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1.
Dunalliella salina (Teodoresco) is a unicellular, wall-less, halotolerant green alga. Previous work has shown that levels of inositol phospholipiils in whole cells of D. salina fluctuate in response to hyper- and hypo-osmotic shock. In this paper, we report the effects of changes in the light environment on levels of phospholipids, including inositol phospholipids, in D. scilina. Utilizing both short-term and long-term labeling of phospholipids with 32PO4, we were able to compare both immediate and long-term changes in lipid metabolism during changes in the light environment. Relative to the other phospholipids. phosphotidic acid and the inositol phospholipids phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were rapidly labeled, even in the dark, suggesting that the metabolism of these compounds is more active than that of the bulk cellular phospholipids. There was little change in inositol phospholipid metabolism when cells were illuminated following a 1 h dark adaptation period, Furthermore, the inositol phospholipid signal transduction pathway did not respond to severe photoinhibition treatment. Apparently this plasma-membrane-based signal transduction pathway, which responds to changes in the external environment, is relatively insensitive to major changes in chloroplast metabolism.  相似文献   

2.
Abstract: Exogenous gangliosides, especially ganglioside GM1 (GM1), seem to potentiate the action of nerve growth factor (NGF). We have examined the possible regulation of the NGF signaling pathway in PC12 cells by the B subunit of cholera toxin (CTB), which binds to endogenous GM1 specifically and with a high affinity. CTB treatment (1 μg/ml) enhanced NGF-induced neurite outgrowth from PC12 cells, NGF-induced activation of ribosomal protein S6 kinase, and NGF-induced stimulation of trk phosphorylation. CTB plus NGF also caused a greater inhibition of [3H]-thymidine incorporation into DNA than did NGF alone. These enhancing effects of CTB were blocked by the presence of cytochalasin B in the culture medium but were not affected by the presence of colchicine or by the depletion of Ca2+ in the medium. 125I-NGF binding experiments revealed that CTB treatment did not affect the specific binding of NGF to the cells. These results strongly suggest that the binding of cell surface GM1 by CTB modulates the pathway of intracellular signaling initiated by NGF and that the association of CTB with a cytoskeletal component is essential for these effects.  相似文献   

3.
Abstract— Glial cells isolated from rabbit cerebral cortex contained approximately one-third more phospholipids per unit protein than the neuronal cell bodies. The pattern of individual phospholipids was rather similar in both cell types. The incorporation of intracisternally administered 32P into neuronal and glial phospholipid classes of rabbit brain was studied at intervals ranging from 5 to 60min. In general, for all investigated phospholipids the incorporation of the label was somewhat faster in neurons than in glial cells. Phosphatidylinositol showed the fastest and ethanolamine plasmalogen the slowest incorporation of 32P in both neurons and glial cells. A lag phase of about 10 min could be observed before labelling of the glial phosphatidylcholine, phosphatidylethanolamine, ethanolamine plasmalogen, phosphatidylserine and sphingomyelin had occurred. Among the neuronal phospholipids a lag phase was found only for the labelling of the ethanolamine plasmalogen. Norepinephrine increased the incoropration of 32P into phosphatidylinositol of both glia and neurons but had no effect on the specific radioactivity of ethanolamine plasmalogen and sphingomyelin. Labelling of phosphatidylcholine was slightly inhibited in both cell types by the administration of norepinephrine.  相似文献   

4.
Abstract— The incorporation in vivo of 32P1 was significantly increased in all glycerophosphatide of preparations of denervated muscle membrane in frogs. There was no increase in incorporation of 32P1 into sphingomyelin. Disuse induced by tenotomy did not significantly increase incorporation of 32P1 into phospholipids of the muscle membrane. The phospholipid content of muscle membranes remained unchanged as a result of denervation or tenotomy. Denervation produced an increase in the incorporation of [2-3H]glycerol into all glycerophosphatides in parallel with the increase in 32P1 incorporation. Although the stimulated incorporation of 32P1 was increased in the regions of the muscle membrane rich in endplates, the most marked effect was in the endplate-poor region where activity in phosphatidylserine was most markedly increased.  相似文献   

5.
1. Phosphorus (P) uptake by macrophytes and epiphytes from the LaPlatte River (VT) was examined in the laboratory by adding 32PO4‐P to recirculating stream microcosms.
2. Water, plugs of sediment and plants were removed from the river and placed into the microcosms. 32PO4‐P was then added either to the water or the sediment, and its incorporation into plants and epiphytes was monitored over 3 days. Uptake was examined at both ambient (5 μg L–1) and increased (50 μg L–1) soluble reactive phosphorus (SRP) concentrations. A computer program was developed to fit curves to the radiotracer data and calculate rate constants for the simultaneous transfer of 32P among compartments.
3. Both macrophytes and epiphytes removed P from the water, but epiphyte uptake of P was more rapid. Phosphate enrichment stimulated P uptake by both macrophytes and epiphytes. Macrophytes also obtained P from the sediment. The relative contribution of P to macrophytes from the water vs. that from the sediment appeared to vary with SRP in the overlying water. Accurate estimates of rates of P uptake from sediments by macrophytes were difficult to obtain however, due to very low and highly variable unit rate constants for P uptake and uncertainty about the magnitude of the phosphate pool available for uptake.
4. SRP concentrations were greater in the overlying water than in the sediment pore water of stream microcosms in the present study. Numerous reports in the literature have suggested that this condition favours uptake by macrophyte stems and leaves rather than by roots.
5. Phosphate uptake from the water by macrophytes in shallow streams may be more common than for macrophytes in lakes.  相似文献   

6.
Abstract— Cultured pineal glands incorporated 32P into membrane phospholipids. Treatment of cultured glands with norepinephrine, which is known to stimulate membrane- bound pineal adenyl cyclase and to increase the production and secretion of melatonin, stimulated the incorporation of 32P into a phospholipid fraction of membranes and particulates containing phosphatidyl serine and phosphatidyl inositol. The labelling of other phospholipid fractions and the total 32P in the gland were not changed by norepinephrine treatment. Experiments with chronically-denervated pineal glands indicated that the effect of norepinephrine on the [32P]labelling of phospholipids occurred at a postsynaptic site. When norepinephrine-stimulated secretion of melatonin was partially inhibited by p -chlorophenylalanine (a compound which blocks the synthesis of melatonin precursors), the norepinephrine-stimulated labelling of phospholipids was still observed. Conversely, when melatonin secretion was stimulated in the absence of norepinephrine by treatment with the immediate precursor of melatonin, N -acetylserotonin, a stimulation of 32P- labelling of phospholipids did not occur. These observations suggest that the increased [32P]- labelling of a phospholipid fraction caused by the norepinephrine treatment is not related to the secretion of melatonin. This effect on phospholipids may be associated with the interaction of norepinephrine with a membrane-bound postsynaptic receptor. Stimulation by norepinephrine of [32P]-incorporation into phospholipids has not been previously reported to occur in a tissue in which cholinergic fibres are absent.  相似文献   

7.
Abstract: The Ca2+ requirement for α-agonist stimulation of 32P incorporation into acidic phospholipids (the phosphatidylinositol effect) of dispersed pineal cells was evaluated by means of several different compounds that interfere with Ca2+ disposition. Simple omission of Ca2+ led to slight increases in basal and norepinephrine-stimulated phosphatidyl-CMP (CDP-diacylglycerol) and phosphatidylglycerol labeling without affecting phosphatidylinositol labeling. In the absence of Ca2+, EGTA (200 μM) or the ionophore for divalent cations A23187 (10 μM) elicited large increases in phosphatidic acid, phosphatidyl-CMP, and phosphatidylglycerol labeling while strongly inhibiting the phosphatidylinositol effect. The Ca2+ translocation inhibitor LaCI3 also reduced the magnitude of this effect. The phosphatidylinositol effect is, however, not induced by increased Ca2+ entry into the cytosol, since A23187 did not mimic the effect of norepinephrine. Under conditions where membrane Ca2+ was lowered, the addition of 1 mM-inositol greatly reduced phosphatidic acid, phosphatidylglycerol, and phosphatidyl-CMP labeling with concomitant increases in basal and norepinephrine-stimulated phosphatidylinositol labeling approaching that observed in the presence of norepinephrine and 2.5 mM-Ca2+. In the presence of 2.5 mM-Ca2+, inositol had negligible effects on phosphatidylinositol labeling. It was concluded that changes in membrane Ca2+ availability and/or disposition alter phospholipid metabolism and concurrently reduce the magnitude of the phosphatidylinositol effect, perhaps by making the pool of readily available inositol in pinealocytes rate-limiting.  相似文献   

8.
Abstract: Muscarinic receptors found in the N IE-115 mouse neuroblastoma cell line were tested for their ability to mediate stimulation of phosphatidylinositol (PI) turnover. This study was facilitated by the development of a new solvent system (acetone: butanol: acetic acid: water, 5: 5: 1: 1) for the rapid and consistent separation of PI by one-dimensional thin-layer chromatography. Cholinergic stimulation caused as much as a 680% increase in the incorporation of 32P into PI. Enhanced incorporation of 32P into PI could be measured as early as 4 min after stimulation began. By 20 min, the rate of incorporation by stimulated cells had decreased to that of unstimulated cells, indicating desensitization. The magnitude of the response was dependent on the extent of receptor occupancy and the response elicited by a saturating dose of carbamylcholine was blocked completely by 10−7 M at-ropine, a specific muscarinic antagonist. Chronic stimulation, known to cause a loss of receptor binding sites, led to a 90% decrease in the maximum response even after a 40-min withdrawal period. Replacement of Na+ ions in the medium with choline or K+ severely impaired the ability of the cells to incorporate added 32P into PI (90 and 50%, respectively). Removal of the putative second messenger Ca2+ for short periods of time by the addition of excess EGTA did not alter either basal or muscarinic-stimulated PI turnover.  相似文献   

9.
The break of dormancy and the early development of Artemia are known to occur in the absence of any DNA and RNA synthesis. The presence and function of preformed messengers in the developing embryos were studied using 32PO4 to track the RNA species that turnover. The rapid labelling of the poly(A) tails of the particulate RNA by 32PO4 is found to be the predominant metabolic event accompanying initiation of development. Although these RNA populations represent a meagre percentage of the total poly(A) RNA of the cells, they nevertheless constitute more than 60% of the labelled poly(A) populations at early stages of development. Moreover the rise in the poly(A) RNA levels of the embryos observed during the first four hours of development could be attributed to the increase in the particulate poly(A) RNA. Prelabelled RNA of this fraction remained rather firmly associated with this fraction in chase experiments, indicating that once processed these RNA species function in association with membranes. The observed shift in the size of these RNAs from low to high molecular weight species further implies that they are being activated to take part in the early developmental programme.  相似文献   

10.
Abstract Signature lipids from the phospholipid esterlinked fatty acids (PELFA) of cell membranes were used to describe benthic microbial communities of 4 Antarctic sediments. Metabolic activities of the communities were determined by incorporation of [3H]thymidine into bacterial DNA and sodium [14C]acetate into membrane lipids. Biomass measurements from extractable phospholipid fatty acids per g dry wt. ranged between 6 to 76 nmol, or when converted to number of bacteria, 3.7 × 108 to 4.5 × 109 cells per g dry wt. The West Sound site at New Harbor contained the lowest biomass, while Cape Evans on the East Sound contained the greatest. A marked difference was also noted between sites in their sediment microbial community structure. The East Sound sites at Cape Armitage and Cape Evans contained a greater abundance of diatom marker lipids, whilst both sides of the Sound contained approximately the same relative amounts of bacterial groups distinguished using PELFA. Activity of sediment microorganisms measured by radiolabel incorporation under ambient conditions followed the trends of the biomass measurements. The East Sound sites were more active by an average of 45–73% for [3H]thymidine and possibly also for sodium [14C]acetate.  相似文献   

11.
Abstract: The addition of either carbachol or muscarinic agonists to cultured bovine adrenal chromaffin cells results in a selective stimulation of phosphatidate (PhA) and phosphatidylinositol (PhI) labeling from 32Pi and [3H]glycerol that can be inhibited by the inclusion of atropine, but not d -tubocurarine. In contrast, increased catecholamine secretion is observed on the addition of carbachol or nicotinic agonists and is inhibited by d -tubocurarine but not by atropine. Added calcium is essential for catecholamine secretion but not for stimulated phospholipid labeling. Chelation of endogenous Ca2+ with EGTA does, however, inhibit the stimulated phospholipid labeling. These results suggest that stimulated phospholipid labeling in the bovine chromaffin cell and catecholamine secretion are separate and distinct processes.  相似文献   

12.
Abstract: The ability of muscarinic cholinergic receptors to activate phosphoinositide turnover following agonist-induced internalization has been investigated. Incubation of SH-SY5Y neuroblastoma cells with oxotremorine-M resulted in a time-dependent endocytosis of both muscarinic receptors and α subunits of Gq and G11, but not of isoforms of phosphoinositide-specific phospholipase C, into a subfraction of smooth endoplasmic reticulum (V1). Agonist-induced increases in diacylglycerol mass and in 32P-phosphatidate labeling, much of which was of the tetraenoic species, were also observed in the V1 fraction, but these increases persisted when the agonist-induced translocation of receptors into the V1 fraction was blocked. All enzymes of the phosphoinositide cycle were detectable in the V1 fraction. However, with the exception of phosphatidylinositol 4-kinase, none was enriched when compared with cell lysates. Both 32P-labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinositol 4,5-bisphosphate, whereas the synthesis of phosphatidylinositol 4-phosphate is robust. These results indicate that endocytosed receptors do not appear to retain their ability to activate phosphoinositide turnover. The availability of the substrate for phospholipase C, phosphatidylinositol 4,5-bisphosphate, may be one factor that limits the activity of muscarinic receptors in this subcellular compartment.  相似文献   

13.
Abstract— Regulation of muscarinic acetylcholine receptor concentration by receptor activity in neuron-like NG108-15 hybrid cells is a highly specific process. Receptor levels, monitored by binding of [3H]quinuclidinyl benzilate ([3H]QNB), decreased 50-75% following 24-h incubation of cells with muscarinic agonists, but none of the following cellular processes was altered by this chronic receptor stimulation: (1) glycolytic energy metabolism, measured by [3H]deoxy- d -glucose ([3H]DG) uptake and retention; (2) rate of cell division; (3) transport, measured by [3H]valine and [3H]uridine uptake; (4) RNA biosynthesis, measured by [3H]uridine incorporation; (5) protein biosynthesis, measured by [3H]valine and [35S]methionine incorporation into total protein and into protein fractions obtained by polyacrylamide gel electrophoresis. In contrast, chronic stimulation did cause a threefold decrease in the capacity of carbachol to stimulate phosphatidylinositol (PI) turnover, a receptor-mediated response. In addition to cholinomimetics, the neuroeffector adenosine (1 m m for 24 h) also caused a decrease in [3H]QNB binding levels, but chronic stimulation of α -adrenergic, opiate, prostaglandin E1, and prostaglandin F receptors found on NG108-15 cells caused no changes. The data indicate that loss of muscarinic receptors caused by receptor stimulation is not a consequence of fundamental changes evoked in overall cellular physiology but reflects a specific regulation of cholinoceptive cell responsiveness.  相似文献   

14.
Abstract: Several α-adrenergic receptor agonists and antagonists were used to strengthen the earlier findings that the stimulation by (-)-norepinephrine of 32P1 incorporation into acidic phospholipids, especially phosphatidylinositol, in the rat pineal gland is mediated through α-adrenergic receptors. Dopamine was able to induce similar stimulation, although always to a smaller extent than equimolar concentrations of norepinephrine. The dopaminergic agonists apomorphine and piribedil did not increase phosphatidylinositol labeling. A number of antagonists considered to act primarily at dopaminergic or α-adrenergic receptors respectively completely prevented dopamine from exerting its effect. Both types of antagonists also were able to inhibit in varying degree the elevation of phospholipid labeling induced by norepinephrine. Dopamine increased phosphatidylinositol turnover without first being converted to norepinephrine, inasmuch as dopamine β-hydroxylase inhibitors had no influence on dopamine activity. Dopamine and α-agonists competitively activated the receptors involved in the phospholipid effect. The conclusion drawn from the several lines of evidence is that only α-adrenergic receptors are concerned with the changes in pineal phospholipid metabolism brought about by the various agonists used and that the action of dopamine occurs through these receptors rather than through discrete dopaminergic receptors.  相似文献   

15.
The main gap in our knowledge about what determines the rate of CH4 oxidation in forest soils is the biology of the microorganisms involved, the identity of which remains unclear. In this study, we used stable-isotope probing (SIP) following 13CH4 incorporation into phospholipid fatty acids (PLFAs) and DNA/RNA, and sequencing of methane mono-oxygenase ( pmoA ) genes, to identify the influence of variation in community composition on CH4 oxidation rates. The rates of 13C incorporation into PLFAs differed between horizons, with low 13C incorporation in the organic soil and relatively high 13C incorporation into the two mineral horizons. The microbial community composition of the methanotrophs incorporating the 13C label also differed between horizons, and statistical analyses suggested that the methanotroph community composition was a major cause of variation in CH4 oxidation rates. Both PLFA and pmoA -based data indicated that CH4 oxidizers in this soil belong to the uncultivated 'upland soil cluster α'. CH4 oxidation potential exhibited the opposite pattern to 13C incorporation, suggesting that CH4 oxidation potential assays may correlate poorly with in situ oxidation rates. The DNA/RNA-SIP assay was not successful, most likely due to insufficient 13C-incorporation into DNA/RNA. The limitations of the technique are briefly discussed.  相似文献   

16.
ABSTRACT. Females of Aedes aegypti (L.) (Diptera, Culicidae) were not receptive to mating 1 h after the initial insemination or at later times throughout the first gonotrophic cycle. A significant proportion of these females (75–90%) were inseminated again prior to the second gonotrophic cycle. Insemination was determined by the detection in females of sperm labelled with 3 H(G)-adenine or 32PO4 after matings with radiolabeled males. Females deprived of males after the first insemination displayed an abnormal reduction in fertility throughout successive gonotrophic cycles.  相似文献   

17.
Abstract: K-252b potentiates the neurotrophic effects of neurotrophin-3 (NT-3) in primary cultures of rat central cholinergic and peripheral sensory neurons and in a rat pheochromocytoma PC12 cell line. The ligand and receptor specificity, and role of the low-affinity neurotrophin receptor (p75NTR) in the potentiation response induced by K-252b, are unknown. To address the issues of ligand and receptor specificity of K-252b potentiation, we have examined neurotrophin-induced DNA synthesis ([3H]thymidine incorporation) in NIH3T3 cells expressing trkA, trkB, or trkC . Neither NT-3 nor K-252b alone could stimulate mitogenic activity in the trkA -overexpressing clone. However, coaddition of K-252b (EC50 of ∼2 n M ) with 10–100 ng/ml NT-3 led to incorporation of [3H]thymidine in trkA expressing cells to a level induced by optimal concentrations of nerve growth factor (NGF). The K-252b- and NT-3-induced [3H]thymidine incorporation correlated with an increase in the tyrosine autophosphorylation of the trkA receptor as well as tyrosine phosphorylation of trk -associated phospholipase C-γ1 and SH2-containing proteins. K-252b did not potentiate submaximal doses of NGF, or maximal doses of brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-4/5) in trkA -expressing cells. Furthermore, K-252b did not potentiate DNA synthesis by submaximal doses of BDNF, NT-4/5, or NT-3 in trkB - or trkC -expressing NIH3T3 cells, suggesting that the potentiation profile for K-252b was specific for NT-3 in trkA -expressing cells. We found no expression of p75NTR in the trk -expressing NIH3T3 cells. This is the first demonstration that K-252b potentiates a trkA -mediated biological nonneuronal response by NT-3 that occurs independent of p75NTR and appears to be both ligand and receptor specific.  相似文献   

18.
Abstract— Rat sympathetic ganglia, vagus nerve and sciatic nerve were each incubated with inorganic 32P for various lengths of time and the resultant labelling of their inositol lipids was measured.
At all times up to 3 hr phosphatidylinositol was the most highly labelled lipid of ganglia, while triphosphoinositide was the most active lipid of vagus and sciatic nerves. Removal of calcium ions from the incubation media had no significant effect on the incorporation of phosphate into any of the inositol lipids of sympathetic ganglia.  相似文献   

19.
Abstract: Cell dissociates from embryonic chick dorsal root ganglia, incubated for 6 h with 22Na+, accumulated four to six times more radioactivity in the absence than in the presence of Nerve Growth Factor (NGF). The accumulation of radioactivity paralleled the external Na+ concentration, indicating that the cells may have been reaching equilibrium with the medium. Delayed presentation of NGF to 22Na+-loaded cells caused a rapid loss of radioactivity, even with extracellular 22Na+ still present, demonstrating that NGF caused an overall efflux of Na+ rather than an accelerated equilibration. The Na+ exclusion from 22Na+-loaded cells was dependent upon NGF concentration. Use of nutrient-rich medium, serum, and certain hormones and other proteins did not prevent the Na+ accumulation in the absence of NGF or its reversion by delayed NGF administration. Incubation of the ganglionic cells with ouabain or dinitrophenol during the 22Na+ loading period (no NGF) increased the rate, but not the magnitude, of loading. The same incubation carried out in a Na+-free medium and followed by 22Na+ presentation resulted in fast radioactive loading that was identical to that occurring in drug-free, NGF-deprived cells and was not prevented by presentation of NGF together with the 22Na+. These data are consistent with a model in which NGF acts through a Na+ pump rather than by restricting Na+ influxes.  相似文献   

20.
RAPID TRANSPORT OF FUCOSYL GLYCOPROTEINS TO NERVE ENDINGS IN MOUSE BRAIN   总被引:4,自引:3,他引:1  
Abstract— Mice were injected intracerebrally with mixtures of [3H]fucose and [14C]gluco-samine, and incorporation into macromolecules in various subcellular fractions of brain was studied at 1, 2, 3 and 4 h after administration of the precursors. There was a lag of several hours between the incorporation of [3H]fucose into the glycoproteins of the whole brain fractions and of that into the soluble and particulate glycoproteins of the nerve ending fractions. In contrast, no lag was observed between the incorporation of [14C]glucosamine into the macromolecules of the whole brain fractions and of that into the soluble macro-molecules of the nerve ending fraction. We conclude that fucosyl glycoproteins of the nerve ending fraction were synthesized in the nerve cell bodies and transported to nerve endings by rapid axoplasmic transport, whereas a substantial proportion of the glucosamine in the soluble macromolecules of the nerve ending fraction was incorporated by the nerve endings themselves. In addition, our evidence indicates that cyclobeximide inhibited fucose incorporation into brain glycoproteins by inhibiting the synthesis of acceptor proteins rather than fucosyl transferase.  相似文献   

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