Single-Molecule Investigation of the Influence Played by Lipid Rafts on Ion Transport and Dynamic Features of the Pore-Forming Alamethicin Oligomer

In this experimental work we employed single-molecule electrical recordings on alamethicin oligomers inserted in lipid bilayers made of brain sphingomyelin (bSM), palmitoyloleoylphosphatidylcholine (POPC) and cholesterol (chol) to unravel novel aspects regarding lipid raft interactions with pore-forming peptides. We probed the effect of lipid rafts on electrical properties of inserted alamethicin oligomers, and our data convincingly prove that the single-channel electrical conductance of various subconductance states of the alamethicin oligomer (1) increases in the presence of raft-containing ternary lipid mixtures (POPC-chol-bSM) compared to cases when bilayers were made of POPC-chol and POPC and (2) decreases in the presence of raft-containing ternary lipid mixtures compared to nonraft ternary mixtures which favor the fluid and liquid ordered phases alone. Our data demonstrate that the presence of lipid rafts leads to a slower association kinetics of alamethicin oligomers, seemingly reflecting a slower lateral diffusion process of such peptide aggregates compared to the case of nonraft, binary lipid mixtures. Furthermore, we show that the electrical capacitance of ternary lipid mixtures (POPC-chol-bSM) decreases in the presence of raft domains by comparison to nonraft binary phases (POPC-chol) or POPC alone, and this could constitute an additional mechanism via which macroscopic electrical manifestations of eukaryotic cells are modulated by the coexistence of gel and fluid domains of the plasma membrane.     

Structure of a sliding clamp on DNA

The structure of the E. coli beta clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the beta ring. The pronounced 22 degrees angle of DNA through beta may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other.     

Retinal pigment epithelium-retinal G protein receptor-opsin mediates light-dependent translocation of all-trans-retinyl esters for synthesis of visual chromophore in retinal pigment epithelial cells

Visual perception begins with the absorption of a photon by an opsin pigment, inducing isomerization of its 11-cis-retinaldehyde chromophore. After a brief period of activation, the resulting all-trans-retinaldehyde dissociates from the opsin apoprotein rendering it insensitive to light. Restoring light sensitivity to apo-opsin requires thermal re-isomerization of all-trans-retinaldehyde to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle in retinal pigment epithelial (RPE) cells. Vertebrates can see over a 10(8)-fold range of background illumination. This implies that the visual cycle can regenerate a visual chromophore over a similarly broad range. However, nothing is known about how the visual cycle is regulated. Here we show that RPE cells, functionally or physically separated from photoreceptors, respond to light by mobilizing all-trans-retinyl esters. These retinyl esters are substrates for the retinoid isomerase and hence critical for regenerating visual chromophore. We show in knock-out mice and by RNA interference in human RPE cells that this mobilization is mediated by a protein called "RPE-retinal G protein receptor" (RGR) opsin. These data establish that RPE cells are intrinsically sensitive to light. Finally, we show that in the dark, RGR-opsin inhibits lecithin:retinol acyltransferase and all-trans-retinyl ester hydrolase in vitro and that this inhibition is released upon exposure to light. The results of this study suggest that RGR-opsin mediates light-dependent translocation of all-trans-retinyl esters from a storage pool in lipid droplets to an "isomerase pool" in membranes of the endoplasmic reticulum. This translocation permits insoluble all-trans-retinyl esters to be utilized as substrate for the synthesis of a new visual chromophore.     

The contribution of naturally occurring polymorphisms in altering the biochemical and structural characteristics of HIV-1 subtype C protease

Fourteen subtype B and C protease variants have been engineered in an effort to study whether the preexistent baseline polymorphisms, by themselves or in combination with drug resistance mutations, differentially alter the biochemical and structural features of the subtype C protease when compared with those of subtype B protease. The kinetic studies performed in this work showed that the preexistent polymorphisms in subtype C protease, by themselves, do not provide for a greater level of resistance. Inhibition analysis with eight clinically used protease inhibitors revealed that the natural polymorphisms found in subtype C protease, in combination with drug resistance mutations, can influence enzymatic catalytic efficiency and inhibitor resistance. Structural analyses of the subtype C protease bound to nelfinavir and indinavir showed that these inhibitors form similar interactions with the residues in the active site of subtype B and C proteases. It also revealed that the naturally occurring polymorphisms could alter the position of the outer loops of the subtype C protease, especially the 60's loop.     

Determination of superoxide dismutase-like activity in some divalent metal saccharinates

The superoxide-dismutase-like activity of a series of divalent metal saccharinates of general stoichiometry [M^II(Sac)₂(H₂O)₄]·2H₂O (with M^II=Mn,Fe,Co,Ni,Cu,Zn) has been investigated using the nitroblue tetrazolium O ₂ ⁻ reduction assay. The results show that all these complexes possess the capability to dismutate the superoxide anion generated in the xanthine/xanthine oxidase system. Interestingly, the greatest activity is shown by the corresponding copper complex. The results are discussed and compared with those obtained for native superoxide dismutase, which was tested under the same experimental conditions. Dedicated to Prof. Pedro J. Aymonino on the occasion of his 65^th birthday.     

<Emphasis Type="Italic">NmEXT</Emphasis> Extensin Gene: a Positive Regulator of Resistance Response Against the Oomycete <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis>

The oomycete pathogens produce important diseases in many plant species. To identify extensin genes expressed during the oomycete Phytophthora nicotianae-Nicotiana megalosiphon interaction, we used the SuperSAGE technology. Using this approach, we detected a N. megalosiphon extensin gene (NmEXT) triggered during the interaction. The extensin gene accumulation induced by the pathogen correlated with disease resistance in different Nicotiana species. Transient expression of NmEXT gene in susceptible Nicotiana tabacum enhanced the resistance to P. nicotianae. Our date indicated that NmEXT gene served a positive role in N. tabacum resistance against P. nicotianae.     

Selective choice of sucrose solution concentration by the hovering hawk mothMacroglossum stellatarum

To determine the preference of the hovering hawk mothMacroglossum stellatarum for different sugar concentrations, the foraging behavior of adults were analyzed under laboratory conditions. Six sucrose concentrations (range, 10–60%, w/w) were simultaneously offered in six artificial ab libitum feeders. The number of feeding bouts and the duration of each visit were automatically recorded and stored in a computer. Results showed that the frequency of visits to the feeders did not vary among the different solutions offered, but the gathered volume by the group attained a maximum at between 20 and 50% (w/w). Moths invested more time in front of the feeder with the more concentrated sugar solutions. It was assumed that factors different from maximizing energy intake, such as water balance and viscosity of concentrated nectars, have to be considered in order to understand the observed patterns of nectar choice.     

Lifetime philopatry in the blue-footed booby: a longitudinal study

Philopatry over the lifetime and its relationship with reproductivesuccess were examined using longitudinal records of nest locationand reproduction of individual blue-footed boobies. Males showedshorter natal dispersal than females, and natal dispersal distanceof both sexes were unrelated to either first reproductive successor lifetime reproductive success. Throughout the early lifetime,males and females nested closer to their first breeding sitesthan to their natal sites, and comparison with a simulationof successive breeding dispersals in random directions showedthat male and female blue-footed boobies are philopatric tothe first breeding site. Therefore, throughout the early lifetime,the first breeding site seems to function as a point of referencefor breeding site use together with the previous season's site.Males and females with shorter natal dispersal distances showedstronger lifetime philopatry to their first breeding sites,suggesting stable individual variation in competitive abilityor dispersal phenotype. However, early lifetime philopatry tofirst breeding sites was unrelated to annual breeding success.Compared with simple fidelity to previous breeding sites, lifetimephilopatry to first breeding sites should result in increasedkin interactions and greater selection for kin recognition,altruism and inbreeding avoidance, as well as long-term familiaritywith neighbors.     

Lymphatic Clearance of the Brain: Perivascular,Paravascular and Significance for Neurodegenerative Diseases

The lymphatic clearance pathways of the brain are different compared to the other organs of the body and have been the subject of heated debates. Drainage of brain extracellular fluids, particularly interstitial fluid (ISF) and cerebrospinal fluid (CSF), is not only important for volume regulation, but also for removal of waste products such as amyloid beta (Aβ). CSF plays a special role in clinical medicine, as it is available for analysis of biomarkers for Alzheimer’s disease. Despite the lack of a complete anatomical and physiological picture of the communications between the subarachnoid space (SAS) and the brain parenchyma, it is often assumed that Aβ is cleared from the cerebral ISF into the CSF. Recent work suggests that clearance of the brain mainly occurs during sleep, with a specific role for peri- and para-vascular spaces as drainage pathways from the brain parenchyma. However, the direction of flow, the anatomical structures involved and the driving forces remain elusive, with partially conflicting data in literature. The presence of Aβ in the glia limitans in Alzheimer’s disease suggests a direct communication of ISF with CSF. Nonetheless, there is also the well-described pathology of cerebral amyloid angiopathy associated with the failure of perivascular drainage of Aβ. Herein, we review the role of the vasculature and the impact of vascular pathology on the peri- and para-vascular clearance pathways of the brain. The different views on the possible routes for ISF drainage of the brain are discussed in the context of pathological significance.