Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis

The poly(ADP-ribose) polymerase-like thermozyme purified from Sulfolobus solfataricus was characterised with respect to some physico-chemical properties. The archaeal protein exhibited a scarce electrophoretic mobility at both pH 2.9 and pH 7.5. Determination of the isoelectric point (pI=7.0-7.2) allowed us to understand the reason for the limited migration at pH 7.5, while amino acid composition analysis showed a moderate content of basic residues, which reduced mobility at pH 2.9. With respect to the charge, the archaeal enzyme behaved differently from the eukaryotic thermolabile poly(ADP-ribose) polymerase, described as a basic protein (pI=9.5). Well known inhibitors of the mesophilic polymerase like Zn(2+), nicotinamide and 3-aminobenzamide exerted a smaller effect on the enzyme from S. solfataricus, reducing the activity by at most 50%. Mg(2+) was a positive effector, although in a dose-dependent manner. It influenced the fluorescence spectrum of the archaeal protein, whereas NaCl had no effect.     

Monolayer black membranes from bipolar lipids of archaebacteria and their temperature-induced structural changes

The membrane of Caldariella acidophila, an extreme thermophilic archaebacterium, is characterized by unusual bipolar complex lipids. They consist of two nonequivalent polar heads, linked by a C40 alkylic component. The molecular organization of these lipids in the plasma membrane is still a matter of study. In this paper, we present current-voltage measurements on artificial bipolar lipid membranes, indicating that molecules are indeed organized as a covalently bound bilayer, in which each molecule is completely stretched and spans its entire thickness. Furthermore, conformational transitions of these artificial membranes (which could be formed only above 70 degrees C from a lipid/squalene dispersion) are analyzed in the 80 to 15 degrees C temperature range. Abrupt variations in capacitance and valinomycin-induced conductance seem to indicate the occurrence of at least two structural changes. Measurements are also extended to different solvent systems. Results are consistent with the picture of a monolayer bipolar lipid membrane in which few solvent molecules align themselves parallel to the lipophilic chains. The amount of solvent as well as the temperature at which conformational transitions occur, depend on the solvent system in which the lipid is dispersed.     

Improvement of catalytic properties of Escherichia coli penicillin G acylase immobilized on glyoxyl agarose by addition of a six-amino-acid tag

A tag of three lysines alternating with three glycines was added to the C-terminal end of the beta chain of penicillin G acylase (PGA). This modification improved the immobilization efficiency of PGA on glyoxyl agarose and the catalytic properties of the PGA derivative, although it impaired the posttranslational steps of overexpressed protein maturation.     

A twisted tRNA intermediate sets the threshold for decoding

Putting together consistent cryo-EM structure, transient kinetic and mutant tRNA suppressor data, it appears that a deformed or waggling aminoacyl-tRNA is the critical transitional structure examined by the ribosome during decoding at the A site. The unusual conformation may be required for effective proofreading of the codon-anticodon complex.     

Evaluation of chromogenic medium (ORSAB) for routine identification of methicillin resistant Staphylococcus aureus (MRSA)

Performance of chromogenic medium (ORSAB) for routine detection of methicillin resistant S. aureus (MRSA) was evaluated on 510 specimens collected from patients suspected of MRSA infection or colonization. Addition of ORSAB plates to the routine protocol allowed MRSA identification in 24 hours from samples plating. In 18 samples MRSA colonies were identified only on ORSAB plates, those cases would have been missed by routine protocol alone.     

Thrombopoietin complements G(i)- but not G(q)-dependent pathways for integrin {alpha}(IIb){beta}(3) activation and platelet aggregation

Binding of thrombopoietin (TPO) to the cMpl receptor on human platelets potentiates aggregation induced by a number of agonists, including ADP. In this work, we found that TPO was able to restore ADP-induced platelet aggregation upon blockade of the G(q)-coupled P2Y1 purinergic receptor but not upon inhibition of the G(i)-coupled P2Y12 receptor. Moreover, TPO triggered platelet aggregation upon co-stimulation of G(z) by epinephrine but not upon co-stimulation of G(q) by the thromboxane analogue U46619. Platelet aggregation induced by TPO and G(i) stimulation was biphasic, and cyclooxygenase inhibitors prevented the second but not the first phase. In contrast to ADP, TPO was unable to induce integrin alpha(IIb)beta(3) activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of integrin alpha(IIb)beta(3) was blocked by antagonists of the G(q)-coupled P2Y1 receptor but was completely restored by the simultaneous co-stimulation of cMpl receptor by TPO. Inside-out activation of integrin alpha(IIb)beta(3) induced by TPO and G(i) stimulation occurred independently of thromboxane A(2) production and was not mediated by protein kinase C, MAP kinases, or Rho-dependent kinase. Importantly, TPO and G(i) activation of integrin alpha(IIb)beta(3) was suppressed by wortmannin and Ly294002, suggesting a critical regulation by phosphatidylinositol 3-kinase. We found that TPO did not activate phospholipase C in human platelets and was unable to restore ADP-induced phospholipase C activation upon blockade of the G(q)-coupled P2Y1 receptor. TPO induced a rapid and sustained activation of the small GTPase Rap1B through a pathway dependent on phosphatidylinositol 3-kinase. In ADP-stimulated platelets, Rap1B activation was reduced, although not abolished, upon blockade of the P2Y1 receptor. However, accumulation of GTP-bound Rap1B in platelets activated by co-stimulation of cMpl and P2Y12 receptor was identical to that induced by the simultaneous ligation of P2Y1 and P2Y12 receptor by ADP. These results indicate that TPO can integrate G(i), but not G(q), stimulation and can efficiently support integrin alpha(IIb)beta(3) activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol 3-kinase and the small GTPase Rap1B.     

Effects of nutrient additions on plant biomass and diversity of the herbaceous-subshrub layer of a Brazilian savanna (Cerrado)

The Brazilian Cerrado is a diversity hotspot due to its high level of endemism and rapid loss of habitats. It is estimated that the number of herbaceous species is four times higher than that of woody species. Increasing levels of nitrogen additions to natural ecosystems have been indicated as a determinant of biodiversity loss. We investigated the effects of nutrient additions on the productivity (aboveground and belowground) and on diversity of the herbaceous-subshrub layer of a Brazilian savanna (cerrado stricto sensu). The experiment was carried out in the IBGE Ecological Reserve, near Brasília, Brazil. Between 1998 and 2006, N, P, N plus P, or Ca were applied to sixteen 225 m² plots, arranged in a completely randomized design. Aboveground biomass was compared 1 year after the first fertilization and 10 years later. Floristic diversity was significantly different (P < 0.01) between the treatments. The highest and lowest species richness were presented in control and NP, respectively. The addition of P alone or in combination with N induced invasion by Melinis minutiflora (exotic C₄ grass). The aboveground biomass of this species was higher in NP and P plots. In the N treatment, Echinolaena inflexa (native C₃ grass) presented elevated cover and biomass but M. minutiflora was absent. The invasion by alien species resulted in negative impacts on native grass species. Besides changes in aboveground biomass, addition of N and P also led, although to a lesser extent, to changes in the root morphology and biomass, but these responses were modulated by seasonal variation in soil moisture. The results suggest that environmental changes in nutrient availability can lead to important consequences for diversity and functioning of this savanna where the numerous rare species have more chance to persist under dystrophic conditions as some species that tend to be dominant would be less competitive.     

Utilizing the intramolecular Fukuyama-Mitsunobu reaction for a flexible synthesis of novel heterocyclic scaffolds for peptidomimetic drug design

We report the synthesis of the novel scaffolds pyrazino[1,2-b]isoquinoline and pyrrolo[1,2-a]pyrazine displaying the somatostatin pharmacophores. Both classes of compounds contain a pyrazine heterocycle, which can be prepared in a straightforward manner utilizing an intramolecular Fukuyama-Mitsunobu reaction. As both the families derive from amino acids, they can be accessed in high optical purity.