Asthma: where beyond steroids?

The prevalence of asthma is increasing dramatically despite major changes in monitoring and treatment of this disease. Currently available bronchodilators and anti-inflammatory drugs are effective in most patients, although these can have side effects and are mainly symptomatic. Many drugs are now in development for the treatment of asthma. Most of these new therapies are aimed at inhibition of the inflammatory components, with better safety profiles than steroids.     

Conformations of tissue plasminogen activator (tPA) orchestrate neuronal survival by a crosstalk between EGFR and NMDAR

Tissue-type plasminogen activator (tPA) is a pleiotropic serine protease of the central nervous system (CNS) with reported neurotrophic and neurotoxic functions. Produced and released under its single chain form (sc), the sc-tPA can be cleaved by plasmin or kallikrein in a two chain form, tc-tPA. Although both sc-tPA and tc-tPA display a similar fibrinolytic activity, we postulated here that these two conformations of tPA (sc-tPA and tc-tPA) could differentially control the effects of tPA on neuronal survival. Using primary cultures of mouse cortical neurons, our present study reveals that sc-tPA is the only one capable to promote N-methyl-D-aspartate receptor (NMDAR)-induced calcium influx and subsequent excitotoxicity. In contrast, both sc-tPA and tc-tPA are capable to activate epidermal growth factor receptors (EGFRs), a mechanism mediating the antiapoptotic effects of tPA. Interestingly, we revealed a tPA dependent crosstalk between EGFR and NMDAR in which a tPA-dependent activation of EGFRs leads to downregulation of NMDAR signaling and to subsequent neurotrophic effects.Tissue-type plasminogen activator (tPA) is secreted by endothelial cells and promotes fibrinolysis via the conversion of fibrin-bound plasminogen into plasmin.¹ Neurons and some glial cells also secrete tPA.^{2, 3, 4, 5} tPA is secreted as a single-chain form (sc-tPA), which can be processed into a two-chain form (tc-tPA) by plasmin or kallikreins.^{6, 7} Interestingly, sc-tPA is proteolytically active even without proteolytic processing. In addition to its vascular functions, tPA displays critical roles in the brain parenchyma with roles in cell migration, neuronal plasticity and survival,^{8, 9, 10, 11, 12, 13, 14} acting either as an enzyme or as a cytokine-like molecule. Among its actions, tPA is well described to promote neurotoxicity, likely through promotion of N-methyl-D-aspartate receptor (NMDAR) activity.^{15, 16, 17} Recently, we reported that only sc-tPA can promote NMDAR signaling and neurotoxicity.¹⁸ Interestingly, data from wild-type mice,¹⁹ transgenic mice overexpressing tPA in neurons²⁰ or in vitro²¹ also report neuroprotective effects of tPA.^{9, 10} The proposed mechanisms involved a tPA-dependent and non-proteolytic activation of either epidermal growth factor receptors (EGFRs)²² on oligodendrocytes or NMDARs.²⁰Here we explored a link between tPA conformations (sc-tPA and tc-tPA), EGFR- and NMDAR-dependent signaling pathways. Our findings identify sc-tPA as a selective positive modulator of NMDAR signaling in neurons when present at high concentrations and both sc-tPA and tc-tPA as positive modulators of EGFR signaling, this even at low concentrations. We also reveal a crosstalk between these two families of receptors, with the tPA-dependent activation of EGFRs reducing NMDAR signaling. By these mechanisms, sc-tPA and tc-tPA control neuronal death and survival.     

Leaf breakdown in an Atlantic Rain Forest stream

The hypothesis of this study was that colonizers in decaying leaf litter prefer native species (Erythrina verna) to exotic ones (Eucalyptus camaldulensis and Protium heptaphyllum). Therefore, native species are expected to show higher breakdown rates, increased biomass, richness and density of invertebrate species, and increased biomass of decomposer fungi. Breakdown of leaf litter from these three species was assessed in an Atlantic Rain Forest stream. Four samples were collected during a period of 90 days and washed on a sieve to separate the invertebrates. Then, a series of leaf disks were cut to determine ash‐free dry mass and fungal biomass, and the remaining material was oven‐dried to determine the dry weight. Eucalyptus camaldulensis and E. verna showed higher breakdown rates than P. heptaphyllum, due to differences in leaf physical and chemical characteristics. The harder detritus (P. heptaphyllum) broke down more slowly than detritus with high concentrations of labile compounds (E. camaldulensis). The density of the invertebrates associated with detritus increased with time. There were no differences in density, taxonomic richness or biomass of invertebrates among the leaf types, which indicated that the invertebrates did not distinguish between exotic and native detritus. Fungal colonization varied among samples; E. camaldulensis showed the lowest ergosterol concentrations, mainly due to a high concentration of total phenolics. The detritus with the highest hardness value was colonized most slowly by fungi. These results showed that leaf breakdown in Atlantic Rain Forest streams could be affected either by changes in riparian vegetation, or by becoming more savanna‐like process due to climate change.     

Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing

Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP-associated splicing factors SF3a and SF3b. While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome. Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex. TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex. Subunits of this complex were not essential for viability. However, they are required for efficient splicing in vitro and in vivo. Furthermore, inactivation of this complex causes pre-mRNA leakage from the nucleus. The corresponding complex was named pre-mRNA REtention and Splicing (RES). The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved.     

Stabilization of internal charges in a protein: water penetration or conformational change?

The ionizable amino acid side chains of proteins are usually located at the surface. However, in some proteins an ionizable group is embedded in an apolar internal region. Such buried ionizable groups destabilize the protein and may trigger conformational changes in response to pH variations. Because of the prohibitive energetic cost of transferring a charged group from water to an apolar medium, other stabilizing factors must be invoked, such as ionization-induced water penetration or structural changes. To examine the role of water penetration, we have measured the 17O and 2H magnetic relaxation dispersions (MRD) for the V66E and V66K mutants of staphylococcal nuclease, where glutamic acid and lysine residues are buried in predominantly apolar environments. At neutral pH, where these residues are uncharged, we find no evidence of buried water molecules near the mutation site. This contrasts with a previous cryogenic crystal structure of the V66E mutant, but is consistent with the room-temperature crystal structure reported here. MRD measurements at different pH values show that ionization of Glu-66 or Lys-66 is not accompanied by penetration of long-lived water molecules. On the other hand, the MRD data are consistent with a local conformational change in response to ionization of the internal residues.     

Age-related impairment of mitochondrial matrix aconitase and ATP-stimulated protease in rat liver and heart.

Mitochondrial matrix proteins are sensitive to oxidative inactivation, and oxidized proteins are known to accumulate during ageing. The Lon protease is believed to play an important role in the degradation of oxidized matrix proteins such as oxidized aconitase. We reported previously that an age-related accumulation of altered proteins occurs in the liver matrix of rats and that the ATP-stimulated proteolytic activity, referred as to Lon-like protease activity, decreases considerably in 27 month-old rats, whereas no concomitant changes in the levels of Lon protein expression occur in the liver. Here, we report that this decline is associated with a decrease in the activity of aconitase, an essential Krebs' cycle enzyme. Contrary to what we observed in the liver, the ATP-stimulated protease activity was found to remain constant in the heart mitochondrial matrix during ageing, and the levels of expression of the Lon protease increased in the older animals in comparison with the younger ones. Although the ATP-stimulated protease activity remained practically the same in older animals as in younger ones, a decrease in the level of aconitase activity was still observed. Altogether, these results indicate that matrix proteins, such as the critical enzymes aconitase and Lon protease, are inactivated with ageing and that the effects of ageing vary from one organ to another.     

Physiology of maize II: Identification of physiological markers representative of the nitrogen status of maize (Zea mays) leaves during grain filling

To illustrate the development of the source-to-sink transition in maize leaves during the grain-filling period, an integrated physiological-agronomic approach is presented in this study. The evolution of physiological markers such as total leaf nitrogen (N), chlorophyll, soluble protein, amino acid and ammonium contents was monitored from silking to a period close to maturity in different leaf stages of three maize genotypes grown at high and low levels of N fertilization. In addition, the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH), two enzymes known to play a direct or an indirect role during leaf N remobilization, were measured. In the three genotypes examined, we found that a general decrease of most metabolic and enzyme markers occurred during leaf ageing and that this decrease was enhanced when plants were N starved. In contrast, such variations were not observed between different sections of a single leaf even at an advanced stage of leaf senescence. We found that there is a strong correlation between total N, chlorophyll, soluble protein and GS activity, which is not dependent upon the N fertilization level, which indicates the N status of the plant, either in a single leaf or during ageing. In contrast, ammonium, amino acids and GDH activity were not subject to such variations, thus suggesting that they are indicators of the metabolic activity of the whole plant in response to the level of N fertilization. The use of these markers to predict the N status of maize as a function of both plant development and N availability is discussed.