School of Kinesiology, University of Illinois at Chicago, Chicago, Illinois 60608
Submitted 7 August 2003 ; accepted in final form 13 November 2003
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ABSTRACT |
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skeletal muscle injury; heat shock protein 25; B-crystallin; dystrophin; desmin
Lengthening contractions can produce immediate damage to muscle cell structures including z-disks and other force-bearing cytoskeletal elements (16, 39). Muscle cross sections show loss of the intermediate filament desmin in specific fibers immediately after lengthening contractions in rabbit tibialis anterior (TA) and extensor digitorum longus (EDL) muscles (30). In addition, dystrophin staining becomes discontinuous at the membrane in certain fibers, but desmin staining is not lost, immediately after lengthening contractions in rat TA muscles (26). Immediately after downhill running in human vastus lateralis muscle, Western blot analysis showed decreased levels of -sarcoglycan but not of actin or desmin (15). The disparate findings between studies may be due to differences in injury protocols, assay methods, or species. The first aim of the present study was to determine whether, in the mouse EDL muscle, lengthening contractions produce immediate loss of specific cytoskeletal elements associated with the z-disk and force transmission from the myofibril to the membrane.
Small heat shock proteins (HSPs), B-crystallin, and HSP25 are constitutively expressed in skeletal muscle, but their precise functions in muscle remain largely undefined. Small HSPs are thought to play a role in muscle development (5) and their protein levels are increased after low-frequency electrical stimulation (38) and eccentric exercise (15, 47), whereas protein levels are decreased after hindlimb suspension (4). In other cell types, small HSPs protect against cytoskeletal disruption, as overexpression of small HSPs protect against disruption induced by a variety of agents (27, 43, 48, 49).
B-crystallin knockout mice have been reported to develop late-onset skeletal muscle degeneration (6), supporting a role for
B-crystallin in maintaining muscle integrity. A review of the literature revealed no studies on the function of small HSPs in skeletal muscle immediately after injurious lengthening contractions. The second aim of this study was to determine whether small HSPs translocate to structures prone to disruption in skeletal muscle after lengthening contractions.
Because cytoskeletal disruption is thought to occur after lengthening contractions and the small HSPs have been shown in other cell types to limit disruption of cytoskeletal elements, small HSPs may be activated during lengthening contractions to limit damage to muscle structures. The purposes of this study were to determine whether, immediately after a protocol of lengthening contractions, 1) levels of specific force-transmitting cytoskeletal proteins (myosin, -actinin, plectin, desmin, dystrophin,
-spectrin, and
-dystroglycan) are reduced in skeletal muscle cells and 2) cytosolic small HSPs (
B-crystallin and HSP25) translocate to the cytoskeleton at structures prone to disruption. Western blot analysis was used as a semiquantitative assay for cytoskeletal protein levels after lengthening contractions, and for small HSP localization in soluble and insoluble fractions of muscle homogenates. Immunofluorescence analysis was used to support the Western blot data and to provide further information about small HSP localization.
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MATERIALS AND METHODS |
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Injury protocol. Mouse EDL muscles were exposed to lengthening or isometric contractions as described by Koh and Brooks (25a). Briefly, mice were anesthetized with an intraperitoneal injection of 2% avertin (tribromoethanol; 400 mg/kg). Boosters of avertin were given if the mouse responded to a toe pinch. A small incision was made at the right ankle, and the distal tendons of the EDL muscle were exposed. The mouse was then placed on a heated platform maintained at 37°C. The hindlimb was stabilized by fixing the distal femur between sharpened screws and securing the foot with tape to the platform. The intact tendon was tied with 4-0 silk to the lever arm of a servomotor (model 305B; Aurora Scientific), which controlled the length of the muscle and measured the force developed by the muscle. The EDL muscle was activated via the peroneal nerve by using an isolated stimulator (model S44; Grass Instruments), and needle electrodes were placed transcutaneously adjacent to the nerve. Pulse duration was kept constant at 0.2 ms, whereas stimulation voltage, frequency, and muscle length (Lo) for maximum isometric force (PO) were determined separately for each mouse. Optimal muscle fiber length (Lf) was calculated by multiplying Lo by the Lf/Lo ratio of 0.44 (33). EDL muscles were then exposed in situ to 75 lengthening or isometric contractions of 300-ms duration performed at 0.25 Hz for a total exercise duration of 5 min. Lengthening contractions were initiated from the plateau of an isometric contraction elicited by 150-Hz stimulation at Lo and were of 20% strain relative to Lf, at a velocity of 1 Lf/s. Isometric contractions were elicited by 150-Hz stimulation at Lo. Force deficits were calculated as the decrease in PO produced 10 min after the protocol compared with PO before injury expressed as a percentage of preinjury values. Immediately after the experiment, muscles were collected, and mice were killed by cervical dislocation while anesthetized.
Sample preparation. EDL muscles were dissected, blotted dry, weighed, and processed for analysis by 1) SDS-PAGE or isoelectric focusing (IEF) followed by Western blotting, 2) oligomerization, or 3) immunofluorescence. For SDS-PAGE and IEF, muscles were homogenized by using a Dounce homogenizer in TE buffer (50 mM Tris·HCl, pH 7.4, with 5 mM EDTA) supplemented with phosphatase and protease inhibitors (1 mM Na3VO4, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, and 0.3 µM aprotinin). Samples were centrifuged at 100,000 g for 30 min at 4°C, the soluble fraction was collected, and the insoluble fraction was resuspended in TE buffer. For oligomerization assays, muscles were homogenized in TE buffer with 1 mM DTT, and 10% (vol/vol) glycerol was supplemented with phosphatase and protease inhibitors. Samples were centrifuged at 10,000 g for 20 min at 4°C, and soluble fractions were collected for assays. For immunofluorescence, muscles were mounted in tissue-freezing media, and were frozen in isopentane chilled with dry ice.
SDS-PAGE. Cytoskeletal protein and small HSP levels in soluble and insoluble fractions of muscle homogenates were determined by using SDS-PAGE. Soluble and insoluble fractions were mixed with concentrated SDS-PAGE sample buffer (to final concentrations of 50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, and 1 mM glycerol) and boiled, and protein concentrations determined (35). Equal amounts of protein (10 µg for small HSPs and desmin; 20 µg for all other proteins) were separated on 6 or 10% SDS-PAGE gels and transferred to nitrocellulose membranes in SDS transfer buffer (25 mM Tris, 192 mM glycine, 0.05% SDS, and 20% methanol).
IEF. Small HSP phosphorylation in soluble and insoluble fractions was assessed by using IEF (13). Three bands are detected when probing for small HSPs in IEF Western blots. The most basic band on the IEF blot is the nonphosphorylated form, whereas the most acidic band is the most phosphorylated (17, 24). Soluble and insoluble fractions were mixed with concentrated IEF sample buffer [to final concentrations of 7 M urea, 1.5% Triton X-100, 1.5% Nonident P-40 (NP-40), 1.5% CHAPS, 3% 6/8 ampholytes, 0.75% 3/10 ampholytes, and 100 mM -mercaptoethanol]. Equal amounts of sample were separated on IEF gels (5% acrylamide, 7 M urea, 2% Triton X-100, 2% NP-40, 2% CHAPS, 6% 6/8 ampholytes, and 1.5% 3/10 ampholytes) by using running buffer composed of 25 mM L-arginine and 25 mM L-lysine for the upper chamber and 25 mM phosphoric acid for the lower chamber and then transferred to nitrocellulose membranes in IEF transfer buffer (0.7% acetic acid, 10% methanol).
Western blotting. After transfer, membranes were stained with Ponceau-S to confirm equal loading and to determine myosin concentrations in muscle homogenates. Membranes were then blocked in 5% milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20) overnight. Membranes were then incubated 3 h with primary antibodies against -actinin, desmin (both 1:500; Sigma), plectin (1:3,000; a kind gift from Dr. Gerhard Wiche, University of Vienna),
-spectrin (1:2,000; a kind gift from Dr. Robert Bloch, University of Maryland), dystrophin (COOH terminus or rod domain epitope, both 1:500, Novocastra),
-dystroglycan (1:500; Novocastra), HSP25,
B-crystallin, HSP72 (1:3,000, StressGen), or HSPB2 (a kind gift from Dr. Atsushi Suzuki, Yokohama City University School of Medicine). Blots were washed with TBST and then incubated with secondary antibody conjugated to horseradish peroxidase (1:25,000; Pierce). After another wash, protein bands were detected by using enhanced chemiluminescence (Amersham), and band densities were determined by image analysis (Bio-Rad FluorS). For cytoskeletal proteins, individual band densities were normalized to the average of the control lanes for the insoluble fraction and multiplied by 100%. For small HSPs, individual band densities were normalized to the sum of the band densities of the soluble and insoluble fractions and multiplied by 100%.
Oligomerization. Oligomerization of small HSPs was determined by using size exclusion chromatography. Soluble fractions of the homogenates of four EDL muscles (1.85 mg protein) were applied to a size exclusion column (TosoHaas TSK G3000 SWXL, 7.8-mm ID x 300 mm, separation range 10-500 kDa) equilibrated with buffer containing 100 mM potassium phosphate, pH 6.8, and 150 mM NaCl. The column was calibrated by using thyroglobulin (669 kDa), apoferritin (443 kDa), b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and lysozyme (14 kDa). Samples were separated at a flow rate of 1.0 ml/min and 2-ml fractions were collected. Twenty microliters of the collected fractions were spotted onto nitrocellulose membranes and small HSPs were detected by using procedures identical to those used for Western blotting. Small HSP antibodies were highly specific, staining only a single band in SDS-PAGE Western blots. Thus this spot blot method was deemed sufficient for detecting HSPs in fractions from the size exclusion column. Spot densities of small HSPs were determined for each fraction by image analysis, normalized to the sum of the densities of all fractions and multiplied by 100%.
Immunofluorescence. To provide support for the Western blot analysis data on altered levels of cytoskeletal proteins and HSPs, and to provide further information about their localization, immunofluorescence analysis was performed on muscle cryosections. Cross sections and longitudinal sections were cut at 10 µm from tissue blocks with a cryostat. Sections were fixed in acetone and blocked for 1 h with a solution containing 0.2% gelatin and 3% bovine serum albumin. Sections were then incubated with primary antibody overnight (1:100 for dystrophin, -dystroglycan, 1:200 for all other antibodies), washed with phosphate buffered saline, and incubated with secondary antibody (1:200) conjugated with fluorescein label. Sections were then washed and mounted in antiphotobleaching medium (Vector) and viewed by using epifluorescence (Nikon Labophot-2). For double labeling, before mounting sections were incubated with a second primary antibody overnight, washed, and incubated with secondary antibody conjugated with rhodamine. Double-labeled sections were viewed by using laser scanning confocal microscopy (Zeiss LSM 510). For semiquantitative analysis, two independent observers examined two sections each from three control muscles and from three muscles exposed to lengthening contractions. For each section, each observer counted the numbers of fibers that demonstrated overtly altered staining patterns after lengthening contractions (reduced staining of cytoskeletal proteins, altered localization of small HSPs), normalized this number to the total number of fibers in the section, and normalized values averaged were between observers.
Statistical analysis. Densitometry data passed tests for normality and equal variance (SigmaStat). Data for cytoskeletal proteins were analyzed with t-tests to determine whether lengthening contractions resulted in decreased levels of cytoskeletal proteins. Data for small HSPs were analyzed with analysis of variance to determine whether lengthening contractions induced translocation of small HSPs from soluble to insoluble fractions of muscle homogenates. The 0.05 level was used to indicate statistical significance.
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RESULTS |
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Densitometric analysis of Western blots demonstrated decreased levels of specific force-bearing cytoskeletal proteins in homogenates of muscle exposed to lengthening contractions compared with contralateral control muscles (Fig. 1). Lengthening contractions produced decreases in levels of the z-disk proteins -actinin and plectin and in the membrane scaffolding proteins dystrophin and
-spectrin, in the insoluble fraction of muscle homogenates. In contrast, lengthening contractions produced little or no change in levels of myosin, desmin, or
-dystroglycan. Decreases in dystrophin levels were similar whether blots were probed with a monoclonal antibody targeting the COOH terminus or the rod domain. Decreases in protein levels in the insoluble fraction were not associated with increases in the soluble fraction. Muscles exposed to isometric contractions showed no evidence of changes in levels of cytoskeletal proteins (not shown).
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Muscle cross sections stained with haematoxylin and eosin demonstrated little evidence of gross injury immediately after lengthening contractions; the most noticeable alterations were separation of fibers in specific parts of the muscle (Fig. 2). This separation of fibers suggests disruption of elements connecting fibers with the extracellular matrix. Supporting the Western blot data demonstrating cytoskeletal disruption, -actinin, and plectin showed overt loss of cytoplasmic staining in 1.0 ± 0.2 and 1.5 ± 0.7%, respectively, of muscle fibers after lengthening contractions. Dystrophin and
-spectrin showed overt loss of staining at the membrane in 13 ± 4 and 14 ± 7%, respectively, of muscle fibers. Because these proteins showed fiber-to-fiber variability in staining intensity in control muscles, only fibers with complete loss of staining in at least part of the fiber were counted in this analysis. That the percent decrease in protein level as determined by Western blotting was greater than the percentage of fibers demonstrating loss of protein in immunostained sections suggests that a number of fibers experienced loss of protein that was not clearly evident in these sections. The loss of staining in specific fibers is consistent with the idea that not all fibers are injured to the same extent after lengthening contractions.
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In control muscles, small HSPs were localized primarily in the soluble fraction of muscle homogenates (Fig. 3). Immediately after lengthening contractions, small HSPs showed translocation from the soluble to the insoluble fraction of homogenates; HSP25 showed equal distribution between soluble and insoluble fractions after lengthening contractions (Fig. 3, A and B), whereas B-crystallin showed more complete translocation to the insoluble fraction (Fig. 3, A and C). Muscle exposed to isometric contractions showed no evidence of HSP translocation (not shown). Lengthening contraction-induced translocation appeared to be specific to HSP25 and
B-crystallin as another small HSP, HSPB2, and the widely studied HSP72, did not show similar translocation (Fig. 3A). In longitudinal and cross sections of contralateral control muscles, HSP25 and
B-crystallin showed diffuse cytoplasmic staining (Fig. 4), with faint localized staining at striations in 5 ± 3 and 7 ± 3%, respectively, of muscle fibers. After lengthening contractions, HSP25 and
B-crystallin showed localization at striations in 40 ± 2 and 53 ± 4%, respectively, of muscle fibers in longitudinal sections and at specific sites in the cytoplasm and the membrane in 45 ± 7 and 49 ± 6%, respectively, of fibers in cross sections. Confocal microscopic analysis of longitudinal sections double stained for each small HSP and desmin suggested that HSP25 and
B-crystallin translocated to sites near the z-disk after lengthening contractions and the small HSPs appeared to colocalize with each other (Fig. 5).
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Translocation of small HSPs was associated with their phosphorylation and some evidence of a reduction in oligomer size. In control muscles, small HSPs were primarily in the nonphosphorylated form, the most basic band identified in IEF Western blots (Fig. 6A). After lengthening contractions, HSP25 showed reduced levels of the nonphosphorylated form and increased levels of phosphorylated forms associated with their translocation to the insoluble fraction (Fig. 6B). B-crystallin showed lesser amounts of lengthening contraction-induced phosphorylation, but phosphorylated forms were only detected in the insoluble fraction (Fig. 6C). In control muscle, cytoplasmic HSP25 and
B-crystallin were eluted from a size exclusion column predominantly at apparent molecular masses corresponding to monomers and small oligomers (Fig. 7). Because small HSPs are typically found in large oligomers (200-800 kDa) in unstressed cell cultures of various cell types (23, 25), we sought to determine whether small HSPs were also found in small oligomers in cultured skeletal muscle cells (C2C12 myotubes). Small HSPs in unstressed C2C12 myotubes (4 days postdifferentiation in DMEM supplemented with 2% horse serum) eluted at masses corresponding to large oligomers (
600 kDa) consistent with unstressed cultured cells of other types. These data suggest that the assay methods did not cause dissociation of large oligomers and indicate a unique localization of small HSPs to small oligomers in unstressed skeletal muscle. Lengthening contractions were associated with some evidence of a further reduction in localization of small HSPs to large oligomers and an increase in localization to small oligomers (Fig. 7).
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DISCUSSION |
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The z-disk links neighboring sarcomeres in series and transmits force produced by contractile proteins both longitudinally and laterally. A number of studies have demonstrated disruption of z-disks immediately after lengthening contractions (16, 39). -Actinin is a major component of z-disks and has recently been identified as a component of the dystrophin complex at the membrane (20). Plectin is an intermediate filament cross-linking protein thought to mediate interactions of desmin with z-disk and membrane proteins (1). Plectin-deficient mice die 2-3 days after birth with evidence of skeletal muscle degeneration. The loss of
-actinin and plectin immediately after lengthening contractions is consistent with z-disk disruption and sarcomere disorganization demonstrated previously (16, 39). In addition, because both
-actinin and plectin are also found at the membrane, loss of
-actinin and plectin may also reflect damage to the membrane induced by lengthening contractions (34).
Dystrophin and spectrin are membrane scaffolding proteins thought to stabilize the membrane against mechanical stresses. Dystrophin-deficient muscle is particularly susceptible to contraction-induced membrane rupture and such mechanical fragility is thought to be a primary mechanism for the initiation of the muscle degeneration in muscular dystrophy (40, 46). A previous study (26) demonstrated loss of dystrophin at the membrane in some muscle fibers immediately after lengthening contractions, and our results suggest that substantial amounts of dystrophin and spectrin are degraded immediately after lengthening contractions. Similar decreases in dystrophin were found when probing with monoclonal antibodies targeting either the COOH terminus or the rod domain, supporting the idea that lengthening contractions induced degradation of dystrophin. The loss of dystrophin and spectrin may have a number of consequences including increased membrane fragility and disrupted signaling pathways (40, 41), which may contribute to the injury process after lengthening contractions.
Desmin intermediate filaments connect z-disks of neighboring myofibrils to each other and are thought to help maintain sarcomere and myofibril organization and desmin knockout mice show disruption of skeletal muscle architecture (8, 29). Previous studies (30) have demonstrated loss of desmin staining in specific muscle fibers after 150 lengthening contractions of rabbit dorsiflexor muscles. However, loss of desmin was not observed after 240 lengthening contractions of rat dorsiflexor muscles (26). In the present study, desmin staining was reduced in specific fibers, but no changes in desmin levels were observed in Western blots immediately after lengthening contractions. These findings suggest that desmin filaments may be disrupted in specific fibers in our model, but that desmin protein degradation is either absent or below detectable levels in Western blots.
The loss of dystrophin, spectrin, -actinin, and plectin immediately after lengthening contractions may be a result of calpain proteolysis. Calpains are calcium-activated neutral proteases thought to be responsible for initiating degradation of many cytoskeletal proteins (11). Dystrophin, spectrin, and plectin have been previously identified as calpain substrates in vitro (10, 12, 37), and
-actinin is released from z-disks by calpain (19). Previous studies have demonstrated that resting intracellular calcium concentrations are increased after lengthening contractions (22, 31), and that calpain activity is increased in the insoluble fraction of muscle homogenates after 60 min of treadmill running (3). Such an increase in calpain activity after lengthening contractions in the mouse EDL muscle could contribute to the loss of cytoskeletal proteins in the present study.
Because lengthening contractions induce cytoskeletal disruption and HSP25 and B-crystallin have been shown to protect different types of cells against cytoskeletal disruption induced with various agents (27, 43, 48, 49), these small HSPs may help to protect muscle cells against contraction-induced injury. Immediately after lengthening contractions, small HSPs showed translocation from the soluble to the insoluble fraction of muscle homogenates, with more complete translocation of
B-crystallin, and translocation from diffuse cytoplasmic staining to striations coinciding with z-disks in longitudinal sections and to the membrane in cross sections. Because the z-disk and the membrane have been identified as sites prone to disruption after lengthening contractions in this and previous studies (16, 39), small HSP translocation to these sites may reflect a protective role against further disruption or a role in repair of damaged structures. A previous study (4) demonstrated loss of
B-crystallin from skeletal muscle during hindlimb suspension-induced atrophy and the authors speculated that
B-crystallin may help to maintain z-disk and myofibrillar integrity.
Similar to the situation in skeletal muscle after lengthening contractions, ischemia induces z-disk disruption in cardiac muscle and translocation of small HSPs from soluble to insoluble fractions of tissue homogenates, and increases localization of these small HSPs to sites near the z-disk (13, 18). After ischemia, B-crystallin was found colocalized with the T21 epitope of titin, suggesting that
B-crystallin may bind titin near the z-disk after ischemia (18). The localization of small HSPs near z-disks after lengthening contractions in the present study is also consistent with binding of titin although there are certainly other candidate binding partners (e.g., desmin, actin).
Lengthening contractions were associated with phosphorylation of both HSP25 and B-crystallin, with greater phosphorylation of HSP25. In general, the role of phosphorylation on small HSP function is unclear, with some studies showing that phosphorylation is required for cytoprotective functions (21, 28) and other studies showing that phosphorylation has little effect on cytoprotection (32, 42). These disparate results may be due to the use of different cell types, different modes of stress, and/or the specific cytoprotective function measured.
Phosphorylation of small HSPs may trigger their protective activity toward the cytoskeleton. In Chinese hamster CCL39 cells, overexpression of wild-type HSP27 (the homologue of HSP25) conferred thermotolerance and increased actin filament stability to heat, cytochalasin D, and hydrogen peroxide, whereas overexpression of nonphosphorylatable mutant HSP27 conferred only limited protection (21, 28). In another study (43), overexpression of wild-type HSP27 and phosphorylation-mimicking mutant HSP27 protected actin filaments in Chinese hamster ovary cells from cholecystokinin-induced disruption, but overexpression of nonphosphorylatable mutant HSP27 did not protect these cells. Thus phosphorylated HSP27 may stabilize actin filaments or other cytoskeletal elements in skeletal muscle against disruption after lengthening contractions.
Oligomerization of HSP25 and B-crystallin may also play a role in regulating their activity. These small HSPs are typically found in unstressed cells as large oligomers of unphosphorylated monomers of 200-800 kDa (
40 monomers), whereas stress events result in HSP phosphorylation and reduction in oligomer size (23, 25). However, in control skeletal muscle, the small HSPs were found in monomers or small oligomers, despite being predominantly in the nonphosphorylated form. This finding contrasts with the situation in cultured C2C12 myotubes, in which small HSP oligomer sizes were similar to those previously reported for other cell types (200-800 kDa). The localization to small oligomers may be specific to skeletal muscle, because
B-crystallin was localized in large oligomers in cardiac muscle tissue (13). The functional significance of localization to small oligomers is unclear, but small HSPs localized in small oligomers may be the form that interacts with cytoskeletal elements and protects the cytoskeleton from stress (49).
In conclusion, force-bearing cytoskeletal structures are disrupted immediately after lengthening contractions, including loss of z-disk and membrane scaffolding proteins. Lengthening contractions also induce phosphorylation and translocation of HSP25 and B-crystallin to the z-disk and membrane. Because these small HSPs have been shown previously to stabilize cytoskeletal elements, HSP25 and
B-crystallin may act to protect the z-disk and membrane from further injury or aid in their repair.
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NOTE ADDED AT PROOF |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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