Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada
Submitted 14 May 2004 ; accepted in final form 6 October 2004
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ABSTRACT |
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Ca2+-handling proteins; immunocytochemistry; colocalization with caveolin-1; interstitial cells of Cajal pacing
The understanding of the role of ICC in intestinal motor activity has come mostly from studies of mutant mice lacking ICC and from studies in which ICC from neonatal intestine or other sources were isolated, cultured, and studied (16, 17, 23, 27). However, we have recently found that intact segments of intestine do not always behave as predicted from findings predicted form studies of isolated, cultured ICC, or isolated intestine. For example, longitudinal segments from mice lacking an ICC network in the myenteric plexus (MP) often had regular contractions not abolished by block of voltage-dependent Ca2+ channels or reduced in frequency by emptying of endoplasmic reticulum (ER)-Ca2+ (2, 5; G. Boddy and E. E. Daniel, unpublished observation). Moreover, some of the findings about the role of release of intracellular Ca2+ from ICC in pacing could not be confirmed. Recently, we have found, too, that L-type Ca2+ (L-Ca2+) channels play a more important role in pacing by ICC than predicted by models derived from studies of isolated, cultured ICC (G. Boddy and E. E. Daniel, unpublished observation). We also found that depletion of Cav1, by extracting cholesterol, markedly reduced the number of caveolae and reduced the frequency of pacing (6). In addition, we have obtained evidence that argues against the importance of gap junctions in transmission of pacing activity from ICC-MP to muscle (4, 810, 20, 24, 25).
ICC located in the deep muscular plexus (DMP) of intestine and in intramuscular sites throughout the gastrointestinal tract appear also to be the sites of innervation by intrinsic enteric nerves in both intestine and LES (3, 30, 31). Although we found no evidence that depletion of Cav1 and loss of caveolae from ICC disrupted neurotransmission to longitudinal muscle segments (6), there is no evidence about ICC of the DMP or in the LES.
Therefore, the aim of this study was to determine the localization and colocalization with Cav1 of signaling molecules, which may play a role in the function of ICC of the MP and of the DMP and smooth muscle in mouse intestine. Moreover, Cav1 knockout mice have recently become commercially available from Jackson Laboratories, and a secondary purpose of this study was to determine which signaling molecules and pathways are likely to be affected by the absence of Cav1. We also analyzed the relationships between Cav1 and ICC of the LES and LES muscle, because evidence suggests that the ICC in this region are the targets for both nitrergic and cholinergic innervation and transmit neural messages through gap junctions to muscle (30, 32).
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MATERIALS AND METHODS |
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Mice (Balb/c, 810 wk) were killed by cervical dislocation in accordance with a protocol approved by the University of Alberta Animal Care Committee and following the guidelines of the Canada Council on Animal Care. The abdomen was opened along the median line. The lower esophagus containing the stomach and the small intestine were removed and put into ice-cold oxygenated (95% O2-5% CO2) Krebs-Ringer buffer (pH 7.4) containing (in mM) 115.5 NaCl, 4.6 KCl, 1.16 MgSO4·7H2O, 21.9 NaHCO3, 2.5 CaCl2·2H2O, 1.16 NaH2PO4·H2O, and 11.1 glucose. To isolate the LES, the stomach was opened along the greater curvature and the lesser curvature to reveal the junction between the lower esophagus and the fundus of the stomach. We studied both the LES and the region just proximal, which has mixed smooth and striated muscle bundles. The jejunum was opened along the mesenteric border and pinned on a petri dish of Sylgard silicon rubber, mucosa side down.
For cryosection preparation, the jejunum and LES were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 4 h at room temperature. The fixed tissues were washed in PB for 30 min x 8 and were cryoprotected in graded sucrose solution (10, 20% sucrose in PB) for 2 h each, placed in 30% sucrose in PB overnight at 4°C, and were then stored at 80°C until sectioned. For whole mount preparation, the jejunum was microdissescted, fixed, and treated as in the cryosection preparation for 4 h at room temperature. The fixed tissues were washed in PB for 30 min eight times, dehydrated and cleared in DMSO for 10 min three times, and were rehydrated in PB for 15 min four times at room temperature.
Double-Immunofluorescent Labeling for Cryosection
Frozen tissues were sectioned by a cryostat (Leitz 1720 digital cryostat, Germany) to make sections of 10-µm thickness. The sections were attached on slide glasses coated with 2% 3-aminopropyltriethoxysilane (Sigma, St. Louis, MO) in acetone, and were dried overnight at 4°C. The sections were washed in PBS (pH 7.0) containing 0.4% Triton X-100 (TX-100; 0.4% in PBS) for 15 min three times. To reduce nonspecific binding proteins, the sections were blocked with 10% normal sera that were raised in the host of the secondary antibody for 1 h at room temperature. For immunohistochemistry, primary antibodies, Cav1, neuronal NOS (nNOS; COOH- or NH2-terminal epitope), Na+/Ca2+ exchanger 1 (NCX1), connexin-43, connexin-40, c-kit (CD117 or ACK4), L-Ca2+ channel, plasma membrane (PM) Ca2+ pump, calreticulin, large conductance Ca2+-activated K+ (BK-Ca2+) channel, and calsequestrin were incubated for 1920 h at 4°C or room temperature.
The sections were washed in 0.4% TX-100 in PBS for 15 min three times. Secondary antibodies, immunoglobulins conjugated with Cy3, FITC, or Alexa488, were incubated for 1 h at room temperature. The sections were washed in 0.4% TX-100 in PBS for 15 min two times and were then washed in PBS for 15 min one time. The sections mounted with aqueous mounting medium with antifading agents (Biomeda, Foster, CA).
Immunofluorescent Labeling for Whole Mount Preparation
Muscle (circular and longitudinal muscle) layers of the jejunum were separated from the mucosa and submucosa layers, and then the circular muscle layer was separated from the longitudinal muscle layer with the MP under the dissection microscope. The muscle layers were washed vigorously in PBS containing 0.5% TX-100 (0.5% in PBS) for 15 min four times on orbital shaker. The muscle layers were blocked with 10% normal sera that were raised in the host of secondary antibody for 1.5 h at room temperature. For immunohistochemistry, primary antibodies used in the cryosection were used for 48 h at 4°C. The muscle layers were washed in 0.5% TX-100 in PBS for 15 min four times. For immunofluorescent labeling secondary antibodies, immunoglobulins conjugated with Cy3, FITC, or Alexa488 were used for 1.5 h at room temperature. The sections were washed in 0.5% TX-100 in PBS for 15 min three times and were then washed in PBS for 15 min one time. The sections were mounted with aqua-mount medium.
To determine specificity of immunostaining, primary antibody was omitted or when the antigen was available, it was used to saturate the primary antibody. The primary and secondary antibodies as well as the antigens used are summarized in Table 1.
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Single and double-immunolabeled sections were observed with confocal laser scanning microscope (Zeiss CLSM 1500) equipped with an argon and helium/neon laser. Most of the images obtained were adjusted by brightness and contrast, and a few were reconstructed to three-dimensional image with the scanning of 1-µm serial images in depth. All results are based on studies from three or more animals.
Evaluation of Extent of Colocalization
All images for the colocalization study of proteins in ICC and smooth muscle of mouse jejunum tissue and the LES study were originally 512 x 512 pixels of image size obtained from Carl Zeiss confocal laser scanning microscope 1500 and LSM 510 software. The images have not only well-focused interesting areas such as ICC and smooth muscle membrane, but also uninteresting areas such as mucosa and submucosa or unfocused interesting areas due to differences of tissue thickness, immunostaining, or artifacts. Almost every image was cut and adjusted to be enhanced using brightness and contrast of LSM 510, and they were also edited using Adobe PhotoShop to organize for submission. Thus sizes of the images were decreased, and brightness and contrast were changed.
Analysis of the colocalized proteins in ICC and smooth muscle membrane was shown by scatter diagram. The scatter diagram was obtained from the modified image, but not 512 x 512 pixels of the original image. With the use of the colocalization toolbar of the laser scanning microscope software (Zeiss LSM 510), each scatter diagram was created and displayed. Consequently, initial points of two intensity channels on the scatter diagram appear sometimes moved to the extent that the brightness and contrast were changed. However, the analyzed scatter diagram shows the general pattern of colocalization distributing along the diagonal line running from the bottom to the top when colocalization was good.
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RESULTS |
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Distribution of Cav1 in smooth muscle and ICC of jejunum, LES. Cav1 was present in both circular and longitudinal smooth muscle of jejunum. Cav1 immunoreactivity (IR) was punctate at the cell periphery of the cross-sectioned circular muscle cells and appeared along the periphery of longitudinal-sectioned longitudinal muscle cells (Fig. 1A). In LES, Cav1 was also present in the cell periphery of the sphincter muscle cells but was almost absent in skeletal muscle cells (Fig. 1, B and C). In addition to its presence at the periphery of smooth muscle cells of the jejunum and LES, Cav1-IR was present at the cell periphery of ICC in MP (ICC-MP), ICC in DMP (ICC-DMP), and ICC-S (Fig. 1A) but was not clearly punctate in its distribution in ICC. These cells marked with asterisks are ICC based on colocalization studies.
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NCX1, colocalized with Cav1, not with Connexin-40. The NCX1-IR was punctate in the cell periphery of the circular muscle cells and located densely along PM of the longitudinal smooth muscle cells. Connexin (Cx)40-IR was distributed in the circular muscle but was not colocalized with NCX1 (Fig. 2, AC). NCX1 was previously reported to be distributed in smooth muscles, ICC-MP, ICC-DMP, and ICC-S of jejunum (see Fig. 2A). Apparent colocalization of Cav1 and NCX1 appeared also in some ICC around myenteric ganglia and a few ICC-DMP of jejunum (Fig. 2, DF).
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Colocalization of Cx43 and Cx40. Cx43 was colocalized with Cx40 in circular muscle cell membranes of jejunum. Some Cx43-IR was sparsely distributed at the inner circular muscle cell layer without colocalization with Cx40, but it was distributed and colocalized with Cx40 in the main circular muscle cell layer (Fig. 2, MO).
Colocalization of nNOS-C and Cx40. nNOS identified when the antibody against the epitope to the COOH-terminal end (nNOS-C) was applied, was colocalized occasionally with Cx40 in jejunum circular muscle. Cx40 was located in the cell periphery of the circular muscle cells immunostained with nNOS-C and showed significant colocalization, based on the presence of punctate yellow dots. Interestingly, nNOS-C-IR was localized in ICC-S of the jejunum (Fig. 2, PR).
Colocalization of Cav1 and nNOS-C. Cav1 appeared to be colocalized with the nNOS-C in smooth muscle of jejunum. In studies using double immunofluorescences, Cav1 antibody conjugated with Cy3 and nNOS-C antibody conjugated with FITC showed nearly complete colocalization, based on the presence of punctate yellow coloring in the cell periphery of both circular muscle and longitudinal muscle of jejunum. Similar colocalization also appeared as dense yellow IR in ICC-DMP (Fig. 3, AC).
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Three-dimensional images of ICC-MP and ICC-DMP. Figure 3, J and K, show three-dimensional confocal images of ICC-MP and ICC-DMP of jejunum. Cell bodies of ICC-MP were distributed near and under myenteric neurons immunostained with an antibody against an epitope to the NH2-terminal end (nNOS-N), and cell processes of ICC-MP were connected to other ICC-MP. ICC-DMP were distributed along the circular muscle layer.
Colocalization of Cav1 and L-Ca2+ channel.
In whole mount preparations of smooth muscle of jejunum, the L-Ca2+, 1c-subunit, channel was distributed in ICC-MP and ICC-DMP and also in MP and DMP (Fig. 3, L and M). Immunoreactivity to the L-Ca2+ channel was distributed over smooth muscle, MP, DMP, and serosa of jejunum. In smooth muscle, L-Ca2+ channel was present not only in cell periphery but also in cell center of the circular and longitudinal muscle and was colocalized with Cav1 in the cell periphery. Dense immunoreactivity to the L-Ca2+ channel was colocalized with Cav1 in MP, DMP, and serosa (Fig. 3, NP).
Localization of plasmalemmal (PM) Ca2+ pump and colocalization with Cav1 and c-kit. In jejunum, the PM-Ca2+ pump was distributed in the periphery of smooth muscle, and in ICC-MP and ICC-DMP, colocalized with Cav1. In smooth muscle, the Ca2+ pump was present in both the cell periphery and the center of the circular muscle cell (Fig. 3, QS). c-kit Was colocalized with the PM-Ca2+ pump in ICC-MP, what appeared to be ICC-DMP and ICC-S (Fig. 4, AC).
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Colocalization of Cav1 and calsequestrin (Ca-q) in jejunum. Only weak immunoreactivity to Ca-q appeared in smooth muscle cells, but dense immunoreactivity was observed in some ICC-MP, a few ICC-DMP, and ICC-S of jejunum. Colocalization with Cav1 was noted in ICC-MP, ICC-DMP, and ICC-S, and colocalization with c-kit was found in ICC-MP and ICC-S (Fig. 4, JO).
LES Smooth Muscle
nNOS-C and NCX1 in smooth muscle of LES, colocalized with smooth muscle Cav1. nNOS-C-IR and NCX1 were densely present in the cell periphery of LES cells. nNOS-C appeared to be colocalized with Cav1 in the cell periphery of smooth muscle cells (Fig. 5, AC), as was NCX1 (Fig. 5, DF).
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Colocalization of Cav1 and Ca-r and Ca-q in LES. In the LES, Ca-r-IR was distributed in the cell membrane and cytoplasm of sphincter muscle cells and colocalized, in part, with Cav1 in the cell periphery (Fig. 7, AC). In skeletal muscle, there was Ca-r-IR in sarcolemma and the Z-line of skeletal muscle cell as well as in ICC-IM in associated small smooth muscle bundles. When ICC-IM were present, Ca-r was colocalized, in part, with Cav1.
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Colocalization of Cav1 and Ca-q and Ca-r. In LES, very weak immunoreactivity to Ca-q was present in sphincter muscle, but ICC-IM showed not only dense immunoreactivity to Ca-q but also colocalization with Cav1 (Fig. 7, G and I). Colocalization with Cav1 appeared to be with ICC-IM, and this was confirmed by findings of colocalization with c-kit in ICC-IM of LES (Fig. 7, JL). In contrast, Ca-r was present as in smooth muscle of jejunum, as shown above, in the cytoplasm of smooth muscle bundles amidst skeletal muscle. It, similar to Ca-q, was colocalized with Cav1 in what appear to be ICC-IM (Fig. 7, AC).
LES Region with Mixed Smooth and Skeletal Muscle
In general, the region just proximal to the LES had smooth muscle bundles intermixed with skeletal muscle, and ICC-IM had the same associated proteins as in LES proper. The smooth muscle cells had Cav1, but skeletal muscle cells lacked immunoreactivity to both Cav1 and nNOS-C (Fig. 8, AC). Small smooth muscle bundles among skeletal muscle often had c-kit immunoreactive cells, presumably ICC-IM, which also were Cav1 immunoreative (Fig. 8, DF).
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Images of Cav1 and NCX1 (Fig. 10, A and E), Cav1 and nNOS-C (Fig. 10, B and F), Cav1 and L-Ca2+ (Fig. 10, C and G), and Cav1 and PM-Ca2+ pump (Fig. 10, D and H) were analyzed to visualize intensity distributions of colocalization in both jejunum and LES. Scatter diagrams of colocalization of Cav1 with NCX1, nNOS, and PM-Ca2+ were centered around the diagonal, indicating good general colocalization. In contrast, the scatter diagram for Cav1 with the L-Ca2+ channel was poorly centered, suggesting less good colocalization. Studies using the inverted ellipse function indicated that L-Ca2+ channels were best colocalized with Cav1 in smooth muscle membranes and ICC (not shown).
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DISCUSSION |
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Many findings were consistent with the structural and functional data we have obtained. For example, we found that the L-Ca2+ channels appeared to be associated with Cav1 in ICC, consistent with the important role we have recently found that these channels play in allowing a gradient of frequencies, higher proximally, along the intestine (G. Boddy and E. E. Daniel, unpublished observation). The apparent colocalization of these channels with Cav1 in smooth muscle and ICC is also consistent with the effects we observed when caveolae and Cav1 distribution were affected by manipulation of cholesterol using cyclodextrin6. We found that removal of cholesterol affected Cav1 and caveolae in ICC and smooth muscle, an effect associated with a decrease in ICC pacing frequencies and contractions. These changes largely reversed on restoring cholesterol. Conversely, adding excess cholesterol affected Cav1 distribution, but not the numbers of caveolae in ICC and muscle, and also decreased pacing frequency and affected contractions. We suspect that these changes were related to the altered functions of L-Ca2+ channels when Cav1 and L-Ca2+ channels are disassociated. However, the coincidence between L-type Ca2+ channels and Cav in ICC and smooth muscle was only partial, as shown by quantitative evaluation (see Fig. 10). This hypothesis can be tested functionally now that Cav1-deficient mice are available.
In general, our findings strongly suggest that, in canine intestine, LES, as in airway smooth muscle (7, 11), besides L-Ca2+ channels, other Ca2+-handling molecules, PM-Ca2+ pump, NCX1, and sometimes Ca-r, were near or colocalized with Cav1. When we examined the degree of colocalization semiquantitatively, it was apparent that the PM-Ca2+ channel, nNOS, and NCX1 were closely colocalized with Cav1 in smooth muscle and ICC, whereas neither Ca-r or Ca-q was closely colocalized with Cav1 in smooth muscle (Fig. 10). Given these findings, it is probable that Cav1 and Ca-r are side by side rather than in the same organelle in LES and intestine. However, the close overlap of Cav1 and the PM-Ca2+ pump, the nNOS and the NCX-IR with Cav1, leads to the hypothesis that they are all associated with caveolae of both smooth muscle and ICC. For the L-Ca2+ channel, the colocalization is quantitatively less good than for other Ca2+-handling molecules, and this may mean that is near but not bound to Cav1. This allows the further hypothesis that the L-Ca2+ channel near caveolae of ICC is very close to the ER and functions to restore ER-Ca2+ when Ca2+ is released from ER and lost from the ICC or smooth muscle cells via the PM-Ca2+ pump or NCx. We have recently reported that the L-Ca2+ channel plays a crucial role in pacing and is essential for the intrinsic frequency gradient (G. Boddy and E. E. Daniel, unpublished data).
Other findings raised important questions about why certain signaling molecules are present associated with Cav1. In smooth muscle of jejunum and LES, as in canine jejunum and LES and canine airway smooth muscle, Cav1 was associated with nNOS using an antibody (7, 11) that recognized an epitope located in the COOH-terminal end of the molecule. Earlier in canine (7) LES, we found that nNOS appeared to function to modulate tone (21, 22) by releasing NO in response to increased intracellular Ca2+ concentration. The role of Cav1 in control of eNOS in endothelium has been described as inhibition of its function by binding it until Ca2+-calmodulin occupies the NOS molecule and releases it. A similar role can be proposed for nNOS in smooth muscle (12) and ICC. However, we found no change in frequency of pacing in longitudinal muscle segments of mouse intestine when L-NNA was applied either alone or with TTX. In circular muscle segments5,6, when the tonic neural inhibition was abolished either by TTX or L-NNA, frequency increased, but not to the level of adjacent longitudinal muscle segments. Studies are required of a possible role of this nNOS in modulating contractions in response to agonists that raise Ca2+ levels near the membrane.
In the ICC of the DMP, Cav1 was also colocalized with nNOS. Again, the implications of this for function of these ICC as secondary pacing molecules, as in the canine intestine, or as intermediaries in neurotransmission are unexplored.
In the case of NCX1, the sodium-calcium exchanger, we found it colocalized semiquantitatively with Cav1 in smooth muscle of jejunum and LES and in some ICC-MP and ICC-DMP. However, so far we have not been able to define a role for this exchanger. Removal or marked reduction of external sodium levels by replacement with LiCl markedly reduced pacing frequency but did not usually cause tone increase as expected on abolition of the Na+ gradient if the exchanger was a major pathway for Ca2+ removal after contraction (E. E. Daniel, G. Boddy, W. J. Cho, and A. Willis, unpublished observation). The effect on frequency could be an effect on inward current in ICC carried mainly by sodium. A selective inhibitor of NCX1 had no effect on the frequency of pacing in longitudinal or circular muscle segments (2).
We found that Cx43 and -40 were closely colocalized with each other but were not quantitatively associated with Cav1 in circular muscle of the jejunum. This was similar to our findings in canine intestine (7, 29). Both were absent from longitudinal muscle, consistent with ultrastructural data. We have found that block of formation of functional gap junctions using peptides (4, 26) from the external loops of Cx40 and -43 had no effect on pacing frequencies driven from ICC (M. Mannarino, W. J. Cho, G. Boddy, and E. E. Daniel, unpublished observation). The last finding is consistent with the lack of evidence that low-resistance contacts through gap junctions are essential for transmission of slow waves and pacing from ICC to muscle (4, 810, 24). Although occasional punctate sites of immunoreactivity to Cx40 appeared to be colocalized with c-kit in ICC-S and very rarely in ICC-MP, there was no evidence that gap junction proteins were present between ICC and smooth muscle. The absence of either Cx40 or Cx43 from LES was discussed above. There were several other findings for which there is, to date, no functional correlation. The plasmalemmal Ca2+ pump was present and colocalized with Cav1 in smooth muscle, ICC-MP, and ICC-DMP. Its role in Ca2+ handling in these cell types and how this might be modulated by binding to Cav1, if it is, remains unclear. Studies have been limited by the absence of a selective inhibitor of pump function. The BK-Ca2+ channel is known to play a role in modulation of smooth muscle contractile function. Its role in ICC functions are less well understood. It (14, 15, 17) was present throughout the structures of the intestine and LES and was colocalized with Cav1 in ICC-DMP, ICC-MP, and ICC-S of jejunum and ICC-IM of LES. Ca-r is reported to be the main Ca2+ sequestering protein of the sarcoplasmic reticulum (SR) of smooth muscle (20). It was present in smooth muscle of intestine and LES and in ICC of all classes. Only in the latter muscle was it clearly located in the periphery where peripheral SR should be located. Its presence in the cytosol might relate to a function in the folding and sorting of proteins after synthesis in ER (19, 28). It was also present modestly in skeletal muscle in the expected locations. Ca-q, the main Ca2+-sequestering protein of skeletal muscle, was virtually absent from smooth muscle and present in skeletal muscle, as expected (20). Surprisingly, it was also present in ICC of all classes. The presence of Ca-r and Ca-q in ICC raises the possibility that they play a role in Ca handling in these cells (33).
In conclusion, these findings provide support for several previous functional findings and provide motivation for examining the roles of several proteins in smooth muscle contraction and ICC pacing in intestine and in LES function in Cav1 knockout mice because of their presence in muscle and ICC and because of their colocalization with caveolin-1, a major modulator of signaling processes and a site localizing Ca2+-handling proteins.
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GRANT |
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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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REFERENCES |
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