Regulation of nicotinic acetylcholine receptors by protein phosphorylation

Neurotransmitter receptors and ion channels play a critical role in the transduction of signals at chemical synapses. The modulation of neurotransmitter receptor and ion channel function by protein phosphorylation is one of the major regulatory mechanisms in the control of synaptic transmission. The nicotinic acetylcholine receptor (nAcChR) has provided an excellent model system in which to study the modulation of neurotransmitter receptors and ion channels by protein phosphorylation since the structure and function of this receptor have been so extensively characterized. In this article, the structure of the nAcChR from the electric organ of electric fish, skeletal muscle, and the central and peripheral nervous system will be briefly reviewed. Emphasis will be placed on the regulation of the phosphorylation of nAcChR by second messengers and by neurotransmitters and hormones. In addition, recent studies on the functional modulation of nicotinic receptors by protein phosphorylation will be reviewed.     

Preservation of the diffusible cations for secondary ion mass spectrometry. II. Artefacts in material embedded in araldite or melamine.

Flotation on hot water (about 60 degrees C) which is frequently employed to stretch semithin sections on substrates for SIMS (secondary ion mass spectrometry) microscopy, is the cause of numerous artefacts. In the case of epoxy resin-embedded tissue, one observes loss of potassium and sodium and accumulation of calcium. The relative contrast of cell nuclei in the ionic images, is rapidly affected by these ion migrations. After prolonged contact with hot water, tissue becomes uniformly emissive. In the case of hydrosoluble resin-embedded tissue, potassium and sodium do not appear to be affected by the action of water, which suggests that they are covalently bound with chelating sites buried beneath the layer of water bound to the surface of the macromolecules. Calcium accumulates, probably on widely exposed anionic sites. Moreover, the domains observed in hydrosoluble resin-embedded tissue shrink differently according to the proportion of water removed by melamine; this can provide interesting information on the initial equilibrium between water, ion sand macromolecules. Our results seem to support the assumption that bound water should play an important role in the preservation of both macromolecular architecture and ion distributions.     

Separation of insect hemolymph proteins by cascade-mode multi-affinity chromatography.

The hemolymph of the adult female Manduca sexta was fractionated by cascade-mode multi-affinity chromatography (CASMAC) on a main-line tandem column chain containing Zn(2+)-TED, T-gel, Ni(2+)-DPA, and phenylsepharose and a side-line column containing Zn(2+)-DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph.     

Prior temperature exposure affects subsequent chilling sensitivity

The chilling sensitivity of small discs or segments of tissue excised from chillingsensitive species was significantly altered by prior temperature exposure subsequent to holding the tissue at chilling temperatures as measured by a number of physiological processes sensitive to chilling. This temperature conditioning was reversible by an additional temperature exposure before chilling, and mature-green and red-ripe tomato tissue exhibit similar chilling sensitivities. Exposing pericarp discs excised from tomato fruit (Lycopersicon esculentum Mill. cv. Castelmart), a chilling-sensitive species, to temperatures from 0 to 37°C for 6 h before chilling the discs at 2.5°C for 4 days significantly altered the rate of ion leakage from the discs, but had no effect on the rate of ion leakage before chilling and only a minimal effect on discs held at a non-chilling temperature of 12°C. Exposing chillingsensitive tissue to temperatures below that required to induce heat-shock proteins but above 20°C significantly increased chilling sensitivity as compared to tissue exposed to temperatures between 10 and 20°C. Rates of ion leakage after 4 days of chilling at 2.5°C were higher from fruit and vegetative tissue of chilling-sensitive species (Cucumis sativus L. cv. Poinsett 76, and Cucurbita pepo L. cv. Young Beauty) that were previously exposed for 6 h to 32°C than from similar tissue exposed to 12°C. Exposure to 32 and 12°C had no effect on the rate of ion leakage from fruit tissue of chilling tolerant species (Malus domestica Borkh. cv. Golden Delicious, Pyrus communis L. cv. Bartlett). Ethylene and CO₂ production were higher and lycopene synthesis was lower in chilled tomato pericarp discs that were previously exposed for 6 h to 32°C than the values from tissue exposed to 12°C for 6 h before chilling. Increased chilling sensitivity induced by a 6 h exposure to 32°C could be reversed by subsequent exposure to 12°C for 6 h.     

Factors affecting cation-anion uptake balance and iron acquisition in peanut plants grown on calcareous soils

Dicotyledonous plants subjected to Fe-deficiency stress can decrease pH in the rhizosphere by proton excretion and reduce ferric iron by an activated reduction system in the plasma membranes of the root or by reductants released from the roots. The efficiency by which these plants take up Fe may strongly depend on their cation-anion balance. This study presents results of two experiments conducted to evaluate the effect of K, growth stage and cultivar on ionic balance and Fe acquisition of peanut (Arachis hypogaea L.) plants.Potassium applications to the high calcareous soil (30.3% CaCO₃) favoured proton release, but did not ameliorate plant Fe acquisition. At the earliest stages of plant growth, anion uptake exceeded cation uptake due to intensive N uptake. With time, a shift in the ionic balance was observed as a result of predominant cation uptake. It appears that the relationship between H/OH-ion release and Fe nutrition of peanut plants is actually a complex phenomenon under soil conditions and depends on some soil parameters, such as CaCO₃ content. Even by enhanced H-ion release Fe nutrition of plants can be impaired if soil CaCO₃ is too high.     

Antiport mediated rounding and endocytosis are enhanced by sulphate

Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na+/H+ antiporter via HCO3(-)-dependent H+ pumping, is significantly enhanced by the addition of Na2SO4 (FTR + SO4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive. At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol.wt which is a 455-fold diversity. This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol.wt species that measures up to 5 microns in width. Two million mol.wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion. Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages. However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz. epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202). The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change. However the cell surface reversion is not associated with efflux of FDx which are sorted out into 'granular patches', the later stage endosomes without membrane outlines in AME. FDx-loaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions. Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches. AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an alternate membrane recycling capability where granular patches are instrumental in sorting. It appears to be not a simple endocytosis-exocytosis pathway.     

Chemiosmotic systems in bioenergetics: H+-cycles and Na+-cycles

The development of membrane bioenergetic studies during the last 25 years has clearly demonstrated the validity of the Mitchellian chemiosmotic H⁺ cycle concept. The circulation of H⁺ ions was shown to couple respiration-dependent or light-dependent energy-releasing reactions to ATP formation and performance of other types of membrane-linked work in mitochondria, chloroplasts, some bacteria, tonoplasts, secretory granules and plant and fungal outer cell membranes. A concrete version of the direct chemiosmotic mechanism, in which H⁺ potential formation is a simple consequence of the chemistry of the energy-releasing reaction, is already proved for the photosynthetic reaction centre complexes.Recent progress in the studies on chemiosmotic systems has made it possible to extend the coupling-ion principle to an ion other than H⁺. It was found that, in ceertain bacteria, as well as in the outer membrane of the animal cell, Na⁺ effectively substitutes for H⁺ as the coupling ion (the chemiosmotic Na⁺ cycle). A precedent is set when the Na⁺ cycle appears to be the only mechanism of energy production in the bacterial cell. In the more typical case, however, the H⁺ and Na⁺ cycles coexist in one and the same membrane (bacteria) or in two diffeerent membranes of one and the same cell (animals). The sets of and generators as well as and consumers found in different types of biomembranes, are listed and discussed.     

Phenylarsine oxide induces the cyclosporin A-sensitive membrane permeability transition in rat liver mitochondria

This paper reports an investigation on the effects of the hydrophobic, bifunctional SH group reagent phenylarsine oxide (PhAsO) on mitochondrial membrane permeability. We show that PhAsO is a potent inducer of the mitochondrial permeability transition in a process which is sensitive to both the oxygen radical scavanger BHT and to cyclosporin A. The PhAsO-induced permeability transition is stimulated by Ca²⁺ but takes place also in the presence of EGTA in a process that maintains its sensitivity to BHT and cyclosporin A. Our findings suggest that, at variance from other known inducers of the permeability transition, PhAsO reacts directly with functional SH groups that are inaccessible to hydrophilic reagents in the absence of Ca²⁺.     

Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method

Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 °C, 5 min) prior to transfection.