Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase

Soluble guanylate cyclase (sGC) is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the alpha- or beta-subunit of sGC show that the sGC beta-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.     

Turn stability in beta-hairpin peptides: Investigation of peptides containing 3:5 type I G1 bulge turns

The turn-forming ability of a series of three-residue sequences was investigated by substituting them into a well-characterized beta-hairpin peptide. The starting scaffold, bhpW, is a disulfide-cyclized 10-residue peptide that folds into a stable beta-hairpin with two antiparallel strands connected by a two-residue reverse turn. Substitution of the central two residues with the three-residue test sequences leads to less stable hairpins, as judged by thiol-disulfide equilibrium measurements. However, analysis of NMR parameters indicated that each molecule retains a significant folded population, and that the type of turn adopted by the three-residue sequence is the same in all cases. The solution structure of a selected peptide with a PDG turn contained an antiparallel beta-hairpin with a 3:5 type I + G1 bulge turn. Analysis of the energetic contributions of individual turn residues in the series of peptides indicates that substitution effects have significant context dependence, limiting the predictive power of individual amino acid propensities for turn formation. The most stable and least stable sequences were also substituted into a more stable disulfide-cyclized scaffold and a linear beta-hairpin scaffold. The relative stabilities remained the same, suggesting that experimental measurements in the bhpW context are a useful way to evaluate turn stability for use in protein design projects. Moreover, these scaffolds are capable of displaying a diverse set of turns, which can be exploited for the mimicry of protein loops or for generating libraries of reverse turns.     

Development of inflammation in proteoglycan-induced arthritis is dependent on Fc gamma R regulation of the cytokine/chemokine environment

FcgammaRs are specialized cell surface receptors that coordinately regulate immune responses. Although FcgammaR expression is a prerequisite for the development of several immune complex-mediated diseases, the mechanism responsible for FcgammaR-dependent regulation in autoimmunity remains unclear. Therefore, we assessed FcgammaR-dependent regulation of inflammation in proteoglycan-induced arthritis (PGIA) using FcgammaR(-/-) mice. FcgammaRIIb(-/-) mice developed arthritis at an earlier time point and with a greater severity than wild-type (WT) mice. In gamma-chain(-/-) (FcgammaRI(-/-) and FcgammaRIII(-/-)) mice, no clinical or histological evidence of inflammation was observed. Exacerbation of arthritis in FcgammaRIIb(-/-) mice correlated with enhanced PG-specific Ab production, but did not significantly affect PG-specific T cell priming. In gamma-chain(-/-) mice, the absence of arthritis did not correlate with serum Ab responses, as PG-specific Ab production was normal. Although PG-specific T cell proliferation was diminished, spleen cells from gamma-chain(-/-) mice successfully adoptively transferred arthritis into SCID mice. Our studies indicated that the mechanism responsible for FcgammaR regulation of PGIA development was at the level of inflammatory cytokine and beta-chemokine expression within the joint. FcgammaRIIb regulated the development of PGIA by controlling the initiation of cytokine and chemokine expression within the joint before the onset of arthritis, whereas the expression of FcgammaRI and or FcgammaRIII controlled cytokine and chemokine expression late in the development of PGIA during the onset of disease. These results suggest that FcgammaRs are critical for the development of inflammation during PGIA, possibly by maintaining or enhancing inflammatory cytokine and beta-chemokine production.     

Calcium microdomains in aspiny dendrites

Dendritic spines receive excitatory synapses and serve as calcium compartments, which appear to be necessary for input-specific synaptic plasticity. Dendrites of GABAergic interneurons have few or no spines and thus do not possess a clear morphological basis for synapse-specific compartmentalization. We demonstrate using two-photon calcium imaging that activation of single synapses on aspiny dendrites of neocortical fast spiking (FS) interneurons creates highly localized calcium microdomains, often restricted to less than 1 microm of dendritic space. We confirm using ultrastructural reconstruction of imaged dendrites the absence of any morphological basis for this compartmentalization and show that it is dependent on the fast kinetics of calcium-permeable (CP) AMPA receptors and fast local extrusion via the Na+/Ca2+ exchanger. Because aspiny dendrites throughout the CNS express CP-AMPA receptors, we propose that CP-AMPA receptors mediate a spine-free mechanism of input-specific calcium compartmentalization.     

Akt activation induced by an antioxidant compound during ischemia-reperfusion

Molecular mechanisms of cardioprotection afforded by modified mexiletine compounds were investigated during ischemia-reperfusion (IR) in Langendorff perfused hearts. Rat hearts were subjected to a global 25 min ischemia followed by reperfusion, either untreated or treated with mexiletine, or three substituted mexiletine derivates (5 muM). A modified mexiletine derivative (H-2693) promoted best the recovery of myocardial energy metabolism (assessed by (31)P NMR spectroscopy) compared to untreated and mexiletine-treated hearts. H-2693 also preserved cardiac contractile function and attenuated the IR-induced lipid peroxidation (TBARS formation) and protein oxidation (carbonyl content). Western blot revealed that H-2693 propagated the phosphorylation of Akt (activation) and its downstream substrate glycogen synthase kinase-3beta (GSK-3beta, inactivation) compared to untreated IR. Parallel treatment with the phosphatidylinositol-3-kinase (upstream activator of Akt) inhibitor wortmannin (100 nM) abolished the beneficial effects of H-2693 on energetics and function, and reduced Akt and GSK-3beta phosphorylation. As a result of the antiapoptotic impacts of Akt activation, H-2693 decreased caspase-3 activity, which was neutralized by wortmannin. Here we first demonstrated that a free radical-entrapping compound could activate the prosurvival Akt pathway beyond its proven ability to scavenge reactive oxygen species. In conclusion, the favorable influence of H-2693 on signaling events during IR may have considerably contributed to its cardioprotective effect.     

Measurement of Inositol 1,4,5-Trisphosphate in Living Cells Using an Improved Set of Resonance Energy Transfer-Based Biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP₃) sensors were created to follow intracellular InsP₃ changes in single living cells and in cell populations. Similar to previous InsP₃ sensors the new sensors are based on the ligand binding domain of the human type-I InsP₃ receptor (InsP₃R-LBD), but contain a mutation of either R265K or R269K to lower their InsP₃ binding affinity. Tagging the InsP₃R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP₃ in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP₃ concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP₃ sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP₃ after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP₃ and Ca²⁺ levels in BRET experiments, the Cameleon D3 Ca²⁺ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P₂ resulted in the fall of InsP₃ level, followed by the decrease of the Ca²⁺-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca²⁺-signal preceded the fall of InsP₃ level indicating an InsP₃-, independent, direct regulation of capacitative Ca²⁺ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP₃ sensor can be used to monitor both the increase and decrease of InsP₃ levels in live cells suitable for high-throughput BRET applications.     

Women Favour Dyadic Relationships,but Men Prefer Clubs: Cross-Cultural Evidence from Social Networking

The ability to create lasting, trust-based friendships makes it possible for humans to form large and coherent groups. The recent literature on the evolution of sociality and on the network dynamics of human societies suggests that large human groups have a layered structure generated by emotionally supported social relationships. There are also gender differences in adult social style which may involve different trade-offs between the quantity and quality of friendships. Although many have suggested that females tend to focus on intimate relations with a few other females, while males build larger, more hierarchical coalitions, the existence of such gender differences is disputed and data from adults is scarce. Here, we present cross-cultural evidence for gender differences in the preference for close friendships. We use a sample of ∼112,000 profile pictures from nine world regions posted on a popular social networking site to show that, in self-selected displays of social relationships, women favour dyadic relations, whereas men favour larger, all-male cliques. These apparently different solutions to quality-quantity trade-offs suggest a universal and fundamental difference in the function of close friendships for the two sexes.