Translocation of soil-derived phosphorus in mycelial cord systems in relation to inoculum resource size

Abstract: Uptake of ³²P phosphorus from soil was investigated in mycelial cord systems of Phanerochaete velutina, Hypholoma fasciculare, Tricholomopsis platyphylla and Phallus impudicus which extended from 0.5, 2, 4 or 8 cm³ beech ( Fagus sylvatica ) inocula. Cord systems accumulated between 4.8 and 18.7% of phosphorus supplied to soil, according to species and size of inoculum. Phosphorus translocation to newly-colonized 2 cm³ beech baits, determined non-destructively, was characterized by an initial steady phase, of 2.5 to 32 nmol P day⁻¹ which lasted at least 12 days for all four species. After the initial steady phase, translocation rates declined. Initial mycelial extension and wood decay rates also varied with species and inoculum size. There was no clear relationship between phosphorus translocation rates, wood decay or the distribution of soil-derived phosphorus in cord system components. However, with increasing inoculum size, P. velutina systems allocated a significantly greater proportion of available phosphorus to newly-colonized baits. The degree to which distribution of soil-derived phosphorus in cord systems is related to nutrient conservation or metabolic demand in the fungi is discussed.     

Translocation of 32P between interacting mycelia of a wood-decomposing fungus and ectomycorrhizal fungi in microcosm systems

Interactions between saprotrophic and ectomycorrhizal fungi have been largely ignored, although their mycelia often share the same microsites. The mycelial systems show general similarity to each other and, although the enzymatic potential of the saprotrophic fungi is generally considered to be higher, the importance of organic nutrient sources to ectomycorrhizal fungi is now widely accepted. In the experiments described here, nutritional interactions involving transfer of elements from one mycelium to the other have been monitored dynamically using radioactive tracers and a non-destructive electronic autoradiography system. Microcosms were used in which mycelial systems of the ectomycorrhizal fungi Suillus variegatus and Paxillus involutus , extending from Pinus sylvestris host plants, were confronted with mycelia of the saprotroph Hypholoma fasciculare extending from wood blocks. The fungi showed a clear morphological confrontation response. The mycorrhizal mycelium often formed dense patches over the Hypholoma mycelia. Up to 25% of the ³²P present in the Hypholoma mycelium was captured by the mycorrhizal fungi and translocated to the plant host within 30 d. The transfer of ³²P to the saprotroph from labelled mycorrhizal mycelium was one to two orders of magnitude lower. The significance of this transfer as a 'short cut' in nutrient cycling is discussed.     

NOREPINEPHRINE INCREASES THE [32P]LABELLING OF A SPECIFIC PHOSPHOLIPID FRACTION OF POST-SYNAPTIC PINEAL MEMBRANES

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.     

Pathway and regulation of phosphate translocation to the pods of soybean explants

We investigated the degree to which developing fruit compete directly with leaves for mineral nutrients, e.g. phosphate coming up from the roots. When soybean ( Glycine max (L.) Merrill cv. Anoka) explants cut at mid-late podfill were given a 15-min pulse of ³²Pi via the cut stem and then transferred to distilled water, 75% of the ³²P accumulated in the leaves and 21% in stem and petiole during the first hour. The amount of ³²P entering the seeds was low (1%) initially, but thereafter increased to 30% in 48 h. An accumulation of ³²P in the seed coats preceded its entry into the embryos. Disruption (with hot steam) of the phloem between the leaf and the pods after pulse labelling indicated that more than 80% of the ³²Pi pulse moved to the leaf before redistribution to the pods. Increasing "sink" size by adjusting the pod load from 1 to 2–3 did not increase the ³²P accumulated by the pods proportionally. Conversely, excision of the seeds after pulse labelling did not prevent translocation of ³²P out of the leaves. These results suggest that the rate of transport of phosphate to the pods at mid-late podfill is controlled primarily by factors in the leaves. The results are consistent with the observation that the relative size of the sink (pod load) does not regulate leaf senescence.     

EFFECTS OF DENERVATION ON THE INCORPORATION OF 32P INTO PHOSPHATIDYL INOSITOL AND OTHER PHOSPHOLIPIDS OF RAT DIAPHRAGM

Abstract— At 24 h after denervation of the rat hemidiaphragm, incorporation of ³²P into phosphatidyl inositol was depressed relative to incorporation of ³²P into phosphatidyl choline (measured 75 min after injection of the isotope intraperitoneally). The ratio of the specific radioactivity of phosphatidyl choline to the specific radioactivity of P_i was unaffected by denervation which implies that denervation had depressed incorporation of isotope into phospatidyl inositol. Denervation did not cause a measurable change in the pool size of phosphatidyl inositol relative to that of phosphatidyl choline. The effect of denervation on incorporation of ³²P into phosphatidyl inositol was not entirely a direct consequence of the cessation of ACh release at the motor end-plate since the effect was clearly manifest in strips of muscle not containing motor end-plates, but the magnitude of the denervation effect was slightly greater in the strips of denervated hemidiaphragm which contained motor end-plates.     

Receptor-mediated increases in phosphatidylinositol turnover in neuron-like cell lines

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.     

Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

Phosphorus translocation in salt-stressed cotton

The effect of salinity on plants has usually been studied at high inorganic P concentration ([Pi]) in the nutrient solution, and salinity × Pi interactions have been examined at much higher [Pi] than found in soil solutions. Short-term ³²Pi experiments were carried out to study the effect of salinity (150 m M NaCl) on phosphorus translocation in cotton plants ( Gossypium hirsutum L. cv. Acala SJ-2) grown in nutrient solutions containing 10 μ M [Pi]. The effect of additional Ca to a concentration of 10 μ M was also tested. Salinity inhibited ³²P translocation from root to shoot. This inhibition was more evident at higher [Pi] in the root medium. Increasing [Pi] 33-fold in the solution resulted in a 4.3-fold increase in [³²P] in the root under saline conditions, but only in a 1,8-fold increase in the shoot. In older shoot tissues total [P] was elevated in the salinized plants. In the young tissues, however, total P concentration was higher in control plants. Inhibition of ³²P translocation by salinity was greater from root to young leaves than to mature shoot tissues. Salinity also decreased ³²P recirculation from the cotyledons to the young leaf. Inhibition by salinity of both ³²P translocation and recirculation to young leaves was fully reversed by increasing Ca supply from 1 to 10     

Effects of Calcium, Strontium, and Barium Ions on Phosphorylation of Hippocampal Proteins In Vitro

Abstract: Calcium ion alone or in the presence of added calmodulin stimulated in vitro transfer of ³²P from [γ³²P]ATP into several proteins of mitochondrial and synaptosomal particulate fractions from rat brain. Strontium ion was capable of substituting for calcium ion in this stimulation, but barium ion lacked this capacity. These results bring into question the hypothesis that calciumdependent protein phosphorylation of synaptic proteins is intrinsic to neurotransmitter release during neurotransmission, but they do not rule out that possibility.     

Alteration of Tubulin-Gi Protein Interaction in Rat Cerebral Cortex with Aging

Abstract: The ability of the tubulin dimer to interact with and to modulate the G_i function inhibiting adenylyl cyclase was examined in cerebral cortex membranes from 2-month-old and 24-month-old rats. The hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp)-dependent inhibition of adenylyl cyclase was significantly decreased in cerebral cortex membranes from 24-month-old rats. Tubulin, prepared from rat brains by polymerization with GppNHp, caused inhibition of adenylyl cyclase (∼28%) in 2-month-old rats. Tubulin-GppNHp-dependent inhibition of adenylyl cyclase in 24-month-old rats was significantly attenuated (∼15%). In 2-month-old rats, when tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [³²P] P ³(4-azidoanilido)- P ¹-5'-GTP ([³²P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to G_iα. Transfer of AAGTP from tubulin to G_iα was reduced in 24-month-old rats. Furthermore, photoaffinity labeling of [³²P]AAGTP to G_iα in cortex membranes was significantly decreased in 24-month-old rats. No differences were observed in the amounts of G_sα, G_iα, or G_β subunits and tubulin, estimated by immunoblotting, in cortex membranes from 2-month-old and 24-month-old rats. These results suggest that the ability of tubulin to interact with G_i and thereby modulate the inhibitory regulation of adenylyl cyclase is reduced in the cerebral cortex of 24-month-old rats.     

NUCLEAR CHROMATIN PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER: SYNTHESIS AND PHOSPHORYLATION

Abstract— In order to investigate synthesis and phosphorylation of the various fractions of nuclear proteins. [³H]leucine and [³²P] phosphate incorporation were studied with tissue slices in vitro. Cerebral cortex and cerebellum were used to delineate the similarity and dissimilarity within CNS, and liver was taken to compare the extraneural organ. There were significant differences in [³H]leucine incorporation into nuclear proteins among those tissue sources examined, while [³²P]phosphate incorporation showed very similar results among them. Although the acidic chromatin protein demonstrated high activity in each tissue source for both synthesis and phosphorylation, 0.14M-NaCl soluble protein showed the activity as high as or even higher than the acidic chromatin protein. Both [³H]leucine incorporation and [³²P]phosphate incorporation were relatively low in histone. When the acidic chromatin protein was further fractionated with SDS-acrylamide gel electrophoresis, significant difference was found between CNS tissue and liver for synthesis and phosphorylation. However, considerable difference was also observed even between cerebral cortex and cerebellum. The present investigation demonstrated complicity and diversity of nuclear chromatin proteins in different organs, not only for their protein constituents but also for their synthesis and phosphorylation.     

INCORPORATION OF 32P INTO THE PHOSPHOLIPIDS OF NEURONAL AND GLIAL CELL ENRICHED FRACTIONS ISOLATED FROM RABBIT CEREBRAL CORTEX: EFFECT OF NOREPINEPHRINE

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.     

Relative feeding rates of Brevicoryne brassicae and Myzus persicae on Brussels sprout plants in relation to their susceptibility to systemic insecticides

Brevicoryne brassicae and Myzus persicae removed similar quantities of ³²P-labelled material from Brussels sprout leaves whether they fed for 24 or 48 h periods. They also removed similar quantities from untreated leaf disks as from leaf disks treated with a sub-lethal dose of menazon. When a lethal dose was used, the uptake of ³²P by B. brassicae was significantly less than by M. persicae. M. persicae excreted a greater proportion of ³²P label in the honeydew than B. brassicae and a greater proportion of the amount absorbed was lost in the progeny of this aphid than in B. brassicae.
B. brassicae was 6.2 times more susceptible than M. persicae to dimethoate acting systemically. When it was applied topically the aphids were equally susceptible.
Considerable variation in uptake of ³²P occurred between replicates and the factors that could influence this are discussed.     

EFFECTS OF ACETYLCHOLINE ON THE INCORPORATION OF [32P]ORTHOPHOSPHATE IN VITRO INTO THE PHOSPHOLIPIDS OF NERVE-ENDING PARTICLES FROM GUINEA PIG BRAIN

Abstract— Guinea pig brain nerve-ending particles (synaptosomes) were incubated with [³²P]orthophosphate in a medium with or without 10⁻⁴M-acetylcholine and 10⁻⁴ M-eserine. Phospholipids were then extracted and separated by chromatography. About 60 per cent of the ³²P was found in phosphatidic acid and about 20 per cent in triphosphoinositide. Acetylcholine significantly increased the specific radioactivity of phosphatidic acid but had no effect on that of phosphatidylinositol or the nucleotide fraction. Labelling of the other phospholipids, including diphosphoinositide and triphosphoinositide, was not altered significantly by acetylcholine. Labelling of the nucleotide fraction and the polyphosphoinositides reached a peak at 40 min, that of phosphatidic acid at 80 min, while that of phosphatidylinositol was still rising at 160 min.     

Phosphate transport in potato cuttings: Effect of gibberellic acid and abscisic acid

Apical cuttings of Solanum tuberosum L. cv. Sirtema were used al different stages of development to study long-distance transport of phosphate. The effects of two hormones, gibberellic acid (GA₃) and abscisic acid (ABA), on this process were also investigated. Before tuberization, phosphate (³²P) supplied to a single leaf was transported preferentially in the young and growing parts of the plant: apical bud, young leaves and roots. After tuberization, the tuber became the principal site of phosphate accumulation. GA³ treatment (10⁻⁴ M) of the tuber as well as of the leaves led to reduced transport of ³²P into the tuber. By contrast, treatment of the tuber with ABA (10⁻⁴M) did not change the ³²P distribution within the plant, while foliar spray with ABA greatly increased the transport into the tuber. The opposite effects of the two hormones on phosphate accumulation by tubers are discussed with regard to their opposite effects on the tuberization process.     

Histones and the first cell cycle in maize germination

The timing of the onset of cell division during seed germination in maize and the role of histones for this process have been studied. Embryonic axes of maize seeds ( Zea mays L. hybrid H-30) were incubated in a sterile nutrient medium for different periods of time. For some experiments putrescine was also added. Mesocotyl, root tip and scutellar node were dissected at specific periods after incubation and the mitotic indices were determined in these tissues. Embryonic axes were incubated in the same medium either with [¹⁴C]-lysine or [³²P]-phosphate. The incorporation of either ¹⁴C or ³²P into histones was followed, both in postribosomal supernatant and in nuclei. It was found that during germination, there is specific timing for meristematic cells entering into cell division. Among the tissues tested, the mesocotyl meristem was the first to initiate this process. De novo synthesis of histones was detected as early as after 6 h of imbibition and the rate increased up to 12 h. Putrescine stimulated cell division and phosphorylation of the histones. The implications of these findings are discussed.     

Phosphorus transformations in the epilimnion of humic lakes: abiotic interactions between dissolved humic materials and phosphate

SUMMARY. 1. Sephadex gel filtration of filtered water from small, Finnish forest lakes demonstrated abiotic movement of ³³P from added PO₄ to two higher molecular weight fractions. This movement was most pronounced in waters with high humic content which also had high iron content. The two fractions which took up ¹³P had nominal molecular weights of > 100,000 and 10,000-20,000.
2. An equilibrium existed between free PO₄ and the two fractions. However, one fraction, at least, appeared to exist in two phases, with one phase in rapid equilibrium with free PO₄ but the other in only slow equilibrium.
3. Additions of ferric iron up to 1 mg Fe l⁻¹ to the filtered lake water stimulated movement from free PO₄, provided high concentrations of humic materials were present. In the absence of humic materials even 0.1 mg Fe 1⁻¹ would precipitate all added ³³PO₄.
4. The high molecular weight P was only partially reactive with standard molybdate reagents. Exposure of the high molecular weight P to sunlight caused a small release of PO₄ under the experimental conditions employed.
5. Possible implications for biological phosphorus demand of such sequestration of free PO₄ by humic materials in combination with iron are discussed.     

Myelin P0 Glycoprotein: Identification of the Site Phosphorylated In Vitro and In Vivo by Endogenous Protein Kinases

Abstract: Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [³²P]PO₄³⁻ from [γ-³²P]ATP into protein constituents. Among these, P₀ glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P₀ protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two ³²P-phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P₀ sequence revealed, besides a Lys¹⁷² to Arg substitution, that in the first peptide, two serine residues (Ser¹⁷⁶ and Ser¹⁸¹) were phosphorylated, Ser¹⁷⁶ appearing to be modified subsequently to Ser¹⁸¹. In the second peptide, Ser¹⁹⁷, Ser¹⁹⁹, and Ser²⁰⁴ were phosphorylated. All these serines are clustered in the C-terminal region of P₀ protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P₀. We found that, in vivo, Ser¹⁸¹ and Ser¹⁷⁶ are not phosphorylated, whereas Ser¹⁹⁷, Ser¹⁹⁹, Ser²⁰⁴, Ser²⁰⁸, and Ser²¹⁴ are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.     

Neuronal Localization of Ca2+-dependent Protein Phosphorylation in Brain

Abstract: The cellular localization of two Ca²⁺-dependent protein phosphorylation systems was investigated using the kainic acid lesioning technique for the selective destruction of neurons. In one of these systems, a crude synaptosomal (P₂) fraction was preincubated with ³²P_j for 30 min; the phosphorylation of several proteins was increased during a short subsequent incubation with veratridine plus Ca²⁺. In the second system, crude synaptosomal membranes isolated from the P₂ fraction were incubated with [γ-³²P]ATP; in this system, the phosphorylation of several proteins was increased in the presence of a "calcium-dependent regulator" plus Ca²⁺. Kainic acid lesioning greatly reduced the amount of Ca-⁺-dependent protein phosphorylation in both systems. The results indicate a predominantly neuronal localization for both Ca²⁺-dependent protein phosphorylation systems.     

Participation of Tubulin in the Stimulatory Regulation of Adenylyl Cyclase in Rat Cerebral Cortex Membranes

Abstract: This study examined effects of tubulin on the activation of adenylyl cyclase in rat cerebral cortex membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of the enzyme by ∼156% under conditions in which stimulation rather than inhibition of the enzyme was favored. Tubulin-GppNHp activated isoproterenol-sensitive adenylyl cyclase, potentiated forskolin-stimulated activity of the enzyme, and reduced agonist binding affinity for β-adrenergic receptors. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [³²P] P ³(4-azidoanilido)- P ¹-5'-GTP ([³²P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to G_sα as well as G_iα. These results suggest that, in rat cerebral cortex membranes, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to G_sα, as well as affecting the G_i-mediated inhibitory pathway.