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.     

Lipid acyl chain-dependent effects of sterols in Acholeplasma laidlawii membranes.

Acholeplasma laidlawii was grown with different fatty acids for membrane lipid synthesis (saturated straight- and branched-chain acids and mono- and di-unsaturated acids). The ability of 12 different sterols to affect cell growth, lipid head group composition, the order parameter of the acyl chains, and the phase equilibria of in vivo lipid mixtures was studied. The following two effects were observed with respect to cell growth: with a given acyl chain composition of the membrane lipids, growth was stimulated, unaffected, reduced, or completely inhibited (lysis), depending on the sterol structure; and the effect of a certain sterol depended on the acyl chain composition (most striking for epicoprostanol, cholest-4-en-3-one, and cholest-5-en-3-one, which stimulated growth with saturated acyl chains but caused lysis with unsaturated chains). The three lytic sterols were the only sterols that caused a marked decrease in the ratio between the major lipids monoglucosyldiglyceride and diglucosyldiglyceride and hence a decrease in bilayer stability when the membranes were enriched in saturated (palmitoyl) chains. With these chains correlations were found for several sterols between the glucolipid ratio and the order parameter of the acyl chains, as well as the lamellar-reversed hexagonal phase transition, in model systems. A shaft experiment revealed a marked decrease in the ratio of monoglucosyldiglyceride to diglucosyldiglyceride with the lytic sterols in unsaturated (oleoyl) membranes. The two cholestenes induced nonlamellar phases in in vivo mixtures of oleoyl A. laidlawii lipids. The order parameters of the oleoyl chains were almost unaffected by the sterols. Generally, the observed effects cannot be explained by an influence of the sterols on the gel-to-liquid crystalline phase transition.     

Catabolism of neurotensin in the epithelial layer of porcine small intestine

The mammalian small intestine is both a source and a site of degradation of neurotensin. Metabolites produced by incubation of the peptide with dispersed enterocytes from porcine small intestine were isolated by high-performance liquid chromatography and identified by amino-acid analysis. The principal sites of cleavage were at the Tyr-11-Ile-12 bond, generating neurotensin-(1-11), and at the Pro-10-Tyr-11 bond, generating neurotensin-(1-10). The corresponding COOH-terminal fragments, neurotensin-(11-13) and -(12-13) were metabolized further. Formation of neurotensin-(1-11) and -(1-10) was completely inhibited by phosphoramidon (Ki = 6 nM), an inhibitor of endopeptidase 24.11, but not by captopril, an inhibitor of peptidyl dipeptidase A. Incubation of neurotensin with purified endopeptidase 24.11 from pig stomach also resulted in cleavage of the Tyr-11-Ile-12 and Pro-10-Tyr-11 bonds. A minor pathway of cell-surface-mediated degradation was the phosphoramidon-insensitive cleavage of the Tyr-3-Glu-4 bond, generating neurotensin-(1-3) and neurotensin-(4-13). No evidence for specific binding sites (putative receptors) for neurotensin was found either on the intact enterocyte or on vesicles prepared from the basolateral membranes of the cells. Neurotensin-(1-8), the major circulating metabolite, was not formed when neurotensin(1-13) was incubated with cells, but represented a major metabolite (together with neurotensin-(1-10] when neurotensin-(1-11) was used as substrate. The study has shown that degradation of neurotensin in the epithelial layer of the small intestine is mediated principally through the action of endopeptidase 24.11, but this enzyme is probably not responsible for the production of the neurotensin fragments detected in the circulation.     

Between-year variation in flowering and fruit set in frost-prone and frost-sheltered populations of dioecious Rubus chamaemorus

Summary The flowering and fruiting patterns of the dioecious perennial herb Rubus chamaemorus L. were studied in frost-prone (open) and frost-sheltered (Shaded) habitats in northern Sweden over 6 years. The number of ramets with flower buds, the proportion of flower buds that opened, and fruit set varied markedly between years. In the frost-prone populations, the occurrence or absence of detrimental frosts during the development of flowers and fruit could explain much of the variation, both in the proportion of flower buds that developed into flowers, and in fruit set. In the frost-sheltered populations, most female flowers that did not develop into fruit aborted without any signs of physical damage and before any ovules had begun to enlarge. Flower mortality caused by herbivores feeding on reproductive parts was commonly low, but reached values higher than 10% in one of the shaded populations. Hand-pollination increased the proportion of ovules producing seeds in the mature fruits by about 20%, and in one year also increased fruit set significantly in one population. Fruit-producing female ramets had a higher mortality and a lower probability of flowering in the subsequent year than male ramets and non-fruiting female ramets. In R. chamaemorus, the conditions for fruit maturation are highly unpredictable at the time of flower initiation. It is suggested that the apparent over-initiation of flower buds is advantageous, as it allows the plant to attain a high reproductive success in years favourable for flowering and fruit development.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

Enzyme patterns during substrate shifts between phenol,acetate and glucose in continuous culture of Trichosporon cutaneum

Summary The soil yeast Trichosporon cutaneum was grown in continuous culture on phenol, acetate or glucose as sole carbon source. The activities of enzymes participating in the tricarboxylic acid cycle, glyoxylate cycle, 3-oxoadipate pathway, pentose phosphate pathway and glycolysis were determined in situ during shifts of carbon sources. Cells grown on phenol or glucose contained basal activity of the glyoxylate-cycle-specific isocitrate lyase. The derepression of the glyoxylate cycle enzymes was partly hindered in the presence of phenol but not in the presence of low levels of glucose. Phenol and glucose caused repression of isocitrate lyase. In the presence of either phenol or glucose, acetate accumulation in the medium increased. However, part of the supplied acetate was utilized simultaneously with phenol or glucose, the utilization rate of either carbon source being reduced in the presence of the other carbon source. Acetate caused repression but not inactivation of the phenol-degrading enzymes, phenol hydroxylase and catechol 1,2-dioxygenase. The simultaneous utilization of phenol and other carbon sources in continuous culture as well as the observed repression-derepression patterns of the involved enzymes reveal T. cutaneum to be an organism of interest for possible use in decontamination processes. Offprint requests to: H. Y. Neujahr Offprint requests to: H. Y. Neujahr     

Transformations of Halogenated Aromatic Aldehydes by Metabolically Stable Anaerobic Enrichment Cultures

Metabolically stable enrichment cultures of anaerobic bacteria obtained by elective enrichment of sediment samples from the Baltic Sea and Gulf of Bothnia have been used to study the oxidation and reduction of the aldehyde group of various halogenated aromatic aldehydes. During the transformation of 5- and 6-chlorovanillin, 6-bromovanillin, 3-chloro-4-hydroxybenzaldehyde, 3,5-dichloro-4-hydroxybenzaldehyde, and 3,5-dibromo-4-hydroxybenzaldehyde, it was shown that synthesis of the corresponding carboxylic acids, which were the principal metabolites, was invariably accompanied by partial reduction of the aldehyde to a hydroxymethyl group in yields of between 3 and 30%. Complete reduction to a methyl group was observed with some of the halogenated vanillins, but to an extremely limited extent with the halogenated 4-hydroxybenzaldehydes. One consortium produced both the hydroxymethyl and methyl compounds from both 5- and 6-chlorovanillin: it was therefore assumed that the methyl compound was the ultimate reduction product. On the basis of the kinetics of formation of the metabolites, it was concluded that the oxidation and reduction reactions were mechanistically related. In addition to these oxidations and reductions, dehalogenation was observed with one of the consortia. In contrast to the transformations of 5- and 6-chlorovanillin, which produced chlorinated methylcatechols, the corresponding compounds were not observed with 5- and 6-bromovanillin: the former was debrominated, forming 4-methylcatechol, whereas the latter produced 6-bromovanillyl alcohol without demethylation. Similarly, although 3-chloro-4-hydroxybenzaldehyde formed the chlorinated carboxylic acid and the benzyl alcohol, the 3-bromo compound was debrominated with formation of 4-hydroxybenzoic acid and, ultimately, phenol. On prolonged incubation, the halogenated carboxylic acids were generally decarboxylated, so that the final products from these substrates were halogenated catechols or phenols. Reductive processes of the type revealed in this study might therefore plausibly occur in the environment during anaerobic transformation of halogenated aromatic aldehydes containing hydroxyl and/or methoxyl groups.     

Metabolic changes of membrane lipid composition in Acholeplasma laidlawii by hydrocarbons, alcohols, and detergents: arguments for effects on lipid packing

The packing of lipids into different aggregates, such as spheres, rods, or bilayers, is dependent on the hydrophobic volume, the hydrocarbon-water interfacial area, and the hydrocarbon chain length of the participating molecules, according to the self-assembly theory [Israelachvili, J. N., Marcelja, S., & Horn, R. G. (1980) Q. Rev. Biophys. 13, 121-200]. The origin of the participating molecules should be of no importance with respect to their abilities to affect the above-mentioned parameters. In this investigation, Acholeplasma laidlawii, with a defined acyl chain composition of the membrane lipids, has been grown in the presence of three different classes of foreign molecules, known to partition into model and biological membranes. This results in an extensive metabolic alteration in the lipid polar head group composition, which is expressed as changes in the molar ratio between the lipids monoglucosyldiglyceride (MGDG) and diglucosyldiglyceride (DGDG), forming reversed hexagonal and lamellar phases in excess water, respectively. The formation of nonlamellar phases by A. laidlawii lipids depends critically upon the MGDG concentration [Lindblom, G., Brentel, I., Sj?lund, M., Wikander, G., & Wieslander, A. (1986) Biochemistry (preceding paper in this issue)]. The foreign molecules tested belong to the following groups: nonpolar organic solvents, alcohols, and detergents. Their effects on the gel to liquid crystalline phase transition temperature (Tm), on the order parameter of the acyl chains, and on the phase equilibria between lamellar and nonlamellar liquid crystalline phases in lipid-water model systems are known in several instances.(ABSTRACT TRUNCATED AT 250 WORDS)