Proteomic analyses of early response of unicellular eukaryotic microorganism Tetrahymena thermophila exposed to TiO2 particles.

Key biological functions involved in cell survival have been studied to understand the difference between the impact of exposure to TiO2 nanoparticles (TiO2-NPs) and their bulk counterparts (bulk-TiO2). By selecting a unicellular eukaryotic model organism and applying proteomic analysis an overview of the possible impact of exposure could be obtained. In this study, we investigated the early response of unicellular eukaryotic protozoan Tetrahymena thermophila exposed to TiO2-NPs or bulk-TiO2 particles at subtoxic concentrations for this organism. The proteomic analysis based on 2DE + nLC-ESI-MS/MS revealed 930 distinct protein spots, among which 77 were differentially expressed and 18 were unambiguously identified. We identified alterations in metabolic pathways, including lipid and fatty acid metabolism, purine metabolism and energetic metabolism, as well as salt stress and protein degradation. This proteomic study is consistent with our previous findings, where the early response of T. thermophila to subtoxic concentrations of TiO2 particles included alterations in lipid and fatty acid metabolism and ion regulation. The response to the lowest TiO2-NPs concentration differed significantly from the response to higher TiO2-NPs concentration and both bulk-TiO2 concentrations. Alterations on the physiological landscape were significant after exposure to both nano- and bulk-TiO2; however, no toxic effects were evidenced even at very high exposure concentrations. This study confirms the relevance of the alteration of the lipid profile and lipid metabolism in understanding the early impact of TiO2-NPs in eukaryotic cells, for example, phagocytosing cells like macrophages and ciliated cells in the respiratory epithelium.

Acclimation of Tetrahymena thermophila to bulk and nano-TiO2 particles by changes in membrane fatty acids saturation.

We provide experimental evidence that changes in the membrane fatty acid profile of Tetrahymena thermophila incubated with nano- or bulk TiO(2) particle are not accompanied by ROS generation or lipid peroxidation. Consequently these changes are interpreted as acclimation to unfavorable conditions and not as toxic effects. T. thermophila cells were exposed to TiO(2) particles at different concentrations for 24h at 32°C. Treatment of cultures with nano- and bulk TiO(2) particles resulted in changes of membrane fatty acid profile, indicating increased membrane rigidity, but no lipid peroxidation or ROS generation was detected. There were no differences in membrane composition when T. thermophila was exposed to nanosized or bulk-TiO(2) particles. We also observed reversible filling of food vacuoles, but this was different in case of nano- or bulk TiO(2) exposure. Our results suggest that interactions of particles and cell membranes are independent of oxidative stress.

Adaptation of adhesion test using Caco-2 cells for anaerobic bacterium Pseudobutyrivibrio xylanivorans, a probiotic candidate.

Pseudobutyrivibrio xylanivorans strain Mz5(T), an anaerobic bacterium (originating from the rumen of a Holstein-Friesian cow), has some attributes that make it a possible probiotic strain (very active hydrolases, bacteriocin and conjugated linoleic acid production). For the estimation of its adhesion ability, the adhesion test on Caco-2 cells was introduced and adapted. The adhesion was performed in an anaerobic glove box in standard 24-well plates at neutral pH for 30 min. The best method for separation of the adhered bacteria from Caco-2 cells appeared to be homogenization with an automatic pipette. The number of adhered bacteria was too small to be determined microscopically, so a new approach, i.e. detection of the apparent lag phase in liquid growth medium was tested. Under the selected assay conditions 1.04 bacterial cells from the late exponential phase adhered to one Caco-2 cell, which confirms the adhesion capability of P. xylanivorans Mz5(T). The adapted adhesion test using Caco-2 cells is suitable for estimation of adhesion capability of anaerobic bacteria.

Xyn11A, a multidomain multicatalytic enzyme from Pseudobutyrivibrio xylanivorans Mz5T.

The rumen bacterium Pseudobutyrivibrio xylanivorans Mz5T has a potent xylanolytic enzyme system. A small native peptide (approximately 30-kDa, designated Xyn11A) from the bacterium was first isolated and characterized by Edman degradation. The gene coding for Xyn11A was identified using PCR amplification with consensus primers. It was then fully sequenced to reveal an open reading frame of 1809 bp. The predicted N-terminal domain exhibited xylanolytic activity and was classed to the family 11 of glycosyl hydrolases; it is followed by a region with homology to a family 6 cellulose binding module. The C-terminal domain codes for a putative NodB-like polysaccharide deacetylase which is predicted to be an acetyl esterase implicated in debranching activity in the xylan backbone. As similar domain organization was also found in several other xylanases from a diverse range of bacteria, a common ancestor of such a xylanase is considered to be present and spread, possibly by horizontal gene transfer, to other microorganisms from different ecological niches.

Interference of Salmonella enteritidis and Lactobacillus spp. with IL-8 levels and transepithelial electrical resistance of enterocyte-like Caco-2 cells.

Caco-2 cells (exhibiting characteristics of mature villus enterocytes) were used to determine bacteria (Salmonella enteritidis causing human gastroenteritis)-intestinal cell interactions. The interference of bacteria with the transepithelial electrical resistance (TEER) of filter-grown Caco-2 cells and the production of IL-8 after exposure of the cells to S. enteritidis 857 and/or Lactobacillus strains (L. gasseri LF221 and L. rhamnosus BGT10) was evaluated. The strain 857 decreased TEER of filter-grown Caco-2 cells; in contrast, lactobacilli had a little or no effect. The effect of S. enteritidis on the TEER decreased if Caco-2 cells were pre-incubated with lactobacilli. This strain induced high levels of IL-8 (which can lead to cell damage). Compared to the IL-8 synthesis after exposure of Caco-2 cells to S. enteritidis 857, simultaneous exposure of Caco-2 cells to S. enteritidis and lactobacilli inhibited the IL-8 synthesis after short recovery periods.

Detection and quantification of genotoxicity in wastewater-treated Tetrahymena thermophila using the comet assay.

In the present study, the comet, or single-cell, gel electrophoresis assay was adapted for use with the ubiquitous unicellular protozoan Tetrahymena thermophila, and the method was evaluated for its ability to detect DNA damage induced by known genotoxins and wastewater samples. The original comet assay protocol was substantially modified (e.g., lower concentrations of detergents were used in the lysis buffer; electrophoresis time was reduced). Using the modified method, T. thermophila were subjected to short exposures of phenol, hydrogen peroxide, and formaldehyde, leading to concentration-dependent increases in DNA damage. The genotoxic potential of influent and effluent water samples from a local municipal wastewater treatment plant was evaluated. The results indicated that the influent wastewater was genotoxic and that the genotoxicity in the effluent water was substantially reduced. We assume employing T. thermophila in the use of the comet assay may become a cost-effective and reliable tool for genotoxicity screening and monitoring of wastewater and similar systems.

Is Pseudobutyrivibrio xylanivorans strain Mz5T suitable as a probiotic? An in vitro study.

Rumen bacterium Pseudobutyrivibrio xylanivorans strain Mz5T possessed a potent xylanolytic enzyme system consisting of at least 7 different xylan hydrolases with molar mass 27-145 kDa. Three of them were successfully isolated in active native form. This strain produced butyrate and lactate on different saccharides. cis-9, trans-11-Conjugated linoleic acid was also detected in the culture medium. Bacteriocin-like inhibitory substances of Mz5T were active against some strains of rumen bacteria and against selected Salmonella and E. coli isolates from poultry meat. The strain Mz5T retained viability and xylanolytic activity also under not fully anaerobic conditions; its cells attached to the Caco-2 cells so that its successful association with gut epithelial cells may be expected. These in vitro results confirmed several probiotic traits of the isolate Mz5T and justified further in vivo experiments to test its ability to improve animal health and performance.

Wheat bran and oat bran effectively reduce oxidative stress induced by high-fat diets in pigs.

AIM: To evaluate the influence of wheat bran and oat bran on the oxidative stress induced by a high proportion of fat in the diet. METHODS: Forty-eight growing pigs were penned individually and after an adaptation period divided into four groups. All groups received isocaloric daily rations composed of basal diet which was then supplemented with: starch (controls; CONT), linseed oil (OIL), linseed oil and wheat bran, or linseed oil and oat bran. The experimental period lasted 14-days. The oxidative stress was evaluated by measuring the malondialdehyde (MDA) concentration in blood plasma, the 48-hour urinary MDA excretion, and the degree of leukocyte nuclear DNA damage. RESULTS: In comparison with the CONT group, a significant increase in the MDA concentration in blood plasma and in the MDA excretion in urine was found in the OIL group. The degree of DNA damage in the OIL group was also significantly higher. In comparison with the OIL group, the wheat bran and oat bran supplementation significantly reduced the 24-hour MDA excretion in urine and reduced the degree of DNA damage in leukocytes to the level of the CONT group. CONCLUSION: The results of the experiments confirmed that a high wheat bran and oat bran intake effectively reduces oxidative stress induced by a high-fat diet.

Allelic differences in bovine kappa-CN gene which may regulate gene expression.

The most common kappa casein (kappa-CN) variants, kappa-CN A and kappa-CN B, are synthesised differentially in the lactating mammary gland of heterozygous animals (kappa-CN AB). In this study we evaluated several approaches for quantification of allele specific mRNA transcripts. The most consistent results were obtained using allele specific RT-PCR and capillary electrophoresis. On average, 13.4% more allele B specific than A specific transcripts were found. DNA sequencing of the proximal promoter region in several homozygous animals (kappa-CN AA, BB, EE) did not reveal any allele specific polymorphisms. Using the EMSA and DNase I footprinting we confirmed functional binding sites for three transcription factors (AP-2, NF1 and MGF) within the kappa-CN proximal promoter region. Sequence analysis of the 3'-UTR of the kappa-CN gene revealed seven allele specific sites. Two of these allelic differences were close to previously identified 3'-end regulatory sequences. In addition, allele specific differences in length between mRNAs of both variants were found. The two later findings suggest a possible post translational control determining content differences of kappa-CN in milk.

Allelic differences in bovine kappa - CN gene which may regulate gene expression.

The most common kappa casein (κ-CN) variants, κ-CN A and κ-CN B, are synthesised differentially in the lactating mammary gland of heterozygous animals (κ-CN AB). In this study we evaluated several approaches for quantification of allele specific mRNA transcripts. The most consistent results were obtained using allele specific RT-PCR and capillary electrophoresis. On average, 13.4% more allele B specific than A specific transcripts were found. DNA sequencing of the proximal promoter region in several homozygous animals (κ-CN AA, BB, EE) did not reveal any allele specific polymorphisms. Using the EMSA and DNase I footprinting we confirmed functional binding sites for three transcription factors (AP-2, NF1 and MGF) within the κ-CN proximal promoter region. Sequence analysis of the 3'-UTR of the κ-CN gene revealed seven allele specific sites. Two of these allelic differences were close to previously identified 3'-end regulatory sequences. In addition, allele specific differences in length between mRNAs of both variants were found. The two later findings suggest a possible post translational control determining content differences of κ-CN in milk.

Degradation and utilization of xylans by the rumen anaerobe Prevotella bryantii (formerly P. ruminicola subsp. brevis) B(1)4.

Freshly harvested whole cells from cultures of P. bryantii B(1)4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantii B(1)4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation with P. bryantii showed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens gave little increase in overall pentose utilization suggesting that external P. bryantii xylanases are as effective as the cloned R. flavefaciens enzymes in releasing products that can be utilised by P. bryantii cells. The xylanase system of P. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.

Isolation of genes encoding beta-D-xylanase, beta-D-xylosidase and alpha-L-arabinofuranosidase activities from the rumen bacterium Prevotella ruminicola B1(4).

Prevotella ruminicola B1(4) is a strictly anaerobic, Gram-negative, polysaccharide-degrading rumen bacterium. Xylanase activity in this strain was found to be inducible, the specific activity of cells grown on xylan being increased at least 20-fold by comparison with cells grown on glucose. Ten bacteriophage clones expressing xylanase activity were isolated from a lambda EMBL3 genomic DNA library of P. ruminicola B1(4). These clones were shown to represent four distinct chromosomal regions, based on restriction enzyme analysis and DNA hybridisation. Three groups of clones encoded activity against oat spelt xylan but not carboxymethylcellulose (CMC). In one of these groups, represented by clone 5, activities against pNP-arabinofuranoside and pNP-xyloside were found to be encoded separately from endoxylanase activity. The fourth region encoded activity against CM cellulose and lichen, in addition to xylan, and contains an endoglucanase/xylanase gene isolated previously.