Pénétration et migration du 59Fe appliqué sur les feuilles de Maïs; effet du dimáthylsulfoxyde

The uptake and translocation of ⁵⁹Fe applied to leaves of Zea mays L. is studied with special reference to the effect of dimethylsulfoxide (DMSO). ⁵⁹Fe is deposited on corn leaves as droplets of solution of ferrous sulfate or ferric nitrate (1 mM. The uptake of ⁵⁹Fe is affected by the associated anion; the penetration is more important with the nitrate than with the sulfate. The translocation of ⁵⁹Fe from the treated part during 24 hours is very low in all the experiments. The exsorption of ⁵⁹Fe taken up, from the site of application in different solutions (FeSO⁴, 7H₂O; EDTA Na) concerns only a low percentage of ⁵⁹Fe present in the treated part. DMSO (0.5 and 1 %) increases the uptake but not the translocation of ⁵⁹Fe applied as sulfate; it seems to have no effect when iron is applied as nitrate. The increase during 24 hours reaches between 32 % and 53 % in seven experiments with sulfate. The effect also appears in an experiment conducted during four weeks, with several applications of ferrous sulfate during this time. This effect of DMSO is discussed: it is mainly explained by the great hygroscopicity of this solvent, therefore the effect would in part depend on the solubility of salts in concentrated DMSO and of the climatic conditions: relative humidity and temperature of the air.     

Translocation of leaf-fed 32P in pea plants as influenced by foliar and root applications of CCC and Phosfon

In controlled environment growth chambers, the effects of foliar and root applications of 2-chloroethyltrimethylammonium chloride (CCC) and 2,4-dichlorobenzyltributylphosphonium chloride (Phosfon) on the translocation of³²P fed to leaves, were investigated. When applied to leaves or to root, CCC had no effect on the relative amounts of³²P radioactivity retained by the fed leaf 5, 20 and 80 h after feeding. At 20 and 80 h after feeding, Phosfon concentrations of 0.01 and 0.1mg l^?1 reduced retention of the applied³²P. 80 h after³²P feeding, CCC concentration of 1 mg l^?1 applied as a foliar spray or to the root enhanced the downward movement of³²P. Phosfon at low concentrations, particularly at 0.1 mg l^?1, on the other hand, favoured an upward transport of the applied³²P. Foliar applications of CCC and Phosfon at high concentrations had no significant effect on³²P transport to the root and the shoot below the fed leaf, while root applications of CCC and of Phosfon inhibited downward transport. Root applications generally caused greater alterations in³²P distribution patterns than did foliar applications. On the basis of total active ingredient uptake, Phosfon was more effective than CCC in altering translocation patterns.     

Senescence of rice leaves at the vegetative stage as affected by growth substances

In rice (Oryza saliva L. ev. Java), the first (younger) leaf senesced later than the second (older) leaf as shown by the decline in chlorophyll and protein contents. Kinetin treatment significantly retarded senescence of leaves, while abscisic acid (ABA) treatment promoted it. The second leaf exported more³²P to the newly emerged growing leaf at the early stages than the first leaf, which always showed higher retention of³²P than the second one. Kinetin treatment lengthened the duration of³²P export and also increased the retention capacity of both leaves, while ABA had the opposite effect. The second leaf showed a higher depletion of nitrogen and phosphorus but à lower depletion of potassium than the first leaf. Kinetin treatment retarded the decline in nutrient content (N and P) while ABA treatment hastened it. Neither growth substance had any effect on potassium content. The content(s) of endogenous eytokinin-like substance(s) decreased while ABA-like substance(s) increased in the two leaves with senescence: these changes in the second leaf took place earlier than in the first leaf.     

Phosphopeptide and phosphoprotein metabolism in brain

Phosphopeptide and phosphoprotein phosphorylation was studied in rat brain microsomes and rat brain slices which were incubated in the presence of [γ-³²P] ATP under various experimental conditions. Radioactive phosphoserine was isolated from phosphopeptides and phosphoproteins.Na⁺, K⁺, Mg²⁺ and cyclic AMP had a stimulating effect on the labelling of phosphopeptides. Ouabain and Ca²⁺ lowered the level of ³²P incorporation into the phosphopeptides.The phosphoproteins behaved similarly to the phosphopeptides except for the potassium effect.Chase experiments showed a faster decrease in the labelling of phosphopeptides than in phosphoproteins. We suggest that both compounds may be involved in active transport phenomena.     

Membrane Functions and Tolerance to Aromatic Hydrocarbon Fungitoxicants in Conidia of Fusarium solani

With the use of ³²P-labeled phosphate and ⁴²K₂CO₃ the effect of diphenyl on permeability and uptake properties of the cytoplasmic membrane in wild type and diphenyl-tolerant mutant conidia of Fusarium solani f. cucurbitae was studied. No general damage to the membrane with unspecific leakage of cell constituents was demonstrated under conditions in which diphenyl prevents germination of wild type conidia. The fresh conidia do not require exogenous supply of energy for the uptake of phosphate or of potassium. In the wild type the entry of ³²P is inhibited but that of ⁴²K strikingly stimulated by diphenyl. Independently of the tolerant mutant gene present, the mutant conidia are significantly less sensitive to the phosphate uptake inhibition and not affected at all by diphenyl with respect to the uptake of potassium. The latter difference from the wild type seems to indicate genetic control of some property of the potassium transport system in this fungus.     

Effects of physiological manipulation on the kinetics of mitochondrial phosphate transport in Saccharomyces cerevisiae

The kinetics of [³²P]phosphate uptake has been studied in different types of Saccharomyces cerevisiae mitochondria. Mitochondria were isolated from yeast grown aerobically on 2% lactate (Lac-mitochondria), 2% galactose (Gal-mitochondria), 5.4% glucose (Glu-mitochondria) or from yeast grown anaerobically on 2% galactose (Promitochondria). The effect of chloramphenicol was also studied by adding it to the growth medium of yeast grown aerobically on 2% galactose (chloramphenicol-mitochondria).[³²P]Phosphate uptake followed an oscillatory pattern in Lac, Gal-mitochondria and Promitochondria.Saturation kinetics were detected in fully differenciated mitochondria and in Promitochondria, but not in chloramphenicol-mitochondria.Glu-mitochondria did not translocate phosphate as shown both by lack of [³²P]phosphate uptake and lack of swelling in isoosmotic potassium solution.Repressed yeast cells were incubated in a resting cell medium and mitochondria were isolated at different times of incubation. The rate of respiration and the oligomycin-sensitive ATPase increased during the course of the incubation. After 2h, a mitochondrial mersalyl-sensitive swelling in an isoosmotic potassium phosphate solution was detected.As expected, no increase of the rate of respiration was observed when chloramphenicol was added in the derepression medium. But the oligomycin-sensitive ATPase decreased. Chloramphenicol did not affect the phosphate transport activity as measured by the swelling of mitochondria, but the [³²P]phosphate uptake did not follow saturation kinetics. A complete derepression of the inorganic phosphate-carrier activity was achieved by a 4 h incubation of the repressed cells in the presence of chloramphenicol, followed by a 6 h incubation in presence of cycloheximide.These data strongly suggest that the mitochondrial protein-synthesis system is required for the normal function of the inorganic phosphate-carrier.     

Phosphorus cycling between the colonial cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and attached bacteria, <Emphasis Type="Italic">Pseudomonas</Emphasis>

Phosphorus (P) transfer between Microcystis aeruginosa and the attached bacterium Pseudomonas was studied using radioactive P (³²P) and green fluorescence protein-labeled Pseudomonas. M. aeruginosa in P-starved condition took up most ³²P (70%) in water and about 50% of ³²P in ³²P-saturated bacteria in individual experiments. However, only 26% of ³²P in the ³²P-saturated M. aeruginosa was transferred to P-starved bacteria. The P-starved M. aeruginosa had an advantage to take up P over the bacteria and its growth rates and abundance were higher in combined cultures, with bacteria as the biotic P source. The rate of P transfer from bacteria to the cyanobacteria was slow. P cycles predominantly between M. aeruginosa and Pseudomonas with little variation in the water. This ability is very useful for the colony-forming M. aeruginosa, especially if phosphate concentrations in water are low during water bloom periods.     

Uptake, Distribution and Translocation of 32P Absorbed through Roots of Cotton Seedlings as Affected with CCC Treatment

The effects of CCC on the ³² P amounts absorbed by cotton seedlings were studied. CCC was applied to the seedlings either as spray or as addition to the nutrient solution in concentrations of 0, 25, 50, 100 and 200 mg/1. ³²P was added to the medium as KH₂³²P O₄ in the concentration of 40 μCi/50 ml. During the experimental period, no morphological changes were observed. The total ³²P uptake was inhibited in CCC treated seedlings. The application of CCC, both as spray and as addition to the medium, led to an accumulation of ³²P in stem, but to a decrease in root. The leaves showed different responses to different methods of application; spraying increased, while an addition of CCC to the medium decerases the ³²P content in the leaves. It is concluded that CCC inhibits ³²P uptake, whereas it ac accelerates the ³²P translocation from root to the aerial parts.     

Estradiol distribution and penetration in rat skin after topical application,studied by high resolution autoradiography

Summary Transdermal pathways and targets in the skin for estradiol were investigated using dry-mount autoradiography.³H-estradiol-17 was applied at doses of 30.1 pmol, 120.4 pmol and 301 pmol/cm² to shaved rat skin in the dorsal neck region. Vehicles were DMSO, ethylene glycol or sesame oil. After 2 h of topical treatment with 30.1 pmol³H-estradiol×cm^–2 dissolved in DMSO a distinct cellular distribution was apparent. Target cells with concentrations of radioactivity were found in epidermis, sebaceous glands, dermal papillae of hair and fibroblasts. After treatment with 120.4 and 301 pmol/cm², a penetration gradient of radioactivity was recognizable however it masked specific cellular and subcellular uptake. The stratum corneum accumulated and retained radioactivity, apparently forming a depot for the hormone. Strong concentration and retention of the hormone was conspicious in sebaceous glands for more than 24 h, suggesting that sebaceous glands serve as a second storage site for the hormone. In all autoradiograms two penetration pathways to the dermis were visible: one through the stratum corneum and epidermis, the other through the hair canals and hair sheaths.This work was supported by US PHS grant NS09914     

Effect of the amputation of the cotyledon and of the application of growth regulators on the transport of32P in decapitated pea seedlings

When the epicotyl and one cotyledon is cut off from pea seedlings, only the axillary of the amputated cotyledon is known to grow. When³²P is applied to the roots of such plants, then a higher radioactivity appears in the axillary of the amputated cotyledon already 24 hrs. after amputation of one cotyledon, although this axillary is of the same size at this time as that of the remaining cotyledon. This fact indicates a more extensive material transport to the axillary bud of the amputated cotyledon already during the first day after amputation The effect of individual regulators on the³²P transport was investigated in an experiment where pea seedlings cultivated in the dark were decapitated and a 0.5% paste, containing the regulatory compounds was placed either on the cutting surface in the apical part of the epicotyl stump or in its central part. After a week the plant roots were supplied with³²P and its transport to the upper part of the epicotyl stump was followed. This transport increased about 10-fold in the case of a paste, containing indolyl acetic acid, when the paste was spread on the apical cutting surface of the stump. However, the transport was inhibited when the paste was applied in the central part of the stump. These results indicate that only the transport of³²P towards the paste with indolyl acetic acid is accelerated, whereas it is decelerated above this paste. A paste, containing triodobenzoic acid inhibited³²P transport only when applied to the apical cutting surface of the epicotyl stump and not when spread over the middle part. In this case³²P transport was more rapid above the paste than towards the paste. The situation was similar in the case of gibberellin and kinetin.     

Effects of bafilomycin A1 and amiloride on the apical potassium and proton gradients in Drosophila Malpighian tubules studied by X-ray microanalysis and microelectrode measurements

The intracellular distribution of potassium in Malpighian tubules from Drosophila larva was measured by electron probe X-ray microanalysis of freeze-dried cryosections. Application of amiloride alone to the haemolymph space had no effect on the intracellular potassium concentration in the region of intermediate cytoplasm (between the basal region of basal membrane infoldings and the apical brush border), whereas a potassium increase as well as a chloride increase was observed after simultaneous blocking of the potassium conductance of the basal membrane with barium. Injected bafilomycin and amiloride applied in the haemolymph caused an increase of the potassium content in the basal cytoplasm but not in the microvilli. In addition, the intracellular water portion was decreased by bafilomycin. pH measurements in isolated larval anterior tubules with proton-selective microelectrodes showed that bafilomycin added to the bathing solution caused a decrease in intracellular pH. Addition of amiloride had no significant effect on intracellular pH, but the pH of the luminal fluid was decreased within 1 min by 0.5 pH units. The amiloride-induced luminal pH decrease could be inhibited by the metabolic blocker KCN as well as by bafilomycin. Furthermore, removing potassium from the bathing saline caused a slow luminal acidification, which could be blocked by KCN. Our results support the hypothesis of a functionally coupled transport system in the apical membrane consisting of a bafilomycin-sensitive V-ATPase and a K⁺-dependent, amiloride-sensitive K⁺/H⁺ exchange system.Abbreviation C _a element concentration related to water - C _d element content related to dry weight - dw dry weight - DMSO dimethylsulphoxide - emf electromotive force - NBD-Cl 7-chloro-4-nitrobenz-2-oxa-1,3-diazole - NEM N-ethylmaleimide - NMDG⁺ N-methyl-d-glucamine - PD potential difference - pH_i intracellular pH value - pH_lu luminal pH value - pmf protonmotive force - SD standard deviation - SE standard error - STEM scanning transmission electron microscopy - V _a apical potential difference - V _b basal potential difference - V _t transepithelial potential difference     

Action du diméthylsulfoxyde sur l'absorption du 42K et du glucose-14C (U) par des disques foliaires de Zea mays et Pelargonium zonale

⁴²K and ¹⁴C (U)-glucose uptake by foliar discs of maize (Zea mays L.) and pelargonium (Pelargonium zonale L.) is inhibited by dimethyl sulfoxide (DMSO) at concentrations of 5 and 10%. This effect increases with the duration of the absorption and appears with varied concentrations in K or glucose. In most cases, this inhibitory effect is less with aged discs than with freshly cut discs. Besides, the DMSO (10%) enhances the exsorption of ⁴²K by foliar discs of maize. The effects of DMSO and two osmotic agents, mannitol and polyethylene glycol 1000, on the uptake and water content of discs are compared. It seems that the effect of DMSO on the uptake is not only an osmotic effect. It may also be related to other actions of this substance in affecting an active uptake.     

Direct application of carbendazim and propiconazole at field rates to the external mycelium of three arbuscular mycorrhizal fungi species: effect on 32P transport and succinate dehydrogenase activity

 The influence of the systemic fungicides propiconazole (Tilt 250E) and carbendazim (Bavistin) at field application rates on the functioning of three arbuscular mycorrhizal fungi was studied. Short-term fungal ³²P transport and succinate dehydrogenase (SDH) activity in external hyphae of Glomus intraradices Schenck and Smith, G. claroideum Schenck and Smith and G. invermaium Hall in symbiosis with pea (Pisum sativum L.) were measured. In the experimental system used, the hyphae grew into two root-free hyphal compartments (HCs). The fungicides were applied to each HC 24 days after sowing and ³²P was added to one HC of each pot. Four days later, the fungicide effect on fungal P transport was measured as the difference in ³²P content of treated and untreated plants. SDH activity in fungal hyphae was determined in the HCs given no ³²P. Carbendazim severely inhibited ³²P transport and SDH activity in external hyphae at an application rate of 0.5 μg g^–1 soil. The ergosterol inhibitor propiconazole affected none of these parameters. The fungicides had similar effects on all three fungal species, although P transport efficiency and SDH activity differed markedly between the fungi. Accepted: 12 December 1996     

Studies on the penetration of mammalian cells by deoxyribonucleoside-5′-phosphates

We have tested the ability of [5′-³²P]-deoxyribonucleoside monophosphates (dNMPs) to penetrate living mouse fibroblast L cells and human HeLa cells. Under the conditions of our experiments, small numbers of apparently intact dNMP molecules appeared to penetrate into the interior of L cells and be incorporated into DNA. This incorporation was not due to mycoplasma contamination nor to extracellular hydrolysis of the dNMPs followed by resynthesis inside the cell. Under these same conditions, penetration of HeLa cells by intact dNMPs did not occur to a significant extent. However, HeLa cells were capable of hydrolyzing extracellular dNMPs to Pi and deoxyribonucleosides at a much faster rate than L cells. These experiments provide a starting point for attempts to specifically label the DNA in intact, living eukaryotic cells with [³²P]-dNMPs.     

Transdermal delivery of diclofenac sodium through rat skin from various formulations

The aim of this study was to evaluate and compare the in vitro and in vivo transdermal potential of w/o microemulsion (M) and gel (G) bases for diclofenac sodium (DS). The effect of dimethyl sulfoxide (DMSO) as a penetration enhancer was also examined when it was added to the M formulation. To study the in vitro potential of these formulations, permeation studies were performed with Franz diffusion cells using excised dorsal rat skin. To investigate their in vivo performance, a carrageenan-induced rat paw edema model was used. The commercial formulation of DS (C) was used as a reference formulation. The results of the in vitro permeation studies and the paw edema tests were analyzed by repeated-measures analysis of variance. The in vitro permeation studies found that M was superior to G and C and that adding DMSO to M increased the permeation rate. The permeability coefficients (Kp) of DS from M and M+DMSO were higher (Kp=4.9×10⁻³±3.6×10⁻⁴ cm/h and 5.3×10⁻³±1.2×10⁻³ cm/h, respectively) than the Kp of DS from C (Kp=2.7×10⁻³±7.3×10⁻⁴ cm/h) and G (Kp=4.5×10⁻³±4.5×10⁻⁵ cm/h). In the paw edema test, M showed the best permeation and effectiveness, and M+DMSO had nearly the same effect as M. The in vitro and in vivo studies showed that M could be a new, alternative dosage form for effective therapy.     

Changes in phosphorus metabolism in alfalfa plants induced by bacterial wilt

Radioactive phosphate was applied to the roots of intact alfalfa plants (Medicago sativa L.) on the 49th day after inoculation withCorynebacterium insidiosum (Me Culloch) Jensen and the³²P contents in different fractions of phosphoric compounds were determined. In inoculated plants, susceptible to bacterial wilt, the inorganio phosphate contents (³²Pinorg) was increased markedly and the³²P bound in organic compounds soluble in acids (³²Porg) decreased as compared with control. In roots of the same plants the³²P contents in phospholipid fraction and DNA were decreased. In tolerant inoculated plants the³²Pinorg increase and³²Porg decrease as compared with those changes in susceptible plants were less expressive. No expressive changes in determined³²P fractions have been proved in resistant plants without any visible disease symptoms.     

Effect of Prolonged Geo-stimulation and Presence of 32P on the Elongation of Lateral Shoots of Decapitated Pea Seedlings

The effects of prolonged geo-stimulation and presence of ³²P on 3-leaf decapitated pea (Pisum sativum L. cv. Alaska) seedlings were studied with regard to lateral bud elongation as well as accumulation and recovery of isotope. Prolonged geo-stimulation favoured the dominance of basally located, upwardly directed buds and suppressed the elongation of subapical bud — a gravimorphic response similar to the one shown by intact geo-stimulated branches of woody plants. Basal bud 2 was an exception as it dominated both in the above- and below-axis position. Accumulation of ³²P in buds was the result of their own growth, whereas in the other parts of the seedlings accumulation of isotope was not necessarily related to growth. Geo-stimulation affected ³²P recovery in redistribution experiments, whereas it had no effect in distribution experiments. In the former, the decrease in the isotope level in the seedling suggests a loss of ³²P. Prolonged presence of ³²P in the seedlings had a deleterious effect on bud elongation.     

oxLDL antibody inhibits MCP‐1 release in monocytes/macrophages by regulating Ca2+/K+ channel flow

oxLDL peptide vaccine and its antibody adoptive transferring have shown a significantly preventive or therapeutic effect in atherosclerotic animal model. The molecular mechanism behind this is obscure. Here, we report that oxLDL induces MCP‐1 release in monocytes/macrophages through their TLR‐4 (Toll‐like receptor 4) and ERK MAPK pathway and is calcium/potassium channel‐dependent. Using blocking antibodies against CD36, TLR‐4, SR‐AI and LOX‐1, only TLR‐4 antibody was found to have an inhibitory effect and ERK MAPK‐specific inhibitor (PD98059) was found to have a dramatic inhibitory effect compared to inhibitors of other MAPK group members (p38 and JNK MAPKs) on oxLDL‐induced MCP‐1 release. The release of cytokines and chemokines needs influx of extracellular calcium and imbalance of efflux of potassium. Nifedipine, a voltage‐dependent calcium channel (VDCC) inhibitor, and glyburide, an ATP‐regulated potassium channel (K⁺_ATP) inhibitor, inhibit oxLDL‐induced MCP‐1 release. Potassium efflux and influx counterbalance maintains the negative potential of macrophages to open calcium channels, and our results suggest that oxLDL actually induces the closing of potassium influx channel – inward rectifier channel (K_ir) and ensuing the opening of calcium channel. ERK MAPK inhibitor PD98059 inhibits oxLDL‐induced Ca²⁺/K_ir channel alterations. The interfering of oxLDL‐induced MCP‐1 release by its monoclonal antibody is through its FcγRIIB (CD32). Using blocking antibodies against FcγRI (CD64), FcγRIIB (CD32) and FcγRIII (CD16), only CD32 blocking antibody was found to reverse the inhibitory effect of oxLDL antibody on oxLDL‐induced MCP‐1 release. Interestingly, oxLDL antibody specifically inhibits oxLDL‐induced ERK MAPK activation and ensuing Ca²⁺/K_ir channel alterations, and MCP‐1 release. Thus, we found a molecular mechanism of oxLDL antibody on inhibition of oxLDL‐induced ERK MAPK pathway and consequent MCP‐1 release.     

Permeabilization of plant cells: 31P NMR studies on the permeability of the tonoplast

A suspension culture of Catharanthus roseus has been used to study the permeability of cell membranes after treatment with various concentrations of a permeabilizing agent (DMSO). The uptake and release (after permeabilization) of inorganic phosphate (P_i) by cells have been investigated by ³²P radiotracer and non-invasive phosphorus-31 NMR experiments. These studies have demonstrated that measurements of the P_i-efflux from plant cells provide a reliable measure of the permeability of the tonoplast.Abbreviations DMSO dimethylsulfoxide - NMR nuclear magnetic resonance - P_i inorgainic phosphate     

Simple synthesis of <Superscript>32</Superscript>P-labelled inositol hexakisphosphates for study of phosphate transformations

Background and aims

In many soils inositol hexakisphosphate in its various forms is as abundant as inorganic phosphate. The organismal and geochemical processes that exchange phosphate between inositol hexakisphosphate and other pools of soil phosphate are poorly defined, as are the organisms and enzymes involved. We rationalized that simple enzymic synthesis of inositol hexakisphosphate labeled with ³²P would greatly enable study of transformation of soil inositol phosphates when combined with robust HPLC separations of different inositol phosphates.

Methods

We employed the enzyme inositol pentakisphosphate 2-kinase, IP5 2-K, to transfer phosphate from [γ-³²P]ATP to axial hydroxyl(s) of myo-, neo- and 1D-chiro-inositol phosphate substrates.

Results

³²P-labeled inositol phosphates were separated by anion exchange HPLC with phosphate eluents. Additional HPLC methods were developed to allow facile separation of myo-, neo-, 1D-chiro- and scyllo-inositol hexakisphosphate on acid gradients.

Conclusions

We developed enzymic approaches that allow the synthesis of labeled myo-inositol 1,[³²P]2,3,4,5,6-hexakisphosphate; neo-inositol 1,[³²P]2,3,4,[³²P]5,6–hexakisphosphate and 1D-chiro-inositol [³²P]1,2,3,4,5,[³²P]6-hexakisphosphate. Additionally, we describe HPLC separations of all inositol hexakisphosphates yet identified in soils, using a collection of soil inositol phosphates described in the seminal historic studies of Cosgrove, Tate and coworkers. Our study will enable others to perform radiotracer experiments to analyze fluxes of phosphate to/from inositol hexakisphosphates in different soils.