Isolation and characterization of gas vesicles from Microcyclus aquaticus

Intact gas vesicles of Microcyclus aquaticus S1 were isolated by using centrifugally accelerated flotation of vesicles and molecular sieve chromatography. Isolated gas vesicles were cylindrical organelles with biconical ends and measured 250×100 nm. The gas vesicle membrane was composed almost entirely of protein; neither lipid nor carbohydrate was detected, although one mole of phosphate per mole of protein was found. Amino acid analysis indicated that the protein contained 54.6% hydrophobic amino acid residues, lacked sulfur-containing amino acids, and had a low aromatic amino acid content. The protein subunit composition of the vesicles was determined by gel electrophoresis in (i) 0.1% sodium dodecyl sulfate at pH 9.0 and (ii) 5 M urea at pH 2.0. The membrane appeared to consist of one protein subunit of MW 50 000 daltons. Charge isomers of this subunit were not detected on urea gels. Antiserum prepared against purified gas vesicles of M. aquaticus S1 cross-reacted with the gas vesicles of all other gas vacuolate strains of M. aquaticus, as well as those of Prosthecomicrobium pneumaticum, Nostoc muscorum, and Anabaena flos-aquae, indicating that the gas vesicles of these widely divergent organisms have some antigenic determinants in common.Abbreviations SDS sodium dodecyl sulfate - MW molecular weight - Tris tris(hydroxymethyl)aminomethane - EDTA disodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - TCA trichloroacetic acid - P _c pressure necessary to collapse gas vesicles     

Effects of cryoprotective agents and freezing on abdominal ganglia of aplysia.

Isolated Aplysia depilans abdominal ganglia were exposed to 10 and 20% dimethylsulphoxide (Me₂SO) or glycerol at room temperature. Results indicate that Me₂SO induced an irreversible depression of extracellularly recorded ganglionic spontaneous spike generation while glycerol proved to be non-toxic. Intracellular recordings of individual nerve cell spontaneous activity during exposure to the cryoprotective agents were obtained in a few preliminary experiments. Both Me₂SO and glycerol induced a decrement in the nerve cell membrane potential. The main difference between the action of the two cryoprotectants was in the rate and the amount of depolarization, both being higher in the case of Me₂SO exposure.The Aplysia abdominal ganglia were frozen to ?20 °C and to ?196 °C. In all but one ganglia frozen to ?20 °C, including the preparations frozen in the absence of any cryoprotective agent, functional recovery was obtained after thawing. However, only the application of 20% glycerol improved the recovery of the preparations to a significant extent. In ganglia protected with 20% glycerol a full recovery of the action potential amplitude and frequency was obtained. In ganglia protected with 20% glycerol intracellular recordings of individual nerve cells demonstrated spontaneous spike activities before freezing and after thawing.No functional recovery was observed in ganglia frozen to ?196 °C in the absence of a cryoprotective agent. While in most preparations frozen with a cryoprotectant spontaneously generated spikes were recorded after thawing. However, the action potential frequency and amplitude were significantly depressed. It is concluded that further investigation is required to improve the freezing technique so that Aplysia ganglia may be preserved at low temperatures. It is suggested that intracellular exploration of the effects of cryoprotectants and freezing on identified nerve cell membrane may prove to be useful in future investigations.     

Na/K-ATPase assay in the intact guinea pig liver submitted to in situ perfusion

We describe an assay for the enzyme Na/K-ATPase in intact guinea pig livers perfused through the portal vein with modified Hank’s solution. The model uses the measurement of non-radioactive rubidium ion incorporation by liver cells, both in the absence and in the presence of the specific Na/K-ATPase inhibitor ouabain, followed by a rinsing procedure with cold saline. The concentration of Rb⁺ in acid-digested liver lobes was measured by atomic emission spectrometry and Na/K pump activity was calculated by the difference between the incorporation of Rb⁺ in the absence and in the presence of ouabain. The optimal conditions for Rb⁺ incorporation were: perfusion flow rate, 3 ml/min per liver; perfusion time at 37 °C, 60 min; rinsing time with cold saline, 5-10 min; and concentration of ouabain, 3 mM. The calculated ouabain IC₅₀ was 100 μM. The major advantage of this model is the possibility of testing experimental drugs affecting this enzyme in conditions close to those in the intact organ.     

Spatial variation in vegetation structure coupled to plant available water determined by two-dimensional soil resistivity profiling in a Brazilian savanna

Tropical savannas commonly exhibit large spatial heterogeneity in vegetation structure. Fine-scale patterns of soil moisture, particularly in the deeper soil layers, have not been well investigated as factors possibly influencing vegetation patterns in savannas. Here we investigate the role of soil water availability and heterogeneity related to vegetation structure in an area of the Brazilian savanna (Cerrado). Our objective was to determine whether horizontal spatial variations of soil water are coupled with patterns of vegetation structure across tens of meters. We applied a novel methodological approach to convert soil electrical resistivity measurements along three 275-m transects to volumetric water content and then to estimates of plant available water (PAW). Structural attributes of the woody vegetation, including plant position, height, basal circumference, crown dimensions, and leaf area index, were surveyed within twenty-two 100-m² plots along the same transects, where no obvious vegetation gradients had been apparent. Spatial heterogeneity was evaluated through measurements of spatial autocorrelation in both PAW and vegetation structure. Comparisons with null models suggest that plants were randomly distributed over the transect with the greatest mean PAW and lowest PAW heterogeneity, and clustered in the driest and most heterogeneous transect. Plant density was positively related with PAW in the top 4 m of soil. The density-dependent vegetation attributes that are related to plot biomass, such as sum of tree heights per plot, exhibited spatial variation patterns that were remarkably similar to spatial variation of PAW in the top 4 m of soil. For PAW below 4 m depth, mean vegetation attributes, such as mean height, were negatively correlated with PAW, suggesting greater water uptake from the deep soil by plants of larger stature. These results are consistent with PAW heterogeneity being an important structuring factor in the plant distribution at the scale of tens of meters in this ecosystem.     

Nanopore sensors: From hybrid to abiotic systems

This paper provides an overview of different nanostructured architectures utilised in electrochemical devices and their application in biosensing and bioelectronics. Emphasis is placed on the fabrication of nanostructured films based on a layer-by-layer (LBL) films approach. We discuss the theory and the mechanism of charge transfer in polyelectrolyte multilayer films (PEM), as well as between biomolecules and redox centres, for the development of more sensitive and selective biosensors. Further, this paper presents an overview of topics involving the interaction between nanostructured materials, including metallic nanoparticles and carbon materials, and their effects on the preservation of the activity of biological molecules immobilised on electrode surfaces. This paper also presents examples of biological molecules utilised in film fabrication, such as DNA, several kinds of proteins, and oligonucleotides, and of the role of molecular interaction in biosensing performance. Towards the utilisation of LBL films, examples of several architectures and different electrochemical approaches demonstrate the potential of nanostructured LBL films for several applications that include the diagnosis and monitoring of diseases. Our main aim in this review is to survey what can assist researchers by presenting various approaches currently used in the field of bioelectrochemistry utilising supramolecular architectures based on an LBL approach for application in electrochemical biosensing.