The block of Shaker K+ channels by kappa-conotoxin PVIIA is state dependent.

kappa-conotoxin PVIIA is the first conotoxin known to interact with voltage-gated potassium channels by inhibiting Shaker-mediated currents. We studied the mechanism of inhibition and concluded that PVIIA blocks the ion pore with a 1:1 stoichiometry and that binding to open or closed channels is very different. Open-channel properties are revealed by relaxations of partial block during step depolarizations, whereas double-pulse protocols characterize the slower reequilibration of closed-channel binding. In 2.5 mM-[K+]o, the IC50 rises from a tonic value of approximately 50 to approximately 200 nM during openings at 0 mV, and it increases e-fold for about every 40-mV increase in voltage. The change involves mainly the voltage dependence and a 20-fold increase at 0 mV of the rate of PVIIA dissociation, but also a fivefold increase of the association rate. PVIIA binding to Shaker Delta6-46 channels lacking N-type inactivation or to wild phenotypes appears similar, but inactivation partially protects the latter from open-channel unblock. Raising [K+]o to 115 mM has little effect on open-channel binding, but increases almost 10-fold the tonic IC50 of PVIIA due to a decrease by the same factor of the toxin rate of association to closed channels. In analogy with charybdotoxin block, we attribute the acceleration of PVIIA dissociation from open channels to the voltage-dependent occupancy by K+ ions of a site at the outer end of the conducting pore. We also argue that the occupancy of this site by external cations antagonizes on binding to closed channels, whereas the apparent competition disappears in open channels if the competing cation can move along the pore. It is concluded that PVIIA can also be a valuable tool for probing the state of ion permeation inside the pore.     

Gender differences in brain areas involved in silent counting by means of fMRI

Background

Pattern of brain asymmetries varies with handedness, gender, age, and with variety of genetic and social factors. Large-scale neuroimaging analyses can optimize the detection of asymmetric features and confirm the factors that might modulate pattern of brain asymmetries. We attempted to evaluate eventual differences between genders in hemodynamic responses to a simple language task.

Methods

12 healthy right-handed volunteers (age 24-46), 6 men and 6 women underwent fMRI scanning while performing the simple cognitive - language processing task – silent number counting in Serbian.

Results

Group analysis of hemodynamic responses shows activation in expected brain language areas of inferior frontal gyrus (IFG) and superior temporal gyrus (STG) in both hemispheres. In the male group, aside from dedicated language areas in IFG and STG, activation was noted in right frontal region and interhemispheric supplementary motor area. On the other hand, in the female group, besides activation in dedicated language areas, activation was noted, in right hippocampus, limbic brain and cerebellum bilaterally.

Conclusions

Our results on differences in silent counting by means of fMRI suggest that those differences may be based on different brain pattern activation in men and women. The relation between performance, strategies and regional brain activation should be the topic of further studies when considering not only gender differences in language processing but also differences that may be attributed to the variations in the task details, stimuli, and the stimulus presentation methods.

     

Evolution of Conus Peptide Genes: Duplication and Positive Selection in the A-Superfamily

A remarkable diversity of venom peptides is expressed in the genus Conus (known as “conotoxins” or “conopeptides”). Between 50 and 200 different venom peptides can be found in a single Conus species, each having its own complement of peptides. Conopeptides are encoded by a few gene superfamilies; here we analyze the evolution of the A-superfamily in a fish-hunting species clade, Pionoconus. More than 90 conopeptide sequences from 11 different Conus species were used to build a phylogenetic tree. Comparison with a species tree based on standard genes reveals multiple gene duplication events, some of which took place before the Pionoconus radiation. By analysing several A-conopeptides from other Conus species recorded in GenBank, we date the major duplication events after the divergence between fish-hunting and non-fish-hunting species. Furthermore, likelihood approaches revealed strong positive selection; the magnitude depends on which A-conopeptide lineage and amino-acid locus is analyzed. The four major A-conopeptide clades defined are consistent with the current division of the superfamily into families and subfamilies based on the Cys pattern. The function of three of these clades (the κA-family, the α4/7-subfamily, and α3/5-subfamily) has previously been characterized. The function of the remaining clade, corresponding to the α4/4-subfamily, has not been elucidated. This subfamily is also found in several other fish-hunting species clades within Conus. The analysis revealed a surprisingly diverse origin of α4/4 conopeptides from a single species, Conus bullatus. This phylogenetic approach that defines different genetic lineages of Conus venom peptides provides a guidepost for identifying conopeptides with potentially novel functions.     

Non‐homogeneous biofilm modeling applied to bioleaching processes

A two‐dimensional non‐homogeneous biofilm model is proposed for the first time to study chemical and biochemical reactions at the microorganism scale applied to biological metal leaching from mineral ores. The spatial and temporal relation between these reactions, microorganism growth and the morphological changes of the biofilm caused by solid inorganic precipitate formation were studied using this model. The model considers diffusion limitations due to accumulation of inorganic particles over the mineral substratum, and allows the study of the effect of discrete phases on chemical and microbiological mineral solubilization. The particle‐based modeling strategy allowed representation of contact reactions between the microorganisms and the insoluble precipitates, such as those required for sulfur attack and solubilization. Time‐dependent simulations of chemical chalcopyrite leaching showed that chalcopyrite passivation occurs only when an impervious solid layer is formed on the mineral surface. This mineral layer hinders the diffusion of one kinetically determinant mineral‐attacking chemical species through a nearly irreversible chemical mechanism. Simulations with iron and sulfur oxidizing microorganisms revealed that chemolithoautotrophic biofilms are able to delay passivation onset by formation of corrosion pits and increase of the solid layer porosity through sulfur dissolution. The model results also show that the observed flat morphology of bioleaching biofilms is favored preferentially at low iron concentrations due to preferential growth at the biofilm edge on the surface of sulfur‐forming minerals. Flat biofilms can also be advantageous for chalcopyrite bioleaching because they tend to favor sulfur dissolution over iron oxidation. The adopted modeling strategy is of great interest for the numerical representation of heterogeneous biofilm systems including abiotic solid particles. Biotechnol. Bioeng. 2010;106: 660–676. © 2010 Wiley Periodicals, Inc.     

NMR structure determination of alpha-conotoxin BuIA, a novel neuronal nicotinic acetylcholine receptor antagonist with an unusual 4/4 disulfide scaffold

We have determined a high-resolution three-dimensional structure of alpha-conotoxin BuIA, a 13-residue peptide toxin isolated from Conus bullatus. Despite its unusual 4/4 disulfide bond layout alpha-conotoxin BuIA exhibits strong antagonistic activity at alpha6/alpha3beta2beta3, alpha3beta2, and alpha3beta4 nAChR subtypes like some alpha4/7 conotoxins. alpha-Conotoxin BuIA lacks the C-terminal beta-turn present within the second disulfide loop of alpha4/7 conotoxins, having only a "pseudo omega-shaped" molecular topology. Nevertheless, it contains a functionally critical two-turn helix motif, a feature ubiquitously found in alpha4/7 conotoxins. Such an aspect seems mainly responsible for similarities in the receptor recognition profile of alpha-conotoxin BuIA to alpha4/7 conotoxins. Structural comparison of alpha-conotoxin BuIA with alpha4/7 conotoxins and alpha4/3 conotoxin ImI suggests that presence of the second helical turn portion of the two-turn helix motif in alpha4/7 and alpha4/4 conotoxins may be important for binding to the alpha3 and/or alpha6 subunit of nAChR.     

Metabolite of SIR2 reaction modulates TRPM2 ion channel

The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death. Sirtuins are protein deacetylases that regulate gene expression, apoptosis, metabolism, and aging. These NAD+-dependent enzymes catalyze a reaction in which the acetyl group from substrate is transferred to the ADP-ribose portion of NAD+ to form deacetylated product, nicotinamide, and the metabolite OAADPr, whose functions remain elusive. Using cell-based assays and RNA interference, we show that puromycin-induced cell death is greatly diminished by nicotinamide (a potent sirtuin inhibitor), and by decreased expression of sirtuins SIRT2 and SIRT3. Furthermore, we demonstrate using channel current recordings and binding assays that OAADPr directly binds to the cytoplasmic domain of TRPM2 and activates the TRPM2 channel. ADP-ribose binds TRPM2 with similarly affinity, whereas NAD+ displays almost negligible binding. These studies provide the first evidence for the potential role of sirtuin-generated OAADPr in TRPM2 channel gating.     

The Ixodes scapularis salivary protein, salp15, prevents the association of HIV-1 gp120 and CD4

Ixodes scapularis salivary protein, Salp15, inhibits CD4(+) T cell activation by binding to the most-extracellular domains of the CD4 molecule, potentially overlapping with the gp120-binding region. We now show that Salp15 inhibits the interaction of gp120 and CD4. Furthermore, Salp15 prevents syncytia formation between HL2/3 (a stable HeLa cell line expressing the envelope protein) and CD4-expressing cells. Salp15 prevented gp120-CD4 interaction at least partially through its direct interaction with the envelope glycoprotein. A phage display library screen provided the interacting residues in the C1 domain of gp120. These results provide a potential basis to define exposed gp120 epitopes for the generation of neutralizing vaccines.