United States Department of Agriculture, Agricultural Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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The flooding dose method, which is used to measure tissue protein synthesis, assumes equilibration of the isotopic labeling between the aminoacyl-tRNA pool and the tissue and blood free amino acid pools. However, this has not been verified for a phenylalanine tracer in an in vivo study. We determined the specific radioactivity of [3H]phenylalanine in the aminoacyl-tRNA and the tissue and blood free amino acid pools of skeletal muscle and liver 30 min after administration of a flooding dose of phenylalanine along with [3H]phenylalanine. Studies were performed in neonatal pigs in the fasted and refed states and during hyperinsulinemic-euglycemic-amino acid clamps. The results showed that, 30 min after the administration of a flooding dose of phenylalanine, there was equilibration of the specific radioactivity of phenylalanine among the blood, tissue, and tRNA precursor pools. Equilibration of the specific radioactivity of the three precursor pools for protein synthesis occurred in both skeletal muscle and liver. Neither feeding nor insulin status affected the aminoacyl-tRNA specific radioactivity relative to the tissue free amino acid specific radioactivity. The results support the assumption that the tissue free amino acid pool specific radioactivity is a valid measure of the precursor pool specific radioactivity and thus can be used to calculate protein synthesis rates in skeletal muscle and liver when a flooding dose of phenylalanine is administered.
protein synthesis; skeletal muscle; liver; insulin
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INTRODUCTION |
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TISSUE PROTEIN MASS is regulated by changes in the relative rates of protein synthesis and protein degradation. To determine rates of tissue protein synthesis, isotopic tracers are used to measure the rate of incorporation of a labeled amino acid into tissue protein, with adjustment for the level of labeling of the precursor pool at the site of protein synthesis. Although the amino acids acylated to tRNA are the immediate precursors of protein synthesis, only a few in vivo studies (5, 21, 34, 37) have measured the labeling (specific radioactivity or enrichment) of aminoacyl-tRNA because of the technical difficulty of its isolation. Aminoacyl-tRNA is present in low concentration within tissues and is extremely labile (23).
The flooding dose method, which is frequently used to measure tissue protein synthesis, involves the relatively rapid administration of a large dose of unlabeled amino acid along with the amino acid tracer (18). The method assumes that this expansion of the amino acid pools results in equilibration of the specific radioactivities among the aminoacyl-tRNA and the tissue and blood free amino acid pools. Equilibration between the aminoacyl-tRNA and the intracellular and/or extracellular amino acid pools was demonstrated in some (9, 24) but not all (3, 32) in vitro studies in which a high concentration of the labeled amino acid was present in the medium. The limited studies performed in vivo suggested that the aminoacyl-tRNA specific radioactivity equilibrates with that of the intracellular and extracellular amino acid pools when a flooding dose of leucine or proline is used (20, 28). Equilibration of the specific radioactivities between the aminoacyl-tRNA pool and the intracellular free amino acid pool has not been verified with a flooding dose of the amino acid tracer phenylalanine in an in vivo study. Phenylalanine is used commonly as an amino acid tracer because it has a relatively small pool size, compared with leucine, and therefore a "flooding" condition is more easily achievable.
The flooding dose technique is used frequently in studies to determine how nutritional and hormonal factors influence tissue protein synthesis. It is essential to establish, therefore, whether the equilibration of the aminoacyl-tRNA with the tissue free amino acid pool changes with different nutritional and hormonal conditions, and whether this varies among tissues when the flooding dose method is used to measure tissue protein synthesis. Recently, studies using the constant infusion method, in which the label is administered at tracer levels, suggested that the relationship between aminoacyl-tRNA enrichment and the enrichment of surrogate precursor pools changes during meal feeding (21) but does not change during insulin infusion (37). In addition, when the constant infusion method is used, the relationship between aminoacyl-tRNA and other precursor pools can differ among tissues under the same study conditions (5, 37).
The aim of this study was to determine whether the specific radioactivity of aminoacyl-tRNA equilibrates with that of the blood and tissue free amino acid pools when a flooding dose of phenylalanine is used to measure tissue protein synthesis in vivo. In addition, we wished to determine whether the relationship between the aminoacyl-tRNA and surrogate precursor pools is altered by changes in the nutritional and hormonal state and is different among tissues. To this end, we determined the specific radioactivity of phenylalanine acylated to tRNA and the phenylalanine specific radioactivity in the tissue and blood free amino acid pools in skeletal muscle and liver 30 min after the administration of a flooding dose of phenylalanine. Studies were performed in neonatal pigs in the fasted and refed states and during hyperinsulinemic-euglycemic-amino acid clamps.
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METHODS |
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Animals and experimental design. Six conventional crossbred pregnant sows were housed in free-standing farrowing crates in individual, environmentally controlled rooms 1 wk before farrowing. Sows were fed a commercial diet (5084; PMI Feeds, Richmond, IN) during the lactation period. After farrowing, piglets remained with the sow and were not given supplemental feed.
In experiment 1, the effect of feeding was studied in 7-day-old pigs. Two pigs from each of three litters were randomly assigned to one of two treatment groups: they were either fasted for 24 h or refed for 2.75 h after a 24-h fast. Jugular vein catheters were inserted at the beginning of the fasting period. After surgery, pigs were placed in individual cages and provided water throughout the remainder of the fasting period. Pigs that were refed after the 24-h fast were given three gavage feeds of 30 ml/kg body weight of porcine mature milk at 60-min intervals. In experiment 2, the effect of insulin infusion was studied in 7-day-old pigs. Two pigs from each of three litters were randomly assigned to one of two treatment groups, and hyperinsulinemic-euglycemic-amino acid clamps were performed at insulin doses of 0 and 100 ng · kgProtein synthesis in vivo.
In both experiments 1 and 2, tissue
protein synthesis was measured in vivo using the flooding dose
technique described by Garlick et al. (18). Thirty minutes before the
end of the study, pigs were injected via the jugular vein catheter with
10 ml/kg body weight of a flooding dose of phenylalanine (Amersham,
Arlington Heights, IL), which provided 1.5 mmol phenylalanine/kg body
weight and 1 mCi of
L-[4-3H]phenylalanine/kg
body weight. Samples of whole blood were taken 5, 15, and 30 min after
the injection of
[3H]phenylalanine for
measurement of the specific radioactivity of the extracellular free
pool of phenylalanine. Immediately after the 30-min blood sample was
taken, pigs were given a lethal injection of pentobarbital sodium (50 mg/kg body weight). Samples of the longissimus dorsi muscle and liver
were rapidly removed, blotted, and frozen in liquid nitrogen. The time
from injection of the pentobarbital to the freezing of the muscle was
<1.5 min and for the liver was <3 min. The time from the injection
of the labeled phenylalanine to the freezing of the tissue was recorded
accurately and considered in the calculation of protein synthesis.
Samples were stored at 70°C until analyzed.
Aminoacyl-tRNA isolation. Tissue aminoacyl-tRNA was isolated using a modified method of Baumann et al. (5). Standard precautions were taken throughout the procedure to eliminate RNase contamination. All procedures were carried out at 4°C. Frozen tissue (5 g) was finely powdered in liquid nitrogen and then homogenized in 10 volumes of a 1% SDS-0.05 M cacodylic acid buffer, pH 5.5. The RNA in the homogenate was extracted with an equal volume of phenol-SDS-cacodylate buffer (4:1), followed by centrifugation (10,000 g for 30 min at 4°C). After further extraction of the RNA with the phenol-SDS-cacodylate buffer, RNA was precipitated with 2.5 volumes of chilled ethanol in the presence of 2% potassium acetate, pH 6.5. The pellet was washed three times in 100% ethanol, dried under a stream of nitrogen, and then dissolved in diethyl pyrocarbonate-treated water. RNA integrity was verified by the presence of discrete bands of 28S and 18S rRNA on a 0.6 M formaldehyde-agarose gel stained with ethidium bromide (15). The tRNA was deacylated with 0.12 M KOH to pH 9.0 and incubated for 1 h at 37°C. The amino acids were separated from tRNA by acidification with 0.5 M HCl to pH 2.0 and centrifugation (3,000 g for 30 min). The supernatant containing the amino acids was dried (Speedvac, Savant Instruments, Farmingdale, NY) and reconstituted in 1.0 ml of 1 M acetic acid. The amino acids were purified by cation exchange (AG 50W-X8 resin, 100-200 mesh, H+ form, Bio-Rad Laboratories, Richmond, CA).
To determine whether the phenylalanyl-tRNA isolates were free of contamination from the intracellular and extracellular free phenylalanine, 0.5 mCi of a solution of [3H]phenylalanine (1 mCi/1.5 mmol) was added to "cold" tissue homogenates from pigs that had not been injected with any radioisotope. The aminoacyl-tRNA was isolated as described above.Isolation of protein-bound amino acids and tissue and blood free amino acids. Tissues were homogenized in 0.2 M perchloric acid (PCA) as previously described (11). The homogenate supernatants containing the tissue free amino acid pools were separated from the PCA-insoluble precipitates and neutralized. The acid-insoluble precipitates were washed three times and hydrolyzed for 24 h with 6 M HCl. Blood proteins were precipitated with PCA, and the supernatants were neutralized.
Analysis of specific radioactivity. Protein hydrolysates, tissue supernatants containing free amino acids, whole blood supernatants containing free amino acids, and amino acids separated from tRNA were dried, washed, and resuspended in water for determination of phenylalanine specific radioactivity. Phenylalanine in the four fractions was separated from other amino acids using anion exchange chromatography (AS8 column; Dionex, Sunnyvale, CA) as described previously (11). Amino acids were postcolumn derivatized with orthophthalaldehyde reagent and detected with a fluorimeter. Fractions were collected, and the radioactivity associated with the phenylalanine peak was measured in a liquid scintillation counter (TM Analytic, Elk Grove Village, IL). The intra-assay coefficients of variation for the blood free amino acid pool, the tissue free amino acid pool, and the aminoacyl-tRNA pool specific radioactivities were 1.2, 1.4, and 5.8%, respectively. The interassay coefficients of variation for the blood free amino acid pool, the tissue free amino acid pool, and the aminoacyl-tRNA pool specific radioactivities were 2.0, 2.6, and 11.4%, respectively.
Calculations. The fractional rate of protein synthesis (KS) was calculated as KS (%/day) = [(SB/SA) × (1,440/t)] × 100, where SB is the specific radioactivity of the protein-bound phenylalanine, SA is the specific radioactivity of the precursor pool, and t is the time of labeling in minutes.
Statistics. Analysis of variance for repeated measures was used to determine the difference between precursor pools, the effect of nutritional or hormonal state, and whether the difference between precursor pools was dependent on nutritional or hormonal state (interaction) with respect to specific radioactivity or fractional protein synthesis rates (31). Linear regression analysis was used to determine whether there were changes in the blood specific radioactivities with time. Results are presented as means ± SE. Probability values of <0.05 were considered statistically significant and are not reported in the text.
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RESULTS |
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Isolation of tRNA. To determine whether the aminoacyl-tRNA isolates were free of contamination from the intracellular and blood free amino acid pools, [3H]phenylalanine was added to tissue homogenates from pigs that had not been injected with any radioisotope. There were no counts that were greater than background in the RNA isolates.
The yield of phenylalanyl-tRNA, as measured by HPLC, was 0.61 ± 0.09 and 0.53 ± 0.06 nmol/g in skeletal muscle and liver, respectively. There were no effects of refeeding or insulin infusion on the concentration of phenylalanyl-tRNA.Blood, tissue, and aminoacyl-tRNA specific radioactivity.
The specific radioactivity of the blood tended to decrease
~0.26%/min (Table 1). However, this
decline was not statistically significant. At the time of tissue
sampling, i.e., 30 min after administration of the flooding dose of
[3H]phenylalanine, the
specific radioactivities of the tissue free amino acid pool and
phenylalanyl-tRNA pool in skeletal muscle were ~98% of those in
blood (Table 2). The specific
radioactivities of the tissue free amino acid pool and
phenylalanyl-tRNA pool in liver were ~95% of those in blood. There
were no significant differences in the phenylalanine specific
radioactivities among the blood, tissue, and aminoacyl-tRNA precursor
pools in skeletal muscle and liver. Neither refeeding nor the insulin
infusion altered the specific radioactivities of the precursor pools in
skeletal muscle and liver.
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Fractional rates of tissue protein synthesis.
The specific radioactivities in the blood, tissue, and the
phenylalanyl-tRNA precursor pools were used to calculate the fractional rates of protein synthesis in skeletal muscle and liver (Table 3). There were no differences among the
estimated fractional protein synthesis rates when the different
precursor pools were selected for either skeletal muscle or liver.
Protein synthesis rates were ~65% greater in skeletal muscle and
~20% greater in liver of refed pigs compared with fasted controls
(experiment 1). The infusion of
insulin while glucose and amino acid concentrations were maintained at
the fasting level increased protein synthesis in skeletal muscle by
~60% but did not stimulate protein synthesis in liver compared with
that of saline-infused controls (experiment 2).
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DISCUSSION |
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Aminoacyl-tRNA isolation. The accurate determination of the rate of protein synthesis requires measurement of the specific radioactivity of the tracer amino acid in tissue protein and in the precursor pool. However, the determination of aminoacyl-tRNA specific radioactivity is not routinely performed because of the technical difficulties of its isolation. Tissue tRNA concentrations are low, and the concentration of each amino acid bound to the tRNA, therefore, is even lower (29). This fact necessitates the analysis of relatively large amounts of tissue, which frequently is not possible. Moreover, the isolation and purification procedure for aminoacyl-tRNA is tedious and laborious, requiring extreme caution to avoid contamination with both amino acids and RNases. The short half-life of aminoacyl-tRNA necessitates that the tissues be acquired and subsequently frozen very rapidly.
The amounts of phenylalanine acylated to tRNA in skeletal muscle and liver in the current study were similar to those reported by most investigators for skeletal muscle, liver, heart, and brain (14, 23, 24, 30, 34), indicating that the yield of phenylalanyl-tRNA from the isolation procedure was adequate. Moreover, the presence of clear 18S and 28S bands on the total RNA isolate was indicative of intact RNA in the total RNA isolates. We verified also that the aminoacyl-tRNA isolates were free from contamination with intracellular and extracellular free amino acids by detecting no radioactivity in extracts from cold tissues to which the [3H]phenyalanine was added only during the homogenization procedure.Specific radioactivities of different precursor pools with the flooding dose method. Because of the technical difficulty of the isolation of tissue aminoacyl-tRNA, numerous techniques have been devised to approximate the labeling of the precursor pool for protein synthesis. The two most common methods of determining tissue protein synthesis rates, the flooding dose and the constant infusion methods, usually estimate the labeling of aminoacyl-tRNA from the labeling of the free amino acids in the tissue or blood. In the constant infusion method, a trace amount of isotopically labeled amino acid is usually infused over a period of 4-8 h during which the animal is in a metabolic and isotopic steady state. In the flooding dose method, a large amount of the unlabeled amino acid is administered along with labeled amino acid to "flood" the free amino acid pools. Because the flooding dose technique allows the determination of acute changes in tissue protein synthesis over a period of 5-30 min, the method is particularly attractive for use in conditions of metabolic nonsteady state, such as acute feeding and hormone infusion studies (17). Moreover, in very small animals, it is the only feasible method.
The flooding dose method was developed to minimize differences in the isotopic labeling between the blood and tissue free amino acid pools and the aminoacyl-tRNA pool. Although it is assumed that there is equilibration among the three pools, few studies have tested the validity of this assumption. Studies performed in vitro and in situ have reported conflicting data. Some report equilibration among aminoacyl-tRNA, tissue, and/or blood free amino acid pools when a high concentration of the labeled amino acid is present in the medium or perfusate (9, 24); others report that the specific radioactivity of aminoacyl-tRNA is less than that in the medium (3, 32). A lack of equilibration among the three precursor pools was reported in in vivo studies that used insufficient quantities of unlabeled phenylalanine and leucine to achieve true flooding conditions during the administration of the labeled amino acids (4) or that administered unlabeled valine 1 h before injecting the labeled amino acid (30). The limited studies performed in vivo using the flooding dose technique suggest that leucyl- and prolyl-tRNA pools in heart become equilibrated with those in plasma after a flooding dose of these amino acids (20, 28). The current study suggests that, 30 min after the injection of a flooding dose of phenylalanine along with a labeled phenylalanine tracer, there is equilibration of the specific radioactivities among the tRNA, tissue, and blood precursor pools in both skeletal muscle and liver. However, the study does not indicate how rapidly the aminoacyl-tRNA becomes equilibrated with other precursor pools. Delays in the equilibration of aminoacyl-tRNA with other precursor pools would underestimate protein synthesis, and this underestimation would be more pronounced the shorter the period of labeling. Although the time course of the phenylalanyl-tRNA labeling was not determined in the current study, the decrease in the specific radioactivity of the blood was minimal, ~8% over the course of the 30-min labeling period, and was not statistically significant. In addition, it has been previously demonstrated (11) that there is equilibration between the blood and tissue free amino acid pools in skeletal muscle by 2 min, and others (1, 26) have found equilibration by 5 min after the injection of the [3H]phenylalanine or [3H]valine in the young rat and lamb. This suggests that the use of specific radioactivity values derived from the tissue free pool would likely be satisfactory for calculating fractional protein synthesis rates. Recently, the labeling of aminoacyl-tRNA has been assessed during studies in which the constant infusion method was used to measure tissue protein synthesis (5, 21, 34, 37). Some of these studies suggest that the enrichment of aminoacyl-tRNA is intermediate between enrichments of blood and the tissue free amino acid pools (34, 37). Tissue free amino acid pool enrichment has also been suggested to be the best predictor of aminoacyl-tRNA enrichment during constant infusions of labeled amino acids (5, 21), particularly under different physiological perturbations such as meal feeding, when the ratio of the enrichment values of aminoacyl-tRNA to other surrogate pools changes (21). Because tRNA labeling may change under different physiological conditions during constant infusion studies (21), in the current study we addressed whether the hyperinsulinemia and hyperaminoacidemia that accompany refeeding, and the hyperinsulinemia with concurrent euaminoacidemia during hyperinsulinemic-euglycemic-amino acid clamps, alter the relationship among the precursor pools for protein synthesis. The results suggest that these hormonal and nutritional perturbations do not alter the relationships among the aminoacyl-tRNA and the tissue and blood free amino acid pool specific radioactivities when a flooding dose of phenylalanine is administered.Tissue protein synthesis rates using different precursor pools in fed and insulin-treated pigs. A number of studies have examined whether feeding or acute increases in plasma insulin stimulate protein synthesis, particularly in skeletal muscle. Most (2, 8, 25) but not all (27, 33) in vivo studies performed in adult humans or animals suggest that feeding has little effect on protein synthesis. Likewise, most (2, 13, 19, 22) but not all (7) studies performed in adult humans and animals show little, if any, response of muscle protein synthesis to increases in insulin within the physiological range. However, studies conducted in growing rats and pigs suggest that protein synthesis can be stimulated both by feeding (10, 12, 16) and insulin (16). This suggests that the effects of feeding and insulin on protein synthesis may be age dependent. Moreover, we have demonstrated that the stimulation of skeletal muscle protein synthesis by feeding (10, 12) and the infusion of insulin (36) declines during early postnatal development. However, interpretation of the results of these studies is made difficult by the different methodologies used to measure protein synthesis. Most studies conducted in adults have utilized the constant infusion method, whereas studies conducted in growing animals have utilized the flooding dose method. In addition, most studies have not determined the labeling of the true precursor for protein synthesis, aminoacyl-tRNA.
The results of the present study demonstrate that the labeling of the aminoacyl-tRNA and the tissue and blood free amino acid pools when a flooding dose of [3H]phenylalanine was administered was similar in the fasted and refed states and during hyperinsulinemic-euglycemic-amino acid clamps. Thus, consistent with the results of our previous studies, in which the tissue free amino acid pools were used to calculate fractional rates of protein synthesis (10, 36), the results of the current in vivo study suggest that 1) feeding stimulates protein synthesis in skeletal muscle and liver of neonatal pigs, 2) the effect of feeding is greater in skeletal muscle than in liver, and 3) physiological elevations of insulin, in the presence of fasting levels of amino acids and glucose, increase protein synthesis in skeletal muscle by a magnitude similar to that which occurs with feeding. Young et al. (37) reported that insulin did not influence aminoacyl-tRNA enrichment during a constant tracer infusion of phenylalanine and that, when aminoacyl-tRNA was used as the precursor pool, insulin did not stimulate protein synthesis in skeletal muscle and heart of adult rats. Thus compilation of the results of studies in which the labeling of the true precursor for protein synthesis was determined, i.e., the current study and that of Young et al. (37), suggests that the effect of insulin on skeletal muscle protein synthesis is age dependent. In addition, the results of the current study suggest that the infusion of insulin while amino acids and glucose are maintained at the fasting level does not reproduce the stimulation of liver protein synthesis that occurs with feeding in neonatal pigs. This suggests that dietary and/or hormonal factors other than insulin likely regulate the stimulation of liver protein synthesis by feeding in the neonate.Conclusions. The results of the present study demonstrate that, 30 min after the injection of a flooding dose of phenylalanine along with a labeled phenylalanine tracer, there is equilibration of the phenylalanine specific radioactivities among the blood free amino acid pool, the tissue free amino acid pool, and the aminoacyl-tRNA pool. Neither feeding nor insulin status affected the aminoacyl-tRNA specific radioactivity relative to the tissue free amino acid specific radioactivity. Equilibration of the specific radioactivity of the precursor pools occurred in both skeletal muscle and liver. The results suggest that the aminoacyl-tRNA and the tissue or blood free amino acid pool specific radioactivities can be used to calculate the fractional rates of protein synthesis in skeletal muscle and liver when a flooding dose of phenylalanine is administered. Given that the calculated fractional protein synthesis rates are similar when any one of the three precursor pools is used, and that the determination of the aminoacyl-tRNA specific radioactivity is difficult, the values derived from the tissue free pool may be considered satisfactory for the calculation of fractional protein synthesis rates in skeletal muscle and liver when a flooding dose of phenylalanine is administered.
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ACKNOWLEDGEMENTS |
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We thank Peter J. Reeds for helpful discussions, E. O'Brian Smith for statistical assistance, L. Loddeke for editorial review, A. Gillum for graphics, and M. Alejandro for secretarial assistance. We acknowledge Eli Lilly Co. for generous donation of porcine insulin, and McGaw, Inc. for the generous donation of TrophAmine.
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FOOTNOTES |
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This work is a publication of the USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, TX. This project has been funded in part by National Institute of Arthritis and Musculoskeletal and Skin Diseases Institute Grant R01-AR44474 and the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement no. 58-6250-6-001. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products or organization imply endorsement by the US Government.
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. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: T. A. Davis, USDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, 1100 Bates St., Houston, TX 77030 (E-mail: tdavis{at}bcm.tmc.edu).
Received 14 August 1998; accepted in final form 9 March 1999.
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