Review articles

AMP-activated protein kinase:

the energy charge hypothesis revisited
D. Grahame Hardie* and Simon A. Hawley

Summary
The AMP-activated protein kinase cascade is a sensor of
cellular energy charge, and its existence provides strong
support for the energy charge hypothesis first proposed
by Daniel Atkinson in the 1960s. The system is activated
in an ultrasensitive manner by cellular stresses that
deplete ATP (and consequently elevate AMP), either by
inhibiting ATP production (e.g., hypoxia), or by accelerating ATP consumption (e.g., exercise in muscle). Once
activated, it switches on catabolic pathways, both acutely
by phosphorylation of metabolic enzymes and chronically by effects on gene expression, and switches off
many ATP-consuming processes. Recent work suggests
that activation of AMPK is responsible for many of the
effects of physical exercise, both the rapid metabolic
effects and the adaptations that occur during training.
Dominant mutations in regulatory subunit isoforms
(g2 and g3) of AMPK, which appear to increase the basal
activity in the absence of AMP, lead to hypertrophy of
cardiac and skeletal muscle respectively. BioEssays
23:11121119, 2001. 2001 John Wiley & Sons, Inc.
Introduction
One of the most fundamental parameters that any healthy cell
must maintain is a high ratio of ATP to ADP (of the order of
10:1). Almost all energy-requiring processes in the cell are
driven, either directly or indirectly, by hydrolysis of one or other
of the acid anhydride bonds in ATP, yielding ADP or AMP
(reactions 1 and 2, Box 1). Healthy cells maintain the reactants
and products of these two reactions many orders of magnitude
away from their equilibrium ratios. This is why ATP hydrolysis
is able to perform useful work when coupled to processes
requiring an input of energy. A useful analogy can be drawn
between the adenine nucleotides in a living cell and the
chemicals in an electrical cell or battery (strictly, the latter term
refers to a number of cells arranged in series). The ``battery'' of
the cell is charged up by catabolism or photosynthesis,
converting ADP and Pi to ATP (reaction 3, Box 1). Almost all
other cellular processes are coupled to ATP breakdown and

Division of Molecular Physiology, Dundee University, DUNDEE,

Scotland.
*Correspondence to: D. Grahame Hardie, Wellcome Trust Biocentre,
Division of Molecular Physiology, School of Life Sciences, Dundee
University, DUNDEE DD1 5EH, Scotland, UK.
E-mail: d.g.hardie@dundee.ac.uk

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Box 1: Reactions interconverting ATP, ADP,

and AMP
ATPases:
ATP!ADP Pi.....................(1)
Ligases:
ATP!AMP PPi...................(2)
ATP synthases: ADP Pi!ATP.............................(3)
Adenylate kinase: 2ADP$ATP AMP.................(4)
If adenylate kinase is at equilibrium:
ATPAMP
ADP2

;ATPAMP K  ADP2
Divide both sides of the equation by [ATP]2:


AMP
ADP 2
;
/
ATP
ATP
i.e., the AMP:ATP ratio varies as the square of the
ADP:ATP ratio.

therefore tend to discharge the ``battery''. Given the critical

importance to the cell of the maintenance of appropriate ratios
of ATP:ADP and ATP:AMP, it is not surprising that sophisticated mechanisms to regulate these ratios have evolved.
Work over the last decade has highlighted the key role of the
AMP-activated protein kinase cascade in this process.(13)
That the nucleotides themselves should be the signals that
mediate this regulation is, with hindsight, rather obvious, but it
appears to have been first proposed by Daniel Atkinson in
1964.(4) By the early 1960s a small number of metabolic
enzymes (e.g., muscle phosphorylase and phosphofructokinase) had been shown to be allosterically regulated by
adenine nucleotides, with AMP and ATP tending to act in
reciprocal directions. Atkinson extrapolated from these initial
examples and proposed that adenine nucleotides would
regulate all branch points between anabolism and catabolism.
He called this the adenylate control hypothesis or (drawing on
the analogy with an electrical cell described above) the energy
charge hypothesis. The idea stimulated considerable interest
at the time, but rather few additional enzymes regulated
directly by adenine nucleotides were subsequently discov-

BioEssays 23:11121119, 2001 John Wiley & Sons, Inc.

DOI 10.1002/bies.10009

Review articles
ered. Thus the idea remained somewhat in abeyance until the
arrival on the scene of the AMP-activated protein kinase.
At this point, it is worth discussing why AMP, rather than
ADP, should be the key regulatory molecule. Eukaryotic cells
have a very active adenylate kinase that interconverts ATP,
ADP and AMP (reaction 4, Box 1), and maintains this reaction
close to equilibrium. If it is at equilibrium, the AMP:ATP ratio
will vary as the square of the ADP:ATP ratio (see lower part of
Box 1). Healthy cells under ideal conditions maintain an
ADP:ATP ratio of the order of 1:10 due to the operation of ATP
synthases (reaction 3). Under these conditions, adenylate
kinase will operate from right to left, keeping AMP very low
(AMP:ATP 1:100). If a cellular stress causes the rate of
ATPases (reaction 1) to exceed that of the ATP synthases
(reaction 3), however, the ADP:ATP ratio will rise, and the
adenylate kinase reaction will operate from left to right,
generating AMP. If the ADP:ATP ratio rises by 5-fold, the
AMP:ATP ratio will rise 25-fold. Thus, as pointed out by Hans
Krebs in 1963,(5) the cellular concentrations of AMP change
much more dramatically than do those of ATP or ADP. It
therefore makes sense for any system that monitors cellular
energy status to respond to AMP (or the AMP:ATP ratio) rather
than ADP (or the ADP:ATP ratio).
Structure and regulation of AMP-activated
protein kinase
AMP-activated protein kinase was originally discovered by its
ability to inactivate HMG-CoA reductase(6) and acetyl-CoA
carboxylase.(7) In 1980 Kim's group reported that an acetylCoA carboxylase kinase that they were studying was
stimulated by 50 -AMP, and suggested that it might inhibit fatty
acid synthesis in response to falling energy charge.(8) Five
years later Hegardt's group reported that an HMG-CoA
reductase kinase was also stimulated by AMP.(9) Shortly after
this our laboratory provided evidence that a single protein
kinase could account for both of these observations.(10) When
it became clear that the kinase had multiple physiological
substrates we renamed it the AMP-activated protein kinase
(AMPK) after its allosteric activator.(11) AMPK is now known to
exist as heterotrimeric complexes comprising a, b and g
subunits (Fig. 1). In mammals, each subunit is encoded by two
or three genes (a1, a2, b1, b2, g1, g2, g3) and at least 12
heterotrimeric combinations are possible.(1214) The a subunit
(63 kDa) contains the kinase domain at the N terminus, plus a
C-terminal regulatory domain containing an autoinhibitory
region that inhibits the kinase in the absence of AMP.(15) The b
subunits appear to be the scaffold on which a and g assemble,
via binding to their conserved KIS and ASC domains, respectively.(13) The g subunits contain four tandem repeats of a
structural module called a CBS domain, examples of which are
also found in various other proteins.(16) The functions of CBS
domains are not known, although the example in cystathione
b-synthase (after which they are named) appears to be

involved in allosteric activation by S-adenosyl methionine.(17)

It is therefore tempting to speculate that one or more of the
CBS domains of the g subunit of AMP is involved in binding the
adenosine moiety of AMP. In support of this, the photoaffinity
analogue 8-azido-[32P]AMP labels the g subunit of rat liver
AMPK, and this is specifically prevented by the presence of
AMP.(14) A current model to explain AMP activation(14)
proposes that AMP binds in the interface between the a and
g subunits, preventing association of the autoinhibitory segment of the a subunit with the kinase domain on the same
subunit (Fig. 1). However, the exact location of the AMPbinding site is currently not well defined.
The degree of activation by AMP depends on the particular
isoform of a and g present in the complex,(14) and can be up to
13-fold with recombinant a2b1g1 complex.(18) This allosteric
activation is only a part, however, and indeed a small part, of
the activation mechanism. AMPK is activated 50- to 100-fold
by an upstream kinase (AMP-activated protein kinase
kinase or AMPKK, whose molecular identity remains unclear)

Figure 1. Model for the changes in interdomain interactions

in the AMPK complex, based in part on two-hybrid analysis in
the yeast system.(65,66) In both the inactive and active
conformations, the b subunit acts as a ``scaffold'' that binds
a and g via the conserved KIS and ASC domains. In the
inactive, T conformation (top) the kinase domain of a is
inhibited by interactions with the autoinhibitory region on the
same subunit. In the active, R conformation (bottom), this
interaction is prevented because the autoinhibitory region on a
now interacts with the CBS domains on g, instead of with the
kinase domain. AMP promotes this conformation by stabilizing the a$g interaction, while ATP binding at the allosteric site
would disrupt it. In the active conformation, the kinase domain
is free to be phosphorylated and activated by the upstream
kinase, and to phosphorylate downstream targets.

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which phosphorylates a threonine residue (Thr-172) within the
``activation loop'' of the kinase domain.(18,19) AMP promotes
this phosphorylation both by binding to dephospho-AMPK and
making it a better substrate, and by activating AMPKK itself.(20)
In addition, AMP binding to phospho-AMPK almost completely
prevents dephosphorylation of Thr-172.(21) This complex
mechanism of AMP activation is summarized in Figure 2.
The multiple effects of AMP, coupled with a very low Km of
AMPKK for AMPK, make the AMPK cascade an ultrasensitive
system in which, over a critical range of concentrations, there
is a large response to a small increase in AMP. Our laboratory
recently showed that there is a sigmoidal (i.e., ultrasensitive)
response to the concentration of the activating nucleotide in
intact cells.(22)
Through the action of adenylate kinase (Box 1), AMP and
ATP almost always vary in reciprocal directions in the cell.
Although both AMPK and AMPKK require low concentrations
of ATP to function as protein kinases, high (mM) concentrations of ATP inhibit activation of the system by antagonizing
binding of AMP at the allosteric site(s) on AMPK. In a manner
highly analogous to the effects of these nucleotides on muscle
phosphorylase and phosphofructokinase, rising AMP and
falling ATP activate the AMPK system, which therefore acts
as an ``energy charge sensor''. AMPK is also allosterically
inhibited by phosphocreatine at concentrations that lie within
the physiological range.(23) Since phosphocreatine in muscle
and some other cells acts as a phosphagen, i.e., a short-term
reservoir of ATP, this fits in well with the energy sensor
concept. Phosphocreatine appears to have no effect on
phosphorylation of AMPK by AMPKK (DGH and SAH,
unpublished).

Figure 2. Model for the regulation of AMPK by AMP and by

phosphorylation, based on the classical Monod/Wyman/
Changeux model for allosteric enzymes.(67) AMPK is proposed to exist in two conformations, i.e., R and T, each of
which can also exist in phosphorylated and dephosphorylated
forms, making four states in all. AMP binding promotes the
T!R transitions by stabilizing the R states (see Fig. 1). Only
the R state is a substrate for AMPKK, while only the T state is
a substrate for the protein phosphatase. The figures in square
boxes indicate the approximate kinase activity of that form,
relative to that of the phosphorylated T state. The activity of
the phosphorylated R state (bottom right) relative to that of the
phosphorylated T state (bottom left) varies according to the
identity of the a and g subunit isoform. Redrawn from Ref. 2

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BioEssays 23.12

Physiological regulation of AMP-activated

protein kinase
A misconception sometimes encountered by the authors is
that adenine nucleotides cannot be important cellular regulators because their concentrations never vary. On inspection of
the pathways and processes by which ATP is produced and
consumed, however, it is not at all obvious why they should
remain so perfectly balanced. Observations that the levels of
ATP, ADP and AMP are usually very constant in cells may
merely reflect the success of the systems that preserve this
balance. Because of this, the easiest way to observe AMPK in
action is to look at situations of cellular stress where this
normal balance has been disturbed. Thus, AMPK is activated
by any stress treatment that interferes with ATP production.
Such stresses include heat shock,(24) metabolic poisons,(24)
glucose deprivation (hypoglycaemia),(25) oxygen deprivation
(hypoxia),(26) and interruption of the blood supply (ischaemia,
which can be regarded as a combination of hypoxia and
hypoglycaemia).(27) These are all abnormal, pathological
events, but a physiological stress that activates AMPK by
increasing ATP consumption is exercise in skeletal muscle, as
demonstrated in both animals(28) and humans.(29,30) Using
transgenic mice in which AMPK activity was almost abolished
in muscle by expression of a dominant negative mutant,
Birnbaum's group have recently shown that the effect of
contraction on muscle glucose uptake was partly eliminated,
while the effect of hypoxia was completely eliminated.(31) The
mice were also ``lazy'' in that they took less voluntary exercise,
presumably because their muscles became fatigued more
easily. This work elegantly confirms the important role of
AMPK in the response of muscle to exercise and hypoxia.
Much of the recent growth in interest in the AMPK system has
stemmed from this aspect of its function, and its potential role
in mediating the beneficial effects of exercise on humans with
Type 2 diabetes(32) (see below).
Homologues in other species
Recent genome sequencing has revealed that there are
homologues of the a, b, and g subunits of AMPK throughout
the eukaryotic domain, including Arabidopsis, Dictyostelium,
Drosophila and C. elegans. However the non-mammalian
species in which an AMPK homologue is best characterized is
the yeast Saccharomyces cerevisiae, where the homologues
of the a and g subunits are termed Snf1p and Snf4p,(33) which
form complexes with one of three alternate b subunits termed
Sip1p, Sip2p and Gal83p. Deletion of either SNF1 or SNF4
(or all three b subunit genes) produces a similar phenotype
characterized by failure to grow on carbon sources other than
glucose. In the presence of glucose (the preferred carbon
source) many genes are repressed, and functional SNF1
complexes are required for their derepression. When glucose
is removed from cells in mid-log phase, there is a dramatic
activation of the SNF1 kinase due to phosphorylation(34) that is

Review articles
associated with very large increases in AMP and decreases in
ATP. However, AMP does not appear to activate the SNF1
kinase in cell-free assays, so it remains unclear whether a rise
in the AMP:ATP ratio is the signal that switches on the yeast
kinase in vivo.(34) What is known is that derepression of many
glucose-repressed genes involves a direct phosphorylation of
the repressor protein Mig1 by the SNF1 kinase, causing the
repressor to become inactive and to be exported from the
nucleus.(35,36) This provides an interesting model to explain
how AMPK might regulate gene expression in mammals
(see below).
Targets of AMP-activated protein kinase
In his paper in 1964(4) Atkinson wrote: ``These indications that
the level of AMP, or the AMP to ATP ratio, may regulate the
metabolic direction (towards energy release or towards energy
storage) ...... suggest that the level of AMP may function more
generally as a mediator of energy metabolism'' (my italics). In
the last few years there has been an explosive growth in our
knowledge of the downstream targets of the AMPK system,
and this has fully vindicated Atkinson's far-sighted proposal.
Much of this progress came from the use of 5-aminoimidazole4-carboxamide (AICA) riboside. This nucleoside is taken up
into cells and phosphorylated by adenosine kinase to the
monophosphorylated nucleotide, usually referred to as ZMP.
ZMP mimics all four effects of AMP on the AMPK cascade(37)
and, although it is much less potent than AMP itself, in most
cells it accumulates to sufficiently high concentrations when
they are incubated with the riboside. This represents a method
for activating AMPK without disturbing the cellular levels of
ATP, ADP or AMP, thus avoiding the many possible sideeffects of the latter. Its absolute specificity remains uncertain
and, like any pharmacological approach, the results should be
interpreted with caution. A novel alternative approach has
been developed recently, in which a constitutively active form
of the AMPK kinase domain is expressed from an adenoviral
vector.(38) This has its own drawbacks: one is overexpressing
a form of the kinase lacking the accessory subunits, which may
therefore not be correctly localized in the cell. However, a role
for AMPK is strengthened when the same results are obtained
using both AICA riboside and the constitutively active mutant,
as has been done in the case of inhibition of transcription of
lipogenic genes in liver.(38)
The many proposed downstream responses to AMPK
activation are summarized in Table 1. A full description of them
is beyond the scope of this article, but I will make a few general
comments and discuss a few specific examples. In most
cases, the effects have been demonstrated using AICA
riboside only, although in some of these the effects are
nevertheless convincing because the target protein and the
site of phosphorylation on the protein has been identified and
found to correspond to the site phosphorylated by AMPK
in vitro. Many of the effects would change the balance between

anabolism and catabolism, exactly as predicted in Atkinson's

energy charge hypothesis. For examples involving anabolism,
AMPK activation acutely inhibits fatty acid, triglyceride and
sterol synthesis while, in the longer term, it inhibits the
expression of enzymes involved in fatty acid synthesis and
gluconeogenesis. For examples involving catabolism, AMPK
activation acutely stimulates glucose uptake (via both GLUT1
and GLUT4), glycolysis (at least in the heart), and fatty acid
oxidation while, in the longer term, it increases the expression
of GLUT4, hexokinase, and mitochondrial enzymes involved
in the TCA cycle and respiratory chain in muscle (see Table 1
for details). Intriguingly, the latter long-term effects are also
seen in response to exercise training, raising the important
possibility that training improves athletic performance in part
by causing regular AMPK activation. Whether AMPK also
increases expression of other muscle proteins (e.g., contractile proteins), thus explaining the hypertrophic response to
training, remains to be tested.
Atkinson does not appear to have anticipated that the
systems that monitor cellular energy charge would regulate
processes other than metabolism. In principle, any cellular
process that consumes ATP, and is not essential for shortterm survival, is a potential target for AMPK. Early examples of
non-metabolic processes that may be regulated by the system
include autophagy and apoptosis (Table 1). The evidence for
AMPK involvement in these cases is based entirely on the use
of AICA riboside and the targets for phosphorylation have not
been identified, although it has been proposed that the effect
on apoptosis in astrocytes is mediated by inhibition of ceramide synthesis.(39)
Involvement of AMPK in metabolic disorders
Diabetes is characterized by abnormally high blood glucose.
The type 2 form (which accounts for > 90% of all cases) is not
caused by a primary deficiency in insulin (as in type 1) but by
reduced insulin sensitivity of the major glucose-metabolizing
tissues, especially muscle, and increased glucose production
by the liver. Treatment of type 2 diabetes and its complications
are currently estimated to account for 1020% of all healthcare spending in developed countries, and the incidence is
rising world-wide, with projections of 200 million people being
affected by the end of this decade.(40) This increase is partly
due to the ageing population, but is also thought to be due to
changes in lifestyle that are associated with obesity, i.e., more
frequent consumption of high calorie foods and lack of physical
exercise. Many of the metabolic abnormalities associated with
type 2 diabetes would be expected to be reversed by activation
of AMPK.(32) Indeed, this has been confirmed by chronic
administration of AICA riboside to animal models of diabetes/
insulin resistance such as the obese Zucker rat, which in
one study improved glucose tolerance,(41) and in another
lowered plasma triglycerides and fatty acids, and reduced
endogenous glucose production, presumably by inhibiting

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Table 1. Proposed downstream physiological effects of AMPK activation

Pathway affected
A) Rapid, Acute effects:
Inhibition of anabolism:
# sterol/isoprenoid synthesis
# fatty acid synthesis
# triacylglycerol synthesis
Stimulation of catabolism:
" fatty acid oxidation
" glucose uptake
via GLUT4 translocation
via GLUT1 activation
" glycolysis (heart)
Other effects:
# lipolysis (adipocytes)
" nitric oxide production
# apoptosis
# autophagy
Gene affected

Direct AMPK target?

Approach

(24,37)

HMG-CoA reductase
Acetyl-CoA carboxylase
Glycerol-P acyl transferase?

AR,* others
AR,* others
AR*

Acetyl-CoA carboxylase

AR*

(52,53)

Endothelial NO synthase?
?
6-phosphofructo-2-kinase

AR*
AR*
Ischaemia, oligomycin

(54,55)

Hormone sensitive lipase

Endothelial NO synthase
? (Ceramide synthesis(39)?)
?

AR*
Ischaemia
AR*
AR*

(37,57)

Pathway affected?

Approach

B) Chronic effects via changes in gene expression:

Inhibition of gene expression:
# acetyl-CoA carboxylase
# Fatty acid synthesis
# fatty acid synthase
# Fatty acid synthesis
# S14
# Lipogenesis?
# L-pyruvate kinase
# Glycolysis, lipogenesis
# PEP carboxykinase
# Gluconeogenesis
Stimulation of gene expression (all effects below in skeletal muscle):
" GLUT4
" Glucose uptake
" hexokinase
" Glycolysis
" mitochondrial enzymes
" Oxidative phosphorylation
" UCP3
" Protection against oxidative damage?
*

Reference

(24,37)
(51)

(56)
(26)

(58)
(39,59,60)
(61)

Reference

CAM**
CAM**
CAM**
CAM**
arsenite

(38)

AR*
AR*
AR*
AR,* hypoxia

(44)

AR,*
AR,*
AR,*
AR,*
AR,*

(38)
(38)
(38)
(62)

(44)
(63)
(64)

AR AICA riboside.

gluconeogenesis.(42) Chronic AICA riboside treatment also

increases the insulin-sensitivity of glucose uptake in muscles
isolated from normal rats(43) (possibly because of increased
expression of GLUT4, Ref. 44) and prevented the development of glucose-induced insulin resistance in vitro.(45) Since
AMPK is activated by exercise,(28) it might explain the
beneficial effects of physical exercise on patients with type 2
diabetes.(46) There is therefore much current interest in the
development of AMPK-activating drugs as potential treatments for Type 2 diabetes. Equally there is a danger that such
drugs would be abused by athletes!
Intriguing links between the AMPK system and disease
have also come from recent studies on naturally occurring
mutations. Three severe forms of familial hypertrophic
cardiomyopathy, a hereditary heart disorder characterized
by thickened heart muscle that results in premature deaths,
have been traced to mutations in the gene encoding the g2
subunit of AMPK (which is expressed at high levels in the
heart, Ref. 14). Two of these (R302Q and H383R) involve
mutations affecting basic residues in equivalent positions in
the CBS1 and CBS2 domains, while the third inserts an extra

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BioEssays 23.12

residue (leucine) between a conserved Arg-Glu in the linker

between CBS1 and CBS2.(47,48) Intriguingly, a mutation in g3,
an isoform that is highly expressed in skeletal muscle,
produces a strain of pigs with a high carcass meat content,
i.e., another hypertrophic response.(49) This mutation,
R200Q, is in CBS1 of g3 and aligns perfectly with the
R302Q and H383R mutations in CBS1 and CBS2 of g2. Also,
a mutation in cystathione b-synthase that affects activation by
S-adenosyl methionine occurs in an equivalent position in its
single CBS domain (see alignment in Fig. 3). Whether these
mutations cause loss of function or gain in function in AMPK is
an important question. This is not known for g2 or g3, but an
engineered mutation in g1 (R70Q, equivalent to R302Q (g2)
or R200Q (g3)) results in a ``constitutively active'' form of
the a1b1g1 complex that is less dependent on AMP and is
more highly phosphorylated, and therefore more active,
under basal conditions.(50) According to the model shown
in Fig. 1, this mutation may stabilize the activating a$g
interaction even in the absence of AMP. Alternatively, it might
prevent binding of the inhibitory nucleotide, ATP, that
normally disrupts this interaction. It is therefore conceivable

Review articles

Figure 3. Alignment of selected CBS domains from cystathionine b-synthase (CBS) and g subunits of AMPK, showing the location of
naturally occurring and engineered mutations. Residues that tend to be conserved across all CBS domains (bold type), and the likely
locations of b strands (bbbb...) and a-helices (hhhh...), are as discussed by Bateman.(16)

that all of these mutations cause forms of AMPK that are more
active under basal conditions in the absence of a rise in AMP.
It is easier to explain the dominant nature of these mutations
if they cause constitutive activation, rather than inhibition, of
the kinase activity. If AMPK activation does indeed cause
muscle hypertrophy as discussed earlier, these findings could
also help to explain the hypertrophy in both cardiac (g2)
and skeletal muscles (g3), as the muscle would respond
as if it sensed a depletion of ATP even when none had
happened.
These are exciting times for those of us working in the
AMPK field, with new papers appearing almost weekly. An
idea that started as a theoretical concept with Daniel Atkinson
has been provided with a firm experimental basis and is now
turning out to have great relevance to important clinical
conditions.
Acknowledgments
Work in the authors' laboratory is supported by the Wellcome
Trust, Diabetes UK and the Medical Research Council. We
thank Lee Witters and Bruce Kemp for permission to cite their
work prior to its publication.
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