Effect of Phytochrome on the Uptake of Gibberellin by Protoplasts of Nicotiana glutinosa L.

The effect of red light on gibberellin uptake by Nicotiana glutinosaL. protoplasts was determined. Five minutes of red light causedover a 50% inhibition of GA₃ uptake within 2 minutes. Five minutesof far red light completely reversed the red light effect. Theantagonistic effect of far red light indicates that gibberellinuptake is under phytochrome control. The rapid inhibition of gibberellin uptake indicates that phytochromeregulates the permeability properties of the plasma membraneas an initial response and not the intracellular binding ofgibberellin. ¹Department of Plant Pathology, University of Missouri. (Received October 28, 1985; Accepted April 23, 1986)     

Manganese Uptake and Efflux in Cultured Rat Astrocytes

Astrocytes play a central role in manganese (Mn) regulation in the CNS. Using primary astrocyte cultures from neonatal rat brains, these studies demonstrate a specific high-affinity transport system for Mn2+. Saturation kinetics are clearly indicated by both 1/v versus 1/s plots (Km = 0.30 +/- 0.03 microM; Vmax = 0.30 +/- 0.02 nmol/mg of protein/min) and plots of v versus [s]. Several divalent cations (Co2+, Zn2+, and Pb2+) failed to inhibit the initial rate of 54Mn2+ uptake. In contrast, extracellular Ca2+ at 10 microM decreased 54Mn2+ uptake. Exchange with extracellular Mn2+ was not obligatory for the efflux of 54Mn2+ into extracellular medium because efflux occurred into Mn(2+)-free extracellular medium, but efflux of 54Mn2+ was enhanced when astrocytes were equilibrated in the presence of unlabeled Mn2+. Efflux of 54Mn2+ was biphasic with both a rapid and a slow component. Efflux was most rapid during the first 10 min of incubation, with 27.5 +/- 2.2% of 54Mn2+ transported extracellularly, and 37.2 +/- 1.2% of preloaded 54Mn2+ was retained by the astrocytes at 120 min. These studies show, for the first time, that mammalian astrocytes can transport Mn via a specific transport system.     

Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines

Background

BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism.

Aim

The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes.

Methods and Results

Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake.

Conclusions

Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.     

Redox Cycling in Iron Uptake,Efflux, and Trafficking

Aerobic organisms are faced with a dilemma. Environmental iron is found primarily in the relatively inert Fe(III) form, whereas the more metabolically active ferrous form is a strong pro-oxidant. This conundrum is solved by the redox cycling of iron between Fe(III) and Fe(II) at every step in the iron metabolic pathway. As a transition metal ion, iron can be “metabolized” only by this redox cycling, which is catalyzed in aerobes by the coupled activities of ferric iron reductases (ferrireductases) and ferrous iron oxidases (ferroxidases).     

Fluctuations in Water Uptake Into and Vapour Efflux From Leaves

Water uptake by leaves, and water vapour efflux from leaves,were found to exhibit short-term fluctuations. These fluctuationswere of an inconstant period and the fluctuations in influxand efflux had an inconstant phase relationship.     

Interleukin-10 Facilitates Both Cholesterol Uptake and Efflux in Macrophages

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by scavenger receptor in macrophages initiates the chronic proinflammatory cascade and necrosis core formation that characterize atherosclerosis. We report here that a cytokine considered to be anti-atherogenic, interleukin-10 (IL10), promotes cholesterol uptake from modified lipoproteins in macrophages and its transformation into foam cells by increasing the expression of scavenger receptor CD36 and scavenger receptor A. Although uptake of modified lipoproteins is considered proatherogenic, we found that IL10 also increases cholesterol efflux from macrophages to protect against toxicity of free cholesterol accumulation in the cell. This process was PPARγ-dependent and was mediated through up-regulation of ABCA1 (ATP-binding cassette transporter A1) protein expression. Importantly, expression of inflammatory molecules, such as tumor necrosis factor-α, intercellular adhesion molecule-1, and MMP9 as well as apoptosis were dramatically suppressed in lipid-laden foam cells treated with IL10. The notion that IL10 can mediate both the uptake of cholesterol from modified lipoproteins and the efflux of stored cholesterol suggests that the process of foam cell formation is not necessarily detrimental as long as mechanisms of cholesterol efflux and transfer to an exogenous acceptor are functioning robustly. Our results present a comprehensive antiatherogenic role of IL10 in macrophages, including enhanced disposal of harmful lipoproteins, inhibition of inflammatory molecules, and reduced apoptosis.     

Simultaneous Influx and Efflux of Nitrate during Uptake by Perennial Ryegrass

Experiments with intact plants of Lolium perenne previously grown with ¹⁴NO₃⁻ revealed significant efflux of this isotopic species when the plants were transferred to solutions of highly enriched ¹⁵NO₃⁻. The exuded ¹⁴NO₃⁻ was subsequently reabsorbed when the ambient solutions were not replaced. When they were frequently replaced, continual efflux of the ¹⁴NO₃⁻ was observed. Influx of ¹⁵NO₃⁻ was significantly greater than influx of ¹⁴NO₃⁻ from solutions of identical NO₃⁻ concentration. Transferring plants to ¹⁴NO₃⁻ solutions after a six-hour period in ¹⁵NO₃⁻ resulted in efflux of the latter. Presence of Mg²⁺, rather than Ca²⁺, in the ambient ¹⁵NO₃⁻ solution resulted in a decidedly increased rate of ¹⁴NO₃⁻ efflux and a slight but significant increase in ¹⁵NO₃⁻ influx. Accordingly, net NO₃⁻ influx was slightly depressed. A model in accordance with these observations is presented; its essential features include a passive bidirectional pathway, an active uptake mechanism, and a pathway for recycling of endogenous NO₃⁻ within unstirred layers from the passive pathway to the active uptake site.     

Cryptochrome and Phytochrome Cooperatively but Independently Reduce Active Gibberellin Content in Rice Seedlings under Light Irradiation

In contrast to a wealth of knowledge about the photoregulation of gibberellin metabolism in dicots, that in monocots remains largely unclear. In this study, we found that a blue light signal triggers reduction of active gibberellin content in rice seedlings with simultaneous repression of two gibberellin 20-oxidase genes (OsGA20ox2 and OsGA20ox4) and acute induction of four gibberellin 2-oxidase genes (OsGA2ox4-OsGA2ox7). For further examination of the regulation of these genes, we established a series of cryptochrome-deficient lines through reverse genetic screening from a Tos17 mutant population and construction of knockdown lines based on an RNA interference technique. By using these lines and phytochrome mutants, we elucidated that cryptochrome 1 (cry1), consisting of two species in rice plants (cry1a and cry1b), is indispensable for robust induction of the GA2ox genes. On the other hand, repression of the GA20ox genes is mediated by phytochromes. In addition, we found that the phytochromes also mediate the repression of a gibberellin 3-oxidase gene (OsGA3ox2) in the light. These results imply that, in rice seedlings, phytochromes mediate the repression of gibberellin biosynthesis capacity, while cry1 mediates the induction of gibberellin inactivation capacity. The cry1 action was demonstrated to be dominant in the reduction of active gibberellin content, but, in rice seedlings, the cumulative effects of these independent actions reduced active gibberellin content in the light. This pathway design in which different types of photoreceptors independently but cooperatively regulate active gibberellin content is unique from the viewpoint of dicot research. This redundancy should provide robustness to the response in rice plants.     

Apparent Bicarbonate Uptake and Possible Plasmalemma Proton Efflux in Chara corallina

It is shown that the apparent uptake of bicarbonate by cells of Chara corallina could be the result of a proton efflux coupled to extracellular production of CO₂ from bicarbonate, with CO₂ being taken up by the cell. The theoretical results presented here show that the influx of CO₂ across the plasmalemma can be much greater than previously thought, if there is a large efflux of protons across the plasmalemma, and that, if this occurs, there would be a much steeper gradient of pH near the cell surface than previously thought possible.     

Effect of Auxin and Gibberellin on Sporulation in Yeast

Effect of auxin and gibberellic acid on sporulation of a yeast, Saccharomyces ellipsoideus, was studied. When added to the sporulation media, gibberellic acid promoted sporulation. The sporulation rate was higher in the medium SGV with vitamins than in the vitamin-free SG, but the effect of gibberellic acid was more pronounced in the latter. Auxin (IAA, 2,4-D, and NAA) inhibited sporulation in SGV, but promoted it in SG. This sporulation-promoting effect of IAA was reversed by an antiauxin, 2,4,6-T. Preculturing in the presence of added IAA increased sporulation. Added to the preculture medium, gibberellic acid alone showed little effect on sporulation, but in combination with IAA it enhanced sporulation conspicuously. IAA and gibberellic acid were effective in sporulation promotion only when added before the nuclear enlargement occurred in sporulation culture.     

The Effect of Calcium on Non-heme Iron Uptake,Efflux, and Transport in Intestinal-like Epithelial Cells (Caco-2 Cells)

It has been suggested that calcium inhibits the absorption of dietary iron by directly affecting enterocytes. However, it is not clear if this effect is due to a decreased uptake of iron or its efflux from enterocytes. We studied the effect of calcium on the uptake, efflux, and net absorption of non-heme iron using the intestinal-like epithelial cell line Caco-2 as an in vitro model. Caco-2 cells were incubated for 60 min in a buffer supplemented with non-heme iron (as sulfate) and calcium to achieve calcium to iron molar ratios ranging from 50:1 to 1,000:1. The uptake, efflux, and net absorption of non-heme iron were calculated by following a radioisotope tracer of ⁵⁵Fe that had been added to the buffer. Administration of calcium and iron at molar ratios between 500 and 1,000:1 increased the uptake of non-heme iron and decreased efflux. Calcium did not have an effect on the net absorption of non-heme iron. At typical supplementary doses for calcium and non-heme iron, calcium may not have an effect on the absorption of non-heme iron. The effect of higher calcium to iron molar ratios on the efflux of non-heme iron may be large enough to explain results from human studies.     

Effect of Gibberellin Biosynthesis Inhibitors on Native Gibberellin Content, Growth and Floral Initiation in Sorghum bicolor

CCC, uniconazol, ancymidol, prohexadione-calcium (BX-112), and CGA 163′935, which represent three groups of gibberellin (GA) biosynthesis inhibitors, were applied as a soil drench to Sorghum bicolor cultivars 58M (phyB-1, phytochrome B-deficient mutant) and 90M (phyB-2, equivalent phenotypically to wild type, PHYB, except for small differences in flowering dates). The inhibitors that block steps before GA₁₂ (CCC, uniconazol, and ancymidol) lowered the concentrations of all endogenous early-C13α-hydroxylation pathway GAs found in sorghum: GA₁₂, GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, and GA₈. In contrast, the inhibitors that block the conversion of GA₂₀→ GA₁, (CGA 163′935 and BX-112) drastically reduced GA₁ and GA₈ levels, but they either did not change or caused accumulation of intermediates from GA₁₂ to GA₂₀. Combinations of pre-GA₁₂ inhibitors and GA₃ plus GA₁ strongly reduced GAs other than GA₁ and GA₃. Each of these compounds inhibited shoot growth in both cultivars and delayed floral initiation in 90M. Floral initiation of 58M was also delayed by CCC, uniconazol, and ancymidol but not by CGA 163`935 and BX-112. This separation of shoot elongation from floral initiation in sorghum is novel. Both inhibition of shoot growth and delayed floral initiation were almost completely relieved by a mixture of GA₃ and GA₁ in both 58M and 90M. This observation, plus the much lower levels of endogenous GA₃ than of GA₁ observed in these experiments, implies that GA₁ is the major endogenous GA active in shoot elongation. CGA 163′935 and BX-112 also failed to promote tillering in 58M, whereas inhibitors active before GA₁₂ did so. The possibility that the GA₂₀→ GA₁ inhibitors fail to block flowering and promote tillering in 58M because biosynthetic intermediates between GA₁₂ and GA₂₀ accumulate and/or because 58M is altered in GA metabolism in this same region of the biosynthetic pathway is discussed. Received April 7, 1998; accepted July 31, 1998     

Uptake and Active Efflux of Polycyclic Aromatic Hydrocarbons by Pseudomonas fluorescens LP6a

The mechanism of transport of polycyclic aromatic hydrocarbons (PAHs) by Pseudomonas fluorescens LP6a, a PAH-degrading bacterium, was studied by inhibiting membrane transport and measuring the resulting change in cellular uptake. Three cultures were used: wild-type LP6a which carried a plasmid for PAH degradation, a transposon mutant lacking the first enzyme in the pathway for PAH degradation, and a cured strain without the plasmid. Washed cells were mixed with aqueous solutions of radiolabelled PAH; then the cells were removed by centrifugation, and the concentrations of PAH in the supernatant and the cell pellet were measured. The change in the pellet and supernatant concentrations after inhibitors of membrane transport (azide, cyanide, or carbonyl cyanide m-chlorophenyl hydrazone) were added indicated the role of active transport. The data were consistent with the presence of two conflicting transport mechanisms: uptake by passive diffusion and an energy-driven efflux system to transport PAHs out of the cell. The efflux mechanism was chromosomally encoded. Under the test conditions used, neither uptake nor efflux of phenanthrene by P. fluorescens LP6a was saturated. The efflux mechanism showed selectivity since phenanthrene, anthracene, and fluoranthene were transported out of the cell but naphthalene was not.     

Uptake of Gibberellin Methyl Esters by Spores and Induction of Dark Germination in Lygodium japonicum

In the fern Lygodium japonicum, the effect of the exogenousapplication of two gibberellin methyl esters, gibberellin A₄methyl ester (GA₄Me) and gibberellin A₂₀ methyl ester (GA₂₀Me)on spore germination in the dark and uptake of GA₄Me and GA₂₀Meby spores was investigated. Tritiated GA₄Me and GA₂₀Me wereprepared and used as radioactive tracers. The activity of GA₄Mewas more than 100-fold that of GA₂₀Me for the induction of sporegermination. When treated for 24 h, the activity for inducingspore germination remained after removal of the gibberellinmethyl esters from the medium. The amount of GA₄Me taken upby spores was more than three times that of GA₂₀Me throughoutthe 24 h time course of treatment. The uptake of both gibberellinmethyl esters was proportional to the external concentrationfor the range of concentrations between 10^–9 M and 10^–6M. When treated with the tritiated gibberellin methyl estersat 10^–6 M and 10^–7 M for 24 h, most of the gibberellinmethyl esters taken up by the spores were not metabolized. Althoughthe uptake of the two gibberellin methyl esters differed by3- to 5- fold, their abilities to induce spore germination differedby more than 100-fold. Therefore, the difference in the activityof the two gibberellin methyl esters regarding the inductionof spore germination could not be explained solely by the differencein their uptake. (Received January 11, 1988; Accepted May 26, 1988)     

Effect of Gibberellin on Mature Euonymus europaeus L. Seeds

The release from dormancy of Euonvmus europaeus L embryos bya brief treatment with GA₃ has been studied During 48 h incubationof dormant embryos in GA-free medium, phospholipid levels increasedat first, then declined sharply over the last 6 h When the embryoswere placed in GA₃ medium during this 6 h period levels of totalphospholipids as well as of phosphatidylethanolamine increasedwhilst phosphatidylinositol and phosphatidylcholine declinedslightly Fine structural changes stimulated by a brief GA₃ treatmentwere of different character depending on tissue region (1) ‘destructive’changes occurred in the superficial procortical parenchyma onthe hypocotyl/radicle boundary, involving autolysis and decompartmentationof organelles, (2) ‘positive’ changes occurred inregions close to root and shoot apical meristems, involvingdegradation of protein bodies and their conversion into vacuoles,and the proliferation of various organelles A number of differenceswere noted when the changes in GA₃-treated embryos were comparedwith those induced by low temperature, which also overcomesdormancy The results suggest that germination is accompaniedby different cytological events depending on whether it is inducedby cold or GA₃ The growth of embryos in which dormancy was overcomeby GA3 was due to the activation of the apical root meristemclose to the quiescent centre, whilst in embryos in which germinationwas induced by low temperature, the periphery of hypotocotyl/radicleboundary was the site of activation Euonymus europaeus L, dormant embryo, fine structure, phospholipids, GA₃ and cold treatments     

Effect of Cycocel Derivatives and Gibberellin on Choline Kinase and Choline Metabolism

Cycocel stimulated the activity of partial purified choline kinase from spinach or squash leaves, but it inhibited the activity of yeast choline kinase. The activity of different Cycocel analogs on plant growth corresponded to their stimulatory effect on the isolated choline kinase. Cycocel had no effect upon the activity of a plant phosphatase which hydrolyzed phosphorylcholine nor upon adenosine triphosphatase from wheat roots or leaves.Gibberellin A(3) inhibited choline kinase activity and reversed the stimulatory effect of Cycocel on the kinase.Total choline kinase activity per squash plant was not greatly increased by Cycocel treatment. However, on the basis of fresh weight, total kinase activity was increased by Cycocel treatment. Gibberellin A(3) partially reversed these increases. Treatment with Cycocel plus indoleacetic acid resulted in a large increase in choline kinase activity.The same distribution of tracer among phosphorylcholine, choline and betaine was observed when either phosphorylcholine-C(14) or choline-C(14) was fed to barley or wheat roots. Cycocel stimulated the incorporation of choline-C(14) into the insoluble fraction and into lipids. Cycocel inhibited phosphorylcholine uptake by roots.Thus Cycocel stimulated choline kinase activity and the utilization of choline-C(14). The effect of Cycocel upon kinase activity in vivo and in vitro was reversed by gibberellin A(3).