RhoGDI facilitates geranylgeranyltransferase-I-mediated RhoA prenylation

Protein prenylation is a post-translational modification where farnesyl or geranylgeranyl groups are enzymatically attached to a C-terminal cysteine residue. This modification is essential for the activity of small cellular GTPases, as it allows them to associate with intracellular membranes. Dissociated from membranes, prenylated proteins need to be transported through the aqueous cytoplasm by protein carriers that shield the hydrophobic anchor from the solvent. One such carrier is Rho GDP dissociation inhibitor (RhoGDI). Recently, it was shown that prenylated Rho proteins that are not associated with RhoGDI are subjected to proteolysis in the cell. We hypothesized that the role of RhoGDI might be not only to associate with prenylated proteins but also to regulate the prenylation process in the cell. This idea is supported by the fact that RhoGDI binds both unprenylated and prenylated Rho proteins with high affinity in vitro, and hence, these interactions may affect the kinetics of prenylation. We addressed this question experimentally and found that RhoGDI increased the catalytic efficiency of geranylgeranyl transferase-I in RhoA prenylation. Nevertheless, we did not observe formation of a ternary RhoGDI∗RhoA∗GGTase-I complex, indicating sequential operation of geranylgeranyltransferase-I and RhoGDI. Our results suggest that RhoGDI accelerates Rho prenylation by kinetically trapping the reaction product, thereby increasing the rate of product release.     

Genetic Drift Suppresses Bacterial Conjugation in Spatially Structured Populations

Conjugation is the primary mechanism of horizontal gene transfer that spreads antibiotic resistance among bacteria. Although conjugation normally occurs in surface-associated growth (e.g., biofilms), it has been traditionally studied in well-mixed liquid cultures lacking spatial structure, which is known to affect many evolutionary and ecological processes. Here we visualize spatial patterns of gene transfer mediated by F plasmid conjugation in a colony of Escherichia coli growing on solid agar, and we develop a quantitative understanding by spatial extension of traditional mass-action models. We found that spatial structure suppresses conjugation in surface-associated growth because strong genetic drift leads to spatial isolation of donor and recipient cells, restricting conjugation to rare boundaries between donor and recipient strains. These results suggest that ecological strategies, such as enforcement of spatial structure and enhancement of genetic drift, could complement molecular strategies in slowing the spread of antibiotic resistance genes.     

The membrane anchor R7BP controls the proteolytic stability of the striatal specific RGS protein, RGS9-2

A member of the RGS (regulators of G protein signaling) family, RGS9-2 is a critical regulator of G protein signaling pathways that control locomotion and reward signaling in the brain. RGS9-2 is specifically expressed in striatal neurons where it forms complexes with its newly discovered partner, R7BP (R7 family binding protein). Interaction with R7BP is important for the subcellular targeting of RGS9-2, which in native neurons is found in plasma membrane and its specializations, postsynaptic densities. Here we report that R7BP plays an additional important role in determining proteolytic stability of RGS9-2. We have found that co-expression with R7BP dramatically elevates the levels of RGS9-2 and its constitutive subunit, Gbeta5. Measurement of the RGS9-2 degradation kinetics in cells indicates that R7BP markedly reduces the rate of RGS9-2.Gbeta5 proteolysis. Lentivirus-mediated RNA interference knockdown of the R7BP expression in native striatal neurons results in the corresponding decrease in RGS9-2 protein levels. Analysis of the molecular determinants that mediate R7BP/RGS9-2 binding to result in proteolytic protection have identified that the binding site for R7BP in RGS proteins is formed by pairing of the DEP (Disheveled, EGL-10, Pleckstrin) domain with the R7H (R7 homology), a domain of previously unknown function that interacts with four putative alpha-helices of the R7BP core. These findings provide a mechanism for the regulation of the RGS9 protein stability in the striatal neurons.     

Kinetics of the interactions between yeast elongation factors 1A and 1Balpha, guanine nucleotides, and aminoacyl-tRNA

The interactions of elongation factor 1A (eEF1A) from Saccharomyces cerevisiae with elongation factor 1Balpha (eEF1Balpha), guanine nucleotides, and aminoacyl-tRNA were studied kinetically by fluorescence stopped-flow. eEF1A has similar affinities for GDP and GTP, 0.4 and 1.1 microm, respectively. Dissociation of nucleotides from eEF1A in the absence of the guanine nucleotide exchange factor is slow (about 0.1 s(-1)) and is accelerated by eEF1Balpha by 320-fold and 250-fold for GDP and GTP, respectively. The rate constant of eEF1Balpha binding to eEF1A (10(7)-10(8) M (-1) s(-1)) is independent of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A.GTP binds Phe-tRNA(Phe) with a K(d) of 3 nm, whereas eEF1A.GDP shows no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu.     

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca²⁺-activated K⁺ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK^–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O₂^– and H₂O₂ production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn²⁺, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca²⁺ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca²⁺ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK^–/– and BK^+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity. killing assay; reactive oxygen species; BK-deficient mice; mice infection     

Effects of local and landscape scale and cattle grazing intensity on Orthoptera assemblages of the Hungarian Great Plain

The aims of this study were to test the influence of grazing intensity, effects of local and landscape parameters, and regional effects on orthopteran assemblages. We made our investigations on extensively and intensively grazed cattle pastures in three regions of the Hungarian Great Plain. The regions differed in landscape complexity; one region was situated in a structurally simple landscape with large landscape units, one in a structurally complex landscape with marshy patches and trees in the grasslands and one in a landscape with intermediate structural complexity. In each region we had seven pairs of differently managed grasslands, which differed in grazing intensity. Grasshoppers were recorded once in July 2003 using sweepnet catches and visual and acoustic observations in two 95 m long transects at each site (84 transects in total). Botanical surveys and measurements of other local factors were also made for each transect. After samplings, we digitised the most important land-use types using aerial photographs to produce landscape scale parameters within 100 and 500 m circles around every site. Analysing the management, regional, landscape and local effects on species richness with linear mixed models, we showed only strong significant regional differences. Linear mixed models for Orthoptera abundance yielded significant regional effects and marginal management effects. However, after including local and landscape parameters in a separate model a marginal local effect was found instead of a management effect in addition to the significant regional effect. Logistic regression models of 15 species also revealed the importance of local factors, particularly the importance of grass height, which is highly dependent on grazing intensity. We conclude that management intensity has indirect effects on Orthoptera species richness and abundance. Landscape scale parameters are also important, at least for some species.     

Multimode selective detection of mercury by chiroptical fluorescent sensors based on methionine/cysteine

Two multimode Hg(II) sensors, L‐MethBQA and L‐CysBQA, were obtained by fusing methionine or S‐methyl cysteine, into a bis‐quinolyl amine‐based chiral podand scaffold. Quinolyl groups serve as the fluorophore and possess nitrogen lone pairs capable of chelating metal ions. On exposure to Hg²⁺ or Zn²⁺, these sensors show signal enhancement in fluorescence. However, Cu²⁺ quenches their fluorescence in 30:70 acetontrile/water. L‐CysBQA complexes with Hg²⁺, producing an exciton‐coupled circular dichroism spectrum with the opposite sign to the one that is produced by Cu²⁺ or Zn²⁺ complexation. L‐CysBQA binds Hg²⁺ more strongly than Zn²⁺ and is shown to differentiate Hg²⁺ from other metal ions, such as Zn²⁺, Cu²⁺, Ni²⁺, and Pb²⁺, exceptionally well. The synergistic use of relatively soft sulfur, quinoline‐based chiral ligands and chiroptically enhanced fluorescence detection results in high sensitivity and selectivity for Hg²⁺. Chirality, 2011. © 2011 Wiley‐Liss, Inc.