Human Molecular Genetics, 2000, Vol. 9, No. 19 2789-2797
© 2000 Oxford University Press
Huntingtin: an iron-regulated protein essential for normal nuclear and perinuclear organelles
Molecular Neurogenetics Unit, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, USA and 1Howard Hughes Medical Institute and Skirball Institute for Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA
Received 7 July 2000; Revised and Accepted 25 September 2000.
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ABSTRACT |
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Huntington’s disease (HD), with its selective neuronal cell loss, is caused by an elongated glutamine tract in the huntingtin protein. To discover the pathways that are candidates for the protein’s normal and/or abnormal function, we surveyed 19 classes of organelle in Hdhex4/5/Hdhex4/5 knock-out compared with wild-type embryonic stem cells to identify any that might be affected by huntingtin deficiency. Although the majority did not differ, dramatic changes in six classes revealed that huntingtin’s function is essential for the normal nuclear (nucleoli, transcription factor-speckles) and perinuclear membrane (mitochondria, endoplasmic reticulum, Golgi and recycling endosomes) organelles and for proper regulation of the iron pathway. Moreover, upmodulation by deferoxamine mesylate implicates huntingtin as an iron-response protein. However, excess huntingtin produced abnormal organelles that resemble the deficiency phenotype, suggesting the importance of huntingtin level to the protein’s normal pathway. Thus, organelles that require huntingtin to function suggest roles for the protein in RNA biogenesis, trafficking and iron homeostasis to be explored in HD pathogenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntingtin is a novel protein which was discovered because of the elongation of an N-terminal glutamine tract of >37 residues which triggers the loss of striatal neurons in Huntington’s disease (HD), a dominantly inherited disorder (1,2). The expansion confers on the mutant protein a novel attribute (3,4) that may initiate disease by changing an activity of huntingtin or an interacting protein, assuming that these are critical to the targeted neurons. Alternatively, it may act independently of the protein’s normal activity, perhaps by disrupting the function of a cellular constituent that is not a normal interactor.
Conservation in evolution (5) suggests an essential function for the 
350 kDa protein, although this is not evident from its novel sequence which features only multiple HEAT protein interaction domains (6). A broad subcellular distribution, however, implies a function that may involve multiple intracellular sites. The bulk of the protein resides in the cytoplasm (7–12), where some is loosely associated with the membrane (7), but a fraction is also found in the nucleus (11,12). Antibodies have distinguished alternate versions of the protein that are detected in distinct subsets of nuclear and cytoplasmic organelles (13,14), each consistent with a different subset of huntingtin’s binding partners that have implied roles for huntingtin in RNA biogenesis (15–18) and in vesicle trafficking (15,19–21).
Homozygous inactivation of the mouse HD gene, Hdh (22–26) has demonstrated that huntingtin’s function is required for normal embryonic development, during gastrulation (22–25), for extra-embryonic tissue (25) and in neurogenesis (26). In contrast, the protein appears to be dispensable for the growth, viability (22,23,25) and the neuronal differentiation (27) of cultured ‘double knock-out’ embryonic stem (ES) cells, but intriguingly is needed for the production of hematopoietic progenitor cells (28). Although revealing the protein’s essential nature, these analyses of the consequences of huntingtin deficiency at the whole animal and cellular levels have not yielded specific candidate pathways for huntingtin function.
Consequently, we have sought clues to huntingtin’s activity by the identification of organelles that may require the protein. We have conducted a comprehensive survey of the consequences of huntingtin deficiency at the subcellular level, comparing Hdhex4/5/Hdhex4/5 knock-out and wild-type ES cells. We have assessed 19 classes of organelle and have found 6 that require huntingtin for normal morphology and function. These organelles reveal a role for huntingtin in the response to hypoxia and also implicate huntingtin function in specific cellular processes that can be investigated in HD pathogenesis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Select nuclear and perinuclear organelles are abnormal in Hdhex4/5/Hdhex4/5 ES cells
To probe huntingtin function we have investigated whether complete deficiency for this novel protein would perturb organelles that may require its activity. Consequently, we compared parental ES cells that express huntingtin by immunoblot analysis (22) and targeted Hdhex4/5/Hdhex4/5 knock-out ES cells which lack the protein (22), using confocal antibody or lectin staining with a total of 24 markers that detect 19 different classes of organelle. The results of this survey are summarized in Table 1.
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For the majority of the markers, Hdhex4/5/Hdhex4/5 and wild-type ES cells exhibited similar staining patterns. The plasma and nuclear membranes, nuclear coil bodies or splicing-speckles detected by two huntingtin partners, HYPI/symplekin and HYPA/FBP-11, the cytoskeleton, centrosome and distinct endosomes (early, sorting, late) or lysosomes all appeared relatively unaffected by the absence of huntingtin. In contrast, 10 of the markers that probed six kinds of organelle exhibited dramatically different staining patterns. Two of these were nuclear: nucleoli and transcription-speckles; and four others were perinuclear: mitochondrial clusters, the endoplasmic reticulum (ER), Golgi complex and recycling vesicles.
Abnormal nuclear organelles involved in RNA biogenesis
The abnormal marker staining patterns reflected aberrant organelle morphology, typically a reduced size or an altered intracellular distribution. This is illustrated for the affected nuclear organelles in Figure 1. The nucleoli were stained only weakly for fibrillarin and were collapsed necklaces rather than robust clusters. These were located within the nuclei bounded by the lamin A-reactive nuclear envelope. However, transcription factor-speckles defined by huntingtin partners HYPB and N-CoR, in each case, were abnormally localized to the cytoplasm, indicating that huntingtin is needed for the normal nuclear localization of these complexes.
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As these organelles are involved in rRNA and mRNA biosynthesis, we tested cellular attributes that are determined by normal gene expression and protein synthesis. Consistent with the normal growth properties of the knock-out cells, flow cytometry indicated that huntingtin deficiency did not alter either DNA content or cell size, although mini-nuclei were found in rare Hdhex4/5/Hdhex4/5 but not wild-type ES cells (data not shown). Thus, although the protein is essential for normal nucleoli and transcription factor-speckles, huntingtin deficiency appears not to globally disrupt nuclear function.
Exportin 1-dependent export of nuclear versions of huntingtin to the cytoplasm
To characterize the nuclear versions of huntingtin that were implicated by the abnormal nuclear organelles, we tested whether the export to the cytoplasm might involve exportin 1 (crm1) by treating cells with the inhibitor leptomycin B (29,30). To assess the nuclear amino-terminal-accessible version of the protein (14), we first stained wild-type ES cells with reagent AP229. However, the low level of signal in these cells was not suited to the confocal format. Consequently, we examined STHdh+/Hdh+ mouse striatal cells, which exhibit readily detectable AP229-reactive N-terminal-accessible protein in splicing-speckles (14). The results indicated that leptomycin B prevented the cytoplasmic AP229-reactive speckles that were evident in the untreated cells, indicating exportin 1-dependent nuclear export (Fig. 2). This implies nuclear export signals (NES) in huntingtin that are involved in the nuclear–cytoplasmic localization of the 350 kDa protein.
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Abnormal perinuclear mitochondrial clusters
In the cytoplasm our survey detected abnormally distributed mitochondria in the absence of huntingtin. The results of staining for Grp75, a mitochondrial matrix protein, are shown in Figure 3. Perinuclear clusters, that are associated with replication and coordinate transcription of mitochondrial and nuclear genes involved in energy biogenesis (31), were evident in wild-type cells. In contrast, Hdhex4/5/Hdhex4/5 ES cells did not exhibit clusters but instead displayed linear mitochondrial arrays. These arrays were abundant in all cells, however, suggesting normal segregation of the mitochondria after cell division. Consistent with this possibility, staining for
-tubulin demonstrated that the cytoskeleton and microtubule organizing center, which are involved in both segregation and perinuclear cluster formation, were not noticeably altered by huntingtin deficiency (Fig. 3). This finding implies that the normal assembly of mitochondria around the nucleus has some specific requirement for huntingtin.
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Abnormal ER and Golgi in the absence of huntingtin
The size of each perinuclear component of the secretory apparatus was reduced by huntingtin deficiency. Figure 4a illustrates the hearty perinuclear Golgi clusters detected by GM130 and by vicia villosa lectin (VVL) in the wild-type cells. In contrast, the knock-out cells exhibited weak, disperse Golgi membrane (cis and trans) that were, however, located near the lamin B-stained nuclear membrane. Perinuclear signals for the Golgi membrane fusion proteins, ß-COP coatmer protein and Arf1 ADP-ribosylation factor, were also reduced, suggesting impaired trafficking (data not shown). Consistent with this possibility, the Concanavalin A (ConA)-‘stained’ rough ER (Fig. 4b) appeared to be diminished and did not properly extend toward the edges of the cell. To directly test ER–Golgi membrane trafficking, we co-stained cells that had been treated with Brefeldin A, which is an inhibitor of Arf activation (32). The results (Fig. 4b) revealed the expected intermixing of Golgi and ER vesicles in wild-type cells. In contrast, Hdhex4/5/Hdhex4/5 cells exhibited engorged ConA-filled ER balloons, ringed by GM130-reactive dots, that indicated abnormal ER–Golgi membrane fusion.
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A re-orientation assay (33) demonstrated impaired perinuclear translocation of the Golgi apparatus in the absence of huntingtin. The VVL signals in the wild-type ES cells bordering a scrape in the monolayer are aligned, reflecting a repositioning of the Golgi to a perinuclear location that is nearest the extending edge of the cell (Fig. 4c). In contrast, the knock-out ES cells were unable to rapidly shift their weak Golgi, indicating that perinuclear membrane trafficking was impaired.
Abnormal perinuclear recycling endosomes
Perinuclear recycling endosomes were detected by the transferrin receptor, and were also reduced in the cells that lack huntingtin (Fig. 5a), although an over-abundance of signal was found throughout the cytoplasm. Brefeldin A treatment to inhibit membrane fusion revealed diminished perinuclear membrane in the knock-out cells compared with the wild-type cells.
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To determine whether this deficit was restricted to the perinuclear recycling endosomes, we tested the uptake and transport of extracellular FITC-tagged transferrin by ES cells that had been stimulated by the iron chelator, deferoxamine mesylate (34). The results (Fig. 5b) confirmed trafficking of FITC–transferrin to both the early and sorting endosomes in the knock-out ES cells, although a weak perinuclear ligand signal indicated impaired transport to perinuclear recycling endosomes compared with the wild-type cells. Furthermore, consistent with the normal low density lipoprotein (LDL) receptor staining found in the marker survey, the receptor-mediated uptake and transport of extracellular Dil-tagged LDL to the lysosomes via the early–late and sorting endosomes (34) was indistinguishable in knock-out and wild-type ES cells. Of the endosomal compartments, the absence of huntingtin affects primarily the perinuclear recycling endosomes. Thus, huntingtin function is implicated in a perinuclear process that is essential both for the normal trafficking of secretory membrane and for the assembly of mitochondria near the nucleus.
Huntingtin and the cellular iron pathway
The abnormalities in perinuclear transferrin receptor trafficking and mitochondrial cluster-tethering also suggested that iron metabolism might be abnormal in the absence of huntingtin. Therefore, we tested the levels of transferrin receptor in naïve and deferoxamine treated ES cells by immunoblot analyses. Typical results are shown in Figure 6a; these revealed an expected 3.9-fold increase (n = 3) in transferrin receptor levels in the ‘treated’ compared with the ‘untreated’ wild-type lysate. In contrast, the naïve knock-out cell extract exhibited a strong band that was 4.2 fold (n = 3) increased compared with the naïve wild-type lysate. Furthermore, this abnormally increased level was only marginally elevated (1.3-fold; n = 3) by deferoxamine mesylate, implicating huntingtin in the normal regulation of the iron pathway.
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Consequently, we assessed the iron modulation of huntingtin itself by probing the immunoblots in Figure 6a with huntingtin reagent monoclonal antibody (mAb) 2166 (Fig. 6). The
350 kDa protein was not detected in the Hdhex4/5/Hdhex4/5 ES cell extract as expected (22). However, the huntingtin band detected in the naïve wild-type proteins was increased with deferoxamine mesylate treatment by 4.5-fold (n = 3), indicating that huntingtin was upregulated by stimulation of the iron pathway. We then searched the 5' and 3' non-coding regions of the mouse, rat and human HD genes for canonical CAGUGX motifs (35) but we failed to find any that were likely to form the ‘hair-pin’ iron-responsive element (IRE) implicated in the mRNA stabilization of iron response proteins (35). However, searches of the MatInspector matrices (http://www.gsf.de/cgi-bin/matsearch ) with the promoter region sequences (36) identified a core binding site for the HIF-1 hypoxia-inducible transcription factor (AHRARNT). As shown in Figure 6b, this sequence is conserved in the mouse, the rat and the human HD homologs, suggesting a hypoxia response element (HRE). This element is also found in HIF-1 target genes such as those encoding the glucose transporter and transferrin receptor (37), suggesting that this hypoxia transcription factor may also coordinately regulate huntingtin levels.
Overexpressed protein produces a phenotype that resembles Hdh deficiency
To explore whether the level of huntingtin is important for its cellular pathway in ES cells and in striatal cells that are targeted in HD, we assessed the impact of excess protein on organelles that were found to require its function. The wild-type and the double knock-out ES cells and the STHdh+/Hdh+ striatal cells were transiently transfected with HD1-3144Q23, which drives expression of full-length normal huntingtin (37). The results of co-staining of huntingtin with HF1 and either fibrillarin or GM130 reagents to detect nucleoli and Golgi membrane, respectively, are shown in Figure 7. These images demonstrated that both the wild-type ES cells and the striatal cells that overexpressed huntingtin exhibited collapsed nucleoli and abnormal fragmented Golgi compared with their untransfected neighbors. In addition, the overexpressed huntingtin also worsened the abnormal organelles that characterized the knock-out ES cells. In these experiments staining for perinuclear transferrin receptor also demonstrated a reduction in the recycling endosome compartment in all the cell types overexpressing huntingtin (data not shown). Thus, overexpressed huntingtin produced a set of nuclear and perinuclear abnormalities that mirrored huntingtin deficiency, strongly suggesting a dominant-negative impact on huntingtin’s pathway that may reflect the overwhelming of a critical limiting component.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntingtin’s novel sequence does not predict the protein’s physiological role or reveal the mechanism by which the expanded polyglutamine segment in the mutant protein triggers the selective degeneration of striatal neurons. To uncover these processes we have conducted a survey to determine which cellular organelles are chiefly affected by the huntingtin deficiency. Our findings indicate that huntingtin is an iron-regulated protein that is essential for normal nuclear and perinuclear organelles that implicate the protein in iron homeostasis, RNA biogenesis and trafficking, providing a variety of candidates for the protein’s normal and/or abnormal pathway.
Although abnormal columnar epithelial cells in huntingtin-deficient embryos (25) and impaired erythroid progenitors from knock-out ES cells (28) have previously implied a connection, our findings demonstrate an essential role for huntingtin function in iron homeostasis. Huntingtin was required for normal regulation of a key iron protein, transferrin receptor, and in response to iron need was modulated with it. This may involve an HIF-1 binding site that suggests coordinate regulation of huntingtin with diverse hypoxia response proteins at the transcriptional level. Interestingly, normal perinuclear mitochondrial clustering also required huntingtin function, implying a role for perinuclear versions of the protein in properly localizing mitochondria that are importing nuclear products for the linked energy–iron pathways.
Our survey has also revealed a role for huntingtin function in normal membrane trafficking of perinuclear portions of the secretory apparatus (ER, Golgi, recycling endosomes), that may be the same activity that is involved in mitochondrial clustering. This role is consistent with results of antibody localization (7,14) and with a subset of huntingtin-interacting proteins that participate in membrane function (15,19–21). Intriguingly, a version of huntingtin with ‘internal-accessible’ epitopes that colocalizes with perinuclear membranes also resides in the nucleolus (14) and may be important to normal nucleolar morphology that was uncovered in our survey. Moreover, these locations imply that this form of huntingtin may be involved in a process that is essential to nucleoli and to the arrangement of membrane near the nucleus.
The necessity for the function of huntingtin in the normal nuclear localization of its transcription factor partners (HYPB and N-coR), however, may involve an alternate version of huntingtin with amino-terminal-accessible epitopes (11,12,14). This form of the protein colocalizes with nuclear-speckles and the nuclear matrix, consistent with huntingtin’s pre-mRNA splicing and polyadenylation complex factors (15–17) that have supported a role for the protein in RNA biogenesis. Our data indicate that huntingtin is exported from the nucleus to the cytoplasm via an exportin 1-dependent pathway. This may entail conformational properties of huntingtin (14) that may be involved in masking/unmasking of NES motifs, determining the proper distribution of huntingtin in the nucleus and the cytoplasm.
Essential nuclear and perinuclear organelles that require huntingtin function were also disrupted by excess protein. This finding suggests that some critical constituent of huntingtin’s pathway is limiting, implying the importance of regulated huntingtin levels. Exploration of the protein’s normal and abnormal functions, therefore, may require accurately expressed protein. Indeed, the organelles that require huntingtin function implicate specific pathways involved in essential cellular processes, including rRNA and mRNA biogenesis, perinuclear membrane trafficking and iron metabolism, to be investigated in HD patient tissue and in model systems.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell culture and cell assays
R1 wild-type and Hdhex4/5/Hdhex4/5 ES cells have been described previously (22) and were maintained on gelatinized dishes in ES culture medium supplemented with 106 U/l leukemia inhibitory factor (LIF) (ESGRO; Life Technologies, Gaithersburg, MD). Twenty-four hours before an uptake experiment the medium was replaced with fresh ES culture medium containing 4 µM deferoxamine mesylate (Sigma, St Louis, MO). Uptake of fluorescent transferrin and LDL was performed at steady state levels over 30 min as described previously (37). Brefeldin A (Calbiochem, La Jolla, CA) treatment (5 µM) was for 90 min. STHdh+/Hdh+ striatal progenitor cells and their growth at 33°C have been described (14). Leptomycin B treatment was for 2 h (100 ng/ml) at 33°C. Wounding–healing entailed a toothpick scrape through a 90% confluent monolayer (33), followed by incubation at 37°C for 1 h.
Antibodies and fluorescent labels
Ligands used in this study were as follows: fluoroscein-labeled transferrin, Dil-labeled LDL, Texas Red-conjugated ConA and biotinylated VVL (Molecular Probes, Eugene, OR). Antibody reagents used in this study were as follows: GM130, caveolin, NuMA, symplekin (Transduction Laboratories, San Diego, CA); lamin A, lamin B, N-CoR, Rabs 1A, 5A and 6 (Santa Cruz Biotechnology, Santa Cruz, CA); AF-1 for HYPA and BF-1 for HYPB (16); fibrillarin, actin, - and 
-tubulin, dynein and ß-COP (Sigma); anti-syntaxin 1A (StressGen Biotechnologies, Victoria, Canada); huntingtin mAb 2166 (Chemicon, Temecula, CA), HF1 (37) and spectrin (Chemicon); rat mAb transferrin receptor (Tfn R; Biosource International, Camarillo, CA). Secondary antibodies conjugated to horseradish peroxidase or fluorescent labels were from Amersham Lifesciences (Piscataway, NJ) and Jackson ImmunoResearch (West Grove, PA), respectively.
Immunofluorescence, fluorescence-activated cell sorting (FACS) and confocal microscopy
ES cells were fixed in 4% paraformaldehyde, permeabilized for 5 min in 0.1% Triton X-100 in phosphate-buffered saline (PBS), treated for 10 min with blocking solution (1% bovine serum albumin in PBS) and incubated for 90 min in blocking solution containing primary antibodies/lectins. After several washes in PBS, cells were incubated for a further 1 h in blocking solution containing secondary antibodies and then rinsed in PBS. Cells were examined with a BioRad (Hercules, CA) MRC-1024 laser confocal microscope using 20x and 40x objective lenses. Digitized images for each field were saved as separate files for each channel and were merged using Adobe PhotoShop.
Transfection
Full-length huntingtin constructs HD1-3144Q23 and HD1-3144Q113 (40) were introduced into ES cells seeded at densities of 5 x 103 cells per 24-well plate using the SuperFect Transfection kit (Qiagen, Valencia, CA) and immunofluorescence was examined 72 h after transfection.
Immunoblot analysis
Soluble proteins were extracted from PBS-washed ES cells by needle sheering in buffer containing 50 mM Tris–Cl pH 7.5, 10% glycerol, 5 mM magnesium acetate, 0.2 mM EDTA and Complete Protease Inhibitors (Roche Diagnostics, Indianapolis, IN), followed by three freeze–thaw cycles and centrifugation for 2 min at 17 110 g. Supernatants (25 µg) were boiled for 5 min in Laemmli loading buffer, separated on a 6% SDS–polyacrylamide gel and transferred onto nitrocellulose membranes. Proteins were detected by chemiluminescence (KPL Laboratories, Gaithersburg, MD) following incubation with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Quantitation was by densitometry of transferrin receptor or huntingtin band (on non-saturating exposures) and normalization to the spectrin (fodrin) band in the same lanes.
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ACKNOWLEDGEMENTS |
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We thank Drs J.F. Gusella and T. Greenamyre for critical discussion, Drs A. Bernards and B. Terns for the gifts of E-cadherin and fibrillarin antibodies and Dr M. Yoshida for leptomycin B. L.A.P. is supported by a fellowship from the Hereditary Disease Foundation. The research was supported by NIH grants NS32765 and NS16367 (Huntington’s Disease Center Without Walls), a grant from the Foundation for the Care and Cure of Huntington’s Disease and Telethon, Italy.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 617 726 5089; Fax: +1 617 726 5735; Email: macdonam@helix.mgh.harvard.edu
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REFERENCES |
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