Ratiometric bimolecular beacons for the sensitive detection of RNA in single living cells

Numerous studies have utilized molecular beacons (MBs) to image RNA expression in living cells; however, there is growing evidence that the sensitivity of RNA detection is significantly hampered by their propensity to emit false-positive signals. To overcome these limitations, we have developed a new RNA imaging probe called ratiometric bimolecular beacon (RBMB), which combines functional elements of both conventional MBs and siRNA. Analogous to MBs, RBMBs elicit a fluorescent reporter signal upon hybridization to complementary RNA. In addition, an siRNA-like double-stranded domain is used to facilitate nuclear export. Accordingly, live-cell fluorescent imaging showed that RBMBs are localized predominantly in the cytoplasm, whereas MBs are sequestered into the nucleus. The retention of RBMBs within the cytoplasmic compartment led to >15-fold reduction in false-positive signals and a significantly higher signal-to-background compared with MBs. The RBMBs were also designed to possess an optically distinct reference fluorophore that remains unquenched regardless of probe confirmation. This reference dye not only provided a means to track RBMB localization, but also allowed single cell measurements of RBMB fluorescence to be corrected for variations in probe delivery. Combined, these attributes enabled RBMBs to exhibit an improved sensitivity for RNA detection in living cells.     

Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells

There have been a growing number of studies where molecular beacons (MBs) are used to image RNA expression in living cells; however, the ability to make accurate measurements can be hampered by the generation of false-positive signals resulting from non-specific interactions and/or nuclease degradation. In the present study, we found that such non-specific signals only arise in the nucleus of living cells. When MBs are retained in the cytoplasmic compartment, by linking them to quantum dots (QDs), false-positive signals are reduced to marginal levels. Consequently, MB–QD conjugates were used to measure the expression of the endogenous proto-oncogene c-myc in MCF-7 breast cancer cells by quantifying the total fluorescent signal emanating from individual cells. Upon the addition of tamoxifen, measurements of MB fluorescence indicated a 71% reduction in c-myc expression, which correlated well with RT-PCR measurements. Variations in MB fluorescence resulting from instrumental fluctuations were accounted for by imaging fluorescent calibration standards on a daily basis. Further, it was established that measurements of the total fluorescent signal were not sensitive to the focal plane. Overall, these results provide evidence that accurate measurements of RNA levels can be made when MBs are retained in the cytoplasm.     

Microporation is a valuable transfection method for efficient gene delivery into human umbilical cord blood-derived mesenchymal stem cells

Background

Mesenchymal stem cells (MSCs) are an attractive source of adult stem cells for therapeutic application in clinical study. Genetic modification of MSCs with beneficial genes makes them more effective for therapeutic use. However, it is difficult to transduce genes into MSCs by common transfection methods, especially nonviral methods. In this study, we applied microporation technology as a novel electroporation technique to introduce enhanced green fluorescent protein (EGFP) and brain-derived neurotropfic factor (BDNF) plasmid DNA into human umbilical cord blood-derived MSCs (hUCB-MSCs) with significant efficiency, and investigated the stem cell potentiality of engineered MSCs through their phenotypes, proliferative capacity, ability to differentiate into multiple lineages, and migration ability towards malignant glioma cells.

Results

Using microporation with EGFP as a reporter gene, hUCB-MSCs were transfected with higher efficiency (83%) and only minimal cell damage than when conventional liposome-based reagent (<20%) or established electroporation methods were used (30-40%). More importantly, microporation did not affect the immunophenotype of hUCB-MSCs, their proliferation activity, ability to differentiate into mesodermal and ectodermal lineages, or migration ability towards cancer cells. In addition, the BDNF gene could be successfully transfected into hUCB-MSCs, and BDNF expression remained fairly constant for the first 2 weeks in vitro and in vivo. Moreover, microporation of BDNF gene into hUCB-MSCs promoted their in vitro differentiation into neural cells.

Conclusion

Taken together, the present data demonstrates the value of microporation as an efficient means of transfection of MSCs without changing their multiple properties. Gene delivery by microporation may enhance the feasibility of transgenic stem cell therapy.     

Sub-cellular trafficking and functionality of 2′-O-methyl and 2′-O-methyl-phosphorothioate molecular beacons

Molecular beacons (MBs) have shown great potential for the imaging of RNAs within single living cells; however, the ability to perform accurate measurements of RNA expression can be hampered by false-positives resulting from nonspecific interactions and/or nuclease degradation. These false-positives could potentially be avoided by introducing chemically modified oligonucleotides into the MB design. In this study, fluorescence microscopy experiments were performed to elucidate the subcellular trafficking, false-positive signal generation, and functionality of 2′-O-methyl (2Me) and 2′-O-methyl-phosphorothioate (2MePS) MBs. The 2Me MBs exhibited rapid nuclear sequestration and a gradual increase in fluorescence over time, with nearly 50% of the MBs being opened nonspecifically within 24 h. In contrast, the 2MePS MBs elicited an instantaneous increase in false-positives, corresponding to ∼5–10% of the MBs being open, but little increase was observed over the next 24 h. Moreover, trafficking to the nucleus was slower. After 24 h, both MBs were localized in the nucleus and lysosomal compartments, but only the 2MePS MBs were still functional. When the MBs were retained in the cytoplasm, via conjugation to NeutrAvidin, a significant reduction in false-positives and improvement in functionality was observed. Overall, these results have significant implications for the design and applications of MBs for intracellular RNA measurement.     

Characterization of a dextran-based bifunctional calcium indicator immobilized in cells by the enzymatic addition of isoprenoid lipids

Cellular processes can be controlled by cell-wide increases in the cytosolic Ca2+ concentration or, alternatively, by localized Ca2+ signals in micro- and nano-domains. The experimental characterization of such localized Ca2+ signals would be facilitated using an immobilized Ca2+ indicator, which could prevent the accelerated spatial spreading of Ca2+ ions that is mediated by binding to diffusible indicators. Here we characterize a dextran-based Ca2+ indicator (CAAX-green) that becomes immobilized in the cytosol by an enzyme-mediated addition of a geranylgeranyl lipid group. CAAX-green consists of a dextran backbone with an attached Ca(2+)-green as well as an 11 residue peptide ending in a C-terminal CAAX-motif. Once introduced into cells by microporation, geranylgeranyl lipid groups are attached to the CAAX peptides by cytosolic enzymes. Measurements in tumor mastcells, myocytes and fibroblasts showed that the indicator becomes membrane attached between 30 min and 1 h following incorporation into the cytoplasm. A time-dependent 10-fold reduction of the diffusion coefficient and a parallel increase in the cytosolic retention after permeabilization indicates that at least 90% of cellular CAAX-green is immobilized. The KD of the indicator in permeabilized cells is 0.65 microM. Overall, these properties make CAAX-green well suited for the investigation of localized Ca2+ signals in a variety of cell types.     

Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.     

Isolation of Rare Tumor Cells from Blood Cells with Buoyant Immuno-Microbubbles

Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells.     

Visualization and detection of infectious coxsackievirus replication using a combined cell culture-molecular beacon assay

Rapid detection of infectious viruses is of central importance for public health risk assessment. By directly visualizing newly synthesized viral RNA with molecular beacons (MBs), we have developed a generalized method for the rapid and sensitive detection of infectious viruses from cell culture. An MB, CVB1, specifically targeting the 5' noncoding region of the enterovirus genome was designed and synthesized. Introduction of MB CVB1 into permeabilized cells highly infected with coxsackievirus B6 resulted in brightly fluorescent cells that can be easily visualized with a fluorescence microscope. In contrast, no detectable signal was observed with noninfected cells or with nonspecific MBs. The number of fluorescent cells also increased in a dose-responsive manner, enabling the direct quantification of infectious viral dosages by direct counting of fluorescent foci. As little as 1 PFU of infectious coxsackievirus B6 was detected within 6 h postinfection. When combined with nuclease-resistant MBs, this method could be useful not only for the real-time detection of infectious viruses but is also useful to study the life cycle of viral processing in vivo.     

Visualization and Detection of Infectious Coxsackievirus Replication Using a Combined Cell Culture-Molecular Beacon Assay

Rapid detection of infectious viruses is of central importance for public health risk assessment. By directly visualizing newly synthesized viral RNA with molecular beacons (MBs), we have developed a generalized method for the rapid and sensitive detection of infectious viruses from cell culture. An MB, CVB1, specifically targeting the 5′ noncoding region of the enterovirus genome was designed and synthesized. Introduction of MB CVB1 into permeabilized cells highly infected with coxsackievirus B6 resulted in brightly fluorescent cells that can be easily visualized with a fluorescence microscope. In contrast, no detectable signal was observed with noninfected cells or with nonspecific MBs. The number of fluorescent cells also increased in a dose-responsive manner, enabling the direct quantification of infectious viral dosages by direct counting of fluorescent foci. As little as 1 PFU of infectious coxsackievirus B6 was detected within 6 h postinfection. When combined with nuclease-resistant MBs, this method could be useful not only for the real-time detection of infectious viruses but is also useful to study the life cycle of viral processing in vivo.     

Gene delivery to human bone marrow mesenchymal stem cells by microporation

Electroporation has been considered one of the most efficient non-viral based methods to deliver genes regardless of frequently observed high cell mortality. In this study we used a microporation technique to optimise the delivery of plasmid DNA encoding green fluorescence protein (GFP) to human bone marrow mesenchymal stem cells (BM-MSC). Using resuspension buffer (RB) and as low as 1.5 × 10⁵ cells and 1 μg of DNA, we achieved 40% of cells expressing the transgene, with cell recovery and cell viabilities of 85% and 90%, respectively. An increase in DNA amount did not significantly increase the number of transfected cells but clearly reduced cell recovery. A face-centered composite design was used to unveil the conditions giving rise to optimal plasmid delivery efficiencies when using a sucrose based microporation buffer (SBB). The BM-MSC proliferation kinetics were mainly affected by the presence of plasmid and not due to the microporation process itself although no effect was observed on their immunophenotypic characteristics and differentiative potential. Based on the data shown herein microporation demonstrated to be a reliable and efficient method to genetically modify hard-to-transfect cells giving rise to the highest levels of cell survival reported so far along with superior gene delivery efficiencies.     

Characterization of TAT-mediated transport of detachable kinase substrates

The conjugation of peptides derived from the HIV TAT protein to membrane-impermeant molecules has gained wide acceptance as a means for intracellular delivery. Numerous studies have addressed the mechanism of uptake and kinetics of TAT translocation, but the cytosolic concentrations and bioavailability of the transported cargo have not been well-characterized. The current paper utilizes a microanalytical assay to perform quantitative single-cell measurements of the concentration and accessibility of peptide-based substrates for protein kinase B (PKB) and Ca(2+)/calmodulin-activated kinase II. The substrate peptide and TAT were conjugated through a releasable linker, either a disulfide or photolabile bond. Free substrate peptide concentrations of approximately 10(-20)-10(-18) moles were attainable in a cell when substrates were delivered utilizing these conjugates. The substrate peptides delivered as a disulfide conjugate were often present in the cytosol as several oxidized forms. Brief exposure of cells loaded with the photolabile conjugates to UVA light released free substrate peptide into the cytosol. Substrate peptide delivered by either conjugate was accessible to cytosolic kinase as demonstrated by the efficient phosphorylation of the peptide when the appropriate kinase was active. After incubation of the conjugated substrate with cells, free, kinase-accessible substrate was detectable in less than 30 min. Release of the majority of loaded substrate peptide from sequestered organelles occurred within 1 h. The utility of the photocleavable conjugates was demonstrated by measuring the activation of PKB in 3T3 cells after addition of varying concentrations of platelet-derived growth factor.     

Cell binding, uptake and cytosolic partition of HIV anti-gag phosphodiester oligonucleotides 3'-linked to cholesterol derivatives in macrophages

The purpose of this study is to evaluate the cell interactions of a new class of compounds composed of phosphodiester oligonucleotides linked to the cholesterol group at position 3, 7, or 22 of the steroid structure. The resulting conjugates were assessed for their capacity to bind, penetrate and partition in the cytoplasmic compartment of murine macrophages. The results showed that lipophilic conjugates bind to cells much faster (t(1/2) < or = 10 min) than do underivatized oligomers. Oligomers tethered to the cholesterol at positions 3 and 7 (PO-GEM-3-Chol and PO-GEM-7-Chol) interacted more efficiently with cell membranes and were better internalized than oligomers attached to the cholesterol moiety at position 22 (PO-GEM-22-Chol). The cytosolic fraction of internalized oligomers was studied by a digitonin-based membrane permeabilization method. The recovered fraction of oligomers that can freely diffuse from the cytosol was comparable for GEM-91, a phosphorothioate congener, and for PO-GEM-7-Chol (50-60% of the internalized oligomers), while that of PO-GEM-3-Chol was less (30% of the internalized oligomers) indicating a higher membrane affinity of the latter derivative as compared to the other investigated compounds. Membrane binding and cell internalization correlated well with the hydrophobicity of the conjugates as characterized by their partition coefficients in a water-octanol system. Due to their capacity of rapid binding and cytosolic partition in cells, cholesterol-derivatized oligonucleotides at position 3 or 7 of the steroid molecule appeared as good candidates for systemic delivery of anti-HIV antisense compounds.     

Mitochondrial uncoupling does not influence the stability of the intracellular signal activating plasma membrane calcium channels.

The effects of various concentrations of thapsigargin, a specific inhibitor of Ca2+-ATPase in the endoplasmic reticulum (ER) membrane, on calcium homeostasis in lymphoidal T cells (Jurkat) were investigated. Preincubation of these cells suspended in nominally calcium-free medium with 0.1 microM thapsigargin resulted in a complete release of Ca2+ from intracellular calcium stores. When the medium was supplemented with 3 mM CaCl2 the cells maintained constantly elevated level of cytosolic Ca2+. However, thapsigargin applied at lower concentration produced only a partial depletion of the stores. For example, in the cells pretreated with 1 nM thapsigargin and suspended in calcium-free medium approximately 75% of the calcium content was released from the intracellular stores. The addition of 3 mM CaCl2 to such cell suspension led to a transient increase in cytosolic calcium concentration, followed by a return to a lower steady-state. This phenomenon, related to the refilling of the ER by Ca2+, allowed to estimate the half-time for the process of cell recovery after activation of store-operated calcium channels. By this approach we have found that carbonyl cyanide m-chlorophenylhydrazone, which has been documented to inhibit calcium entry into Jurkat cells, does not influence the stability of the intracellular signal involved in the activation of store-operated calcium channels.     

Optimization of gene delivery to HEK293T cells by microporation using a central composite design methodology

Microporation is an efficient method for delivering plasmid DNA molecules into cultured cells. Herein, we present the optimization of gene delivery by microporation using a Central Composite Design methodology. It was given relevance not only to the transfection efficiency but also to the cell recovery. Different amounts of DNA (1 and 3 μg) mainly affected cell viabilities and cell recoveries, which decrease from 93 to 76% and from 47 to 25% respectively, when higher DNA quantity is used. With this work we suggest an easy methodology to improve transfection of mammalian cells underlining the feasibility to achieve 60% of gene delivery efficiencies whilst recovering 50% of cells, with 90% of viability.     

Quantitative analysis of protein synthesis inhibition and recovery in CRM107 immunotoxin-treated HeLa cells

Previously a mathematical model was proposed that quantitatively related protein synthesis inhibition kinetics of antitransferrin receptor-gelonin immunotoxins to the cellular trafficking of the targeting agent. That work is here extended to describe protein synthesis inhibition kinetics of immunotoxins containing the diphtheria toxin mutant CRM107. CRM107 differs from gelonin in both translocation and ribosomal inactivation mechanisms. Targeting agents used were antitransferrin monoclonal antibodies 5E9 and OKT9, OKT9Fab, and transferrin. CRM107 conjugates inhibited protein synthesis at substantially lower concentrations than gelonin conjugates; this effect was attributed to substantially higher translocation rates for CRM107. However, under certain conditions, CRM107 immunotoxin-treated cells were able to recover completely; this behavior was never observed with gelonin immunotoxins. To quantitatively capture this phenomenon, extracellular and cytosolic degradation of the toxin as well as growth-related recovery from toxin-induced damage were incorporated into the mathematical model. Translocation and cytosolic degradation rate constants were determined for each immunotoxin. Unlike the gelonin conjugates, the translocation rate of CRM107 conjugates depended on the targeting molecule. This provided indirect evidence that CRM107 remains disulfide linked to the targeting agent for at least part of the translocation process. Although the CRM107 conjugates all had higher translocation rates and inhibited protein synthesis at lower concentrations than the gelonin conjugates, the cells' ability to recover from protein synthesis inhibition at low immunotoxin concentrations limits the utility of CRM107 conjugates for targeted cell killing.     

Stress-dependent redistribution of abscisic acid (ABA) in Hordeum vulgare L leaves: the role of epidermal ABA metabolism, tonoplastic transport and the cuticle

When ¹⁴C-labelled abscisic acid ([¹⁴C]ABA) was supplied to isolated protoplasts of the barley leaf at pH 6, initial rates of metabolism were about five times higher in epidermal cell protoplasts than in mesophyll cell protoplasts if equal cytosolic volumes were considered. In spite of the fact that epidermal cells make up only about 35% of the total water space in barley leaves, and despite the small cytosolic volume of these cells, in intact leaves all epidermal cells would thus metabolize half as much ABA per unit time as the mesophyll cells (0–27 and 0–51 mmol h^?1 m^?3 leaf water). Therefore, under these conditions epidermal cells seem to be a stronger sink than mesophyll cells for ABA that arrives via the transpiration stream. However, at an apoplastic pH of 7–25, which occurs in stressed leaves, the proportion of total metabolized ABA would be much smaller in epidermal than in mesophyll cells (0–029 and 0–204 mmolh^?l m^?3 leaf water). Our results indicate that under conditions of slightly alkaline apoplastic pH the epidermis may serve as the main source for fast stress-dependent ABA redistribution into the guard cell apoplast. This is partly the result of ABA transport across the epidermal tonoplast, which is dependent on the apoplastic pH and possibly on the cytosolic calcium concentration. The cuticle seems to be of no particular importance in stress-induced apoplastic ABA shifts and cannot be regarded as a significant sink for high ABA concentrations under stress.     

Soluble gangliosides in cultured neurotumor cells

Abstract: The biosynthesis and degradation of glycosphingolipids were studied in cytosolic and membrane fractions obtained from rat glioma C6 cells. Both pools had a similar composition of neutral glycosphingolipids but the soluble pool contained only a few percent of the total. The major ganglioside in C6 cells was GM3, of which only 2% was soluble. Whereas the bulk of the membrane GM3 was accessible to surface labeling procedures, the soluble GM3 was not. Mouse neuroblastoma N18 cells also contained small amounts of cytoplasmic gangliosides corresponding to GM3, GM2, GM1, and GDla. When C6 cells were incubated with medium containing [³H]galactose at 37°C, the specific activity of soluble GM3 initially increased more rapidly than that of membrane GM3; by 4 h, the specific activities in both pools became equal. Total incorporation into the membrane pool, however, was always several-fold greater even at the shortest incubation times examined. The labeling pattern of neutral glycosphingolipids in both soluble and membrane fractions indicated the existence of a precursor-product relationship between glucosylceramide and other glycosphingolipids. When labeled cells were transferred to nonradioactive medium, glucosylceramide disappeared the most rapidly, with a 50% loss within <6 h. The turnover rates of other glycosphingolipids were much slower. Although cytosolic GM3 was degraded more rapidly (t_1/2= 26 h) than membrane-bound GM3 (t_1/2= 44 h), its turnover rate was much slower than the time required for transport of GM3 to the cell surface (20–30 min). Our results are consistent with the existence of a small intracellular pool of soluble gangliosides and neutral glycosphingolipids that is stable and independent of the main membrane-bound pool. Although the role of these cytosolic glycolipids is unknown, they do not appear to represent a transport pool between the site of synthesis and the plasma membrane.     

Translation inhibition reveals interaction of 2′-deoxy and 2′-O-methyl molecular beacons with mRNA targets in living cells

Understanding the interaction between oligonucleotide probes and RNA targets in living cells is important for biological and clinical studies of gene expression in vivo. Here, we demonstrate that starvation of cells and translation inhibition by blocking the mTOR or PI-3 kinase pathway could significantly reduce the fluorescence signal from 2′-deoxy molecular beacons (MBs) targeting K-ras and GAPDH mRNAs in living cells. However, the intensity and localization of fluorescence signal from MBs targeting nontranslated 28S rRNA remained the same in normal and translation-inhibited cells. We also found that, in targeting K-ras and GAPDH mRNAs, the signal level from MBs with 2′-O-methyl backbone did not change when translation was repressed. Taken together, our findings suggest that MBs with DNA backbone hybridize preferentially with mRNAs in their translational state in living cells, whereas those with 2′-O-methyl chemistry tend to hybridize to mRNA targets in both translational and nontranslational states. This work may thus provide a significant insight into probe design for detection of RNA molecules in living cells and RNA biology.     

A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum

Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.