Respiration-dependent Removal of Exogenous H2O2 in Brain Mitochondria INHIBITION BY Ca2+

Abstract In brain mitochondria, state 4 respiration supported by the NAD-linked substrates glutamate/malate in the presence of EGTA promotes a high rate of exogenous H2O2 removal. Omitting EGTA decreases the H2O2 removal rate by almost 80%. The decrease depends on the influx of contaminating Ca2+, being prevented by the Ca2+ uniporter inhibitor ruthenium red. Arsenite is also an inhibitor (maximal effect ∼40%, IC50, 12 μm). The H2O2 removal rate (EGTA present) is decreased by 20% during state 3 respiration and by 60–70% in fully uncoupled conditions. H2O2 removal in mitochondria is largely dependent on glutathione peroxidase and glutathione reductase. Both enzyme activities, as studied in disrupted mitochondria, are inhibited by Ca2+. Glutathione reductase is decreased by 70% with an IC50 of about 0.9 μm, and glutathione peroxidase is decreased by 38% with a similar IC50. The highest Ca2+ effect with glutathione reductase is observed in the presence of low concentrations of H2O2. With succinate as substrate, the removal is 50% less than with glutamate/malate. This appears to depend on succinate-supported production of H2O2 by reverse electron flow at NADH dehydrogenase competing with exogenous H2O2 for removal. Succinate-dependent H2O2 is inhibited by rotenone, decreased ΔΨ, as described previously, and by ruthenium red and glutamate/malate. These agents also increase the measured rate of exogenous H2O2 removal with succinate. Succinate-dependent H2O2 generation is also inhibited by contaminating Ca2+. Therefore, Ca2+ acts as an inhibitor of both H2O2 removal and the succinate-supported H2O2 production. It is concluded that mitochondria function as intracellular Ca2+-modulated peroxide sinks

Copia and Gypsy retrotransposons activity in sunflower (Helianthus annuus L.)

<p>Abstract</p> <p>Background</p> <p>Retrotransposons are heterogeneous sequences, widespread in eukaryotic genomes, which refer to the so-called mobile DNA. They resemble retroviruses, both in their structure and for their ability to transpose within the host genome, of which they make up a considerable portion. <it>Copia</it>- and <it>Gypsy</it>-like retrotransposons are the two main classes of retroelements shown to be ubiquitous in plant genomes. Ideally, the retrotransposons life cycle results in the synthesis of a messenger RNA and then self-encoded proteins to process retrotransposon mRNA in double stranded extra-chromosomal cDNA copies which may integrate in new chromosomal locations.</p> <p>Results</p> <p>The RT-PCR and IRAP protocol were applied to detect the presence of <it>Copia </it>and <it>Gypsy </it>retrotransposon transcripts and of new events of integration in unstressed plants of a sunflower (<it>Helianthus annuus </it>L.) selfed line. Results show that in sunflower retrotransposons transcription occurs in all analyzed organs (embryos, leaves, roots, and flowers). In one out of sixty-four individuals analyzed, retrotransposons transcription resulted in the integration of a new element into the genome.</p> <p>Conclusion</p> <p>These results indicate that the retrotransposon life cycle is firmly controlled at a post transcriptional level. A possible silencing mechanism is discussed.</p

Immersed boundary method: performance analysis of popular finite element spaces

The aim of this paper is to understand the performances of different finite elements in the space discretization of the Finite Element Immersed Boundary Method. In this exploration we will analyze two popular solution spaces: Hood-Taylor and Bercovier- Pironneau (P1-iso-P2). Immersed boundary solution is characterized by pressure discontinuities at fluid structure interface. Due to such a discontinuity a natural enrichment choice is to add piecewise constant functions to the pressure space. Results show that P1 + P0 pressure spaces are a significant cure for the well known “boundary leakage” affecting IBM. Convergence analysis is performed, showing how the discontinuity in the pressure is affecting the convergence rate for our finite element approximation

Mass preserving distributed langrage multiplier approach to immersed boundary method

This research is devoted to mass conservation and CFL properties of the Finite Elements Immersed Boundary Method. We first explore an enhanced higher order scheme applied to the Finite Element Immersed Boundary Method technique introduced by Boffi and Gastaldi. This technique is based on a Pointwise (PW) formulation of the kinematic condition, and higher order elements show better conservation properties than the original scheme. A further improvement with respect to the classical PW formulation is achieved introducing a fully variational Distributed Lagrange Multiplier (DLM) formulation. Numerical experiments show that DLM is not affected by any CFL condition. Furthermore the mass conservation properties of this method are extremely competitive

Prostacyclin and Sodium Nitroprusside Inhibit the Activity of the Platelet Inositol 1,4,5-Trisphosphate Receptor and Promote Its Phosphorylation

Prostaglandin I2 (PGI2) and sodium nitroprusside (SNP) induce a rapid decay of the thrombin-promoted increase of [Ca2+]i in aspirin-treated platelets incubated in the absence of external Ca2+. The mechanism of their effect was studied with a new method which utilizes ionomycin to increase [Ca2+]i, followed by bovine serum albumin (BSA) to remove the Ca2+ ionophore. The rapid decay of [Ca2+]i after BSA is mostly due to the reuptake into the stores, since it is strongly inhibited by the endomembrane Ca2+-ATPase inhibitor thapsigargin. PGI2 and SNP are without effect on the BSA-promoted decay both with and without thapsigargin, showing that they do not affect the activity of the Ca2+-ATPases. The fast decay of [Ca2+]i after BSA is decreased by thrombin which produces the Ca2+ releaser inositol 1,4,5-trisphosphate (InsP3), thus counteracting the activity of the endomembrane Ca2+ pump. When added after thrombin, PGI2 and SNP accelerate the BSA-activated decay of [Ca2+]i. However, under the same conditions, they do not decrease the concentration of InsP3. In saponin-permeabilized platelets, cAMP and cGMP counteract the Ca2+ release induced by exogenous InsP3. Their inhibitory effect disappears at high InsP3 concentrations. This demonstrates that PGI2 and SNP potentiate Ca2+ reuptake by inhibiting the InsP3 receptor. Two bands of approximately 260 kDa are recognized by a monoclonal antibody recognizing the C-terminal region of the InsP3 receptor. Both are phosphorylated rapidly, the heavier more intensely, in the presence of PGI2 and SNP. The phosphorylation of the InsP3 receptor is fast enough to be compatible with its involvement in the inhibition of the receptor by cyclic nucleotides

Different histories of two highly variable LTR retrotransposons in sunflower species

In the Helianthus genus, very large intra- and interspecific variability related to two specific retrotransposons of Helianthus annuus (Helicopia and SURE) exists. When comparing these two sequences to sunflower sequence databases recently produced by our lab, the Helicopia family was shown to belong to the Maximus/SIRE lineage of the Sirevirus genus of the Copia superfamily, whereas the SURE element (whose superfamily was not even previously identified) was classified as a Gypsy element of the Ogre/Tat lineage of the Metavirus genus. Bioinformatic analysis of the two retrotransposon families revealed their genomic abundance and relative proliferation timing. The genomic abundance of these families differed significantly among 12 Helianthus species. The ratio between the abundance of long terminal repeats and their reverse transcriptases suggested that the SURE family has relatively more solo long terminal repeats than does Helicopia. Pairwise comparisons of Illumina reads encoding the reverse transcriptase domain indicated that SURE amplification may have occurred more recently than that of Helicopia. Finally, the analysis of population structure based on the SURE and Helicopia polymorphisms of 32 Helianthus species evidenced two subpopulations, which roughly corresponded to species of the Helianthus and Divaricati/Ciliares sections. However, a number of species showed an admixed structure, confirming the importance of interspecific hybridisation in the evolution of this genus. In general, these two retrotransposon families differentially contributed to interspecific variability, emphasising the need to refer to specific families when studying genome evolution

Variability in LTR-retrotransposon redundancy and proximity to genes between sunflower cultivars and wild accessions.

The sunflower (Helianthus annuus) genome contains a very large proportion of transposable elements, especially long-terminal-repeat retrotransposons. Being knowledge on the retrotransposon-related variability within this species still limited, we performed a quantitative and qualitative survey of intraspecific variation of LTR-retrotransposon fraction of the genome across different genotypes of H. annuus, using next generation sequencing technologies. First, we characterized the repetitive component of a sunflower homozygous experimental line, using 454 reads, and prepared a library of retrotransposon-related sequences. Then, we analysed the LTRretrotransposon fraction of 7 wild accessions and 8 cultivars of sunflowerby mapping Illumina reads of the 15 genotypes onto the library. We observed large variations in redundancy among genotypes, at both superfamily and family levels. In another analysis, we mapped Illumina paired reads of the 15 genotypes onto two sets of sequences, i.e. retrotransposons and protein-encoding sequences, and evaluated the extent of retrotransposon proximity to genes in the 15 genomes by counting the number of paired reads of which one mapped onto a retrotransposon and the other onto a gene. Large variability among genotypes was ascertained also for retrotransposonproximity to genes. Both retrotransposon redundancy and proximity to genes showed different behaviour among retrotransposon families and also between cultivated and wild genotypes, indicating a possible involvement in sunflower domestication

A survey of variability in LTR-retrotransposon abundance and proximity to genes between wild and cultivated sunflower genotypes

Sunflower (Helianthus annuus) is an important crop species of the Asteraceae family. Recent characterization of sunflower repetitive fraction has shown that the genome of this species contains a very large proportion of transposable elements, especially long-terminal-repeat retrotransposons. However, knowledge on the retrotransposon-related variability within this species is still limited. We used next generation sequencing technologies to perform a quantitative and qualitative survey of intraspecific variation of the retrotransposon fraction of the genome across different genotypes of H. annuus. First, we characterized the repetitive component of a sunflower homozygous experimental line, using 454 reads, and prepared a library of retrotransposon-related sequences. Then, we analysed the retrotransposon fraction of 7 wild accessions and 8 cultivars of H. annuus by mapping Illumina reads of the 15 genotypes onto the library. We observed large variations in redundancy among genotypes, at both superfamily and family levels. In another analysis, we mapped Illumina paired reads of the 15 genotypes onto two sets of sequences, i.e. retrotransposons and protein-encoding sequences, and evaluated the extent of retrotransposon proximity to genes in the 15 genomes by counting the number of paired reads of which one mapped onto a retrotransposon and the other onto a gene. Large variability among genotypes was ascertained also for retrotransposon proximity to genes. Both retrotransposon redundancy and proximity to genes showed different behaviour among retrotransposon families and also between cultivated and wild genotypes, indicating a possible involvement in sunflower domestication

A survey of Gypsy and Copia LTR-retrotransposon superfamilies and lineages and their distinct dynamics in the Populus trichocarpa (L.) genome

In this work, we report a comprehensive study of long terminal repeat retrotransposons of Populus trichocarpa. Our research group studied the retrotransposon component of the poplar genome in 2012, isolating 1479 putative full-length elements. However, in that study, it was not possible to identify the superfamily to which the majority of isolated full-length elements belonged. Moreover, during recent years, the genome sequence of P. trichocarpa has been updated, deciphering thek sequences of a number of previously unresolved loci. In this work, we performed a complete scan of the updated version of the genome sequence to isolate full-length retrotransposons based on sequence and structural features. The new dataset showed a reduced number of elements (958), and 21 fulllength elements were discovered for the first time. The majority of retroelements belonged to the Gypsy superfamily (57%), while Copia elements amounted to 41.1% of the dataset. Fulllength elements were dispersed throughout the chromosomes. However, Gypsy and, to a lesser extent, Copia elements accumulated preferentially at putative centromeres. Gypsy elements were more active in retrotransposition than Copia elements, with the exception of during the past million years, in which Copia elements were the most active. Improved annotation procedures also allowed us to determine the specific lineages to which isolated elements belonged. The three Gypsy lineages, Athila, OGRE, and Chromovirus (in the decreasing order), were by far the most abundant. On the other hand, each identified Copia lineage represented less than 1 % of the genome. Significant differences in the insertion age were found among lineages, suggesting specific activation mechanisms. Moreover, different chromosomal regions were affected by retrotransposition in different ages. In all chromosomes, putative pericentromeric regions were filled with elements older than themean insertion age. Overall, results demonstrate structural and functional differences among plant retrotransposon lineages and further support the view of retrotransposons as a community of different organisms in the genome

Genome editing: il futuro (prossimo) del miglioramento genetico delle piante

Genome editing, or genome editing with engineered nucleases, is a technology that, using engineered nucleases, allows site-specific single-base mutations or the insertion, deletion or replacement of DNA sequences in a specific site in the genome of an organism. Genome editing is based on the induction of double strand breaks (DSBs) in the DNA in the locus of interest to introduce mutations in that locus. In fact, after DSB induction, the damage will be repaired by processes (the non-homologous end joining and/or the homology-directed repair), that occur naturally in the cells and during which mutations may occur. DSBs can be induced by different nucleases, all capable of specifically recognising a locus in the genome. The most promising is the CRISPR/Cas system, for ease of designing nucleases with sequence specificity and for the fact that it can be used in nearly every organism. In the CRISPR/Cas9 system, the recognition of the DNA sequence to be modified is operated by an RNA sequence. After successful DNA DSB, the cell proceeds with the repair of DNA. Generally, the cell uses non-homologous end joining, which produces substitutions, insertions and deletions of nucleotides in the damaged DNA site, and usually leads to loss of function of the target gene. When using this mode, the genome editing can be considered a biological site-specific mutagenesis, different from the mutagenesis induced by physical or chemical agents which randomly induce mutations through the entire genome. On the contrary, when homologydirected repair is involved, genome editing can be considered a predetermined biological mutagenesis that modifies or corrects the target gene in the sense determined by the investigator. Applying genome editing to plants requires also ancillary technologies, according to the species and cell types. First, in vitro culture techniques, especially protoplast cultures, might be necessary for the production of cells that can be subjected to the nuclease treatment. Then, transformation vectors (Agrobacterium, viruses or biolistic methods) are needed to enable the transfer of the components required for genome editing to the plant cell. The vectors may be stable or transient; in the latter case, both the possible cytotoxicity of constitutively expressed nucleases and the production of transgenic plants would be avoided. Concerning the first results obtained using this technology, mutations in target genes of cultivated plants were obtained mostly through non-homologous end joining for traits related to morphology, quality and to the resistance to pathogens and herbicides, in both herbaceous and woody species. Results were also reported exploiting the homology-directed repair. Overall, the genome editing technology proved suitable to introduce precise and predictable gene mutations directly into elite cultivars, reducing the duration of traditional crossing and backcrossing breeding, with the possibility to modi fy more than one genes per experiment . Although many advances in genome editing technology have been achieved in recent years, some technical problems remain to be solved, including the need for increasing the efficiency of the system, the production of off-target mutations, the influence of chromatin structure on the editing efficiency, the possible side effects on genes lying close to target genes and the efficiency of the technology in polyploid species (where many copies of target genes occur). In conclusion, the CRISPR/Cas system has emerged as the most important tool for the future of genetics because of its simplicity, versatility and efficiency. It will have a major impact on both basic and applied research and will be used to produce cultivars with improved disease resistance, with a higher nutritional value, and able to survive climate changes, more suitable as bioenergy crops, producing useful chemicals and biomolecule