How calmodulin interacts with the adenosine A(2A) and the dopamine D(2) receptors

Receptor heteromerization is a mechanism used by G protein-coupled receptors to diversify their properties and function. We previously demonstrated that these interactions occur through salt bridge formation between epitopes of the involved receptors. Recent studies claim that calmodulin (CaM) binds to an Arg-rich epitope located in the amino-terminus of the dopamine D(2) receptor third intracellular loop. This is the same epitope involved in adenosine A(2A)-D(2) receptor heteromerization, through Coulombic interaction between the Arg residues and a phosphorylated serine (pS) located in the medial segment of the C-terminus of the A(2A) receptor. Mass spectrometric analysis indicates that an electrostatic interaction involving the D(2) receptor Arg-rich epitope and several CaM acidic epitopes are mainly responsible for the D(2) receptor-CaM binding. CaM could also form multiple noncovalent complexes by means of electrostatic interactions with an epitope localized in the proximal segment of the C-terminus of the A(2A) receptor. Ca(2+) disrupted the binding of CaM to the D(2) but not to the A(2A) receptor epitope, and CaM disrupted the electrostatic interactions between the D(2) receptor epitope and the more distal A(2A) receptor epitope. A model is introduced with the possible functional implications of A(2A)-D(2)-CaM interactions. These in vitro findings imply a possible regulatory role for CaM in receptor heteromers formation.     

Natural selection fails to optimize mutation rates for long-term adaptation on rugged fitness landscapes

The rate of mutation is central to evolution. Mutations are required for adaptation, yet most mutations with phenotypic effects are deleterious. As a consequence, the mutation rate that maximizes adaptation will be some intermediate value. Here, we used digital organisms to investigate the ability of natural selection to adjust and optimize mutation rates. We assessed the optimal mutation rate by empirically determining what mutation rate produced the highest rate of adaptation. Then, we allowed mutation rates to evolve, and we evaluated the proximity to the optimum. Although we chose conditions favorable for mutation rate optimization, the evolved rates were invariably far below the optimum across a wide range of experimental parameter settings. We hypothesized that the reason that mutation rates evolved to be suboptimal was the ruggedness of fitness landscapes. To test this hypothesis, we created a simplified landscape without any fitness valleys and found that, in such conditions, populations evolved near-optimal mutation rates. In contrast, when fitness valleys were added to this simple landscape, the ability of evolving populations to find the optimal mutation rate was lost. We conclude that rugged fitness landscapes can prevent the evolution of mutation rates that are optimal for long-term adaptation. This finding has important implications for applied evolutionary research in both biological and computational realms.     

Molecular modeling and dynamics simulations of PNP from Streptococcus agalactiae

This work describes for the first time a structural model of purine nucleoside phosphorylase from Streptococcus agalactiae (SaPNP). PNP catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is a potential target for the development of antibacterial drugs. We modeled the complexes of SaPNP with 15 different ligands in order to determine the structural basis for the specificity of these ligands against SaPNP. The application of a novel empirical scoring function to estimate the affinity of a ligand for a protein was able to identify the ligands with high affinity for PNPs. The analysis of molecular dynamics trajectory for SaPNP indicates that the functionally important motifs have a very stable structure. This new structural model together with a novel empirical scoring function opens the possibility to explorer larger library of compounds in order to identify the new inhibitors for PNPs in virtual screening projects.     

PyDeT, a PyMOL plug-in for visualizing geometric concepts around proteins

The fast growing Protein Data Bank (PDB) contains a vast amount of 3-dimensional data on proteins, and nucleic-acid structures obtained by X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy. PyDeT is a PyMOL (molecular visualization software system) plug-in that visualize tessellations derived from the protein structure along with the source protein. PyDeT is released under a GNU General Public License (GPL) and is available from the authors.     

Carbohydrate accumulation and utilization by oocytes of Rhodnius prolixus

The processes of accumulation and mobilization of carbohydrate stores in eggs of Rhodnius prolixus were analyzed. During oogenesis, the total amounts of glycogen, glucose, and trehalose increased with an accumulation of proteins, especially when oocytes grew from 1.0 to 1.5 mm in length. At 2.0 mm length, when oocytes were ready for oviposition, nutrient reserves did not increase appreciably and trehalose content decreased. Mating did not affect the final content of carbohydrates or proteins in oocytes of mated and virgin females. A trehalase activity was detected in follicles containing vitellogenic oocytes, 1.0 and 1.5 mm length, in both mated and virgin females. This activity was extremely low in chorionated, 2.0-mm oocytes. After oviposition, glycogen content decreased in fertilized eggs, but not in unfertilized ones, and some was present in newly hatched nymphs. Glucose content remained constant in unfertilized eggs, but increased in fertilized ones, while total protein amount was constant in both groups after egg laying.     

Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes

Free radical production is implicated in the pathogenesis of diabetes mellitus, where several pathways and different mechanisms were suggested in the pathophysiology of the complications. In this study, we used electron paramagnetic resonance (EPR) spectroscopy combined with in vivo spin-trapping techniques to investigate the sources and mechanisms of free radical formation in streptozotocin-induced diabetic rats. Free radical production was directly detected in the diabetic bile, which correlated with lipid peroxidation in the liver and kidney. EPR spectra showed the trapping of a lipid-derived radical. Such radicals were demonstrated to be induced by hydroxyl radical through isotope-labeling experiments. Multiple enzymes and metabolic pathways were examined as the potential source of the hydroxyl radicals using specific inhibitors. No xanthine oxidase, cytochrome P450s, the Fenton reaction, or macrophage activation were required for the production of radical adducts. Interestingly, inducible nitric oxide synthase (iNOS) (apparently uncoupled) was identified as the major source of radical generation. The specific iNOS inhibitor 1400W as well as L-arginine pretreatment reduced the EPR signals to baseline levels, implicating peroxynitrite as the source of hydroxyl radical production. Applying immunological techniques, we localized iNOS overexpression in the liver and kidney of diabetic animals, which was closely correlated with the lipid radical generation and 4-hydroxynonenal-adducted protein formation, indicating lipid peroxidation. In addition, protein tyrosine nitration occurred in the diabetic target organs. Taken together, our studies support inducible nitric oxide synthase as a significant source of EPR-detectable reactive intermediates, which leads to lipid peroxidation and may contribute to disease progression as well.     

Influence of drought on algal biofilms and meiofaunal assemblages of temperate reservoirs and rivers

The role of hydrological droughts in shaping meiofauna abundance through alterations in biofilm biomass and composition was investigated. In January 2005, continental Portugal was under a moderate to severe drought resulting from a 40% to 60% decrease in rainfall during the previous 12 months relative to the long-term average (1961–1990). Reservoir capacity was reduced by 30–50% relative to average values and the width of streams was reduced by 20–80% in the Zêzere River Basin (central Portugal). Algal biomass and algal class composition of biofilms was assessed through quantification of algal pigments in three reservoir and six river locations. During drought, habitat alterations are expected to be sharp in rivers while, in the absence of water quality deterioration, the habitat characteristics of reservoirs are expected to remain fairly unaffected. Chlorophylls and carotenoid pigments were extracted from biofilm samples and analysed using high performance liquid chromatography (HPLC). In the winter of 2003, during the period of average rainfall, biofilm biomass did not exceed 5 μg chlorophyll a cm⁻² at any location. River biofilm biomass was roughly half of that measured in the reservoirs. In the winter of 2005 (drought), biofilm biomass increased by more than 5-fold in river locations and remained low or decreased in the reservoirs. Algal biofilms were either dominated by Bacillariophyceae or by Chlorophyceae regardless of the existence of drought. The relative contribution of Bacillariophyceae to total biofilm biomass was higher during the drought than under average hydrological conditions. The abundance of harpacticoids, cladocerans and ostracods was favoured by the drought only in the reservoirs where an increase in diatom proportion in biofilms was observed. The increase in the abundance of cyclopoid copepods, turbellarians, nematodes and chironomids in rivers during the drought could be explained by algal class composition and biomass of biofilms and environmental variables (organic matter sediment content, phosphorus availability content and sediment granulometry). The hydrological drought appears to regulate meiofauna abundance only in river locations, possibly through the promotion of the growth of biofilms and the availability of organic matter deposited in rivers during the drought. Handling editor: D. Ryder     

Magnetic properties of six-coordinated high-spin cobalt(II) complexes: Theoretical background and its application

In this contribution we study and analyse the influence of the different parameters involved in the magnetic susceptibility of six-coordinated high-spin Co(II) complexes. We propose an empirical expression to fit the magnetic susceptibility of polycrystalline samples of mononuclear Co(II) complexes with an axial distortion, the variable parameters being Δ (axial distortion), α (orbital reduction factor) and λ (spin–orbit coupling). This expression avoids solving the 12 × 12 matrix associated to the distortion of the ⁴T_1g term. In order to take into account the magnetic coupling (J) in the polynuclear Co(II) complexes, a perturbational approach is proposed to describe their magnetic susceptibility in the whole temperature range (2–300 K) as a function of J, Δ, α and λ. This approach is valid in the limit of the weak magnetic coupling as compared to the spin–orbit coupling, |J/λ| < 0.1. The model allows the treatment of each cobalt(II) ion in axial symmetry as an effective spin S_eff = 1/2. That causes a drastic reduction of the matrix size of the polynuclear compounds from 12ⁿ × 12ⁿ to 2ⁿ × 2ⁿ, n being the number of Co(II) ions in the complex. The main advantage of the model is to make possible the fit of the magnetic susceptibility data of those polynuclear Co(II) complexes whose high nuclearity involved intractable matrices.