Energy dependant plant stress acclimation

Plants may live and grow under suboptimal environmental conditions having certain biochemical and metabolic adaptations that facilitate their survival. Plant “metabolic flexibility” consists of the accomplishment of the same step in a metabolic pathway in a variety of different ways. Pyrophosphate which works as an energy donor when cellular ATP pools become diminished during stresses, alternative glycolytic reactions that bypass ATP-requiring steps, additional pathways for electron transport in plant mithocondria and the salvage pathways are some of the aspects related to “energetic flexibility”. This key feature that plays an important role in plant acclimation to stress can be an important target for engineering enhanced stress tolerance in crop plants.     

Formulation and numerical simulations of a continuum model of avascular tumor growth

In this paper we present a continuum mathematical model for a multicellular spheroid that mimics the micro-environment within avascular tumor growth. The model consists of a coupled system of non-linear convection-diffusion-reaction equations. This system is solved using a previously developed conservative Galerkin characteristics method. In the model considered, there are three cell types: the proliferative cells, the quiescent non-dividing cells which stay in the G₀ phase of the cell cycle and the necrotic cells. The model includes viable cell diffusion, diffusion of cellular material and the removal of necrotic cells. We assume that the nutrients diffuse passively and are consumed by the proliferative and quiescent tumor cells depending on the availability of resources (oxygen, glucose, etc.). The numerical simulations are performed using different sets of parameters, including biologically realistic ones, to explore the effects of each of these model parameters on reaching the steady state. The present results, taken together with those reported earlier, indicate that the removal of necrotic cells and the diffusion of cellular material have significant effects on the steady state, reflecting growth saturation, the number of viable cells, and the spheroid size.     

Matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry- and MALDI biotyper-based identification of cultured biphenyl-metabolizing bacteria from contaminated horseradish rhizosphere soil

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.     

Lysate of probiotic Lactobacillus casei DN-114 001 ameliorates colitis by strengthening the gut barrier function and changing the gut microenvironment

Background

Probiotic bacteria can be used for the prevention and treatment of human inflammatory diseases including inflammatory bowel diseases (IBD). However, the nature of active components and exact mechanisms of this beneficial effects have not been fully elucidated. Our aim was to investigate if lysate of probiotic bacterium L. casei DN-114 001 (Lc) could decrease the severity of intestinal inflammation in a murine model of IBD.

Methodology/Principal Findings

The preventive effect of oral administration of Lc significantly reduces the severity of acute dextran sulfate sodium (DSS) colitis in BALB/c but not in SCID mice. In order to analyze how this beneficial effect interferes with well-known phases of intestinal inflammation pathogenesis in vivo and in vitro, we evaluated intestinal permeability using the FITC-labeled dextran method and analysed tight junction proteins expression by immunofluorescence and PCR. We also measured CD4⁺FoxP3⁺ regulatory T cells proportion by FACS analysis, microbiota composition by pyrosequencing, and local cytokine production by ELISA. Lc leads to a significant protection against increased intestinal permeability and barrier dysfunction shown by preserved ZO-1 expression. We found that the Lc treatment increases the numbers of CD4⁺FoxP3⁺ regulatory T cells in mesenteric lymph nodes (MLN), decreases production of pro-inflammatory cytokines TNF-α and IFN-γ, and anti-inflammatory IL-10 in Peyer''s patches and large intestine, and changes the gut microbiota composition. Moreover, Lc treatment prevents lipopolysaccharide-induced TNF-α expression in RAW 264.7 cell line by down-regulating the NF-κB signaling pathway.

Conclusion/Significance

Our study provided evidence that even non-living probiotic bacteria can prevent the development of severe forms of intestinal inflammation by strengthening the integrity of intestinal barrier and modulation of gut microenvironment.     

Patterns of early gut colonization shape future immune responses of the host

The most important trigger for immune system development is the exposure to microbial components immediately after birth. Moreover, targeted manipulation of the microbiota can be used to change host susceptibility to immune-mediated diseases. Our aim was to analyze how differences in early gut colonization patterns change the composition of the resident microbiota and future immune system reactivity. Germ-free (GF) mice were either inoculated by single oral gavage of caecal content or let colonized by co-housing with specific pathogen-free (SPF) mice at different time points in the postnatal period. The microbiota composition was analyzed by denaturing gradient gel electrophoresis for 16S rRNA gene followed by principal component analysis. Furthermore, immune functions and cytokine concentrations were analyzed using flow cytometry, ELISA or multiplex bead assay. We found that a single oral inoculation of GF mice at three weeks of age permanently changed the gut microbiota composition, which was not possible to achieve at one week of age. Interestingly, the ex-GF mice inoculated at three weeks of age were also the only mice with an increased pro-inflammatory immune response. In contrast, the composition of the gut microbiota of ex-GF mice that were co-housed with SPF mice at different time points was similar to the gut microbiota in the barrier maintained SPF mice. The existence of a short GF postnatal period permanently changed levels of systemic regulatory T cells, NK and NKT cells, and cytokine production. In conclusion, a time window exists that enables the artificial colonization of GF mice by a single oral dose of caecal content, which may modify the future immune phenotype of the host. Moreover, delayed microbial colonization of the gut causes permanent changes in the immune system.     

Effect of normobaric oxygen treatment on oxidative stress and enzyme activities in guinea pig heart

Normobaric oxygen (NBO) therapy is commonly applied for the treatment of various diseases, including myocardial infarctions, but its effectiveness is controversial. Potential adverse effects of hyperoxia are related to excessive formation of free radicals. In the present study we examined the effect of 60-h NBO treatment on lipid peroxidation (LPO), activity of manganese superoxide dismutase (Mn-SOD) and mitochondrial enzymes of energy metabolism in guinea pig heart. NBO treatment resulted in significant accumulation of thiobarbituric acid reactive substances and loss of Mn-SOD activity despite slight elevation of Mn-SOD protein content. Activity of electron transport chain complex III decreased significantly, while activity of complex IV was slightly elevated and citrate synthase was unchanged. LPO, inhibition of Mn-SOD and complex III activities were more pronounced when inhaled oxygen was partially enriched with superoxide radical. In contrast, when O(2) was enriched with oxygen cation (O(2)●+), LPO and loss of Mn-SOD activity were prevented. Complex III activity in the O(2)●+-treated group remained depressed but activities of complex IV and citrate synthase were elevated. These data suggest that NBO treatment is associated with myocardial oxidative damage and attenuation of antioxidant defense, but these adverse effects can be partially attenuated by inhalation of O(2) enriched with oxygen cation.     

Oxidative modifications of cardiac mitochondria and inhibition of cytochrome c oxidase activity by 4-hydroxynonenal

4-hydroxynonenal (HNE) is a highly toxic product of lipid peroxidation (LPO). Its role in the inhibition of cytochrome c oxidase activity and oxidative modifications of mitochondrial lipids and proteins were investigated. The exposure of mitochondria isolated from rat heart to HNE resulted in a time- and concentration-dependent inhibition of cytochrome c oxidase activity with an IC50 value of 8.3 +/- 1.0 microM. Immunoprecipitation-Western blot analysis showed the formation of HNE adducts with cytochrome c oxidase subunit I. The loss of cytochrome c oxidase activity was also accompanied by reduced thiol group content and increased HNE-lysine fluorescence. Furthermore, there was a marked increase in conjugated diene formation indicating LPO induction by HNE. Fluorescence measurements revealed the formation of bityrosines and increased surface hydrophobicity of HNE-treated mitochondrial membranes. Superoxide dismutase + catalase and the HO* radical scavenger mannitol partially prevented inhibition of cytochrome c oxidase activity and formation of bityrosines. These findings suggest that HNE induces formation of reactive oxygen species and its damaging effect on mitochondria involves both formation of HNE-protein adducts and oxidation of membrane lipids and proteins by free radicals.     

2-DE analysis of a new human cell line EM-G3 derived from breast cancer progenitor cells and comparison with normal mammary epithelial cells

We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized clonal cell line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 cells may represent progenitors of the mammary epithelial cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 line with proteins from populations of normal mammary epithelial cells (NME), and determined the phenotype of both types of cells. NME cells are a mixture of both main cell types in breast epithelia, myoepithelial and luminal cells. The EM-G3 breast cancer cell line has a unique basal-like phenotype. We identified proteins that are differently expressed in these cells. Cytokeratin 16, cytokeratin 19, squamous cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intracellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 cells. The proteins up-regulated in EM-G3 cells may represent potential protein markers of mammary epithelial cells progenitors and may be important in early phase of carcinogenesis.     

Mixed chelate copper complex, Casiopeina IIgly, binds and degrades nucleic acids: a mechanism of cytotoxicity

Metal-containing drugs that interact with DNA have been designed and studied for their anticancer activity. In this study, the mixed chelate copper-based anticancer drugs, the casiopeinas, were found to bind to DNA and to degrade DNA and RNA in the presence of reducing agents (e.g. ascorbic acid). Casiopeinas binding to DNA is high affinity, with harsh wash conditions failing to remove the interaction. The reaction requires oxygen, probably involved in the generation of *OH radicals, which would be responsible for the strand breakage. The reaction was diminished by catalase, and was completely abolished by copper chelators (e.g. trientine, EDTA); however, superoxide dismutase (SOD) had no significant effect on casiopeina-mediated DNA degradation. Casiopeina IIgly (casIIgly) in the presence of ascorbate was capable of degrading RNA, plasmid and genomic DNA, and chromatin and intranuclear genetic material. Moreover, catalase and/or SOD partially protected cells, ascorbic acid enhanced and trientine, a copper chelator, abolished the cytotoxicity of casIIgly. The generation of 8-oxodG in cells exposed to casIIgly suggests that the generation of ROS is the major cause of the cytotoxicity observed and underlies the high toxicity and anticancer activity of these compounds.