Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene

Exon 3 of the human apolipoprotein A-II (apoA-II) gene is efficiently included in the mRNA although its acceptor site is significantly weak because of a peculiar (GU)₁₆ tract instead of a canonical polypyrimidine tract within the intron 2/exon 3 junction. Our previous studies demonstrated that the SR proteins ASF/SF2 and SC35 bind specifically an exonic splicing enhancer (ESE) within exon 3 and promote exon 3 splicing. In the present study, we show that the ESE is necessary only in the proper context. In addition, we have characterized two novel sequences in the flanking introns that modulate apoA-II exon 3 splicing. There is a G-rich element in intron 2 that interacts with hnRNPH1 and inhibits exon 3 splicing. The second is a purine rich region in intron 3 that binds SRp40 and SRp55 and promotes exon 3 inclusion in mRNA. We have also found that the (GU) repeats in the apoA-II context bind the splicing factor TDP-43 and interfere with exon 3 definition. Significantly, blocking of TDP-43 expression by small interfering RNA overrides the need for all the other cis-acting elements making exon 3 inclusion constitutive even in the presence of disrupted exonic and intronic enhancers. Altogether, our results suggest that exonic and intronic enhancers have evolved to balance the negative effects of the two silencers located in intron 2 and hence rescue the constitutive exon 3 inclusion in apoA-II mRNA.     

Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene

Strawberry (Fragaria x ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry.     

Viral hemorrhagic septicaemia virus (VHSV) up-regulates the cytotoxic activity and the perforin/granzyme pathway in the rainbow trout RTS11 cell line

A survey of immune-relevant genes that might be up-regulated in response to viral hemorrhagic septicaemia virus (VHSV) in the rainbow trout monocyte-macrophage cell line, RTS11, unexpectedly revealed an increased expression of perforin (PRF) and granzyme (GRZ) genes, which represent components of the major cytotoxic pathway. The natural killer-enhancing factor (NKEF), also known to modulate cytotoxic activity, was up-regulated at the gene but strikingly down-regulated at protein level. The expression of these genes was not affected in head kidney leukocytes (HKLs) infected with VHSV, leading us to evaluate the potential cytotoxic activity of RTS11 and HKLs. For the first time, the cytotoxic activity of RTS11 against xenogeneic targets has been demonstrated, although this was modest relative to HKLs. Yet the activity in RTS11 was significantly increased by VHSV, as in HKLs. This cytotoxic activity elicited by viral infection appeared to require viral gene expression because inactivated VHSV failed to increase RTS11 cytotoxic activity. As for other immune functions, RTS11 cells provide a model for further studying cytotoxic activities of fish monocyte-macrophages.     

Variations in Amazon forest productivity correlated with foliar nutrients and modelled rates of photosynthetic carbon supply

The rate of above-ground woody biomass production, W(P), in some western Amazon forests exceeds those in the east by a factor of 2 or more. Underlying causes may include climate, soil nutrient limitations and species composition. In this modelling paper, we explore the implications of allowing key nutrients such as N and P to constrain the photosynthesis of Amazon forests, and also we examine the relationship between modelled rates of photosynthesis and the observed gradients in W(P). We use a model with current understanding of the underpinning biochemical processes as affected by nutrient availability to assess: (i) the degree to which observed spatial variations in foliar [N] and [P] across Amazonia affect stand-level photosynthesis; and (ii) how these variations in forest photosynthetic carbon acquisition relate to the observed geographical patterns of stem growth across the Amazon Basin. We find nutrient availability to exert a strong effect on photosynthetic carbon gain across the Basin and to be a likely important contributor to the observed gradient in W(P). Phosphorus emerges as more important than nitrogen in accounting for the observed variations in productivity. Implications of these findings are discussed in the context of future tropical forests under a changing climate.     

Restoration of Corticosteroid Sensitivity by p38 Mitogen Activated Protein Kinase Inhibition in Peripheral Blood Mononuclear Cells from Severe Asthma

Background

Severe asthma accounts for a small number of asthmatics but represents a disproportionate cost to health care systems. The underlying mechanism in severe asthma remains unknown but several mechanisms are likely to be involved because of a very heterogeneous profile. We investigated the effects of a p38MAPK inhibitor in corticosteroid sensitivity in peripheral blood mononuclear cells (PBMCs) from severe asthmatics and the profile of its responders.

Methodology/Principal Findings

Corticosteroid sensitivity was determined by measuring dexamethasone inhibition of CD3/28 and TNF-α induced IL-8 production in PBMCs by using ELISA. PBMCs from severe asthmatics were relatively less sensitive to dexamethasone (Dex) as compared to those of non-severe asthmatics and healthy volunteers. The IC₅₀ values of Dex negatively correlated with decreased glucocorticoid receptor (GR) nuclear translocation assessed using immunocytochemistry (r = −0.65; p<0.0005) and with decreased FEV₁ (% predicted) (r = 0.6; p<0.0005). A p38α/β inhibitor (SB203580) restored Dex-sensitivity in a subpopulation of severe asthma that was characterized by a defective GR nuclear translocation, clinically by lower FEV₁ and higher use of oral prednisolone. We also found that SB203580 partially inhibited GR phosphorylation at serine 226, resulting in increased GR nuclear translocation in IL-2/IL-4 treated corticosteroid insensitive U937s.

Conclusions/Significance

p38MAPKα/β is involved in defective GR nuclear translocation due to phosphorylation at Ser226 and this will be a useful biomarker to identify responders to p38MAPKα/β inhibitor in the future.     

Diversity of inorganic carbon acquisition mechanisms by intact microbial mats of Microcoleus chthonoplastes (Cyanobacteriae, Oscillatoriaceae)

The dissolved inorganic carbon (DIC) acquisition mechanisms were researched in intact microbial mats dominated by the cyanobacteria Microcoleus chthonoplastes Thuret, by determining the effect on photosynthesis of different inhibitors. The microbial mats exhibited high affinity for DIC at alkaline pH, with K(m(DIC)) values similar to the ones described for pure cultures of cyanobacteria and algae in which carbon concentrating mechanisms have been researched. Besides, the photosynthesis was non-sensitive to pH changes within the range of 5.6-9.6, indicating that HCO(3)(-) was the main DIC source used for photosynthesis. The M. chthonoplastes mats featured external and internal carbonic anhydrase (CA) activity as measured in intact cells and cell extracts, respectively. Acetazolamide (AZ, which slowly enters the cell and then inhibits mainly the external CA) and ethoxyzolamide (EZ, which inhibits both external and internal CA) reduced significantly the oxygen evolution rates, demonstrating that the CA was implied in the DIC acquisition. Vanadate inhibited photosynthesis by 60% although its application, when CA being inhibited (i.e. after applying AZ + EZ), did not produce any additional effect. It could indicate that ATPase-dependent HCO(3)(-) use occurred and also that this putative mechanism was coupled with CA-like activity at the plasma membrane. The involvement of Na(+)-dependent HCO(3)(-) transporters in DIC acquisition was also inferred as monensin and 4-4'-diisothiocyanatostibilene-2,2'-disulfonate (DIDS) reduced photosynthesis by 70%. DIDS produced a strong inhibitory effect even after application of AZ + EZ + vanadate, indicating that this mechanism was not related to CA activity. The microbial mats become subject to very unfavourable conditions for Rubisco carboxylation at their natural habitats (e.g. external pH of 10.5 and O(2) concentration doubled with respect to saturation concentration); therefore, this putative diversity of DIC acquisition mechanisms could ensure their growth under these extreme conditions.