Mechanism of calcium oscillations in migrating human astrocytoma cells

Numerous studies show that intracellular calcium controls the migration rate of different mobile cell types. We studied migrating astrocytoma cells from two human cell lines, U-87MG and A172, in order to clarify the mechanisms by which calcium potentially influences cell migration. Using the wound-healing model to assay migration, we showed that four distinct components of migration could be distinguished: (i) a Ca(2+)/serum-dependent process; (ii) a Ca(2+)-dependent/serum-independent process; (iii) a Ca(2+)/serum-independent process; (iv) a Ca(2+)-independent/serum-dependent process. In U-87MG cells which lack a Ca(2+)-dependent/serum-independent component, we found that intracellular Ca(2+) oscillations are involved in Ca(2+)-dependent migration. Removing extracellular Ca(2+) greatly decreased the frequency of migration-associated Ca(2+) oscillations. Furthermore, non-selective inhibition of Ca(2+) channels by heavy metals such as Cd(2+) or La(3+) almost completely abolished changes in intracellular Ca(2+) observed during migration, indicating an essential role for Ca(2+) channels in the generation of these Ca(2+) oscillations. However, specific blockers of voltage-gated Ca(2+) channels, including nitrendipine, omega-conotoxin GVIA, omega-conotoxin MVIIC or low concentrations of Ni(2+) were without effect on Ca(2+) oscillations. We examined the role of internal Ca(2+) stores, showing that thapsigargin-sensitive Ca(2+) stores and InsP(3) receptors are involved in Ca(2+) oscillations, unlike ryanodine-sensitive Ca(2+) stores. Detailed analysis of the spatio-temporal aspect of the Ca(2+) oscillations revealed the existence of Ca(2+) waves initiated at the leading cell edge which propagate throughout the cell. Previously, we have shown that the frequency of Ca(2+) oscillations was reduced in the presence of inhibitory antibodies directed against beta3 integrin subunits. A simple model of a Ca(2+) oscillator is proposed, which may explain how the generation of Ca(2+) oscillations is linked to cell migration.     

The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein

MLN64 is a transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein. The steroidogenic acute regulatory protein is located in the inner membrane of mitochondria, where it facilitates cholesterol import into the mitochondria. Crystallographic analysis showed that the START domain of MLN64 is a cholesterol-binding domain. The present work was undertaken to determine which step of the intracellular cholesterol pathway MLN64 participates in. Using immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found specifically in late endosomes. Electron microscopy indicates that MLN64 is restricted to the limiting membrane of late endosomes. Microinjection or endocytosis of specific antibodies shows that the START domain of MLN64 is cytoplasmic. Deletion and mutagenesis experiments demonstrate that the amino-terminal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine- or tyrosine-based targeting signals, we show that a dileucine motif (Leu(66)-Leu(67)) and a tyrosine residue (Tyr(89)) are critical for the targeting or the proper folding of the molecule. Finally, MLN64 colocalizes with cholesterol and Niemann Pick C1 protein in late endosomes. However, complementation assays show that MLN64 is not involved in the Niemann Pick C2 disease which, results in cholesterol lysosomal accumulation. Together, our results show that MLN64 plays a role at the surface of the late endosomes, where it might shuttle cholesterol from the limiting membrane to cytoplasmic acceptor(s).     

Egg fertilisation in a freshwater mussel: effects of distance,flow and male density

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Plasmalemmal voltage-activated K + currents in protoplasts from tobacco BY-2 cells: possible regulation by actin microfilaments?

Summary.  Plasmalemmal ionic currents from enzymatically isolated protoplasts of suspension-cultured tobacco ‘Bright Yellow-2’ cells were investigated by whole-cell patch-clamp techniques. In all protoplasts, delayed rectifier outward K⁺ currents having sigmoidal activation kinetics, no inactivation, and very slow deactivation kinetics were activated by step depolarization. Tail current reversal potentials were close to equilibrium potential E_K when external [K⁺] was either 6 or 60 mM. Several channel blockers, including external Ba²⁺, niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, inhibited this outward K⁺ current. Among the monovalent cations tested (NH₄ ⁺, Rb⁺, Li⁺, Na⁺), only Rb⁺ had appreciable permeation (P_Rb/P_K ⁼ 0.7). In addition, in 60 mM K⁺ solutions, a hyperpolarization-activated, time-dependent, inwardly rectifying K⁺ current was observed in most protoplasts. This inward current activated very slowly, did not inactivate, and deactivated quickly upon repolarization. The tail current reversal potential was very close to E_K, and other monovalent cations (NH₄ ⁺, Rb⁺, Li⁺, Na⁺) were not permeant. The inward current was blocked by external Ba²⁺ and niflumic acid. External Cs⁺ reversibly blocked the inward current without affecting the outward current. The amplitude of the inward rectifier K⁺ current was generally small compared to the amplitude of the outward K⁺ current in the same cell, although this was highly variable. Similar amplitudes for both currents occurred in only 4% of the protoplasts in control conditions. Microfilament-depolymerizing drugs shifted this proportion to about 12%, suggesting that microfilaments participate in the regulation of K⁺ currents in tobacco ‘Bright Yellow-2’ cells. Received December 7, 2001; accepted April 15, 2002; published online July 4, 2002 RID="*" ID="*" Correspondence and reprints: Pharmacologie et Physicochimie, UMR CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur, 74 route du Rhin, BP 24, 67401 Illkirch, France. Abbreviations: TBY-2 Tobacco ‘Bright Yellow-2’; DHCB dihydrocytochalasin B; I_Kin inward rectifier K⁺ current; I_Kout outward K⁺ current; MFs microfilaments; MTs microtubules; NPPB 5-nitro-2-(3-phenylpropylamino)-benzoic acid.     

Demographic characteristics of young-of-year freshwater mussel populations in ponds

We describe demographics of young-of-year (YOY) Pondmussels (Ligumia subrostrata) and Giant Floaters (Pyganodon grandis) in ponds during 2009 and 2011. YOY attained large size by approximately 6 months (Pondmussel, mean = 48.5 mm; Floater = 57.5), most individuals were sexually mature, and most females were gravid. Size and sex ratios varied among ponds. Pondmussel size was negatively related to mussel density, suggesting food competition; Floater size was not related to density. Size was not related to glochidial infestation pressure on fishes, suggesting that acquired immunity did not affect YOY performance. The percentage of gravid female Pondmussels varied between years from 27 to 100%, and 91% of female Floaters were gravid in 2011. Mean fecundity was high (Pondmussel = 34,311; Floater = 38,873). The proportion of gravid females and mean fecundity were not related to male density, showing that fertilization was efficient. Variation in size, sex ratios, and gravidity among ponds suggests that small differences in environmental conditions or demographic stochasticity can have large effects on populations. Rapid growth, early maturity, efficient fertilization, and high fecundity of YOY are contrary to traditional views of mussel life history, but these traits may allow Pondmussels and Floaters to rapidly colonize disturbed, unstable habitats.

     

Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene

We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards.     

Multi‐stage disruption of freshwater mussel reproduction by high suspended solids in short‐ and long‐term brooders

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Observations ultrastructurales sur la parathyroide de souris

Résumé La parathyroïde de souris présente de nettes variations ultrastructurales sous l'influence de traitements activateurs (phosphates) ou inhibiteurs (calcium seul ou associé à la vitamine D₂ ou à la parathormone). Les modifications générales des cellules glandulaires montrent que l'action du régulateur calcique s'exerce sur toutes les structures intervenant dans les processus d'élaboration et de maturation du matériel sécrétaire; elle ne se borne pas à un simple contrôle de l'activité excrétoire.

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Summary The parenchyma cells of the mouse parathyroid show changes in the ultrastructure when under the influence of an activator (indirect action of phosphates) or an inhibitor (calcium on its own or combined with either vitamine D₂ or parathormone). The changes in the glandular cells show that calcium exercises a regulative activity in all stages of formation and maturation of secretory material, and not only by controlling the hormone release.