Thiol-induced oligomerization of α-lactalbumin at high pressure

Denaturation and aggregation of-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding of-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation of-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield of-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers of-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecular-sheets.     

Localization of α1-adrenoceptors in rat and human hearts by immunocytochemistry

The localization of the ai adrenoceptors (₁-AR) in the heart tissues from rat and human and in the cultured heart cells from neonatal rats was studied by indirect immunofluorescence and postembedding electronmicroscopical immuno-gold technique. With antipeptide antibodies directed against the second extracellular loop of the human ₁-AR (AS sequence 192–218), this receptor was found to be localized along the sarcolemma in both human and rat hearts. Similar localization sites were detected in cultivated rat neonatal cardiomyocytes. Beside the localization in cardiomyocytes, ₁-AR were identified in endothelial cells of capillaries and smooth muscle cells of coronary vessels, in neuronal endings, in mast cells of cultivated heart cells but not, or in less amount in fibroblasts. Interestingly, in the right atrium of rat heart the localization of ₁-AR was found to be near or on atrial natriuretic factor (ANF) granules, providing the basis for the -adrenergic influence on ANF release. The immunocytochemical studies further confirm and complete the findings known by using autoradiographic binding studies with specific ligands.     

The mitochondrial inner membrane anion channel is inhibited by DIDS

The mitochondrial inner membrane anion channel (IMAC) is a channel, identified by flux studies in intact mitochondria, which has a broad anion selectivity and is maintained closed or inactive by matrix Mg²⁺ and H⁺. We now present evidence that this channel, like many other chloride/anion channels, is reversibly blocked/inhibited by stilbene-2,2-disulfonates. Inhibition of malonate transport approaches 100% with IC₅₀ values of 26, 44, and 88 M for DIDS, H₂-DIDS, and SITS respectively and Hill coefficients 1. In contrast, inhibition of Cl^– transport is incomplete, reaching a maximum of about 30% at pH 7.4 and 65% at pH 8.4 with an IC₅₀ which is severalfold higher than that for malonate. The IC₅₀ for malonate transport is decreased about 50% by pretreatment of the mitochondria withN-ethylmaleimide. Raising the assay pH from 7.4 to 8.4 increases the IC₅₀ by about 50%, but under conditions where only the matrix pH is made alkaline the IC₅₀ is decreased slightly. These properties and competition studies suggest that DIDS inhibits by binding to the same site as Cibacron blue 3GA. In contrast, DIDS does not appear to compete with the fluorescein derivative Erythrosin B for inhibition. These findings not only provide further evidence that IMAC may be more closely related to other Cl^– channels than previously thought, but also suggest that other Cl^– channels may be sensitive to some of the many regulators of IMAC which have been identified.     

Biochemical and immunocytochemical identification of conglutin γ, a lupin seed lectin,in the roots of young germinating seedlings

Summary Polyclonal antibodies directed against polypeptide epitopes of conglutin , a glycosylated lectin in lupin seed, have been used to identify and quantify this protein in root extracts of germinating lupins. The highest conglutin content was found in protein extracts of root elongation zones after 5 to 7 days germination. Root conglutin showed the same subunit composition, glycosylation pattern, isoelectric point, and lectin activity as the cotyledonary one. Immunolocalization experiments on root thin sections demonstrated that conglutin is chiefly present in the intercellular spaces of the cortical parenchyma, where it forms large aggregates. Labelling of the Golgi complexes and the area between the plasmalemma and cell wall revealed the conglutin pathway from post-synthetic processing to excretion via the secretory system.Abbreviations EDTA ethylene diamino tetraacetic acid - IEF isoelectric focusing - LRW London resin white - NC nitrocellulose - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - P_i inorganic phosphate - SDS sodium dodecylsulphate - TEM transmission electron microscopy     

Predicting badger sett numbers: evaluating methods in East Sussex

Abstract. One way in which a species' numbers may be estimated without direct counting is to predict their dispersion and density from more readily available habitat measures, such as landscape variables measured from maps or vegetation variables measured in the field. We compare the power of ordination and regression techniques for predicting badger ( Meles meles L.) numbers at a local scale, using a land class system, map-read landscape variables and field-derived vegetation variables. Sett density was used as a surrogate of badger density. Multiple linear regression using vegetation and landscape variables together gave the most accurate prediction of sett density, while ordination techniques were of lesser value. The addition of vegetation variables to landscape variables did not substantially improve the power of ordination. Outlier Sett Density was predicted more accurately, and by different variables, to Main Sett Density. The relationship between badger ecology and habitat variables that were useful in predicting sett density is discussed.     

Production of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine by entrapped ACV-synthetase fromStreptomyces clavuligerus

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase fromStreptomyces clavuligerus was studied under conditions that enabled the reuse of the enzyme. Coupling of ACV-synthetase to DEAE-Trisacryl and aminopropyl-glass resulted in an immobilized enzyme product of little or no catalytic activity. However, an enzyme reactor was designed by physical confinement of partially-purified ACV-synthetase in an ultrafiltration cell. This system was stimulated by phosphoenolpyruvate at lower concentrations of ATP, an effect not observed with purified enzyme. Up to 30% conversion of the limiting substrate, cysteine, to ACV occurred under semi-continuous conditions. Reaction products were investigated as potential inhibitors: AMP was the most inhibitory, but only when used at concentrations in excess of those produced in reaction mixtures. Under a nitrogen atmosphere, both product and enzyme stabilities were greatly improved and the enzyme retained 45–46% of its initial activity after five uses at room temperature during a 24-h period. Extrapolations based on these data suggest that 1.3 g partially purified enzyme (0.13 U g^–1) would be capable of producing 411 mg of ACV in a 1-L reaction mixture in this period.     

Production of fuel alcohol from oats by fermentation

Very high gravity (>30 g dissolved solids per 100 ml) mashes were prepared from hulled and hulless oats and fermented at 20° C with active dry yeast to produce ethanol. Excessive viscosity development during mashing was prevented by hydrolyzing -glucan with crude preparations of -glucanase or Biocellulase. Both these preparations possessed endo--glucanase activity. By using these enzymes and by decreasing the water to grain ratio, very high gravity mashes with low viscosity were prepared. Unlike wheat and barley mashes, oat mashes contained sufficient amounts of assimilable nitrogen to promote a fast rate of fermentation. The free amino nitrogen (FAN) content of oat mash could be predicted by the equation, mg FAN L^–1=8.9n wheren is the number of grams of dissolved solids in 100 ml of mash supernatant fluid. Ethanol yields of 353.2±3.7 L and 317.6±1.3 L were obtained per tonne (dry weight basis) of hulless (59.8% starch) and hulled (50.8% starch) oats respectively. The efficiency of conversion of starch to ethanol was the same in normal and very high gravity mashes.     

Ecological optima and tolerances of Thelypteris limbosperma,Athyrium distentifolium,and Matteuccia struthiopteris along environmental gradients in Western Norway

The distribution and abundance of Thelypteris limbosperma, Athyrium distentifolium, and Matteuccia struthiopteris are modelled statistically in relation to 14 environmental variables along the major climatic, topographic, and edaphic gradients in western Norway. The data are from 624 stands from which measurements or estimates of mean January and mean July temperatures, humidity, altitude, aspect, and slope are available. From 182 of these stands eight soil variables have also been measured. The species responses are quantified by two numerical methods: Gaussian logit regression and weighted averaging (WA) regression. The estimated WA optima suggest that A. distentifolium has an ecological preference for low July and January temperatures, high altitudes, and soils of low-medium pH and base content. The species shows statistically significant Gaussian responses with summer temperature, humidity (= Martonnes humidity index), altitude, slope, aspect, pH, cation exchange capacity, and base saturation with optima of 8.7 °C, 188.9, 1220 m, 28°, 29°, 4.8, 13.77 mEq 100 g dry soil^-1, and 13.4%, respectively. These suggest that the occurrence and relative abundance of A. distentifolium are well predicted by summer temperature, topography, and soil pH and base status. T. limbosperma has WA optima that suggest that it favours moderately high winter and summer temperatures, high humidity, medium altitude, and soils of low pH and base content. It has significant Gaussian responses to summer temperature (optimum =12.6 °C), winter temperature (-1.8 °C), humidity (179.2), altitude (459.5 m), slope (22.5°), and Na (0.7 mg 100 g dry soil^-1). These suggest that climatic factors, altitude, and slope are significant predictors for its occurrence and abundance. M. struthiopteris has high WA optima for summer temperature, pH, Ca, Mg, K, Na, cation exchange capacity (CEC), and base saturation, and a low optima for humidity and winter temperature. Of these, summer temperature (16.0 °C), Ca (63.1 mg 100 g dry soil^-1), Mg (41.0 mg 100 g dry soil^-1), K (23.6 mg 100 g dry soil^-1), Na (5.0 mg 100 g dry soil^-1), CEC (60.7 mEq 100 g dry soil^-1), and base saturation (56.3%) have significant Gaussian logit responses, as do aspect (150.2°) and loss-on-ignition (9.4%). These results suggest that the occurrence and relative abundance of M. struthiopteris are well predicted by high soil base cations, a generally southern aspect, low organic content in the soil, and high July temperatures.