Báo cáo khoa học: Double-stranded RNA-dependent protein kinase (PKR) is downregulated by phorbol ester ppt

Double-stranded RNA-dependent protein kinase (PKR)
is downregulated by phorbol ester
Yan Zhou
1
, Barbara I. Chase
1
, Mark Whitmore
2
, Bryan R. G. Williams
2
and Aimin Zhou
1,2
1 Department of Chemistry, Cleveland State University, OH, USA
2 Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic Foundation, OH, USA
The double-stranded (ds) RNA-dependent protein kin-
ase (PKR) is a serine ⁄ threonine kinase that is induced
in mouse and human cells by interferons (IFN). Upon
binding to dsRNA, the activation of PKR by auto-
phosphorylation leads to phosphorylation of the
a-subunit of eukaryotic initiation factor 2 (eIF-2a),
subsequently resulting in an inhibition of protein syn-
thesis. Thus, PKR plays a central role in the antiviral
and cell proliferation inhibitory activities of IFN [1].
Overexpression of wild-type PKR greatly enhances the
ability of cells to resist viral infection, whereas domin-
ant negative PKR suppresses IFN-mediated antiviral
activity [2,3]. PKR null mice succumb to encephalo-
myocarditis virus (EMCV) infection more rapidly than
wild-type mice and display increased susceptibility to
the infections of vesicular stomatitis virus and influ-
enza virus [4,5]. PKR also exerts an antiproliferative

role in cells. Expression of wild-type PKR in yeast
inhibits cell proliferation [6,7]. As predicted, NIH3T3
cells expressing various inactive mutants of PKR
formed tumours in nude mice. In contrast, overexpres-
sion of wild-type PKR in cells does not induce tumour
growth in mice [8,9]. PKR can induce apoptosis in cer-
tain cell types [10]. HeLa cells overexpressing wild-type
PKR undergo apoptosis. However, inactive mutants
did not induce apoptosis when similarly expressed [11].
PKR mediates apoptosis in cells induced by different
stimuli, including dsRNA, viral infection, endotoxin
and cytokines [12–16]. Increased contact hypersensi-
tivity responses in PKR null mice compared with
Keywords
interferon; PKC; PKR; poly I:C; proteasome
Correspondence
A. Zhou, Clinical Chemistry Program,
Department of Chemistry, SI 424, Cleveland
State University, Cleveland, OH 44115, USA
Fax: +1 216 687 9298
Tel: +1 216 687 2416
E-mail:
(Received 7 October 2004, revised 22
December 2004, accepted 18 January 2005)
doi:10.1111/j.1742-4658.2005.04572.x
The double-stranded RNA-dependent protein kinase (PKR) is one of the
key mediators of interferon (IFN) action against certain viruses. PKR also
plays an important role in signal transduction and immunomodulation.
Understanding the regulation of PKR activity is important for the use of
PKR as a tool to discover and develop novel therapeutics for viral infec-

tions, cancer and immune dysfunction. We found that phorbol 12-myristate
13-acetate (PMA), a potent activator of protein kinase C (PKC), decreased
the level of autophosphorylated PKR in a dose- and time-dependent man-
ner in IFN-treated mouse fibroblast cells. Polyinosinic–polycytidylic acid
(poly I:C) treatment enhanced the activity of PKR induced by IFN, but
did not overcome the PMA-induced reduction of PKR autophosphoryla-
tion. Western blot analysis with a monoclonal antibody to mouse PKR
revealed that the decrease of PKR autophosphorylation in cells by PMA
was a result of PKR protein degradation. Selective PKC inhibitors blocked
the degradation of PKR stimulated by PMA, indicating that PKC activity
was required for the effect. Furthermore, we also found that proteasome
inhibitors prevented PMA-induced down regulation of PKR, indicating
that an active proteasome is required. Our results identify a novel mechan-
ism for the post-translational regulation of PKR.
Abbreviations
ds, double-stranded; eIF-2a, a-subunit of eukaryotic initiation factor 2; IFN, interferon; PKC, protein kinase C; PKR, RNA-dependent protein
kinase; PMA, phorbol 12-myristate 13-acetate; poly I:C, polyinosinic–polycytidylic acid.
1568 FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS
wild-type mice has been suggested by a role for PKR
in host immune functions [17].
PKR mediates the activation of signal transduction
pathways by a wide range of proinflammatory factors,
including lipopolysaccharide, tumour necrosis factor-a,
and interleukin-1 [18–21]. Interestingly, the activation
of the nuclear factor jB (NFjB) pathway by dsRNA,
lipopolysaccharide or tumour necrosis factor-a in cer-
tain cells is PKR dependent [22]. PKR is part of the
complex of IjB kinase, an upstream effector kinase
that phosphorylates critical serine residues in the IjB
family of inhibitors [23]. In addition, PKR is also

involved in the activation of the stress-activated pro-
tein kinases (p38) and c-Jun NH2-terminal kinase [24].
Although PKR is not essential in the most of these
pathways, it is required for the maintenance and
amplification of cellular responses to proinflammatory
and apoptotic stimuli. These results demonstrate that
PKR, in addition to its function as a mediator of IFN
action, plays broad roles in cell signalling.
Although PKR plays an important role in the anti-
viral action of IFNs, the control of cell proliferation and
the mediation of signal transduction pathways, relat-
ively little work has been done on the transcriptional
regulation of PKR expression. Furthermore, studies on
the regulation of PKR protein levels by agents other
than IFN are largely lacking. Here, we report that
4b-phorbol 12-myristate 13-acetate (PMA), a potent
activator of protein kinase C (PKC), inhibits PKR activ-
ity with a dose- and time-correlation in IFN-treated
mouse fibroblast cells. Western blot analysis revealed
that the downregulation of PKR activity is due to the
degradation of PKR protein. Pretreatment of cells with
selective proteasome inhibitors prevents the PKR degra-
dation induced by PMA, suggesting the involvement
of an active proteasome. Our results provide the first
evidence that PKR is regulated at the post-translational
level by a tumour-promoting agent.
Results
PKR plays an important role in the inhibition of cell
proliferation and virus replication. Previously, we have
reported that PMA downregulated RNase L, one of

the key enzymes in the 2-5 A system that also func-
tions in IFN-induced antiviral and antiproliferative
activities [25]. To determine if PMA affects PKR activ-
ity, lysates from mouse fibroblast L929 cells treated
with or without PMA in the presence or absence of
1000 UÆml
)1
of IFN-a were analyszed. PKR was isola-
ted using the affinity resin, polyinosinic–polycytidylic
acid (poly I:C) agarose, and the complex was incuba-
ted with a reaction mixture containing [
32
P]ATP[cP].
As shown in Fig. 1, treatment with 10 ngÆml
)1
PMA
resulted in a time-dependent decrease in PKR auto-
phosphorylation. At 60 min, PMA caused a decrease
of the autophosphorylation of PKR to 15% of its
induced level. PMA induced downregulation of PKR
autophosphorylation was dose dependent (Fig. 2), with
maximal reduction observed at 10 ngÆmL
)1
, and lesser
effects at 20 and 30 ngÆmL
)1
, respectively. This result
is consistent with the observation in our previous study
on the effects of PMA on RNase L [25]. Similar results
were also obtained after the treatment of NIH3T3 cells

Fig. 1. Double-stranded RNA-dependent protein kinase (PKR) auto-
phosphorylation is downregulated by PMA in a time-dependent
manner. (A) L929 cells were stimulated with 1000 UÆmL
)1
IFN-a for
14 h and then treated with 10 ngÆmL
)1
PMA for 5, 30 and 60 min.
PKR was isolated using poly I:C–Agarose beads and incubated with
[
32
P]ATP[cP] at 30 °C for 30 min. Phosphorylated PKR was separ-
ated by SDS ⁄ PAGE on 10% gels and subjected to autoradiography.
(B) PKR autophosphorylation was quantified by PhosphorImager
analysis. The results represent data from three separate experi-
ments.
Y. Zhou et al. Control of protein kinase R degradation
FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS 1569
with PMA in the presence of 1000 UÆml
)1
of IFN-a
(Fig. 3). These experiments indicate that PMA treat-
ment decreases the autophosphorylation of PKR and
that this activity is not cell line specific.
Poly I:C is a potent inducer of PKR expression and
activity in different types of cells. We examined the
effect of poly I:C on the downregulation of PKR
autophosphorylation by PMA. L929 cells were incuba-
ted with 1 lgÆml
)1

of poly I:C in the presence of
1000 UÆml
)1
of IFN-a for 14 h and treated with or
without PMA for 60 min. Poly I:C synergistically
induced PKR expression with IFN-a in the cells. How-
ever, the relative PMA-induced decrease of autophos-
phorylated PKR levels in cells treated with (lane 3 vs.
lane 5) or without (lane 2 vs. lane4) poly I:C is almost
same (Fig. 4). There was a 3.6-fold decrease in the
presence of poly I:C and a 3.4-fold decrease in the
absence of poly I:C after PMA treatment. Therefore,
this result showed that the downregulation of PKR
autophosphorylation by PMA is poly I:C-independent.
Following dsRNA binding and autophosphoryla-
tion, PKR phosphorylates several cellular substrates.
The best characterized one of these is eIF-2a.To
determine the effect of PMA-induced PKR downregu-
lation on a biologically relevant substrate, we exam-
ined the phosphorylation status of eIF-2a in L929
cells. Western blot analysis indicated that while the
levels of total eIF-2a and phosphorylated eIF-2a
were essentially equivalent in L929 cells treated with or
without IFN-a, there was a significant increase of
Fig. 2. PKR autophosphorylation is downregulated by PMA in a
dose-dependent manner. (A) L929 cells were stimulated with
1000 UÆmL
)1
IFN-a for 14 h and then treated with 10, 20 and
30 ngÆmL

)1
PMA for 60 min. PKR autophosphorylation was deter-
mined as described above. (B) PKR autophosphorylation was quan-
tified by PhosphorImager analysis. The results represent data from
three separate experiments.
Fig. 3. PMA treatment downregulates PKR autophosphorylation in
NIH3T3 cells. (A) NIH3T3 cells were stimulated with 1000 UÆmL
)1
IFN-a for 14 h, or left unstimulated, and then treated with
10 ngÆmL
)1
PMA for 60 min. PKR autophosphorylation was ana-
lysed as described above. (B) PKR autophosphorylation was quanti-
fied by a densitometry.
Control of protein kinase R degradation Y. Zhou et al.
1570 FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS
phosphorylated eIF-2a, but not eIF-2a protein in the
cells treated with dsRNA. Importantly, treatment of
dsRNA-induced cells with PMA resulted in a signifi-
cant reduction in the level of phosphorylated eIF-2a
(Fig. 5). Thus, the PMA mediated downregulation of
PKR results in reduced activity towards a key endo-
genous substrate.
PMA treatment has temporally distinct effects on
the activation of PKC. A short-term treatment of cells
with PMA results in the activation of PKC, but long-
term incubation of cells with PMA depletes PKC
protein through the ubiquitin-proteasome pathway
[26]. To test if PKC is responsible for the downregula-
tion of PKR, L929 cells were treated with 1000 UÆml

)1
of IFN-a plus 10 ngÆml
)1
PMA for 14 h or IFN-a
alone for 14 h, and then 10 ngÆml
)1
PMA for 60 min.
Short-term treatment of cells with PMA caused a 2.5-
fold of PKR autophosphorylation when compared to
long-term treatment (Fig. 6). This observation suggests
that PKC is involved in the downregulation of PKR
autophosphorylation in the cells. To determine further
the role of PKC in the downregulation of PKR auto-
phosphorylation and investigate the level at which
PKR is regulated by PKC, we used GO
¨
6983, a general
PKC inhibitor that can inhibit several PKC isoforms.
L929 cells were treated with PMA in the presence of
GO
¨
6983, and PKR levels were determined by western
blot analysis probed with a monoclonal antibody to
mouse PKR. Interestingly, PMA treatment induced
the degradation of PKR protein and the PKC inhib-
itor effectively blocked this event, suggesting that
PKC-mediated phosphorylation is responsible for the
decreased levels of autophosphorylated PKR through
the degradation of PKR protein (Fig. 7A). Treatment
of cells with the PKC inhibitor alone did not have any

Fig. 4. Downregulation of PKR autophosphorylation is independent
of PKR activation status. L929 cells were incubated in the presence
of 1000 UÆmL
)1
IFN-a alone or IFN-a plus 1 lgÆmL
)1
poly I:C for
14 h and then treated 10 ngÆmL
)1
PMA for 60 min, or left
untreated. PKR autophosphorylation was analysed as described
above.
Fig. 5. PMA treatment disrupts PKR catalytic function. L929 cells
were incubated with 1000 UÆmL
)1
IFN-a for 14 h before treatment
with 10 ngÆmL
)1
PMA for 60 min. The cells were then transfected
with 1 lgÆmL
)1
poly I:C using Lipofectamine reagent. The phos-
phorylation of eIF-2a was determined by western blot analysis
using monoclonal antibodies to phospho-eIF-2a and total eIF-2a.
Fig. 6. Depletion of PKC reduces the downregulation of PKR auto-
phosphorylation. L929 cells were untreated (lane 1) or treated
with dimethyl sulfoxide (lane2), 1000 UÆ mL
)1
IFN-a (lane 3),
1000 UÆmL

)1
IFN-a plus 10 ngÆ mL
)1
PMA for 14 h (lane 4);
1000 UÆmL
)1
IFN-a for 14 h prior to PMA treatment for 60 min
(lane 5). PKR autophosphorylation was determined as described
above.
Y. Zhou et al. Control of protein kinase R degradation
FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS 1571
effect on PKR levels (Fig. 7B). To confirm this result
further we treated NIH3T3 cells with two additional
PKC inhibitors, GO
¨
6976 and Rottlerin. As shown in
Fig. 7C, GO
¨
6976 effectively blocked the PMA-induced
degradation of PKR, but not Rottlerin, suggesting that
PKC-a and b may play a role in this event.
The degradation of certain proteins by PMA treat-
ment is via a proteasome-dependent mechanism [27]. To
test if the downregulation of PKR levels in L929 cells
by PMA requires an active proteasome, we used the
selective inhibitors of proteasomal proteases, ALLN,
MG132 or PSI. The cells were incubated with these
inhibitors for 4 h prior to PMA treatment. Pre-incuba-
tion of cells with the proteasome inhibitors completely
prevented the degradation of PKR caused by PMA

(Fig. 8). Proteasome inhibitors induce apoptosis in dif-
ferent types of cells [28]. However, these proteasome
inhibitors do not induce apoptosis in L929 cells [25].
These experiments suggest that an active proteasome is
required for the PMA-induced degradation of PKR.
Discussion
Studies have revealed that PKR is involved a wide range
of biological activities. The structure and function of
PKR have been well characterized. However, relatively
less work has been done on the regulation of PKR at the
transcriptional or post-translational levels. Our results
reported here provide the first evidence that PKR
protein can be downregulated by PMA, a well-known
PKC activator, through proteasome-mediated degrada-
tion. As shown in Fig. 7A, GO
¨
6983, a general inhibitor
of PKC completely blocked the degradation of PKR
induced by PMA, implicating the involvement of PKC
activation. We have further confirmed this observation
by using other PKC inhibitors, such as GO
¨
6976 and
Rottlerin. GO
¨
6976 displays a better inhibitory role in
PMA-induced PKR degradation (Fig. 7C), suggesting
the involvement of PKC-a and b. Although we have not
determined if the PKC-mediated PKR degradation is a
direct or indirect effect, our findings raise the possibility

that PKR is a substrate of PKC.
Katze and colleagues have reported that PKR
degradation occurs during poliovirus infection and
demonstrated that preincubation of cell extracts
with poly I:C, a synthetic dsRNA, largely prevented
PKR proteolysis, suggesting that the degradation of
PKR during viral infection does not require PKR
Fig. 7. Activation of PKC is responsible for the degradation of PKR
induced by PMA in L929 cells. (A) L929 cells were incubated in the
presence of 1000 UÆmL
)1
IFN-a for 14 h and treated with or with-
out GO
¨
6983 (10 n
M) for 2 h prior to PMA treatment (10 ngÆmL
)1
)
for various times. PKR was detected by western blot analysis
probed with a monoclonal antibody to mouse PKR. (B) L929 cells
were incubated with 1000 UÆmL
)1
IFN-a for 14 h and then treated
with GO
¨
6983 (10 n
M) for 30, 60 and 90 min. PKR levels were
determined by western blot analysis as described above. (C)
NIH3T3 cells were incubated in the presence of 1000 UÆmL
)1

IFN-a
for 14 h and treated with or without GO
¨
6976 (1 l
M) and Rottlerin
(5 l
M) for 2 h prior to PMA treatment (10 ngÆmL
)1
) for 30 min. PKR
was detected as described above.
Fig. 8. PKR degradation induced by PMA is proteasome-dependent
L929 cells were incubated with different selective proteasome
inhibitors, ALLN (10 l
M), MG132 (10 lM) and PSI (1 lM), for 4 h
prior to PMA treatment of 60 min. PKR protein levels in the cells
were analysed by western blot analysis probed with antibodies to
mouse PKR and b-actin.
Control of protein kinase R degradation Y. Zhou et al.
1572 FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS
activation and autophosphorylation [29]. Further-
more, cells transfected with poly I:C using the Lipo-
fectamine plus reagent for 4 h displayed a markedly
high level of autophosphorylated PKR, suggesting
that autophosphorylated PKR is not the target of
the proteasome and is relatively stable [5]. Viral
infection results in the activation of PKC in cells
[30–32]. Entry of influenza viruses into cells is inhib-
ited by a highly specific PKC inhibitor [33]. Other
viruses including vesicular stomatitis virus, herpes
simplex I and vaccinia virus, are also inhibited by

H7, a broader PKC inhibitor [34], indicating that
the activation of PKC in cells by virus is important
for virus to infect host cells. Taken together, these
observations and our results suggest a possibility that
virus may counteract the antiviral activity of PKR in
cells through the activation of PKC, resulting in the
degradation of PKR protein. Therefore, investigating
the regulation of PKR by PKC may provide useful
information for designing therapeutic methods for
viral infectious diseases.
Autophosphorylation is the first step in converting
PKR to its active form. Several critical autophospho-
rylation sites in human PKR have been identified,
including S242, S448, T255, T258, T446 and T451.
Autophosphorylated PKR in cells infected by virus or
transfected by dsRNA is relatively stable [5,35]. There-
fore, if PKR is indeed a PKC substrate, it is unlikely
that PKC-mediated phosphorylation sites are the same
as the autophosphorylation sites on PKR. Mouse
PKR has 74 serine ⁄ threonine amino acids and human
PKR has 87 serine ⁄ threonine amino acids. Should
PKR prove to be a direct target of PKC, identifying
the location of PKC-phosphorylated serine ⁄ threonine
residues on PKR and mutating these amino acids will
be very important for studying the role of PKR in
growth suppression, apoptosis and antiviral infection.
It is possible that the effect of PMA on the downregu-
lation of PKR is indirect. For example, a PKR interact-
ing protein (several have been identified [1]), may be
phosphorylated in response to PMA and, in turn, recruit

PKR to the proteasome. Phosphorylation of target pro-
teins promotes ubiquitylation and accelerates protea-
some mediated proteolysis. PKC is involved in inducing
a wide range of proteasome-mediated protein degrada-
tion, including p21(WAF1 ⁄ CIP1), STAT3, IjB, estro-
gen receptor and beta-amyloid precursor [26,27,36–39].
Interestingly, we have been unable to observe the higher
molecular weight bands of ubiquitylated PKR in the
presence of proteasome inhibitors after the cells were
treated with PMA. This observation is similar to the
result obtained by Halvorsen and colleagues for the
transcription factor STAT3 [27]. Thus, PMA-induced
PKR degradation is likely to involve proteasome-
dependent degradation. However, whether or not PKR
is directly ubiquitylated requires further investigation.
PMA treatment causes a degradation of STAT2 and
STAT3 in neuroblastoma cells through the PKC-
dependent phosphorylation and the proteasome path-
way, suggesting a role of PMA in the regulation of
cytokine signalling transduction [27]. Furthermore,
Larner and colleagues have reported that the inhibi-
tory effect of PMA on the IFN signalling pathway
results in suppression of the expression of IFN-stimu-
lated genes [40]. Previously, we reported that the deg-
radation of RNase L (an IFN inducible gene) induced
by PMA in L929 cells is PKC dependent [25].
RNase L and PKR are important enzymes in the
molecular mechanism of IFN functions against viral
infection and cellular proliferation. RNase L displays
its antiproliferative effects in cells through regulating

the expression of genes at the post-transcripational
levels, whereas PKR works at the translational levels. It
is possible that the tumour promoting activity of PMA
may be mediated, in part, through downregulating the
products of tumour suppressor genes, such as RNase L
and PKR, providing a preferable environment for
tumour growth. Further study of the disruption of
IFN-stimulated gene products by PMA in cells may
shed new light on the molecular mechanism by which
this agent promotes tumorigenesis.
Experimental procedures
Cell culture and treatments
Murine L929 and NIH3T3 cells (ATCC, Manassas, VA,
USA) were grown in DMEM (Cleveland Clinic Foundation
Core Facility) supplemented with 10% fetal bovine serum
(Biosource, Camarillo, CA, USA) and antibiotics in a
humidified atmosphere of 5% CO
2
at 37 °C. The cells were
grown to 90% confluence and incubated with 1000 UÆmL
)1
murine IFN-a (R & D Systems, Minneapolis, MN, USA)
for 14 h. Cells then were treated with or without 10 ngÆmL
)1
PMA (Sigma, St. Louis, MO, USA) for various times. Cells
were incubated with selective proteasome inhibitors, ALLN,
MG132, and PSI (Calbiochem, La Jolla, CA, USA) for 4 h
prior to PMA treatment as indicated. The PKC inhibitor,
GO
¨

6983, GO
¨
6976 and Rottlerin were purchased from
Calbiochem. Poly I:C was purchased from Sigma.
Preparation of cellular extracts
Cells were harvested by washing twice with ice-cold phos-
phate-buffered saline (NaCl ⁄ P
i
) and collected with a scraper.
Cytoplasmic extracts were prepared by suspension of cell
Y. Zhou et al. Control of protein kinase R degradation
FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS 1573
pellets in NP-40 lysis buffer [10 mm Tris ⁄ HCl pH 8.0, 5 mm
Mg(OAc)
2
,90mm KCl, 0.2 mm phenylmethylsulfonyl fluor-
ide, 100 unitsÆmL
)1
aprotinin, 10 lgÆmL
)1
leupeptin and 2%
NP-40). After centrifugation at 10 000 g in a microcentrifuge
at 4 °C for 10 min, the supernatant was removed and stored
at )80 °C.
Autophosphorylation assay
Cell extracts were incubated with poly I:C–Agarose (Roche)
prewashed with 1 · DBG buffer [20 mm Tris ⁄ HCl pH 7.5,
50 mm KCl, 5 mm Mg(OAc)
2
, 0.2 mm phenylmethylsulfo-

nyl fluoride, 100 unitsÆ mL
)1
aprotinin, 10 lgÆmL
)1
leupep-
tin, 7 mm mercaptoethanol, and 10% glycerol] on ice for
1 h. After centrifugation, the beads were washed three times
with NP-40 lysis buffer as described above. The washed
beads were incubated in NP-40 buffer containing 2 mm
MnCl
2
,1lm cold ATP and 1 lm [
32
P]ATP[cP] (50 CiÆm-
mol
)1
, Amersham, Piscataway, NJ, USA) at 30 °C for
30 min. Proteins were separated by SDS ⁄ PAGE on 10%
gels and the autphosphorylated PKR was detected by auto-
radiography.
Western blot analysis
Cellular extracts (150 lgÆsample
)1
) were fractionated by
SDS ⁄ PAGE on 10% gels and transferred to PVDF mem-
branes (Millipore, Bedford, MA, USA). Western blot
analysis was performed using a monoclonal antibody to
mouse PKR (Santa Cruz Biotechnology, Santa Cruz, CA,
USA) at a dilution of 1 : 2000 and PKR was detected by
a chemiluminescence method according to the manufac-

turer’s specification (Amersham). The phosphorylated
eIF-2a antibody was from Cell Signaling Technology,
Beverly, MA, USA, and the total eIF-2a antibody was
from Santa Cruz Biotechnology.
Acknowledgements
This work was supported by the Start-Up Package
Fund from Cleveland State University to A.Z. and in
part by NIH grant AI34039 to B.R.G.W. We thank
Robert H. Silverman (Cleveland Clinic Foundation)
and Bret A. Hassel (University Maryland Medical
School) for critical reading of the manuscript.
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