Volume 6, Issue 5 , Pages 605-611, October 2010
Caspase-9-dependent decrease of nuclear pore channel hydrophobicity is accompanied by nuclear envelope leakiness
Article Outline
Abstract
Advances in nanomedicine require conceptual understanding of physiological processes. Apoptosis is a fundamental physiological process that is characterized, among other things, by an increased permeability of the nuclear envelope (NE). The latter is a tight transport barrier, known to restrict nuclear delivery rate of therapeutic nanoparticles. Therefore, an understanding of the underlying mechanism that leads to the breakdown of the barrier during apoptosis could stimulate the development of new approaches in gene therapy. We set out to elucidate this mechanism following induction of apoptosis on isolated cell nuclei. We tested the hypothesis whether caspases, mediators of apoptosis, trigger the NE leakiness at the level of the nuclear pore complexes (NPCs) using fluorescence techniques. As the permeability barrier inside the NPC channel is thought to be based on hydrophobic–hydrophobic protein interactions we further investigated the NPC channel hydrophobicity using atomic force microscopy. Caspase-9 was found to induce NE leakiness to large macromolecules. Leakiness was prevented by pretreatment of NPCs with an importin-β mutant, which irreversibly binds and thereby obstructs the NPC channel. Utilizing an ultra-sharp, hydrophobic atomic force microscope tip as a chemical nanosensor that reaches deep into the apoptotic NPC channel, a remarkable decrease of hydrophobic binding sites was detected therein. We conclude that caspase 9 gives rise to NE leakiness by perturbing the hydrophobicity-based barrier inside the NPC channel. This explains the high passive NE permeability in early apoptosis.
From the Clinical Editor
In this study, biological processes taking place in the nucleus during the course of apoptosis have been monitored using atomic force microscopy-based nanosensors. The conclusion was that one of the caspases, caspase 9 perturbes the hydrophobicity-based barrier inside the nuclear pore complex channel causing nuclear envelope leakiness.
Key words: Apoptosis, Atomic force microscopy, Nuclear envelope, Nuclear pore, Nucleocytoplasmic
The eukaryotic nucleus is surrounded by a protective nuclear envelope (NE), a selective transport barrier between the cytosol and the nucleus, referred to as nuclear barrier.1 The latter sequesters the DNA in the nucleus and therefore provides the cell with the opportunity to control access to its pivotal genetic material. The selectivity of the nuclear barrier is provided by supramolecular structures spanning the NE at regular distances, nuclear pore complexes (NPCs), the sole transport pathways between the cytosol and the nucleus.2, 3 The main structural components of the NPC include a central framework (embedded in the double-membraned NE), the cytoplasmic ring moiety with the cytoplasmic filaments (involved in transport into the nucleus), and the nuclear ring moiety, which is decorated by the nuclear basket (involved in export out of the nucleus).4, 5, 6, 7 Enclosed by the central framework is the NPC central channel through which the macromolecular exchange between the cytoplasm and the nucleus proceeds.8, 9, 10, 11, 12, 13 The NPC is composed of a set of ∼30 different proteins,14, 15, 16 called nucleoporins (nups), which allow passage of material in essentially two modes: passive diffusion and facilitated translocation. Passive diffusion does not require any specific interactions between the diffusing cargo and components of the NPC; it is restricted to molecules smaller than 20–40 kDa.17 In contrast, facilitated translocation requires transport receptors. These receptors bind cargo molecules on one side of the NE, translocate through the NPC to the other side, release their cargo, and finally return to the original compartment to mediate another cycle of transport. Receptor-mediated transport cycles can accumulate cargoes against a gradient of chemical activity, which is an energy-consuming task. This energy originates from the chemical potential of the RanGTP gradient.10 The facilitated translocation process per se is, however, not directly coupled to nucleotide hydrolysis. It is based on hydrophobic–hydrophobic interactions between transport receptors and nups containing phenylalanine- and glycine-rich (FG-rich) repeats, which enrich the interior of the NPC central channel. They are thought to form either a meshwork, a so-called selective phase,18, 19, 20 or an entropic barrier.21 In both cases the FG-rich nups pose a barrier to inert molecules, which can collapse transiently if binding of the transport receptors to the FG repeats occurs. This barrier excludes inert or hydrophilic cargoes but favors the transport of relatively hydrophobic cargoes,22 because they can bind to the FG repeats and can thus be translocated to either side of the NPC. As mentioned earlier, the presence of such a barrier is physiologically critical. During the essential physiological process, apoptosis, however, either the NPC channel barrier alone or the nuclear barrier as a whole become leaky.23, 24 It has been suggested that nuclear barrier leakiness contributes to the progression of apoptosis by providing apoptotic factors like endonucleases and caspases access to the nucleus.25 Some of these factors harbor a nuclear localization signal, whereas others do not. Active receptor-mediated nuclear import of apoptosis-promoting factors is questionable, however, because the RanGTP gradient, which is crucial for this mode of transport, is known to collapse early during apoptosis.25 In other words, nuclear barrier leakiness could be essential for the accomplishment of nuclear apoptosis. In this work we addressed the question by which factor nuclear barrier leakiness is triggered. Furthermore, we investigated whether the barrier leakiness is due to potential ruptures in the NE or due to alterations in the NPCs. Finally, we investigated whether the hydrophobic binding sites of the selective barrier inside the NPC central channel are affected during apoptosis. As mentioned above, these hydrophobic binding sites are crucial for the selectivity of the barrier.20 Because the nuclear barrier becomes leaky during apoptosis, its selectivity is lost, and we were interested in investigating whether this loss in selectivity was accompanied by a reduced density of hydrophobic binding sites in the NPC central channel. Our data indicate that alterations of the NPC central channel are the major cause for nuclear barrier leakiness. These alterations are triggered directly or indirectly by caspase-9 and include a decrease in the density of hydrophobic binding sites inside the NPC channel.
Methods
Preparation of oocytes, nuclei, and nuclear envelopes
The preparation of Xenopus laevis oocytes, nuclei, and NEs was according to Kramer et al26 with the exception that poly-l-lysine (Sigma, St. Louis, Missouri)-coated glass coverslips were used. The coating was performed by incubating coverslips with 0.1% (w/v) poly-l-lysine for 30 minutes. NE samples were kept unfixed in fluid at all times.
Microinjection of cytochrome c
Typically, 50 nL of aqueous solution of cytochrome c (Sigma) were microinjected into each oocyte using a microinjector (OocytePipet; Drummond, Broomall, Pennsylvania), resulting in a final concentration of 10 μM. After injection of cytochrome c, oocytes were incubated at 18°C for the indicated times.
Preparation of apoptotic cytosolic extracts
Oocytes were incubated for 2 hours at room temperature (RT, 20-25°C) in oocyte storage solution (87 mM NaCl, 6.3 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, 100 U/100 μg penicillin/streptomycin; pH 7.4) containing 1 mg/mL collagenase (Sigma). Oocytes were washed several times in nuclear isolation medium (NIM; 90 mM KCl, 26 mM NaCl, 5.6 mM MgCl2, 1.5% 40-kDa polyvinylpyrrolidone, and 10 mM HEPES pH 7.4) and sequentially crushed by centrifugation at 14,000 g for 30 minutes. The soluble phase was removed, and cytochrome c (Sigma) was added leading to a final concentration of 10 μM. The extract was incubated for 2 hours at RT and centrifuged again at 14,000 g for 30 minutes. Aliquots of the supernatant were frozen using liquid nitrogen and stored at –80°C.
Assay of DEVDase and LEHDase activity in apoptotic cytosolic extracts
DEVD represents the amino acid sequence asparagine-glutamine-valine-asparagine, which is cleaved by caspase-3. LEHD is the one-letter code for leucine-glutamic acid-histidine-aspartic acid, which is cleaved by caspase-9. The assay of DEVDase and LEHDase activity was performed according to Bhuyan et al.27 The fluorescent caspase substrates Ac-DEVD-AFC and Ac-LEHD-AFC were purchased from Biomol (Lörrach, Germany).
Expression of importin-β 45-462
The expression plasmid28 for importin-β 45-462 was a kind gift from Dirk Görlich (Max-Planck Institute, Göttingen, Germany). Expression and purification of importin-β 45-462 was according to Kramer et al.29
Macromolecule permeability assays
Oocyte nuclei were isolated and incubated for 3.5 hours at RT in apoptotic cytosolic extracts (diluted 1:1 in NIM). For the inhibitor studies, the extracts were preincubated with the inhibitor (40 μM zDEVD-fmk or zLEHD-fmk; Sigma) for 10 minutes at RT. For the application of importin-β 45-462, nuclei were preincubated with 0.5 μM of the mutant transport factor for 30 minutes in NIM at RT. Subsequently, the nuclei were incubated in apoptotic cytosolic extracts containing the same concentration of importin-β 45-462. Nuclei were then mounted onto a chamber on the stage of a confocal laser-scanning microscope (CLSM Fluoview; Olympus, Tokyo, Japan) and superfused with NIM containing 200 μg/mL 70 kDa fluorescein isothiocyanate (FITC)-dextran (Sigma). The light source of the confocal microscope was a diode laser with an excitation wavelength of 488 nm (Melles Griot, Bensheim, Germany). Cell-tak (BD Biosciences, San Jose, California) was used to immobilize the cell nuclei. The diffusion of the 70-kDa FITC-dextran was monitored over 60 minutes with a recording rate of 1 image/min. The ratio between average intranuclear and extranuclear fluorescence was calculated using the Fluoview software. The curves derived from the fluorescence ratio over time can be fitted using the function F(t) = Fmax × e–k × t, where Fmax is the maximum fluorescence ratio at equilibrated concentrations between cytosol and nucleus and k is the first-order rate constant. Because the measured fluorescence intensity was found to be proportional to the FITC-dextran concentration, the intranuclear FITC-dextran concentration cnuc could be derived from the concentration in the surrounding buffer cbuff:
Initial rates of diffusion were derived from the slope during the first 5 minutes of each measurement.
Atomic force microscopy
The application of atomic force microscopy (AFM) to NEs has been described in detail elsewhere.1, 30, 31 SuperSharpShort tips (Nanotools, Munich, Germany) were used. The tips are composed of high-density carbon and are therefore hydrophobic. The settings for the adhesion measurements were as follows: Number of single adhesion measurements was 128 × 128 (x, y), scan rate was 9 Hz (measurements per second), and the trigger threshold was set to 1 nN. All curves of an adhesion measurement were analyzed automatically using the software PUNIAS (P. Carl and P. Dalhaimer, http://site.voila.fr/punias/klmenu/punias0.htm). Adhesion maps were generated and analyzed using the SPIP software (Image Metrology, Hørsholm, Denmark).
Statistics
Data are presented as the mean ± standard error of the mean (SEM). Statistical significance of mean values was tested with the unpaired Student's t-test. P value was < 0.05 (⁎⁎) or < 0.001 (⁎⁎⁎). n indicates the number of nuclei (permeability assays) or number of NPCs investigated (adhesion measurements).
Results
Induction of apoptosis in intact oocytes induces NE leakiness
Xenopus oocytes are widely used to study NPC structure and function. Given their fairly large size and cytosolic content, they can easily be microinjected and a cytosolic extract can readily be harvested. Microinjection of Xenopus oocytes with cytochrome c followed by AFM is an excellent system for studying nuclear apoptosis in vitro, because oocytes have a high NPC density and yield structurally well-conserved NPCs. Given the large size of the oocyte nucleus, it can easily be manually isolated, kept in a cytoslic extract, and next exposed to cytochrome c.
In the present study we triggered apoptosis in X. laevis oocytes by microinjecting them with 10 μM cytochrome c. Cytochrome c injection mimics its release out of mitochondria during apoptosis, where it binds to Apaf-1 and pro-caspase-9 to form the apoptosome.32 Previous studies have demonstrated that cytochrome c induces apoptosis in Xenopus cell-free system33 and in intact oocytes.26, 27 Following injection, oocytes were incubated at 18°C and cell nuclei were isolated after 1.5, 2, and 2.5 hours. Passive nuclear permeability was measured using an FITC-dextran conjugate of 70 kDa, which exceeds the limit for passive diffusion across the NPCs. After 1.5 and 2 hours hardly any influx of FITC-dextran was detectable, but after 2.5 hours the influx increased dramatically (Figure 1).
Figure 1.
Nuclear envelope permeability increases remarkably 2.5 hours after cytochrome c injection into oocytes. Passive diffusion of 70-kDa FITC-dextran into isolated oocyte nuclei at different points in time after cytochrome c injection. Controls were injected with nuclear isolation medium. The small inset shows schematically the measurement setup.
Caspase-9 is a key player in NE leakiness
In a next step we tried to mimic this effect in vitro by incubating isolated nuclei in apoptotic cytosolic extracts. This in vitro approach has two major advantages. First, it is possible to divide the activation cascades of the numerous apoptotic factors and the triggering of nuclear barrier leakiness spatially and chronologically. In doing so it is possible to test various inhibitors of apoptotic factors and to specifically identify the factor x, which finally triggers nuclear barrier leakiness. The second main advantage of the in vitro approach is that it allows one to perform paired measurements of the NE structure before and after exposure to an apoptotic cytosolic extract. We incubated isolated nuclei in apoptotic extracts for 3.5 hours and found indeed a dramatic increase in NE permeability similar to the in vivo situation (Figure 2). Adding the caspase-3 inhibitor zDEVD-fmk at a concentration of 40 μM did not show any effect. Application of the caspase-9 inhibitor zLEHD-fmk, however, fairly reduced the permeability increase by the equivalent of ∼43% (Figure 2, Figure 3). Hence, caspase-9 seems to substantially contribute to nuclear barrier leakiness. To address the question whether ruptures in the NE structure or alterations of the NPCs are the cause of nuclear barrier leakiness, we used the importin-β mutant 45-462. The latter binds specifically and irreversibly to the FG nups and thereby blocks the NPC central channel completely28 (Figure 3, B), making it inaccessible for caspases. Preincubation of isolated nuclei with 0.5 μM of the mutant transport factor for 30 minutes prevented the increase in NE permeability almost completely. This result suggests that indeed alterations of the NPC central channel are responsible for the nuclear barrier leakiness.
Figure 2.
Nuclear barrier leakiness is independent of caspase-3 but is modulated by caspase-9. Passive diffusion of 70-kDa FITC-dextran into isolated oocyte nuclei after incubation in apoptotic cytosolic extracts in presence of caspase inhibitors. Pretreatment of nuclei with importin-β 45-462 prevents the permeability increase almost completely. Error bars represent SEM values.
Figure 3.
Initial rates of diffusion of 70-kDa FITC-dextran after incubation of nuclei in apoptotic extracts. (A, B) Pretreatment of NPCs with importin-β 45-462 leads to a blockage of the NPC channel, which prevents apoptotic nuclear barrier leakiness as shown in C. (C) Initial rates of diffusion after preincubation with importin-β 45-462 and incubation in an apoptotic extract. (D) Rates of diffusion after incubation of nuclei in an apoptotic extract in presence of caspase inhibitors. Asterisks indicate significantly different from “apoptotic extract” condition. ⁎⁎P < 0.05; ⁎⁎⁎P < 0.001.
AFM reveals reduced density of hydrophobic binding sites in apoptotic NEs
To characterize these alterations further we applied AFM to isolated NEs, which were spread on a glass surface. For this purpose an atomic force microscope tip with the following key features was utilized. The tip diameter is 2 nm at the very top and barely 6 nm along a distance of up to 30–50 nm (Figure 4). In addition, the tip is made of a high-density carbon material and is therefore hydrophobic. Hence, the utilized tip not only scans the NE surface at fairly high resolution but readily enters the NPC central channel and can thus act as a chemical sensor therein and on the rest of the NE surface. While scanning, the tip will interact with hydrophobic but barely with hydrophilic spots. The interaction in turn can be observed as adhesion events on adhesion maps generated from the AFM images. We measured adhesion between the atomic force microscope tip and the NE sample before and after exposure to an apoptotic cytosolic extract. Figure 5 shows the result of such a paired measurement. The occurrence of one or more hydrophobic binding events in one spot is shown as a white pixel in the adhesion map. Figure 5, E and F show an overlay of the adhesion map and the topographical image, which are recorded simultaneously. It is apparent that the density of hydrophobic spots on the NPCs decreases after the exposure to the apoptotic cytosolic extract. To quantify, we measured the density of the hydrophobic spots in the NPC central channels (i.e., the fraction of pixels showing adhesion peaks divided by the entity of pixels in the area of the NPC central channels). Figure 6 shows that the density of hydrophobic spots or binding sites in the NPC central channel was reduced by more than 50%.
Figure 4.
Schematic interaction between high-aspect-ratio atomic force microscope tip and nuclear pore channel (true to size comparison). NPC model modified from Patel et al.11 Small inset: SEM image of a high-aspect-ratio tip (image taken from: http://www.nano-tools.com).
Figure 5.
Nuclear envelope (NE) surface hydrophobicity maps. (A, B) Height images of the cytoplasmic face of the NE before and after incubation with an apoptotic cytosolic extract. (C, D) Corresponding hydrophobicity maps. Each white pixel represents an adhesive event between the hydrophobic tip and the sample. (E, F) Overlay of the height images and the hydrophobicity maps. Images are 1.2 × 1.2 μm each.
Figure 6.
The density of hydrophobic spots in the nuclear pore channel (NPC) central channel is decreased after incubation of the nuclear envelope in an apoptotic cytosolic extract. Hydrophobic spot density is measured as a fraction of pixels showing adhesion peaks divided by the entity of pixels in the area of the NPC central channels. Error bars represent SEM values. Upper right inset: Representative adhesion measurement. The hydrophobic atomic force microscope tip is brought into contact with the sample. Upon retraction of the tip an adhesive force is felt by the cantilever, which results in an adhesion peak (dashed circle).
Discussion
For the nuclear manifestation of apoptosis, death signals have to be transmitted to the nucleus. In fact, it has been shown that a crosstalk between the nuclear and cytoplasmic compartments plays an essential role in propagating the death program.25 In this respect, nuclear transport appears to provide an important level of control for regulating apoptosis. Inhibition of the nuclear transport machinery in different ways prevents nuclear apoptosis, suggesting that important mediators of apoptosis are not able to enter the nucleus,34, 35 their place of action. Thus far it remains controversial how cytosolic apoptosis mediators gain access to the nucleus. As the RanGTP gradient collapses early during apoptosis and nuclear transport factors redistribute across the NE,24, 36 it is at least disputable whether active transport plays a role in the delivery of apoptotic factors to the nucleus. Furthermore, it is a feature of active transport to concentrate substrates in one compartment. Faleiro and Lazebnik23 found that at least for the nuclear import of caspase-3 this is not the case. In fact, caspase-3 was found to equilibrate between cytosol and nucleus, which suggests passive diffusion rather than active transport. Moreover, at the same point in time they observed an increase in the passive permeability of the NE. This leakiness of the nuclear barrier has been confirmed later on in several cell types and for different apoptotic triggers.37, 38, 39 The factors triggering nuclear barrier leakiness and the underlying mechanism, however, remain open questions. We set out to answer these questions using our in vitro apoptosis assay.
Caspase-9 but not caspase-3 gives rise to nuclear barrier leakiness
By microinjection of cytochrome c we confirmed that nuclear barrier leakiness during apoptosis also occurs in X. laevis oocytes (Figure 1). Applying caspase inhibitors in combination with our in vitro assay we found that caspase-9 gives rise to nuclear barrier leakiness, whereas caspase-3 fails to do so (Figure 2). This finding is in agreement with a previous study,23 in which the expression of a dominant-negative form of caspase-9 in MCF-7 cells (human breast cancer cell line) was found to inhibit nuclear barrier leakiness, translocation of caspase-3 into the nucleus, and redistribution of Ran. By contrast, Kihlmark et al found a correlation between caspase-3-mediated cleavage of POM121, NPC membrane protein, and a disruption of nuclear compartmentalization.38 Kamada et al found that caspase-2 had an effect on the passive permeability at late stages of apoptosis.37 Additionally, the authors of this study found an early increase in the passive permeability of the NE that seemed to be caspase-9-dependent. In contrast to these observations, Ferrando-May et al measured an early increase in the passive permeability in the presence of the pan caspase inhibitor zVAD-fmk and concluded that the increase is therefore independent of caspase activity.24 However, whereas the concentration of the inhibitor used in this study (20 μM) is high enough to inhibit caspase-3 activity completely, caspase-9 activity might not be inhibited to the same extent. Johnson et al have shown that concentrations between 100 and 200 μM of zVAD-fmk are required to inhibit caspase-9 activity.40 Therefore, caspase-9 may be involved in nuclear barrier leakiness in HeLa cells as well. Whether nuclear barrier leakiness is solely caused by caspase-9, however, remains an open question. Fractionation of apoptotic cytosolic extracts in combination with the established in vitro assay may provide a conclusive answer for this question.
Nuclear barrier leakiness is a consequence of NPC channel leakiness
Previous studies have indicated with electron microscopy that the integrity of the NE remains largely intact during apoptosis.41, 42, 43 In support of these data, we observed previously that apoptotic oocyte nuclei maintain their capability to swell on exposure to hypo-osmotic medium, which is a physiological criterion for the integrity of the nucleus and the NE.26 Yet, we set out to provide conclusive evidence for nuclear barrier leakiness being caused directly through NPC channel leakiness but not potential insubstantial ruptures in the NE. Irreversible obstruction of the NPC channel by importin-β 45-462 prevented nuclear barrier leakiness (Figure 3, C), which proves that the increased diffusion permeability of the apoptotic nucleus was indeed caused by increased diffusion limit of the NPC central channel.
Nuclear barrier leakiness is accompanied by a reduction of hydrophobic binding sites in the NPC central channel
In a next step we set out to determine the mechanism underlying the leakiness of the NPC channel. The NPC channel is known to be a selective transporter. Transport selectivity is established by NPC channel proteins containing FG repeats, which are assumed to form a hydrophobicity-based permeability barrier inside the NPC channel.18, 19, 20 In other words, the permeability barrier of the NPC channel seems to operate via hydrophobic exclusion. Cargoes to be selectively transported through the NPC channel are recognized by hydrophobic receptors in either the cytoplasm or the nucleoplasm. The resulting hydrophobic complex next targets the NPC channel, readily dissolves in the hydrophobicity-based meshwork therein, and is rapidly translocated.20 Consistently, interfering with hydrophobic interactions causes a reversible collapse of the permeability barrier of NPCs.20 With respect to the hydrophobicity based transport barrier inside the NPC channel, we asked the question as to whether the caspase-9-induced NPC channel leakiness is the consequence of diminished channel hydrophobicity. On the other hand, the NPC channel is only 40 nm wide and 50 nm long. Thus, gaining insight into its interior, importantly in physiologically relevant environments, is challenging and has remained only a wishful concept until the development of the powerful nano-approach of AFM.44 This technique, which utilizes a diminutive tip to scan a surface, allows simultaneous imaging of biological samples and probing of their surface properties, importantly in fluid, at the nanoscale.26,45, 46, 47 In this study an atomic force microscope tip was utilized, which readily reaches deep into the NPC channel (Figure 4). At the same time, due to its hydrophobicity it can act as a sensor of hydrophobicity inside the NPC channel. As seen in Figure 6, the apoptotic NPC channel reveals remarkably reduced density of hydrophobic binding sites as compared with the control NPC channel. From this observation it clearly follows that the permeability barrier, which is thought to be based on interactions between the hydrophobic FG repeats inside the NPC channel, is seriously perturbed.
All in all, the in vitro assay developed in the present study provides unique insight into understanding the mechanisms underlying the physiologically critical process of apoptosis. We conclude that caspase-9 gives rise to nuclear barrier leakiness during apoptosis. Caspase-9 exerts this action by causing, directly or indirectly, a reduction of hydrophobic binding sites inside the NPC channel. This, in turn, is probably caused by cleavage of NPC channel proteins, rich in hydrophobic domains, which make up the barrier inside the NPC channel.
Implications for nanomedicine
Determining the mechanism underlying NPC channel dilation, as is the case during apoptosis, will have direct implications to gene therapy. The latter has long since taken center stage in the realm of nanomedicine. On the other hand, the efficiency of gene therapy in nondividing cells is marginal. This is caused by a poor nuclear delivery rate of therapeutic macromolecules due to their size-dependent exclusion from NPC passage. NPC dilation may thus pave the way to increasing the efficiency of gene therapy in nondividing cells, a widespread aspiration that we hope will become reality upon understanding how to cause nuclear barrier leakiness.
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This study was supported by grants from the Innovative Medizinische Forschung (no. SH-110315, SH-520404, and SH-120613) and the Deutsche Forschungsgemeinschaft (Graduate School Molecular Basis of Dynamic Cellular Processes) of the SFB629, International Graduate School, Interaction of pathogens with biotic and abiotic surfaces GRK1409 and OB 63/16-1, and Interdisciplinary Center of Clinical Research (IZKF) Münster, project no. Küh3/064/04.
PII: S1549-9634(10)00154-1
doi:10.1016/j.nano.2010.04.006
© 2010 Elsevier Inc. All rights reserved.
Volume 6, Issue 5 , Pages 605-611, October 2010
