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Cerulo Plasm in A
Cerulo Plasm in A
I. INTRODUCTION
Copper and zinc metabolism became easier to study after science provided
two basic research tools: isotopic tracers and atomic absorption spectropho-
tometry. The recent developments in cell and molecular biology have yielded
an additional array of techniques that have allowed the development of
exciting new insights into the absorption, metabolism, and function of these
trace elements.
Interest in these nutrients has grown dramatically in the last decade.
There has been an increased awareness that the levels of dietary intake of
copper and zinc may be marginal for patients with particular diseases or
for entire population groups. On the other end of the spectrum, pharma-
cological doses of these nutrients have been reported to have therapeutic
properties for specific diseases. In the past the total copper or zinc content
of a tissue was used to make judgments about biochemical changes and
metabolic fluxes of these metals. However, little was known about the bio-
logical environment around the metal atom or what influences its distribution.
Recently the perspective has changed dramatically. These metals were shown
to be interactive with intracellular components and influenced by the en-
docrine system in the same manner as the metabolism of the major nutrients.
Two examples of the “biological interactivity” of trace elements are the
The basic aspects of copper and zinc absorption metabolism are shown
in Figures 1 and 2. To review the absorption mechanisms for copper and
Liver
FIG. 1. Basic aspects of mammalian copper metabolism. Dietary copper is absorbed from
stomach and small intestine. In human adults the RDA is -2.5 mg/day. Some copper in intestinal
contents is biliary and serves as the major excretory route. Normally only small quantities of
copper are lost in urine, but this increases during aminoaciduria. Copper is transported in portal
plasma, bound principally to albumin and possibly as amino acid complexes. Hepatic uptake
occurs via a saturable transport process. Systemic transport of copper from liver is primarily
as ceruloplasmin, which appears to donate copper to tissues. Circulating level of ceruloplasmin
increases in response to various stresses and disease-related processes.
240 ROBERT J. COUSINS
Liver/--)
In
It is possible that. both copper and zinc are transported across the brush
border surface of the small intestine bound to one or more absorbable ligands.
The chemical nature of these ligands has not been elucidated, although this
area has been enthusiastically pursued. Also, the kinetics of ion uptake may
argue against the concept of ion-ligand cotransport. Even if free copper or
zinc is transported, specific ligands may present these metals to the brush
Lumen Plasma
Albumin
Ku’
Dietary
8 CU
Albumin
Endogenous \
I
cr”
cu
Zn
cu
Zn
cu 1 Zn
Dietary
0l -.- - . . . . . . -. t
C nrlnntannrlc - \ PO01 / 0000 \
Albumin
\Zn’
Functions Albumin
L
I
FIG. 3. Schematic representation of intestinal mucosal cell and probable sites of dietary copper and zinc interaction. Lumenal copper of dietary E
and/or endogenous origin is transported across brush border membrane surface (A). Transport of endogenous and/or dietary zinc is similarly z
shown in B. Potential interaction between high lumenal concentrations of copper and zinc for a common transport system, endogenous binding
factors, or receptors is shown as C. Once transported, these nutrient metals interact with intracellular pools. Copper and zinc are transferred from
enterocytes bound to albumin, probably at different sites on the molecule. If intracellular concentrations of copper or zinc are very high, because
of high dietary levels or elevated blood levels due to parenteral administration of these nutrients, promoter for the metallothionein gene is activated,
and enhanced transcription of the gene occurs. Normally the promotor appears more sensitive to dietary zinc levels than to dietary copper levels.
As the enhanced level of metallothionein mRNA is translated, cytoplasmic level of thionein polypeptides (wcwy line) is increased, and they bind
available cellular copper and zinc. Because the binding affinity of this protein is greater for copper than for zinc, eventually more copper is bound,
which prevents copper transport across basolateral membrane to plasma, thus reducing absorption. When intracellular level of thionein is sufficiently
high, zinc absorption is also reduced. Copper and zinc may compete for transport across the basolateral membrane. Serosal-to-mucosal fluxes of
copper and zinc are also shown, because this may contribute to intestinal endogenous secretion of these metals.
Copper Absorption
L-Amino acids TT 226
Nitrilotriacetate TT 217
D-Amino acids T 226
Citrate, phosphate, gluconate 369
Oxalate, EDTA 58,226
High dietary protein 94,113; 170, 267
Effects on uptake vs. actual absorption in various species not separated. T, Increase; 0, no
effect; 1, decrease.
April 1.985 COPPER AND ZINC METABOLISM 243
and gluconate complexes may be more easily absorbed than complexes formed
with other ligands (369). Similarly, CuCO:<, Cu(NO&, and Cu-oxalate or Cu-
ethylenediaminetetraacetic acid (EDTA) are better sources of available copper
than CuS04 (58). Dietary phytate (94), ascorbic acid (54, 194, 440), thiomo-
lybdate (88, 421), and fiber (219) appear to complex with copper and limit
its absorption. Dietary fructose may also limit copper absorption and decrease
copper status during a marginal copper intake (152). The chelator nitrilo-
triacetate enhances copper absorption (217), perhaps by forming a complex
that is transported intact across membranes. However, this area requires
further study, particularly at the membrane level.
absorption (441). This relationship may be coupled to the extent that the
proteins are degraded and/or to how tenaciously they bind zinc. Giroux and
Prakash (166) have shown that some zinc-amino acid ligand mixtures stim-
ulate absorption (e.g., lysine, cysteine, glycine) when absorption is measured
as the increase in serum zinc 3 h after oral intubation. Histidine has been
shown to increase zinc absorption in some studies (420,453), but not all (166,
299). Glutamic acid has been suggested as an important ligand for absorption
(261, 264). Heth and Hoekstra (193) demonstrated that high dietary calcium
in practical rations decreases zinc absorption, an observation that has con-
siderable practical application in the swine production industry. In contrast,
and other constituents (e.g., lactose and phosphorus content) in these milks
varies widely (206, 246, 270). The major zinc-binding protein in human milk
has been reported to be &her lactoferrin (27) or albumin (246), whereas
casein is the major zinc-binding protein in cow milk (206, 270). These milks
also differ markedly in free amino acids (160). Based on these differences,
the greater zinc bioavailabilitv in human milk mav be related partlv to
digestibility and binding affinitv of the constituent I;roteins. This proI~osa1
has received experimental s&ort (27, 246). The hioavailabilitv of iron in
human milk was shown to be greater than that in cow milk, and differences
in composition rather than the presence of specific chelators were used to
This situation may occur because only available sites may actually bind
exogenously added ‘5Zn, and these may have little biological relevance. The
findings of Lonnerdal et al. (250) on the distribution of zinc in bile and
pancreatic secretions agree with this interpretation. These authors observed
that when the actual zinc content was measured, rather than 65Zn binding,
zinc was shown to be bound to high-molecular-weight components in pan-
creatic secretions of rats. They found that biliary zinc was associated with
low-molecular-weight moieties, which agrees with the observation that glu-
tathione binds appreciable amounts of zinc in rat bile (3). Glutathione-de-
pleting agents lowered the biliary secretion of probable zinc-glutathione
beling experiments also indicated that this intestinal protein was inducible
by zinc administration (347). Inducibility was confirmed by feeding experi-
ments (t350) and injection studies (310). Little metallothionein was found in
intestines of zinc-depleted rats, whereas dietary repletion with zinc increased
metallothionein in a dose-dependent fashion (350). There is evidence that
high dietary copper will also induce this intestinal protein (178).
The inducible nature of intestinal metallothionein led to the suggestion
that the protein is an integral regulatory component for zinc absorption (75,
348, 349, 352). Although the responsiveness of biosynthesis to zinc is firmly
established, the role this protein has in the absorption process at normal
(75, 276). In some studies “Q-P may exchange with nonradioactive Zn2+,
which could be incorrectly interpreted as an increase in absorption and/or
uptake.
Intestinal metallothionein levels may not be altered to the same extent
in response to moderate dietary zinc or copper changes. In some cases these
dietary changes are not accompanied by simultaneous changes in apparent
absorption. This suggests that altered metallothionein biosynthesis may not
be a major factor in regulating the extent of absorption under normal dietary
conditions. Clear species differences exist, however, as shown by Flanagan
et al. (146) and Olafson (306), because there was no correlation between
The binding ligand(s) that transports copper from the intestine to the
liver has not been extensively studied. Nevertheless it is generally assumed
that albumin carries out that function. Copper is loosely bound to albumin,
(291, 326). A competition between copper and zinc at this point in the ab-
sorption process is thus unlikely (Fig. 3).
The nature of copper and zinc transfer across the basolateral membrane
of the intestine has not been studied directly. Recently, basolateral membrane
vesicles from rat intestine have been used to characterize the kinetics of zinc
transport across this membrane surface (300). The data suggest that a sat-
urable component with a K, of 63 PM and Jmax of 38 nmol Znmg-’ pro-
tein . min-’ is involved and may be regulated by an ATP-driven mechanism.
Aspects of copper and zinc absorption covered in section IIA-C have been
reviewed in considerable detail elsewhere (33, 76, 77, 98, 396).
A. Copper
Only -10-2(X% of the zinc in blood is found in the plasma (30, l317, 335,
373). The remainder is localized within erythrocytes in which carbonic an-
hydrase is the major binding site (199,437;. The erythrocyte membrane may
contain zinc. Bettger and O’Dell (24) have shown that zinc deficiency decreases
the ability of ervthrocytes to resist in vitro hemolysis. It was suggested that
zinc stabilizes t’he erythrocyte membrane. Despite this important function,
erythrocyte zinc uptake may be influenced by many fact,ors, as discussed by
Chesters and Will (67). Leukocytes also incorporate zinc, but there appears
to be no exchange with plasma zinc like that occurring in erythrocytes (97).
COPPER AND ZINC METABOLISM 257
Dreosti et al. (107) found that in contrast to plasma zinc levels, leukocyte c
zinc levels were not responsive to zinc depletion.
Plasma provides a metabolicallv active transport compartment for zinc,
and numerous factors influence the flux of zinc through it (17, 69, 107, 116,
241, 269, 321, 352). Most plasma zinc is associated with proteins, albumin
being the principal binding protein. This distribution varies among species,
but usually about two-thirds of the zinc in plasma is bound to albumin (30,
69, 164, 165, 335, 373). There is a good correlation between the changes in
albumin-bound zinc and plasma zinc concentration that occur during acute
and chronic disease (135,180). This pool of plasma zinc is frequently referred
but the effect may be a factor in the interpretation of data from zinc-deficiencv
studies when food restriction is also part of the experimental protocol. At-
tempts have been made to develop ways to maintain high plasma zinc levels
without continuous zinc administration. For example, systemic lupus ery-
thematosus in mice was markedly reduced when elevated serum zinc con-
centrations were maintained by ‘continuous release of zinc from suhcuta-
neously administered suspensions of zinc pamoate (236 ). Similarly, Brewer
et al. (42) have shown that the plasma zinc concentration can be elevated
for long periods by administering zinc salts as oil suspensions that slowly
release zinc. This may have therapeutic application in human diseases, because
ucagon - - - - +
GI ucocorticoid - - -
: \ /
/ \ / J
I \ \ / Ceruloplasmln Apoceruloplasmin
. /
/‘ - .
/ / -- --
, --
Estrogen /
--- ------ -- -------
Testosterone
Bile Plasma
FIG. 4. Schematic diagram of liver parenchymal cell showing key components of hormonal regulation of copper metabolism and ceruloplasmln
synthesis and secretion. Copper uptake from the portal circulation may involve amino acid- and/or albumin-bound copper or free copper. Cellular
copper is distributed among various compartments, represented here as a copper pool. Epinephrine stimulation increases cellular copper accumulation
more than other hormones. Transcription of the ceruloplasmin gene could be regulated by glucocorticoids, CAMP, and/or copper. Epinephrine and
glucagon increase cellular CAMP levels. The number of exons (dark bars) and introns (light bars) for gene and formation of completed mRNA are
hypothetical. Synthesis of ceruloplasmin involves only membrane-bound polyribosomes. Secretion of ceruloplasmin as a single 1X2,000-dalton
polypeptide (including carbohydrate fragment) and concomitant change in plasma copper content is increased by glucocorticoids and to a lesser
extent by estrogen and testosterone. Interleukin 1 may increase plasma ceruloplasmin directly or indirectly via hormones. Glucocorticoids also
appear to increase biliary excretion of hepatic copper.
2. Zinc
Fluctuations in the dietary zinc supply cause small changes in the hepatic
concentrations of this metal (434). Basic aspects of zinc metabolism by he-
patocytes are shown in Figure 5. The fairly constant supply of intracellular
zinc is undoubtedly maintained through the concerted action of many hor-
monal inputs. There have been relatively few studies carried out to char-
Int erleu
/7
\
kin -7 Receptor\
\ \
‘\ / ,q’ 0 ‘,d I- \Nucleus
Free Polyribosomes
Albumm -Zn
Amino Acid%
lectively represented here as a labile zinc pool that accounts for accumulation, and a zinc-
metalloprotein pool that accounts for exchange. High intracellular levels of zinc, which can
follow increases in changes in plasma zinc concentration, activate the promotor for the me-
tallothionein gene. Expression of this gene seems to be more responsive to changes in dietary
zinc than in dietary copper. Glucocorticoids also activate transcription of this gene. Exons and
introns are approximately as established for the mouse metallothionein I gene (109). Nuclear
metallothionein has been detected (63), and this may serve a regulatory function related to gene
expression. Completed mRNA is 300-400 nucleotides and is translated as free polyribosomes.
Glucagon appears to exert its effect on metallothionein synthesis via both transcriptional and
translational regulation. Epinephrine may exert transcription of regulation as well. Glucagon
and epinephrine probably activate transcription via CAMP. Nascent metallothionein polypeptides
bind intracellular zinc and copper after transport into the cell. Zinc accumulation by liver cells
in response to endotoxin or interleukin 1 may act directly or through glucocorticoids and glucagon.
A variety of functions for metallothionein and thionein (apometallothionein) have been proposed.
These may be related to hormonal regulation of zinc metabolism. Intracellular copper also is
bound to metallothionein. These proteins differ in susceptibility to degradation in the order
thionein > zinc metallothionein > copper metallothionein. Lysosomal proteases appear to be
involved in degradation. Copper metallothionein resistance to proteolysis could partially explain
its accumulation in some genetic disorders, e.g., Wilson’s disease. Plasma metallothionein levels
appear proportional to the extent that the gene is expressed.
268 ROBERT J. COUSINS Volume 65
B. Intracellular Compartmentalization
Both copper and zinc are distributed throughout the various subcellular
fractions of hepatocytes. Smeyers-Verbeke et al. (385) have analyzed the
nuclear, mitochondrial, rough and smooth microsomal, and supernatant frac-
tions of liver for protein, nitrogen, copper, and zinc. The average distribution
of copper in these cellular fractions was 27, 7, 7, 3, and 54%, respectively.
The average zinc distribution in these fractions was 23, 5, 12, 5, and 60%,
respectively. Clearly the supernatant fraction (in this case, 3 h at 100,000 9)
contained the highest percentage of both metals. This corresponded to ~36%
of the total liver protein and 40% of the total nitrogen content. Alfonzo and
Heaton (4) found that liver homogenates averaged 20.5 and 97.4 pg/g dry
matter for copper and zinc, respectively. The supernatant fraction represented
39.3 and 46.7% of the total copper and zinc content, respectively.
The supernatant fraction of liver appears to be metabolically active with
respect to copper and zinc. For example, copper deficiency in rats led to a
reduction in the copper content of all cellular fractions, but the supernatant
accounted for the most pronounced decrease (4, 285). The majority of 67Cu
from [67Cu]ceruloplasmin was shown to be taken up into the soluble fraction
of rat liver (258). However, copper-loading experiments generally produced
an increase in the copper content of the heavier subcellular particles (nuclear
and mitochondrial fractions) (126, 171). Lysosomes also accumulate appre-
ciable copper (447), which may result partly from binding of hepatic copper
April 1985 COPPER AND ZINC METABOLISM 269
1. Zinc
Efflux of copper and zinc from the liver undoubtedly depends on many
factors, specifically intracellular factors that might favor retention of these
April 1985 COPPER AND ZINC METABOLISM 271
metals within cells, and the availability of circulating ligands that could
transfer these metals from hepatocytes. Saltman and Boroughs (361) observed
such a rapid efflux of zinc from fish liver slices into Krebs-Ringer solution
that within 0.5-Z h, only a small portion of the accumulated zinc remained.
This observation is at variance with more recent data from studies with
isolated rat liver parenchymal cells in an albumin-containing medium, which
demonstrated that most of the newly acquired zinc was not readily available
for efflux. It appeared from these studies that efflux did occur, because within
lo-15 h the 65Zn uptake process appeared to plateau (130). Because cells at
this point were still able to take up 65Zn, it was concluded that this plateau
2. Copper
A relatively low efflux rate of 64Cu was observed when rat liver paren-
chymal cells were incubated continuously with 64Cu for 6-12 h in albumin-
containing media (457). It was estimated that -15% of the intracellular
copper was lost in a l-h period, the majority being lost during the first 5-
to 15-min measurement period. This may represent exchange of copper with
a rapidly turning-over pool. In contrast to these studies, which were performed
with purified parenchymal cells that were maintained at 37°C and exhibited
physiological responsiveness, Schmitt et al. (367) have shown that hepatocytes
maintained in suspension culture lose 35-40% of accumulated 64Cu during a
40-min preincubation period. These studies, however, were carried out at
272 ROBERT J. COUSINS b%wne 65
ZO”C, and significant efflux was also evident at 4”C, which questions phys-
iological significance. Efflux rates of 64Cu from rat liver slices reported by
Saltman et al. (360) were comparable to these. Using a liver perfusion system,
Owen and Hazelrig (315) found that a steady state between 64Cu influx and
efflux could be reached. Their efflux data were more suggestive of those
observed with isolated cultured liver cells (457).
Because glucagon increased copper uptake by cultured liver cells while
glucocorticoids increased copper secretion, the collective effect of these com-
pounds may be to enhance copper flux through the hepatocytes in the direction
of copper efflux as secreted ceruloplasmin. This may be important, because
VI. METALLOTHIONEIN
A. Properties
In the past decade metallothionein has been one of the most widely
studied metalloproteins. The reason for this, apart from its unique protein
chemistry, relates to its induction by heavy metals and hormones. Depending
on animal species and tissue from which it is isolated, this protein is found
to bind a variety of metals, particularly cadmium, copper, mercury, and zinc.
Equine kidney contains relatively large amounts of cadmium. In examining
the reason for the accumulation of this nonnutrient metal, a hitherto un-
discovered protein was isolated and characterized, which Kagi and Vallee
(210) termed metallothionein. The salient features of the protein are as follows.
I) It is a single polypeptide chain of 61 amino acids. 2) It has a metal-binding
capacity of between 5 and 7 g atoms/mol. 3) Twenty-five to 30% of the amino
acid residues are cysteine, and in the native protein there are no disulfide
bonds. (There is some evidence that under aerobic conditions and extensive
April 1985 COPPER AND ZINC METABOLISM 273
B. Synthesis
I. Regulation by metals
Inducer Ref.
Physiological
Development 7, 20, 38, 40, 220, 305, 467
Dietary zinc 36, 61, 144, 146, 178, 269, 276, 306, 349, 350
Infection 394
Starvation 35, 349
Stress 303, 305, 364
Experimental
Adjuvant arthritis 426
Alkylating agents 234
Cadmium 209”
Carbon tetrachloride 51, 303
Copper 36, 37, 255, 278, 459, 465
Diabetes 133
Endotoxin 100, 117, 318
Epinephrine 31
Glucocorticoids 115, 116, 130, 131, 177, 213, 265, 342, 459
Glucagon 99, 115, 237
Interleukin lt 100, 102
Isopropanol 427
Retinoic acid 426
Turpentine 392, 393
Zinc 35, 37, 61, 109, 137, 178, 255, 302, 306, 310, 347, 348, 349, 350, 351,
352, 372, 378, 379, 380, 388, 406, 407, 410, 425, 430, 459
* General reference for induction by cadmium and metals other than copper or zinc.
t Also called leukocytic endogenous med iator or endogenous pyrogen.
April 1.tw.s COPPER AND ZINC METABOLISM 275
of gene expression. A major advance in this direction was the isolation and
characterization of the mouse metallothionein I gene by Durnam and co-
workers (110). To accomplish this a double-stranded cDNA was synthesized
from a mouse liver mRNA population that was enriched in metallothionein
mRNA. This DNA was subsequently inserted into the plasmid vector pBR322
and cloned in transformed Escherichia coli. A plasmid containing a 380 base-
pair fragment, which included the entire coding region, was used to identify
metallothionein I genomic clones prepared from a mouse embryo DNA library.
These clones were nick-translated with 32P-dNTPs and used as DNA probes
in hybridization experiments to quantitate metallothionein mRNA levels.
2. Dietary control
3. Hormonal regulation
of zinc may be transferred to the liver and other tissues; then metallothionein
synthesis is induced. How this event would relate to hypozincemia associated
with IL-1 administration is not clear (18). However, hypozincemia may be
a transient response to the initial induction of hepatic metallothionein syn-
thesis.
The influence of hormonal status and chronic hormonal imbalance on
copper and zinc metabolism and on hepatic and renal metallothionein has
been shown with the streptozotocin-induced diabetic rat. Failla and Kiser
(133) found that the concentration of both copper and zinc in these tissues
increased within a few days after treatment with this diabetogenic drug.
C. Degradation
D. Function
VII. CERULOPLASMIN
Holmberg and Laurel1 (196) named the blue protein from plasma ce-
rulorplasmin. Recent detailed reviews of the physical and chemical properties
of this cuz-globulin are available (155, 240). Only general properties of ce-
ruloplasmin are described here. Ceruloplasmin from human plasma is a gly-
coprotein of -132,000 daltons containing 6 copper atoms per molecule and
78% carbohydrate. Proteolytic cleavage of intact ceruloplasmin may occur
Inducer Ref.
Physiological
Cancer 72
Chronic inflammation 73, 96, 153, 282, 424
Development 57, 126, 327, 468, 470
Exercise 106, 182
Pregnancy 147
Experimental
ACTH 5, 154, 411
Copper 125, 243, 458, 462
Endotoxin 87, 117, 154, 355
Epinephrine 154, 281, 458, 459
Estrogen 72, 119, 147, 281, 330, 356
Glucocorticoids 5, 87, 411, 459
Inflammation
Adjuvant arthritis 426
Carrageenen 282, 283
Turpentine 281
Interleukin 1’ 452
Retinoic acid 426
Zinc 426
requirements of the fetus (190). Planas and Frieden (330) observed that
estrogens induce ferroxidase activity (presumably ceruloplasmin) in normal
and copper- or iron-deficient roosters. Earlier, estradiol was found to increase
serum ceruloplasmin in rats (281). In humans, elevated testosterone levels
were correlated with increased serum ceruloplasmin (208, 470).
Adrenal hormones have received particular attention regarding their
effect on ceruloplasmin levels in the blood. The regulation of ceruloplasmin
synthesis and secretion by glucocorticoids is similar to that documented for
other acute-phase proteins, e.g., fibrinogen (173). The early experiments
showing the effects of adrenalectomy on plasma copper and ceruloplasmin
gested that when the extracellular copper content was sufficiently high, ce-
ruloplasmin gene expression may have been increased. This agrees with the
intact-animal data of Linder et al. (243), who demonstrated an inductive
action for exogenous copper when the dietary copper supply was diminished.
In this case, the sudden influx of copper associated with a large dose of
copper may be sufficient to bypass normal controls and activate the gene or
Gl ucogon ! Glucocorticoid
Fe
Plas mo Zn
Cu - Ceruloplasmin \
Toxins’
Detoxified
Product
Tissue Cu Antioxidant
Fe II---- Fe IIf
FIG. 6. Hypothesis for integrated hormonal and mediator-controlled changes in hepatic
copper and zinc metabolism as related to host defense. Stress and trauma increase plasma
glucagon and glucocorticoids as well as interleukin 1, which in turn increase metallothionein
levels in liver cells and promote ceruloplasmin secretion. Increased metallothionein and zinc
redistribution in liver could relate to altered enzyme activity, membrane stabilization, and/or
detoxification mechanisms. Elevated plasma ceruloplasmin relates to increases in ferroxidase
activity, copper transport to tissues, and serum antioxidant properties, which may preclude
deleterious oxidative damage. Macrophages also synthesize metallothionein in response to glu-
cocorticoids (318). This change is correlated to the ability of the macrophage to combat the
inhibitory influence of endotoxin on phagocytosis. Integral role of interleukin 1 in these stress-
related phenomena and fever is indicated.
296 ROBERT J. COUSINS Vdume 65
VIII. CONCLUSION
Aside from the well-documented roles for copper and zinc in metal-
loenzyme activity, there have been few attempts to combine observations
I thank Ann C. Coutu for typing the manuscript, Walter Jones for drawing some of the
figures, and various colleagues who have critically evaluated the manuscript and made valuable
suggestions. Research from my laboratory discussed in this review was supported by National
Institutes of Health Grants AM-31127, AM-31651, and ES-03103. This review is Florida Agric.
Exp. Station Journal Ser. No. 5796.
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