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Articles |
1
Research and Development and Manufacturing Department for Diagnostics, Diagnostic Science Division, Shionogi & Co., Ltd., 2-5-1, Mishima, Settsu, Osaka 566-0022, Japan.
2
First Department of Internal Medicine, Miyazaki Medical
College, 5200, Kihara, Kiyotake, Miyazaki 889-1601, Japan.
3
National Cardiovascular Center Research Institute,
5-7-1, Fujishirodai, Suita, Osaka 565-0873, Japan.
a Address correspondence to this author at: R & D Manufacturing Department for Diagnostics, Diagnostic Science Division, Shionogi & Co., Ltd., 2-5-1, Mishima, Settsu, Osaka 566-0022, Japan. Fax 81 6 6319 4109; e-mail hideki.ohta{at}shionogi.co.jp.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Development of the RIA revealed that a considerable amount of AM was present in several types of nondiseased human tissues, among which it was especially abundant in the adrenal medulla, lungs, kidneys, and cardiac atrium and ventricle (4). Cultured endothelial cells and vascular smooth muscle cells actively produced AM, and the AM gene was also expressed in intact rat aorta (5)(6). In addition, it was reported that AM circulates in the blood (7), and its concentration was increased in patients with essential hypertension (7), heart failure (8)(9), renal failure (10), and sepsis (11) when compared with healthy subjects. It was conjectured that AM is synthesized as AM-Gly, which is an intermediate that has a glycine residue attached to the C terminus, and that the C terminus is amidated enzymatically for conversion into a biologically active form, hereafter called "mature-type" AM (m-AM) (3)(12). Recently, m-AM and AM-Gly have been reported to be present in plasma (12). However, competitive RIAs, which were used in previous studies, cannot distinguish between m-AM and AM-Gly, include an extraction step, which is tedious and time-consuming, and require as much as 1–5 mL of plasma sample (7)(8)(9)(10)(11)(12)(13)(14).
Here, we describe the establishment of a simple one-step two-site IRMA specific for m-AM, with two kinds of monoclonal antibodies, and the development of an assay kit, which does not require extraction and which is rapid and precise enough for routine determination of m-AM in human plasma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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peptide synthesis
Human AM fragments AM(12-25), AM(46-52)NH2,
AM(46-52), AM(38-52), and AM(30-41) were synthesized using a solid
phase method with a peptide synthesizer (431A; Applied Biosystems) and
using 4-hydroxymethyl-phenoxymethyl-copolystyrene–1% divinylbenzene
resin as a starting material according to the strategy using
9-fluorenylmethoxycarbonyl. All coupling reactions were carried
out by the dicyclohexylcarbodiimide-1-hydroxybenzotriazole method, with
1-methyl-2-pyrrolidone as the solvent. After the chain
elongation was completed, the protected peptide resins (1.06–1.28 g)
were cleaved with a mixture containing 10 mL of trifluoroacetic acid
(TFA), 750 mg of phenol, 500 µL of thioanisole, 500 µL of
H2O, and 250 µL of 1,2-ethanedithiol for
3.5–13 h at room temperature. The crude products were extracted by
washing with TFA. After the volatile materials were removed under
reduced pressure, ethyl acetate was added to the crude
residue. The resulting precipitate was washed with ethyl acetate to
remove trace amounts of scavengers. For disulfide bond formation of
AM(12-25), the peptide was first reduced with dithiothreitol;
the disulfide bridge was then formed by means of air oxidation, with
the conditions set for a sample concentration of 0.5 g/L in 0.1 mol/L
NH4OAc solution (pH 8.1) for 18 h at room
temperature. The crude peptides were purified by reversed-phase
HPLC on a YMC-GEL ODS-AM 120-S50 column (4 x 25 cm) with a
mixture of H2O and acetonitrile as solvent
(containing 1 mL/L TFA) systems at a flow rate of 1 mL/min. The
concentrations of acetonitrile on purification of AM(12-25), AM(46-52),
AM(46-52), AM(38-52) and AM(30-41) by reversed-phase HPLC were 180,
120, 140, 180, and 170 mL/L, respectively. The amino acid
composition of the synthetic peptides was determined by amino acid
analysis with an Hitachi L-8500 amino acid analyzer after hydrolysis in
6 mol/L HCl containing trace amounts of phenol at 110 °C for 20
h.
conjugation of haptens with bovine thyroglobulin
Human AM fragments [AM(12-25) and
AM(46-52)NH2] were allowed to react with bovine
thyroglobulin and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride in 0.05 mol/L sodium phosphate buffer (pH 7.5)
solution for 4 h at room temperature. The resulting reaction
mixtures were dialyzed against water (4 x 1000 mL) at 4 °C
overnight. Lyophilization of the solutions yielded the conjugate of AM
fragments as a white fluffy powder. The molar ratio of the hapten
(total binding) to the protein in the conjugate was determined by amino
acid analysis. The molar ratios of AM(12-25) and
AM(46-52)NH2 were 93 and 42, respectively.
monoclonal anti-am(12-25) IgG1(
) AdR-1 for
biotinylation and monoclonal anti=am(46-52)NH2IgG1() AdC-1 for iodination
BALB/c mice were immunized five times with bovine
thyroglobulin-conjugated AM(12-25) or
AM(46-52)NH2. Hybridomas were prepared by fusion
of mouse myeloma cells (X63-Ag8.653) with spleen cells from the
immunized mice, using 500 g/L polyethylene glycol 4000, and
cloned by the limiting dilution technique. The resulting monoclonal
antibodies, AdR-1 and AdC-1, recognized the ring structure and the
amidated C-terminal structure of AM, respectively. These two antibodies
belonged to the IgG1 subclass and were prepared from ascites by use of
Protein A (Bio-Rad Laboratories).
preparation of anti-biotin antibody
Solid sodium sulfate
(Na2SO4; 0.36
g) was added to 2 mL of anti-biotin antiserum and dissolved. After
standing for 10 min at room temperature, the mixture was centrifuged at
10 000g for 20 min. The precipitate was dissolved in 0.8 mL
of 17.5 mmol/L sodium phosphate buffer, pH 6.3, and dialyzed against
300 mL of the above buffer. The dialyzed sample was applied to a
DEAE-cellulose column (DE52, 2 mL; Whatman International Ltd.)
equilibrated with the above buffer and washed with the same buffer. The
pass-through fractions were pooled, and the concentration was
determined.
anti-biotin antibody-coated polystyrene beads
Polystyrene beads (6.5 mm diameter; Immuno Chemical Inc.) were
incubated in 50 mmol/L phosphate buffer, pH 7.1 (buffer A), containing
15 mg/L of anti-biotin antibody for 20 h at 25 °C and for
3 h at 4 °C. The antibody solution was then removed from the
beads, which were washed three times with buffer A. The remaining
protein-binding sites on the beads were blocked by incubation with
buffer A containing 250 mL/L Block Ace (Dainippon Pharmaceutical
Co., Ltd., ) overnight at 4 °C. After aspiration, the beads were
stored in buffer B (0.1 mol/L phosphate buffer, pH 6.8, containing 0.3
mol/L NaCl, 1 mmol/L EDTA, 0.2 mmol/L cystine, 1 g/L bovine
serum albumin, and 1 g/L NaN3) at
4 °C.
biotinylation of AdR-1
IgG (AdR-1) was digested with pepsin (IgG:pepsin ratio = 1
mg:0.05 mg) for 3 h at 37 °C in 100 mmol/L citrate buffer (pH
4.1) containing 100 mmol/L NaCl and F(ab')2 was
purified on a Sephadex G-150 column (1.5 x 100 cm; Amersham
Pharmacia Biotech) from undigested IgG or other digested peptides. Fab'
was then prepared by reduction with 10 mmol/L 2-mercaptoethylamine in
0.1 mol/L sodium phosphate buffer, pH 6.0, containing 5 mmol/L EDTA,
followed by purification on a Sephadex G-25 column (1 x 47 cm,
Amersham Pharmacia Biotech) in the same buffer. The resulting Fab' was
biotinylated in the above buffer by incubation with a 10-fold molar
excess of 3-(N-maleimidylpropionyl)biocytin (Molecular
Probes, Inc.) in dimethyl sulfoxide, and the free
3-(N-maleimidylpropionyl)biocytin was removed on a Sephadex
G-25 column (1 x 47 cm) in 0.1 mol/L sodium phosphate buffer, pH
7.4, containing 0.15 mol/L NaCl. The biotinyl-Fab' fractions were
pooled and diluted with buffer C (50 mmol/L phosphate buffer, pH 7.4,
containing 80 mmol/L NaCl, 5 mmol/L EDTA, 0.5g/L Triton X-100, 2.5 g/L
bovine serum albumin, and 0.5 g/L NaN3)
to give a final concentration 22 pmol/L; the pooled fractions were
stored in aliquots at -40 °C.
synthesis of
4-(n-maleimidomethyl)cyclohexanecarboxylic
acid[2-(4-hydroxyphenyl)ethyl]amide
Tyramine (0.74 g, 5.4 mmol) was reacted with succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (1.8 g, 5.4
mmol) and 1-hydroxybenzotriazole (0.82 g, 5.4 mmol) in
dimethylformamide (36 mL) at room temperature overnight. The solvent
was evaporated, and then the residue was dissolved in ethyl acetate,
washed with 0.5 mol/L hydrochloric acid and distilled water, and dried
over magnesium sulfate. The solvent was evaporated, and the residue was
crystallized from a mixture of methylene chloride and methanol to yield
4-(N-maleimidomethyl)cyclohexanecarboxylic acid
[2-(4-hydroxyphenyl)ethyl]amide (1.31 g; yield, 68%; decomposition
at 213.5–215 °C). We called this reagent as maleimido-tyramine.
iodination of AdC-1
4-(N-maleimidomethyl)cyclohexanecarboxylic acid
[2-(4-hydroxyphenyl)ethyl]amide (37.5 µg) was first iodinated with
Na125I (370 MBq) and chloramine T (10 µg) in
0.2 mol/L phosphate buffer, pH 7.0. The reaction mixture was
fractionated by reversed-phase HPLC on a Nucleosil
C18 column (4.6 x 300 mm; Chemco Scientific
Co.) with acetonitrile–methanol–1 mL/L trifluoroacetic acid (3:2:5,
by volume) at a flow rate of 1 mL/min to give the desired compound,
iodinated 4-(N-maleimidomethyl)cyclohexanecarboxylic
acid[2-(4-hydroxyphenyl)ethyl]amide (296 MBq,
125I-maleimido-tyramine), which eluted from the
column at 15 min.
Anti-AM(46-52)NH2 Fab' (196.4 µg, 4.27 nmol) prepared by pepsin digestion and reduction as described above from IgG (AdC-1) was incubated with 125I-maleimido-tyramine (296 MBq, 4.27 nmol) at 25 °C for 1 h. To separate the labeled Fab' from unreacted 125I-maleimido-tyramine, the reaction mixture was gel-filtered on an Ampure SA column (Amersham Pharmacia Biotech) in 0.1 mol/L sodium phosphate buffer, pH 6.5, containing 0.15 mol/L NaCl, followed by further purification on phenyl-Sepharose CL-4B column (0.5 x 3.5 cm, Amersham Pharmacia Biotech) in the above buffer. The flow-through fractions of labeled antibody were pooled and diluted with buffer C to give a final concentration of 210 kBq/5 mL; the pooled fractions were store in 5-mL aliquots in vials.
irma for human m-AM
Before being assayed, 0.5 mL of biotinylated anti-AM(12-25) Fab'
was added to 5 mL of iodinated anti-AM(46-52)NH2
Fab' (final concentration of biotinylated anti-AM(12-25) Fab' and
iodinated anti-AM(46-52)NH2 Fab' were 2 pmol/L
and 2 000 000 cpm/mL, respectively). Calibrator solutions of human AM
at concentrations of 0–300 pmol/L were prepared with buffer C
containing 20 g/L human serum albumin.
In the typical assay procedure, 200 µL each of calibrators or plasma samples was placed in polystyrene tubes. The mixture of labeled antibodies (100 µL, 200 000 cpm/tube) and one bead were added (total volume, 300 µL), and the mixture was incubated at 4 °C for 20 h. After removal of the incubation mixture, the beads were washed twice with 2 mL of distilled water, and then the radioactivities were measured with a gamma counter (ARC-600, Aloka Co., Ltd.). Experiments were performed in duplicate except where noted otherwise. Human m-AM concentrations were expressed as m-AM-like immunoreactivity.
plasma samples
Blood samples were withdrawn into plastic syringes and quickly
transferred to chilled tubes containing EDTA (1.5 g/L blood) and
aprotinin (50 000 kIU/L blood), and then centrifuged at
1600g at 4 °C for 20 min. The plasma samples thus
obtained were kept frozen below -20 °C until determination. Human
sample acquisition was conducted in accordance with the policies and
procedures of the Institutional Review Board for the use of human
subjects in research at the Diagnostic Science Division, Shionogi &
Co., Ltd.
characterization of am in human plasma
Plasma from the healthy subject (10 mL) was loaded onto a Sep-Pak
C18 cartridge (Millipore-Waters) equilibrated
with 5 mL of saline. After the cartridge was washed with 5 mL of saline
and 100 mL/L acetonitrile in 1 mL/L TFA, the adsorbed materials
were eluted with 4 mL of 600 mL/L acetonitrile in 1 mL/L TFA and
lyophilized. The plasma extract was dissolved in 100 mL/L acetonitrile
in 1 mL/L TFA and was analyzed by a reversed-phase HPLC using a TSK ODS
120A column (4.6 x 150 mm; Tosoh). Each fraction was lyophilized
and dissolved with 350 µL of buffer C containing 20 g/L human serum
albumin, followed by measurement of the m-AM concentration by the IRMA
for m-AM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the characterization of these antibodies. The association
constants were 3.2 x 1011 and 7.4 x
1010 L/mol, respectively, and clearly showed that AdR-1 and
AdC-1 were highly specific for the ring structure and the amidated
C-terminal structure of AM, respectively.
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development of irma for m-AM
We developed a two-site IRMA for human m-AM with two kinds of
monoclonal antibodies. Fig. 1
shows the principle of this IRMA for human m-AM. One of the
monoclonal antibodies, which was biotinylated, recognizes the ring
structure and binds to the rabbit anti-biotin antibody immobilized onto
polystyrene beads, and the other, which recognizes the amidated
C-terminal structure, is radioiodinated by the Hinge procedure.
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analytical evaluation
Calibration curve and sensitivity.
Calibration
curves were prepared for the standard assay system, using synthetic
human AM(1–52)NH2 as a calibrator. Fig. 2
shows a typical calibration curve of human AM, assayed ~1
month after the iodination of AdC-1. The lower detection limit
of this IRMA, defined as the concentration at the mean ± 2 SD of
10 determinations of the zero calibrator, was 0.5 pmol/L, and the
working range (CV <15%) was 1–300 pmol/L.
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Precision.
The reproducibility of our IRMA was estimated using
three plasma samples having different AM concentrations. The within and
between-day CVs were 4.4–8.2% (n = 10) and 5.5–8.3% (n =
12), respectively, as shown in Table 2
.
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Dilution and recovery tests.
The effect of dilution of the
plasma samples was investigated with three samples. As shown in Fig. 3
, the calculated AM values were linear with dilution. Recoveries
of exogenously added AM from plasma samples containing three different
concentrations of endogenous AM were estimated. The recovery ranged
from 91% to 118%, as shown in Table 3
.
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Interferences and cross-reactivity.
We examined the influence
of bilirubin (0.41 g/L), total lipids (1.76 g/L), and hemoglobin (1
g/L). These components of human plasma had no influence on the assay
under the conditions described in Materials and Methods.
We also examined the cross-reactivity with peptides (fragments) derived
from AM, AM-Gly, and other peptide hormones that were related to AM or
similar to AM. As shown in Fig. 4
, the assay was not cross-reactive with AM-Gly. In addition, no
cross-reaction was observed with partial peptide fragments of AM, such
as AM(12-25), AM(30-41), AM(38-52), AM(46-52), and
AM(46-52)NH2 (Fig. 4A
). Moreover, this IRMA had no
cross-reactivities with pro-AM N-terminal 20 peptide, amylin, CGRP,
calcitonin, and neuropeptide Y (Fig. 4B
). The cross-reactivity with rat
AM was <1.2% on a molar basis.
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AM concentration in plasma.
The m-AM concentration in plasma
obtained from 61 healthy volunteers was 1.18 ± 0.65 pmol/L
(mean ± SD; Table 4
). Table 4
(experiment 2) shows the plasma concentrations of
m-AM in patients with heart failure when they were classified according
to the New York Heart Association (NYHA) functional class and with
chronic renal failure (CRF). The plasma m-AM concentration in the NYHA
I or II groups was significantly higher than in the control group
(P <0.02), and the concentration in the NYHA III or IV
groups was significantly higher than in the NYHA I or II groups
(P <0.001). Similarly, the plasma concentration in the
group with CRF was significantly higher than in the control group
(P <0.001).
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characterization of plasma am
The elution profile by reversed-phase HPLC of a plasma extract
from a healthy subject is shown in Fig. 5
. One major peak emerged at an elution position identical to
that of authentic human AM(1–52)NH2.
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stability of am in edta plasma containing aprotinin
Both high and low concentrations of exogenously added AM in EDTA
plasma containing aprotinin was stable at least for 2 months at
-20 °C (Fig. 6
B). When stored at 4 °C, the AM concentration remained mostly
unchanged for 24 h (>90%); however, under more prolonged
storage, the AM concentration decreased gradually (Fig. 6A
). When
stored at 25 or 37 °C, AM was not stable and the immunoreactivity of
AM decreased to ~40–50% at 2 days (Fig. 6A
). Five freeze-thaw
cycles had no effect on plasma AM (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, we showed a typical
calibration curve for AM, which was assayed ~1 month after iodination
of Fab'. Nevertheless, at 1 pmol/L, the CV is <15%. Moreover,
the recovery of AM added to human plasma and the reproducibility of the
assay gave satisfactory results.
In the present study, we were able to specifically measure the
concentration of biologically active AM, m-AM, in the plasma of healthy
volunteers by one-step direct assay without extraction for the first
time. Unexpectedly, the concentration of m-AM was lower (1.18 ±
0.65 pmol/L) than any "AM" concentration measured previously by
RIAs (5.05 ± 0.21 pmol/L) (13). In addition, HPLC of a
plasma extract from a healthy subject showed that our IRMA was specific
for m-AM. With previously reported RIA systems, m-AM and AM-Gly could
not be distinguished, and therefore, the values of the AM concentration
measured by these RIAs may represent the sum of m-AM and AM-Gly. Our
results indicate that the m-AM concentration is very low in circulating
blood, suggesting that there is much more biologically inactive AM-Gly
than m-AM. This agrees with the findings of Kitamura et al.
(12) that the immunoreactivity of m-AM in plasma treated
with peptidylglycine 
-amidation enzyme was approximately fourfold
higher than before treatment. They concluded that the major form of AM
circulating in blood was biologically inactive AM-Gly.
Lewis et al. (14) recently reported that the immunoreactivity of exogenous AM added to plasma decreased up to 70% over four freeze-thaw cycles, whereas endogenous AM was stable when measured by RIA. In our study, exogenous AM was stable over five freeze-thaw cycles when measured with our IRMA. The difference between the RIA and IRMA results may have arisen because the RIA is likely to be influenced by plasma components and the recovery of AM is not complete in the extraction step.
As described above, the AM concentration in most samples from healthy
subjects was ~1 pmol/L. At this point, the CVs for our IRMA
were rather high; however, the m-AM concentrations in plasma from
patients with heart failure, CRF (Table 4
), and sepsis (unpublished
data) were much higher than that of the healthy subject, which probably
does not pose a serious problem for the clinical study of AM. Our
observations are compatible with previous reports that used competitive
RIA (7)(8)(9)(11)(13). In this study,
it is most important that we could first detect plasma m-AM without
extraction. However, more detailed data are needed to clarify the
relationship between plasma AM and the diseases described above.
We conclude that the major advantages of our IRMA method for m-AM can be summarized as follows: (a) it can simply and specifically detect biologically active AM; (b) it needs only a small amount of plasma sample; (c) it is suitable for measurement of large numbers of samples; and (d) it should be useful for physiological and clinical studies of AM.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Biochem Biophys Res Commun 1994;203:719-726.
[ISI][Medline]
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),
rat AM (
), AM-Gly (
), AM(12-25) (
), AM(30-41) (
), AM(38-52)
(
), AM(46-52) (
), and AM(46-52)NH2,(
).
(B), cross-reactivity was examined with AM (
