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Food Science and Human Nutrition Department and Center for Nutritional Sciences, University of Florida, Gainesville, FL 32611-0370
2To whom correspondence should be addressed at 201 FSHN, P.O. Box 110370.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • zinc deficiency • diarrhea • gene regulation • uroguanylin • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) that was originally isolated from urine and duodenal
mucosa and identified by its unique biochemical properties and
physiological actions, similar to the Escherichia coli
heat-stable enterotoxin STa (Hamra et al. 1993
).
Autocrine and paracrine actions of UG and guanylin stimulate the
transepithelial secretion of both Cl- and
HCO3- anions in the intestine (Forte 1999
, Joo et al. 1998
). In addition, UG peptides
are secreted into the blood, where the circulating hormone is also
involved in the regulation of urinary NaCl excretion (Forte et al. 2000
). These actions occur via activation of guanylate
cyclase C-type receptors in the gastrointestinal epithelium and
kidney (Currie et al. 1992
, Fan et al. 1997b
). UG is more potent and effective in stimulating anion
secretion across the proximal duodenum when the mucosal surface is
exposed to acidic conditions (Hamra et al. 1997
,
Joo et al. 1998
).
UG mRNA is very abundant in the proximal small intestine of rats, with
progressively decreasing amounts in the lower small intestine, colon,
thymus, stomach, kidney, pancreas, lung and testis (Blanchard and Cousins 1997
, Fan et al. 1997a
, Li et al. 1997
). Through in situ hybridization and
immunohistochemical methods, respectively, UG mRNA and protein have
been identified in rat intestine enterochromaffin (EC) cells, the most
abundant type of enteroendocrine cells (Nakazato et al. 1998
, Perkins et al. 1997
).
Diarrheal disease has long been recognized as a major international
health problem and is one of the major causes of infant mortality,
especially in developing countries (Golden and Golden 1981
). In addition, diarrhea has been recognized as one of the
gastrointestinal symptoms of zinc deficiency in humans (Golden and Golden 1981
, Hambidge 1992
, Okada et al. 1976
). The incidence of diarrhea can be markedly reduced by
zinc supplementation, as demonstrated in several large-scale
intervention studies (Rosada et al. 1997
, Sazawal et al. 1995
). The causes of diarrhea are diverse and include
infection, genetic disorders and malnutrition; however, for many of
these disorders, the exact mechanisms responsible for regulating the
hypersecretion of water are unknown. For example, zinc-deficient
(-Zn) rats challenged with interleukin-1
show a much higher
frequency of diarrhea, which is accompanied by a profound expression of
inducible nitric oxide synthase (Cui et al. 1997
). This
suggests that an immunological cascade component may play a significant
role. Identification, through mRNA differential display, of the
upregulation of preprouroguanylin (pre-PUG) mRNA in the small intestine
during zinc deficiency suggests a potential mechanistic link between
zinc deficiency and the fluid secretion of the diarrhea that
accompanies it (Blanchard and Cousins 1996
). However,
whether upregulation of UG gene expression results in increased UG
peptide has not been determined.
In the current study, we identified the cells in the -Zn rat intestine responsible for greater PUG production by using in situ hybridization and immunohistochemical methods. We also used Western blot analysis to examine the level of PUG in small intestine. The data support the hypothesis that zinc deficiency produces an upregulation of UG expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male, 5- to 6-wk-old Sprague-Dawley rats (Harlan, Indianapolis, IN)
that weighed 150–175 g were individually housed in hanging stainless
steel cages on a 12:12-h light/dark cycle and had free access to
distilled, deionized water. Rats were fed an AIN-76a–based pelleted
diet (AIN 1977
) in which casein was replaced with
spray-dried egg white as the protein source (Research Diets, New
Brunswick, NJ) as described previously (Blanchard and Cousins 1996
). After being fed the normal zinc diet with 30 mg Zn/kg
for 1 wk, the rats were randomly assigned to one of two groups. One
group continued to receive the zinc adequate (30 mg Zn/kg; +Zn) diet,
whereas the other group received a zinc-deficient (<1 mg Zn/kg;
-Zn) diet. A pair-fed group was not included in these experiments
because it was shown previously that UG expression is not significantly
influenced by food restriction (Blanchard and Cousins 1997
). After 15 d, the rats were anesthetized with
methoxyflurane and killed by exsanguination between 0900 and 1200 h for most experiments. Blood was collected via cardiac puncture, and
the serum zinc concentration was measured with flame atomic absorption
spectrophotometry (Blanchard and Cousins 1996
). All
animal procedures were approved by the University of Florida
Institutional Animal Care and Use Committee.
In situ hybridization.
Rat pre-PUG clone 1783-1, which contains the 3' end cloned into
pCRII (Invitrogen, Carlsbad, CA) (Blanchard and Cousins 1997
), was subjected to restriction digestion with
HindIII or XbaI and used as a template
for sense and antisense riboprobe synthesis. Digoxigenin (DIG)-labeled
RNA probes were prepared from the linearized plasmid using SP6 and T7
RNA polymerases and a DIG RNA labeling kit (Roche, Indianapolis, IN).
Excised rat intestinal tissues were immediately fixed with buffered 4% paraformaldehyde and sealed in paraffin, and cross sections (4 µm) were mounted on slides. These were washed twice with xylene and then with 95% and 70% ethanol, followed with phosphate-buffered saline (PBS) containing 0.3% Triton X-100. Sections were permeabilized with Proteinase K (Roche) (100 mmol Tris-HCl/L, 50 mmol EDTA/L, 20 mg/L Proteinase K, pH 8.0), fixed with 4% paraformaldehyde and washed twice with PBS. After incubation with 0.1 mol triethanolamine/L, pH 8.0, containing 0.25% (v/v) acetic anhydride (Sigma Chemical Co., St. Louis, MO), the sections were incubated with prehybridization buffer [4x standard saline citrate (SSC), 50% (v/v) deionized formamide].
Hybridization was performed overnight in 50% deionized formamide, 10%
dextran sulfate, 1x Denhardt’s solution, 4x SSC, 10 mmol
dithiothreitol/L, 1 g/L yeast tRNA and 1 g/L denatured, sheared salmon
sperm DNA containing 
200 µg/L labeled RNA probe. Sense and
antisense probes were always applied to adjacent sections for control
purposes. After hybridization, sections were washed once in 2x SSC,
three times in 1x SSC plus 50% formamide and once in buffer 1 (100
mmol Tris/L, pH 7.5, and 150 mmol NaCl/L). Blocking for nonspecific
hybridization was performed in buffer 2 (buffer 1 containing 0.1%
Triton X-100 and 2% normal sheep serum). Next, a 1:2000 dilution of an
alkaline phosphatase–conjugated anti-DIG antibody (Roche) in
buffer 2 was added. The sections were washed twice in buffer 1 at room
temperature and incubated in 100 mmol Tris/L, pH 9.5, 100 mmol NaCl/L
and 50 mmol MgCl2/L until the desired color was achieved.
Color development was stopped by washing with 10 mmol Tris-HCl/L, pH
8.1, 1 mmol EDTA/L, followed briefly with distilled water. Fast green
FcF (0.02%) was used for counterstaining. Cover slides were applied
with an aqueous mounting solution (Aqua-Mount; Lerner Laboratories,
Pittsburgh, PA).
Photomicrographs were obtained with a Zeiss Axiovert S100 microscope (Carl Zeiss, Thornwood, NY) fitted with a SPOT digital CCD camera (Diagnostic Instruments, Sterling Heights, MI) for image analysis.
Antibody production.
UG peptide (TDECELCINVACTGC) was synthesized, and the composition,
showing three possible disulfide configurations, was verified by mass
spectrometry. This peptide was conjugated at the amino terminal by
glutaraldehyde to keyhole limpet hemocyanin as directed by the
manufacturer (Pierce, Rockford, IL). Keyhole limpet
hemocyanin–conjugated peptide was then injected into a New Zealand
White rabbit for production of polyclonal antiserum. A total IgG
fraction was prepared from whole serum through differential
precipitation with caprylic acid and ammonium sulfate (Dankert et al. 1985
). Peptide-specific antibody was then isolated
from this total IgG fraction by affinity chromatography with an
immobilized UG peptide column (Sulfo-Link; Pierce). The unbound IgG
fraction from affinity purification [flow through (FT)] and preimmune
serum (PIS) were used as negative controls.
Dot blotting and Western blotting.
UG peptide (1–5 µg) was dotted onto strips of nitrocellulose transfer membrane (MSI, Westboro, MA). The strips were incubated with affinity-purified (AP) antibody after being blocked with 5% nonfat dry milk in PBS-T (PBS, 0.05% Tween-20, pH 7.5). After washing in PBS-T, anti-rabbit IgG horseradish peroxidase conjugate (Sigma Chemical Co.) was applied and detected by Renaissance Chemiluminescence (NEN, Boston, MA) with X-ray film.
Rats for Western blot experiments were killed between 1330 and
1500 h, alternating between the two dietary groups to minimize any
diurnal variation in PUG expression (Scheving and Jin 1999
). For semiquantitative Western analysis of intestinal
proteins, the lumen was flushed with ice-cold 0.9% saline, and the
mucosal layer from duodenum and proximal jejunum was removed by
scraping and then immediately homogenized in 4 volumes of 20 mmol
HEPES/L, pH 7.4, 1 mmol EDTA/L and 300 mmol mannitol/L, containing 5%
protease inhibitor cocktail (P2714; Sigma Chemical Co.) added
immediately before use. After centrifugation at 225,000 x g, the cytosolic fraction (supernatant) was collected
and analyzed for protein content by colorimetric assay. Equal amounts
of cytosolic protein (300 µg) were resolved on a 15% Tris/tricine
SDS–polyacrylamide gel (Hempe and Cousins 1991
) and
electroblotted onto Immobilon-P (Millipore, Bedford, MA) submerged
in 192 mmol glycine/L, 10 mmol Tris/L, 0.05% SDS and 20% methanol.
Blots were stained with amido black, and immunodetection was performed
as described earlier.
Immunohistochemistry.
Immunohistochemical detection was performed with a Histostain Kit (Zymed Laboratories, South San Francisco, CA). Tissue sections were prepared as described earlier. After deparaffinization with xylene, 4-µm sections were hydrated in 95 and 70% ethanol and water, followed by incubation in 3% H2O2. Slides were rinsed three times with PBS-T, blocked with 10 g/L bovine serum albumin and then blocked with 10% normal goat serum. Finally, the sections were incubated overnight at 4°C with the AP UG antibody (1:100 = 10 mg/L) or FT (negative control) diluted in 1% normal goat serum/PBS. After washing in PBS, all sections were incubated with biotinylated goat anti-rabbit IgG at room temperature and rinsed with PBS, followed by streptavidin-peroxidase conjugate to develop the red AEC chromagen. Hematoxylin counterstaining was performed before mounting. Microscopy and image analysis was performed as described earlier.
Statistical analysis.
Data are expressed as means ± SD. Differences between groups were determined using a two-tailed Student’s t test. A value of P < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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, E
) and -Zn (Fig. 1C
, G
) groups. Pre-PUG
mRNA signals were primarily localized in a subset of villus cells
within the duodenum and jejunum of rats fed both levels of zinc.
However, the number of cells demonstrating a pre-PUG signal in the
sections was substantially greater in -Zn rats (duodenum 57 ± 10
cells/mm2, jejunum 43 ± 13
cells/mm2) than in +Zn rats (duodenum 5 ± 0
cells/mm2, jejunum 6 ± 0
cells/mm2; P < 0.001;
n = 3). No signals were observed in adjacent sections
treated with the sense-strand riboprobe (Fig. 1B
, D
, F
, H
).
|
). Specificity of the antibody for PUG was further examined
by Western blot analysis of intestinal cytosolic proteins (Fig. 2B
). The AP fraction produced a major signal for a single
protein band at 8 kDa in cytosol prepared from intestinal mucosa of
a -Zn rat. This size corresponds to the calculated molecular mass of
rat PUG as determined from its deduced amino acid sequence. When the AP
was incubated with excess UG peptide before the antibody was used for
Western blotting, this band was not detected. The FT fraction did not
produce a major signal from any protein band. Based on these data, the
AP polyclonal IgG is specific for proteins containing the UG epitope,
and the intestinal protein band detected is most likely PUG.
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). In contrast, positive staining cells were identified
throughout the entire villus, and some cells were very strongly stained
in sections from the -Zn rat (Fig. 3C
). The morphology and
distribution of these cells are identical to those of EC cells,
which typically have a large, basolateral compartment in
contact with the basal lamina and a thin apical process that provides a
small cytoplasmic compartment adjacent to the lumen. There was no
specific staining of cells using FT as the negative control (Fig. 3B
, D
). Similarly, the positive cells could be seen only at
the tip of the jejunal villi of the +Zn rat (Fig. 3E
).
However, the specific staining was scattered along the entire villus of
the -Zn rat jejunum with the same staining time (Fig. 3G
).
Again, a subset of cells was very heavily stained. There was no
specific staining of cells using FT in place of the AP (Fig. 3
F, H
). In addition, no specific staining with the AP
antibody was found in lymphatic nodes or in lymphocytes distributed in
the intestinal villi or submucosal layers.
|
) suggested signals using the pre-PUG antisense
riboprobe (Fig. 4B
) and immunoreactive PUG (Fig. 4C
) are present in a subset of intestinal epithelial cells.
These cells appeared to have a unique morphology (Fig. 4A
),
and may be EC cells. The number of cells demonstrating a pre-PUG
mRNA signal (43–57 cells/mm2) was similar to the
number of cells displaying PUG and/or UG immunoreactivity (37
cells/mm2) in -Zn rats. However, these values
were different in +Zn rats. The exact explanation for this discrepancy
is not known. However, a difference in the incubation time for color
development of these two methods may play a role. It took hours for in
situ hybridization of the mRNA but only minutes for
immunohistochemistry.
|
8 kDa is believed to represent the PUG
peptide before processing to the active UG hormone. In both regions of
the small intestine, mucosa PUG expression was greatly elevated in zinc
deficiency (Fig. 5
), and although not readily visible on the short exposure shown in this
figure, basal levels of PUG were readily detected in normal zinc rat
intestine. The terminally processed, 15- to 16-amino-acid UG peptides
were not detectable under the Western blot conditions used. This likely
was due to multiple factors, including the low abundance of mature
hormone compared with precursor peptides, plus the inability of small
peptides to resolve on a gel of the composition used and to transfer
efficiently during electroblotting. In an attempt to circumvent this
difficulty, the cytosol fractions used for the Western blots were also
passed through a Microcon-3 membrane (Millipore; 3-kDa cutoff) and slot
blotted to a nitrocellulose membrane before immunodetection, as was
performed for the Western blots. Signals were detected that presumably
represent terminally cleaved UG peptides (data not shown). Analysis by
densitometry showed that more putative UG was present in intestinal
cytosol from the -Zn rats than from the +Zn rats. The arbitrary units
were 10 ± 9 and 7 ± 3 in the duodenum and jejunum from -Zn
rats and 2 ± 0 and 2 ± 2 from +Zn rats, respectively. This
is consistent with the hypothesis of UG overproduction in zinc
deficiency.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Perkins et al. 1997
), in situ hybridization localized
pre-PUG transcripts predominately to EC-like cells. Intestine
from -Zn rats showed significantly greater numbers of cells producing
a UG mRNA-specific signal than did intestine from +Zn rats.
The increased pre-PUG transcript-positive cell count alone is
likely sufficient to account for the increased levels of pre-PUG
mRNA previously observed in total RNA from intestinal homogenates
(Blanchard and Cousins 1997
). Two possible explanations
could account for these data. The first is that given the fact that UG
transcript-positive cells seem to be a subpopulation of
enteroendocrine cells in the villus, zinc deficiency converts some of
the UG-negative cells into UG-expressing cells. Specifically,
zinc deficiency turns on pre-PUG mRNA expression in a subset of
enteroendocrine cells that were previously not expressing it.
Alternatively, zinc deficiency does not directly affect the
transcription of the pre-PUG gene but indirectly increases
pre-PUG mRNA within the organ by stimulating the intestinal stem
cells to produce more of the enteroendocrine cell type that normally
produces PUG. In this manner, zinc deficiency could alter the relative
abundance of certain subpopulations of intestinal cells, a phenomenon
already observed in immune cell classes during zinc deficiency
(Beck et al. 1997
). Given the committed terminal
differentiation of intestinal villus cells, coupled with their very
rapid turnover, 4 d, the latter is a distinct possibility. PUG was
shown, using immunohistochemistry, to be scattered throughout EC-like
cells of the intestinal villi in -Zn rats, compared with a limited
localization to the apical portion of the villus in +Zn control rats.
Upregulation of PUG protein was further confirmed by Western blot
analysis. These data are also consistent with the observation that zinc
deficiency increases intestinal UG precursor expression in rats
(Blanchard and Cousins 1997
).
A synthetic peptide of the carboxyl-terminal 15 residues of rat UG
was used to prepare the rabbit polyclonal antibody used in these
studies. Although the cysteine-rich native UG hormone appears to
have a single biologically active conformation maintained by two
disulfide bonds (Forte 1999
), the synthetic peptide
consists of approximate equal mass ratios of the three possible
conformations produced by disulfide formation. The antiserum to this
synthetic peptide antigen will not distinguish biologically active
hormone from inactive peptides, but it should detect the
carboxyl-terminal end of all translation products from the
pre-PUG gene. Our dot blotting results indicate that the
immunoreactivity of the IS, total IgG fraction and AP antibody was UG
specific, because both the PIS and unbound antibody fraction from
affinity purification were free from reactivity. The AP antibody
clearly recognized a protein band of 8 kDa from polyacrylamide gel
electrophoresis of the cytosol of intestinal mucosa that corresponds to
the approximate expected size of the second UG precursor peptide, i.e.,
PUG. The location of this band on Western blots is consistent with
reports from other laboratories (Perkins et al. 1997
).
Using in situ hybridization to compare the distribution of UG and
guanylin mRNAs in murine intestine, Whitaker et al. (1997)
demonstrated that pre-PUG transcripts were localized
to villus cells of small intestine, whereas preproguanylin transcripts
are found in cells of crypts and villi of the small intestine and in
surface enterocytes of the colon. We did not examine whether the whole
IS, total IgG fraction or AP antibody raised against the UG peptide and
used in the current study reacts with guanylin. However, it was
unlikely that a cross-reaction with guanylin played an important
role in an explanation of the current results. Guanylin activity in the
colon of rats is highest in a subset of goblet cells and absorptive
cells (Li and Goy 1993
). Using equal staining times and
the AP antibody, we found there was little positive staining in the
colon and ileum compared with proximal small intestine (unpublished
data). Furthermore, our localization of UG protein–positive cells
suggests high specificity of the AP antibody, because there was no
immunoreactivity in goblet cells where guanylin is detected (Li and Goy 1993
). UG was found in a subset of EC cells of rat
intestine and was colocalized with serotonin in the jejunum
(Nakazato et al. 1998
, Perkins et al. 1997
). These findings clearly establish that UG and guanylin
have distinctly different patterns of expression in major segments of
the gastrointestinal epithelium and in different cell types within the
mucosa, suggesting that unique regulatory mechanisms exist for each
peptide. Our in situ hybridization and immunohistochemical analysis
results indicate that UG distribution was rather restricted. The cells
that reside in the upper intestine and exhibit the UG-specific
labeling appear to be those responsible for the increase observed in
zinc deficiency. It is unlikely that the intestinal lymphocytes in
lymph nodes or scattered throughout the intestine that produce
lymphoguanylin (Forte et al. 1999
) are responsible
for the labeling produced with our AP antibody to UG, because no
labeling was found at sites at which lymphocytes are located (see Fig. 1
).
There is increasing evidence that the signaling pathway for UG activity
includes binding to and activating the guanylyl cyclase C receptor,
increasing intracellular cGMP, phosphorylating the cystic fibrosis
transmembrane conductance regulator Cl- channel
and ultimately stimulating intestinal Cl- and
HCO3- secretion (Fan et al. 1997b
, London et al. 1997
). Upregulation of
UG may provide a compensatory mechanism for regulation of
water–electrolyte metabolism as well as acid–base homeostasis in zinc
deficiency. An example of this effect could be the demonstration that
zinc deficiency produces a negative intestinal fluid balance in chicks
and rats (Bettger et al. 1981
, Ghishan 1984
).
Because increases in UG production would shift water balance toward
secretion into the intestinal lumen and therefore toward diarrhea,
upregulation of this hormone may begin to explain the observed effect
of zinc supplementation in reducing diarrhea in studies of humans
(Rosado et al. 1997
, Sazawal et al. 1995
). On the other hand, diarrhea is not a usual finding in
experimentally induced -Zn rats. In the current study, dietary zinc
deficiency for 2 wk reduced serum zinc concentration by 80%
compared to control rats. A previous study demonstrated that diarrhea
in rats did not occur even if the plasma zinc concentration decreased
by 90% in rats fed a -Zn diet for 4 wk (Cui et al. 1997
). Therefore, rats are not normally the experimental animal
of choice for such studies because of their ability to reabsorb large
amounts of intestinal fluid via the cecum. However, intestinal
infection most likely accompanies human zinc deficiency (Golden and Golden 1981
, Hambidge 1992
); therefore, the
finding that the treatment of -Zn rats with interleukin-1
produces diarrhea is relevant (Cui et al. 1997
).
Consequently, the rat may be a good model for studying mechanisms
leading to zinc deficiency–induced diarrhea. Investigation of the UG
signaling pathway and production of cGMP could be the next steps in
understanding the disturbance in water homeostasis associated with zinc
deficiency.
In conclusion, the results presented in this report demonstrate that dietary zinc deficiency increases PUG peptide expression in the intestine. This is limited to a subset of cells lining the villi, most likely EC cells. Overexpression of PUG may contribute to altered fluid balance and explain in part the mechanism of zinc deficiency–associated diarrhea.
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FOOTNOTES |
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3 Abbreviations used: AP, affinity-purified; DIG, digoxigenin; EC, enterochromaffin; FT, flow through; IS, immune serum; PBS, phosphate-buffered saline; PIS, preimmune serum; PUG, prouroguanylin; SSC, standard saline citrate; UG, uroguanylin; -Zn, zinc deficient; +Zn, zinc adequate.
Manuscript received June 9, 2000. Initial review completed June 23, 2000. Revision accepted July 12, 2000.
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