... more Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce the risk of developing chronic diseases. b-Caryophyllene (CAR) is a common constituent of the essential oil of numerous plants, vegetables, fruits and medicinal herbs, and has been used as a flavouring agent since the 1930 s. Here, we report the antioxidant activity of CAR, its protective effect on liver fibrosis and its inhibitory capacity on hepatic stellate cell (HSC) activation. CAR was tested for the inhibition of lipid peroxidation and as a free radical scavenger. CAR had higher inhibitory capacity on lipid peroxidation than probucol, a-humulene and a-tocopherol. Also, CAR showed high scavenging activities against hydroxyl radical and superoxide anion. The activity of 5-lipoxygenase, an enzyme that actively participates in fibrogenesis, was significantly inhibited by CAR. Carbon tetrachloride-treated rats received CAR at 2, 20 and 200 mg/kg. CAR significantly improved liver structure, and reduced fibrosis and the expression of Col1a1, Tgfb1 and Timp1 genes. Oxidative stress was used to establish a model of HSC activation with overproduction of extracellular matrix proteins. CAR (1 and 10 mM) increased cell viability and significantly reduced the expression of fibrotic marker genes. CAR, a sesquiterpene present in numerous plants and foods, is as a natural antioxidant that reduces carbon tetrachloride-mediated liver fibrosis and inhibits hepatic cell activation.

British Journal of Nutrition, Oct 8, 2007

Several studies have reported differing data on the effect of exogenous nucleosides and nucleotid... more Several studies have reported differing data on the effect of exogenous nucleosides and nucleotides on the proliferation and differentiation in various intestinal cell lines and explants. To study whether exogenous nucleosides modulate intestinal cell differentiation, IEC-6 cells were differentiated in the presence or absence of a nucleoside mixture (cytidine, uridine, guanosine and inosine, 30 mM each), and the concentrations of nucleoside derivatives were determined by HPLC. Cell differentiation was assessed by electron microscopy, alkaline phosphatase activity and Rnd3 gene expression. The present results showed that uridine, guanosine and inosine were cleared from culture media (up to 32, 63 and 100 % in proliferating cells, and 31, 80 and 94 % in differentiated cells, respectively) whereas cytidine concentrations increased. Differentiation of IEC-6 cells was associated with a significant increase in intracellular nucleotide concentrations. Clearance of nucleosides correlated with a significant increase in the intracellular nucleotide pool in proliferating and differentiated IEC-6 cells. Intracellular guanosine nucleotides increased 2•5-and 5-fold in nucleoside-supplemented proliferating and differentiated cells, respectively. At 24 h, nucleoside-supplemented differentiated IEC-6 cells had significantly higher energy charge and GTP levels than non-supplemented ones. These modifications paralleled changes in cell differentiation as indicated by increased alkaline phosphatase activity, prolonged microvilli formation and accelerated down-regulation of Rnd3 gene expression. The present findings suggest that exogenous nucleosides were selectively taken up by IEC-6 cells, increased the intracellular nucleotide pool, GTP and energy charge, and favoured cell morphological and functional changes during differentiation.

The Journal of nutrition, 2007

Active hexose correlated compound (AHCC) is a product prepared from the mycelium of edible Basidi... more Active hexose correlated compound (AHCC) is a product prepared from the mycelium of edible Basidiomycete fungi that contains oligosaccharides. Here we have studied the antiinflammatory effect of AHCC in the trinitrobenzenesulfonic acid (TNBS) model of colitis in rats. Rats received AHCC (100 or 500 mg/kg) daily starting 2 d before (pretreatment) colitis induction and were killed 6 d after the TNBS challenge. The status of the rats was assessed by morphological and biochemical methods. The effect of AHCC on the colonic microflora was also assessed by studying the bacteria profile in feces by standard culture techniques. AHCC administration attenuated colonic inflammation, improving rat weight, food intake, damage score, extension of necrosis, colonic weight, colonic weight-to-length ratio, myeloperoxidase and alkaline phosphatase activities, glutathione concentration, and the expression of proinflammatory cytokines and chemokines (IL-1beta, IL-1 receptor antagonist, TNF, and monocyte...

Gene, 2003

An immunomodulatory membrane protein, CD200R displays an expression pattern restricted to myeloid... more An immunomodulatory membrane protein, CD200R displays an expression pattern restricted to myeloid cells in mice. It is the receptor for a ligand, CD200, expressed by a broad range of cell types. In this study, we describe the cloning and characterization of the human homologue of the CD200R gene. This gene maps closely to the CD200 gene on human chromosome 3q12-13. The human CD200R gene spans a region of 52 kb, consists of nine exons, and encodes a 348-amino-acid cell-surface protein consisting of two IgFF domains in a typical V/C2 arrangement. The 59-amino-acid cytoplasmic domain has two tyrosine residues, one of which is contained within a NPXY motif. In common with other IgSF genes, the CD200R gene can generate different protein isoforms through alternative splicing. An alternative spliceout form, which has not yet been described in mice, encodes a 188-amino-acid truncated soluble polypeptide containing only the V immunoglobulin domain. In contrast to murine CD200R protein, the human membrane-bound and soluble CD200R proteins have an insertion of 23 amino acids at position 23, encoded by exon 2, which generates a putative dihydroxyacid dehydratase domain. The splicing of exon 2 generates two new isoforms, encoding the membrane and soluble proteins but lacking the dyhydroxyacid dehydratase domain. Northern-blot analysis shows that both membrane-bound and soluble isoforms are expressed in the thymus, liver, spleen and placenta. By RT-PCR, we have analyzed the expression of the four transcript variants in human placenta, spleen, liver, brain and kidney.

Fems Microbiology Reviews, 2009

The world of microorganisms comprises a vast diversity of live organisms, each with its individua... more The world of microorganisms comprises a vast diversity of live organisms, each with its individual set of genes, cellular components and metabolic reactions that interact within the cell and communicate with the environment in many different ways. There is a strong imperative to gain a broader view of the wired and interconnected cellular and environmental processes as a whole via the systems microbiology approach in order to understand and predict ecosystem functioning. On the other hand, currently we experience a rise of metagenomics as an emerging tool to study communities of uncultured microorganisms. In this review, we conducted a survey of important methodologies in metagenomics and describe systems microbiology-like approaches for gaining a mechanistic understanding of complex microbial systems to interrogate compositional, evolutionary and metabolic properties. The review also discusses how metagenomics can be used as a holistic indicator for ecosystem response in terms of matter, nutrient and energy sources and functional networking.

Microbial biotechnology, Oct 13, 2008

In recent years, the application of approaches for harvesting DNA from the environment, the so-ca... more In recent years, the application of approaches for harvesting DNA from the environment, the so-called, 'metagenomic approaches' has proven to be highly successful for the identification, isolation and generation of novel enzymes. Functional screening for the desired catalytic activity is one of the key steps in mining metagenomic libraries, as it does not rely on sequence homology. In this mini-review, we survey high-throughput screening tools, originally developed for directed evolution experiments, which can be readily adapted for the screening of large libraries. In particular, we focus on the use of in vitro compartmentalization (IVC) approaches to address potential advantages and problems the merger of cultureindependent and IVC techniques might bring on the mining of enzyme activities in microbial communities.

Isolation, identification and characterisation of strains with probiotic activity, from faeces of infants fed exclusively with breast milk

Molecular Methods to Study Complex Microbial Communities

Methods in Molecular Biology, 2010

Microbes, which constitute a major fraction of the total biomass, are the main source of biodiver... more Microbes, which constitute a major fraction of the total biomass, are the main source of biodiversity on our Planet and play an essential role in maintaining global processes, which ultimately regulate the functioning of the Biosphere. Recent emergence of "metagenomics" allows for the analysis of microbial communities without tedious cultivation efforts. Metagenomics approach is analogous to the genomics with the difference that it does not deal with the single genome from a clone or microbe cultured or characterized in laboratory, but rather with that from the entire microbial community present in an environmental sample; it is the community genome. Global understanding by metagenomics depends essentially on the possibility of isolating the entire bulk DNA and identifying the genomes, genes, and proteins more relevant to each of the environmental sample under investigation. Following on this, in this chapter, we provide an analysis of methods available to isolate environmental DNA and to establish metagenomic libraries that can further be used for extensive activity screens.

Yeast, 1994

The LUC gene coding for Photinuspyralis firefly luciferase was cloned in different yeast episomal... more The LUC gene coding for Photinuspyralis firefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as an in vivo reporter gene. Activity of the enzyme in transformed cells in vivo was measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a tenfold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast-regulated promoters (ADH1, GAL1-10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for the in vivo sensitive determination of gene expression and promoter strength in yeast.

British Journal of Nutrition, 2007

Several studies have reported differing data on the effect of exogenous nucleosides and nucleotid... more Several studies have reported differing data on the effect of exogenous nucleosides and nucleotides on the proliferation and differentiation in various intestinal cell lines and explants. To study whether exogenous nucleosides modulate intestinal cell differentiation, IEC-6 cells were differentiated in the presence or absence of a nucleoside mixture (cytidine, uridine, guanosine and inosine, 30 μmeach), and the concentrations of nucleoside derivatives were determined by HPLC. Cell differentiation was assessed by electron microscopy, alkaline phosphatase activity andRnd3gene expression. The present results showed that uridine, guanosine and inosine were cleared from culture media (up to 32, 63 and 100 % in proliferating cells, and 31, 80 and 94 % in differentiated cells, respectively) whereas cytidine concentrations increased. Differentiation of IEC-6 cells was associated with a significant increase in intracellular nucleotide concentrations. Clearance of nucleosides correlated with ...

Science, 2009

Metabolite Arrays Methods suitable for the biochemical analysis of multiple metabolic pathways in... more Metabolite Arrays Methods suitable for the biochemical analysis of multiple metabolic pathways in mixed samples are in short supply. Beloqui et al. (p. 252 ) report a method to sample the global metabolic state of an organism or mixture of organisms using an array of more than 1500 metabolites linked to a glass slide. The substrates are linked to the plate so that the reaction of an enzyme with one of the metabolites releases a fluorescent dye, which allows sensitive detection of the enzymatic activity. From a sample with small numbers of a mixture of bacteria, the authors were able to collect DNA, amplify it in a host bacterium, and measure its encoded metabolic activity with the array. Furthermore, by coating the substrates on nanoparticles with a specially designed linker, the authors could trap and purify enzymes that reacted with the immobilized substrate. The metabolite array may be useful in the characterization of environmental samples, in diagnostic procedures, and in enzym...

Pediatric Allergy and Immunology, 2010

Ninety per cent of the human body is composed of prokaryote cells, and this group of microorganis... more Ninety per cent of the human body is composed of prokaryote cells, and this group of microorganisms is known as microbiota (1). Microbiota is concentrated in the gut and plays a key role in the preservation of the hostÕs health. It can be viewed as a metabolic ÔorganÕ that is exquisitely tuned to our physiology and performs specific functions, including the production of Thompson-Chagoyan OC, Vieites JM, Maldonado J, Edwards C, Gil A. Changes in faecal microbiota of infants with cowÕs milk protein allergy-a Spanish prospective case-control 6-month follow-up study.

Microbiology, 2011

Although it is well established that early infant feeding has a major influence on the establishm... more Although it is well established that early infant feeding has a major influence on the establishment of the gut microbiota, very little is understood about how the introduction of first solid food influences the colonization process. This study aimed to determine the impact of weaning on the faecal microbiota composition of infants from five European countries (Sweden, Scotland, Germany, Italy and Spain) which have different lifestyle characteristics and infant feeding practices. Faecal samples were collected from 605 infants approximately 4 weeks after the introduction of first solid foods and the results were compared with the same infants before weaning (6 weeks of age) to investigate the association with determining factors such as geographical origin, mode of delivery, previous feeding method and age of weaning. Samples were analysed by fluorescence in situ hybridization and flow cytometry using a panel of 10 rRNA targeted group- and species-specific oligonucleotide probes. The...

Molecular Methods to Study Complex Microbial Communities

Methods in molecular biology, 2010

Microbes, which constitute a major fraction of the total biomass, are the main source of biodiver... more Microbes, which constitute a major fraction of the total biomass, are the main source of biodiversity on our Planet and play an essential role in maintaining global processes, which ultimately regulate the functioning of the Biosphere. Recent emergence of "metagenomics" allows for the analysis of microbial communities without tedious cultivation efforts. Metagenomics approach is analogous to the genomics with the difference that it does not deal with the single genome from a clone or microbe cultured or characterized in laboratory, but rather with that from the entire microbial community present in an environmental sample; it is the community genome. Global understanding by metagenomics depends essentially on the possibility of isolating the entire bulk DNA and identifying the genomes, genes, and proteins more relevant to each of the environmental sample under investigation. Following on this, in this chapter, we provide an analysis of methods available to isolate environmental DNA and to establish metagenomic libraries that can further be used for extensive activity screens.

Metagenomic Mining of Enzyme Diversity

Springer eBooks, 2010

ABSTRACT

Microbial biotechnology, Jul 17, 2009

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structura... more Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k cat/Km)]CE/ [(k cat/Km)]FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys 281 , Asp 282 , Asn 316 and Lys 317 ) situated close to the catalytic core (Ser 143 /Asp 273 /His 305 ) and a deletion of a 34-AA-long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k cat/Km values) and enzymes with inverted specificity ((k cat/Km)CE/(kcat/Km)FAE ratio of ~0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol -1 ), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol -1 ) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of 'hot spot' mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.

PLOS ONE, Jun 25, 2012

Microbial communities from cow rumen are known for their ability to degrade diverse plant polymer... more Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional bxylosidase, a-xylanase, a-L-arabinase and a-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit b-1,4 xylosidase, a-1,5 arabinofur(pyr)anosidase, b-1,4 lactase, a-1,6 raffinase, a-1,6 stachyase, b-galactosidase and a-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose-and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose-and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose-and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.

Isolation, identification and characterisation of three novel probiotic strains (Lactobacillus paracaseiCNCM I-4034,Bifidobacterium breveCNCM I-4035 andLactobacillus rhamnosusCNCM I-4036) from the faeces of exclusively breast-fed infants

British Journal of Nutrition, Jan 29, 2013

British Journal of Nutrition, May 1, 2012

Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce ... more Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce the risk of developing chronic diseases. b-Caryophyllene (CAR) is a common constituent of the essential oil of numerous plants, vegetables, fruits and medicinal herbs, and has been used as a flavouring agent since the 1930 s. Here, we report the antioxidant activity of CAR, its protective effect on liver fibrosis and its inhibitory capacity on hepatic stellate cell (HSC) activation. CAR was tested for the inhibition of lipid peroxidation and as a free radical scavenger. CAR had higher inhibitory capacity on lipid peroxidation than probucol, a-humulene and a-tocopherol. Also, CAR showed high scavenging activities against hydroxyl radical and superoxide anion. The activity of 5-lipoxygenase, an enzyme that actively participates in fibrogenesis, was significantly inhibited by CAR. Carbon tetrachloride-treated rats received CAR at 2, 20 and 200 mg/kg. CAR significantly improved liver structure, and reduced fibrosis and the expression of Col1a1, Tgfb1 and Timp1 genes. Oxidative stress was used to establish a model of HSC activation with overproduction of extracellular matrix proteins. CAR (1 and 10 mM) increased cell viability and significantly reduced the expression of fibrotic marker genes. CAR, a sesquiterpene present in numerous plants and foods, is as a natural antioxidant that reduces carbon tetrachloride-mediated liver fibrosis and inhibits hepatic cell activation.

British Journal of Nutrition, Oct 8, 2007

Several studies have reported differing data on the effect of exogenous nucleosides and nucleotid... more Several studies have reported differing data on the effect of exogenous nucleosides and nucleotides on the proliferation and differentiation in various intestinal cell lines and explants. To study whether exogenous nucleosides modulate intestinal cell differentiation, IEC-6 cells were differentiated in the presence or absence of a nucleoside mixture (cytidine, uridine, guanosine and inosine, 30 mM each), and the concentrations of nucleoside derivatives were determined by HPLC. Cell differentiation was assessed by electron microscopy, alkaline phosphatase activity and Rnd3 gene expression. The present results showed that uridine, guanosine and inosine were cleared from culture media (up to 32, 63 and 100 % in proliferating cells, and 31, 80 and 94 % in differentiated cells, respectively) whereas cytidine concentrations increased. Differentiation of IEC-6 cells was associated with a significant increase in intracellular nucleotide concentrations. Clearance of nucleosides correlated with a significant increase in the intracellular nucleotide pool in proliferating and differentiated IEC-6 cells. Intracellular guanosine nucleotides increased 2•5-and 5-fold in nucleoside-supplemented proliferating and differentiated cells, respectively. At 24 h, nucleoside-supplemented differentiated IEC-6 cells had significantly higher energy charge and GTP levels than non-supplemented ones. These modifications paralleled changes in cell differentiation as indicated by increased alkaline phosphatase activity, prolonged microvilli formation and accelerated down-regulation of Rnd3 gene expression. The present findings suggest that exogenous nucleosides were selectively taken up by IEC-6 cells, increased the intracellular nucleotide pool, GTP and energy charge, and favoured cell morphological and functional changes during differentiation.

The Journal of nutrition, 2007

Active hexose correlated compound (AHCC) is a product prepared from the mycelium of edible Basidi... more Active hexose correlated compound (AHCC) is a product prepared from the mycelium of edible Basidiomycete fungi that contains oligosaccharides. Here we have studied the antiinflammatory effect of AHCC in the trinitrobenzenesulfonic acid (TNBS) model of colitis in rats. Rats received AHCC (100 or 500 mg/kg) daily starting 2 d before (pretreatment) colitis induction and were killed 6 d after the TNBS challenge. The status of the rats was assessed by morphological and biochemical methods. The effect of AHCC on the colonic microflora was also assessed by studying the bacteria profile in feces by standard culture techniques. AHCC administration attenuated colonic inflammation, improving rat weight, food intake, damage score, extension of necrosis, colonic weight, colonic weight-to-length ratio, myeloperoxidase and alkaline phosphatase activities, glutathione concentration, and the expression of proinflammatory cytokines and chemokines (IL-1beta, IL-1 receptor antagonist, TNF, and monocyte...

Gene, 2003

An immunomodulatory membrane protein, CD200R displays an expression pattern restricted to myeloid... more An immunomodulatory membrane protein, CD200R displays an expression pattern restricted to myeloid cells in mice. It is the receptor for a ligand, CD200, expressed by a broad range of cell types. In this study, we describe the cloning and characterization of the human homologue of the CD200R gene. This gene maps closely to the CD200 gene on human chromosome 3q12-13. The human CD200R gene spans a region of 52 kb, consists of nine exons, and encodes a 348-amino-acid cell-surface protein consisting of two IgFF domains in a typical V/C2 arrangement. The 59-amino-acid cytoplasmic domain has two tyrosine residues, one of which is contained within a NPXY motif. In common with other IgSF genes, the CD200R gene can generate different protein isoforms through alternative splicing. An alternative spliceout form, which has not yet been described in mice, encodes a 188-amino-acid truncated soluble polypeptide containing only the V immunoglobulin domain. In contrast to murine CD200R protein, the human membrane-bound and soluble CD200R proteins have an insertion of 23 amino acids at position 23, encoded by exon 2, which generates a putative dihydroxyacid dehydratase domain. The splicing of exon 2 generates two new isoforms, encoding the membrane and soluble proteins but lacking the dyhydroxyacid dehydratase domain. Northern-blot analysis shows that both membrane-bound and soluble isoforms are expressed in the thymus, liver, spleen and placenta. By RT-PCR, we have analyzed the expression of the four transcript variants in human placenta, spleen, liver, brain and kidney.

Fems Microbiology Reviews, 2009

The world of microorganisms comprises a vast diversity of live organisms, each with its individua... more The world of microorganisms comprises a vast diversity of live organisms, each with its individual set of genes, cellular components and metabolic reactions that interact within the cell and communicate with the environment in many different ways. There is a strong imperative to gain a broader view of the wired and interconnected cellular and environmental processes as a whole via the systems microbiology approach in order to understand and predict ecosystem functioning. On the other hand, currently we experience a rise of metagenomics as an emerging tool to study communities of uncultured microorganisms. In this review, we conducted a survey of important methodologies in metagenomics and describe systems microbiology-like approaches for gaining a mechanistic understanding of complex microbial systems to interrogate compositional, evolutionary and metabolic properties. The review also discusses how metagenomics can be used as a holistic indicator for ecosystem response in terms of matter, nutrient and energy sources and functional networking.

Microbial biotechnology, Oct 13, 2008

In recent years, the application of approaches for harvesting DNA from the environment, the so-ca... more In recent years, the application of approaches for harvesting DNA from the environment, the so-called, 'metagenomic approaches' has proven to be highly successful for the identification, isolation and generation of novel enzymes. Functional screening for the desired catalytic activity is one of the key steps in mining metagenomic libraries, as it does not rely on sequence homology. In this mini-review, we survey high-throughput screening tools, originally developed for directed evolution experiments, which can be readily adapted for the screening of large libraries. In particular, we focus on the use of in vitro compartmentalization (IVC) approaches to address potential advantages and problems the merger of cultureindependent and IVC techniques might bring on the mining of enzyme activities in microbial communities.

Isolation, identification and characterisation of strains with probiotic activity, from faeces of infants fed exclusively with breast milk

Molecular Methods to Study Complex Microbial Communities

Methods in Molecular Biology, 2010

Microbes, which constitute a major fraction of the total biomass, are the main source of biodiver... more Microbes, which constitute a major fraction of the total biomass, are the main source of biodiversity on our Planet and play an essential role in maintaining global processes, which ultimately regulate the functioning of the Biosphere. Recent emergence of "metagenomics" allows for the analysis of microbial communities without tedious cultivation efforts. Metagenomics approach is analogous to the genomics with the difference that it does not deal with the single genome from a clone or microbe cultured or characterized in laboratory, but rather with that from the entire microbial community present in an environmental sample; it is the community genome. Global understanding by metagenomics depends essentially on the possibility of isolating the entire bulk DNA and identifying the genomes, genes, and proteins more relevant to each of the environmental sample under investigation. Following on this, in this chapter, we provide an analysis of methods available to isolate environmental DNA and to establish metagenomic libraries that can further be used for extensive activity screens.

Yeast, 1994

The LUC gene coding for Photinuspyralis firefly luciferase was cloned in different yeast episomal... more The LUC gene coding for Photinuspyralis firefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as an in vivo reporter gene. Activity of the enzyme in transformed cells in vivo was measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a tenfold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast-regulated promoters (ADH1, GAL1-10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for the in vivo sensitive determination of gene expression and promoter strength in yeast.

British Journal of Nutrition, 2007

Several studies have reported differing data on the effect of exogenous nucleosides and nucleotid... more Several studies have reported differing data on the effect of exogenous nucleosides and nucleotides on the proliferation and differentiation in various intestinal cell lines and explants. To study whether exogenous nucleosides modulate intestinal cell differentiation, IEC-6 cells were differentiated in the presence or absence of a nucleoside mixture (cytidine, uridine, guanosine and inosine, 30 μmeach), and the concentrations of nucleoside derivatives were determined by HPLC. Cell differentiation was assessed by electron microscopy, alkaline phosphatase activity andRnd3gene expression. The present results showed that uridine, guanosine and inosine were cleared from culture media (up to 32, 63 and 100 % in proliferating cells, and 31, 80 and 94 % in differentiated cells, respectively) whereas cytidine concentrations increased. Differentiation of IEC-6 cells was associated with a significant increase in intracellular nucleotide concentrations. Clearance of nucleosides correlated with ...

Science, 2009

Metabolite Arrays Methods suitable for the biochemical analysis of multiple metabolic pathways in... more Metabolite Arrays Methods suitable for the biochemical analysis of multiple metabolic pathways in mixed samples are in short supply. Beloqui et al. (p. 252 ) report a method to sample the global metabolic state of an organism or mixture of organisms using an array of more than 1500 metabolites linked to a glass slide. The substrates are linked to the plate so that the reaction of an enzyme with one of the metabolites releases a fluorescent dye, which allows sensitive detection of the enzymatic activity. From a sample with small numbers of a mixture of bacteria, the authors were able to collect DNA, amplify it in a host bacterium, and measure its encoded metabolic activity with the array. Furthermore, by coating the substrates on nanoparticles with a specially designed linker, the authors could trap and purify enzymes that reacted with the immobilized substrate. The metabolite array may be useful in the characterization of environmental samples, in diagnostic procedures, and in enzym...

Pediatric Allergy and Immunology, 2010

Ninety per cent of the human body is composed of prokaryote cells, and this group of microorganis... more Ninety per cent of the human body is composed of prokaryote cells, and this group of microorganisms is known as microbiota (1). Microbiota is concentrated in the gut and plays a key role in the preservation of the hostÕs health. It can be viewed as a metabolic ÔorganÕ that is exquisitely tuned to our physiology and performs specific functions, including the production of Thompson-Chagoyan OC, Vieites JM, Maldonado J, Edwards C, Gil A. Changes in faecal microbiota of infants with cowÕs milk protein allergy-a Spanish prospective case-control 6-month follow-up study.

Microbiology, 2011

Although it is well established that early infant feeding has a major influence on the establishm... more Although it is well established that early infant feeding has a major influence on the establishment of the gut microbiota, very little is understood about how the introduction of first solid food influences the colonization process. This study aimed to determine the impact of weaning on the faecal microbiota composition of infants from five European countries (Sweden, Scotland, Germany, Italy and Spain) which have different lifestyle characteristics and infant feeding practices. Faecal samples were collected from 605 infants approximately 4 weeks after the introduction of first solid foods and the results were compared with the same infants before weaning (6 weeks of age) to investigate the association with determining factors such as geographical origin, mode of delivery, previous feeding method and age of weaning. Samples were analysed by fluorescence in situ hybridization and flow cytometry using a panel of 10 rRNA targeted group- and species-specific oligonucleotide probes. The...