In mammals, complex-I (NADH-ubiquinone oxidoreductase) of the mitochondrial respiratory chain has 31 supernumerary subunits in addition to the 14 conserved from prokaryotes to humans. Multiplicity of structural protein components, as well as of biogenesis factors, make complex-I a sensible pace-maker of mitochondrial respiration. The work reviewed here shows that the Alternative Splicing and Nonsense Mediated Decay pathways regulate the transcription products of different nuclear genes encoding subunits of complex I. Complex-I dysfunction has been found to be associated with several human diseases. Involvement of altered pattern of transcription products of complex-I genes in pathogenetic mechanisms of these diseases is examined

Recently has been acknowledged the healthy use of Bacillus and related bacteria as probiotics. A mixture reported to contain four probiotic strains of Bacillus clausii is marketed as an OTC (Over The Counter) medicinal supplement for human use. Their poliantibiotic resistant property, useful for restoring the gut microbiota during antibiotic treatment, raises the question about the risk of resistance transfer. In order to better assess the risk-benefit ratio it is important to always monitoring the pattern and stability of resistance spectra in these bacteria. In this work, we have extensively redefined the antibiotic susceptibility profile of these four probiotic strains. Resistance phenotype has been determined by screening a large number of antibiotics, including natural products (such as penicillin, vancomycin and erythromycin), and completely synthetic molecules (such as fluoroquinolones). Extensive comparison with a wild type strain belonging to the normal intestinal microbiome was carried out. The molecular basis of some resistances was determined. Observed antibiotic resistances were correlated with previous and new data in safety evaluations of these strains for human use

The developmental competence of in vitro-produced embryos is strictly related to oocyte quality. Analyses of energy and redox status parameters are emerging technologies useful for further oocyte quality characterisation. Mitochondrial (mt) activity is a necessary feature involved in cytoplasmic maturation, and the primary function of mitochondria is adenosine triphosphate (ATP) production. Mitochondria distribution pattern and ATP content are important parameters in the evaluation of oocyte metabolic activity, particularly activities driving microtubules dynamics leading to chromosomes segregation. Superoxide dismutase (SOD), a first-line antioxidant enzyme, has also been hypothesised as being associated to oocyte quality. The aim of the present study was to analyse ATP content and SOD activity in single equine oocytes examined before and after in vitro maturation. Cumulus–oocyte complexes surrounded by a compact cumulus oophorus were recovered from the ovaries of slaughtered mares and analysed before or after in vitro maturation (Ambruosi et al. 2009 Theriogenology 71, 1093–1104). After cumulus cell removal, all oocytes underwent evaluation of signs of meiotic maturation, and only those oocytes showing cumulus expansion, regular ooplasmic size (4160 mm in diameter) and morphology, and 1st polar body extrusion were selected for analysis. Adenosine triphosphate intracellular levels were analysed by luciferin-luciferase bioluminescent reaction (ATPlite, PerkinElmer, Monza, Italy). Quantification of SOD activity was performed by spectro- photometrical assay with WST1 and by polyacrylamide native gel and nitro blue tetrazolium reduction method. Intracellular ATP levels were influenced by meiotic stage in that oocytes at the germinal vesicle stage (GV, n 1⁄4 15) showed 1.25 ` 0.8 pmol cell1, whereas metaphase II (MII) oocytes (n1⁄415) showed significantly higher levels (2.29`1.69 pmolcell1; Po0.05). This is in line with our previous observations on mt distribution pattern analysed by Mitotracker Orange CMTM Ros staining and confocal microscopy (Ambruosi et al. 2009). In vitro-matured MII oocytes showed significantly higher rates of perinuclear mt distribution pattern, indicating mt aggregation around meiotic metaphase spindle, compared with GV oocytes (3/12, 25% v. 0/13, 0% in GV oocytes; Po0.05). Superoxide dismutase spectrophotometrical activity was 0.72 ` 0.55 U mg1 prot in GV oocytes (n 1⁄4 4) and 2.33 ` 0.33 U mg1 prot in MII oocytes (n 1⁄4 2; P o 0.001). In native gel SOD activity was 16 285.05 arbitrary densitometric units (ADU) in a GV oocyte and 22 501.35 ADU in a MII oocyte. To our knowledge, this is the first study reporting intracellular SOD activity in single oocytes in mammals. Moreover, this is the first study reporting ATP content in single equine oocytes. Observed quantitative differences seem to be related to meiotic stage.

A comparative proteomic approach, using two dimensional gel electrophoresis and mass spectrometry, has been developed to compare and elucidate the differences among the cellular proteomes of four closely related isogenic O/C, SIN, N/R and T, B. clausii strains during both exponential and stationary phases of growth. Image analysis of the electropherograms reveals a high degree of concordance among the four proteomes, some proteins result, however, differently expressed. The proteins spots exhibiting high different expression level were identified, by mass-spectrometry analysis, as alcohol dehydrogenase (ADHA, EC1.2.1.3; ABC0046 isoform) aldehyde dehydrogenase (DHAS, EC 1.2.1.3; ABC0047 isoform) and flagellin-protein of B. clausii KSM-k16. The different expression levels of the two dehydrogenases were confirmed by quantitative RT-PCR and dehydrogenases enzymatic activity. The different patterns of protein expression can be considered as cell proteome signatures of the different strains. (C) 2011 Elsevier B.V. All rights reserved.

Background: The spore-bearing alkaliphilic Bacillus species constitute a large, heterogeneous group of microorganisms, important for their ability to produce enzymes, antibodies and metabolites of potential medical use. Some Bacillus species are currently being used for manufacturing probiotic products consisting of bacterial spores, exhibiting specific features (colonization, immune-stimulation and antimicrobial activity) that can account for their claimed probiotic properties. In the present work a comparative proteomic study was performed aimed at characterizing the secretome of four closely related isogenic O/C, SIN, N/R and T B. clausii strains, already marketed in a pharmaceutical mixture as probiotics. Results: Proteomic analyses revealed a high degree of concordance among the four secretomes, although some proteins exhibited considerable variations in their expression level in the four strains. Among these, some proteins with documented activity in the interaction with host cells were identified, such as the glycolytic enzyme enolase, with a putative plasminogen-binding activity, GroEL, a molecular chaperone shown to be able to bind to mucin, and flagellin protein, a structural flagella protein and a putative immunomodulation agent. Conclusion: This study shows, for the first time, differences in the secretome of the OC, SIN, NR and T B. clausii strains. These differences indicate that specific secretome features characterize each of the four strains despite their genotypic similarity. This could confer to the B. clausii strains specific probiotic functions associated with the differentially expressed proteins and indicate that they can cooperate as probiotics as the secretome components of each strain could contribute to the overall activity of a mixed probiotic preparation.

In: Reproduction, Fertility and Development, Vol 25(1), 2013 Juvenile in vitro embryo transfer in farm animals reduces the generation interval and increases the rate of genetic gain. In human reproductive medicine, it enables the preservation of female fertility of young patients affected by cancer or by premature ovarian failure. The developmental competence of in vitro-produced juvenile embryos is strictly related to oocyte quality. The aim of the present study was to analyse the developmental potential and the mitochondrial/oxidative status of ovine prepubertal oocytes matured in vitro to clarify their suitability in juvenile in vitro embryo transfer programs. Oocytes from the ovaries of slaughtered prepubertal lambs (less than 6 months of age) were analysed after in vitro maturation. After cumulus cell removal, oocytes at the metaphase II stage (MII) underwent either IVF plus in vitro embryo culture (Experiment 1; n 1⁄4 200; Bogliolo et al. 2011 Reprod. Fert. Dev. 23, 809–817) or confocal analysis of mitochondria (mt) and reactive oxygen species (ROS) fluorescence distribution, intensity, and colocalization (Experiment 2; n 1⁄4 30; Martino et al. 2012 Fertil. Steril. 97, 720–728) or scavenger enzyme [superoxide dismutase (Ambruosi et al. 2011 PLoS ONE 6, e27452) and catalase (Beers and Sizer 1952 J. Biol. Chem. 195, 133–140)] activity analyses in cell lysates of individual oocytes (Experiment 3; n 1⁄4 7). In Experiment 1, 150 of 200 MII oocytes (75%) cleaved after 30 h of in vitro embryo culture, and 36 of 150 2- to 4-cell-stage embryos (24%) reached the blastocyst stage at Day 8. In Experiment 2, 60 of 111 (54%) oocytes selected for in vitro maturation culture reached the MII stage, and 30 of them (50%) with a regular size (.150 mm in diameter) and morphology were analysed for bioenergy/redox parameters. Fourteen of 30 oocytes (47%) showed a heterogeneous (perinuclear, pericortical, or both) mt distribution pattern, and the remaining 16 of 30 oocytes (53%) showed a homogeneous distribution of small mt aggregates. Intracellular ROS were uniformly distributed, thus not corresponding to the mt distribution pattern. Fluorescent intensity of mt and ROS labelling, expressed as arbitrary densitometric units, were 821.4 ` 274.7 and 737.6 ` 226.5 in oocytes with a heterogeneous pattern and 723.7 ` 371.6 and 831.7 ` 263.7 in oocytes with a homogeneous pattern, respectively (not significant). The mt/ROS colocalization (Pearson correlation coefficient) did not differ between heterogeneous (0.47`0.2) and homogeneous (0.51`0.09; not significant) oocytes. In Experiment 3, superoxide dismutase (n 1⁄4 4) and catalase activity (n 1⁄4 3) values were 1.09 ` 0.03 and 10.63 ` 1.96 IU mg1 of protein, respectively. This study provides basal values of bioenergy/redox parameters in prepubertal lamb MII oocytes as related to their developmental potential and may increase the knowledge of prepubertal oocyte physiology compared with their young adult counterparts.

Current hypothesis suggest that tumors can originate from adult cells after a process of 'reprogramming' driven by genetic and epigenetic alterations. These cancer cells, called cancer stem cells (CSCs), are responsible for the tumor growth and metastases. To date, the research effort has been directed to the identification, isolation and manipulation of this cell population. Independently of whether tumors were triggered by a reprogramming of gene expression or seeded by stem cells, their energetic metabolism is altered compared with a normal cell, resulting in a high aerobic glycolytic 'Warburg' phenotype and dysregulation of mitochondrial activity. This metabolic alteration is intricately linked to cancer progression.The aim of this work has been to demonstrate the possibility of differentiating a neoplastic cell toward different germ layer lineages, by evaluating the morphological, metabolic and functional changes occurring in this process. The cellular differentiation reported in this study brings to different conclusions from those present in the current literature. We demonstrate that 'in vitro' neuroblastoma cancer cells (chosen as experimental model) are able to differentiate directly into osteoblastic (by rapamycin, an mTOR inhibitor) and hepatic lineage without an intermediate 'stem' cell step. This process seems owing to a synergy among few master molecules, metabolic changes and scaffold presence acting in a concerted way to control the cell fate.

ABSTRACT - The aim of the present study was to analyze the effects of in vitro exposure to Di-(2-ethylhexyl) phthalate (DEHP), an industrial plasticizer, on cumulus-oocyte maturation and energy/oxidative status in the horse. After in vitro maturation (IVM) in presence of 0.12, 12 and 1200 μM DEHP, cumulus cells (CCs) were removed and evaluated for apoptosis and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase-II stage; MII) oocytes were further evaluated for cytoplasmic energy/oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 μM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 μM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial (mt) distribution patterns, apparent energy status, intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with the antioxidant N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without improving oocyte maturation. In conclusion, in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy/oxidative parameters in matured oocytes.

Juvenile in vitro embryo production from oocytes of prepubertal subjects is a promising assisted reproductive technology (ART). In farm animals, it reduces the generation gap and in human reproductive medicine, it helps to overcome premature ovarian failure. Oxidative stress in germ cell in vitro culture and cryopreservation procedures is an emerging problem in ART and studies aimed to evaluate the potentially antioxidant activity of natural bioactive compounds, such as phenolic compounds and polyphenols, present in plant-derived by-products would be beneficial to improve cryopreservation and in vitro culture protocols of prepubertal oocytes. Verbascoside (VB) or acteoside is a phenolic bioactive compound with known antioxidant activity, which is present in a good amount in olive oil mill wastewater (1). The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. Previously published methods were used for: VB extraction, purification, HPLC analysis and uptake by the cumulus-oocyte complex (1); in vitro oocyte maturation (IVM) and assessment of bioenergy redox biomarkers, such as mitochondrial distribution pattern and activity; intracellular reactive oxygen species (ROS) levels; mitochondria/ROS colocalization; catalase and total superoxide dismutase activities (2); oocyte vitrification (3); in vitro fertilization and embryo development (4). VB effects were tested at microM concentrations (1.03, 2.06 and 4.11). In fresh oocytes, 4.11 microM VB exerted pro-oxidant short-term effects, i.e. catalase activity increase and uncoupled increases of ooplasmic mitochondria and reactive oxygen species (ROS) specific fluorescence signals. It also induced pro-oxidant long-term effects, i.e reduced blastocyst formation rate, thus indicating an affected developmental competence of exposed oocytes. In vitrified oocytes, 1.03 microM VB increased ROS levels. Pro-oxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as being almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of IVM culture, may have contributed to significantly increase oocyte oxidation. In conclusion, VB, added at microM concentrations in a continuative 24 hours IVM exposure protocol, acts as a pro-oxidant molecule by impairing bioenergetic potential, oxidative status and embryo developmental competence of prepubertal lamb oocytes. Further studies are ongoing aimed to identify suitable conditions, lower concentrations and/or shorter exposure times, to figure out VB antioxidant effects in juvenile ARTs.

Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 µM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos even though further studies are necessary to confirm this possibility.

Increased ROS (cellular reactive oxygen species) are characteristic of both fibrosis and tumour development. ROS induce the trans-differentiation to myofibroblasts, the activated form of fibroblasts able to promote cancer progression. Here, we report the role of ROS produced in response to dysfunctions of mitochondrial complex I, in fibroblast activation and in tumour progression. We studied human fibroblasts with mitochondrial dysfunctions of complex I, leading to hyperproduction of ROS. We demonstrated that ROS level produced by the mutated fibroblasts correlates with their activation. The increase of ROS in these cells provides a greater ability to remodel the extracellular matrix leading to an increased motility and invasiveness. Furthermore, we evidentiated that in hypoxic conditions these fibroblasts cause HIF-1 alpha stabilization and promote a proinvasive phenotype of human melanoma cells through secretion of cytokines. These data suggest a possible role of deregulated mitochondrial ROS production in fibrosis evolution as well as in cancer progression and invasion.

The NDUFS4 subunit of complex I of the mammalian respiratory chain has a fully conserved carboxy-terminus with a canonical RVSTK phosphorylation site. Immunochemical analysis with specific antibodies shows that the serine in this site of the protein is natively present in complex I in both the phosphorylated and non-phosphorylated state. Two-dimensional IEF/SDS-PAGE electrophoresis, (32)P labelling and immunodetection show that "in vitro" PKA phosphorylates the serine in the C-terminus of the NDUFS4 subunit in isolated bovine complex I. (32)P labelling and TLC phosphoaminoacid mapping show that PKA phosphorylates serine and threonine residues in the purified heterologous human NDUFS4 protein.

Verbascoside (VB) is a bioactive compound from olive oil mill wastewater with known antioxidant activity. Oxidative stress in germ cell in vitro culture and cryopreservation procedures is an emerging problem in assisted reproductive technology (ART). Juvenile in vitro embryo production from oocytes of prepubertal subjects is a promising ART because, in farm animals, it reduces the generation gap and in human reproductive medicine, it helps to overcome premature ovarian failure. The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. In fresh oocytes, when used at high concentration, VB exerted pro-oxidant short-term effects, i.e. catalase activity increase and uncoupled increases of ooplasmic mitochondria and reactive oxygen species (ROS) specific fluorescence signals and long-term effects, i.e reduced blastocyst formation rate. In vitrified oocytes, it increased ROS levels. Pro-oxidant VB effects in ovine prpubertal oocytes could be related to higher VB accumulation, which was found as being almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of in vitro maturation (IVM) culture, may have contributed to significantly increase oocyte oxidation. Further studies are needed to identify suitable conditions, lower concentrations and/or shorter exposure times, to figure out VB antioxidant effects in juvenile ARTs.

Background The 14-3-3 proteins family consists of seven proteins that are highly conserved molecular chaperones with roles in the regulation of metabolism, signal transduction, cell cycle control, protein trafficking and apoptosis. Their role in several pathologies has been reported. In this study, we investigated the mRNA and protein expression of the 14-3-3s in rat brain and liver in the early stage of Type-1 diabetes (T1D). Material and methods Diabetes was induced by a single intraperitoneal injection (70 mg/kg bw) of freshly prepared streptozotocin (STZ), and, after 3 weeks of treatment, brain and liver nuclei and cytosolic extracts were prepared. Quantitative real-time PCR and Western blotting analyses were performed to evaluate mRNA and protein expression for each of the seven 14-3-3s. Results In nondiabetic control rats, the expression profile of 14-3-3s revealed a tissue-specific distribution, and the expression level of each isoform was found higher in the brain than in the liver. In the diabetic brain, mRNA and protein levels of the 14-3-3b, e, f, g and h were lower; 14-3-3r mRNA significantly increased while its protein level decreased. In the diabetic liver, the mRNA of 14-3-3c, 14-3-3h and 14-3-3r significantly increased, but only the 14-3-3c protein level increased. Overall, in diabetic animals, the changes in the expression levels of brain 14-3-3s were much more pronounced than in the liver. Conclusion Our results indicate that during the early phase of STZ-induced T1D, the 14-3-3 proteins are affected in an isoform- and tissue-specific way

OBJECTIVES: Different nuclear genes are thought to be involved in the regulation of the complex phenotype of metabolic syndrome (MS) and their number is increasing. A mutation in mitochondrial DNA (mtDNA), T4291C in transfer RNA isoleucine (tRNAile), has been associated with MS in a large American family. In addition, a mtDNA T16189C variant, already known to be associated with insulin resistance and type 2 diabetes mellitus in Caucasians, seems to underlie susceptibility to MS in the Chinese population. Our aim was to verify the T4291C and T16189C variants in subjects affected by different phenotypes of MS. METHODS: Seventy patients with MS and 35 healthy individuals were investigated for the presence of the mtDNA variants by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: The T4291C variant was absent in patients and in controls. The T16189C variant was more frequent in patients with MS than in control subjects (21.4% versus 5.7%, P<0.04) and was associated with hypertension (P=0.01), waist circumference (P=0.02), body mass index (P=0.009), visceral fat thickness (P=0.04), homeostasis model assessment (P=0.03), and the number of MS diagnostic criteria (P=0.01). CONCLUSION: The mtDNA T16189C variant is associated with MS and its different clinical expressions. Prospective studies are warranted to establish the clinical relevance of this association. Copyright © 2011 Elsevier Inc. All rights reserved.

2-Arachidonoylglicerol (2-AG) is an endocannabinoid that mimics the pharmacological effects of Delta(9) tetrahydrocannabinol, the psychoactive component of the plant Cannabis sativa. It is present in many mammalian tissues, such as brain, liver, spleen, heart and kidney, where it exerts different biological effects either receptor mediated or independently of receptor activation. This work analyzes the effects of 2-AG on liver mitochondrial functions. It is shown that 2-AG causes a relevant decrease of calcium induced cyclosporine A sensitive cytochrome c release from mitochondria, a process representing an early event of the apoptotic program. Cyclosporin sensitive matrix swelling and ROS production measured under the same conditions are, on the contrary, almost unaffected or even enhanced, respectively, by 2-AG. Furthemore, 2-AG is found to stimulate resting state succinate oxidase activity and to inhibit oligomycin sensitive FoF1 ATP synthase activity. All these effects are apparently associated with 2-AG dependent alteration in the fluidity of the mitochondrial membranes, which was measured as generalized polarization of laurdan fluorescence.

In this study the impact of hUPF1 and hUPF2 knockdown on alternative splicing (AS) isoforms of different genes encoding subunits of respiratory chain complex I and complex IV is described. As expected, loss of both hUPF1 and hUPF2 led to impairment of nonsense-mediated mRNA decay (NMD) and accumulation of PTC-containing NMD substrates generated by both complex I and complex IV genes. The levels of some complex I splice variants, which did not contain PTC as well as the level of some complex I canonical transcripts were, however, affected only by hUPF1 knockdown. This finding confirms that NMD plays a role in the maintenance of the transcriptome integrity and reveals a specific impact of hUPF1 on the regulation of complex I genes.

A comparative analysis of terminal respiratory enzymes has been performed on four strains of Bacillus clausii used for preparation of a European probiotic. These four strains originated most probably from a common ancestor through early selection of stable clones for industrial propagation. They exhibit a low level of intra-specific diversity and a high degree of genomic conservation, making them an attractive model to study the different bioenergetics behaviors of alkaliphilic bacilli. The analysis of the different bioenergetics responses has been carried out revealing striking differences among the strains. Two out of the four strains have shown a functional redundancy of the terminal part of the respiratory chain. The biochemical data correlate with the expression level of the mRNA of cytochrome c oxidase and quinol oxidase genes (heme-copper type). The consequences of these different bioenergetics behaviors are also discussed.