1 Department of Pathology, St. Louis University Medical School, St. Louis, Missouri 63104; and Departments of 2 Pathology, 3 Pharmacology and Toxicology, and 4 Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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ABSTRACT |
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Diabetes-induced changes in phospholipase A2 (PLA2) activity have been measured in several tissues but are undefined in diabetic myocardium. We measured ventricular PLA2 activity in control, streptozotocin-induced diabetic, and insulin-treated diabetic rats and characterized myocardial phospholipids to determine whether diabetes altered myocardial phospholipid metabolism. Increased membrane-associated Ca2+-independent PLA2 (iPLA2) activity was observed in diabetes that was selective for arachidonylated phospholipids. Increased iPLA2 activity was accompanied by an increase in choline lysophospholipids. Diabetes was associated with marked alterations in the phospholipid composition of the myocardium, characterized by decreases in esterified arachidonic and docosahexaenoic acids and increases in linoleic acid. The decrease in polyunsaturated fatty acids was confined to diacylphospholipids, whereas the relative amount of these fatty acids in plasmalogens was increased. Diabetes-induced changes in PLA2 activity, lysophospholipid production, and alterations in phospholipid composition were all reversed by insulin treatment of diabetic animals. Diabetes-induced changes in membrane phospholipid content and phospholipid hydrolysis may contribute to some of the alterations in myocardial function that are observed in diabetic patients.
diabetes; myocardium; phospholipase A2
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INTRODUCTION |
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DIABETES MELLITUS IS ASSOCIATED with increased cardiovascular morbidity and mortality that can occur independently of coronary artery disease (9). Diabetes is reported to elicit several changes in the properties of the myocardium, such as abnormalities in contractile and regulatory proteins and defects in intracellular ionic handling, especially calcium handling, through alterations in various sarcoplasmic reticular and sarcolemmal membrane calcium transporters (9). Prolonged diabetes has been shown to induce an essential fatty acid deficiency state that leads to a number of disease states, such as atherosclerosis (4), neuropathy (7), and glomerular hyperfiltration (16). A decrease in arachidonylated phospholipids and an increase in phospholipids containing linoleic acid have been observed in the phospholipid composition of membranes from several tissues from rat models of diabetes, including heart, kidney, and liver (17).
Diabetes is also associated with increased circulating levels of lysophosphatidylcholine (LPC), free fatty acids (25), and stable derivatives of prostaglandin E2, prostaglandin I2, and thromboxane A2 (2, 3, 24), indicating increased phospholipase A2 (PLA2)-catalyzed hydrolysis of phospholipids. It appears likely that diabetes-induced changes in PLA2 activity may be different in different tissues. For example, PLA2 activity in the plasma and liver was found to be reduced (27), whereas PLA2 activity in skeletal muscle and platelets was found to be increased by diabetes (29, 30). Diabetes-induced changes in phospholipid fatty acid composition (28) and alterations in PLA2 activity (27, 30) have been demonstrated to be reversed by insulin treatment.
Because both phospholipid composition (8, 31) and accumulation of metabolites of PLA2-catalyzed phospholipid hydrolysis (19, 21, 23) can influence the molecular dynamics and biophysical properties of membranes, this study was performed to examine the effects of streptozotocin (STZ)-induced diabetes and insulin treatment on the activity and expression of myocardial PLA2 isoforms and the composition of plasmalogen and diacylphospholipids in the diabetic rat myocardium. Diabetes-induced changes in the membrane composition may be responsible for alterations in the function of integral membrane proteins, contributing to some of the changes observed in the diabetic myocardium.
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MATERIALS AND METHODS |
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Animal Model: Streptozotocin-Induced Diabetes
All experimental protocols for animal studies were approved by the Animal Care and Use Committee at the University of Arkansas for Medical Sciences. Male Sprague-Dawley rats (300-350 g) were housed in the Division of Laboratory Animal Medicine at the University of Arkansas for Medical Sciences (UAMS) and allowed free access to food and water. They were randomly separated into 3 groups: 1) controls, 2) diabetics, and 3) diabetics treated with insulin. Diabetes was induced by intravenous administration of 50 mg/kg of STZ dissolved in 0.05 M of citrate buffer, pH 4.5; controls received an equivalent volume (1 ml/kg) of diluent. Blood glucose and body weight were measured immediately before injection of STZ and at weekly intervals thereafter. Blood was obtained from a tail vein, and glucose concentrations were determined by means of an Accu-Chek II blood glucose monitor (Boehringer Mannheim Diagnostics, Indianapolis, IN). The presence of diabetes was confirmed by sustained blood glucose values ofMeasurement of PLA2 Activity
Animals were anesthetized with ether, and the hearts were rapidly removed. The ventricles from control, diabetic, and insulin-treated rat hearts were homogenized at 4°C in homogenization buffer containing (in mmol/l) 250 sucrose, 10 KCl, 10 imidazole, 5 EDTA, and 2 dithiothreitol with 10% glycerol, pH 7.8. The homogenate was centrifuged at 14,000 g for 10 min to remove unbroken cells, nuclei, and mitochondria. The resultant supernatant fraction was centrifuged at 100,000 g for 60 min to separate the membrane fraction (pellet) from the cytosolic fraction (supernatant). The membrane fraction was washed twice with buffer to remove residual cytosol and resuspended in ice-cold homogenization buffer. The membrane fraction is comprised predominantly of microsomes, including vesicles of sarcolemma and sarcoplasmic reticulum, with >70% recovery of sarcolemma and sarcoplasmic reticulum marker enzyme activities (Na+/K+-ATPase, Ca2+-ATPase) in this fraction with minimal cytosolic contamination (absence of creatine kinase, lactate dehydrogenase). Based on protein measurements, the total yield of membrane and cytosol fractions was similar in each experimental group. PLA2 activity in the subcellular fractions was assessed by incubating enzyme (8 µg membrane protein or 300 µg cytosolic protein) with 100 µM of plasmenylcholine or phosphatidylcholine radiolabeled with oleate (18:1) or arachidonate (20:4) at the sn-2 position and containing a saturated 16C aliphatic moiety at the sn-1 position (16:0), as described previously (20, 22). Incubations were performed in assay buffer containing 10 mM Tris, 10% glycerol, pH 7.0, with either 4 mM EGTA or 10 mM Ca2+ at 37°C for 5 min in a total volume of 200 µl. Reactions were terminated by the addition of 100 µl of butanol and then vortexed and centrifuged at 2,000 g for 5 min. Released radiolabeled fatty acid was isolated by application of 25 µl of the butanol phase to channeled silica gel G plates and development in petroleum ether-diethyl ether-acetic acid (70:30:1, vol/vol/vol) and was subsequently quantified by liquid scintillation spectrometry.Measurement of Choline Lysophospholipids
Lysophosphatidylcholine (LPC) and lysoplasmenylcholine (LPlasC) were measured as described previously (22). The procedure involves the extraction of lipids from the myocytes by the method of Bligh and Dyer (5), followed by the separation of the lysophospholipids from other phospholipids by HPLC. The purified LPC and LPlasC fractions, as well as known amounts of LPC and LPlasC standards, were then acetylated with [3H]acetic anhydride by using 0.33 M of dimethylaminopyridine as a catalyst. The acetylated lysophospholipid was then separated by thin-layer chromatography, and radioactivity was quantified by liquid scintillation spectrometry. Standard curves were constructed, and LPC and LPlasC content was derived for all samples.Immunoblot Analysis of PLA2
Rat myocardium was homogenized in lysis buffer containing (in mmol/l) 20 HEPES (pH 7.6), 250 sucrose, 2 dithiothreitol, 2 EDTA, 2 EGTA, 10Extraction, Separation, and Analysis of Phospholipid Classes
Cellular phospholipids were extracted from homogenized rat ventricles with chloroform and methanol by the method of Bligh and Dyer (5) at 0-4°C. Phospholipids were separated into different classes by injecting them onto an Ultrasphere-Si (5 µm silica), 4.6 × 250 mm HPLC column (Beckman Instruments, Fullerton, CA) and by using gradient elution with hexane-isopropanol-water (10). Fractions were collected corresponding to the following phospholipid classes (listed in order of elution): diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin. Phospholipid classes were quantified by measurement of lipid phosphorus by microphosphate assay (6). The fatty acid composition of glycerophospholipids was determined by gas-liquid chromatography (GLC) analysis of the fatty acid methyl ester and dimethylacetal derivatives produced after acid-catalyzed methanolysis (8, 13). The alkylacyl content of phosphatidylcholine and phosphatidylethanolamine was determined by quantification of lipid phosphorus in the lysophospholipid fraction remaining after sequential exhaustive base- and acid-catalyzed hydrolysis of the diradylphospholipids (13).Separation and Quantification of Individual Choline and Ethanolamine Glycerophospholipid Molecular Species
Individual choline glycerophospholipid (CGP) and ethanolamine glycerophospholipid (EGP) molecular species were isolated by reverse-phase HPLC with the use of an Ultrasphere ODS (5 µm, C-18) column, 4.6 × 250 mm (Beckman Instruments). Individual molecular species were separated by means of gradient elution with acetonitrile-methanol-water with 20 mM of choline chloride (21). The molecular identity of individual molecular species was established by GLC characterization (21). Quantification of individual phospholipid molecular species was achieved by determination of lipid phosphorus in reverse-phase HPLC column effluents. For lipid phosphorus determination, column effluents were taken to dryness under N2 and electrically heated at 150°C for 2 h with 400 µl of HClO4. The samples were allowed to cool to room temperature, and excess perchloric acid was neutralized by addition of 1 ml of 4.5 N KOH. The samples were centrifuged at 2,000 g for 10 min to sediment the KClO4 precipitate, and 600 µl of the supernatant were removed for assay of lipid phosphorus by the method of Itaya and Ui (18).Statistics
Statistical comparison of values was performed by the Student's t-test or analysis of variance with the Fisher multiple-comparison test, as appropriate. All results are expressed as means ± SE. Statistical significance was considered to be P < 0.05. ![]() |
RESULTS |
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Body Weight, Blood Glucose, and Ketones
Changes in body weight and blood glucose in control and diabetic animals are shown in Table 1. Body weight was found to be significantly lower in 50 mg/kg STZ-treated diabetic animals than in controls when measured 8 wk after administration of STZ or diluent. Blood glucose was elevated 8 wk after administration of STZ. Serum ketone levels were only slightly elevated in the diabetic animals. In a group of eight rats, three showed ketone levels to be negative (10 mg/dl), four had values in the low range (~20 mg/dl), and one showed a moderate level (30-40 mg/dl); none of the animals had ketone concentrations in the high range (80-100 mg/dl). The assay for ketones was always negative in control animals. Animals treated with STZ and given insulin daily gained more weight than the diabetic animals; however, at the end of 8 wk, their body weight was still significantly lower than that of control animals. Daily insulin treatment of diabetic animals significantly lowered blood glucose levels compared with diabetic animals.
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PLA2 Activity in Control and Diabetic Myocardium
PLA2 activity in cytosolic and membrane fractions isolated from the myocardium of control, diabetic, and insulin-treated rats was measured in the presence and absence of Ca2+ with (16:0, [3H]18:1) and (16:0, [3H]20:4) plasmenylcholine (Fig. 1A) and phosphatidylcholine (Fig. 1B) substrates. The majority of total PLA2 activity in control rat hearts was found to be membrane associated compared with activity in the cytosol or mitochondria (Table 2). The presence of Ca2+ in the assay buffer did not significantly increase PLA2 activity with use of either plasmenylcholine (Fig. 1A) or phosphatidylcholine (Fig. 1B) substrates, indicating that the majority of PLA2 activity was Ca2+ independent (iPLA2). Both membrane-associated (Fig. 1) and cytosolic (data not shown) PLA2 activities were selective for arachidonylated substrates. Membrane-associated iPLA2 activity measured with plasmenylcholine and phosphatidylcholine substrates was found to be significantly increased by diabetes (Fig. 1). The diabetes-induced increase in membrane-associated iPLA2 activity was reversed by treating diabetic animals with insulin (Fig. 1). The presence of diabetes had no effect on cytosolic PLA2 activity measured with plasmenylcholine or phosphatidylcholine substrates (data not shown). Thus diabetes is associated with an insulin-reversible increase in membrane-associated iPLA2, which demonstrates a preference for arachidonylated phospholipid substrates.
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Immunoblot Analysis of PLA2 Isoforms in Rat Myocardium
Immunoblot analysis was used to detect the presence of different PLA2 isoforms in control, diabetic, and insulin-treated myocardium and to determine whether the level of expression of any of the PLA2 isoforms was altered in diabetes. Separation of subcellular fractions and immunoblotting of cytosol and membranes revealed the presence of iPLA2 in the membrane fraction only, with no detectable iPLA2 signal in the cytosol. Immunoblot analysis of purified iPLA2 protein (Genetics Institute) demonstrated the presence of iPLA2 at the same molecular mass (~85 kDa, positive control). The presence of cPLA2 was detected in the cytosolic fraction from rat myocardium that migrated at the same molecular mass as that of purified cPLA2 protein (Genetics Institute; 110 kDa, positive control). The presence of cPLA2 was not detected in the membrane fraction. The presence of sPLA2 was detected in the cytosolic fraction, but the signal was very weak. No sPLA2 was detected in the membrane fraction. Thus rat myocardium possesses different PLA2 isoforms that are located in different subcellular compartments.After initial detection of PLA2 isoforms in the rat
myocardium, membrane and cytosolic protein samples isolated from
control, diabetic, and insulin-treated rat hearts were subjected to
immunoblot analysis by use of sPLA2, cPLA2 and
iPLA2 antibodies (Fig.
2A). The density of the
iPLA2 bands in membrane samples from diabetic myocardium
were approximately twofold greater than control myocardium (Fig. 2,
A and B). No significant change in the density of
cPLA2 or sPLA2 bands in the cytosolic fraction
was observed in diabetic myocardium (Fig. 2). The presence of diabetes
did not alter the subcellular localization of any of the
PLA2 isoforms. Thus diabetes causes a selective increase in
iPLA2 protein expression in the membrane fraction of rat
myocardium. Insulin treatment of the diabetic animals resulted in
reversal of the increased iPLA2 expression in the membrane
fraction (Fig. 2).
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Diabetes-Induced Changes in Myocardial Phospholipid Composition
Total phospholipid phosphorus in rat myocardium was found to be 94 ± 4 nmol/mg protein in control animals (n = 6), 117 ± 4 nmol/mg protein in diabetic animals (n = 5, P < 0.05), and 98 ± 5 nmol/mg protein in insulin-treated animals (n = 4). The major phospholipid classes were found to be CGP and EGP (Table 3). Plasmalogen phospholipids containing an sn-1 vinyl ether linkage were found exclusively in the choline and ethanolamine phospholipid classes (Table 3). Choline plasmalogens were significantly decreased in diabetic animals, whereas plasmenylethanolamine was increased by diabetes (Table 3). A significant increase in alkylacyl glycerophosphorylcholine was also observed in diabetic animals (Table 3). The total mass of EGP was found to be 32% greater in diabetic than in control hearts. Comparison of the contribution of phosphatidylethanolamine and plasmenylethanolamine to the total EGP mass demonstrated that the increase in mass was due entirely to an increase in plasmalogen phospholipids (Table 3). There was no increase in the mass of phosphatidylethanolamine in diabetic animals (Table 3). Insulin treatment of diabetic animals reversed the diabetes-induced changes in phospholipid classes (Table 3).
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After separation of phospholipids into individual classes, the
choline and ethanolamine phospholipids were separated by reverse-phase HPLC, and individual molecular species were collected and quantified (Table 4). Arachidonic acid (20:4) and
docosahexaenoic acid (22:6) were esterified exclusively at the
sn-2 position of CGP and EGP. Linoleic acid (18:2) was found
predominantly at the sn-2 position; however, up to 15% of
the total esterified linoleate was found at the sn-1
position of diacylglycerophospholipids. The majority (78%) of
esterified oleic acid (18:1) was located at the sn-2 position of choline diacylglycerophospholipids but was found
predominantly (81%) at the sn-1 position of ethanolamine
diacylglycerophospholipids. As shown in Table 4, diabetes resulted in a
remarkable alteration in the distribution of fatty acyl moieties
esterified to the sn-2 position of both CGP and EGP. There
was a marked reduction in the amount of docosahexaenoate (22:6)
esterified to the sn-2 position of both CGP and EGP. In CGP,
the content of esterified arachidonate (20:4) was diminished by a
striking 61% and was accompanied by an even more striking threefold
increase in the amount of linoleate (18:2) esterified at the
sn-2 position. The increase in the amount of esterified
linoleate at the sn-2 position corresponded to the decrease
in esterified arachidonate and thus may represent a compensatory alteration. In EGP, there was also an increase in sn-2
linoleate, accompanied by a corresponding decrease in arachidonic and
docosahexaenoic acids esterified at the sn-2 position of
diacylphospholipids, based exclusively on measurements of absolute
phospholipid mass (Table 4). In fact, diabetes resulted in a marked
increase in esterified arachidonate in plasmenylethanolamine
phospholipids (Table 4). When comparing the total esterified
sn-2 fatty acid composition of CGP and EGP, the selective
loss of 20:4 and 22:6 and increase in 18:2 fatty acids in
diacylphospholipids and conservation of esterified 20:4 and 22:6 fatty
acids in plasmalogens is clearly evident (Table
5). Treatment of diabetic animals with
insulin resulted in reversal of diabetes-induced changes in total
phospholipid classes (Table 3). However, some changes in individual
molecular species were only partially resolved (Table 4).
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Because diabetes results in an increase in myocardial iPLA2
activity, we measured the LPlasC and LPC content in control and diabetic rat myocardium. Total choline lysophospholipid content in the
diabetic myocardium was significantly greater than in control myocardium (0.72 ± 0.11 vs. 0.37 ± 0.08 nmol/mg protein,
P < 0.05, n = 8). The increase in
choline lysophospholipid mass was a result of increases in both LPasC
and LPC content in the diabetic myocardium (Fig.
3). Treatment of diabetic animals with
daily insulin inhibited the diabetes-induced increase in choline
lysophospholipids.
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Thus diabetes is associated with a significant loss of polyunsaturated fatty acids from the sn-2 position of diacylphospholipids, whereas polyunsaturated fatty acids in plasmalogens are conserved. This redistribution of fatty acids is largely reversed by treatment of diabetic animals with insulin. The diabetic heart is also associated with an increase in the activity of a membrane-associated iPLA2, which in turn leads to increased choline lysophospholipid content in the diabetic myocardium.
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DISCUSSION |
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This is the first study to characterize fully the substrate specificity, subcellular distribution, and effects of Ca2+ and the presence of diabetes on myocardial PLA2 activity. The majority of myocardial PLA2 activity in control, diabetic, and insulin-treated animals was found to be membrane associated, Ca2+ independent, and selective for arachidonylated substrates. The rat myocardium was found to contain multiple PLA2 isoforms with specific subcellular localization; iPLA2 was detected in the membrane only, and cPLA2 and sPLA2 were detected only in the cytosol. In diabetic animals, we have demonstrated increased expression of iPLA2 and increased PLA2 activity in the membrane fraction that is evident in the absence of Ca2+. No change in expression or subcellular localization of cPLA2 or sPLA2 was detected in diabetic myocardium.
Changes in the fatty acid composition of membrane phospholipids in several tissues from the diabetic rat show that profound alterations develop gradually (17). Because diabetes is a long-standing disease in humans, it is likely that changes in membrane phospholipids in human myocardium may be even more pronounced than those in experimental animals. The most consistent changes that have been described in several diabetic tissues are a reduction in arachidonic and docosahexaenoic acids and an increase in linoleic acid (1, 17, 26). We have observed the same changes in fatty acid composition in our diabetic rat heart model. However, in previous studies, the redistribution of fatty acids was not quantified separately in plasmalogen and diacylphospholipids. In this study, we measured a significant increase in ethanolamine phospholipids that was due to a selective increase in plasmenylethanolamine. An increase in ethanolamine plasmalogens has been described previously in the plasma of diabetic patients (12). When examining changes in esterified fatty acid composition, we discovered that the reduction in polyunsaturated fatty acids observed in the diabetic myocardium reflected a selective loss of these fatty acids from the sn-2 position of diacylphospholipids, whereas the content of polyunsaturated fatty acids in plasmalogens was preserved in the diabetic state. The net result of these changes is that the overall percentage of arachidonic and docosahexaenoic acids in plasmalogen phospholipids is dramatically increased in diabetic animals.
Plasmalogen phospholipids are the predominant phospholipids found in the sarcolemma and sarcoplasmic reticulum of myocardial cells (13, 14). The selective enrichment of these phospholipids in electrically excitable membranes suggests that they may be involved in the regulation of ionic channel function. Plasmalogens have been shown to play an important role in regulating transmembrane concentration gradients, myocardial excitability (31), and ion transport (11), and arachidonylated plasmalogens have been demonstrated to preserve transmembrane ion gradients while providing arachidonic acid for signal transduction pathways (31). Thus the retention of arachidonic acid at the sn-2 position of plasmalogens in the diabetic myocardium at the expense of diacylphospholipids may serve as a protective mechanism to attempt to minimize abnormal ion channel function.
Increased iPLA2 activity in the diabetic myocardium is associated with increases in both lysoplasmenylcholine and lysophosphatidylcholine content. A twofold increase in myocardial choline lysophospholipid content has been implicated in the initiation of arrhythmogenesis (23). We have demonstrated that choline lysophospholipids directly alter the electrophysiological properties of cardiac myocytes (22, 23). In particular, lysoplasmenylcholine at low concentrations (0.3-1 µM) is a potent modulator of multiple membrane ionic currents (19). Thus the accumulation of choline lysophospholipids at the level measured in the diabetic myocardium may contribute to increased arrhythmia vulnerability in diabetic patients.
Diabetes-associated changes in both PLA2 activity and membrane phospholipids appear to be related to a lack of insulin, because insulin-treated diabetic animals exhibit a reversal of these changes. For example, insulin treatment restored PLA2 activity to normal levels in the liver, plasma, and skeletal muscle of experimental animals (27, 31). Insulin treatment of diabetic animals also resulted in normalization of diabetes-induced changes in phospholipid composition in erythrocytes and liver microsomes (28). In this study, we have demonstrated that daily insulin treatment in diabetic animals effectively reduces diabetes-induced growth retardation, elevated blood glucose levels, changes in myocardial Ca2+-independent PLA2 activity, and redistribution of phospholipid fatty acids in the myocardium, although it is not known at this time whether these latter corrections contribute to the alleviation of diabetic heart disease by adequate control of type 1 diabetes.
In conclusion, this study emphasizes the importance of detailed phospholipid characterization when studying changes in esterified fatty acid composition in phospholipid pools. Our results also demonstrate for the first time that diabetes is associated with increased myocardial Ca2+-independent PLA2 activity and expression with little change in expression of cPLA2 or sPLA2 isoforms. The increase in membrane-associated Ca2+-independent PLA2 activity, coupled with alterations in membrane phospholipid composition, may contribute to some of the observed diabetes-induced changes in myocardial function as a result of alterations in myocardial membrane properties.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. McHowat, Dept. of Pathology, St. Louis Univ. School of Medicine, 1402 S. Grand, St. Louis, MO 63104 (E-mail: Mchowatj{at}slucare1.sluh.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 14 September 1999; accepted in final form 3 February 2000.
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