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.     

The measuring of cell features

The usefulness of various feature sets for discriminating between different cell populations cannot be assessed without considering the entire automated cytology system, from specimen preparation through scanning, cell search, cell segmentation, artifact rejection and feature extraction to object classification and multivariate data analysis methods. These different stages in the analysis procedure are discussed in this paper and illustrated by examples from the author's experience of cell analysis studies, ranging from single-cell classification experiments to a test of a complete automated system under realistic conditions on a set of 397 cell specimens.     

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.     

Growth and metabolic flexibility in groundwater bacteria

Groundwater bacteria isolated from an oligotrophic-saturated soil showed a mixed strategy of economized metabolism and migration when grown in a continuous-flow column system simulating poor or nutrient-amended growth conditions. The cells were generally <0.5m in diameter in pure groundwater, but doubled in size when the concentration of dissolved organic carbon and phosphate in groundwater was increased 10-fold. The biomass, estimated from analysis of muramic acid (MuAc) in cell wall peptidoglucans, increased at the same time by a factor of 5 when the solid support in the columns was gravel and by a factor of 10 when it was glass beads. Bacteria in pure groundwater stored 10 times more of the energy-rich polysaccharide, poly--hydroxybutyric acid (PHB), than bacteria in enriched groundwater, and those cells that were attached to the gravel stored 10 times as much as cells in the interstitial pore water. Once phosphate was added to groundwater, stored PHB was metabolized. The proportion of free-living to attached bacteria was 2 to 10 times higher in enriched compared with pure groundwater indicating a mass transport of cells as the carrying capacity of their habitat rose.     

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.