membrane lipid peroxidation. In this study, we examined the accumulation of 4-HNE- protein adducts in phagosomes of neutrophils obtained from a male patient.
lmmunocytochemical detection of lipid peroxidation in phagosomes of human neutrophils: correlation with expression of flavocytochrome b Mark
T. Quinn
and Algirdas 4Departments Department
Abstract: activated targets (4-HNE).
John
G.
Linner*,
Daniel
Siemsen4,
Edward
A.
Dratzt,
E. Stephen
BuescherS,
J. Jesaitis* of Microbiology
of Pediatrics,
and Eastern
Chemistry Virginia
and
Biochemistry,
Medical
School,
Oxidants generated by the NADPH oxidase of neutrophils can react with a number of tissue to form toxic metabolites such as 4-hydroxynonenal 4-HNE is a lipid peroxidation product gener-
ated by free radical attack on w-6 polyunsaturated fatty acids and is a marker for membrane lipid peroxidation. In this study, we examined the accumulation of 4-HNEprotein adducts in phagosomes of neutrophils obtained from a male patient with homozygous X-linked, flavocytochrome b-deficient chronic granulomatous disease ( CGD), his heterozygous mother, and his normal father. Specific polyclonal antibodies recognizing 4-HNEprotein adducts and gp9l-phox (flavocytochrome b large subunit) were prepared and used to immunocytochemically detect these antigens in cryofixed, molecular distillationdried neutrophils. No 4-HNE-protein adducts were detected in fiavocytochrome b-deficient cells from the homozygous patient or from the heterozygous CGD carncr. However, in gp9l-phox-positive cells from both the normal and heterozygous CGD carrier, significant 4-HNE-protein adduct labeling was observed, primarily in the phagosomes. When data from singleand doublelabeled cells were combined, the frequency distribution of the labels in phagosomes supported this observation, showing that neutrophils from the heterozygous CGD carncr were 71% 4-HNE-protein adduct-positive and 56% gp9l-phox-positive, while cells from the normal father were > 97% positive for both 4-HNE-protein adducts and gp9l-phox. These results confirmed the nitroblue tetrazolium tests of 100%, 60 ± 2%, and 0% positive for the father’s, mother’s, and son’s cells, respectively, and demonstrated that 4-HNE-protein adduct antibodies are useful and accurate probes of the occurrence of lipid peroxidation in vivo. We conclude that 4-HNE and resulting 4-HNE-protein adducts are generated as a result of NADPH oxidase activity in the phagosomes of human neutrophils and that these lipid peroxidation products may contribute to microbial killing and/or damage of neutrophil phagolysosomal proteins. J. Lcukoc Biol. 57: 415-421; 1995. Key Words: 4-hydroxynonenal . electron microscopy leukocyte
chronic NADPH .
.
granulomatous oxidase
disease
Montana Norfolk,
State
University,
Bozeman,
Montana
and
Virginia
alkenes, and alkanes peroxidation results peroxide, hydrogen acid, etc.) produced
[2, 3]. At sites of inflammation, lipid when activated oxygen species (e.g. , superoxide, hydroxyl radical, hypochlorous by phagocytes attack polyunsaturated
fatty acids in the nearby membranes [2, 4]. Lipid peroxidation appears to contribute to a number ofdisturbances in structure and function, some of which may augment ability of the phagocyte to kill invading microbes. These turbances include alterations in cellular morphology [5], cium homeostasis [6], ATP levels [7, 8], and glutathione
cell the discal-
levels
and
[9],
as
well
as
inhibition
of
enzyme
activity
[10]
DNA damage [11]. Although the free radical generating systems on the phagocyte are designed to restrict oxidant production to the region where the pathogen is located [12], reactive oxidants tend to leak into the surrounding areas where they have the capacity to inflict tissue damage at sites of inflammation [13]. Free radical-mediated tissue damage appears to contribute to many inflammatory diseases, including rheumatoid arthritis [14, 15], cancer [16], atherosclerosis [17], and adult respiratory distress syndrome [18, 19]. The most abundant polyunsaturated fatty acids in most cells are in the w-6 family (i.e., linoleic and arachidonic acids). One of the major lipid peroxidation products generated by free radical attack on w-6 polyunsaturated fatty acids is 4-hydroxynonenal (4-HNE) [2-4], and 4-HNE has been detected in micromolar concentrations in tissue exudates isolated from early stages of both experimental [20] and spontaneous [21] inflammation models. 4-HNE exhibits a number of biological effects, including chemotactic activity toward neutrophils in vitro and in vivo [22-24], modification of low-density lipoprotein [25, 26], inhibition of enzymes [10], and inhibition of DNA and protein synthesis [8]. In many cases, the effects of 4-HNE are thought to be due to the ability of 4-HNE to react with sulfhydryl groups [3, 4,
27] or with 28]. Studies
lysine c-amino groups on target by Steinbrecher et al. [29] and have identified 4-HNE-protein conjugates density lipoprotein in vitro and more recent et al. [26] and Yl#{228}-Herttuala et al. [28] has 4-HNE-protein conjugates in vivo. Free radicals are produced by neutrophils lated by a variety of soluble and particulate ing opsonized bacteria [31, 32]. Bacteria are
proteins [25, 26, Hoff et al. [30] in modified lowwork by Palinski identified such when stimustimuli, includphagocytosed by
INTRODUCTION Abbreviations:
Free which variety
radical-mediated unsaturated of products
lipid peroxidation lipids become oxidatively [1], including ketones,
is
a process in degraded to a esters, aldehydes,
CGD, Reprint tana
State
Received
Journal
4-HNE.
chronic
4-hydroxynonenal:
granulomatous
requests:
Mark
University,
July
disease; T.
Quinn,
Bozeman,
22,
of Leukocyte
1994;
accepted
Biology
MT
BSA,
NBT,
bovine
nitroblue
Department
serum
of
Microbiology,
59717. November
Volume
1,
57,
albumin;
tetrazolium.
1994.
March
1995
Mon-
the neutrophil and become sequestered in the phagosome where the neutrophil can deliver a concentrated burst of oxidants and lysosomal enzymes to destroy the microorganism [12, 33, 34]. One of the primary activated oxygen species produced in the phagosome is superoxide anion (O2). 02 is generated by a multicomponent enzyme complex that assembles on the phagosomal membrane [35, 36]. This system, known as the NADPH oxidase, is composed of a number of membrane and cytosolic proteins, including a flavocytochrome b that is thought to be the terminal donor ofelectrons to oxygen [37, 38]. Previously, we demonstrated that flavocytochrome b and an associated small GTP-binding protein (RaplA), were localized to the phagosomal membrane in phagocytosing neutrophils [39, 40], where the assembled NADPH oxidase could generate oxidants against engulfed bacteria. Thus, one would expect that 4-HNE-protein adducts would be present in fairly high concentrations in the phagosome and, therefore, could serve as markers of membrane lipid peroxidation. 4-HNE would bacterial and
Additionally, high local concentrations result in the modification and inactivation phagosomal proteins. However, it is unknown
of of
(n = 5), and the son’s neutrophils were 0% NBT positive (n = 3). Neutrophils from the son also failed to produce H2O2, as measured by a scopoletin/horseradish peroxidase assay [45], confirming the complete absence of NADPH oxidase activity in the son’s cells. In addition, Western blots of membranes prepared from the son’s cells confirmed the absence of flavocytochrome b558 (data not shown). Neutrophil phagocytosis of heat-killed Staphylococcus aureus was performed as previously described [39, 40]. Briefly, purified cells ( 107/ml) and boiled S. aureus ( 109/ml) were combined in fresh autologous serum and immediately allocated (5 l) onto thermonox cover slips. These aliquots were then held at 37#{176}C(5% CO2) for 15 mm before cryofixation as described below. Neutrophils in serum alone and S. aureus in serum alone were also cryofixed as controls and showed no labeling above background levels (data not shown).
Anti-4-HNE-protein
adduct
antibody
preparation
Purified 4-HNE was incubated with KLH (Sigma, St. Louis, MO) to generate KLH-HNE adducts following the procedure of Curzio et al. [46]. These adducts were injected into rabbits according to standard [47] procedures to generate rabbit anti-4-HNE-KLH adduct antibodies.
to what extent neutrophil generated oxidants do act locally and whether the neutrophil membranes are also themselves damaged. To examine these possibilities, we produced antibodies specific for 4-HNE-protein adducts and used immunoelectron microscopy to analyze resting and phagocytosing human neutrophils for sites of lipid peroxidation and protein damage. We found that the staining of 4-HNE correlated with the expression of flavocytochrome b in normal neutrophils and in neutrophils exhibiting X-linked heterozygous and homozygous flavocytochrome b-deficient chronic granulomatous disease (CGD), which is a disease in which neutrophils cannot produce O2 because of a defect in gp9l-phox (flavocytochrome b large subunit) [41]. These results confirm the absence of flavocytochrome b as the genetic defect in this family and show that protein adducts of the lipid peroxidation product 4-HNE can provide a subcellular marker for the oxidative injury of membranes that results from the inflammatory response of activated human neutrophils.
20#{176}Cusing 9% polyacrylamide slab gels as described previously [47], and Western blotting was performed as described previously [48]. Transfers were blotted with primary antibody for 3 h at 20#{176}C,followed by alkaline phosphatase conjugated goat anti-rabbit IgG secondary antibody (BioRad, Richmond, CA) for 1 h at 20#{176}C,and developed using an alkaline phosphatase development kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
MATERIALS
ded
AND METHODS
and Western
SDS-polyacrylamide
gel
Chemicals and reagents used in these studies were of the highest grade commercially available. 4-hydroxy-2,3-transnonenal (4-HNE) was kindly provided by Dr. Hermann Esterbauer (Institute of Medical Biochemistry, University of Graz, Austria). Affinity purified rabbit antibody to a peptide from gp9l-phox (residues 546-58) has been previously described [42] and used to immunolocalize cytochrome b in cryofixed neutrophils 39, 40].
electrophoresis
was
carried
out
at
Purified neutrophils, either resting or phagocytosing, were cryofixed by slam-freezing as described previously using a LifeCell CF-100 (LifeCell Corp., The Woodlands, TX) and stored under liquid N2 until dried [39, 40]. Cryofixed cells were dried using molecular distillation drying and embedin resin
for
sectioning
as described
previously
[39,
40].
microscopy
Grid-mounted sections were either single-labeled with specific antibodies or double-labeled as described previously [39, 40] by labeling on one side with anti-flavocytochrome b antibody (1:100 dilution overnight at 4#{176}C)followed by protein A conjugated with colloidal gold (20 nm) and then labeling the other side ofthe section with anti-4-HNE-protein adduct antibody (1:100 dilution overnight at 4#{176}C)followed by biotinylated goat anti-rabbit IgG and streptavidinconjugated ously [39,
preparations
blotting
Cryofixation
Immunoelectron
Materials
Neutrophil
Electrophoresis
colloidal 40], there
gold (10 nm). As we established is normally no cross-reactivity
previbetween
Heparinized venous blood was obtained from a male patient with X-linked recessive, cytochrome b558-negative CGD and from his mother and father, and the neutrophils were
the two confirmed periments or both
purified from heparinized peripheral blood by a FicollHypaque/dextran sedimentation/hypotonic lysis method [43]. Using a phorbol myristate acetate-stimulated nitroblue tetrazolium (NBT) slide test [44], the father’s peripheral neutrophils were 100% NBT positive (n = 1), while the mother’s peripheral neutrophils were 60 ± 2% NBT positive
tions with preimmune serum replacing one or both of the primary antibodies. In each case, there was no labeling when preimmune serum was used or when primary antibodies were omitted, and no cross-reactivity was observed between the first and second labels in the double-labeling experiments.
416
Journal
of Leukocyte
Biology
Volume
57,
March
1995
labels in these double-labeling experiments, and we that no cross-reactivity was occurring in the cxreported here by (1) processing sections with one primary antibodies omitted and (2) processing sec-
RESULTS
low
density
lipoprotein
epitopes
Antibody
on
adducts
a variety
lmmunocytochemical
dation product 4-HNE with amino or sulfhydryl neighboring proteins, we required an antibody was specific for 4-HNE-protein adducts. This prepared by conjugating 4-HNE to KLH and
groups probe antibody injecting
However,
after
these
immunization
rabbits
recognized BSA alone 4-HNE-BSA recognized
4-HNE-BSA (right blot, was also specifically
4-HNE-KLH
with
produced
antibodies
that
adducts (right blot, lane B). A small amount present in this sample, by the anti-4-HNE-protein
determine
To
on that was the
KLH-HNE adduct into rabbits as described in Materials and Methods. To test the specificity of this antibody, we prepared 4-HNE adducts of bovine serum albumin (BSA) and analyzed the ability of the antibody to recognize this 4-HNE-protein adduct using Western blotting. As shown in Figure 1, preimmune rabbit serum did not recognize native BSA (left blot, lane B) or its 4-HNE adduct (left blot, lane ducts,
ad-
lane A) but not of aggregated and it too was adduct
the
ultrastructural
localization
conjugated
des
as
with
described
in
As shown father actively 4-HNE-protein
difl#{233}rent
in Figure phagocytosed adducts
brane
that
antibodies
generated
against
4-HNE-
oxidase
complex,
where
bacterial
Prebleed
Immune
Serum
surface.
only
Mr
A
AB
B
and,
consequently, #{176}2
cells
obtained
43
(Fig.
labeling
labeling
(data
singlecells
ing
In
(lescrih(-cl
in
blot
) or ant
i ng
an
alkaline
)hOSphataSe used
with
1)ata
are
Nlaterials -4- H N E- K
on and
Nlethuds
gels,
representative
antI
kit the of
.
Prestat
nsolecular five
5(p44t(
gels
using
preirnilitintblot
ned
antiseruiii.
BSA
B
) (75 jig/lane
) sst-n
and
( right
phusphatase-conjugated (levelopnnni
all
#{182})polvacrvlainide
1.1-1 antiseruus
ad(luct
).
hlottetl ral)hit
Blots antibody
tuolecular
weight
blots
of the frons
( left
and
us-
alkaline
standards
standards t’\’()
seitirit (l(s’slf)((l
secondary
weights
as
are
ilnulunize(l
Quinn
were
in(licated. rabbits.
et al.
(10
nm
None
of
by
peaking
at labeling
duct
of 11
each from
with
(panels
displayed
a
populations tions
ranges
(Fig. con-
labeling
for
mother’s dells showed of the phagosomes were only backgrou nd 4- H NE
all
both
anti-gp9l-phox
labeling
(
95 %
29
neutrophils
and
with
h
the
deficient
but
mother,
flavocytochrome
labeling
cell
cot
separated
are
oxidase
lipid
the
neutrophils not
NADPH
heavily
2d).
in
Resting
of
30-40% that
markers
labeled.
of
for
other
phagosomal
( lane
from
which bacteria
adducts
subsequent
heterozygous
3)
and
the
I-I istogramns
alone
obtained
son,
4-HNE-protein
absence
labeled
phagosomes
mixed
lISA
for
the
Fig.
were
while
both
or
neutrophils
b-deficient phagocytosed
and
from
gold)
nm
tamed
68
A)
contrast,
the
(see
2c),
wit h 4- H N E ( lane
In
production)
that
SOII1C5
(10
con)ugate(l
parti-
Double-labeling (20 nm gold) 40], showing that the the rest of the NADPH on the phagosomal mem02 in close apposition to the
localized
labeling
(i.e.,
60-70%
98
of anti-4-bydroxynonenal-protein
gold
membranes. antibodies
generate
cytochrome killing, had
background
ofcolloidal
gold) and gp9l-pha (20 nm gold; Fig. 2b, top and botu)m panels). The lack of labeling with these antibodies is consistent with the genetic absence of flavocytochrome b in these cells
200
1 . Specificity
was
it could
homozygous, in bacterial
sizes
and Methods. 2a, neutrophils from the normal bacteria, and heavy labeling of (10 nm gold) was O1)ser’.’ed on both
[261
found
4-HNE-
Materials
the phagosomal and bacterial the sections with anti-gp9l-phox confirmed our previous results flavocytochrome, and presumably
who
of
protein adducts in resting and phagocytosing neutrophils, immunocytochemical studies were performed on neutrophils obtained from three members of a family with demonstrated X-linked recessive CGD [41]. These members included the normal father, heterozygous mother, and homozygous, cytochrome b553-deficient son who manifested the full symptoms of X-linked CGD [41]. Purified neutrophils were allowed to phagocytose heat-killed Staphylococcus aureus and then prepared for sectioning by cryofixation and molecular distillation drying as described. Thin sections of these cells were then double-labeled with anti-4-HNE-protein adduct antibodies and anti-flavocytochrome b (i.e., anti-gp9l-phox) antibodies
specifically
localization
antiserum (see higher Mr band on the right blot, lane A) and not by preimmune serum. Thus, the antibody is specific for 4-HNE-protein adducts and would be expected to recognize them on a number ofdifferent proteins. The reactivity of this type of antibody is consistent with studies by Palinski et al.
Fig.
4-UNE-lysine
proteins.
specificity
To analyze possible subcellular localizations of aldehydeprotein adducts generated by conjugation of the lipid peroxi-
A).
recognized
of different
mother’s with
frequency and
limits
the
heavily
mentioned
phagosomes
cells two
cell
distribulabeled
above,
417
Fig.
2.
Itiiinunocytuchemical
phagocvtusing with
labeled nal
71%
o1
prepared
anti-4-HNE-protein
magnifications
protein
localization
neutrophils are:
the
aclduct (a)
mother’s
adduct
of
from
x 65,000,
the
antiserum (h)
cells and
56%
positive
measure
adducts
father (10
x 85.000.
were
positive
4-hvdroxynonenal normal
nm
(c)
calculated
(panel gold)
be
the
60%
slide
test
for
that
4-HNE-protein
curate and
probes of the occurrence of lipid peroxidation that highly reactive 4-HNE generated in
phagosomes
sequent proteins
418
oxidation in the
Journal
cells.
adduct
as
determined
heterozygous
a result
of
antibodies
NADPH
of membrane bacteria-containing
of Leukocyte
Biology
the are
(d)
(panel
antiserum
b in b).
and
(20
nm
phagocytosing heterozygous gold)
human
neutrophils.
mother
(panels
as described
in Materials
Thin c and and
sections
d) were
of
double-
Methods.
Origi-
x 40,000.
DISCUSSION
4-HNEthe
results
useful
57,
son
anti-gp9l-p/tox
NBT show
and
ac-
in vivo neutrophil oxidase activity and sublipids forms adducts with phagosome.
Volume
flavocytochrome
agreeing
using
Thus,
and homozygous
and
positive,
with
the
and
x 65,000.
to
gp9l-phox
a),
March
1995
Chronic granulomatous disease is a clear demonstration that the generation of superoxide anion and its associated toxic oxygen metabolites is required for optimal neutrophil microbicidal function. Lipid peroxidation has been shown to occur
during
studies nature main
by Shohet, Stossel, and others of the lipid peroxides formed unknown. Our studies suggest
killing
of
phagocytosed
pneumococci
in
[49, 50]; however, in the phagosome that toxic lipid
early
the realde-
16 14 12
10 8 6 4 2 0 16 14 U)
12
0
0
10
8
E
z
6
4 2 0
F
16 14 12 10. 8. 6. 4.
111iJli11.
2
0
10
5
15
20
25
3.
the
Distribution
anti-4-HNE for
of 4-HNE-protein
homozygous each
hydes
son
adduct donor,
(e.g.,
and
(panels
antiserum the
4-HNE)
adduct
A and
(panels
number
ofgrains
accumulate
D),
and
Havocytochrome
heterozygous
A-C)
and
present
in the
mother
B and
antiserum
phagosome
surface
0
b labeling (panels
anti-gp9l-phox per
30
were
of
counted
phagocy-
ci
1
in
5
4
3
2
phagocytosing
E),
and
(panels
tosed bacteria. 4-HNE is a major product of lipid peroxidation [2-4] that can contribute to the bacterial killing process through its ability to inactivate important proteins [7, 10, 51], alter cell morphology [3, 4, 52], inhibit DNA synthesis [8], and act as a chemoattractant to recruit more neutrophils [22-24, 52]. There is increasing evidence that toxic oxygen species are causally involved in the pathogenesis of many diseases [53] and also contribute to aging itself [54,
Quinn
I
6
per Phagosome
Grains Fig.
I.IIII.IIIIIIMIIIL
hi
0
al
1)-F)
kr
father
One
target such
4-HNE has
of attack
and,
injury
in
and
Detection
the
of
present
lipid
neutrophils
single-
Methods.
and
40-69
radicals
fatty
acids
indicator
that
4-HNE
readily
would
serve
as
on
microorganisms.
studies
we
support
in
neutrophil
peroxidation
priparirl
reacts
from
double-labeled
cells
with analyzed
were
staining
free
that 4-HNE-protein concentrations at sites or
or
anti-gp9l-phox
these
to be a reliable
therefore, tissues
and
for
of
were
polyunsaturated
occurred,
ation
F)
adduct
as
appears
sections
and
in Materials
we hypothesized present in higher
In
C
anti-4-HNE-protein
lipids non
Thin
(panels
as described
boils
1
neutrophils.
normal
in each
cell.
is membrane [2].
Because
lipid
peroxida-
with
proteins,
adducts would be of free radical gener-
a marker this
for
oxidative
hypothesis
phagosomes
by
419
demonstrating
that
anti-4-HNE-protein
adduct
ACKNOWLEDGMENTS
antibodies
can be used to detect 4-HNE-protein adducts that are intensely concentrated at sites of oxidant production in neutrophil phagosomes. In phagosomes where NADPH oxidase activity is absent due to a genetic defect, i.e., in neutrophils from a donor with X-linked CGD, 4-HNE-protein adducts are also absent. These results are supported by studies showing that formation of 4-HNE by phagocytosing neutrophils in vitro correlates directly with the rate of superoxide formation [21]. In previous studies, 4-HNE has been reported to be an indicator of lipid peroxidation in vivo [2-4]. Elevated 4-H NE levels have been measured in tissues from vitamin Edeficient rats [56, 57], in livers from bromobenzenepoisoned mice [58], in retinas from dogs with neuronal retinal ceroidosis [59], in tissues from patients with post-surgical trauma [21], and in brains from normal mice compared to transgenic mice carrying the human superoxide dismutase gene [60]. In all of these studies, 4-HNE was measured by extraction from tissues using organic solvents, followed by mass spectrometry 2,4-dinitrophenyihydrazones chromatography and phy [3]. The above
[57]
or
by derivitization to form and analysis with thin layer high-performance liquid chromatograanalyses cannot detect 4-HNE con-
jugated to protein sulffiydryl groups, which may be among the most toxic 4-HNE products [56]. In the studies presented here, we report a method that has the ability to detect 4-HNE-protein adducts and which can be used to identify the presence 4-HNE-protein adducts in situ so that the relative distributions oflipid peroxidation markers can be visualized in intact cells and ultimately in tissues. We report the immunocytochemical detection of 4-HNEprotein adducts in phagosomes of human neutrophils; however, the protein or proteins that are conjugated with 4-HNE have not yet been identified. Because 4-HNE can react readily with thiol or free c-amino groups on proteins, it seems likely that a number of bacterial and phagosomal proteins would form 4-HNE-protein adducts, some which may play important functions in the cell [3, 4]. As summarized in the Introduction, 4-HNE has been shown to have many biological effects, and presumably these effects are mediated through the modification and/or inactivation of a number of important proteins in the cell. One target protein that is sensitive to sulfhydryl modification by 4-HNE is the calcium ATPase [10]. Inhibition of Ca2-ATPase function results in increased intracellular calcium levels and eventually cell death [10]. Another group of proteins that would be especially sensitive to sulfhydryl modification are GTP binding proteins. GTP binding proteins play an important role in neutrophil signal transduction [61], including two low molecular weight GTP binding proteins, RaplA and Racl/2, that are associated with the NADPH oxidase system [42, 62, 63], and recent studies by Rossi and coworkers [52] implicated the GTP binding protein involved in phospholipase C activation as a target of 4-HNE attack in rat neutrophils. The results shown here demonstrate that 4-HNE production is correlated with the presence of functional flavocytochrome b (and 02 production) in human neutrophils and confirm that CGD neutrophils are unable to mediate the formation of lipid peroxides. These results are consistent with earlier studies showing lipid peroxidation during bacterial phagocytosis and its absence in cells from patients with CGD [49, 50] and begin of the lipid peroxidation in neutrophil lipid peroxidation species involved.
to define phagosomes
420
Volume
Journal
of Leukocyte
Biology
the
locatton and the
We
thank
Grant
March
1995
Hermann
Esterbauer
This
2198R
from
rk
the
for
was
his
supported
Council
for
National Institutes of Health and FIRST Award AR40929 and an Arthritis Foundation cal Science Grant (M.TQ).
generous
in part
Tobacco
Research (M.TQ), Investigator
gift
of
by Research
Research
(A.J.J.),
Grant A126711 (Aj.J.) a USDA Award (E.A.D.), Award and Biomedi-
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