Impact of interrupted leptin pathways on ventilatory control.

Leptin deficiency in ob/ob mice produces marked depression of the hypercapnic ventilatory response, particularly during sleep. We now extend our previous findings to determine whether 1) leptin deficiency affects the hypoxic ventilatory response and 2) blockade of the downstream excitatory actions of leptin on melanocortin 4 receptors or inhibitory actions on neuropeptide Y (NPY) pathways has an impact on hypercapnic and hypoxic sensitivity. We have found that leptin-deficient ob/ob mice have the same hypoxic ventilatory response as weight-matched wild-type obese mice. There were no differences in the hypoxic sensitivity between agouti yellow mice and weight-matched controls, or NPY-deficient mice and wild-type littermates. Agouti yellow mice, with blocked melanocortin pathways, exhibited a significant depression of the hypercapnic sensitivity compared with weight-matched wild-type controls during non-rapid eye movement sleep (5.8 +/- 0.7 vs. 8.9 +/- 0.7 ml x min(-1) x %CO(2)(-1), P < 0.01), but not during wakefulness. NPY-deficient transgenic mice exhibited a small increase in the hypercapnic ventilatory response compared with wild-type littermates, but this was only present during wakefulness. We conclude that interruption of leptin pathways does not affect hypoxic sensitivity during sleep and wakefulness but that melanocortin 4 blockade is associated with depressed hypercapnic sensitivity in non-rapid eye movement sleep.     

Studies of anti-fibrillogenic activity of phthalocyanines of zirconium containing out-of-plane ligands

Series of phthalocyanines of zirconium containing lysine, citric, nonanoic acid residues and dibenzolylmethane groups as out-of-plane ligands are firstly studied as inhibitors of fibrillogenesis using cyanine-based fluorescent inhibitory assay. It was shown that studied phthalocyanines at concentration of 20μM inhibited aggregation reaction on 38.5-57.6% and inhibitory activity of phthalocyanines depended on the chemical nature of out-of-plane ligand. For the most active compound PcZrLys(2) (zirconium phthalocyanine containing lysine fragment) the efficient inhibitor concentration was estimated to be 37μM. AFM studies have shown that in the presence of PcZrLys(2) the inhibition of fibrils formation and formation of spherical oligomeric aggregates took place. Due to the ability of phthalocyanines to decrease efficiently protein aggregation into the amyloid fibrils, modification of phthalocyanine molecules via out-of-plane substitutions was proposed as approach for design of anti-fibrillogenic agents with required properties.     

The annual cycle of a trans-equatorial Eurasian-African passerine migrant: different spatio-temporal strategies for autumn and spring migration

The small size of the billions of migrating songbirds commuting between temperate breeding sites and the tropics has long prevented the study of the largest part of their annual cycle outside the breeding grounds. Using light-level loggers (geolocators), we recorded the entire annual migratory cycle of the red-backed shrike Lanius collurio, a trans-equatorial Eurasian-African passerine migrant. We tested differences between autumn and spring migration for nine individuals. Duration of migration between breeding and winter sites was significantly longer in autumn (average 96 days) when compared with spring (63 days). This difference was explained by much longer staging periods during autumn (71 days) than spring (9 days). Between staging periods, the birds travelled faster during autumn (356 km d(-1)) than during spring (233 km d(-1)). All birds made a protracted stop (53 days) in Sahelian sub-Sahara on southbound migration. The birds performed a distinct loop migration (22 000 km) where spring distance, including a detour across the Arabian Peninsula, exceeded the autumn distance by 22 per cent. Geographical scatter between routes was particularly narrow in spring, with navigational convergence towards the crossing point from Africa to the Arabian Peninsula. Temporal variation between individuals was relatively constant, while different individuals tended to be consistently early or late at different departure/arrival occasions during the annual cycle. These results demonstrate the existence of fundamentally different spatio-temporal migration strategies used by the birds during autumn and spring migration, and that songbirds may rely on distinct staging areas for completion of their annual cycle, suggesting more sophisticated endogenous control mechanisms than merely clock-and-compass guidance among terrestrial solitary migrants. After a century with metal-ringing, year-round tracking of long-distance migratory songbirds promises further insights into bird migration.     

The differential engagement of arrestin surface charges by the various functional forms of the receptor

G-protein-coupled receptor signaling is terminated by arrestin proteins that preferentially bind to the activated phosphorylated form of the receptor. Arrestins also bind active unphosphorylated and inactive phosphorylated receptors. Binding to the non-preferred forms of the receptor is important for visual arrestin translocation in rod photoreceptors and the regulation of receptor signaling and trafficking by non-visual arrestins. Given the importance of arrestin interactions with the various functional forms of the receptor, we performed an extensive analysis of the receptor-binding surface of arrestin using site-directed mutagenesis. The data indicated that a large number of surface charges are important for arrestin interaction with all forms of the receptor. Arrestin elements involved in receptor binding are differentially engaged by the various functional forms of the receptor, each requiring a unique subset of arrestin residues in a specific spatial configuration. We identified several additional phosphate-binding elements in the N-domain and demonstrated for the first time that the active receptor preferentially engages the arrestin C-domain. We also found that the interdomain contact surface is important for arrestin interaction with the non-preferred forms of the receptor and that residues in this region play a role in arrestin transition into its high affinity receptor binding state.     

Kinetics of rhodopsin deactivation and its role in regulating recovery and reproducibility of rod photoresponse

The single photon response (SPR) in vertebrate phototransduction is regulated by the dynamics of R* during its lifetime, including the random number of phosphorylations, the catalytic activity and the random sojourn time at each phosphorylation level. Because of this randomness the electrical responses are expected to be inherently variable. However the SPR is highly reproducible. The mechanisms that confer to the SPR such a low variability are not completely understood. The kinetics of rhodopsin deactivation is investigated by a Continuous Time Markov Chain (CTMC) based on the biochemistry of rhodopsin activation and deactivation, interfaced with a spatio-temporal model of phototransduction. The model parameters are extracted from the photoresponse data of both wild type and mutant mice, having variable numbers of phosphorylation sites and, with the same set of parameters, the model reproduces both WT and mutant responses. The sources of variability are dissected into its components, by asking whether a random number of turnoff steps, a random sojourn time between steps, or both, give rise to the known variability. The model shows that only the randomness of the sojourn times in each of the phosphorylated states contributes to the Coefficient of Variation (CV) of the response, whereas the randomness of the number of R* turnoff steps has a negligible effect. These results counter the view that the larger the number of decay steps of R*, the more stable the photoresponse is. Our results indicate that R* shutoff is responsible for the variability of the photoresponse, while the diffusion of the second messengers acts as a variability suppressor.     

Fast and precise protein tracking using repeated reversible photoactivation

Photoactivatable fluorescent proteins opened principally novel possibilities to study proteins' movement pathways. In particular, reversibly photoactivatable proteins enable multiple tracking experiments in a long-drawn work with a single cell. Here we report 'protein rivers tracking' technique based on repeated identical rounds of photoactivation and subsequent images averaging, which results in dramatic increase of imaging resolution for fast protein movement events.