<i>In vitro</i> study of the effect of <i>Bifidobacterium bifidum </i> probiotic strain DNA on the cell concentration and colonization properties of intestinal microsymbionts

Aim. To estimate in vitro the effect of DNA isolated from the probiotic strain Bifidobacterium bifidum 791 on the cell concentration and adhesive properties of fecal isolates of bifidobacteria and opportunistic microorganisms of different species.Materials and methods. DNA was isolated from the probiotic strain Bifidobacterium bifidum 791. Biomass containing bifidobacteria was washed from the nutrient medium. The suspension of bacteria in the buffer solution was subjected to ultrasonic disintegration with a frequency of 40 kHz three times for 30 minutes, followed by centrifugation. The supernatants were combined and purified chromatographically on CL-4B Sepharose. B. breve, B. bifidum, B. infantis, Staphylococcus aureus, Escherichia coli lac-, Enterococcus faecalis, and Candida albicans were used as test cultures, isolated from the intestines of conditionally healthy adults. Results. The nucleic acid solution with a concentration of 3.54 |jg/ml did not affect the cell number of bifidobacteria (p = 0.61). The DNA content in the solution of 14.15-21.23 jg/ml increased the titers of B. bifidum and B. breve by 2 lg CFU/ml compared to the control (p = 0.01), but did not affect the titers of S. aureus, E. coli lac-, E. faecalis, C. albicans (p = 0.73). The DNA solution stimulated the self-aggregation of bifidobacteria in 1.5-2.0 times. The ability to autoaggregate under the influence of bifidobacterial DNA in S. aureus, E. faecalis, C. albicans did not change, in E. coli lacincreased 2.3 times (p = 0.05).Conclusion. A DNA solution of the probiotic strain B. bifidum 791 with a content 14.15-21.23 jg/ml stimulates the reproduction and autoaggregation of fecal B. breve, B. bifidum

A family of silicon transporter structural genes in a pennate diatom Synedra ulna subsp. danica (Kütz.) Skabitsch.

Silicon transporters (SIT) are the proteins, which capture silicic acid in the aquatic environment and direct it across the plasmalemma to the cytoplasm of diatoms. Diatoms utilize silicic acid to build species-specific ornamented exoskeletons and make a significant contribution to the global silica cycle, estimated at 240 ±40 Tmol a year. Recently SaSIT genes of the freshwater araphid pennate diatom Synedra acus subsp. radians are found to be present in the genome as a cluster of two structural genes (SaSIT-TD and SaSIT-TRI) encoding several concatenated copies of a SIT protein each. These structural genes could potentially be transformed into "mature" SIT proteins by means of posttranslational proteolytic cleavage. In the present study, we discovered three similar structural SuSIT genes in the genome of a closely related freshwater diatom Synedra ulna subsp. danica. Structural gene SuSIT1 is identical to structural gene SuSIT2, and the two are connected by a non-coding nucleotide DNA sequence. All the putative "mature" SITs contain conserved amino acid motifs, which are believed to be important in silicon transport. The data obtained suggest that the predicted "mature" SIT proteins may be the minimal units necessary for the transport of silicon is S. ulna subsp. danica. The comparative analysis of all available multi-SITs has allowed us to detect two conservative motifs YQXDXVYL and DXDID, located between the "mature" proteins. Aspartic acid-rich DXDID motif can, in our opinion, serve as a proteolysis site during the multi-SIT cleavage. The narrow distribution of the distances between CMLD and DXDID motifs can serve as additional evidence to the conservation of their function

α-Tocopherol at Nanomolar Concentration Protects Cortical Neurons against Oxidative Stress

The aim of the present work is to study the mechanism of the α-tocopherol (α-T) protective action at nanomolar and micromolar concentrations against H2O2-induced brain cortical neuron death. The mechanism of α-T action on neurons at its nanomolar concentrations characteristic for brain extracellular space has not been practically studied yet. Preincubation with nanomolar and micromolar α-T for 18 h was found to increase the viability of cortical neurons exposed to H2O2; α-T effect was concentration-dependent in the nanomolar range. However, preincubation with nanomolar α-T for 30 min was not effective. Nanomolar and micromolar α-T decreased the reactive oxygen species accumulation induced in cortical neurons by the prooxidant. Using immunoblotting it was shown that preincubation with α-T at nanomolar and micromolar concentrations for 18 h prevented Akt inactivation and decreased PKCδ activation induced in cortical neurons by H2O2. α-T prevented the ERK1/2 sustained activation during 24 h caused by H2O2. α-T at nanomolar and micromolar concentrations prevented a great increase of the proapoptotic to antiapoptotic proteins (Bax/Bcl-2) ratio, elicited by neuron exposure to H2O2. The similar neuron protection mechanism by nanomolar and micromolar α-T suggests that a “more is better” approach to patients’ supplementation with vitamin E or α-T is not reasonable

α-Tocopherol at Nanomolar Concentration Protects PC12 Cells from Hydrogen Peroxide-Induced Death and Modulates Protein Kinase Activities

The aim of this work was to compare protective and anti-apoptotic effects of α-tocopherol at nanomolar and micromolar concentrations against 0.2 mM H2O2-induced toxicity in the PC12 neuronal cell line and to reveal protein kinases that contribute to α-tocopherol protective action. The protection by 100 nM α-tocopherol against H2O2-induced PC12 cell death was pronounced if the time of pre-incubation with α-tocopherol was 3–18 h. For the first time, the protective effect of α-tocopherol was shown to depend on its concentration in the nanomolar range (1 nM < 10 nM < 100 nM), if the pre-incubation time was 18 h. Nanomolar and micromolar α-tocopherol decreased the number of PC12 cells in late apoptosis induced by H2O2 to the same extent if pre-incubation time was 18 h. Immunoblotting data showed that α-tocopherol markedly diminished the time of maximal activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and protein kinase B (Akt)-induced in PC12 cells by H2O2. Inhibitors of MEK 1/2, PI 3-kinase and protein kinase C (PKC) diminished the protective effect of α-tocopherol against H2O2-initiated toxicity if the pre-incubation time was long. The modulation of ERK 1/2, Akt and PKC activities appears to participate in the protection by α-tocopherol against H2O2-induced death of PC12 cells. The data obtained suggest that inhibition by α-tocopherol in late stage ERK 1/2 and Akt activation induced by H2O2 in PC12 cells makes contribution to its protective effect, while total inhibition of these enzymes is not protective

X-Ray Fluorescence Imaging: A New Tool for Studying Manganese Neurotoxicity

<div><p>The neurotoxic effect of manganese (Mn) establishes itself in a condition known as <em>manganism</em> or Mn induced parkinsonism. While this condition was first diagnosed about 170 years ago, the mechanism of the neurotoxic action of Mn remains unknown. Moreover, the possibility that Mn exposure combined with other genetic and environmental factors can contribute to the development of Parkinson's disease has been discussed in the literature and several epidemiological studies have demonstrated a correlation between Mn exposure and an elevated risk of Parkinson's disease. Here, we introduce X-ray fluorescence imaging as a new quantitative tool for analysis of the Mn distribution in the brain with high spatial resolution. The animal model employed mimics deficits observed in affected human subjects. The obtained maps of Mn distribution in the brain demonstrate the highest Mn content in the globus pallidus, the thalamus, and the substantia nigra pars compacta. To test the hypothesis that Mn transport into/distribution within brain cells mimics that of other biologically relevant metal ions, such as iron, copper, or zinc, their distributions were compared. It was demonstrated that the Mn distribution does not follow the distributions of any of these metals in the brain. The majority of Mn in the brain was shown to occur in the mobile state, confirming the relevance of the chelation therapy currently used to treat Mn intoxication. In cells with accumulated Mn, it can cause neurotoxic action by affecting the mitochondrial respiratory chain. This can result in increased susceptibility of the neurons of the globus pallidus, thalamus, and substantia nigra pars compacta to various environmental or genetic insults. The obtained data is the first demonstration of Mn accumulation in the substantia nigra pars compacta, and thus, can represent a link between Mn exposure and its potential effects for development of Parkinson's disease.</p> </div

XRF imaging of the substantia nigra of control and treated samples.

<p>XRF images of Mn (<b>A</b>) and Fe (<b>B</b>) for treated and control samples. Note that the maximum Mn intensity for the control sample is 30% of the treated maximum intensity. Numbers given are in µg/g. (<b>C</b>) Tri-colored image of the SN where red, green, and blue represent Mn, Fe, and Cu respectively (same scale for Mn). Scale bar represents a length of 1 mm. (<b>D</b>) Confocal images of tyrosine hydroxylase stained SN area of adjunct sections recorded in identical experimental conditions.</p

Average concentrations for particular brain areas (mean ± SEM).

*<p>Denotes a significant difference from control (p<0.05).</p><p>AB, axon bundle; CPu, caudate putamen; GP, globus pallidus; SN, substantia nigra; Th, thalamus±.</p

Scatter plots and correlations for control and treated groups.

<p>Data points calculated by taking the average of the brain areas of interest for control and treated samples. We performed cluster analysis (2–5 clusters) on the data obtained from treated samples to objectively identify similar data points in terms of metal concentrations. Clustering was done using Cu/Mn, Fe/Mn, or Zn/Mn at equal weights. We found that there was no clear grouping according to Fe/Mn while according to Cu/Mn or Zn/Mn two clusters were appropriate (Group1 and Group2). For Zn/Mn, cluster analysis resulted in two exclusive groups in terms of brain structures. Color matched Pearson's correlation coefficients and <i>p-values</i> are also given. Linear regression parameters and statistical analysis results are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048899#pone.0048899.s011" target="_blank">Table S4</a>.</p

Mn and Fe distributions in the choroid plexus.

<p>XRF images of the Fe (<b>A</b>) and Mn (<b>B</b>) distributions in the choroid plexus (CP) within the lateral ventricle (lv) as identified by increased Fe signal. Images are Bregma −0.48 mm coronal sections of untreated rats (top) and Mn treated (bottom). Note that the images are not displayed on the same intensity scale, in order to see Mn contrast in the control image. Yellow dashed lines indicate the boundary between the ventricle and the labeled structures (CPu and HPC, hippocampal formation). The Fe signal shows the presence of CP (containing blood) within the ventricle. Mn concentration in the ventricle is lower than in adjacent brain structures of the CPu and HPC indicating clearance of Mn from the CP. All values given are in µg/g. Scale bar represents a length of 2 mm.</p