Studies on the Interaction of Ca2+ Ions with Some Fractions of the Neurospecific S-100 Protein

Abstract: Fractions of neurospecific S-100 protein were purified from bovine brain and their physicochemical properties were studied. Conformational changes caused by the binding of calcium to S-100 protein fractions were detected by means of differential and fluorescence spectroscopy. Fractions demonstrating opposite shifts of their spectra also differ in the distribution in double-phase system. The number of calcium-binding centers and their association constants were determined by means of equilibrium dialysis and gel filtration. The nature of the differences in the interaction of various S-100 protein fractions with calcium is discussed.     

Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L.

From compartmental analysis of radioisotope elutin measurements, fluxes of Ca²⁺ were estimated for cortical cells in root segments of onion, Allium cepa L., relative to complete nutrient solutions containing a range of calcium concentrations ([Ca₀]) from 2 eq l^-1 to 20 meq l^-1, increasing in 10-fold steps for Ca²⁺. Except for the calcium counter-ion (usually NO ₃ ^- , sometimes Cl^- at the highest [Ca₀]), the composition of the nutrient solution was other-wise the same at all calcium concentrations. Compartmental analysis indicated that the cytoplasm had a high content of exchangeable Ca²⁺ but, in the light of evidence from animal studies, ionic activity of calcium in the cytoplasm was assumed to be no greater than 0.002 eq ml^-1. With the Ussing-Teorell flux equation as the criterion, it was concluded that at all values of [Ca₀] tested, Ca²⁺ entered the cytoplasm passively and was actively pumped back into the external solution. Entry of calcium to the vacuole from the cytoplasm was active in all cases. The conclusions regarding the character of ion transport across the plasmalemma were the same as when the whole calcium content of the cytoplasm was taken to contribute to the ionic activity. However, the electrochemical activity gradient was very much steeper than formerly estimated. Calcium was transported to the stele in proportion to the calcium content of the cytoplasm and moved in the xylem almost exclusively in the basipetal direction.     

Tyrosine Hydroxylase Regulation: Apparent Kinetic Alterations Following Incubation of Brain Slices in a Sodium-free Medium

In an attempt to determine if alterations in intraneuronal Ca2+ may regulate tyrosine hydroxylase activity, brain slices were subjected to experimental manipulations known to increase the intraneuronal concentration of free Ca2+ ions. Incubation of either striatal or olfactory tubercle slices in a Na+-free medium for 15 min at 37 degrees resulted in a marked increase in the activity of tyrosine hydroxylase present in the 20,000 g supernatant fraction of homogenates prepared from the slices. Tyrosine hydroxylase isolated from slices previously incubated in a Na+-free, choline-enriched medium or in a Na+-free, sucrose-enriched medium exhibited maximal activities when assayed at pH 6.0 and 7.0, respectively. However, the percentage stimulation of enzyme activity induced by incubation of the slices in a Na+-free medium was maximal when the enzyme assays were performed at pH 7.0. The observed increase in enzyme activity seems to be mediated by a decrease in the apparent Km of the enzyme for pteridine cofactor, regardless of whether the kinetic enzyme analyses were conducted at pH 6.0 or 7.0, and by an increase in the Ki of the enzyme for end-product inhibitor dopamine. The apparent kinetic changes in the enzyme do not seem to result from alterations in the endogenous dopamine content of the slices, and they are independent of any increase in dopamine release that might have occurred as a response to the augmented intraneuronal Ca2+ concentration. Furthermore, the activation of tyrosine hydroxylase produced by incubating slices in a Na+-free medium is observed even in slices depleted of dopamine by pretreatment of rats with reserpine 90 min before preparation of brain slices. The activation of tyrosine hydroxylase observed under these experimental conditions does not seem to be mediated by cAMP or by a cAMP-dependent phosphorylation process. It is suggested that the changes in tyrosine hydroxylase reported are mediated primarily by a rise in the free Ca2+ concentration within the nerve tissue. These observations are consistent with the hypothesis that the kinetic activation of tyrosine hydroxylase produced after depolarization of central dopaminergic neurons may occur through a Ca2+-dependent even other than transmitter release.     

Effects of acid irrigation and liming on two clones of Norway spruce

Summary The effect of acid irrigation on the growth of rooted cuttings ofPicea abies (L.) Karst, was investigated in a pot experiment lasting 3 years. It involved two clones of Norway spruce, H 253 Bogstad I and H 254 Bogstad II. Irrigation water of pH 5.4, 4.0, 3.0 and 2.5 was used. Liming was included in the experiment.After the experimental period, the plants of all treatments were growing reasonably well. However, those plants irrigated at pH 2.5 were slightly discoloured. The plant mortality was only 3% throughout the experiment, and was not connected to acid irrigation. The limiting growth factor was N. All other nutrient elements measured in the plants were close to optimal concentration. Plants irrigated at pH 2.5, and to some extent at pH 3, contained excessively high concentrations of Al, t-S and SO₄. The total amount of Ca, Fe and Mn taken up by the plants decreased with increasing soil acidity. The increased growth of clone H 254 relative to H 253, produces a corresponding impression on soil characteristics. Soil acidity is governed by acid irrigation and CaCO₃ application, but the clonal effects are also of importance. Norway spruce appears to be tolerant to Al concentrations as high as 50 mmol/kg in the needles.     

砂培条件下施加钙、砷对蜈蚣草吸收砷、磷和钙的影响

在砂培条件下 ,研究施加钙、砷对蜈蚣草生长和砷、磷和钙的吸收及转运的影响。添加砷对蜈蚣草的生物量 (根、叶柄和羽叶的干物重 )虽未达到显著影响 (p<0 .0 5) ,但添加 0 .1 mmol/L砷时 ,表现出刺激生长效应。提高介质中钙浓度明显抑制蜈蚣草根系生长 ,钙浓度过高还会显著限制地上部生长。供应 0 .0 3mmol/L钙时 ,蜈蚣草羽片砷浓度为 42 1 8mg/kg,明显高于 2 .5和 5 mmol/L钙处理下相应的砷浓度。砷的转运系数 (羽片 /根 )随着介质中砷浓度的升高而增大 ,随着介质中钙浓度的升高而减少。这说明一定范围内提高介质中砷浓度促进砷向地上部运输 ,而钙却明显抑制砷向地上部转运。钙和砷浓度过高时 ,植株均会出现中毒症状。钙中毒表现为叶脉变褐和叶肉坏死 ;而砷中毒现象表现在叶尖和叶缘变褐。介质中砷限制蜈蚣草根部对磷的吸收 ,但对地上部磷浓度无显著影响。介质中添加砷 ,植物体内钙浓度升高 ,可能起缓解砷毒的作用。钙、砷对蜈蚣草羽片砷累积量和总累积量均有极显著的交互作用 ,钙是负交互效应 ,砷是正交互效应。添加 2 .5和 5.0 mmol/L钙时 ,相对于 0 .0 3 mmol/L钙处理分别减少地上部砷累积量 2 0 .8%和73.1 %。这表明在应用蜈蚣草进行植物修复时 ,介质中出现过高浓度的钙是不利于提高土壤修复效率     

Dopaminergic Inhibition of Prolactin Release and Calcium Influx Induced by Neurotensin in Anterior Pituitary Is Independent of Cyclic AMP System

The present study demonstrates that 3,4-dihydroxyphenylethylamine (DA, dopamine) prevents neurotensin (NT) stimulation of both prolactin (PRL) release and calcium influx by interacting with specific receptors that are functionally linked to calcium channels. As shown by the studies with dispersed cells from rat anterior pituitary, the pharmacology of the control of PRL release and calcium influx, both induced by NT, was found to be typical of a DAergic process. This was demonstrated by the order of potency of agonists in inhibiting PRL release and calcium influx (DA greater than epinephrine greater than norepinephrine much greater than isoproterenol); by the high affinity of antagonists such as haloperidol and fluphenazine for this process; and by the high degree of stereoselectivity of sulpiride. Specific D2 receptor agonists, such as bromocriptine and lisuride, and the specific D2 receptor antagonist (-)-sulpiride were found to be highly potent on the DA receptors negatively coupled with calcium channels and PRL release. DA was found to lack the capacity to change the influx of calcium induced by either the sodium channel activator veratridine or high extracellular potassium levels, thus indicating a specific action of this amine on calcium channels sensitive to NT. In a range of concentrations that are effective in inhibiting either the calcium influx or the PRL release, both induced by NT, DA did not alter the cyclic AMP generating system. DA (from 1.0 nM to 50 nM) did not affect adenylate cyclase activity in rat pituitary gland homogenates and did not modify intracellular cyclic AMP levels in pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mode of action of thidiazuron: auxins,indoleamines, and ion channels in the regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.     

PRIP (Phospholipase C-related but Catalytically Inactive Protein) Inhibits Exocytosis by Direct Interactions with Syntaxin 1 and SNAP-25 through Its C2 Domain

Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K⁺-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation.     

Ionophore A23187, Verapamil, Protonophores, and Veratridine Influence the Release of γ-Aminobutyric Acid from Synaptosomes by Modulation of the Plasma Membrane Potential Rather than the Cytosolic Calcium

The release of GABA induced by veratridine shows no correlation with the synaptosomal Ca content and is therefore not mediated by the release of mitochondrial Ca. Instead, with both Ca-repleted and -depleted synaptosomes, the extent of GABA efflux is correlated with the decrease in plasma membrane potential. The slow release of GABA induced by protonophores and the Ca-dependent release induced by ionophore A23187 are also consequences of the depolarization of the plasma membrane, rather than of elevated cytosolic Ca. Finally, the ability of verapamil to inhibit the release of GABA induced by low veratridine concentrations is due to the ability of the Ca channel inhibitor to antagonize the action of veratridine, rather than to inhibit Ca entry into the synaptosome. It is concluded that it is essential to monitor plasma membrane potentials in experiments in which amino acid efflux from synaptosomes is induced.