From the Departments of Medicine and
¶ Pharmacology and the
Center for Molecular Genetics,
University of California at San Diego, La Jolla, California 92093 and
the § Department of Immunology, The Scripps Research
Institute, La Jolla, California 92121
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ABSTRACT |
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p38 mitogen-activated protein (MAP) kinase
activities were significantly increased in mouse hearts after chronic
transverse aortic constriction, coincident with the onset of
ventricular hypertrophy. Infection of cardiomyocytes with adenoviral
vectors expressing upstream activators for the p38 kinases, activated mutants of MAP kinase kinase 3b(E) (MKK3bE) and MAP kinase kinase 6b(E)
(MKK6bE), elicited characteristic hypertrophic responses, including an
increase in cell size, enhanced sarcomeric organization, and elevated
atrial natriuretic factor expression. Overexpression of the activated
MKK3bE in cardiomyocytes also led to an increase in apoptosis.
The hypertrophic response was enhanced by co-infection of an
adenoviral vector expressing wild type p38, and was suppressed by
the p38
dominant negative mutant. In contrast, the MKK3bE-induced cell death was increased by co-infection of an adenovirus expressing wild type p38
, and was suppressed by the dominant negative p38
mutant. This provides the first evidence in any cell system for divergent physiological functions for different members of the p38 MAP
kinase family. The direct involvement of p38 pathways in cardiac
hypertrophy and apoptosis suggests a significant role for p38 signaling
in the pathophysiology of heart failure.
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INTRODUCTION |
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A variety of pathophysiological stimuli, such as myocardial infarction, hypertension, valvular diseases, viral myocarditis, and dilated cardiomyopathy can lead to an increase in cardiac workload and elevated mechanical stress on cardiomyocytes. In response to hemodynamic overload, an adaptive hypertrophic response is triggered, which is characterized by an increase in the mass and volume of individual myocytes, resulting in an increase of heart weight without an increase in the number of cardiomyocytes (reviewed in Refs. 1 and 2). During the hypertrophic response, cardiomyocytes activate a distinct pattern of gene expression that eventually results in qualitative and quantitative alterations in contractile protein content and the induction of an embryonic gene program (3, 4). As hemodynamic overload persists, the stressed heart enters a critical transition from compensatory hypertrophy to decompensated heart failure. Chamber dilatation, excitation-contraction uncoupling, abnormal interstitial morphology, sarcomeric disorganization, altered energy metabolism, and the loss of viable myocytes are common features found in end-stage failing hearts (5). Signaling molecules that transduce the signals from this extracellular stress to different cellular compartments play central roles in mediating the hypertrophic process and the transition to heart failure. Accordingly, the identification and characterization of these signaling molecules have been the focus of intense study in recent years (6).
One recently identified group of signaling molecules that mediates environmental stress responses in various cell types is the family of p38 mitogen-activated protein (MAP)1 kinases. The p38 MAP kinase activity is activated by dual phosphorylation on a Thr-Gly-Tyr motif in response to endotoxin, cytokines, physical stress (such as hyperosmolarity), and chemical stress (such as hydrogen peroxide) (7-12). In non-cardiac cells, p38 MAP kinases have been implicated in gene regulation, morphological alterations, and cell survival in response to various environmental stimuli (13-20). Recently, it has been reported that in ischemia/reperfusion-treated hearts, p38 MAP kinase activities are elevated in association with the onset of hypertrophy and programmed cell death (30, 31). In addition, p38 kinase activities are also significantly induced in transgenic mouse hearts expressing activated Ha-Ras(V12), correlating with the onset of cardiac hypertrophy.2 However, the specific function of p38 in the development of cardiac hypertrophy and cardiac cell apoptosis have not yet been directly demonstrated.
The intracellular activation cascade for p38 MAP kinases under
physiological conditions is still unclear, but several upstream MAP
kinase kinases (MKKs) have been identified from in vitro
analysis, including MKK3b and MKK6b (24-26). In the family of p38 MAP
kinases, at least four isoforms have been identified thus far (8, 12, 27-29). Two well characterized isoforms, and
, share extensive sequence similarity and a broad range of tissue distribution, including
relatively high levels in the heart (8, 27). Although different
isoforms of p38 have similar kinase activities in vitro on a
given substrate, their specific functions under in vivo
physiological conditions are largely unknown.
The present study was designed to critically assess the potential
function of the p38 pathway in the onset of features that relate to
cardiac muscle cell hypertrophy and failure. The p38 MAP kinase
activities are activated during hypertrophy following in
vivo pressure overload, suggesting their potential role in signal
transduction of mechanical stimuli. To dissect specific functions of
the p38 MAP kinases, we utilized recombinant adenoviruses to achieve
efficient expression of the p38 signaling molecules in cultured
cardiomyocytes, which allowed biochemical as well as morphological
analysis on entire cell populations. Forced activation of p38
activity results in characteristic features of hypertrophy, whereas the activation of p38
activity leads to the induction of
myocyte apoptosis. The opposing effects of the p38 MAP kinase isoforms
suggest that the activation of the p38 pathway may contribute to the
development of hypertrophy and the transition to overt heart
failure.
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EXPERIMENTAL PROCEDURES |
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Transverse Aortic Constriction Surgery-- Transverse thoracic aortic constriction was performed as described previously (32, 33) on 8-week-old adult mice (C57/BL6XSJL, Jackson Laboratories). Briefly, in the anesthetized animals, a 7-0 nylon suture ligature was tied against a 27-gauge needle at the transverse aorta to produce a 65-70% constriction following the removal of the needle. At 4 h or 7 days after surgery, animals from the experimental and sham-operated groups were killed and the hearts removed. Ventricular chambers were weighed and quickly frozen in liquid nitrogen for protein extraction.
Recombinant Adenovirus Vectors--
Recombinant adenoviruses
expressing activated MKK3bE, MKK6bE, wild type p38, wild type p38
and their corresponding dominant negative (TGY
AGF) mutants,
p38
dn and p38
dn, driven by a cytomegalovirus promoter were
generated as described previously (22, 34). Similarly, recombinant
adenoviruses expressing GFP and Ha-Ras-v were generated using cDNAs
from pEGFP (CLONTECH) and mutant Ha-Ras(V12) (35).
All recombinant adenoviruses were tested for transgene expression in
cardiac myocytes by reverse transcriptase-polymerase chain reaction,
Western blot, or kinase assays. The concentrated recombinant
adenoviruses were prepared and titered as described (34).
Cardiomyocyte Culture and Adenoviral Infection-- Neonatal cardiomyocytes were prepared using a Percoll gradient method as described previously (36). Myocytes from 1-2-day-old Sprague-Dawley rats were plated in serum-containing medium (4:1 Dulbecco's modified Eagle's medium:medium 199, 10% horse serum, 5% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10 mM glutamine) overnight. Subsequently, the cells were changed into low serum medium containing 1% horse serum and 0.5% fetal bovine serum, and infected with adenoviruses at a multiplicity of infection of 50-100 particles/cell for 12 h. The cells were then cultured in serum-free medium for an additional 36-70 h before morphological or biochemical analysis.
MAP Kinase Assays--
Protein extracts from heart or myocytes
were prepared and assayed for kinase activities, as described
previously (37). Briefly, crushed frozen heart tissue or cells were
harvested in lysis buffer (25 mM HEPES, pH 7.6, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 3 mM -glycerophosphate, 100 mM
Na3VO4, 1% Nonidet P-40, 1 mM
dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride. p38 kinases were
immunoprecipitated using rabbit polyclonal anti-p38 antiserum (from J. Han (Scripps Research Institute, La Jolla, CA) and Santa Cruz
Biotechnology, Inc.) conjugated to protein A-Sepharose. The kinase
assays were then performed at 30 °C using [
-32P]ATP
and myelin basic protein (Sigma) or glutathione
S-transferase-ATF2 as a substrate. The phosphorylated
substrate was separated by SDS-polyacrylamide gel electrophoresis, and
visualized by autoradiography. The incorporated
32Pi in the substrate was quantified by
radioanalytic scanning (AMBIS). A similar protocol was used to assay
kinase activities of ERK1 and c-Jun N-terminal kinase (JNK) using
myelin basic protein and c-Jun as substrates, as described previously
(37).
Immunohistochemical Assay--
Cells were fixed in 3.7%
formaldehyde and permeabilized in 0.3% Triton X-100. The atrial
natriuretic factor (ANF) protein was detected using rabbit anti-rat
-ANF polyclonal antibody (Peninsula Laboratories) and fluorescein
isothiocyanate-conjugated goat anti-rabbit secondary antibody (Amersham
Life Science). The F-actin was detected using rhodamine-conjugated
phalloidin (Sigma). The tropomyosin was detected using a monoclonal
antibody CH1 (a kind gift from Dr. Jim Lin, University of Iowa) and a
tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse
secondary antibody (Amersham Life Science).
Ribonuclease Protection Assays--
The ANF and EF-1 mRNA
were detected using a Direct-Protect kit according to manufacturer's
recommendations (AMBION). Briefly, the rat ANF cDNA (pGEM-ANF) (54)
or EF-1
cDNA (45) was linearized with XhoI or
DdeI and used as templates for generation of radiolabeled riboprobes using polymerase T7 and SP 6, respectively. The protected fragments were separated on a 7% denaturing polyacrylamide gel, visualized by autoradiography, and quantitated by radioactive scanning
(AMBIS).
Cell Survival Assay and Apoptosis Analysis-- Cell survival was analyzed using the 3-(4,5-dimethylthiaziazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) method (38) as reported previously. Briefly, myocytes were cultured in triplicate in 24-well tissue culture plates and infected with adenoviruses as described above. The cells were then stained with 500 µg/ml MTT (Sigma) for 4 h, and the positively stained cells were counted as living cells. For analysis of apoptotic cells, a DNA fragmentation assay was performed as described previously using a DNA isolation kit (Purogene) and standard agarose gel electrophoresis (49). Fragmented and condensed nuclei in apoptotic cells were also identified by staining cultured cells with Hoescht 33258 dye as described (49).
Statistical Analysis-- Analyses between two groups were performed using unpaired two-tailed t tests, with p values less than 0.001 as being significantly different.
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RESULTS |
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In Vivo Activation of the p38 MAP Kinase Activities during Pressure Overload-induced Hypertrophy-- To establish the functional role of the p38 pathway in the development of cardiac hypertrophy, we measured the p38 MAP kinase activity in mouse hearts that had been exposed to pressure overload following transverse aortic constriction (TAC). Previous studies have established that chronic TAC in mice can induce several conserved phenotypic features of ventricular hypertrophy with concentric enlargement of the ventricular chamber, an increase in heart weight/body weight ratio, and a concomitant activation of immediate early genes and embryonic marker genes (such as ANF) expression (32, 33). In hearts, isolated 4 h after TAC, the p38 MAP kinase activities were not significantly different from the basal level activity (Fig. 1). Seven days after surgery, however, a marked elevation of p38 activity was observed in TAC animal hearts as compared with those from the sham-operated group. Activation of the p38 MAP kinase activities during the development of hypertrophy suggested a potential role for this pathway in mediating defined features of the hypertrophic response.
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Expression of p38 Signaling Molecules in Cultured
Cardiomyocytes by Adenoviral Vector-mediated Gene Transfer--
To
study the function of the p38 pathway in neonatal cardiac myocytes, we
utilized recombinant adenoviruses as an efficient gene delivery vector
to express various p38 signaling molecules (22, 34, 45). As
demonstrated using a recombinant adenovirus expressing the green
fluorescent protein (GFP) as a reporter, greater than 90% of the
myocytes expressed the transgene, a much higher level of expression as
compared with conventional calcium phosphate methods (Fig.
2A). The constitutively
activated mutants of two upstream activators for the p38 kinases,
MKK3bE and MKK6bE, as well as the wild type and dominant negative
mutants of the p38 MAP kinase and
isoforms, were constructed in
recombinant adenoviruses (22), and their expression in infected cardiac myocytes was detected at comparable levels by Western blot analysis (Fig. 2B). When cardiomyocytes were infected with vectors
expressing MKK3bE and MKK6bE, the endogenous p38 MAP kinase activities
were induced 12.2-fold and 3.0-fold, respectively (Fig. 2C).
In contrast, the endogenous JNK activity and ERK activity were not
activated by either virus (data not shown). This result was consistent
with previous studies that have established MKK3b and MKK6b as specific upstream activators of the p38 MAP kinases (26, 27).
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Activation of the p38 Pathway in Cardiomyocytes Induces Several Independent Features of the Hypertrophic Response and Cell Death-- To assess the effects of the activated p38 pathway on cardiomyocytes, a number of independent effects on cellular morphology were assayed including surface area, F-actin organization, and sarcomere organization. The expression of a marker of the hypertrophic response, ANF, was also monitored by immunohistochemistry and RNase protection assays. In comparison to uninfected cells, MKK3bE- and MKK6bE-infected cells displayed an increase in cell surface area, enhanced organization of sarcomeric units with increased nonstriated myofibrils, and induction of ANF expression (Table I and Fig. 3). Levels of ANF mRNA, as quantified by RNase protection, were elevated approximately 2.9-fold by MKK3bE and 4.6-fold by MKK6bE (Fig. 4). These are all well characterized features of myocardial cell hypertrophy in this in vitro assay system induced by other bona fide hypertrophic stimuli (3, 4). To determine whether the effects of MKK3bE and MKK6bE were indeed mediated by the p38 MAP kinases, cardiomyocytes were treated with SB202190, a pyridinyl imidazole compound that specifically inhibits p38 kinase activity (12, 22). As shown in Figs. 3 and 4, addition of the p38 inhibitor suppressed both morphological changes and ANF expression in either MKK3bE- or MKK6bE-infected myocytes. Based on morphological and biochemical criteria, these results suggested that activation of the p38 MAP kinase pathway was sufficient to induce hypertrophy in cardiomyocytes.
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Differential Effects of p38 and p38
on Cardiac Muscle
Cells--
Although it has been suggested from previous in
vitro studies that p38
and p38
isoforms may have
preferential upstream activators and different downstream target
substrates (27, 28, 29), their specific functions under physiological
conditions has not yet been demonstrated. To dissect the specific roles
of these two isoforms in mediating cardiac muscle cell hypertrophy and cell death, adenoviruses expressing the wild type and dominant mutants
of p38
or p38
were co-infected along with recombinant adenoviruses that direct the expression of MKK3bE or MKK6bE.
p38-mediated Cardiac Muscle Cell Death Involves Apoptotic
Pathways--
To determine whether the observed cell death in myocytes
involved apoptosis, a programmed genetic process, we performed DNA fragmentation assays to detect the presence of internucleosomal laddering in the genomic DNA, which is the hallmark of apoptosis (see
Fig. 6). DNA fragmentation was observed
in samples from myocytes that were infected with the MKK3bE vector, and
the DNA laddering was significantly induced in samples from myocytes
co-infected with wild type p38
or the dominant negative mutant of
p38
vectors. In comparison, DNA laddering was not detected from
control myocytes or myocytes infected with MKK6bE. We also analyzed the
integrity of myocyte nuclei by Hoescht dye staining in myocytes.
Chromosomal condensation and fragmentation of nuclei, another
characteristic feature of apoptotic cells, was also observed in a high
percentage of tropomyosin-positive cardiac muscle cells co-infected
with MKK3bE and wild type p38
or the dominant negative mutant p38
(shown by arrows in Fig. 7).
Taken together, these data suggested that cell death induced by the
activation of the p38 MAP kinase pathway was an apoptotic process.
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DISCUSSION |
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Cardiac hypertrophy is an adaptive process to cellular stress that
involves changes in both gene expression and sarcomeric organization
(1, 2). It is believed to be mediated by signaling molecules that
transduce the stress signals from the environment into different
cellular compartments (for a review, see Ref. 39). In this report, we
document that the p38 MAP kinase activities are induced during the
onset of in vivo hypertrophy in an experimental pressure
overload model. In cultured cardiomyocytes, activation of the p38
pathway induces several independent characteristic features of myocyte
hypertrophy, including an increase in cell surface area, enhanced
sarcomeric organization, and expression of an embryonic marker gene,
ANF. In the case of MKK3bE-infected myocytes, activation of the p38
pathway is also able to induce cell death. In addition, we have been
able to dissect the specific roles of the p38 family members and
demonstrate that the p38 isoform mediates the hypertrophic response,
whereas the p38
isoform is involved in an apoptotic process.
The distinct phenotypes of hypertrophy and cell death in
MKK3bE-infected cardiac muscle cells appears to be dictated by the balance of the relative activity between two different p38 isoforms. This observation could be the result of a quantitative difference between the ability of the two isoforms to activate a common signaling pathway or a qualitative difference in the activation of divergent bona
fide pathways for apoptosis and hypertrophy. In other cell types, the
p38 kinase has been implicated as part of the Fas-induced apoptotic
pathway involving ICE/Ced-3 proteases, suggesting a direct role for the
p38 kinase in apoptotic responses (21, 22). Our data with the dominant
negative mutants of p38
and p38
also supports a qualitative
difference between the p38
- and p38
-mediated responses in
apoptosis and hypertrophy, respectively. The final outcome of p38
activation in myocytes, either programmed cell death or hypertrophy,
may be determined by the competing downstream pathways as suggested by
previous studies of other MAP kinase pathways (48). Interestingly,
MKK3b- and MKK6b-induced responses are differentially affected by the
overexpression of p38 molecules. Both MKK3b and MKK6b are able to
phosphorylate and activate different p38 kinases in vitro,
although p38
has been suggested as a preferred substrate for MKK6b
rather than MKK3b (25, 26). Therefore, the difference between the
effects of MKK3bE and MKK6bE in cardiomyocytes may result from their
different specificities to various members of the p38 MAP kinase
family. This conclusion remains to be tested when isoform-specific
monoclonal antibodies for p38 kinases become available.
Activation of gene expression and the change of cellular morphology in p38 activated cardiac myocytes could be mediated by several distinct down-stream target molecules that have been identified in other cell types. A number of transcription factors, including ATF-2, ELK-1, CREB (13, 14), and MEF-2C, have been shown to be activated upon phosphorylation by p38 (16). Although the involvement of these transcriptional factors in hypertrophy is unclear, several of these factors, particularly MEF-2C, are known to play important roles in regulating cardiac gene expression and development (15). p38 can also activate some members of the small heat-shock proteins, including hsp25 and hsp27, through phosphorylation of MAPKAP kinase 2/3 (11, 18, 19, 20). Interestingly, it has been shown in non-cardiac cells that p38-mediated activation of hsp27 can induce F-actin reorganization and vinculin recruitment to the focal adhesion complex (46). Additional studies are needed to identify the specific activators as well as downstream targeting molecules of different p38 isoforms and to dissect out the relationship among the effectors of this signaling pathway in vivo.
Two other groups of MAP kinases mediate signal transduction in parallel with the p38 pathway, including ERK and JNK pathways (25-27, 40-44). Previous studies have documented that ERK activation is not sufficient to initiate a hypertrophic response in vitro (44), whereas in vivo its activation is not associated with the hypertrophic phenotype in the Ras transgenic mice (37). On the other hand, the JNK pathway is also activated in the Ras transgenic animals and its activation is essential for a hypertrophic response in vitro (37). It is highly likely that p38 and JNK are both required to generate a hypertrophic or an apoptotic response in overloaded hearts. Therefore, the potential interactions between the p38 pathway and the JNK or other signaling pathways in cardiac muscle cells is worthy of further investigation.
The finding that a stress-activated signaling pathway may play direct
roles in inducing apoptosis has significant implications. There is an
increasing body of evidence which suggests that apoptotic cells are a
clear feature of heart failure in various animal models, in
ischemia/reperfusion-treated hearts, as well as in human end-stage failing hearts (recently reviewed in Ref. 47). Programmed cell death
may therefore serve as one of the underlined mechanisms for the
transition from hypertrophy to decompensated heart failure. The
implication of p38 in apoptosis of cardiomyocytes thereby provides a
potential signaling pathway for such an apoptotic response. It will
become of interest to determine if p38 pathways play any role in the
cell survival effects mediated by cardiotrophin-1 (49) via
GP130-dependent pathways (50). It will also be of interest
to determine if manipulation of the p38 MAP kinase pathway and their
downstream target molecules in vivo would have an effect in
animal models of heart failure that are associated with apoptosis.
In conclusion, activation of p38 MAP kinase activities during hypertrophy, and the opposing effects of hypertrophy and cell death mediated by the two members of p38 MAP kinase family suggest a potential role of the p38 pathway in the onset of hypertrophy and heart failure. As presented in Fig. 8, a working model can now be constructed whereby hemodynamic stress, as a result of mechanical overload or chronic ischemia, can activate the p38 MAP kinase activities, which subsequently contribute to the hypertrophic response in the initial compensatory phase. As the stress stimulus persists, the balance between hypertrophic and apoptotic signaling is disrupted and, as a consequence, cardiomyocytes lose cellular viability and structural integrity and enter the cell death pathway. The loss of contractile function and viable cells eventually places more stress on the surviving myocytes and initiates the irreversible deterioration of cardiac function, resulting in overt heart failure. The recent development of miniaturized physiological technology (51), strategies for conditional transgenesis and ventricular chamber-restricted gene targeting in the murine heart (54), and genetically based mouse models of distinct forms of concentric and asymmetric hypertrophy (52) and failure (53) should allow a rigorous assessment of the in vivo validity of this model.
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ACKNOWLEDGEMENTS |
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We thank Dr. Lan Mao for surgical assistance and Mahmoud Itani for technical help. We are in debt to members of the Chien laboratory for their input and critical review of this manuscript.
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FOOTNOTES |
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* This work was supported by grants from the American Heart Association (AHA) and National Institutes of Health (NIH) (to K. R. C.), by AHA Grant-in-aid 95007690 and NIH Grants GM51417 and AI 41637 (to J. H.), and by NIH Grants HL28143 and HL46345 (to J. H. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Medicine and Center for Molecular Genetics, Mail Box 0613-C, 9500 Gilman Dr., University of California at San Diego, La Jolla, CA 92093. Tel.: 619-534-4801; Fax: 619-534-8081; E-mail: kchien{at}ucsd.edu.
1 The abbreviations used are: MAP, mitogen-activated protein; TAC, transverse aortic constriction; MKK, MAP kinase kinase; ANF, atrial natriuretic factor; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; GFP, green fluorescent protein; MTT, 3-(4,5-dimethylthiaziazol-2-yl)2,5-diphenyl tetrazolium bromide.
2 J. J. Hunter, M. Shimizu, J. Brown, V. P. Sah, K. Gottshall, C. Milano, R. Lefkowitz, J. H. Brown, and K. R. Chien, submitted for publication.
3 Y. Wang and K. R. Chien, unpublished observations.
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REFERENCES |
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