Original Article

Regulation of rat hepatic 3ß-hydroxysterol ⁷-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation

Correspondence to: S. Shefer.

	ABSTRACT

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The mechanism for the catalytic reduction of the double bond at C-7,8 in 7-dehydrocholesterol by 3ß-hydroxysterol ⁷-reductase was investigated by testing structurally related sterols as substrates and potential inhibitors. The hepatic smooth endoplasmic reticulum was identified as the site of enzyme activity. All putative substrates contained 27 carbons, but differed from 7-dehydrocholesterol by the addition of either an ethyl substituent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl substituent at C-24 (ergosterol), epimerization of the hydroxyl from the 3ß- to 3-configuration (7-dehydroepicholesterol), or a saturated double bond at C-5,6 (lathosterol). Two non-steroidal compounds that inhibit 3ß-hydroxysterol ⁷-reductase in vivo (AY 9944 and BM 15.766) were also tested. Ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol were reduced at C-7,8 to form brassicasterol, sitosterol, and epicholesterol, respectively, but 75% less efficiently than 7-dehydrocholesterol. Increasing concentrations of these sterols competitively inhibited 3ß-hydroxysterol ⁷-reductase activity. The double bond at C-7,8 in lathosterol was not reduced. AY 9944 and BM 15.766 inhibited 3ß-hydroxysterol ⁷-reductase activity non-competitively. 3ß-Hydroxysterol-⁷-reductase activity declined after microsomes were exposed to alkaline phosphatase, and enzyme activity was increased by phosphorylation with Mg²+, and ATP.

These results demonstrate that the reduction of the double bond at C-7,8 requires binding of the enzyme protein with the B-ring of the sterol substrate that contains a double bond at C-5,6. The reaction is hindered by substituents located on the apolar side-chain and epimerization of the hydroxyl group in ring A to a 3-configuration. 3ß-Hydroxysterol ⁷-reductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form.—Shefer, S., G. Salen, A. Honda, A. K. Batta, L. B. Nguyen, G. S. Tint, Y. A. Ioannou, and R. Desnick. Regulation of rat hepatic 3ß-hydroxysterol ⁷-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation. J. Lipid Res. 1998. 39: 2471–2476.

Supplementary key words: Smith-Lemli-Opitz syndrome, cholesterol biosynthesis

	INTRODUCTION

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3ß-Hydroxysterol ⁷-reductase catalyzes the last reaction in the cholesterol biosynthetic pathway, the reduction of the double bond at C-7,8 in 7-dehydrocholesterol to form cholesterol ( Figure 1). This enzyme, also known as 7-dehydrocholesterol ⁷-reductase, is inherited defectively in the Smith-Lemli-Opitz syndrome, a recessive birth defect (1) (2). Homozygotes show a characteristic clinical phenotype with abnormal brain development, microcephaly, facial dysmorphism, syndactyly and polydactyly of the fingers and toes, and congenital anomalies of the heart and kidneys. Low plasma and tissue cholesterol levels with the accumulation of the precursor, 7-dehydrocholesterol, and its 8-dehydrocholesterol isomer are prominent biochemical features (3) (4) (5).

View larger version (14K): [in this window] [in a new window]   Figure 1. Flow diagram that shows key reactions in the biosynthesis of cholesterol. The conversion of HMG-CoA to mevalonic acid is catalyzed by HMG-CoA reductase and is considered rate controlling. The last reaction, the conversion of 7-dehydrocholesterol to cholesterol is catalyzed by 3ß-hydroxysterol 7-reductase. This enzyme is inherited abnormally in the Smith-Lemli-Opitz syndrome (2).

To better understand the enzymatic defect in the Smith-Lemli-Opitz syndrome, where 3ß-hydroxysterol ⁷-reductase activity is markedly inhibited (2), we examined the mechanism for the reduction of the double bond at C-7,8 as catalyzed by 3ß-hydroxysterol ⁷-reductase. The hepatic subcellular location of the enzyme, substrate specificity, competitive and non-competitive inhibition, and short-term regulation by phosphorylation/dephosphorylation were examined in rat liver. A major aim was to gain insight into the ⁷-reduction mechanism and its regulation to better understand gene mutations that might be responsible for the Smith-Lemli-Opitz syndrome.

	METHODS

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Preparation of hepatic subcellular fractions
Male Sprague-Dawley rats (250 g) were fed Purina rodent chow and killed at 10 AM at the nadir of the diurnal cycle of cholesterol biosynthesis (6). Livers were excised and mitochondria, microsomes and the 100,000 g cytosolic fractions were isolated by differential ultracentrifigation in the presence of sodium chloride (2). Protein concentrations were determined by the method of Lowry et al. (7). Cholesterol 7-hydroxylase activity was assayed in each fraction as a marker of microsomal contamination (8). Briefly, the assay is performed by measuring the conversion of [¹⁴C]cholesterol to 7-hydroxycholesterol (9).

3ß-Hydroxysterol ⁷-reductase assay
3ß-Hydroxysterol ⁷-reductase activity was assayed using the method previously described by Shefer et al. (2) with minor modifications. Microsomes (0.5 mg of protein) were incubated in a final volume of 300 µl buffer (pH 7.3) containing 100 mM K₂HPO₄, 1 mM DTT, 30 mM nicotinamide, 0.1 mM EDTA and NADPH generating system: 3.4 mM NADP⁺, 30 mM glucose-6-phosphate, and 0.3 IU glucose-6-phosphate dehydrogenase. The reaction was initiated by the addition of [³H]7- dehydrocholesterol (30 nmol, 8,000 cpm) solubilized with 15 µl of a 13% solution of ß-cyclodextrin (Pharmatec Inc., Alachua, FL). The reaction was stopped after 30 min incubation at 37°C with the addition of 1 ml of 1 N ethanolic NaOH, and the mixture was allowed to stand for 1 h at 37°C. After adding 0.5 ml water, the products were extracted twice with 2 ml n-hexane, separated by argentation thin-layer chromatography, and radioactivity was measured by liquid scintillation spectroscopy (2).

Assay of 3ß-hydroxysterol ⁷-reductase activity with structurally different substrates
Incubations were performed with the following substrates: ergosterol, 7-dehydrositosterol, 7-dehydroepicholesterol, or lathosterol substituting for 7-dehydrocholesterol ( Figure 2) using the same cofactors and conditions as described in the previous section. Each substrate was solubilized with 15 µl of 13% solution of ß-cyclodextrin. The reaction was stopped by adding 1 ml of 1 N ethanolic NaOH, and after extraction with n-hexane as described above, trimethylsilyl-ether derivatives were formed. Quantitation was carried out by gas–liquid chromatography–mass spectrometry with selected-ion monitoring (SIM) using a Hewlett-Packard model 5988 mass spectrometer. A nonpolar CP-Sil 5CB (25 m x 0.25 mm ID) capillary column (Chrompack, Raritan, NJ) was used with a flow-rate of helium carrier gas of 1.0 ml/min. The column oven temperature was programmed to increase from 100°C to 265°C at 35°C/min, after a 2-min delay from the start time. The mass spectral resolution was about 1000. The multiple ion detector was focused on m/z 363 for ergosterol, m/z 380 for brassicasterol, m/z 325 for 7-dehydroepicholesterol, m/z 329 for epicholesterol, m/z 484 for 7-dehydrositosterol, m/z 486 for sitosterol, m/z 458 for lathosterol, and m/z 445 for cholestanol (Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 2. Putative substrates for 3ß-hydroxysterol 7-reductase and reaction products. Assay conditions are described in Methods.

Inhibition of 3ß-hydroxysterol ⁷-reductase by structurally different sterols
The inhibitory effects of ergosterol, 7-dehydrositosterol, 7-dehydroepicholesterol, and lathosterol on hepatic 3ß-hydroxysterol ⁷-reductase activities were determined by measuring catalytic activities in rat hepatic microsomal fractions in the presence of increasing concentrations of the inhibitors. Microsomes (0.5 mg of protein) were incubated in a final volume of 300 µl buffer (pH 7.3) containing an NADPH generating system and increasing concentrations of the test compounds (ergosterol, 7-dehydrositosterol, 7-dehydroepicholesterol, and lathosterol) solubilized with 15 µl of a 13% solution of ß-cyclodextrin. The reaction was initiated by the addition of [³H]7-dehydrocholesterol (30 nmol, 8,000 cpm) and incubated for 30 min at 37°C. The reaction was stopped and the product [³H]cholesterol was extracted and quantitated as described above in the 3ß-hydroxysterol ⁷-reductase assay.

To determine the mechanism of inhibition, rat hepatic microsomes were assayed for 3ß-hydroxysterol ⁷-reductase activities after incubations with increasing concentrations of the ³H-labeled 7-dehydrocholesterol substrate in the presence of 100 and 300 µM inhibitors, and the Lineweaver-Burk double reciprocal plots were analyzed.

Effects of AY 9944 and BM 15.766 on 7-dehydrocholesterol ⁷-reductase activity
AY 9944 [1,4-bis(2-dichlorobenzylaminomethyl) cyclohexane] and BM 15.766 [4-(2-[1-(4-chlorocinnamyl) piperazin-4-yl] ethyl) benzoic acid] ( Figure 3) were solubilized in a 13% solution of ß-cyclodextrin and added to the incubation mixture described above. The effect of increasing concentrations of the non-steroidal compounds on the activity of 3ß-hydroxysterol ⁷-reductase was evaluated, and the mechanism of inhibition was defined by the Dixon plot, of 1/V versus the increasing concentrations of the inhibitors.

View larger version (12K): [in this window] [in a new window]   Figure 3. Structures of non-steroidal inhibitors of 3ß-hydroxysterol 7-reductase. AY 9944, 1,4-bis (2-dichlorobenzylaminomethyl) cyclohexane and BM 15.766, 4-(2-[1-(n-chlorocinnamyl) piperazin-4-yl] ethyl) benzoic acid.

Effects of activation/inactivation (phosphorylation/ dephosphorylation) on 3ß-hydroxysterol ⁷-reductase activity
Hepatic microsomes were prepared by differential ultracentrifugation with 50 mM NaCl or 50 mM NaF (10). In the experiments of phosphorylation, microsomes (0.5 mg of protein) prepared with buffer containing NaCl were preincubated for 60 min at 37°C in a final volume of 150 µl. Tris-HC1 buffer (50 mM), pH 7.4, containing 0.3 M sucrose, 1 mM DTT, 1 mM EDTA, 5 mM MgC1₂, 5 mM ATP. In some experiments, 50 µM cAMP and 20 units of cAMP-dependent protein kinase were also included in the preincubation mixture. For dephosphorylation experiments, microsomes (0.5 mg of protein) prepared with NaF were preincubated for 60 min at 37°C in a final volume of 150 µl, Tris-HC1 buffer (50 mM), pH 7.4, containing 0.3 M sucrose, 1 mM DTT, 1 mM EDTA, and 10 units of E. coli alkaline phosphatase. The reactions were started by the addition of [³H]7-dehydrocholesterol (30 nmol, 8,000 cpm) solubilized with 15 µl of a 13% solution of ß-cyclodextrin, and were stopped after 30 min, and [³H]cholesterol was isolated and quantitated as described above.

	RESULTS

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Figure 4 shows 3ß-hydroxysterol ⁷-reductase activity in rat liver whole homogenate and three subcellular fractions: microsomes, mitochondria, and 100,000 g cytosolic fraction prepared by differential ultracentrifugation (2) (11). Proof of purity was based on the recovery of cholesterol 7-hydroxylase activity in the various fractions. 3ß-Hydroxysterol ⁷-reductase was most active in the microsomal fraction. Whole homogenate, which contains about 30% microsomes (12), was also active but the total activity reflected the dilution of the microsomes by other cellular protein. Virtually no activity was detected in the cytosolic fraction, and the mitochondria that were contaminated with 1.8% microsomes (13) showed very low activity. Thus, 3ß-hydroxysterol ⁷-reductase is almost exclusively located in the microsomal fraction.

View larger version (31K): [in this window] [in a new window]   Figure 4. Subcellular localization of 3ß-hydroxysterol 7-reductase. Enzyme activity was located almost exclusively in hepatic microsomes (Micro.). Low levels of activity in whole homogenates (WH) and mitochondria (Mito.) reflect small amounts of microsomes in these fractions. The percent contamination was ascertained by the recovery of cholesterol 7-hydroxylase activity (8) (9) and reflects 30% and 1.8% microsomes in whole homogenate and mitochondria, respectively. No 3ß-hydroxysterol 7-reductase activity was detected in the cytosolic fraction (Cyto.). The results reflect mean ± SEM from 5 different rat livers.

In order to gain some insight into the properties of 3ß-hydroxysterol ⁷-reductase, we tested the enzyme's substrate specificity, competitive and non-competitive inhibition, and short-term regulation by phosphorylation/dephosphorylation.

Figure 2 illustrates the structures of the substrates tested and the products that were formed. Only sterols that have a double bond at C-7,8, but differ in the orientation of the hydroxyl group in ring A (7-dehydroepicholesterol), the absence of the double bond at C-5 in ring B (lathosterol) or the addition of substituents and double bonds in the side- chain (ergosterol, 7-dehydrositosterol), were tested. Ergosterol differs from 7-dehydrocholesterol by having an additional double bond at C-22 and a methyl group at C-24; 7-dehydrositosterol, contains an extra ethyl substituent at C-24; 7-dehydroepicholesterol, has an -orientation of the hydroxyl group at C-3; and lathosterol contains only a single double bond in ring B, at C-7,8.

Figure 5 shows 3ß-hydroxysterol ⁷-reductase activity as measured by pmoles of reduced products (Figure 2) formed per mg microsomal protein per min. The highest activity was observed with 7-dehydrocholesterol as substrate. The activity was more than 4-times higher than for ergosterol, 7-dehydrositosterol, or 7-dehydroepicholesterol as substrates. There was virtually no reduction of the double bond at C-7,8 when lathosterol was used as a substrate. Note that lathosterol is the only substrate tested that does not have a double bond at C-5,6. Thus, a double bond at C-5,6 in ring B of the substrate is essential for the reduction of the double bond at C-7,8. In addition, substitutions on the side-chain and reversal of the orientation of the hydroxyl group at C-3 reduced the catalytic efficiency of the 3ß-hydroxysterol ⁷-reductase.

View larger version (26K): [in this window] [in a new window]   Figure 5. Substrate specificity of 3ß-hydroxysterol 7-reductase. Compared to 7-dehydrocholesterol (7DHC), the reduction of the double bond at C-7,8 in ergosterol (Ergo), 7-dehydrositosterol (7DHS), and 7-dehydroepicholesterol (7DHEC) was 75% lower. The double bond at C-7,8 in lathosterol (Latho.) was not reduced. Each bar represents the mean ± SEM for 5 rat liver preparations.

Figure 6 shows the effects of ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol on the activity of 3ß-hydroxysterol ⁷-reductase. In these experiments, incubations were carried out with increasing amounts of unlabeled ergosterol, 7-dehydrositosterol, or 7-dehydroepicholesterol solublized in ß-cyclodextrin added to 0.5 mg of rat hepatic microsomal protein. [³H]7-dehydrocholesterol was the substrate and [³H]cholesterol was the product measured. The results show that ergosterol and 7-dehydroepicholesterol are more potent inhibitors than 7-dehydrositosterol. Ergosterol and 7-dehydroepicholesterol in a concentration of 400 µM inhibited 3ß-hydroxysterol ⁷-reductase activity about 30%, whereas 400 µM of 7-dehydrositosterol inhibited the same enzyme by only 17%. In separate experiments, 200 µM and 400 µM unlabeled lathosterol were tested and did not inhibit 3ß-hydroxysterol ⁷-reductase activity.

View larger version (20K): [in this window] [in a new window]   Figure 6. Inhibition of 3ß-hydroxysterol 7-reductase. Increasing concentrations of ergosterol (x–x) 7-dehydrositosterol (–), and 7-dehydroepicholesterol (–) inhibited the conversion of 7-dehydrocholesterol to cholesterol. Ergosterol and 7-dehydroepicholesterol are more potent inhibitors than 7-dehydrositosterol. Each point and bars represent mean ± SEM from 5 rat liver microsomal preparations. The addition of lathosterol (–) from 100–400 µM did not inhibit 7- reductase activity.

Figure 7 shows 3ß-hydroxysterol ⁷-reductase activities with increasing concentrations of [1, 2-³H]7-dehydrocholesterol substrate, in the absence and presence of 200 or 400 µM concentrations of unlabeled ergosterol. The Lineweaver-Burk double reciprocal plots (1/V vs. 1/S) all intersect at the same point on the ordinate, which indicates competitive inhibition of 3ß-hydroxysterol ⁷-reductase activity by ergosterol. Similar results were obtained with either 7-dehydrositosterol or 7-dehydroepicholesterol but not lathosterol. These three structurally similar sterols that undergo reduction of the double bond at C-7,8 competitively inhibit the reduction of 7-dehydrocholesterol by binding at the enzyme active site.

View larger version (14K): [in this window] [in a new window]   Figure 7. Competitive inhibition of 3ß-hydroxysterol 7-reductase activity. Lineweaver-Burk double reciprocal plots of the conversion of increasing concentrations (1/S) of 7-dehydrocholesterol to cholesterol catalyzed by 3ß-hydroxysterol 7-reductase in the presence of ergosterol (0, 200, and 400 µM). Each point represents the mean ± SEM from 5 different rat liver microsomal preparations.

Figure 3 illustrates the structures of AY 9944 and BM 15.766, two additional inhibitors of 3ß-hydroxysterol ⁷-reductase activity. These non-steroidal compounds are structurally and chemically unrelated to 7-dehydrocholesterol and show different physical properties, but are powerful inhibitors of 3ß-hydroxysterol ⁷-reductase in vivo and produce congenital anomalies similar to the Smith- Lemli-Opitz syndrome when fed to pregnant rats. (14) (15). Both are more powerful inhibitors than the competitive inhibitors, ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol [AY 9944 is 500 times more potent than BM 15.766; (I₅₀ = 5 x 10^-8 vs. 1 x 10^-6, respectively)]. These concentrations are several orders of magnitude lower than the concentrations of the competitive inhibitors that produced only up to 30% inhibition of 3ß-hydroxysterol ⁷-reductase activity.

To define the mechanism of enzyme inhibition, we have analyzed the activity (1/V) of 3ß-hydroxysterol ⁷-reductase with increasing concentrations of the inhibitor in the presence of 10, 20, and 40 µM concentrations of the substrate ( Figure 8, Dixon plots). The two compounds (AY 9944 and BM 15.766) showed similar patterns of inhibition as illustrated for AY 9944. All three lines intersected at the same point on the abcissa which indicated that AY 9944 is a non-competitive inhibitor. Similar results were obtained for BM 15.766. In other words, these inhibitors bind either to the free enzyme or to the enzyme–substrate complex at a site on the enzyme, other than the active site.

View larger version (19K): [in this window] [in a new window]   Figure 8. Non-competitive inhibition of 3ß-hydroxysterol 7-reductase activity. Dixon plots of the conversion of 7-dehydrocholesterol (10, 20, and 40 µM) to cholesterol catalyzed by 3ß-hydroxysterol 7-reductase in the presence of increasing concentrations of AY 9944. Each point represent mean ± SEM from 5 different rat liver microsomal preparations.

Table 1 summarizes the results of experiments to determine whether 3ß-hydroxysterol ⁷-reductase activity is regulated by phosphorylation/dephosphorylation. Preparation of microsomes in the presence of 50 mM NaF, which is known to inhibit endogenous phosphatases, led to a significant increase (+30%, P < 0.005) in the activity. Preincubation of microsomes with 5 mM MgC1₂ and 5 mM ATP significantly increased the activity (+150%, P < 0.005) compared with 5 mM MgC1₂ alone. Mg-ATP was enough to activate the enzyme and the addition of 50 µM cAMP and 20 units of cAMP-dependent protein kinase to the preincubation mixture did not lead to more activation. In contrast, preincubation of microsomes with 10 units of alkaline phosphatase significantly reduced the activity (-13%, P < 0.05) compared with 10 units of boiled alkaline phosphatase. The addition of more than 10 units of alkaline phosphatase did not cause greater inhibition. These results indicate that 3ß-hydroxysterol ⁷-reductase is inhibited by dephosphorylation and that enzyme activity can be increased by phosphorylation. Thus, 3ß-hydroxysterol ⁷-reductase is short-term regulated by phosphorylation/dephosphorylation so that dephosphorylation inactivates the enzyme and phosphorylation increases its activity.

View this table: [in this window] [in a new window]   Table 1. Effects of NaF and alkaline phosphatase on 3ß-hydroxysterol 7-reductase activities

	DISCUSSION

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The results of this investigation demonstrate specific structural requirements for the sterol substrate to bind to the enzyme protein for the reduction of the double bond at C-7,8. The reaction is catalyzed by the enzyme, 3ß-hydroxysterol ⁷-reductase that is located in the microsomes. When the substrate, 7-dehydrocholesterol, is modified by adding substituents or a double bond to the apolar side-chain or epimerizing the 3ß-hydroxy to a 3-configuration in ring A, ⁷-reductase activity is hindered (Figure 5). However, the most specific requirement for the reduction of the double bond at C-7,8 is the presence of a double bond at C-5,6. Lathosterol which is 5-dihydrosaturated and lacks the C-5,6 double bond does not interact with the enzyme protein and its double bond at C-7,8 is not reduced. Increasing concentrations of ergosterol, 7-dehydrositosterol, or 7-dehydroepicholesterol competitively inhibited the conversion of 7-dehydrocholesterol to cholesterol as illustrated in Figure 6. Lineweaver-Burk double reciprocal plots all intersected at the same point on the Y-axis (Figure 7). In contrast, AY 9944 and BM 15.766 (Figure 3which are non-competitive inhibitors, reacted with the enzyme protein at a different site so that additional substrate did not displace these inhibitors from the inhibitor–enzyme–substrate complex. As a result, the double bond at C-7,8 was not catalytically reduced by the enzyme. The Dixon plots (Figure 8) illustrate the kinetics of this inhibition where the plots of increasing inhibitor concentrations all intersect together on the X-axis.

An important new observation was the short-term regulation of 3ß-hydroxysterol ⁷-reductase activity by phosphorylation/dephosphorylation. Exposure of the microsomes to alkaline phosphatase (dephosphorylation) significantly decreased ⁷-reductase activity. The addition of Mg²+ and ATP (phosphorylation) increased ⁷-reductase activity 2.5-fold. Thus, 3ß-hydroxysterol ⁷-reductase activity undergoes short-term regulation similar to cholesterol 7-hydroxylase (10), but opposite to HMG -CoA reductase, where dephosphorylation activates and phosphorylation inhibits enzyme activity (14).

Another important implication is that ergosterol, which is chemically more stable than 7-dehydrocholesterol, can be substituted for 7-dehydrocholesterol in the assay to measure the reduction of the double bond at C-7,8. Although enzyme activities are lower with the ergosterol substrate, significant differences in ⁷-reductase activities between homozygotes, heterozygotes, and controls could be demonstrated in fibroblasts (15).

In summary, we have defined the structural requirements necessary to reduce the double bond at C-7,8. A double bond at C-5,6 is an absolute requirement, while substitutions in the side-chain and epimerization of the 3ß- to a 3-hydroxy configuration significantly reduced ⁷-reductase activity. Moreover, 3ß-hydroxysterol ⁷-reductase is short-term regulated by dephosphorylation (inhibition) and phosphorylation (activation).

	ACKNOWLEDGMENTS

This work was supported by a grant from the National Institutes of Health, DK-26756, the Smith-Lemli-Opitz Research Fund, a Grant-in-Aid from the American Heart Association, NJ Affiliate Inc., and the Research Service Veterans Administration, Washington DC. The excellent technical assistance of S. Hauser and B. Rouse is greatly appreciated.

Manuscript received February 2, 1998; and in revised form July 13, 1998.

	REFERENCES

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