Measurement of the Membrane Potential of Isolated Nerve Terminals by the Lipophilic Cation [3H]Triphenylmethylphosphonium Bromide

Abstract: The lipophilic cation [³H]triphenylrnethylphosphonium bromide ([³H]TPMP⁺) was investigated as a measure of the membrane potential of synaptosomes. Conditions under which [³H]TPMP⁺ achieved an equilibrium distribution were tested. The toxicity of TPMP has been studied relative to its inhibitory effects on [³H]y-aminobutyric acid ([³H]GABA) transport. In some experiments the distribution of ⁸⁶RbZ⁺ and [³H]TPMP⁺ was changed upon incubation in the presence of elevated levels of K⁺, ouabain, or KCN, or at 0°C in a way that would be expected from the membrane potential. In normal incubation conditions a membrane potential of ∼−60 mv was calculated.     

Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin

Binding constants for the binding of high-affinity heparin to antithrombin at different ionic strengths were determined by fluorescence titrations and were also estimated from dissociation curves of the heparin-antithrombin complex. These curves were monitored by near-ultraviolet circular dichroism or fluorescence. The dependence of the binding constant on the activity of NaCl suggested that maximally 5–6 charged groups are directly involved in the interaction between the two macromolecules. Major pH-dependent changes of the interaction, as evident by changes of the spectroscopic properties of the complex between the molecules, were found to occur below pH 5.5 and above pH 8.5. The acid change, which was irreversible, was most likely caused by an irreversible conformational change of antithrombin. At alkaline pH, however, the gross conformation of antithrombin was stable up to pH 12, while the affinity of high-affinity heparin for antithrombin began to decrease markedly at pH 8.5. The dissociation curve, which was reversible, had a midpoint around pH 9.5. This is compatible with the loss of affinity being caused by either a local conformational change, by ionization of tyrosine or by titration of one or more amino groups.     

Upstream migratory behaviour in landlocked Arctic charr

Synopsis Extensive upstream migration of landlocked Arctic charr during spring floods was recorded in several tributaries of an oligotrophic lake in north-west Sweden. Migration was confined to a period of about two weeks and residence in most creeks was of short duration. Only fish migrating to two small productive lakes remained in the new habitat over the summer. Repeated annual migrations were only recorded in the creek leading to these lakes and no straying was observed among repeat migrants. Water temperatures provided the primary cues for initiation and direction of migration, although an ability to detect productive habitats by odour was indicated. Creek size, feeding opportunities during migration and conspeeific odour were subordinate guiding factors.     

Comparison of diagnostic methods for detection of low infestation levels ofVarroa jacobsoni in honey-bee (Apis mellifera) colonies

Thirty-five honey-bee colonies, originally free fromVarroa jacobsoni (Oudemans) were monitored approximately every third week for the presence of the mite during 16 months following an initial introduction of five to eight adultVarroa females in early July. Investigations of hive debris detected the presence ofV. jacobsoni in 22 colonies (63%) within three months of the mite introduction. During the first winter period (October–April), mites were found in the hive debris of 13 colonies (37%). In terms of detectingVarroa during the summer in colonies with sealed brood, investigations of hive debris were more effective than sampling of brood. Brood sampling was more effective than sampling of live bees. In colonies without sealed brood, investigations of hive debris or of live bee samples seemed approximately equally efficient. The highest correlation between sampling methods was found between daily mite downfall and mites per live bee (r=0.81) in colonies with sealed brood. During the winter, investigations of dead bees and hive debris were approximately equally efficient in detectingVarroa.     

Concentration and annual accumulation values of heavy metals in lake sediments: Their significance in studies of the history of heavy metal pollution

The first part of the paper discusses the significance of using either concentration or accumulation values for expressing the results of investigations of lake sediment cores aimed at studying the history of heavy metal pollution. Neither the values for heavy metal concentration in the lake sediment, whether expressed per gram dry sediment or per gram soluble (organic) sediment, nor the values for their total annual accumulation per unit area of the lake bottom, can, on their own, provide an accurate picture of past pollution conditions, but when considered in combination they render a fairly reliable and detailed interpretation. The second part of the paper deals with Pb, Cd and Hg analyses of cores of varved sediments from several lakes in N. Sweden. Pb and Cd pollution increased during the second half of the 19th century. In most lakes, Hg pollution seems first to have started during the 20th century. Marked increases in both concentration and accumulation of heavy metals took place during the 20th century. For recent decades, the estimated accumulation rates of heavy metals from anthropogenic sources are: Pb 0.5–1.5 µg cm^–2 yr^–1, Cd 15–30 ng cm^–2 yr^–1 and Hg 1–2 ng cm^–2 yr^–1, Higher values were recorded in lakes affected by local emissions.     

Oxidation of omega-hydroxylated fatty acids and steroids by alcohol dehydrogenase

Recrystallized alcohol dehydrogenase from horse liver was found to oxidize 17-hydroxystearic acid into 17-oxostearic acid, the 17-L-enantiomer faster than the 17-D-enantiomer. Alone at high pH or in combination with aldehyde dehydrogenase, the alcohol dehydrogenase also catalyzed conversion of 18-hydroxystearic acid into 1, 18-octadecadioic acid and 5β-cholestane-3α,7α,12α,26-tetrol into 3α,7α,12α-trihydroxy-5β-cholestanoic acid. All the activities as well as the ethanol dehydrogenase activity disappeared after specific carboxymethylation of a single cystein residue at the active site of alcohol dehydrogenase. These results conclusively show that alcohol dehydrogenase itself has ω-hydroxyfatty acid dehydrogenase activity and ω-hydroxysteroid dehydrogenase activity.     

Identification of glycoproteins on nitrocellulose membranes and gels

In this article we describe a procedure for the detection of glycoproteins on gels employing the periodic acid-Schiff’s reagent. In addition, a number of staining protocols and direct binding ELISA, employing antibodies and lectins, are described for the identification and quantitation of glycoproteins after their immobilization by dot, slot, or Western blotting onto nitrocellulose membranes. We document, in detail, the conditions (i.e., the effect of solvent and detergents) for the immobilization of one specific family of O-linked glycoproteins, namely mucins. However, taking into account our suggestions, these procedures should be applicable to other types of glycoprotein.     

VIP (Vasoactive Intestinal Polypeptide)-immunoreactive neurons in the retina of the rat

Summary Immunoreactive vasoactive intestinal polypeptide (VIP) was detected in a population of amacrine cells in the retina of the rat. Processes of these cells reach both the inner and outer half of the inner plexiform layer where they form sublayers. The VIP neurons are different from previously known amacrine cell types.     

Structure of adsorbed fibrinogen obtained by scanning force microscopy

It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three-dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces; a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM-results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule.     

Mechanism of inactivation of trypsin by antithrombin

General aspects of the mechanism of antithrombin action were elucidated by a comparison of the inactivation of trypsin by antithrombin with the inactivation of coagulation proteinases by the inhibitor. Bovine antithrombin and bovine trypsin were shown to form an inactive equimolar complex. A non-complexed, proteolytically modified form of antithrombin, electrophoretically identical with that formed in the reaction with coagulation proteinases, was also produced in the reaction with trypsin. In the absence of heparin, the inactivation of trypsin by antithrombin was 20 times faster than the inactivation of thrombin; the second-order rate constant was 1.5 x 10(5)m(-1).s(-1) at 25 degrees C and pH 7.4. However, the inhibition of thrombin was accelerated about 30 times more efficiently by small amounts of heparin than was trypsin inhibition. Dissociation of the antithrombin-trypsin complex at pH 7.4 followed first-order kinetics with a half-life for the complex of about 80h at 25 degrees C. The complex was rapidly and quantitatively dissociated at pH 11, resulting in the liberation of a modified two-chain form of the inhibitor, cleaved at the same Arg-Ser bond as in modified antithrombin released from complexes with thrombin, Factor Xa and Factor IXa. This supports the previous proposal that this bond is the active-site bond of antithrombin. Antisera specific for thrombin-modified antithrombin reacted with purified antithrombin-trypsin complex, indicating that the inhibitor was present in the complex in a form immunologically identical with thrombin-modified antithrombin. The results thus suggest a common mechanism, but different kinetics, for the inhibition of trypsin and coagulation proteinases by antithrombin.