Gastroenterology and Nutrition Laboratory, Biomedical Center B11, Lund University Hospital, S-22184 Lund, Sweden
Submitted 7 April 2004 ; accepted in final form 28 May 2004
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ABSTRACT |
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bile salt; intestine; mass spectrometry; octanoyl-sphingosine; palmitoyl-sphingosine
Ceramide, sphingosine, and S-1-P are signaling substances that influence numerous cell functions, including growth, differentiation, and apoptosis (8, 22). Ceramide and sphingosine commonly favor cell differentiation or growth inhibition, and S-1-P often has antagonistic effects (42). Dietary sphingolipids were shown to counteract chemically induced colon carcinogenesis and tumor development in a chemical carcinogenesis model and in multiple intestinal neoplasia (MIN) mice with an inactivating mutation in the familial adenomatous polyposis (FAP) gene (46, 48). In cultures of colon carcinoma HT29 cells, sphingosine induced apoptosis and inhibited proliferation by decreasing the level of free -catenin (48). Purified intestinal Alk-SMase (34) from the brush border of the gut inhibited proliferation of colon carcinoma HT29 cells (18). Dietary sphingolipids may also interact with bile acids to counteract their cocarcinogenic action in colon (30) and with sterols to cause inhibition of cholesterol absorption and delayed SM digestion (37). To understand the sequence of events involved in the mediation of these effects, knowledge about the participating enzymes is crucial.
Whereas Alk-SMase was recently purified and cloned (5, 9, 10), the intestinal ceramidases have not been purified and fully characterized in the purified form. Early studies demonstrated the presence of ceramidase with neutral pH optimum in the mucosa, in brush-border preparations, and in intestinal content (35). Later, purified bile salt-stimulated lipase (BSSL) in pancreas and human milk was shown to have lipoamidase (19) and ceramidase activity (38). Most of the intestinal neutral ceramidase activity cannot, however, be attributed to BSSL. The longitudinal distribution of ceramidase activity in the rat intestine was different (25), and neutral ceramidase of rat and human intestinal content was separated from and differed in properties from BSSL (11). Furthermore, mucosal neutral ceramidase activity was present in BSSL(/) mice (20). In this study, we further examined the identity of the enzyme accounting for the major digestive ceramidase activity in the gut.
The first possibility was that it is a member of the highly conserved neutral/alkaline ceramidase family. Members of this family have been purified and/or cloned from a number of tissues such as kidney (29), rat brain (13), mouse liver (40, 50), human liver (14), and endothelial cells (44) and from bacteria (41), yeast (26, 27), and Drosophila (53). During the progress of this work, Choi et al. (6) cloned mouse neutral ceramidase and reported a high expression of neutral ceramidase in mouse intestine and kidney. The second possibility was that the intestinal ceramidase activity is accounted for by a brush- border enzyme related to another enzyme family, e.g., peptidases or lipases. This possibility was not considered unlikely, since we recently found that Alk-SMase is not homologous to any other known SMase but to a new tissue-specific ectoenzyme related to the alkaline nucleotidase/phosphodiesterase (NPP) family (9). We have purified neutral ceramidase from a luminal bile salt eluate of rat small intestine and found that the enzyme is identical or closely related to the neutral ceramidase 2 from rat kidney (29) and highly homologous to the mouse neutral ceramidase recently shown to be expressed also in the intestine (6). Characterization of the purified enzyme indicates important physiological roles of the enzyme in ceramide digestion.
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MATERIALS AND METHODS |
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[14C]octanoic acid was purchased from American Radiolabeled Chemicals (St. Louis, MO). D-Erythro-sphingosine was obtained from Matreya (Pleasant Gap, PA). Taurocholate (TC), glycocholate (GC), taurodeoxycholate (TDC), glycodeoxycholate (GDC), taurochenodeoxycholate (TCDC), glycochenodeoxycholate (GCDC), phenylmethylsulfonyl fluoride (PMSF), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), trypsin, chymotrypsin, and octanoyl chloride were from Sigma (St. Louis, MO). PD-10 columns, HiTrap Q XL, HiTrap Butyl FF, and Superdex 200 HR 10/30 were purchased from Amersham Bioscience (Uppsala, Sweden). Protein determination was performed with Bio-Rad Detergent-Compatible protein assay. Anion exchange HQ cartridges were purchased from Bio-Rad (Stockholm, Sweden). 1-[14C]palmitoyl-labeled ceramide was provided by Dr. Peter Ström (AstraZeneca; Lund, Sweden) and was prepared by conversion of 1-[14C]palmitic acid to the corresponding anhydride with palmitoyl chloride, followed by reaction of the anhydride with D-erythro-sphingosine. The labeled ceramide was purified with flash-column chromatography, and the purified ceramide had a specific activity of 15.7 mCi/mmol with radiochemical purity of >97% (38). Synthesis of D-erythro-[14C]octanoyl-sphingosine was performed according to the method of Bielawska et al. (2) after equilibration of [14C]octanoic acid and octanoyl chloride according to the method of Borgström and Krabisch (3). The labeled ceramide was purified on precoated PK5 150A 0.5-mm TLC plates with a mobile phase of chloroform-methanol-2 M ammonium hydroxide (4:1:0.1, vol/vol/vol). The spot of ceramide was identified by iodine staining, scraped, and extracted three times with chloroform-methanol (2:1, vol/vol).
Ceramidase Assay
The [14C]octanoyl-sphingosine was taken to dryness under nitrogen and then sonicated on ice for 2 min in assay buffer, i.e., 50 mM Tris-maleate, pH 7.0, containing 10 mM sodium-TC. The assay was started by mixing 10 µl of sample containing the enzyme with 90 µl of the assay buffer with substrate, and the samples were then incubated at 37°C for 1 h. The added substrate contained 50 nmol [14C]octanoyl-sphingosine (specific activity 440 dpm/nm) except in the experiments where different substrate concentrations were tested. The reaction was interrupted by adding 0.6 ml methanol-chloroform-heptane in proportions of 28:25:20 (vol/vol/vol) and 0.2 ml 0.05 M K2CO3-K2B2O2, pH 10 (1). After mixing, the tubes were centrifuged at 10,000 rpm for 10 s. An aliquot of 200 µl of upper phase was taken for liquid scintillation counting. The total disintegrations per minute in the upper phase were calculated. The distribution of released fatty acids in the upper phase under these condition was 90%, which was considered in calculation of enzyme activity (25). After the detergent effects on the activity of the purified enzyme had been considered, the assay condition was changed to 50 mM Tris·HCl buffer, pH 8.0, with 8 mM CHAPS, because of the lower cost of CHAPS than of TC.
Purification of Ceramidase
Collection of intestinal protein. Thirty rats fasted overnight were anesthetized (im) with Ketalar-Rompun (2:1) (Ketalar, 50 mg/ml, Parke Davis, Detroit, MI; Rompun Vet, 20 mg/ml). A segment of small intestine from 510 cm below pylorus to 510 cm above the cecum was cannulated. The segment was flushed twice with 20 ml 0.9% NaCl containing 1 mM benzamidine, 1 mM PMSF, and 3 mM TDC, which serves to dissociate both Alk-SMase and ceramidase from the intestinal brush border. The eluted solution was centrifuged at 5,000 rpm (3,000 g) at 0°C, and the supernatant was concentrated by ultrafiltration through a YM-30 membrane. The proteins were precipitated by addition of acetone to 50% (vol/vol) at temperatures below 10°C. After centrifugation at 8,000 rpm (7,600 g) for 15 min at 0°C, the protein pellets were dissolved in 50 ml of 20 mM Tris·HCl buffer, pH 8.2, and 0.075 M NaCl containing 1 mM benzamidine. The nondissolved proteins were removed by centrifugation at 15,000 rpm (27,000 g) for 15 min at 0°C. The supernatant was used for purification. All chromatographic procedures were performed at 4°C with the use of a Bio-Logic HR chromatography system. The elution of protein was monitored by ultraviolet light when applicable.
Ion-exchange chromatography.
The acetone precipitate dissolved in 20 mM Tris·HCl, pH 8.2, containing 1 mM benzamidine was loaded on an anion-exchange HQ column preequilibrated with the same buffer. To remove unbound proteins, the column was washed with 15 ml of equilibrating buffer. The column was first eluted with a NaCl gradient from 0 to 0.25 M, which elutes Alk-SMase. The salt concentration was then increased to 0.5 M to elute remaining proteins, including ceramidase. The ceramidase fractions were pooled and applied to a PD-10 column equilibrated with 20 mM Tris·HCl buffer, pH 7.5, containing 1 mM PMSF, 1 mM benzamidine, and 0.005% (wt/vol) Triton X-100. After buffer change, the sample was concentrated by ultrafiltration using Amicon PM-10 Ultrafiltation Discs. The sample containing 32 mg proteins was loaded on a HiTrap Q XL column. The column was equilibrated with the same buffer as the PD-10 column. To elute unbound proteins, the column was washed with 15 ml of equilibrating buffer. The bound proteins were eluted with a NaCl gradient from 0 to 0.4 M at 2 ml/min. The fractions with ceramidase activity were pooled and concentrated by ultrafiltration.
Hydrophobic-interaction chromatography.
The ceramidase sample now containing 1 mg of protein was applied to a HiTrap Butyl FF column. The column was equilibrated with 25 ml of 20 mM Tris·HCl buffer, pH 7.5, containing 1 mM PMSF, 1 mM benzamidine, 0.005% (wt/vol) Triton X-100, and 1 M ammonium sulfate. After loading of the sample, the column was washed with equilibration buffer. The bound proteins were eluted from the column by a gradient of decreasing ammonium sulfate concentration, from 1 M to 0. The proteins were collected in 1-ml fractions. The fractions with ceramidase activity were pooled and concentrated for the next purification step.
Gel-filtration chromatography. An aliquot of 200 µl of the pooled fractions from the hydrophobic-interaction chromatography (HIC) was applied to a Superdex 200 HR 10/30 column equilibrated with 20 mM Tris·HCl buffer, pH 7.5, containing 1 mM PMSF, 1 mM benzamidine, 0.005% (wt/vol) Triton X-100, and 0.1 M NaCl. The proteins were eluted with 30 ml of equilibration buffer and collected in 0.3-ml fractions.
SDS-PAGE.
The progress of the purification was followed by monitoring the protein composition of the peaks containing ceramidase on 10% SDS-PAGE. The protein bands were visualized with silver staining. The migration of the purified ceramidase was compared with standards of known molecular mass. To examine the effect of deglycosylation on migration, ceramidase was deglycosylated with the use of a glycoprotein deglycosylation kit from Calbiochem-Merck (Darmstadt, Germany) according to the provided protocol. The kit contained N-glycosidase F [peptide N4-(acetyl--glucosaminyl)asparagine amidase cloned from Flavobacterium meningosepticum and expressed in Escherichium coli], endo-
-N-acetyl-galactosaminidase, neuraminidase,
-1,4-galactosidase, and
-N-acetyl-glucosaminidase.
Characterization of Ceramidase
Effects of bile salts. Varying concentrations of TC, GC, TDC, GDC, TCDC, GCDC, CHAPS, and Triton X-100 in 0.05 M Tris·HCl buffer, pH 8.0, were incubated with 2.5 ng ceramidase and [14C]octanoyl-sphingosine as substrate. Optimal pH was determined by incubations in 0.1 M acetate buffer at pH 46.5, in 0.1 M phosphate buffer at pH 7.08.0, in 0.05 M Tris·HCl buffer at pH 8.5, and in 0.1 M glycine-NaOH buffer at pH 910. The effects of 0.14.0 mM concentrations of Li2+, Ca2+, Cu2+, Mg2+, Mn2+, and Zn2+ on the ceramidase activity were examined. All buffers contained 8 mM CHAPS.
Effects on octanoyl- and palmitoyl-sphingosine. The rates of reaction with [14C]octanoyl- and [14C]palmitoyl-sphingosine as substrates were compared in assays as above with different concentrations of unlabeled ceramide present. Vmax and Km were calculated from Lineweaver-Burk plots.
Effects of trypsin and chymotrypsin. Purified ceramidase was incubated with 0.05 M Tris·HCl buffer, pH 8.0, containing 8 mM CHAPS and 0, 0.05, 0.5, and 1 mM trypsin and chymotrypsin for 30 min at 37°C. After preincubation the ceramidase activity was measured. The activity of trypsin was verified with the use of benzoyl-DL-arginine-p-nitroanilide as substrate (17).
Mass spectroscopic analysis. The purified enzyme band from the SDS-PAGE was eluted and subjected to trypsin digestion. The fragments were then subjected to mass fragmentographic (MALDI-TOF) analysis. These analyses were performed by methods established at the Swegene Proteomics Unit (Lund, Sweden). Briefly, the gel was stained by GelCode blue and the ceramidase band was cut and subjected to a procedure involving destaining and dehydration/rehydration, reduction/alkylation, trypsin digestion, and extraction of peptides.
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RESULTS |
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The results from the different chromatographic steps are summarized in Table 1. The protein peak that was not retained on the HQ column contained Alk-SMase and was collected and used for purification of this enzyme (5). The ceramidase was retained on the column, and the enzyme was eluted with 0.5 M NaCl. The fractions with ceramidase activity were pooled and desalted on PD-10 columns. On the subsequent HiTrap Q XL column, the ceramidase was retained to the column and was eluted with a NaCl gradient as a broad peak between 0.02 M and 0.3 M NaCl, with maximal ceramidase activity at 0.1 M NaCl (Fig. 1A). During elution thereafter with equilibration buffer containing 0.5% (wt/vol) Triton X-100, a second ceramidase peak was detected that was not further investigated in this paper. The ion-exchange step gave a 13-fold increase of the specific activity with a recovery of 20% (Table 1).
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In the last step, an aliquot of 200 µl of the concentrated ceramidase peak from the preceding step was applied to a Superdex 200 HR 10/13 column with a total volume of 25 ml. Ceramidase was eluted as a distinct peak at an elution volume of 13.5 ml (Fig. 1C). After this step, 5% of the original ceramidase activity remained and the increase in specific activity was 307-fold, compared with the ceramidase containing protein fraction from the initial HQ column (Table 1).
In initial experiments, most of the enzyme activity in hydrophobic interaction and gel filtration steps was lost when no detergent was present. Addition of 0.005% Triton X-100 was found to be sufficient to avoid this problem and did not interfere with the ceramidase assay. This detergent concentration also gave a favorable elution pattern.
The protein composition of the ceramidase-containing peaks was monitored by SDS-PAGE (Fig. 2). The purified ceramidase obtained after Superdex 200 HR appeared as one single band visible with silver staining. The band corresponds to a molecule mass of 116 kDa (Fig. 2, left). After incubation of the native enzyme with glycoprotein deglycosylation kit, a major protein band with a mass of
97 kDa was observed (Fig. 2, right).
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Mass spectroscopic analysis. The MALDI-TOF analysis identified 12 peptide fragments with sequences identical to parts of the sequence for the ceramidase purified from rat kidney by Mitsutake et al. (29). The sequence coverage was 17.5%. Of the sequence between amino acid 107 and 251, 88 out of 144 amino acids, i.e., sequence coverage of 61%, were obtained. At the COOH-terminal end between amino acid 617 and 761, 68 amino acids, i.e., 47%, were covered (Fig. 3, A and B). The sequences exhibit 92% homology with the sequence for mouse ceramidase clones from liver (50), neonatal adipose tissue, central nervous system, adrenal gland, intestine, and kidney (6) and 78.7% homology with human mitochondrial neutral ceramidase (14). Clones from Drosophila melanogaster (53), Arabidophsis thaliana, and Mycobacterium tuberculosis also exhibited significant homology. Unigene search of the mouse ceramidase 2 reveals that a full-length cDNA Riken clone accounts for 0.07% of the adult mouse small intestine cDNA [Mn-104900, db-expressed sequence tag (EST) library ID no. 7218]. The search also indicates that Unigene Cluster Rn-81031, i.e., N-acylsphingosine hydrolase 2 from rat, has homologous EST sequences consisting of up to 400 bases, with 73100% homology. These are derived from libraries from fetal colon, a combined library, dorsal root ganglion, prostate tissue, and hypothalamus.
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DISCUSSION |
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The MALDI-TOF analysis identified the purified enzyme as a member of the neutral/alkaline ceramidase family. Twelve fragments containing 818 amino acids each and a total of 160 amino acids are identical to sequences in the segment containing amino acid 107 to 251 and 617 to 736 in the neutral ceramidase 2 earlier purified and cloned from rat kidney and shown to be located in the apical membrane of the renal tubular cells (Fig. 3A) (29). The sequence of rat kidney neutral ceramidase 2 also exhibits 92% homology with the mouse neutral/alkaline ceramidase 2 (Fig. 3B), which is expressed at mRNA level in several tissues including the small intestine (Mn-104900, dbEST library ID no. 7218, Riken full-length cDNA). Thus the intestinal ceramidase purified in this study is very likely a product of the neutral ceramidase 2 gene. We have not excluded that it is an isoform that may be differently spliced than the kidney enzyme, nor have we excluded that other neutral ceramidases may be expressed in the intestine. During the progress of this work, Choi et al. (6) reported that the highly homologous (92%) mouse neutral ceramidase 2 is strongly expressed in duodenum, jejunum, and ileum, with highest levels in the jejunum. This is in agreement with our studies (25) of the longitudinal distribution of neutral ceramidase in the rat intestine.
The enzymatic properties of the enzyme purified in the present study are also identical to those of the neutral ceramidase from kidney (29). The molecular mass, the broad pH optimum between 6 and 8, and the effects of metal ions are all similar. Thus both enzymes were inhibited by Zn 2+ and Cu2+ ions, the effect of the Zn2+ ions being most pronounced, and were not influenced by Ca2+, Mg2+, or Mn2+ ions. This contrasts with the brain ceramidase that was stimulated by Mn2+ ions (13) and the bacterial (41) and hepatic enzymes (50) that were stimulated by Ca2+.
An important feature of the intestinal ceramidase is its resistance to trypsin and chymotrypsin in its native form. The reason is unknown but may be related to extensive glycosylation. The amino acid sequence of the rat kidney enzyme indicates the presence of nine potential glycosylation sites, and the estimated molecular mass (113 kDa) exceeds the one predicted from the deduced sequence of 761 amino acids (83.5 kDa). This indicates a high degree of glycosylation, and it has been suggested that mannose glycosylation is essential for its association as a type II transmembrane protein with the tubular membrane in the kidney (49). The change in molecular mass observed after deglycosylation in our study (Fig. 2, right) indicates a high degree of glycosylation of the intestinal ceramidase also.
The longitudinal distribution and the resistance to pancreatic proteases make the enzyme suited for a key role in the digestion of ceramide. In our earlier studies, neutral ceramidase was found throughout the small intestine (25) . In BSSL gene knockout mice the total ceramidase was decreased only in the most proximal part, where BSSL is normally abundant, but not in the remaining small intestine (20). The enzyme is thus expected to be most active in the part of the small intestine where most of the lipid digestion has been finished but where the bile salt concentration is still high. In the present study, the activity of the enzyme was found to be influenced by bile salts. The trihydroxy bile salts TC and GC stimulated over a relatively broad range and may have a stimulatory effect in vivo. The dihydroxy bile salts, which have a lower critical micellar concentration, had their optimal effect at lower concentrations and were strongly inhibitory at higher concentrations. To evaluate the role of bile salts for the kinetics of ceramide hydrolysis in vivo is therefore difficult on the basis of the present data.
Protease resistance, inhibition by Zn2+ ions, and ability to act at neutral/alkaline pH are properties that the intestinal ceramidase shares with Alk-SMase. Being active under similar conditions, the enzymes may act in a concerted manner during SM digestion. The choline, the sphingoid base, and the fatty acid parts of SM can thereby be utilized without the need for powerful pancreatic sphingolipidases that might threaten the integrity of the brush border. As a consequence of the moderate capacity of the enzymes, the digestion of SM and ceramide is extended and incomplete, and the colon is exposed to sphingolipid metabolites (34, 39). Because of the protease resistance of both enzymes, significant amounts of both Alk-SMase and ceramidase are also transferred with the luminal content into colon where they may continue to generate sphingolipid metabolites. The factors that regulate the sphingolipid hydrolysis and colon exposure in vivo need further study.
An intriguing question is whether gut Alk-SMase and ceramidase are only digestive enzymes or whether their action also influences mucosal functions. A number of observations indicate that sphingolipid hydrolysis does indeed influence mucosal function and cell growth. Field et al. (15) showed that exposure of Caco-2 cells to SMase decreased cholesterol absorption (15) and lipoprotein and apolipoprotein B secretion (28), and Kirby et al. (20) reported that hydrolysis of ceramide by BSSL influenced the size and composition of intestinal lipoproteins. BSSL(/) mice lacking BSSL had less total ceramidase activity in the upper gut and produced predominantly smaller lipoproteins than the BSSL(+/+) mice. In polarized Caco-2 cells, SMase induced an inhibition of secretion of large lipoproteins that could be reversed by BSSL hydrolyzing ceramide formed (20). Thus SM and ceramide hydrolysis in/at the brush border may influence sterol absorption and lipoprotein secretion. Studies with free sphingoid bases (48) and with purified Alk-SMase (18) indicate that ceramide and sphingosine generated by the combined action of Alk-SMase and ceramidase may influence growth of colon carcinoma cell lines.
Choi et al. (6) recently reported that in the mouse, feeding of C-2 ceramide (N-acetyl-sphingosine) induced epithelial apoptosis in the gut, particularly when the ceramidase inhibitor (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP) was fed simultaneously. Choi et al. (6) proposed that neutral ceramidase in the gut is an essential component of a detoxifying mechanism to prevent ceramide-induced apoptosis. An important consideration is, however, that long-chain ceramides formed from natural dietary and brush-border sphingolipids are expected to permeate much less efficiently into the mucosal cells in intact form than the C-2 ceramide because of their molecular structure and low solubility in bile salt micelles. The question as to whether long-chain ceramides from dietary and brush-border sphingolipids permeate into the mucosal cells has not been solved. Radioactive ceramide is found to be associated with the mucosal tissue after a feeding of radioactive SM (39), but the intracellular location has not been confirmed, and very little dietary ceramide appears intact in chyle (34). There may be alternative mechanisms by which ceramide reaches intracellular signaling targets. Recent data indicated that SM hydrolysis in the outer layer of erythrocyte ghosts and model membranes caused a transmembrane movement of lipids due to transient formation of nonlamellar structural intermediates and loss of asymmetry in the lipid bilayer with a transbilayer movement of ceramide together with other lipids (7). If similar events occur in the brush border, ceramide generated in the outer layer could reach intracellular signaling targets. If so, the mucosal ceramidase must be important to balance signaling effects of ceramide. Interestingly, gut mucosa and isolated crypt and villus cells from intestinal mucosa contain significant amounts of ceramide located in several subcellular fractions (4).
Ceramidase generates free sphingosine, which is absorbed much more efficiently than long-chain ceramides (34). Because of the high levels of sphingosine-1-phosphokinase in the mucosa (16), sphingosine is also rapidly converted to S-1-P, which could act as a signal to favor cell growth and survival in the mucosa. Sphingosine may also be rapidly reacylated to ceramide in the mucosal cell. Hypotheses regarding the biological significance of the intestinal ceramidase must therefore consider the elimination of ceramide, the rapid absorption and metabolism of the sphingosine formed, and the further conversion of S-1-P to palmitaldehyde and ethanolamine by S-1-P-lyase (32), an enzyme that is found in higher levels in the gut than in any other tissue (51).
Because of the significant content of SM in milk, the significance of sphingolipid metabolism in the gut may be particularly important in the suckling newborn. Interestingly, both the rat lactase-phlorizine hydrolase that catalyzes ceramide formation from glycosphingolipids (21, 43) and intestinal neutral ceramidase in the mouse (6) and the Alk-SMase in the rat (24) are promptly expressed during late gestation soon before delivery. All three enzymes are thus developmentally regulated and appear with the differentiated absorptive epithelium just in time to participate in digestion of milk sphingolipids. Recent studies (31) on newborn rats receiving artificial milk containing SM or equivalent amounts of phosphatidylcholine suggested that SM may influence maturation of the small intestine as judged by differences in lactase levels and morphological criteria. The relation of the gut ceramidase to neonatal nutrition and biological effects of suckling is thus an interesting area for further study.
In summary, the neutral ceramidases found in the gut brush border, the apical membrane of the renal tubular cells (29), and the caveolae of endothelial cells (44, 45) are likely to be products of the same gene. The neutral ceramidase 2 is thus an important ectoceramidase, the multiple roles of which deserve further study. Possible functional aspects are related to digestion, to detoxifying and scavenging functions, and to distinct signaling functions.
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GRANTS |
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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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