Stoichiometry of aerobic mineralization (O/C) of aquatic macrophytes leachate from a tropical lagoon (São Paulo –Brazil)

The temporal variation of stoichiometry between consumed oxygen and oxidized carbon was investigated for the aerobic mineralization of leachates from aquatic macrophytes. Seven species of aquatic plants, viz. Cabomba piauhyensis, Cyperus giganteus, Egeria najas, Eichhornia azurea, Salvinia auriculata, Scirpus cubensisand Utricularia breviscapa, were collected from Òleo lagoon located in the floodplain of Mogi-Guacu river (São Paulo State, Brazil). After being collected, the plants were washed, oven-dried and triturated. In order to obtain the leachate, the fragments were submitted to an aqueous extraction (cold). Mineralization chambers were incubated at 20 °C containing leachates dissolved in water samples from Òleo lagoon to a final concentration of ca. 200 mg l^–1on carbon basis. The chambers were maintained under aerobic conditions; the concentrations of the organic carbon (particulate and dissolved) and the dissolved oxygen were measured during approximately 80 days. Elemental analysis of the detritus and the concentrations of the remaining material (DOC and POC) were used to determine the amounts of mineralized organic carbon. The data were analyzed with first-order kinetics models, from which the daily rates of consumption (carbon and oxygen) and the stoichiometry (O/C) were determined. In the early phase of mineralization the O/C rates increased before reaching a maximum, after which they tended to decrease. For the mineralization of leachates from C. giganteus, S. auriculata and U. breviscapa, the decrease was relatively slow. For all substrata the initial values were smaller than 1, and ranged from 0.42 (S. cubensis) to 0.81 (C. piauhyensis). The maximum values were within the range from 0.58 (U. breviscapa) to 23.1 (E. najas) and at their highest 26th (C. piauhyensis) and 106th (C. giganteus) days. These variations are believed to be associated with the chemical composition of the leachates, with their transformations and alterations of metabolic pathways involved in the mineralization.     

Multiple effects of paclitaxel are modulated by a high c-myc amplification level

Paclitaxel affects microtubule stability by binding to beta-tubulin, thus leading to cell accumulation in the G(2)/M phase, polyploidization, and apoptosis. Because both cell proliferation and apoptosis could be somehow regulated by the protooncogene c-myc, in this work we have investigated whether the c-myc amplification level could modulate the multiple effects of paclitaxel. To this aim, paclitaxel was administered to SW613-12A1 and -B3 human colon carcinoma cell lines (which are characterized by a high and low c-myc endogenous amplification level, respectively), and to the B3mycC5 cell line, with an enforced exogenous expression of c-myc copies. In this experimental system, we previously demonstrated that a high endogenous/exogenous level of amplification of c-myc enhances serum deprivation- and DNA damage-induced apoptosis. Accordingly, the present results indicate that a high c-myc amplification level potentiates paclitaxel cytotoxicity, confers a multinucleated phenotype, and promotes apoptosis to a great extent, thus suggesting that c-myc expression level is relevant in modulating the cellular responses to paclitaxel. We have recently shown in HeLa cells that the phosphorylated form of c-Myc accumulates in the nucleus, as distinct nucleolar and extranucleolar spots; here, we demonstrated that, after the treatment with paclitaxel, phosphorylated c-Myc undergoes redistribution, becoming diffused in the nucleoplasm.     

Beta-alanine protection against hypoxic liver injury in the rat

Liver hypoxia still represents an important cause of liver injury during shock and liver transplantation. We have investigated the protective effects of beta-alanine against hypoxic injury using isolated perfused rat livers and isolated rat hepatocyte suspensions. Perfusion with hypoxic Krebs-Henseleit buffer increased liver weight and caused a progressive release of lactate dehydrogenase (LDH) in the effluent perfusate. The addition of 5 mmol/l beta-alanine to the perfusion buffer completely prevented both weight increase and LDH leakage. These findings were confirmed by histological examinations showing that beta-alanine blocked the staining by trypan blue of either liver parenchymal and sinusoidal cells. Studies performed in isolated hepatocytes revealed that beta-alanine exerted its protective effects by interfering with Na+ accumulation induced by hypoxia. The addition of gamma-amino-butyric acid, which interfered with beta-alanine uptake by the hepatocytes or of Na+/H+ ionophore monensin, reverted beta-alanine protection in either hepatocyte suspensions or isolated perfused livers. We also observed that liver receiving beta-alanine were also protected against LDH leakage and weight increase caused by the perfusion with an hyposmotic (205 mosm) hypoxic buffer obtained by decreasing NaCl content from 118 to 60 mmol/l. This latter effect was not reverted by blocking K+ efflux from hepatocyte with BaCl(2) (1mmol/l). Altogether these results indicated that beta-alanine protected against hypoxic liver injury by preventing Na+ overload and by increasing liver resistance to osmotic stress consequent to the impairment of ion homeostasis during hypoxia.     

Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.     

Modulation of immune response in Plasmodium falciparum malaria: role of IL-12, IL-18 and TGF-beta

The interaction between pro- and anti-inflammatory cytokines such as interleukin 12 (IL-12), interleukin 18 (IL-18) and transforming growth factor beta (TGF-beta) plays an important role in malaria pathogenesis and outcome, modulating the immunoresponse in Plasmodium falciparum malaria. In our previous studies, we analyzed the plasmatic levels of IL-12, IL-18 and TGF-beta in 105 African children with different degrees of malaria and we correlated the production of these cytokines with the severity of the disease.The aim of the present study was to analyze with a mathematical model, taking into account all the relationships between these cytokines and the parameter variations involved in malaria pathogenesis that influence the results of each type of treatment or therapeutic protocol on patients at different stages of the disease. A mathematical correlation was demonstrated between the levels of pro-inflammatory and anti-inflammatory cytokines, and from this it was possible to build curves of reference in which each patient was positioned based on IL-12 level.Our data, obtained in patients with mild and severe diseases, demonstrate that the levels of IL-12 represent a reliable parameter to predict the progression of the disease, which may be complemented or modulated by administration of IL-18 and TGF-beta.Our findings provide future implications for an immune therapy against the P. falciparum malaria infection, especially in the early phase of the disease showing that a more aggressive outcome may be due to the lack of a balanced immune response.     

Translational recoding in archaea

Translational recoding includes a group of events occurring during gene translation, namely stop codon readthrough, programmed ±1 frameshifting, and ribosome bypassing, which have been found in organisms from all domains of life. They serve to regulate protein expression at translational level and represent a relatively less known exception to the traditional central ‘dogma’ of biology that information flows as DNA→RNA→protein and that it is stored in a co-linear way between the 5′→3′ of nucleic acids and N→C-terminal of polypeptides. In archaea, in which translational recoding regulates the decoding of the 21st and the 22nd amino acids selenocysteine and pyrrolysine, respectively, only one case of programmed ?1 frameshifting has been reported so far and further examples, although promising, have not been confirmed yet. We here summarize the current state-of-the-art of this field that, especially in archaea, has relevant implications for the physiology of life in extreme environments and for the origin of life.     

Interaction between ephrins/Eph receptors and excitatory amino acid receptors: possible relevance in the regulation of synaptic plasticity and in the pathophysiology of neuronal degeneration

There is increasing evidence that Eph receptors and their transmembrane ligands, named ephrins, interact with glutamate receptors in both developing and adult neurons. EphB receptors interact with proteins that regulate the membrane trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits, and both ephrins and EphB receptors have been found to co-localize with N-methyl-d-aspartate (NMDA) receptors and to positively modulate NMDA receptor function. Moreover, pharmacologic activation of ephrin-Bs amplifies group-I metabotropic glutamate receptor signaling through mechanisms that involve NMDA receptors. The interaction with ionotropic or metabotropic glutamate receptors provides a substrate for the emerging role of ephrins and Eph receptors in the regulation of activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, which are established electrophysiologic models of associative learning. In addition, these interactions explain the involvement of ephrins/Eph receptors in the regulation of pain threshold and epileptogenesis, as well as their potential implication in processes of neuronal degeneration. This may stimulate the search for new drugs that might modulate excitatory synaptic transmission by interacting with the ephrin/Eph receptor system.     

The extremo-α-carbonic anhydrase (CA) from Sulfurihydrogenibium azorense,the fastest CA known,is highly activated by amino acids and amines

The α-carbonic anhydrase (CA, EC 4.2.1.1) from the extremophilic bacterium Sulfurihydrogenibium azorense (SazCA) was recently shown to be the fastest CA known. Here we investigated this enzyme for its activation with a series of amino acids and amines. The best SazCA activators were d-Phe, l-DOPA, l- and d-Trp, dopamine and serotonin, which showed activation constants in the range of 3–23 nM. l- and d-His, l-Phe, l-Tyr, 2-pyridyl-methylamine and L-adrenaline were also effective activators (K_As in the range of 62–90 nM), whereas d-Dopa, d-Tyr and several heterocyclic amines showed activity in the micromolar range. The good thermal stability, robustness, very high catalytic activity and propensity to be activated by simple amino acids and amines, make SazCA a very interesting candidate for biomimetic CO₂ capture processes.     

Computational, spectroscopic, and resonant mirror biosensor analysis of the interaction of adrenodoxin with native and tryptophan-modified NADPH-adrenodoxin reductase

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition.