- Open Access
Serine phosphorylation regulates paxillin turnover during cell migration
© Abou Zeid et al; licensee BioMed Central Ltd. 2006
- Received: 30 June 2006
- Accepted: 22 November 2006
- Published: 22 November 2006
Paxillin acts as an adaptor protein that localizes to focal adhesion. This protein is regulated during cell migration by phosphorylation on tyrosine, serine and threonine residues. Most of these phosphorylations have been implicated in the regulation of different steps of cell migration. The two major phosphorylation sites of paxillin in response to adhesion to an extracellular matrix are serines 188 and 190. However, the function of this phosphorylation event remains unknown. The purpose of this work was to determine the role of paxillin phosphorylation on residues S188 and S190 in the regulation of cell migration.
We used NBT-II epithelial cells that can be induced to migrate when plated on collagen. To examine the role of paxillin serines 188/190 in cell migration, we constructed an EGFP-tagged paxillin mutant in which S188/S190 were mutated into unphosphorylatable alanine residues. We provide evidence that paxillin is regulated by proteasomal degradation following polyubiquitylation of the protein. During active cell migration on collagen, paxillin is protected from proteasome-dependent degradation. We demonstrate that phosphorylation of serines 188/190 is necessary for the protective effect of collagen. In an effort to understand the physiological relevance of paxillin protection from degradation, we show that cells expressing the paxillin S188/190A interfering mutant spread less, have reduced protrusive activity but migrate more actively.
Our data demonstrate for the first time that serine-regulated degradation of paxillin plays a key role in the modulation of membrane dynamics and consequently, in the control of cell motility.
- Focal Adhesion Kinase
- Focal Adhesion
- Cell Spreading
- Serine Phosphorylation
Cell migration is a cyclic multistep event that involves cell polarization and extension of membrane protrusions at the leading edge. At the front of the protrusion, cell-substratum adhesive complexes are formed providing traction points for translocation. Adhesive complexes at the cell rear are then released as the cell continues to move forward .
Integrins are cell-surface adhesion receptors that link the extracellular matrix (ECM) to the intracellular actin cytoskeleton . Integrins are heterodimeric receptors that interact with numerous structural and signaling molecules to form large protein complexes, the focal adhesions. Upon binding to ECM elements, integrins cluster and interact with an array of structural and signaling molecules to form large protein complexes, the focal adhesions. These structures are spatially regulated during cell migration as they assemble at the leading edge to stabilize new membrane protrusions and disassemble at the rear of the cell to allow cell detachment .
The assembly process of focal adhesions at the leading edge has been extensively examined, leading to the conclusion that they are highly dynamic. Their molecular composition varies over time [4, 5], thereby regulating membrane protrusive activity and cell movement. For instance, fast movement of the lamellipodium has been associated with focal complexes containing relatively low levels of paxillin whereas their slower movement with focal complexes rich in paxillin .
Compared with the process of formation, less is known about adhesion breakdown. Adhesive complexes are disassembled at the rear and at the base of dynamic protrusions  in polarized migrating cells. Proteolysis of focal adhesion components has been proposed to regulate the protein content of adhesive complexes resulting in the control of focal adhesion turnover and cell migration. Calpains, cysteine proteases which localize to adhesive structures , have been shown to cleave several focal adhesion proteins, such as focal adhesion kinase (FAK) , talin  and paxillin [9–11]. It is now accepted that these proteases play an essential role in regulating focal adhesion dynamics, membrane protrusion stability and cell migration.
The ubiquitin-dependent proteasomal degradation is another proteolytic pathway that has been implicated in the control of focal adhesion turnover and cell migration. Proteasomal degradation of FAK was shown to inhibit cell migration . Similarly, ubiquitylation of paxillin was shown to affect its localization to focal adhesions and to inhibit cell migration .
Paxillin is a 68 kD adapter protein that localizes to focal adhesions. Paxillin contains many protein-protein interaction domains: five LD motives (Leucine-rich domains) in the amino-terminal part and four LIM domains (double zinc fingers) in the carboxy-terminal part. LIM domains mediate binding to protein tyrosine phosphatase-PEST (PTP-PEST) [14, 15], α- and γ-tubulin . LIM3 is responsible for proper targeting of paxillin to focal adhesions . LD motifs mediate interactions with an array of partners, including FAK  and vinculin .
Regulation of paxillin by phosphorylation is well documented . Tyrosine residues are phosphorylated in response to extracellular signals and serve as docking sites for protein interactions. Phosphorylation of tyrosines 31 and 118 provides binding sites for the SH2 domain of Crk and this interaction is necessary for cell migration . Serine phosphorylation of paxillin is also a prevalent posttranslational modification that has been extensively examined. Paxillin serine phosphorylation is increased by cell adhesion on fibronectin  or vitronectin . In contrast, during cell mitosis, which is associated with an absence of adhesive complexes, paxillin serine phosphorylation coincides with its degradation . ERK and GSK-3 have been reported to phosphorylate serines 126 and 130, respectively, leading to the relocalization of paxillin from focal adhesions to the cytosol [23, 24], while serine 178 phosphorylation by JNK is necessary for cell migration . It appears therefore that the phosphorylation of paxillin at specific serine/threonine residues could regulate its activity and biological functions. A function has still to be attributed to the phosphorylation of serines 188 and 190 of paxillin, the two major phosphorylation sites in response to cell adhesion to an extracellular matrix.
Because paxillin is a focal adhesion adaptor protein, it is possible that specific serine phosphorylation events control its degradation rate, as already suggested , thereby influencing cell movement through the regulation of focal adhesion dynamics. The implication of paxillin in focal adhesion turnover has been recently demonstrated. Horwitz and coworkers showed that the rate of focal adhesion disassembly is slower in cells expressing several phosphorylation mutants or domain-deleted forms of paxillin compared to cells expressing wild-type paxillin .
In this report, we found that paxillin could be ubiquitylated and degraded by the proteasome. Moreover, we show that substrates such as collagen that support cell migration prevented paxillin degradation. Using a S188/190A phosphorylation mutant of paxillin, we were able to demonstrate that phosphorylation of S180/S190 was required for the increased stability of paxillin on collagen. We also showed that paxillin S188/190A double mutant impaired membrane protrusion dynamics as well as cell migration and spreading without affecting cell adhesion on collagen.
Paxillin ubiquitin-dependent proteasomal degradation is regulated by collagen
We next wished to determine whether paxillin degradation could be modified under conditions allowing cell migration. Since NBT-II carcinoma cells are induced to migrate on collagen substrates , cells were labeled with 35S-methionine/cysteine mix and chased on plastic or collagen for 18 h in the presence or absence of lactacystin. Uncoated plastic plates were used as a substrate that does not induce cell migration. As shown in Figure 1B, paxillin protein levels significantly decreased after 18 h chase. Quantification of the radioactive bands migrating with the apparent molecular weight of paxillin indicated an approximate reduction of 70% of the newly synthesized molecules during the incubation time. This decrease was mostly due to proteasomal degradation since it could be prevented by treatment of cells with lactacystin added during the entire chase. In contrast, paxillin levels were not reduced during the chase period on collagen substrate. As a consequence, lactacystin had no obvious effect on paxillin stability under those conditions. The additional bands that are visible below and above the apparent size of paxillin probably represent proteins that co-precipitated with the adaptor protein paxillin. The differences in paxillin half life in cells plated on collagen versus plastic were apparently specific to paxillin as other proteins, such as tubulin, did not exhibit similar variations (data not shown). These experiments indicate that the half-life of paxillin of approximately 7 h in stationary cells on plastic is noticeably increased in motile cells on collagen, suggesting that collagen prevents paxillin proteasomal degradation.
It is well established that ECM protects cells from programmed cell death. To ensure that the accelerated degradation of paxillin in NBT-II cells plated on plastic as compared to collagen was not due to an increase in cell death on plastic, the number of cells plated on each substrate was evaluated over a 5-day period (Fig. 1C). No difference in cell numbers on plastic and collagen was observed. These results suggest that collagen-induced reduction of paxillin degradation is linked to induction of cell motility rather than to protection from cell death.
Phosphorylation of serines 188/190 prevents paxillin from degradation
Serine/threonine phosphorylation events are known to regulate ubiquitin-dependent proteasomal degradation of several proteins [31–33]. Paxillin, originally identified as a tyrosine phosphorylated protein [34, 35], is also heavily phosphorylated on serine/threonine residues [11, 21, 23–25, 36–39]. Phosphorylation of specific serine residues of paxillin has been shown to be implicated in cytoskeleton rearrangement and cell motility [23, 25, 37]. Interestingly, two recent reports have proposed a possible link between serine phosphorylation-dependent degradation of paxillin and cell movement [25, 37]. Two major sites of serine phosphorylation are residues 188/190, previously shown to be heavily phosphorylated upon cell adhesion to ECM . However, the role of these modifications remains undefined.
Mutations on serines 188/190 do not affect cell adhesion but inhibit cell spreading
We next analyzed the effect of the S188/190A paxillin mutant on cell spreading on collagen. Cells expressing EGFP-paxillin or EGFP-S188/190A were plated on collagen (10 μg/ml) for 30, 60, 120 and 240 min, and the number of EGFP-positive spread cells estimated after fixation. Cells expressing the S188/190A mutant spread slower than those expressing wild-type paxillin (Fig. 3B). Two hours after plating cells on collagen, there was a reproducible and significant 15% decrease in the ratio of spread cells expressing EGFP-S188/190A paxillin as compared to EGFP-paxillin (p < 0.001). These results suggest that phosphorylation on serines 188/190 contributes to cell spreading on collagen but not to cell attachment. Furthermore, these data argue for a dominant negative effect of S188/190A paxillin double mutation. The presence of endogenous paxillin may minimize the inhibition due to the mutant molecule. Alternatively, the moderate inhibition is comprehensible, considering that paxillin S188/S190 phosphorylation is only one element of the signaling pathway(s) leading to cell spreading.
Mutations on serines 188/190 enhance random cell migration on collagen
Serine phosphorylations of paxillin have been shown to be involved in cell migration [25, 40]. To determine whether the S188/190A mutant of paxillin affected random cell migration induced by collagen, we used time-lapse videomicroscopy to follow the movement of isolated cells expressing EGFP-tagged paxillin during 10 h. Using Metamorph software, we quantified the migration rate as the total distance traveled by one cell divided by the time of recording. On non-permissive substrates such as plastic plates, the absence of active cell migration of cells expressing the S188/190A paxillin mutant (10.71 μm/h ± 6.07) was in the same range as that of cells expressing EGFP-paxillin (5.46 μm/h ± 1.37). Mutations of paxillin S188/S190 into unphosphorylatable residues significantly increased migration on collagen (27.98 μm/h ± 1.21) as compared to cells expressing wild type paxillin (20.95 μm/h ± 1.09). Therefore, phosphorylation of residues S188/S190 is negatively implicated in cell migration on collagen.
Mutations on S188/S190 inhibit membrane protrusion dynamics
In this study, we provide evidence that paxillin levels can be regulated by proteasomal degradation following polyubiquitylation of the protein. On collagen, which sustains active cell migration of NBT-II cells, paxillin is protected from proteasome-dependent degradation leading to an increase in its half-life. In addition, we demonstrate that phosphorylation of serine residues 188 and 190 is necessary for the protective effect of collagen. When S188/S190 phosphorylation is prevented by mutating the two residues into alanine, several functional consequences can be observed. Cells spread less, have reduced protrusive activity and migrate more actively. Collectively, our data demonstrate for the first time that serine-regulated degradation of paxillin plays a key role in the modulation of membrane dynamics and consequently, in the control of cell motility.
In line with our data, degradation of paxillin by two different mechanisms has already been reported. On one hand, it was shown to depend on a calpain-regulated process [9–11]. On the other hand, ubiquitylation of paxillin was postulated to lead to selective degradation of the focal adhesion-associated insoluble pool but not the cytosolic pool of the protein . This suggests that the ubiquitylated forms of paxillin degraded in the NBT-II cell line used in our study could belong mainly to the adhesive contact-associated pool of the protein. In NBT-II cells, the insoluble pool of paxillin corresponds to approximately 30% of total paxillin (data not shown).
Using a non-phosphorylatable mutant of paxillin at serines 188 and 190, we found that phosphorylation of these sites was necessary for paxillin stabilization by collagen. The mechanism by which this effect is achieved remains elusive. Phosphorylated S188/S190 may constitute a regulated docking site for a protein partner, thereby sequestering paxillin away from the proteasomal machinery of degradation. Alternatively, S188/S190 phosphorylation may prevent other posttranslational modifications of the molecule required for ubiquitylation and/or degradation. In an attempt to investigate the significance of the increased paxillin stability induced by S188/190 phosphorylation on collagen, we demonstrate that phosphorylation on serines 188/190 is not necessary for cell adhesion but is important for cell spreading. It is now accepted that paxillin plays an important role in cell spreading since a number of mutants act in a dominant-negative fashion to reduce this process. CHO.K1 fibroblasts stably expressing a paxillin variant lacking the LD4 motif present a defect in cell spreading on fibronectin . Expression of the ERK/GSK-3 phosphorylation site double mutant, S126/130A, also strongly inhibits cell spreading of paxillin-null fibroblasts on fibronectin . Collectively, these data suggest that paxillin may control cell spreading through multiple signaling pathways.
We also noticed that expression of EGFP-S188/190A paxillin increases cell migration on collagen but not basal cell motility (on plastic), indicating that phosphorylation of serines 188 and 190 regulates collagen-induced but not basal cell migration. To our knowledge, this is the first time that serine phosphorylation of paxillin is shown to be negatively involved in cell motility. These results suggest that the function of paxillin may be orchestrated by an array of negative as well as positive posttranslational modifications. They could be combined on the same molecule leading to an integrated behavior or, alternatively, could arise on molecules localized at different intracellular sites. In the present study, we observe that cells expressing EGFP-S188/190A paxillin mutant spread less but migrate more on collagen than control cells. Reduction in cell spreading induced by the expression of mutant paxillin is consistent with an increase in cell motility, as cells derived from mice deficient in the focal adhesion molecule vinculin likewise show reduced cell spreading and increased cell migration .
In addition to negatively regulating cell migration, phosphorylation of paxillin serines 188/190 is important for the collagen-mediated increase in protein stability. In this respect, paxillin ubiquitylation was previously reported to be associated with the inhibition of cell migration . Proteasomal degradation of another focal adhesion protein, FAK, was also shown to inhibit cell migration . Moreover, we observed an inverse correlation between cell migration and membrane protrusion dynamics. In that sense, it is commonly accepted that in order for a cell to translocate properly, membrane protrusions at the leading edge of the cell need to be stabilized, leading to a reduction of protrusion dynamics .
Our results also show that a decrease in paxillin stability is associated with a decrease in protrusion dynamics. There are conflicting reports about the role of localized proteolysis at the leading edge of cells in regulating lamellipodia dynamics. Calpain activity was found to be necessary for stabilizing protrusions at the leading edge of migrating cells [9, 44]. In contrast, other studies support a role for calpains in limiting membrane extensions [9, 45, 46], suggesting that calpains may function both as a negative and positive regulator of protrusion events. It is required for stabilization of protrusions at the leading edge while suppressing protrusions in other parts of the cell. Other critical mediators of cell motility, including Rho-pathway components, have a similar relationship between their activities and protrusion stabilization/retraction events, serving both as negative and positive regulators of cell motility [47, 48, 49, 50]. We postulate that proteasome-dependent proteolysis of focal-adhesion molecules such as paxillin may serve similar functions. Degradation of paxillin would be inhibited in stabilized protrusions while taking place in labile membrane extensions, leading to polarization of the cell in the direction of movement. Further experiments are required to explore the role of S188/190 phosphorylation in polarization of cell migration and to establish whether the two serines are equally implicated in these effects.
Reagents and antibodies
Lactacystin, used at 1 μM, was from Calbiochem (Darmstadt, Germany). Monoclonal antibodies against paxillin (clone 349) and ubiquitin (clone P4D1) were from Transduction Laboratories (Lexington, KY, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Rat type I collagen was from BD Bioscience (San Jose, CA, USA).
Complementary DNA coding for chicken S188/190A mutant paxillin in the pcDNA3 expression vector was kindly provided by Dr. C.E. Turner (State University of New York, Syracuse, NY, USA). To construct the EGFP-tagged paxillin S188/190A mutant, the pcDNA3-S188/190A plasmid was subcloned in EGFP-C3 plasmid (Invitrogen, Carlsbad, CA, USA) using BamHI and XbaI restriction sites. The wild-type EGFP-paxillin was previously described . All constructs were sequenced prior to use (MWG-Biotech, Ebersberg, Germany).
Cell culture and transfection
NBT-II carcinoma cell line was cultured routinely in DMEM (Gibco BRL-Invitrogen, Carlsbad, CA, USA) supplemented with 1% glutamine and 10% heat-inactivated fetal calf serum as previously described .
For immunoprecipitation experiments, 1.5 × 106 cells plated one day before in 60-mm dishes were transfected with 6 μg DNA and polyethylenimine (Sigma, St. Louis, MO, USA) in DMEM for 3 h 30 as described before .
For all other experiments, 2 × 106 cells plated one day before in 60-mm dishes were transfected with Lipofectamine™ 2000 (Invitrogen) following the manufacturer's recommendations.
Western blots and immunoprecipitations
Cells were trypsinized, resuspended in complete medium for 15 min and replated on plastic dishes or on plates pre-coated overnight at 4°C with collagen (10 μg/ml). For western blotting experiments, cells were lysed with NP-40 buffer (1% NP-40, 50 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA), protease inhibitors (Complete tablets, Roche Diagnostics, Mannheim, Germany) and a cocktail of phosphatase inhibitors (1 mM Na-orthovanadate, 1 mM Na-pyrophosphate, 25 mM β-glycerophosphate, 1 mM Na-fluoride). For immunoprecipitations, cells were lysed with Triton buffer (1% Triton X-100, 10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, Complete proteases and phosphatase inhibitors). Immunoprecipitations were performed on equivalent amounts of proteins as described before .
For Western blot analysis of paxillin, equivalent amounts of proteins were separated by SDS-PAGE electrophoresis and transferred onto polyvinylidene membranes (Immobilon P, Millipore Corp., Bedford, MA, USA). Western blot detection of paxillin was performed essentially as described previously .
Ubiquitylation of paxillin
Cells were lysed in 1% Triton X-100, 1% SDS, 20 mM Tris pH7.5, 150 mM NaCl, 1 mM CaCl2, 10 mM N-ethylmaleimide, protease and phosphatase inhibitors, diluted 10 times in the same buffer without SDS and 1 mg of proteins was used for paxillin immunoprecipitation. Samples were separated by SDS-PAGE and subjected to Western blotting with an anti-ubiquitin antibody.
Pulse chase analysis
Non-transfected or cells transfected with plasmids encoding for EGFP-paxillin or EGFP-S188/190A paxillin were harvested by trypsinization and maintained in suspension in complete medium without methionine and cysteine for 30 min at 37°C. One hundred μCi of 35S-methionine/cysteine mix (Redivue™ PRO-MIX™, Amersham Biosciences, Piscataway, NJ, USA) was added per 1 × 106 cells. Cells were then washed 3 times in radioactive-free medium and plated onto plastic or collagen-coated plates and incubated at 37°C for increasing periods of time prior to cell lysis. 1.5 × 106 cells were lysed and paxillin immunoprecipitation was done as described previously. Proteins were separated by SDS-PAGE electrophoresis, gels were dried and exposed to autoradiographic films (Amersham Biosciences). Quantification of the autoradiographs from three independent experiments was done using Image J free software (NIH, USA). Means and SEM were then calculated. The Student's t test was used to evaluate the data.
Cell proliferation assay
NBT-II cells (7.5 × 104 cells) were plated in 60-mm culture dishes pre-coated with collagen (10 μg/ml) and allowed to grow at 37°C. Each day, cells were trypsinized and counted using a hemocytometer.
Cell adhesion and spreading
NBT-II cells transiently expressing EGFP-paxillin or EGFP-S188/190A paxillin were trypsinized, washed 3 times with DMEM and resuspended in 100 μl of DMEM. Eighty thousand cells were then plated on 96 well plates pre-coated overnight at 4°C with increasing concentrations of collagen and saturated for 1 h at room temperature with 5% BSA (Sigma). Non-specific cell adhesion was measured on wells coated with BSA only. After incubation at 37°C for 1 h, wells were washed 3 times with DMEM to remove non-adherent cells. The remaining adherent cells were fixed for 20 min with 3.8% paraformaldehyde, washed extensively and then stained for 10 min with 5% crystal violet in methanol. Plates were then washed with water until the excess dye was removed. Crystal violet incorporated within the cells was released with 100 μl acetic acid (10% v/v) and the absorbance was quantified at 580 nm. All experiments were performed in triplicate with at least three independent experiments done. Means and SEM were then calculated. The Student's t test was used to evaluate the data.
For cell spreading assays, NBT-II transfected cells were trypsinized, washed 3 times with DMEM, resuspended at a concentration of 0.5 × 106 cells/ml. Cells were then plated on 6 wells plates pre-coated with 10 μg/ml collagen. After incubation at 37°C for the indicated times, attached cells were fixed with 3.8% paraformaldehyde for 20 min at room temperature. Round phase bright, refractile cells were scored as unspread. Phase dark, nonrefractile cells and cells that were elongated were defined as spread. Spreading was calculated as the ratio between spread and attached GFP-positive cells. All experiments were done in duplicate with at least three independent experiments performed. Means and SEM were calculated. The Student's t test was used to evaluate the data.
Cell migration assay
Eighteen hours after transfection with EGFP-tagged constructs, 105 cells were trypsinized and plated on plastic or collagen-coated 35-mm Petri dishes in complete medium. After 4 h, the dish was placed on the motorized stage of a Leica 2D multi-positioning inverted microscope microscopy (Leica Microsystems, Wetzlar, Germany) equipped with a chamber providing controlled temperature and CO2 concentration. Phase-contrast and fluorescent images were acquired from a cool SNAP™ CCD camera (PhotoMetrics, Tucson, AZ, USA). Image acquisition was controlled using the Metamorph software (Universal Imaging Corp., Downingtown, PA, USA). Images of the fluorescently tagged cells were acquired at the beginning of the experiment. Phase-contrast time-lapse movies were then recorded at 4-minutes intervals. Motility of individual cells was evaluated by tracking their movement during 10 h using Metamorph software. The average speed of locomotion (μm/h) was calculated as the total track length divided by the number of hours recorded. For each experimental condition, 50–100 GFP-expressing cells were recorded and analyzed. At least three independent experiments were performed for each condition. Means and SEM were calculated. The Student's t test was used to evaluate the data.
NBT-II cells were trypsinized 24 h after transient transfection and plated on glass cover-slips for an additional 24 h. Cells were then fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 2 min prior to incubation with 100 μM 4'6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA) for 5 min. The cover-slips were mounted in PBS/glycerol+100 mg/ml 1,4-diazabicyclo [2.2.2]octane (DABCO, Sigma) and observed using a 100× objective by 3D immunofluorescence microscopy (Leica Microsystems). Images were deconvoluted and treated using the Metamorph software.
Membrane protrusion dynamics
Membrane protrusion analysis was performed using a 40× objective on a 2D multi-positioning inverted fluorescence microscope (Leica Microsystems). Phase-contrast images were captured at 15 sec intervals for 5 min using a Cool SNAP™ CCD camera. In each experiment, at least 5 fields were analyzed per condition. For each condition, a total of 35–50 cells were scored in 5 independent experiments. Membrane protrusion analysis was done using Image J software and based on a program described elsewhere  with slight modifications. Briefly, a black mask covering the cell was drawn for every plane in the stack. Binary images in which cell was in black against a white background were obtained using the threshold. Subsequent images in the stack were overlaid with each other and a differential image created. Any pixel undergoing a change in gray value was scored as positive. A binary image was then created in which positive pixels were displayed as black against a white background. The total black pixels for a single cell in the 5-minutes interval were then scored. For each experiment, the average number of pixels displaying a change in gray level was determined. It was converted into change in cell area by taking into account the real size of pixels. Two other parameters were analyzed: the number of protrusions per cell was calculated by adding the number of extensions present at the cell periphery in the 21 images of the entire movie. The protrusive index represents the ratio of the change in cell area divided by the number of protrusions.
We thank Dr C. E. Turner for kind gift of paxillin construct and Dr F. Cordelières for development of protrusion analysis plug-in under ImageJ software. This work was supported by the Curie Institute, the Centre National de la Recherche Scientifique and the Astra-Zeneca pharmaceutical company (Manchester, UK).
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