Brain plasmamembrane NA+:,K+-ATPase is inhibited by acetylated tubulin

Membranes from brain tissue contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. The hydrophobic behavior of this tubulin is due to the formation of a complex with the -subunit of Na⁺,K⁺-ATPase. In the present work we show that the interaction of tubulin with Na⁺K⁺-ATPase inhibits the enzyme activity. We found that the magnitude of the inhibition is correlated with: (1) concentration of the acetylated tubulin isoform present in the tubulin preparation used, and (2) amount of acetylated tubulin isoform isolated as a hydrophobic compound. In addition, some compounds involved in the catalytic action of Na⁺K⁺-ATPase were assayed to determine their effects on the inhibitory capability of tubulin on this enzyme. The inhibitory effect of tubulin was only slightly decreased by ATP at relatively low nucleotide concentration (0.06 mM). NaCl (1-160 mM) and KCl (0.2-10 mM) showed no effect whereas inorganic phosphate abolished the inhibitory effect of tubulin in a concentration-dependent manner.     

A sexually dimorphic group of atypical glomeruli in the mouse olfactory bulb

Atypical glomeruli (AtG) are clearly distinguishable from typical ones because of their strong cholinergic innervation. AtG are located in defined positions in the caudal half of the main olfactory bulb of rodents. The AtG partially overlap with other specialized olfactory subsystems, such as the modified glomerular complex, which is close to the accessory olfactory bulb. So far, possible sex differences in these specialised olfactory systems have not been investigated. In this work we have identified AtG in the mouse by means of acetylcholinesterase histochemistry and compared the number and size of these glomeruli between the sexes and also between the two strains that demonstrate intraglomerular synaptic differences, i.e. BALB/c and CD-1 mice. First, we divided the AtG into three types according to their position (I, rostral-most; II, around the accessory olfactory bulb; III, caudal-most) or their reactivity to acetylcholinesterase histochemistry (AtG type II being the least reactive glomeruli). ANOVA analyses revealed differences in the maximum diameter of glomeruli among the three types, but not in their sectional areas, indicating that all three types have different shapes. Moreover, both morphoplanimetric parameters were seen to be different between the two strains studied and also between the sexes: male mice and BALB/c animals had the largest glomeruli. The number of AtG was also significantly different between the sexes and strains, although these factors presented a strong interaction. Thus, the males had higher numbers of AtG in the CD-1 strain whereas in the BALB/c mice males demonstrated fewer AtG than females. These differences in number were largely due to AtG type II. The present work is evidence that AtG type II is a sexually dimorphic group of specialized glomeruli located in the main olfactory bulb.     

New insights into host factor requirements for prokaryotic beta-recombinase-mediated reactions in mammalian cells

The prokaryotic beta-recombinase catalyzes site-specific recombination between two directly oriented minimal six sites in mammalian cells, both on episomic and chromatin-integrated substrates. Using a specific recombination activated gene expression system, we report the site-specific recombination activity of an enhanced green fluorescent protein (EGFP) fused version of beta-recombinase (beta-EGFP). This allows expression of active beta-recombinase detectable in vivo and in fixed cells by fluorescence microscopy. In addition, cellular viability is compatible with a substantial level of expression of the beta-EGFP protein. Using fluorescence-activated cell sorting, we have been able to enrich cell populations expressing this fusion protein. Application of this strategy has allowed us to study in more depth the host factor requirements for this system. Previous work showed that eukaryotic HMG1 protein was necessary and sufficient to help beta-recombinase activity in vitro. The influence of ectopic expression of HMG1 protein in the recombination process has been analyzed, indicating that HMG1 overexpression does not lead to a significant increase on the efficiency of beta-recombinase-mediated recombination both on episomal substrates and chromatin-associated targets. In addition, beta-recombinase-mediated recombination has been demonstrated in HMG1 deficient cells at the same levels as in wild type cells. These data demonstrate the existence of cellular factors different from HMG-1 that can act as helpers for beta-recombinase activity in the eukaryotic environment.     

Distribution of virus-like Particles in an Oligotrophic Marine Environment (Alboran Sea,Western Mediterranean)

Viruses are abundant in a variety of aquatic environments, often exceeding bacterial abundance by one order of magnitude. In the present study, the spatial distribution of viruses in offshore waters of the Alboran Sea (Western Mediterranean) have been studied to determine the relationships between viruses and host communities in this oligotrophic marine environment. Viral abundance was determined using two methods: (i) epifluorescence light microscopy using the dsDNA binding fluorochrome DAPI, and (ii) direct counts by transmission electron microscopy (TEM). The results obtained were significantly different; the highest viral counts were obtained by mean of TEM analyses. In all the samples tested the number of viruses was exceeded by the bacterial concentrations, with a ratio between viral and bacterial titers varying between 1.4 and 20. VLP (virus-like particle) counts were not significantly correlated (p > 0.001) with chlorophyll a concentration or the abundance of cyanobacteria. However, there was a positive and significant correlation with bacterial abundance (p < 0.001). The analysis of size and morphology of viral particles by TEM and the correlation obtained between the numbers of VLP and bacteria suggest that the majority of the viral particles in the Alboran Sea are bacteriophages. None of the indirect evidence suggested that eukaryotic algae or cyanobacteria were important host organisms in these waters.     

Mixed membranes of sphingolipids and glycerolipids as studied by spin-label ESR spectroscopy. A search for domain formation

The temperature dependences of the ESR spectra from different positional isomers of sphingomyelin and of phosphatidylcholine spin-labeled in their acyl chain have been compared in mixed membranes composed of sphingolipids and glycerolipids. The purpose of the study was to identify the possible formation of sphingolipid-rich in-plane membrane domains. The principal mixtures that were studied contained sphingomyelin and the corresponding glycerolipid phosphatidylcholine, both from egg yolk. Other sphingolipids that were investigated were brain cerebrosides and brain gangliosides, in addition to sphingomyelins from brain and milk. The outer hyperfine splittings in the ESR spectra of sphingomyelin and of phosphatidylcholine spin-labeled on C-5 of the acyl chain were consistent with mixing of the sphingolipid and glycerolipid components, in fluid-phase membranes. In the gel phase of egg sphingomyelin and its mixtures with phosphatidylcholine, the outer hyperfine splittings of sphingomyelin spin-labeled at C-14 of the acyl chain of sphingomyelin are smaller than those of the corresponding sn-2 chain spin-labeled phosphatidylcholine. This is in contrast to the situation with sphingomyelin and phosphatidylcholine spin-labeled at C-5, for which the outer hyperfine splitting is always greater for the spin-labeled sphingomyelin. The behavior of the C-14 spin-labels is attributed to a different geometry of the acyl chain attachments of the sphingolipids and glycerolipids that is consistent with their respective crystal structures. The two-component ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled at C-14 of the acyl chain directly demonstrate a broad two-phase region with coexisting gel and fluid domains in sphingolipid mixtures with phosphatidylcholine. Domain formation in membranes composed of sphingolipids and glycerolipids alone is related primarily to the higher chain-melting transition temperature of the sphingolipid component.     

A theoretical framework of the hybrid mechanism of photosystem II photodamage

Photosynthesis Research - Photodamage of photosystem II is a significant physiological process that is prevalent in the fields of photobiology, photosynthesis research and plant/algal stress. Since...     

Thermodynamics of Ca2+ transport through sarcoplasmic reticulum membranes during the transient-state of simulated reactions

The kinetics of a chemical model of Ca2+ transport and coupled ATPase activity in sarcoplasmic reticulum membranes were solved for the transient-state of simulated reactions, using a numerical integration procedure. The simulation conditions reproduced in vitro experiments using either fragmented membranes or vesicles with Ca2+ accumulating ability. The results yielded the concentrations of all the ligands and intermediates of the enzymatic cycle as a function of the reaction time. These results were applied to calculations of several thermodynamic variables: (1) the step by step profile of the standard free energy change of the cycle. (2) The step by profile of the actual free energy change of the cycle, and its evolution with the reaction time. (3) The separate contributions of ATP hydrolysis and Ca2+ transport to the overall free energy change with the reaction. (4) The dependence of the velocity of the free energy change with the reaction time. (5) The efficiency of the transport system, and its change with the reaction time. (6) The separate contributions of the Ca2+ gradient and some enzymatic intermediates as free energy stores. The main findings are: (1) the step by step diagrams of the free energy change calculated from the results of the kinetic analysis better describe the thermodynamic profile of the cycle than previously reported diagrams of the standard free energy and basic free energy changes. The relative contribution of each partial step to the driving force of the whole reactions, as well as their changes upon the advancement of the reactions, are derived from the diagrams. (2) Free energy yielded by ATP hydrolysis is stored by the system, not only as a Ca2+ gradient, but also as enzymatic intermediates of the reaction. The progressive increase of both free energy pools upon the advancement of the reaction is quantitated.     

A Polymer/Oil Based Nanovaccine as a Single-Dose Immunization Approach

The recognized necessity for new antigen delivery carriers with the capacity to boost, modulate and prolong neutralizing immune responses prompted our approach, in which we describe a multifunctional nanocarrier consisting of an oily nanocontainer protected by a polymeric shell made of chitosan (CS), named CS nanocapsules (CSNC). The CS shell can associate the antigen on its surface, whereas the oily core might provide additional immunostimulating properties. In this first characterization of the system, we intended to study the influence of different antigen organizations on the nanocarrier''s surface (using the recombinant hepatitis B surface antigen –rHBsAg– as a model antigen) on their long-term immunopotentiating effect, without any additional immunostimulant. Thus, two prototypes of antigen-loaded CSNC (CSNC+ and CSNC−), exhibiting similar particle size (200 nm) and high antigen association efficiency (>80%), were developed with different surface composition (polymer/antigen ratios) and surface charge (positive/negative, respectively). The biological evaluation of these nanovaccines evidenced the superiority of the CSNC+ as compared to CSNC- and alum-rHBsAg in terms of neutralizing antibody responses, following intramuscular vaccination. Moreover, a single dose of CSNC+ led to similar IgG levels to the positive control. The IgG1/IgG2a ratio suggested a mixed Th1/Th2 response elicited by CSNC+, in contrast to the typical Th2-biased response of alum. Finally, CSNC+ could be freeze-dried without altering its physicochemical properties and adjuvant effect in vivo. In conclusion, the evaluation of CSNC+ confirms its interesting features for enhancing, prolonging and modulating the type of immune response against subunit antigens, such as rHBsAg.