New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology

Background  

Oxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H₂O₂ from 0 to 130 ppm.     

Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice

Unlike the adjustable gastric banding procedure (AGB), Roux-en-Y gastric bypass surgery (RYGBP) in humans has an intriguing effect: a rapid and substantial control of type 2 diabetes mellitus (T2DM). We performed gastric lap-band (GLB) and entero-gastro anastomosis (EGA) procedures in C57Bl6 mice that were fed a high-fat diet. The EGA procedure specifically reduced food intake and increased insulin sensitivity as measured by endogenous glucose production. Intestinal gluconeogenesis increased after the EGA procedure, but not after gastric banding. All EGA effects were abolished in GLUT-2 knockout mice and in mice with portal vein denervation. We thus provide mechanistic evidence that the beneficial effects of the EGA procedure on food intake and glucose homeostasis involve intestinal gluconeogenesis and its detection via a GLUT-2 and hepatoportal sensor pathway.     

Mitochondrial oxygen consumption inhibition importance for TMT-dependent cell death in undifferentiated PC12 cells

The evolving role of mitochondria as a target for different death-inducing noxae prompted us to investigate trimethyltin (TMT)-dependent effects on mitochondrial functionality. For this purpose, we used a homogeneous cell culture model represented by undifferentiated PC12 cells. Mitochondria isolated from PC12 cells treated with TMT for 6, 12 and 24h, showed a time-dependent inhibition of ADP-stimulated oxygen consumption using succinate or glutamate/malate as substrate. Using a fluorescent assay, the effect of TMT on mitochondrial membrane potential (delta Psi) in PC12 cells was also determined. After 24h in culture, a strong loss of mitochondrial membrane potential (delta Psi) was observed in TMT-treated cells. Collapse of mitochondrial membrane potential correlated with an increased expression of bax/bcl-2 ratio, as evaluated by polymerase chain reaction. Western blotting and spectrophotometric analysis showed that cytochrome c release and activation of caspase 3 were concurrently induced. Our findings suggest that inhibition of mitochondrial respiration represents the early toxic event for cell death in PC12 due to trimethyltin.     

Synthetic prions with novel strain-specified properties

Prions are infectious proteins that possess multiple self-propagating structures. The information for strains and structural specific barriers appears to be contained exclusively in the folding of the pathological isoform, PrP^Sc. Many recent studies determined that de novo prion strains could be generated in vitro from the structural conversion of recombinant (rec) prion protein (PrP) into amyloidal structures. Our aim was to elucidate the conformational diversity of pathological recPrP amyloids and their biological activities, as well as to gain novel insights in characterizing molecular events involved in mammalian prion conversion and propagation. To this end we generated infectious materials that possess different conformational structures. Our methodology for the prion conversion of recPrP required only purified rec full-length mouse (Mo) PrP and common chemicals. Neither infected brain extracts nor amplified PrP^Sc were used. Following two different in vitro protocols recMoPrP converted to amyloid fibrils without any seeding factor. Mouse hypothalamic GT1 and neuroblastoma N2a cell lines were infected with these amyloid preparations as fast screening methodology to characterize the infectious materials. Remarkably, a large number of amyloid preparations were able to induce the conformational change of endogenous PrP^C to harbor several distinctive proteinase-resistant PrP forms. One such preparation was characterized in vivo habouring a synthetic prion with novel strain specified neuropathological and biochemical properties.     

Unraveling amyloid toxicity pathway in NIH3T3 cells by a combined proteomic and 1H‐NMR metabonomic approach

A range of debilitating human diseases is known to be associated with the formation of stable highly organized protein aggregates known as amyloid fibrils. The early prefibrillar aggregates behave as cytotoxic agents and their toxicity appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increase in free Ca²⁺ that lead to apoptotic or necrotic cell death. However, specific signaling pathways that underlie amyloid pathogenicity remain still unclear. This work aimed to clarify cell impairment induced by amyloid aggregated. To this end, we used a combined proteomic and one‐dimensional ¹H‐NMR approach on NIH‐3T3 cells exposed to prefibrillar aggregates from the amyloidogenic apomyoglobin mutant W7FW14F. The results indicated that cell exposure to prefibrillar aggregates induces changes of the expression level of proteins and metabolites involved in stress response. The majority of the proteins and metabolites detected are reported to be related to oxidative stress, perturbation of calcium homeostasis, apoptotic and survival pathways, and membrane damage. In conclusion, the combined proteomic and ¹H‐NMR metabonomic approach, described in this study, contributes to unveil novel proteins and metabolites that could take part to the general framework of the toxicity induced by amyloid aggregates. These findings offer new insights in therapeutic and diagnostic opportunities. J. Cell. Physiol. 228: 1359–1367, 2013. © 2012 Wiley Periodicals, Inc.     

Drugs based on natural compounds: recent achievements and future perspectives

Phytochemistry Reviews -