The Electro-osmotic Theory of Phloem Transport in the Light of Recent Measurements on Heracleum Phloem

The bearing, on the electro-osmotic theory, of work on the Onsagercoefficients for Heracleum phloem, currently reported by Tyreeand Fensom (1970), is briefly discussed. As an aid to the discussionthe Onsager resistance coefficients are evaluated in terms ofSpiegler's frictional model. It is concluded that the experimentalevidence, while it does not support the theory, does not invalidateit. Some further suggestions regarding the electro-osmotic theoryare put forward.     

Selective knockdown of AT1 receptors by RNA interference inhibits Val5-ANG II endocytosis and NHE-3 expression in immortalized rabbit proximal tubule cells

Receptor-mediated endocytosis of extracellular ANG II has been suggested to play an important role in the regulation of proximal tubule cell (PTC) function. Using immortalized rabbit PTCs as an in vitro cell culture model, we tested the hypothesis that extracellular ANG II is taken up by PTCs through angiotensin type 1 receptor (AT₁; or AT_1a) receptor-mediated endocytosis and that inhibition of ANG II endocytosis using a selective AT₁ receptor small-interfering RNA (siRNA; AT₁R siRNA) or endocytotic inhibitors exerts a physiological effect on total and apical sodium and hydrogen exchanger isoform 3 (NHE-3) protein abundance. Western blots and live cell imaging with FITC-labeled ANG II confirmed that transfection of PTCs with a human specific AT₁R siRNA for 48 h selectively knocked down AT₁ receptor protein by 76 ± 5% (P < 0.01), whereas transfection with a scrambled siRNA had little effect. In nontransfected PTCs, exposure to extracellular ANG II (1 nM) for 60 min at 37°C increased intracellular ANG II accumulation by 67% (control: 566 ± 55 vs. ANG II: 943 ± 160 pg/mg protein, P < 0.05) and induced mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (163 ± 15% of control, P < 0.01). AT₁R siRNA reduced ANG II endocytosis to a level similar to losartan, which blocks cell surface AT₁ receptors (557 ± 37 pg/mg protein, P < 0.05 vs. ANG II), or to colchicine, which disrupts cytoskeleton microtubules (613 ± 12 pg/mg protein, P < 0.05 vs. ANG II). AT₁R siRNA, losartan, and colchicine all attenuated ANG II-induced ERK1/2 activation and total cell lysate and apical membrane NHE-3 abundance. The scrambled siRNA had no effect on ANG II endocytosis, ERK1/2 activation, or NHE-3 expression. These results suggest that AT₁ receptor-mediated endocytosis of extracellular ANG II may regulate proximal tubule sodium transport by increasing total and apical NHE-3 proteins. extracellular signal-regulated kinase 1/2; kidney; sodium transport; receptor internalization; ribonucleic acid interference     

N-methyl-D-glucamine and propidium dyes utilize different permeation pathways at rat P2X(7) receptors

Activation of membrane P2X₇ receptors by extracellular ATP [or its analog 2',3'-O-(4-benzoylbenzoyl)-ATP] results in the opening within several milliseconds of an integral ion channel that is permeable to small cations. If the ATP application is maintained for several seconds, two further sequelae occur: there is a gradual increase in permeability to the larger cation N-methyl-D-glucamine and the cationic propidium dye quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide (YO-PRO-1) enters the cell. The similarity in the time course of these two events has led to the widespread view that N-methyl-D-glucamine and YO-PRO-1 enter through a common permeation pathway, the "dilating" P2X₇ receptor pore. Here we provide two independent lines of evidence against this view. We studied single human embryonic kidney cells expressing rat P2X₇ receptors with patch-clamp recordings of membrane current and with fluorescence measurements of YO-PRO-1 uptake. First, we found that maintained application of the ATP analog did not cause any increase in N-methyl-D-glucamine permeability when the extracellular solution contained its normal sodium concentration, although YO-PRO-1 uptake was readily observed. Second, we deleted a cysteine-rich 18-amino acid segment in the intracellular juxtamembrane region of the P2X₇ receptor. This mutated receptor showed normal YO-PRO-1 uptake but had no permeability to N-methyl-D-glucamine. Together, the clear differential effects of extracellular sodium ions or of mutation of the receptor strongly suggest that N-methyl-D-glucamine and YO-PRO-1 do not enter the cell by the same permeation pathway. ATP; cation channel; permeability; quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide     

Permeability of human granulocytes to dimethyl sulfoxide

The permeability of the membrane of human granulocytes to the permeating solute dimethyl sulfoxide (DMSO) was studied using the Onsager form of the phenomenological equations derived from the theory of irreversible thermodynamics. Changes in cellular volume were monitored with an electronic particle counter as samples of that population were introduced into hypertonic osmotica. Temperature and concentration sensitivity analyses of the permeability coefficients were carried out. It is shown that the introduction of the Onsager formalism allows further insight into the observed transport phenomena. It was found that DMSO may affect the water permeability properties of the membrane for that population of cells.     

Purification and Characterization of 4,5-Dioxovalerate Reductase (NADPH) from Chlorella regularis

Two types of 4,5-dioxovalerate reductases (NADPH) were partiallypurified and characterized from green alga, Chlorella regularis.The enzyme was separated by DEAE-Sephacel chromatography intotwo peaks: type I (first peak) and type II (second peak). Theactivity ratio of the type II to type I enzyme varied between5 to 7 with a starting cell material. Both enzymes had the samepH optimum at 6.0 and pI value of 4.9. The molecular weightestimated by gel filtration was 33,000 for type I and 99,000for type II enzyme. Both enzymes used only NADPH, but were notspecific for 4,5-dioxovaleric acid (DOVA). Type I enzyme reducedglyoxylate 68-fold faster than DOVA, whereas type II enzymeacted more specifically on a variety of aldehydes than DOVA.It is suggested that these enzymes may not function primarilyas NADPH-DOVA reductases in the metabolic pathway of DOVA. (Received June 15, 1985; Accepted October 14, 1985)     

Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein-associated O-GlcNAc

Increased levels of protein O-linked N-acetylglucosamine (O-GlcNAc) have been shown to increase cell survival following stress. Therefore, the goal of this study was to determine whether in isolated neonatal rat ventricular myocytes (NRVMs) an increase in protein O-GlcNAcylation resulted in improved survival and viability following ischemia-reperfusion (I/R). NRVMs were exposed to 4 h of ischemia and 16 h of reperfusion, and cell viability, necrosis, apoptosis, and O-GlcNAc levels were assessed. Treatment of cells with glucosamine, hyperglycemia, or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate(PUGNAc), an inhibitor of O-GlcNAcase, significantly increased O-GlcNAc levels and improved cell viability, as well as reducing both necrosis and apoptosis compared with untreated cells following I/R. Alloxan, an inhibitor of O-GlcNAc transferase, markedly reduced O-GlcNAc levels and exacerbated I/R injury. The improved survival with hyperglycemia was attenuated by azaserine, which inhibits glucose metabolism via the hexosamine biosynthesis pathway. Reperfusion in the absence of glucose reduced O-GlcNAc levels on reperfusion compared with normal glucose conditions and decreased cell viability. O-GlcNAc levels significantly correlated with cell viability during reperfusion. The effects of glucosamine and PUGNAc on cellular viability were associated with reduced calcineurin activation as measured by translocation of nuclear factor of activated T cells, suggesting that increased O-GlcNAc levels may attenuate I/R induced increase in cytosolic Ca²⁺. These data support the concept that activation of metabolic pathways leading to an increase in O-GlcNAc levels is an endogenous stress-activated response and that augmentation of this response improves cell survival. Thus strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection. hexosamine biosynthesis; calcium; protein O-glycosylation     

Ubiquitination regulates the plasma membrane expression of renal UT-A urea transporters

The renal UT-A urea transporters UT-A1, UT-A2, and UT-A3 are known to play an important role in the urinary concentrating mechanism. The control of the cellular localization of UT-A transporters is therefore vital to overall renal function. In the present study, we have investigated the effect of ubiquitination on UT-A plasma membrane expression in Madin-Darby canine kidney (MDCK) cell lines expressing each of the three renal UT-A transporters. Inhibition of the ubiquitin-proteasome pathway caused an increase in basal transepithelial urea flux across MDCK-rat (r)UT-A1 and MDCK-mouse (m)UT-A2 monolayers (P < 0.01, n = 3, ANOVA) and also increased dimethyl urea-sensitive, arginine vasopressin-stimulated urea flux (P < 0.05, n = 3, ANOVA). Inhibition of the ubiquitin-proteasome pathway also increased basolateral urea flux in MDCK-mUT-A3 monolayers (P < 0.01, n = 4, ANOVA) in a concentration-dependent manner. These increases in urea flux corresponded to a significant increase in UT-A transporter expression in the plasma membrane (P < 0.05, n = 3, ANOVA). Further analysis of the MDCK-mUT-A3 cell line confirmed that vasopressin specifically increased UT-A3 expression in the plasma membrane (P < 0.05, n = 3, ANOVA). However, preliminary data suggested that vasopressin produces this effect through an alternative route to that of the ubiquitin-proteasome pathway. In conclusion, our study suggests that ubiquitination regulates the plasma membrane expression of all three major UT-A urea transporters, but that this is not the mechanism primarily used by vasopressin to produce its physiological effects. ubiquitin-proteasome pathway; urea transport; membrane localization     

Some Experimental and Theoretical Observations Concerning Mass Flow in the Vascular Bundles of Heracleum

The theory and practice of applying the thermodynamics of irreversibleprocesses to mass-flow theories is presented. Onsager coefficientswere measured on cut and uncut phloem and cut xylem strandsof Heracleum muntegazzimum. In 0.3 N sucrose + 1 mN KC1 theyare as follows. In phloem, L_EE = 5 ? 10–⁴ mho cm^–1,L_pE = 9 ? 10^–6 cm³ s^–1 cm^–2 volt^–1 cm,and L_PP = 0.16 cm³ s^–1 cm^–2 (J cm^–3)^–1cm. In uncut phloem strands L_EE is about 1 ? 10^–3 mhocm^–1. In xylem in 2 x 10^–3 N KCI, L_pp = 50 to 225,L_PE = 2 ? 10^–4, and L^EE = 4 ? 10^–3. The measurementsare tentative since the blockage of the sieve plates is an interferingfactor, but if they are valid they lead to the conclusion thatneither a pressure-flow nor an electro-kinetic mechanism envisaginga ‘long distance’ current pathway can be the majormotive ‘force’ for transport in mature phloem. Measurementsof biopotentials along conducting but laterally detached phloembundles of Heracleum suggest, nevertheless, that there may bea small electro-osmotic component of at least 0.1 mV cm^–1endogenous in the phloem.     

The Electrical Resistance and Capacitance of the Membranes of Nitella translucens

The resistance and capacitance of the membranes of Nitella translucenshave been measured by direct current and alternating currentmethods. Current of the order of 10^-7 amp. was injected intothe cell by means of a conventional Ag, AgCl_-3N KCl glass microelectrodeinserted into the vacuole of the cell. The change of potentialacross the membrane was recorded by two other internal microelectrodeswhich had been inserted into the cell at known distances fromthe current-injecting electrode. In the direct-current experimentsthe input current was in the form of a square pulse, while sinusoidalcurrents of frequency 25 cycles per second were used in thealternating current experiments. The cell was treated as a shortlength of coaxial cable and from the measurements the followingparameters could be obtained: the space constant (), the membraneresistance (R_m) and the membrane capacitance (C_m). The valuesof R_m ranged from 6.7 to 36 K ohm cm.² (mean of 21.4 K ohm cm.²)and those of ranged from 1.5 to 5.7 cm. (mean of 2.6 cm.).The capacitance value was about I µF cm.^-2 These results are discussed within the framework of our knowledgeof these parameters for other cells, particularly plant cells.The measured electrical resistance is shown to be at least tentimes less than the value estimated from the passive fluxesof the principal ions K, Na, and Cl. It is suggested that thisdiscrepancy, which is usually attributed to non-independentmovement of these ions, could be partially explained on electro-osmoticgrounds. The value of the capacitance is very close to thatwhich is usually obtained for other cell membranes. One exceptionallylow value for Nitella has been quoted in the literature. Thereason for the gross error in this particular measurement isgiven.     

Respiration and Mitochondrial Activity in Zea mays Roots As Affected by Osmotic Stress

Studies were made of metabolism in highly vacuolated and slightlyvacuolated Zea mays root tissue both during and after plasmolysis. Plasmolysis resulted in decreased respiration and carbon dioxideevolution from glucose and an increased sucrose synthesis. Inhibitionof respiration during plasmolysis in both the highly vacuolatedand slightly vacuolated tissue was not relieved by supply ofglucose, organic acids, or uncouplers of oxidative phosphorylation.Mitochondria isolated from plasmolysed tissue were tightly coupled,but activity in vitro was inhibited by exposure to a high negativeosmotic potential. It is suggested that low TCA cycle activityin vivo must be due either to inhibition of mitochondrial activityor to reduced flow of carbon through the glycolytic pathway. A low potential for TCA cycle activity after deplasmolysis issuggested, as addition of pyruvate stimulated carbon dioxideevolution but not oxygen uptake, which was severely decreased.This was presumably due to severe mitochondrial damage as shownby their activity in vitro. However, it is not clear whetherrespiration in vivo is rate limited by rapid leakage of metabolicintermediate (reported earlier) or by lysis of mitochondria. Deplasmolysis did not damage mitochondria from slightly vacuolatedtissue, a result which was consistent with respiratory measurementsmade in vivo. The data show that mitochondria in vacuolated tissue are damagedduring and after deplasmolysis and not before. It is suggestedthat lysis of mitochondria occurs in vivo as a result of a sharpincrease in the osmotic potential of the cell fluids.     

The Reciprocal Transfer of Radiocarbon between a Developing Tiller and its Parent Shoot in Vegetative Plants of Lolium multiflorum Lam.

The transfer of ¹⁴C-labelled assimilates between a tiller andits parent shoot was examined in young plants of Lolium multiflorumLam. Radiocarbon was exported freely from an expanded laminato sinks within the shoot axis from which it originated andto the root system. Lesser amounts of radiocarbon were exportedto the other shoot. It is suggested that the reciprocal exchangeof radiocarbon between tiller and main shoot occurred principallyvia a direct pathway through stem tissues rather than via apathway involving the roots.     

Quantification of annexin I in subcellular fractions of human neutrophils reveals an exclusive cytosolic localisation

Annexin I is an abundant cytosolic protein in human neutrophils. Besides its intracellular location, annexin I is found as an extracellular protein and the pathway for secretion has been of interest since the protein lacks a signal sequence for secretion. It was recently shown that annexin I is stored in the secretory gelatinase granules of human neutrophils, suggesting that the protein might be released through a granule mobilisation and fusion process resembling classical secretion. In this study we have determined the intracellular localisation of annexin I in human neutrophils using subcellular fractionation, protein separation by SDS-PAGE and immunoblotting, and show that virtually all annexin I is localised in the cell cytosol.     

Cellular and Extracellular Polysaccharides of the Blue green Alga Nostoc

The carbohydrate content of various cellular fractions of theblue-green alga Nostoc was studied as a function of age of theculture. The production of extracellular and intracellular polysaccharideswas higher in actively growing cultures. Mannose and glucosewere the main components of cell wall polysaccharides. Glucosamineand diaminopimelic acid were also detected in the cell walls.The kinetics of incorporation of radioactivity from sodium [¹⁴C]bicarbonate showed that the extracellular polysaccharides werelabelled within 10 min of incubation suggesting the active exudationof polysaccharides by this alga. The selective excretion ofpolysaccharides by the alga Nostoc is also suggested.     

Studies on the Growth in Culture of Plant Cells: XI. THE INFLUENCE OF SHAKING RATE ON THE GROWTH OF SUSPENSION CULTUEES

The influence of shaking rates (expressed as revolutions permin) on orbital shaking platforms (1 in (2.54 cm) diam. rotarymotion) on the growth of cell suspension cultures of Acer pseudoplatanusL. and Atropa belladonna cultivar lutea Döll are described.By following cell growth and respiration and the levels of oxygenand carbon dioxide in the media during the progress of incubationit is concluded that the reduction of growth at sub-optimalshaking rates is not due to oxygen deficiency or toxic accumulationof carbon dioxide. The growth of the Atropa cell suspensionin ‘closed systems’ has been studied by the developmentof modified culture vessels and evidence obtained that the reducedgrowth in the systems is due to the formation by the culturesof an unidentified volatile growth inhibitor and not to eitheroxygen depletion or toxic accumulation of either carbon dioxideor ethylene. It is suggested that the reduced growth in ‘opensystems’ cultures at sub-optimal shaking speeds is eitherdue to retention of this volatile inhibitor or to restrictionof nutrient uptake by the existence of a stationary liquid-phaseboundary to the cells.     

Arachidonic acid induces an increase of β-1,4-galactosyltransferase I expression in MDA-MB-231 breast cancer cells

Arachidonic acid (AA) is a common dietary n‐6 cis polyunsaturated fatty acid that under physiological conditions is present in an esterified form in cell membrane phospholipids, and it might be present in the extracellular microenvironment. AA and its metabolites are implicated in FAK activation and cell migration in MDA‐MB‐231 breast cancer cells, and an epithelial‐to‐mesenchymal‐like transition process in mammary non‐tumorigenic epithelial cells MCF10A. During malignant transformation is present an altered expression of glycosiltransferases, which promote changes on the glycosilation of cell‐surface proteins. The β‐1,4‐galactosyltransferase I (GalT I) is an enzyme that participates in a variety of biological functions including cell growth, migration, and spreading. However, the participation of AA in the regulation of GalT I expression and the role of this enzyme in the cell adhesion process in breast cancer cells remains to be investigated. In the present study, we demonstrate that AA induces an increase of GalT I expression through a PLA2α, Src, ERK1/2, and LOXs activities‐dependent pathway in MDA‐MB‐231 breast cancer cells. Moreover, MDA‐MB‐231 cells adhere to laminin via GalT I expression and pretreatment of cells with AA induces an increase of cell adhesion to laminin. In conclusion, our findings demonstrate, for the first time, that AA promotes an increase of GalT I expression through an AA metabolism, Src and ERK1/2 activities‐dependent pathway, and that GalT I plays a pivotal role in cell adhesion to laminin in MDA‐MB‐231 breast cancer cells. J. Cell. Biochem. 113: 3330–3341, 2012. © 2012 Wiley Periodicals, Inc.     

The Cellular Pathway of Short-distance Transfer of Photosynthates and Potassium in the Elongating Stem of Phaseolus vulgaris L. A Structural Assessment

The potential cellular pathway of radial transfer of photosynthateand potassium delivered in the phloem to the elongation zone(apical 0.5–2.5 cm) of internode 2 ofPhaseolus vulgarisL. seedlings was elucidated. This was achieved using ultrastructuralobservations of the cell types that constitute the radial pathwayand estimates of potential sucrose and potassium fluxes throughthe cross-sectional area of interconnecting plasmodesmata andacross the plasma membrane surface areas of selected cell types.The investigation relied on predicting the relative roles ofthe mature and developing sieve elements as conduits for theaxial delivery of solutes to the elongation zone. In turn, thesepredictions led to formulation of two transport models whichwere subsequently evaluated. It was found that unloading ofsucrose and potassium from the protophloem sieve elements cannotbe through the symplast due to the absence of plasmodesmata.On the other hand, mature metaphloem sieve element-companioncell complexes have the potential capacity to unload eitherthrough the stem symplast or apoplast. The potential symplasticroute is proposed to be via the companion cells to the adjacentlarge phloem parenchyma cells. Continued radial transfer couldoccur either by exchange to the stem apoplast from the largephloem parenchyma cells or continue in the symplast to the groundtissues. It was further predicted that sucrose utilized forthe development of the procambial/small phloem parenchyma cellscould be delivered axially by them and not by the mature sieveelements. Phaseolus vulgaris ; apoplast; elongating stem; photosynthates; potassium; transport; symplast     

Riboflavin uptake by human-derived colonic epithelial NCM460 cells

Normal microflora ofthe large intestine synthesize a number of water-soluble vitaminsincluding riboflavin (RF). Recent studies have shown that colonicepithelial cells posses an efficient carrier-mediated mechanism forabsorbing some of these micronutrients. The aim of the present studywas to determine whether colonic cells also posses a carrier-mediatedmechanism for RF uptake and, if so, to characterize this mechanism andstudy its cellular regulation. Confluent monolayers of thehuman-derived nontransformed colonic epithelial cells NCM460 and[³H]RF were used in the study. Uptake of RF wasfound to be 1) appreciable and temperature and energydependent; 2) Na⁺ independent; 3) saturableas a function of concentration with an apparent K_mof 0.14 µM and V_max of 3.29 pmol · mgprotein¹ · 3 min¹; 4) inhibited by the structural analogslumiflavin and lumichrome (K_i of 1.8 and 14.1 µM,respectively) but not by the unrelated biotin; 5) inhibited ina competitive manner by the membrane transport inhibitor amiloride(K_i = 0.86 mM) but not by furosemide, DIDS, orprobenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RFuptake in RF-deficient and oversupplemented conditions, respectively;and 7) modulated by an intracellularCa²⁺/calmodulin-mediated pathway. These studies demonstratefor the first time the existence of a specialized carrier-mediatedmechanism for RF uptake in an in vitro cellular model system of humancolonocytes. This mechanism appears to be regulated by extracellularsubstrate level and by an intracellularCa²⁺/calmodulin-mediated pathway. It is suggested that theidentified transport system may be involved in the absorption ofbacterially synthesized RF in the large intestine and that this sourceof RF may contribute toward RF homeostasis, especially that of colonocytes.

     

Hypoxia induces apoptosis via two independent pathways in Jurkat cells: differential regulation by glucose

Glucose uptakeand metabolism inhibit hypoxia-induced apoptosis in a varietyof cell types, but the underlying molecular mechanisms remain poorlyunderstood. In the present study, we explore hypoxia-mediated celldeath pathways in Jurkat cells in the presence and absence ofextracellular glucose. In the absence of extracellular glucose, hypoxiacaused cytochrome c release, caspase 3 andpoly(ADP-ribose)polymerase cleavage, and DNA fragmentation; thisapoptotic response was blocked by the caspase 9 inhibitorz-LEHD-FMK. The presence of extracellular glucose during hypoxiaprevented cytochrome c release and activation of caspase 9 but did not prevent apoptosis in Jurkat cells. In theseconditions, overexpression of the caspase 8 inhibitor v-FLIP preventedhypoxia-mediated cell death. Thus hypoxia can stimulate twoapoptotic pathways in Jurkat cells, one dependent on cytochrome c release from mitochondria that is prevented by glucoseuptake and metabolism, and the other independent of cytochromec release and resulting from activation of the deathreceptor pathway, which is accelerated by glucose uptake and metabolism.

     

Pathway of Photosynthate Transfer in the Developing Seed of Vicia faba L: A Structural Assessment of the Role of Transfer Cells in Unloading from the Seed Coat

Photosynthate movement within the coat of the developing seedof Vicia faba occurs radially inward from the restricted vascularsystem and laterally through the non-vascularized region ofthe seed coat prior to exchange to the seed apoplast. Thin-walledparenchyma/transfer cells line the entire inner surface of theseed coat and thus are located at the terminus of the photosynthatetransfer pathway. The principal cellular route of transfer withinthe seed coat and the role of the thin-walled parenchyma/transfercells in membrane exchange to the seed apoplast has been investigated.Sucrose fluxes, computed from estimates of the plasma membranesurface areas of the cell types of the pathway, the plasmodesmatalcross-sectional areas interconnecting contiguous cells and theobserved rate of sucrose delivery to the embryo indicate thatsieve element unloading and subsequent transfer to the thin-walledparenchyma/transfer cells is through the symplast. For the cellsof the ground tissue, plasmodesmatal density is consistentlyhigher on their anticlinal walls. This observation supportsthe reported pattern of lateral transfer through these tissuesin the non-vascular regions of the seed coat. Wall ingrowthsare initiated sequentially in the thin-walled parenchyma cellsto maintain 1–3 rows of thin-walled parenchyma/transfercells. The development of these wall ingrowths results in a58% increase in the plasma membrane surface area of these cellsand provides them with the capacity to act as the principalcellular site for membrane exchange of sucrose to the seed apoplast.This cellular route of symplastic transfer from the sieve elementsto the ground tissues where membrane exchange to the seed apoplastoccurs is consistent with that reported for Phaseolus vulgaris Key words: Cellular pathway, photosynthate transfer, transfer cell, Vicia seed coat     

Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L

The sodium iodide symporter (NIS) mediates iodide (I^–) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I^–-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I^– induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I^– (35 µM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na⁺ dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I^– influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I^– influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I^– also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I^– uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I^– may be a useful tool by which to study the pathophysiology and pharmacology of NIS. thyroid; fluorescence microscopy; FRTL-5 cells