Departments of 2 Kinesiology and Applied Physiology and 1 Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309
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ABSTRACT |
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The fast skeletal IIb gene is the source of most myosin heavy chain (MyHC) in adult mouse skeletal muscle. We have examined the effects of a null mutation in the IIb MyHC gene on the growth and morphology of mouse skeletal muscle. Loss in muscle mass of several head and hindlimb muscles correlated with amounts of IIb MyHC expressed in that muscle in wild types. Decreased mass was accompanied by decreases in mean fiber number, and immunological and ultrastructural studies revealed fiber pathology. However, mean cross-sectional area was increased in all fiber types, suggesting compensatory hypertrophy. Loss of muscle and body mass was not attributable to impaired chewing, and decreased food intake as a softer diet did not prevent the decrease in body mass. Thus loss of the major MyHC isoform produces fiber loss and fiber pathology reminiscent of muscle disease.
myosin; muscle development; locomotion; motor behavior; dystrophy
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INTRODUCTION |
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MYOSIN HEAVY
CHAIN (MyHC) is both structurally and functionally one of the
most important proteins expressed in skeletal muscle. Three adult fast
isoforms of MyHC, along with type I or -MyHC, are expressed in
different types of fibers that are named for the type of MyHC they
express, i.e., IIB, IID, IIA, and type I (13). Fibers
expressing these different isoforms of MyHC have been shown to have
distinct contractile properties, sizes, and metabolic profiles
(2, 3, 14). These studies have suggested that the
different isoforms of MyHC are functionally distinct, despite the
extremely high amino acid identity among the three adult fast genes
(~93-94%; see Ref. 15). In adult murine skeletal muscle three fast isoforms, IIa, IId, and IIb, are expressed in varying
percentages in different muscles. Type IIb MyHC, which accounts for
~70-80% of the total MyHC, is the predominant isoform expressed
in adult mouse skeletal muscle (6). Previously, we reported the generation of mice containing a null mutation in either
the IId or IIb MyHC gene (1). These mice are viable but
demonstrate a number of morphological and physiological alterations, many of which are distinct between the two strains, despite the fact
that total MyHC content was unchanged in either the IIb or IId null
mice (1). Analysis of mice lacking expression of the IId
MyHC gene revealed that the IIa MyHC isoform is upregulated to
compensate for the loss of IId MyHC, resulting in normal total MyHC
content (12). Despite this upregulation of IIa MyHC
expression, IId null mice have considerable muscle pathology and spinal
kyphosis (11). These studies have provided a convincing
argument in favor of the hypothesis that different MyHC isoforms have
unique roles in the growth and morphology of skeletal muscle.
In the present study, we have analyzed mice in which the IIb MyHC isoform gene has been rendered null by homologous recombination to determine the morphological consequences of the loss of this gene. We demonstrate that mean body mass was significantly lower in mice lacking the IIb MyHC gene but that this was not a consequence of inability to chew hard rodent chow, because feeding IIb null mice a softer diet did not abrogate the loss in mean body mass over time. We also demonstrate that loss of the IIb MyHC gene results in a significant decrease in mean total fiber number in the gastrocnemius, tibialis anterior (TA), extensor digitorum longus (EDL), plantaris, and vastus muscles and a corresponding hypertrophy of the remaining fibers in an attempt to compensate for this fiber loss. Finally, we observed evidence of substantial fiber pathology, including degeneration and regeneration, in certain muscles of the hindlimb of IIb null mice. Together these data support a role for IIb MyHC in the maintenance of normal muscle growth and structure.
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MATERIALS AND METHODS |
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Animals.
The generation of IIb MyHC null mice has been described previously
(1). Ten male wild-type mice and ten male mice homozygous for a null mutation in the IIb MyHC gene were weighed and killed at 6 wk of age by cervical dislocation. Muscles of the hindlimb (TA, EDL,
gastrocnemius, soleus, plantaris, quadriceps) and head (tongue,
masseter) were dissected, weighed, and frozen in isopentane cooled in
liquid nitrogen. Muscles were stored at 70°C until use.
Fiber percentages, fiber size, and fiber number.
For immunohistochemical analysis, 10-µm sections were cut from
the muscle midbelly using a cryomicrotome, placed on gelatin-coated slides, and stored at 70°C until use. Slides were air-dried for 30 min at room temperature followed by blocking for 1 h at room temperature in PBS containing 0.12% BSA, 0.1% nonfat dry milk, and 5 mg/ml purified Fab fragments (Jackson Immunoresearch, West Grove, PA).
After several rinses with PBS, sections were incubated either at room
temperature for 1 h or overnight at 4°C in primary antibody
solution. The primary antibodies used were as follows and at the
following dilutions: 1) MHCs, which recognizes type I/
MyHC (Novocastra, Newcastle-on-Tyne, UK), 1:20; 2) SC-71, which recognizes type IIa MyHC (5) 1:3; 3)
BF-F3, which recognizes type IIb MyHC (5), 1:3; and
4) BF-35, which recognizes all but type IId MyHC, and thus
pure IId-expressing fibers are unstained (5). After
primary antibody incubation, the sections were rinsed several times
with PBS and then incubated for 1 h at room temperature in
secondary antibody solution consisting of PBS plus secondary antibody
diluted 1:100. The secondary antibody was either goat anti-mouse
IgG-peroxidase conjugate (for MHCs, SC-71, and BF-35) or goat
anti-mouse IgM-peroxidase conjugate (for BF-F3 and RT-D9), both from
Jackson Immunoresearch. Sections were rinsed several times in PBS and
visualized using a DAB reaction kit with nickel enhancement (Vector
Laboratories, Burlingame, CA), dehydrated by serial washes with
ethanol, and mounted in Permount (Fisher Scientific, Pittsburgh, PA).
Histology. Muscle histology was assessed in the gastrocnemius muscle using immunohistochemical markers for muscle regeneration. Frozen sections were air-dried for 30 min and then rinsed in PBS for 5 min and stained with one of the following antibodies: 1) F1.652, which recognizes embryonic MyHC and 2) MCA-VIM, which recognizes the intermediate filament protein vimentin (Sigma Chemical, St. Louis, MO). After several rinses with PBS, sections were stained with the appropriate secondary antibody conjugated to peroxidase and visualized using either a DAB or an AEC reaction kit (Vector Laboratories).
Electron microscopy. From the histological studies mentioned above, it was evident that the gastrocnemius muscle demonstrated considerable histopathology. We therefore chose the gastrocnemius for ultrastructural analysis. Samples of gastrocnemius from wild-type and IIb null mice (n = 2/group) were processed for electron microscopy following standard protocols. Small pieces from the superficial and deep portions of freshly isolated gastrocnemius muscles were placed in 2% glutaraldehyde for 2 h and then embedded and stained using standard electron microscopy protocols. All samples were processed by the Electron Microscopy Core of the University of Colorado.
Analysis of feeding behavior. Feeding behavior was studied in age-matched wild-type and IIb null mice. Animals were divided into the following five different groups (n = 4/group): wild-type mice fed a standard hard rodent chow, wild-type mice fed a commercially available soft pet food, IIb null mice fed hard rodent chow, IIb null mice fed a powdered form of the standard hard rodent chow, and IIb null mice fed soft pet food. Each day for 2 wk each animal was weighed. In a separate experiment, the amount of food consumed per 24 h was estimated by weighing the food each day for 1 wk.
Statistical analyses.
All results are reported as means ± SD in Tables 1-3 and as
means ± SE in Figs. 1-3. Statistical significance was
assessed using one-way ANOVA combined with Fisher's protected least
significant difference post hoc test to compare wild-type with IIb null
parameters. P < 0.05 was used to indicate statistical
significance.
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RESULTS |
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Fiber percentages. Given that >70% of the skeletal MyHC in the mouse is IIb, we determined the percentage of fibers expressing the different MyHC isoforms in these null mice. Staining with the BF-35 antibody, which recognizes all sarcomeric isoforms except IId, revealed an increase in the number of BF-35-negative fibers. In all muscles except the soleus and the vastus intermedius, the percentage of these BF-35-negative fibers, which we consider to be IID fibers, was significantly increased (Table 1). In the gastrocnemius muscle, the percentage of IID fibers increased from 11 to 82% in the IIb null mice (Table 1). In addition, the percentage of fibers expressing IIa MyHC was also increased in most analyzed muscles, but to a lesser extent (Table 1). In the gastrocnemius muscle, the percentage of MyHC IIa-expressing fibers increased from 6 to 16%; similar increases in the percentage of IIa-expressing fibers occurred in most muscles of the hindlimb (Table 1). An even smaller increase of ~5% occurred in the percentage of fibers expressing type I MyHC in the hindlimb muscles of IIb null mice (Table 1). Thus an increase in the number of IID fibers is the major adaptation to the loss of IIb MyHC gene expression.
Mean body and muscle mass. As previously described (1), mean absolute body mass was significantly smaller in age-matched IIb null mice compared with wild-type mice (Fig. 1A). In addition, the absolute masses of numerous muscles of IIb null mice were also significantly smaller than those of wild-type mice (Fig. 1A). The magnitude of this decrease appeared to depend on the amount of IIb MyHC normally expressed in a given muscle in the wild-type mouse. For example, muscles that contained a high percentage of type IIb MyHC such as the gastrocnemius or quadriceps experienced the greatest decrement in absolute muscle mass (up to 83%). Muscles containing a slightly smaller percentage of type IIb MyHC such as TA and EDL demonstrated a smaller decrement in absolute muscle mass (~50-60%). Mean muscle mass for muscles that in the wild type express very low percentages of type IIb MyHC such as the soleus were not significantly different from the wild type (P > 0.05; Fig. 1A). Examination of relative muscle mass revealed that only muscles containing a high percentage of IIb MyHC in the wild-type mouse, the TA, gastrocnemius, and quadriceps, had significantly lower relative mass compared with the wild type (Fig. 1B). Thus the elimination of IIb MyHC expression results in atrophy to specific muscles in the mouse hindlimb.
Normal feeding and locomotor behavior of IIb null mice.
To examine the possibility that impairment in masticatory muscle
function caused a reduction in food consumption, we examined the effect
of feeding a softer food on the increase in body mass postweaning. Both
wild-type and IIb null mice fed the soft chow increased in body mass to
a greater extent than mice fed the standard hard rodent chow (Fig.
2A). However, IIb null mice
fed soft food, powdered rodent chow, or rodent chow pellets all gained
weight to a lesser extent than wild-type mice under all conditions
(Fig. 2A). We also quantified the amount of food eaten per
24 h and found no significant difference between wild-type and IIb
null mice (3.54 and 3.86 g · mouse1 · day
1,
respectively; Fig. 2B). When food intake was normalized to
body weight, IIb null mice actually consumed more food per body mass (0.159 vs. 0.239 g · mouse
1 · day
1 · g
body mass
1 for wild-type and IIb null mice, respectively).
Fiber CSA is increased in the IIb null mice. Given that muscle mass was significantly lower in IIb null mice compared with wild type mice, one logical hypothesis is that fiber CSA was reduced in the IIb null mice. However, mean fiber CSA was significantly larger in all fiber types in the IIb null mice (Table 2). For example, in the gastrocnemius muscle, the type I, IIA, and IID fibers in the IIb null mice were 20, 56, and 75% larger than the same fiber types in the wild type, respectively (Table 2). CSA of IId fibers of IIb null mice was comparable to that of IIb MyHC-expressing fibers in the wild type (1,607 vs. 1,748 µm2 in the gastrocnemius of IIb null and wild-type mice, respectively). This was also true for the EDL, TA, plantaris, and quadriceps muscles (Table 2).
Fiber number is decreased in IIb null mice. To examine whether a decrease in fiber number was responsible for the decrease in mean muscle mass in the IIb null mice, estimates of total fiber number were made based on values for muscle CSA, interfiber space, mean fiber size, and mean fiber percentages. Total fiber number was significantly lower in all hindlimb muscles in the IIb null mice except the soleus and vastus intermedius muscles (Table 3). The percentage decrease in total estimated fiber number for the gastrocnemius muscle was extremely similar to the percentage decrease in mean muscle mass (66 vs. 64%, respectively), suggesting that the loss in fiber number could account for most of the loss in mean muscle mass. Moreover, direct counts of the number of fiber profiles in an entire cross section through the muscle midbelly of the TA also revealed a significant decrease in total fiber number (2,597 ± 135 vs. 1,919 ± 194 in the wild type and IIb null mice, respectively). Thus the decrease in mean muscle mass appears to be a consequence of a decrease in fiber number rather than fiber size.
A decrease in mean fiber number could come about either as a result of decreased fiber formation during development or by actual loss of fibers after myogenesis is completed. To address which of these was responsible for the decrease in mean fiber number observed in adult IIb null mouse muscles, we examined the hindlimb muscles of IIb null mice at various postnatal time points. Because the muscle fibers are very immature and difficult to distinguish from one another at very early time points, direct fiber counts were extremely difficult to obtain. Nonetheless, qualitative examinations of wild-type and IIb null hindlimb muscles revealed no overt evidence of insufficient fiber formation or muscle pathology at early postnatal time points (data not shown). In addition, we measured type I fiber CSA as an indirect measure of muscle fiber loss, assuming that fiber CSA increased in the IIb null animals as a compensatory hypertrophy as a result of fiber loss. Examination of muscle sections from mice at 1, 5, 10, and 20 days postnatal revealed that type I CSA was not significantly different during the early postnatal period (Fig. 3). Type I CSA was significantly increased compared with the wild type starting at 10 days postnatal (Fig. 3). This suggests that before this time there was no fiber hypertrophy because muscle fiber loss was probably minimal (see below).Ultrastructural and histological analyses reveal pathology in the
IIb null mice.
Immunostaining of adult wild-type hindlimb muscles with antibodies to
laminin revealed a regular pattern of normal-sized muscle fibers (Fig.
4A); however, in certain
hindlimb muscles of the adult IIb null mouse, small profiles
reminiscent of severely atrophied and/or degenerating/regenerating
fibers were observed (Fig. 4D). These heterogeneous fiber
profiles were most evident in hindlimb extensor muscles ordinarily
containing a large percentage of IIB fibers such as the quadriceps and
the gastrocnemius muscles. Moreover, the pathological area in these
muscles was usually restricted to the most superficial regions of the
muscle, which tend to be 100% IIb in the wild-type mouse.
Immunostaining with antibodies to embryonic MyHC and vimentin, two
markers of skeletal muscle regeneration, demonstrated the presence of
embryonic MyHC and/or vimentin positive myotubes in the superficial
quadriceps and gastrocnemius of IIb null mice (Fig. 4, E and
F). The percentage of embryonic MyHC-positive fibers was
very low in the gastrocnemius muscle of IIb null mice; nevertheless,
neither embryonic MyHC nor vimentin immunostaining was ever observed in
muscle fibers of adult wild-type mice at this time point (Fig. 4,
B and C).
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DISCUSSION |
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In the present study, we examined the consequences of a null mutation in the IIb MyHC gene to elucidate the role of this isoform in the generation of normal muscle structure. In mice, the IIb MyHC accounts for ~70-80% of the total MyHC expressed in all muscles and is therefore quantitatively the most prominent of the adult fast isoforms. Our results demonstrate that the null mutation of the IIb MyHC gene results in a number of dramatic effects on skeletal muscle growth and morphology in the adult mouse.
Loss of IIb MyHC results in an increase in IId fibers. Immunohistochemical staining using the BF-35 antibody, which recognizes all except the IId MyHC isoform, demonstrated that the null mutation of the IIb MyHC gene resulted in a significant increase in the percentage of so-called "pure" fibers expressing only IId MyHC (Table 1). Given the lack of an antibody specific to just the IId MyHC, we were unable to evaluate the extent of coexpression of the IIb and IId isoforms in the wild-type mouse. This is important; one way an increase in the number of IID fibers could come about is if there is substantial coexpression of IIb and IId MyHC in the wild type that is lost with loss of the IIb MyHC, resulting in IId fibers by default without any change in IId gene expression. However, this is probably not the case, since we previously showed using quantitative high-resolution gel electrophoresis that IId MyHC levels are significantly increased in the IIb null mouse compared with wild-type mice (Allen and Leinwand, unpublished observation). Thus the increase in type IID fibers is a specific result of an increase in IId MyHC protein expression compared with wild-type mice.
IIb null mice have normal feeding patterns.
Previously, we reported that the normal increase in mean body mass
during postnatal growth is attenuated in IIb null mice. This is first
seen around the age at which mice are weaned and begin to consume hard
rodent chow. In the present study, analysis of body mass in IIb null
mice fed softer food diets revealed that body mass was not rescued by
these presumably more consumable diets, and mean body mass remained
decreased compared with wild-type mice (Fig. 2). Moreover, the absolute
amount of food consumed per 24 h was not significantly different
between wild-type and IIb null mice (3.54 vs. 3.86 g · mouse1 · day
1 for
wild-type and IIb null mice, respectively; Fig. 2B). This was somewhat surprising considering the smaller overall body size of
the IIb null mice. However, it is possible that IIb null mice may have
a higher metabolic rate necessitated by the requirement to regenerate
fibers lost to degeneration (see below). These data suggest that
feeding ability is not overtly impaired by the null mutation in the IIb
gene and that the decrement in mean body mass must arise as a more
direct consequence of the mutation of this gene on the growth of the
muscles themselves.
The decrease in muscle mass is due to a loss of fibers in the IIb null mouse. Mean absolute skeletal muscle mass was significantly lower in a large number of muscles in the IIb null mice compared with wild-type mice (Fig. 1A). Muscles such as the quadriceps and gastrocnemius demonstrated the greatest reduction in mean muscle mass, and muscles such as TA and EDL demonstrated a slightly lower decrease in mean absolute muscle mass compared with the wild type, whereas the soleus muscle was not significantly different from the wild type (Fig. 1A). Thus the magnitude of the decrease in mean absolute muscle mass for a given muscle appears to be related to the percentage of IIb MyHC that would ordinarily be expressed in that muscle. Moreover, relative muscle mass was significantly lower in only three hindlimb muscles, the TA, gastrocnemius, and quadriceps muscles (Fig. 1B). These muscles contain the highest percentage of IIb MyHC in the wild-type mouse and moreover were the muscles demonstrating the most pathology in the IIb null mice.
There are three ways in which a decrease in muscle mass could come about. First, all fiber types could be reduced in size in the IIb null mice compared with the wild type, resulting in decreased muscle mass. However, quantitative measurement of fiber CSA revealed that fiber size was actually increased in types I, IIA, and IID fibers in the IIb null mice compared with wild-type mice (Table 2), suggesting that reduced fiber size cannot account for the loss in muscle mass. Second, it is possible that the fibers that would ordinarily be IIB in the IIb null mice were unable to reach their normal size due to a compensatory shift in MyHC expression in the IIb null mice. This was a particularly attractive hypothesis given that the IIB fibers in the wild-type mouse are ordinarily the largest fibers in most muscles and were significantly larger than IID fibers (Table 2), which are the MyHC isoforms upregulated in the IIb null mice (Table 1). However, our results show that the IID fibers in the IIb null mice were significantly larger than IID fibers in the wild type and comparable in size to IIB fibers in the wild type. The final possibility is that the decrease in mean muscle mass arose as a consequence of a decrease in the number of fibers, and the results of the present study are consistent with this hypothesis. Mean total fiber number was significantly lower in muscles from the IIb null mice compared with wild-type mice. Furthermore, the increase in fiber CSA in all fiber types in the IIb null mice is consistent with a decrease in muscle fiber number; the remaining fibers undergo a compensatory hypertrophy, presumably as a consequence of the increased activity of the remaining fibers due to fiber loss. These data suggest that the compensatory hypertrophy of the remaining fibers is insufficient to prevent a decrease in mean muscle mass brought about by the decrease in mean fiber number in these animals. The decrease in fiber number could conceivably come about in one of two ways. First, it is possible that disruption of the IIb MyHC gene results in disregulation of normal fiber formation during prenatal and early postnatal development, resulting in fewer fibers formed a priori. Second, it is possible that the elimination of IIb MyHC results in some critical defect in fiber function that causes fibers to degenerate and die in the adult animal. Several lines of evidence support the latter conclusion. 1) As mentioned, the decrease in mean body mass (and presumably muscle mass as a result of fiber loss) is progressive, becomes more salient during late postnatal development, and is not evident at birth (1). 2) Histological examination of muscle from early postnatal development revealed no evidence of significant compensatory fiber hypertrophy until ~10-20 days of age (Fig. 3). 3) Immunohistochemical and ultrastructural analysis of muscle from adult IIb null and wild-type mice revealed substantial pathology in these muscles consistent with fiber loss (Figs. 4 and 5). Regarding this latter point, both anti-laminin immunostaining and electron microscopy revealed evidence of fiber pathology in the superficial aspects of limb extensors such as the gastrocnemius (Figs. 4 and 5). In addition, immunostaining with antibodies to vimentin and embryonic MyHC, developmental markers that are ordinarily not expressed in adult muscle except during periods of regeneration, revealed an increase in regenerating myotubes in the muscle of IIb null mice compared with control (Fig. 4). The mechanism(s) responsible for producing this pathological response in the muscles of adult IIb null mice is not currently known. One possibility is that, in the IIb null mice, the muscle fibers that express type IId MyHC but would ordinarily express IIb MyHC are not able to withstand the mechanical loading normally experienced by these fibers. However, why these fibers are less able to withstand strain simply as a consequence of a shift in MyHC isoform expression is not clear, unless this shift is accompanied by a change in expression of other fiber-specific proteins involved in the generation and transmission of contractile force. Specifically, maximum force normalized to CSA tends to be lower in fibers expressing IId MyHC, whereas force normalized to MyHC content is not different between IId and IIb fibers (4). Another possibility is that the shift in MyHC isoform expression causes a mismatch between the contractile components of the muscle and the metabolic mechanisms that provide energy for muscle contraction; this in turn could cause excessive generation of reactive metabolic intermediates, which results in fiber damage and degeneration. We are currently testing both of these hypotheses to further elucidate the mechanisms responsible for this degenerative phenotype. Another intriguing possibility is that loss of the IIb MyHC results in disruption of the establishment and/or maintenance of normal innervation by the motoneurons. Motoneurons typically innervate fibers of the same type (7), and the loss of the predominant MyHC expressed in adult mouse muscle may result in an inability of the motoneurons innervating the IIb fiber pool to recognize the resulting IId MyHC-expressing fibers. The presence of three adult fast MyHC isoforms in the adult mouse has prompted the question of whether these genes are functionally redundant, as is observed in some members of the myogenic regulatory factor family (11), or whether they are functionally distinct. Studies on isolated skeletal muscle fibers have demonstrated differences in contractile parameters in fibers containing different MyHC isoforms (2, 3, 10), suggesting that these fibers were functionally distinct. The present study on IIb null mice and studies on mice lacking the ![]() |
ACKNOWLEDGEMENTS |
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We thank Jill Jones with help in preparation of this manuscript and Laura Sycuro and Christopher Miller with help in genotyping the animals. In addition, we thank Emily Leinwand Krauter for preliminary studies on the effects of commercial pet food on mouse body mass.
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FOOTNOTES |
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D. L. Allen was supported by a Neuromuscular Disease Research Development Grant from the Muscular Dystrophy Association. In addition, this work was supported by National Instute of General Medical Sciences Grant R01 GM-29090-17 to L. A. Leinwand.
Address for reprint requests and other correspondence: L. A. Leinwand, Dept. of Molecular, Cellular, and Developmental Biology, Univ. of Colorado, Boulder, Campus Box 0347, Boulder, CO 80309-0347 (E-mail: leinwand{at}stripe.colorado.edu).
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.
Received 17 August 2000; accepted in final form 10 October 2000.
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