Vol. 277, Issue 4, H1435-H1440, October 1999
Alteration of gene expression for glycolytic enzymes in
aerobic and ischemic myocardium
A. James
Liedtke and
Matthew L.
Lynch
University of Wisconsin Hospital and Clinics, Madison, Wisconsin
53792-3248
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ABSTRACT |
The purpose of
this report was to describe mRNA abundance for the glycolytic enzymes
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase, and
pyruvate dehydrogenase in ischemic and adjacent aerobic myocardium.
Mechanical, metabolic, and mRNA data were acquired in a pig model of
regulated coronary flow using extracorporeal perfusion. Trials of
coronary hypoperfusion included sustained and intermittent exposures of
acute ischemia with or without reperfusion. These were compared
with a chronic 4-day model of partial coronary stenosis. In ischemic
tissues, levels of mRNA, normalized by mRNA for 
-actin, were
increased over control values for GAPDH (range 2.7- to 4.6-fold),
pyruvate kinase (2.9-fold), and pyruvate dehydrogenase (2.1-fold). It
is of interest that increases in mRNA levels over control values were
also observed in adjacent aerobic heart muscle from intervention
hearts, including 3.6- to 4.5-fold elevations in message for GAPDH and
a 2.1-fold increase in signal for pyruvate dehydrogenase. Augmentation
in mRNA abundance occurred in as short a time as 40 min of
ischemia and was maintained for as long as 4 days in partial
coronary stenosis. Whether the former time was of an interval
sufficient to affect protein production is problematic, but the latter
time was ample to influence enzyme concentration, which may in turn
have regulated glycolysis in this condition.
acute ischemia; reperfusion; myocardial
hibernation
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INTRODUCTION |
MYOCARDIAL ISCHEMIA, sometimes in association with
postischemic reperfusion, is a potent stimulus to a variety of gene
signals for protein production in response to stress. mRNA
was reportedly induced or increased for such proteins as -adrenergic
receptor kinase 1, which inactivates -receptors (33);
1-adrenergic receptors per se
(12); heat shock proteins 70 (16, 24, 35), 72 (29), and 90 (27), which
are most intensely expressed in reperfusion, perhaps as a result of
secondary oxidative injury; vascular endothelial growth factor and
endothelin-1 (1, 9, 32); interleukin-6 and intracellular adhesion
molecule (15, 36); insulin-like growth factor II (13); and the
Na+/H+
exchanger (4). Other reports have described the appearance of two new
heart transcripts and three new stress-inducible genes in response to
ischemia (14) and the suppression of mRNA message regulating
Gs and
Gi proteins, which may explain the
observed disruption to the G protein-adenylate cyclase system (28).
We have expanded this survey in a recent report to include an
evaluation of the effects of myocardial ischemia to influence the levels of mRNA regulating the proteins of glucose metabolism (5).
In animals preselected for increased glucose utilization in myocardium
rendered ischemic in several protocols with or without reperfusion, it
was noted that gene signal was increased for GLUT-1, GLUT-4,
hexokinase, phosphofructokinase, and glyceraldehyde-3-phosphate dehydrogenase. Gene signal for several of these proteins was
upregulated in as short a time as 40 min after ischemic exposure and
was maintained for as long as 4 days in a model of partial coronary
stenosis. The initial study focused on characterizing the proximal
enzymes in the glycolytic pathway as well as the glucose transporters and was a descriptive review surveying mRNA abundance for a select animal in each of the interventional conditions. The purpose of the
present study was to describe mRNA levels for the mid-to-distal enzymes
in the glycolytic cascade (glyceraldehyde-3-phosphate dehydrogenase,
pyruvate kinase, and pyruvate dehydrogenase) and included a larger
sampling of representative hearts (n = 3 hearts/intervention). Gene signal for these enzymes was normalized by
mRNA levels of -actin also acquired in aerobic and ischemic
myocardium. mRNA data in hearts were collected and contrasted between
tissue perfused aerobically in the left circumflex (LCF) circulation
and that rendered ischemic with and without reperfusion in the left
anterior descending (LAD) circulation. The same interventional states
that were previously reported (5) will be reviewed. Our test hypothesis is also the same, i.e., inducibility of gene signals for various glycolytic enzymes occurs as a consequence of exposures to acute ischemic syndromes and/or to coronary perfusion states simulating myocardial hibernation.
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METHODS |
Experimental conditions.
The surgical preparation and protocols are described elsewhere (5).
Frozen tissues of myocardium for RNA isolation were obtained from
hearts acquired in several previous studies conducted in the
laboratory. An intact, working pig heart preparation was employed in
which regional coronary flows were controlled and regulated by
extracorporeal perfusion. Physiological and metabolic information for
the several interventional groups were previously reported (7, 8, 18,
19, 21). Mechanical and metabolic data from the subset of animals
representing each of the interventional groups to be reported in this
study are listed in RESULTS.
Conditions included 1) an aerobic
"control" state in which regional coronary flow was maintained at
aerobic levels for 60 min (for further details, see Ref. 21);
2) three conditions of acute
ischemic syndromes, and 3) two
experimental protocols designed to simulate conditions of ischemic
myocardial hibernation. The acute ischemic syndromes included two
protocols of sustained hypoperfusion (60% reduction from aerobic
levels) to the LAD-perfused myocardium for 40 min either with or
without accompanying reperfusion (for further description, see Refs. 18
and 19), designated respectively as "ischemia" and
"ischemia-reflow." A third protocol of acute intermittent
LAD ischemia, termed "intermittent
ischemia-reflow," was also employed (7) in which LAD flow
was cyclically reduced by 60% for 5 min, reperfused aerobically
between cycles for 15 min, rendered ischemic for a total of 4 cycles,
and terminally reperfused for 20 min. The two conditions of ischemic
myocardial hibernation included an acute protocol of mild regional
ischemia (40% decrease in LAD flow) modified after the
experiments of Schulz et al. (30, 31) (for further details, see Ref.
8), designated "mild ischemia," and a chronic 4-day model
of partial coronary stenosis (21), termed "chronic coronary
stenosis." From the original groupings, three hearts were selected
from each experimental condition to be surveyed for mRNA abundance in
this study.
RNA isolation.
Tissue samples (~2 g) from LAD and LCF perfusion beds were quickly
excised, freeze-clamped in liquid nitrogen, and stored at
70°C. With the use of TRIzol reagent (Life Technologies,
Grand Island, NY) and an improved single-step RNA isolation method, based on Chomczynski and Sacchi (2), total RNA was obtained. The total
RNA was quantitated spectrophotometrically at 260 nm. The ratio of
absorption at 260 nm to that at 280 nm was >1.6 for all samples.
Electrophoresis and transfer.
Equal amounts of total RNA (20 µg) were fractionated by
electrophoresis through a denaturing 1% agarose gel with 0.6 M
formaldehyde in 1× MOPS. The RNA was transferred to a Nytran
membrane (Schleicher and Schuell, Keene, NH) using the Turboblotter
system and protocol (Schleicher and Schuell). The transfer was done for
3 h with 20× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium
citrate, pH 7.0). The RNA was fixed to the membrane by
ultraviolet cross-linking at 302 nm.
Probe generation.
To generate the probes for -actin and pyruvate kinase, total RNA
from porcine myocardium was reverse transcribed, and the corresponding
cDNA was amplified by PCR to obtain short sequences for the
NH2-terminal regions of each gene.
AMV (avian myeloblastosis virus) Reverse Transcriptase,
Tfl DNA polymerase, and the primers for -actin were purchased from Promega (Madison, WI). The primers for pyruvate kinase were synthesized by the University of Wisconsin Biotechnology Center (Madison, WI).
The reverse transcription and amplification were carried out using the
Access RT-PCR system (Promega). Total RNA (1 µg) was reverse transcribed and amplified in a 50-µl reaction containing 1× AMV/Tfl reaction buffer, 200 µM dNTP, 1 µM primers, 0.1 U/µl AMV Reverse Transcriptase, and
0.l U/µl Tfl DNA polymerase. The MgSO4 concentration was 1 mM for
the pyruvate kinase reaction and 0.5 mM for the -actin reaction. The
reverse transcription and subsequent amplification were performed in a
Techne Cyclogene HL-1 thermal cycler. The reactions were incubated at
48°C for 45 min for reverse transcription. Amplification by PCR was
followed immediately with 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and elongation at 72°C for 2 min. A final elongation at 72°C for 10 min was added to allow for
complete extension of the products.
The PCR products were gel purified using the QIAquick Gel Extraction
Kit (Qiagen, Valencia, CA). The purified PCR products were used
directly as probes for Northern analysis. Also, the purified products
were cloned into the pCR2.1 vector from Invitrogen (Carlsbad, CA) by
using their Original TA Cloning Kit for sequencing and future use. To
ensure the fidelity of the PCR products, sequencing of the two inserts
was performed by the University of Wisconsin Biotechnology Center
(Madison, WI). Sequencing confirmed the identity of the inserts as
-actin and pyruvate kinase.
The probe for rat glyceraldehyde-3-phosphate dehydrogenase was a
generous gift from Dr. J. M. Aiken (University of Wisconsin, Madison,
WI). The probe for human pyruvate dehydrogenase E1-
-subunit was a
generous gift from Dr. Henrik Dahl (Royal Children's Hospital, Melbourne, Australia). The sequences for these nonporcine probes were
compared with the sequences for their porcine counterparts, and the
sequence similarity was found to be >90%.
Hybridization and detection.
The Renaissance Random Primer Fluorescein Labeling Kit from NEN
(Boston, MA) was used to generate labeled probes using random hexamers,
fluorescein-N6-dATP,
and other nucleotides. The labeled probes were denatured at 95°C
for 5 min, placed on ice for 5 min, and added to hybridization tubes
with the membranes. The hybridization was carried out for 18 h in
Church buffer (3) and 50 µg/ml sheared, sonicated herring sperm DNA.
The hybridization was performed in a Hybaid hybridization oven at
62°C.
After hybridization, the membranes were washed once at 62°C in 125 mM
Na2HPO4,
pH 7.2, 0.05 mM EDTA, and 2.5% SDS for 10 min and twice for 10 min
each at room temperature in 25 mM
Na2HPO4, pH 7.2, 0.05 mM EDTA, and 0.5% SDS. All subsequent washes and incubations were carried out at room temperature. The membranes were
then vigorously washed for 5 min in 0.1 M Tris · HCl,
pH 7.5, and 0.15 M NaCl. The membranes were blocked for 1 h with gentle
agitation in 0.1 M Tris · HCl, pH 7.5, 0.15 M NaCl,
and 0.5% blocking reagent. After blocking, the membranes were
incubated for 1 h with gentle agitation in 0.1 M
Tris · HCl, pH 7.5, 0.15 M NaCl, 0.5% blocking
reagent, and 0.1% antifluorescein-alkaline phosphatase
conjugate. After incubation, the membranes were vigorously washed four times for 5 min each in 0.1 M Tris · HCl,
pH 7.5, and 0.15 M NaCl. The membranes were given two final washes for 5 min each in 0.1 M Tris · HCl, pH 9.5, and 0.1 M
NaCl. From the final wash, the membranes were incubated for 5 min with
the chemiluminescent substrate CDP-Star. The excess CDP-Star was
blotted from the membranes, and the membranes were wrapped in plastic
and exposed to film.
Analysis of mRNA data.
Analysis of the mRNA data was accomplished with the Bio-Rad model
GS-700 Imaging Densitometer and Bio-Rad Molecular Analyst Software
(version 1.4.1; Hercules, CA). The signals were quantified within the
linear range of the film's sensitivity. The densitometric values were
normalized to a signal obtained with the -actin probe to account for
minor differences in RNA integrity. To ensure that equal amounts of RNA
were loaded onto different gels, visual inspection of the gels after
they were stained with ethidium bromide was routinely performed.
Representative gels that show this loading for -actin and
glyceraldehyde-3-phosphate dehydrogenase are included in Fig.
1.
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Fig. 1.
Ethidium bromide-stained gels before blotting. Total RNA (20 µg) was
fractionated by electrophoresis through a denaturing 1% agarose gel
with 0.6 M formaldehyde in 1× MOPS. RNA from left anterior
descending (LAD) beds for intermittent ischemia (animals 71-13, 71-23, and 71-29), ischemia-reflow (animals 64-3, 64-8, and
64-13), and ischemia (animals 48-14, 48-16, and 48-22)
interventions are shown. The gels were transferred and fixed to Nytran
membranes and probed for -actin
(A) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH; B).
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Statistics.
The data are expressed as means ± SE. Despite the large variation
inherent and anticipated with a small data set of animals per
intervention, statistical comparisons for mRNA abundance were made
between and among experimental conditions in an effort to identify
appreciable and meaningful differences between and among groups. For
select comparisons of intervention groups with control hearts,
nonpaired Student t-tests were
employed. For generic comparisons among all experimental groups, ANOVA
was used. Statistical significance was defined for
P values <0.05.
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RESULTS |
Performance data for the animal subsets in control and intervention
groups are listed in Table 1. Reductions in
LAD flow conscribed to the protocol specifics given in
METHODS. Average aerobic flow values
ranged from 4.4 to 6.1 ml · min
1 · g
dry wt1 (average 5.5 ml · min1 · g
dry wt1). Coronary
perfusion in the chronic coronary stenosis group, which was keyed to in
situ resting flow rates after 4 days of partial coronary stenosis, did
not appear as an appreciable outlier and was only 10% lower than the
average for all groups. Systolic shortenings during ischemia
were appreciably compromised from their respective aerobic values in
all intervention groups (range 9 to 60% of initial values) and
were followed by evidence of mechanical stunning in those hearts that
were reperfused. Errors in some subsets were large because of small
sample size and, in the case of the mild ischemia group, no
doubt reflected the modest nature of the stress. Myocardial oxygen
consumption (M
O2) fell by
50% (range 44-55%) in hearts exposed to a 60% reduction in LAD
flow and by 10% in the mild ischemia group. In the chronic coronary stenosis group, MO2
appeared to be maintained at aerobic levels. Exogenous glucose
utilization was calculated from data acquired from steady-state
infusions of
[5-3H]glucose into the
LAD arterial perfusate (for further details, see Refs. 7, 8, 18, 20,
21). Direct measurements of glucose metabolism were not obtained in the
ischemia (without reflow) group, which involved experiments to
describe changes in the radioactive pool size of acetate in moderate
myocardial ischemia (19). However, it may be reasonably assumed
that glycolysis was increased in this group because, as demonstrated by
either indirect or direct estimates of glucose utilization (18, 20), this was the case in other studies using this protocol.
Furthermore, in the animals from that group in this study, other
demonstrable effects of ischemia were noted (Table 1).
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Table 1.
Performance values for mechanical and metabolic functions in hearts
surveyed for mRNA abundance encoding messages for glycolytic enzymes
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mRNA data are presented in Table 2. Gene
signals for glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase,
and pyruvate dehydrogenase are listed for both LAD- and LCF-perfused
myocardium. All data were normalized by mRNA data for -actin, also
measured directly from the same LAD- and LCF-perfused myocardium. This latter signal for cytoskeletal protein is used routinely for
calibration of nucleic acid gels. In comparing the ethidium
bromide-stained gels with those showing Northern hybridization for
-actin, we could find no evidence that this signal was altered
because of ischemia per se (see examples in Figs. 1 and
2). To facilitate comparisons and increase
sample size in Table 2, aerobic values of mRNA in control hearts were
combined from LAD- and LCF-perfused tissue to yield a sample size of
n = 5 (for technical concerns, one LCF
sample was deleted). Nonpaired Student
t-test analysis was used here as a
surveying tool to evaluate shifts in mRNA abundance against control
values. It was noted that in no instance in any of the interventions
was there an appreciable decrease in mRNA signal for any of the enzymes
during acute or chronic ischemia. Conversely, in several
conditions appreciable increases were observed. In LAD-perfused
myocardium, these included a 2.7- and 4.6-fold increase in mRNA
abundance for glyceraldehyde-3-phosphate dehydrogenase in the mild
ischemia and chronic coronary stenosis groups, respectively, a
2.9-fold increase in mRNA levels for pyruvate kinase in the chronic
coronary stenosis group, and a 2.1-fold increase in mRNA signal for
pyruvate dehydrogenase in the ischemia (without reflow) group.
With the exception of the mild ischemia group, all of these increases achieved statistical significance by nonpaired Student t-test analysis but not by ANOVA.
Because of the possible influence of LAD ischemia and its
effects on adjacent aerobic myocardium, we also interrogated
LCF-perfused myocardium for possible shifts in glycolytic mRNA.
Surprisingly, mRNA increases were noted here as well. These included
4.5-, 4.3-, and 3.6-fold increases in mRNA for
glyceraldehyde-3-phosphate dehydrogenase for the ischemia, mild
ischemia, and chronic coronary stenosis groups, respectively, and a 2.1-fold increase in mRNA abundance for pyruvate dehydrogenase in
the ischemia (without reflow) group. The shifts in signal for glyceraldehyde-3-phosphate dehydrogenase in the ischemia and
mild ischemia conditions over control values met statistical
significance by ANOVA. An increase in mRNA signal from either LAD- or
LCF-perfused myocardium in select groups occurred with as little as 40 min of ischemic exposure and was maintained for as long as 4 days of
altered coronary reserve. The exact mechanism responsible for triggering this gene message remains unknown at this time.
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Fig. 2.
Northern hybridization of -actin and GAPDH from LAD beds for
intermittent ischemia (animals 71-13, 71-23, and 71-29),
ischemia-reflow (animals 64-3, 64-8, and 64-13), and
ischemia (animals 48-14, 48-16, and 48-22) interventions.
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DISCUSSION |
The purpose of this report was to evaluate the effect of myocardial
ischemia as a stimulus to trigger alterations in gene signal
responsible for the glycolytic enzymes located in the mid-to-distal portion of the glycolytic cascade. Glucose metabolism was chosen as a
study area because glucose utilization, given a sufficient level of
residual coronary washout (26), is almost invariably increased during
episodes of myocardial ischemia in both clinical and animal
studies. In this report, and in that of a previous study bearing on the
same general topic (5), mRNA levels for glycolytic enzymes and glucose
transporters were never appreciably reduced from aerobic control
values. Conversely, increases in mRNA abundance, sometimes reaching
values severalfold higher than control levels, were noted at several
conditions of ischemia. In select instances these were
accompanied by increases in gene signal in adjacent aerobic myocardium.
The changes in mRNA abundance in either perfusion bed were noted most
frequently for glyceraldehyde-3-phosphate dehydrogenase and to a lesser
extent for pyruvate kinase and pyruvate dehydrogenase. The potency of
this ischemic stimulus was reflected by the abrupt onset of the mRNA
increase (40 min) and the prolonged nature of its augmentation (4 days).
Critique of the methods.
All mRNA data in this study were expressed as a ratio by using the RNA
signal for -actin as a normalizing factor. -Actin is a
cytoskeleton protein found ubiquitously in several cell lines and is
distributed regionally within cells, possibly as part of a signal
transduction pathway for cell motility (11). Certain cytoskeletal
proteins in heart muscle (-actinin, desmin, myomesin, spectrin, and
tubulin) are reportedly altered in settings of myocardial ischemia (10, 23), but we could find no evidence in the
literature that -actin is affected. Furthermore, in our study,
comparison of ethidium bromide-stained gels (Fig. 1) with Northern
hybridization gels for -actin (Fig. 2) suggested no shift in
-actin signal as a function of ischemia per se.
The relationship between gene expression and protein production for the
enzymes reported in this study must remain circumspect at this juncture
not only because of the short time intervals used for some of the
protocols in the acute ischemic groups but also because enzyme
concentrations and kinetics were not directly measured. Moreover, the
increased transcript signal may indicate an increase in either gene
expression or RNA stability. However, by inference, activities in one
or more protein systems must have been increased to account for the
accelerated glycolysis noted in all intervention groups. It does seem
reasonable, given the duration of the protocol in the chronic coronary
stenosis model, that augmented protein production secondary to the
elevated mRNA levels was plausible and likely in the 4-day model of
myocardial hibernation.
The data are expressed as means ± SE. The SEs for certain
mechanical, metabolic, and gene data are sometimes large, consistent with the small sample size of the data subsets. Data trends for gene
signal are presented as multifold shifts over control values. Nonpaired
Student t-test analyses were used only
to qualitatively survey comparisons in this report, and, given the
small sample size, their absolute significance values should not be
overemphasized. The ANOVA by Tukey's test for the LCF comparisons are,
however, valid absolute markers.
The method of measuring mRNA for glycolytic proteins varied from that
of a previous report, i.e., RT-PCR amplification (5) vs. Northern blot
analysis. The previous report characterized the glucose transporters
and proximal enzymes in the glucose utilization pathway, whereas the
present study focused on the mid-to-distal enzymes in the cascade. Both
studies measured mRNA for glyceraldehyde-3-phosphate dehydrogenase, and
it was possible to make comparisons between methods. Results varied,
showing greater increases in mRNA signal for hearts from the acute
ischemic syndromes in the previous study and greater increases in
tissue from the hibernation models in this study. Such variation may
have resulted from differences in sample size and strategies of
normalizing the data. It was also possible to describe variation for
individual animals in these two studies because identical hearts were
employed in both studies. To facilitate comparisons between studies, in
the present report mRNA from LAD tissue in intervention groups was
referenced to control tissue as was done previously (5). With the use of Northern blot analysis, mRNA signal appeared more amplified than
with the use of RT-PCR (average 5.6:1 vs. 1.5:1 for all intervention groups). Correlation between measuring methods was directionally identical for the ischemia, ischemia-reflow, and
intermittent ischemia-reflow groups but not for the mild
ischemia and chronic coronary stenosis groups, in which signal
increase was noted by Northern blot but not by RT-PCR. Variations here
may have been intrinsic to the methods employed and to possible
differences in the quality of the extracted RNA.
In general, the present study adds to the information currently being
developed about the potent affects of ischemia in evoking elevations of gene message to a variety of protein systems. In the
introduction we listed increased gene expression or induction for
-adrenergic receptor kinase 1, 1-adrenergic receptors, heat shock proteins, vascular endothelial growth factor, endothelin-1, interleukin-6, intracellular adhesion molecule, insulin-like growth factor II, and the
Na+/H+
exchanger. To this list may now be added myocardial glucose
transporters and several enzymes of glycolysis. Webster et al. (34)
previously described mRNA increases for glyceraldehyde-3-phosphate
dehydrogenase, pyruvate kinase, and triose-phosphate isomerase in
hypoxic skeletal muscle myogenic cultures, but we are unaware of any
previous information on ischemic myocardium. Signal increases included
those for hexokinase, phosphofructokinase (5), and, in this study,
glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and pyruvate
dehydrogenase. Together with the glucose transporters,
these enzymes regulate the key reactions of carbohydrate utilization in
well-oxygenated hearts and, with respect to glyceraldehyde-3-phosphate
dehydrogenase, the key site of flux inhibition during myocardial
ischemia (25). Possible interpretations of the present and
previous data from our laboratory are that the mRNA increases represent
a random response to some ischemic stimulus, perhaps mechanical or
chemical in nature, or rather a purposeful response to upregulate
glycolysis in an attempt to preserve residual energy production. It is
of added interest that this stimulus was not confined to the
hypoperfused LAD circulation but involved the adjacent aerobic
myocardium as well. Mäki et al. (22) noted brisk increases in
myocardial glucose uptake in normal segments from patients with
chronically dysfunctional (hibernating) myocardium. We had previously
reported (17) a greater preference for glucose utilization in aerobic myocardium adjacent to ischemic, restricted tissue. It has been postulated that, during ischemia, tethering and other
influences place an added burden on aerobic myocardium to compensate
mechanically for the dysfunction occurring in adjacent stressed tissue.
The increase in gene signal noted in the LCF bed may be part of a regulatory mechanism to achieve energy balance for the increased energy
demands that occur during this stress period.
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ACKNOWLEDGEMENTS |
This research was supported in part by the Oscar and Rosalie Mayer
Cardiovascular Research Fund, the Oscar G. and Elsa S. Mayer Charitable
Trust, and the Rennebohm Foundation of Wisconsin as well as National
Institutes of Health and American Heart Association grants.
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FOOTNOTES |
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 reprint requests and other correspondence: A. J. Liedtke,
Univ. of Wisconsin Hospital and Clinics, Cardiology Section H6/349, 600 Highland Ave., Madison, WI 53792-3248 (E-mail:
ajl{at}medicine.wisc.edu).
Received 20 November 1998; accepted in final form 27 April 1999.
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