Human Molecular Genetics, 2000, Vol. 9, No. 19 2845-2851
© 2000 Oxford University Press
The Menkes copper transporter is required for the activation of tyrosinase
Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, 221 Burwood Highway, Burwood 3125, Australia
Received 14 July 2000; Revised and Accepted 14 September 2000.
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ABSTRACT |
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Menkes disease is an X-linked recessive copper deficiency disorder caused by mutations in the ATP7A (MNK) gene. The MNK gene encodes a copper-transporting P-type ATPase, MNK, which is localized predominantly in the trans-Golgi network (TGN). The MNK protein relocates to the plasma membrane in cells exposed to elevated copper where it functions in copper efflux. A role for MNK at the TGN in mammalian cells has not been demonstrated. In this study, we investigated whether the MNK protein is required for the activity of tyrosinase, a copper-dependent enzyme involved in melanogenesis that is synthesized within the secretory pathway. We demonstrate that recombinant tyrosinase expressed in immortalized Menkes fibroblast cell lines was inactive, whereas in normal fibroblasts known to express MNK protein there was substantial tyrosinase activity. Co-expression of the Menkes protein and tyrosinase from plasmid constructs in Menkes fibroblasts led to the activation of tyrosinase and melanogenesis. This MNK-dependent activation of tyrosinase was impaired by the chelation of copper in the medium of cells and after mutation of the invariant phosphorylation site at aspartic acid residue 1044 of MNK. Collectively, these findings suggest that the MNK protein transports copper into the secretory pathway of mammalian cells to activate copper-dependent enzymes and reveal a second copper transport role for MNK in mammalian cells. These findings describe a single cell-based system that allows both the copper transport and trafficking functions of MNK to be studied. This study also contributes to our understanding of the molecular basis of pigmentation in mammalian cells.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Menkes disease is an X-linked recessive disorder of copper metabolism, which is lethal during early childhood (1). Menkes patients suffer a systemic copper deficiency due to malabsorption of dietary copper in the small intestine and defective distribution within the body. Affected individuals have reduced copper levels in the brain, liver and serum, but elevated copper in the kidney and small intestine (2). The clinical features of Menkes disease include severe neurological problems, connective tissue defects, hypothermia and hypopigmentation (2). These pleiotropic symptoms can be attributed to the reduced activities of a range of copper-dependent enzymes, which include cytochrome c oxidase, dopamine ß hydroxylase, lysyl oxidase and tyrosinase (2). The gene defective in Menkes disease (ATP7A; MNK) encodes a protein that has all the features of P-type ATPases, a family of proteins that transfer cations through the lipid bilayer of membranes using the energy derived from the hydrolysis of ATP (3–5). A role for MNK in copper efflux was suggested by observations that cultured fibroblasts from Menkes patients accumulate copper (6). More recent evidence is the association of an increased ability to efflux copper with overexpression of the hamster MNK ATPase in Chinese hamster ovary (CHO) cell lines, selected for copper resistance (7) and in CHO cell lines stably expressing the human MNK cDNA (8). MNK has a steadystate localization at the trans-Golgi network (TGN) (9–11). This finding led to the hypothesis that MNK may have a role in copper transport to copper-dependent enzymes that are synthesized within the secretory pathway. These copper-dependent enzymes include tyrosinase, lysyl oxidase and dopamine ß hydroxylase.
In this study we show that expression of MNK is essential for the activity of tyrosinase, which is a copper-dependent enzyme that is involved in the formation of melanin pigment. Tyrosinase mutations are responsible for oculocutaneous albinism (type 1), a human disorder characterized by reduced or absent skin pigmentation, vision defects due to reduced optical pigmentation, misrouting of optical nerve fibres and enhanced sensitivity to skin and optical cancers (12). Tyrosinase initiates melanin formation by catalyzing the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to DOPA-quinone (12). Tyrosinase is expressed in melanocytes and is localized within specialized organelles known as melanosomes. The enzyme has a single membrane-spanning region and is orientated such that its catalytic copper-binding domain is located on the lumenal side of the secretory pathway (12,13). This catalytic domain has two copper-binding sites and the activity of tyrosinase requires the incorporation of copper into both sites of the apo-enzyme during its synthesis (14). In order for copper to be made available to apo-tyrosinase, it must be transported from the cytoplasm into the secretory pathway. We hypothesized that this copper transport role is carried out by MNK. In this study, tyrosinase was expressed from a cDNA construct in immortalized Menkes disease fibroblasts and was found to have very low levels of activity compared with its activity in normal fibroblast cell lines. Significantly, the tyrosinase activity in Menkes fibroblasts was greatly enhanced when the MNK protein was co-expressed from a cDNA construct. This MNK-dependent tyrosinase activity was impaired by chelation of copper in the cell culture medium and when a mutation predicted to inhibit copper transport was introduced into the highly conserved phosphorylation site of MNK. These observations suggest that MNK has a role in delivering cytoplasmic copper into the secretory pathway for tyrosinase and provide an experimental system in cultured mammalian cells for studying the copper-loading of secreted cuproenzymes. The significance of these findings in our understanding of the aetiology of Menkes disease is discussed.
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RESULTS |
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Tyrosinase immunofluorescence detection and in situ activity in normal and Menkes fibroblasts
Tyrosinase has been recently shown to be active when ectopically expressed from transfected plasmids in non-melanocytic cell types such as fibroblasts, HeLa, COS and CHO cells (14–17). Given our hypothesis that MNK delivers copper to tyrosinase, we predicted that tyrosinase activity would be reduced when tyrosinase was expressed from a plasmid construct in the immortalized Menkes fibroblasts relative to the normal fibroblasts. The tyrosinase cDNA expression plasmid, pcTYR, was transiently transfected into two immortalized Menkes fibroblast cell lines, Me32 and Me52, that have no detectable MNK protein, as well as two immortalized control fibroblasts, GM2069 and GM847, that we had shown previously to express MNK protein (18). Expression of tyrosinase in each cell line was confirmed 48 h after transfection by immunofluorescence microscopy using a monoclonal anti-tyrosinase antibody. In each cell line, tyrosinase was detected predominantly in punctate structures associated with the juxtanuclear region and extended into the cytoplasm (Fig. 1A, C, E and G). These structures were consistent with a lysosomal localization of tyrosinase reported previously in non-melanocytic cell types (17,19). There was no immunoreactivity detected with the anti-tyrosinase antibody in untransfected cells (unpublished data).
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The in situ tyrosinase activity was visualized in the transfected cell lines using the colourimetric L-DOPA reaction of tyrosinase which involves the formation of the brown-coloured DOPA-chrome, from the colourless substrate, L-DOPA (17,20). Many pigmented cells were observed within the population of the normal fibroblast cell lines, GM2069 and GM847 (Fig. 1F and H), indicating substantial tyrosinase activity. However, in the population of both Me32 and Me52 cells, there was no pigmentation of cells after they were incubated in L-DOPA, suggesting a markedly reduced tyrosinase activity in these cells (Fig. 1B and D). Pigmentation was not observed in untransfected cells incubated in L-DOPA (unpublished data). These data suggest a role for the Menkes protein in the activation of tyrosinase by transporting copper into the secretory pathway to apo-tyrosinase.
Analysis of the in situ tyrosinase activity in Me32 cells stably expressing wild-type MNK protein or the MNK D1044E mutation
To test more rigorously for the requirement of MNK in the activation of tyrosinase, we stably transfected Me32 cells with the normal MNK cDNA construct, pCMB117, and tested whether tyrosinase was activated when the pcTYR plasmid was transiently expressed in these cells. The subcellular distribution of tyrosinase was similar between Me32 cells and both clones of Me32 stably expressing normal MNK protein, MeMNK1 and MeMNK2 (Fig. 2A, C and E). After incubation of cells with L-DOPA substrate, the MeMNK1 and MeMNK2 cells were stained intensely (Fig. 2D and F), whereas the Me32 cells remained unstained (Fig. 2B). These observations suggested that there was an MNK-dependent activation of tyrosinase. To characterize this activation further we tested whether a mutant form of MNK, containing a D1044E substitution that was predicted to prevent copper transport activity, could activate tyrosinase. This aspartic acid is conserved in all members of the P-type ATPase family and is phosphorylated during the reaction cycle of these proteins (21). Two Me32 cell lines, MeD/E1 and MeD/E2, stably expressing the D1044E mutant construct were clonally purified and then transiently transfected with the pcTYR plasmid to enable expression of tyrosinase (Fig. 2G and I). In both the MeD/E mutant cell lines there was no pigment formation after incubation of cells in L-DOPA (Fig. 2H and J). This result suggested that the D1044E mutation impaired the MNK-dependent activation of tyrosinase.
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A comparison of tyrosinase activity with the tyrosinase and MNK protein levels
The in situ results suggested a role for MNK in tyrosinase activation. However, it was important to control for levels of tyrosinase and Menkes protein across all cell lines to ensure that protein expression differences were not responsible for the differences in tyrosinase activity. To this end, tyrosinase activity in cell extracts was measured in non-reducing polyacrylamide gels and compared with levels of tyrosinase and MNK protein detected on western blots (Fig. 3A). In both MeMNK1 and MeMNK2 cell lines there was strong DOPA-oxidase activity of tyrosinase, as evidenced by an intense band of activity at the expected size of 70 kDa. However, in the Me32 cell line transiently transfected with the pcTYR plasmid, DOPA-oxidase activity was barely detectable. There was an elevated DOPA-oxidase activity in the MeD/E1 and MeD/E2 cell lines relative to the Me32 cell line, but this was substantially lower than the activity in MeMNK1 and MeMNK2 cells. There was no DOPA-oxidase activity in cells that were not transfected with the pcTYR plasmid (unpublished data). These results suggest there was an MNK-dependent activation of tyrosinase, which was impaired by mutation of the conserved aspartic acid residue 1044 of MNK. Immunoblot experiments on the same samples demonstrated that tyrosinase protein levels were within a similar range across each line (Fig. 3). This was also found for the levels of MNK protein within the MeMNK1, MeMNK2, MeD/E1 and MeD/E2 cell lines (Fig. 3). There was no MNK protein within the Me32 cell line, as shown previously (18). Hence, the differences in tyrosinase activity across the cell lines were not due to large variations in tyrosinase or MNK protein expression.
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Melanin levels were also measured from those cells shown in Figure 3A. Melanin levels in the pcTYR-transfected Me32 cells increased
0.2-fold relative to untransfected Me32 cells, whereas melanin levels increased 8.1- and 7.0-fold in MeMNK1 and MeMNK2 cells, respectively (Fig. 3B). This magnitude of increased melanogenesis in pcTYR-transfected MeMNK1 and MeMNK2 cell lines was consistently observed over five independent experiments that were controlled for tyrosinase protein levels (unpublished data). Melanin levels increased 0.9- and 1.0-fold in both MeD/E1 and MeD/E2 cell lines relative to untransfected Me32 cells, but were substantially lower than in both MeMNK1 and MeMNK2 cells. Collectively, these data support the hypothesis that MNK transports copper into the secretory pathway to allow the formation of active holo-tyrosinase and melanogenesis.
Analysis of the MNK-dependent tyrosinase activity in MeMNK1 cells depleted of copper
The MNK-dependent activation of tyrosinase was further investigated by testing whether this process was copper dependent. We determined whether chelation of copper in the growth medium of MeMNK1 cells affected tyrosinase activity. MeMNK1 cells were transiently transfected with the pcTYR expression plasmid and cultured for 48 h in the presence of the copper chelator, bathocuproein disulphonate (BCS). There was a marked reduction in tyrosinase activity in BCS-treated cells relative to untreated cells (Fig. 4). However, there was no such reduction when cells were incubated in medium containing both BCS and excessive copper. These data suggest that the MNK-dependent tyrosinase activity in MeMNK1 requires copper and are consistent with the hypothesis that MNK delivers copper into the secretory pathway to apo-tyrosinase.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In cultured cells, the MNK protein is localized in the TGN and this finding led to speculation that MNK may transport copper into the secretory pathway to copper-dependent enzymes as they are synthesized (9–11). In the yeast Saccromyces cerevisiae Ccc2p is a copper-transporting P-type ATPase which transports copper into the late-Golgi to Fet3p, a protein involved in iron uptake (22,23). A previous finding that MNK can transport copper to Fet3p and complement the ccc2 yeast mutant provided additional support that the MNK protein may transport copper to secreted cuproenzymes in mammalian cells (24). The results of our study provide the first direct evidence that this role for the MNK protein exists in mammalian cells and that this process is defective in Menkes disease. Our results provide a molecular explanation for the mosaic hypopigmentation defects which occur in human female heterozygotes of Menkes disease and the series of mottled mice, the animal models of Menkes disease (2).
Transient expression of tyrosinase in the immortalized cell lines from Menkes patients resulted in reduced tyrosinase activity compared with the normal fibroblast cell lines (Fig. 1). This reduced tyrosinase activity in Me32 cells was complemented by the expression of the wild-type MNK protein (Figs 2 and 3). The MNK-dependent activation of tyrosinase was prevented by the chelation of copper in the growth medium, suggesting that this process requires copper (Fig. 4). Mutation of the conserved aspartic acid (D1044) within the phosphorylation domain impaired the ability of MNK to activate tyrosinase and resulted in a lower level of melanin formation compared with the wild-type protein (Figs 2 and 3). This suggested that the complete activation of tyrosinase required a catalytically active MNK copper transporter. This result is consistent with a previous finding that the conserved aspartic acid in the MNK homologue of Caenorhabditis elegans is required to complement the yeast ccc2 mutant (25). It was notable, however, that there was a small increase in both tyrosinase activity and melanogenesis in the MeD/E1 and MeD/E2 cells relative to the Me32 parental cells (Fig. 3) and this was consistently observed in five independent experiments (unpublished data). This increase in tyrosinase activation could be due to some residual copper transport activity by the D1044E MNK protein. Alternatively, the overexpression of this protein using our experimental system may permit a limited amount of copper to diffuse through the channel into the secretory pathway to tyrosinase. Nevertheless, these data collectively support the hypothesis that MNK transports copper into the secretory pathway to apo-tyrosinase, thereby allowing the formation of the active holo-enzyme. These findings provide the first direct evidence that the Menkes protein is involved in the biosynthesis of secreted copper-dependent enzymes in mammalian cells.
The enzymatic activity of tyrosinase is dependent on the presence of two copper atoms in the active site and this is underscored by the observation that mutations in the copper-binding site of tyrosinase cause type 1 oculocutaneous albinism (14). Tyrosinase folds through several intermediate conformations in the endoplasmic reticulum (ER) and the ER chaperone, calnexin, is involved in this maturation process (17,26). Recent studies demonstrate that, if the association of tyrosinase with calnexin is prevented, the enzyme is folded more rapidly and fails to acquire copper (26). These findings suggest that the copper loading of tyrosinase requires the enzyme to be pre-folded in the correct conformation. Our findings suggest that the MNK protein delivers copper into the secretory pathway for the copper loading of tyrosinase, but the precise mechanism by which this process occurs is unknown. Recent studies using yeast as a model suggest that copper is unlikely to exist as the free ion in the cytoplasm (27). Copper in this form can generate highly toxic hydroxyl radicals, which can damage membranes and other cellular components (28). Hence, it is unlikely that a pool of free copper ions is used in the activation of copper-dependent enzymes (27). Based on this information, we propose two models for the direct incorporation of copper into copper-dependent enzymes of the secretory pathway such as tyrosinase. In the first model, copper emerging from the MNK protein into the lumen of the secretory pathway may be directly incorporated into apo-tyrosinase that is specifically bound to lumenal regions of the MNK protein. Alternatively, copper entering the secretory pathway may be specifically transferred from the MNK protein to an intermediate copper chaperone, which subsequently surrenders this copper to apo-tyrosinase via a specific interaction. This latter model is analogous to those chaperone-mediated pathways for transferring copper between proteins in the cytoplasm (29,30). Testing whether the lumenal loops of MNK are involved in binding to tyrosinase will be the focus of future studies investigating the mode of MNK-mediated transfer of copper to cuproenzymes of the secretory pathway.
In summary, our findings demonstrate that the MNK protein functions in the activation of copper-dependent enzymes of the secretory pathway in mammalian cells. These data provide a biological basis for the location of MNK in the TGN. Together with its role in regulating copper efflux from the plasma membrane, the discovery of a biosynthetic role for MNK reveals a second function for the copper transporter in mammalian cells and highlights the importance of this molecule in copper metabolism. Our findings support the hypothesis that the underlying pathogenesis of Menkes disease is caused by defective copper transport to cuproenzymes in the secretory pathway, as well as reduced copper efflux from cells. The mammalian cell-based experimental model presented in this study represents a significant advance in the study of MNK, because it allows copper-dependent trafficking of MNK to be assessed along with copper transport to tyrosinase and copper efflux. Hence, this system will enable the identification of the regions of MNK essential for each of these facets of MNK biology and provide an understanding of how these processes are related. Furthermore, the use of this system to identify which aspects of MNK biology are affected by disease-causing MNK mutations may provide a biochemical explanation for the clinical differences observed in classical Menkes disease and the less severe variants, occipital horn syndrome and mild Menkes.
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MATERIALS AND METHODS |
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Plasmid constructs
The MNK mammalian expression construct, pCMB117, used in this study has been described previously (18). A 2.0 kb EcoRV subclone of pCMB117, pCMB129, was used as a template to generate the D1044E mutation using the Transformer mutagenesis kit (Clontech, Palo Alto, CA). The introduced mutation was verified by manual sequencing using the Sequenase kit (Amersham Pharmacia Biotech, Cleveland, OH). The mutated fragment was cloned into the full-length MNK expression construct to create the plasmid pCMB134. The tyrosinase expression plasmid, pcTYR, has been described previously (15).
Cultured cells
All cell lines were maintained at 37°C in Eagle’s basal medium supplemented with 10% fetal bovine serum (Trace Biosciences, Castle Hill, Australia) and 0.2% (w/v) bicarbonate. The Me32 and Me52 cell lines were derived from Menkes patient skin biopsies and were immortalized by SV40 gene transfer as described previously (18). Both Menkes cell lines have no detectable MNK protein. The GM2069 and GM847 control fibroblast cell lines were also SV40-immortalized and were previously shown to express MNK protein (18). BCS (Sigma, St Louis, MO) and CuCl2 were added to the growth medium of cells at 200 and 300 µM, respectively, as indicated in the figure legends.
Transfections and immunofluorescence microscopy
Lipofectamine 2000 (Gibco BRL, Gaithersburg, MD) was used to transfect cells according to the recommended protocol. Cells cultured in 75 cm2 flasks were transiently transfected with 10 µg of the pcTYR plasmid. For stable transfection of MNK constructs, Me32 cells were transfected with 30 µg of pCMB117 (wild-type MNK) or pCMB134 (D1044E mutation) and selected in medium containing 500 µg/ml G418 (Gibco BRL) for 14 days. Colonies were screened for MNK expression by immunofluorescence microscopy using the anti-MNK antibody as described previously (31). Immunofluorescence detection was carried out as previously described on cultured cells fixed on glass coverslips (31). Tyrosinase was detected using the T311 monoclonal anti-tyrosinase antibody (1:300) (32). Anti-MNK and anti-tyrosinase antibodies were detected using Alexa488-conjugated secondary antibodies (1:2000) (Molecular Probes, Eugene, OR). Images were visualized with an Olympus AX70 microscope (Tokyo, Japan) using a x60 oil objective and images were captured using a RT Slider CCD camera (Diagnostic Instruments, Sterling Heights, MI).
In situ tyrosinase activity
In situ tyrosinase activity was colorimetrically detected in cultured cell lines as previously described by assaying the formation of DOPA-chrome (brown) from the oxidation of L-DOPA (colourless) (17). Cells on coverslips were transfected with the pcTYR expression construct, washed twice in phosphate-buffered saline (PBS) and then fixed for 30 s in acetone/methanol (1:1) at –20°C. These fixation conditions do not destroy the activity of tyrosinase (17). Coverslips were then incubated for 1 h at 37°C in phosphate buffer (0.1 M pH 6.8) containing 0.15% (w/v) L-DOPA (Sigma) to allow the formation of the brown DOPA-chrome pigment. Coverslips were mounted on slides and cells were observed by phase contrast light microscopy (x60 objective).
Tyrosinase PAGE assay, melanin assay and immunoblots
The colorimetric DOPA-oxidase assay measuring the second catalytic activity of tyrosinase, the conversion of L-DOPA to DOPA-chrome, was assayed within polyacrylamide gels and has been extensively described previously (17,33). Briefly, 48 h after transfection with the pcTYR plasmid, cells cultured in 75 cm2 flasks were scraped with a rubber policeman into PBS at 4°C and divided evenly into two tubes and pelleted. The cells in one tube were used for melanin measurements, whilst those from the remaining tube were used for the tyrosinase PAGE activity assay and western immunoblots. Cells for the tyrosinase PAGE activity assay were lysed in Laemmli buffer without reducing agents and protein levels were estimated using the BCA Protein Assay kit (Pierce, Rockford, IL). Samples were separated on a 7.5% polyacrylamide SDS gel and gels were equilibrated in 50 mM phosphate buffer (pH 6). Colorimetric staining was carried out by incubating gels for 15 min at 37°C in 10 mM phosphate buffer (pH 6.8) containing 1.5 mM L-DOPA and 4 mM 3-methyl-2-benzothiazolinone hydrazone (Sigma). For immunoblot detection of MNK and tyrosinase proteins, 20 µg of protein lysates were mixed in loading dye containing 100 mM ß-mercaptoethanol and separated using 7.5% SDS–PAGE. Proteins were transferred to nitrocellulose membranes and MNK and tyrosinase proteins were detected using anti-MNK antibodies (1:1000) or anti-tyrosinase antibodies (1:500) using a chemiluminescence detection kit (Boehringer Mannheim, Mannheim, Germany) according to the specified protocol. Melanin content within cells was determined as described previously (20). Briefly, the remaining half of the pelleted cells that were not used for tyrosinase PAGE activity were solubilized in 0.2 M NaOH to provide a protein concentration of 5 µg/µl. Melanin levels were determined spectrophotometrically within 200 µg of each sample by measuring the optical density at 475 nm.
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ACKNOWLEDGEMENTS |
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We thank Richard Spritz for providing the pcTYR plasmid and Julie Stevenson, Michelle Howie, Natalie Barnes and Paul Gilson for critically reading the manuscript. This work was supported by funding from the National Health and Medical Research Council and the International Copper Association.
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FOOTNOTES |
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+ To whom correspondence should be addressed at present address: Department of Nutritional Sciences, Gwynn Hall, University of Missouri-Columbia, Columbia, MO 65211, USA. Tel: +1 573 882 9685; Fax: +1 573 882 0185; Email: petrism@missouri.edu
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