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Journal of Virology, September 1998, p. 7669-7675, Vol. 72, No. 9
Department of Pathology and Laboratory
Medicine,
Received 9 January 1998/Accepted 21 May 1998
Coxsackievirus B3 (CVB3), an enterovirus in the family
Picornaviridae, induces cytopathic changes in cell culture
systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells.
Caspase activation assays confirm that the cleaved caspase 3 is
proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a
cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while
inhibition of caspase activation by ZVAD.fmk in control apoptotic cells
induced by treatment with the porphyrin photosensitizer benzoporphyrin
derivative monoacid ring A and visible light inhibited the apoptotic
phenotype. Caspase activation and cleavage of substrates may not be
responsible for the characteristic cytopathic effect produced by
picornavirus infection yet may be related to late-stage alterations of
cellular homeostatic processes and structural integrity.
Coxsackievirus B3 (CVB3) is an
enterovirus in the family Picornaviridae. Following binding
to the coxsackievirus and adenovirus receptor (6, 64), the
viral RNA enters the cytoplasm, where it is translated into a single
polyprotein by the host translational machinery. The polyprotein is
then proteolytically processed by viral proteases to produce all of the
viral structural and nonstructural proteins. The virus-encoded
RNA-dependent RNA polymerase transcribes negative-strand viral
RNAs, which serve as templates for the synthesis of multiple progeny
genomes. Following viral packaging, the virus is released, potentially
under the influence of the virus-encoded 2B protein (67).
During the replicative process and viral progeny release, the
cytopathic effect (CPE) occurs and the host cell is injured, with
eventual loss of viability.
Multiple host cellular processes are altered during picornavirus
parasitization. Virus protein 2Apro directly cleaves
eukaryotic initiation factor 4 gamma (eIF4G). Cleavage of this
translation initiation factor not only abolishes cap-dependent mRNA
translation (19); the cleavage products are believed to
stimulate translation of uncapped mRNA, such as the noncellular
picornavirus genome (43), which uses a novel internal ribosome entry mechanism to begin protein translation (31,
76). Poliovirus proteins 2Apro and 3Cpro
have been shown to cleave the TATA-binding protein, with
3Cpro also shutting off transcription of RNA polymerases I,
II, and III (11, 72, 74). The transcription factors TFIIIC
(10), CREB (73), and Oct-1 (75) are
also cleaved by 3Cpro during picornavirus infection. CVB3
protein 2B has been shown to modify endoplasmic reticulum and plasma
membrane permeability (14), causing an increase in the
cytosolic free calcium concentration (28, 67) and membrane
lesions which may facilitate viral progeny release. Ionic gradients
collapse (40, 52), and the phospholipase activity is altered
(24, 29). CVB3 infection of HeLa cells results in tyrosine
phosphorylation of two cellular proteins at 4 h postinfection, and
inhibition of these phosphorylations significantly reduces viral
progeny production (27). It is clear that infection is a
dynamic cellular process in which timely interactions between viral and
host proteins determine the outcome for both the virus and the host
cells.
It is now clear that cysteine proteases in the caspase family of
enzymes are key effector molecules in apoptotic cell death. Once
activated, caspases cleave specific substrates, including poly(ADP-ribose) polymerase (PARP) (35), DNA fragmentation
factor (DFF) (37), gelsolin (34), lamin A
(58), sterol regulatory element-binding proteins
(68), Many viruses possess biochemical mechanisms to evade and/or induce
apoptosis in cells in which they reside (for reviews, see references
49 and 60). Different viruses
interact at different stages of the apoptotic death pathway. Viruses
have evolved strategies targeting the Fas ligand-Fas or tumor necrosis
factor alpha (TNF- To determine if caspases are activated and responsible for the CPE
observed following CVB3 infection, HeLa cells (American Type Culture
Collection, Rockville, Md.) were either infected, at a multiplicity of
infection (MOI) of 5, with CVB3 (generously provided by Charles Gauntt,
University of Texas Health Sciences Center, San Antonio) or sham
treated with minimum essential medium (MEM) lacking fetal bovine serum
(FBS) for 45 min. Cells were washed with phosphate-buffered saline
(PBS), and complete MEM containing 10% FBS was then substituted. A
positive apoptosis control consisted of HeLa cells treated with the
photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA) for
1 h and then exposed to visible light as previously described
(22, 23). Cultures were examined and harvested at 0, 1, 3, 5, 6, 7, 8, 9, 10, and 12 h postinfection. Cells were washed two
times in cold PBS and lysed in 1 ml of lysis buffer (20 mM Tris [pH
8], 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 0.15 U of aprotinin per ml) per
75-cm2 culture area. After a 20-min incubation on ice,
supernatants were collected following centrifugation at 10,000 × g and stored at The temporal pattern of production of CVB3 viral proteins, progeny
virus, and the evolution of HeLa cell degenerative morphological changes were considered in conjunction with an examination of host cell
death proteins. Cell lysate samples were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Proteins were transferred
to nitrocellulose (Hybond ECL nitrocellulose membranes; Amersham). The
membranes were incubated for 1 h at room temperature in blocking
buffer (PBS with 0.1% Tween 20 and 5% powdered nonfat milk).
Following two washings in wash buffer (PBS with 0.1% Tween 20), the
membranes were incubated with antibody against CVB3 (rabbit polyclonal
anti-CVB3, 1:1,000; Accurate Chemicals). The membranes were washed
three times in wash buffer and incubated with a donkey anti-rabbit
immunoglobulin secondary antibody (Amersham). The membranes were washed
three times, and the horseradish peroxidase-conjugated secondary
immunoglobulins were detected by the enhanced chemiluminescence method
(ECL, Amersham) and exposed to Hyperfilm (Amersham) autoradiography film. Significant increases in viral protein synthesis could be detected between 3 and 5 h postinfection (Fig.
1B). The viral proteases cleave viral as
well as host proteins early following infection. By immunoblot analysis
with mouse monoclonal anti-eIF4G (1:1,000; Transduction Laboratories),
it was found that eIF4G is cleaved by viral protease 2A beginning
within 1 h postinfection, with further loss of detection of the
220-kDa protein by 5 h postinfection (Fig. 1C). The amount of CVB3
in the cell supernatant (released virus) was determined on monolayers
of HeLa cells by the agar overlay plaque assay method as previously
described (3). Briefly, sample supernatant was serially
diluted 10-fold, the dilutions were overlaid on 90 to 95% confluent
monolayers of HeLa cells in six-well plates (Costar), and the overlaid
cells were incubated for 1 h (5% CO2, 37°C). Medium
containing nonbound virus was removed, and warm complete MEM containing
0.75% agar was overlaid in each well. The plates were incubated 36 to
48 h (5% CO2, 37°C), fixed with Carnoy's fixative
(95% ethanol-acetic acid [3:1]), and stained with 1% crystal
violet. Progeny virus was present in the supernatant at basal levels
between 1 and 5 h. By 6 h postinfection there was a
detectable increase in supernatant virus levels, and exponential virus
production began at 9 h postinfection as determined by plaque assays (Fig. 1A). HeLa cells exhibited marked changes in morphology, including cellular condensation, rounding up, and release from the
culture monolayer, between 6 and 7 h following infection, as noted
by contrast microscopy (Fig. 1D).
To determine whether the host cell death machinery is activated
following CVB3 infection, immunoblot analysis of lysate collected at
specific time points was performed. Caspase 3, which is present in
cells as a precursor protein with a molecular mass of 32 kDa, is a
primary molecule involved in the execution of cell death. Using mouse
monoclonal anti-caspase 3 (1:1,000; Transduction Laboratories), it was
determined that uninfected cells contained the 32-kDa precursor protein. Following CVB3 infection, the level of the 32-kDa precursor protein began to diminish between 7 and 8 h postinfection, and it
was almost completely undetectable by 12 h postinfection (Fig. 2). To determine whether the depleted
pro-caspase 3 had been proteolytically processed from a single-chain
zymogen to its active two-chain enzyme, HeLa cell lysates were
incubated with caspase 3 fluorescent substrates as previous described
(23). Briefly, cellular lysates were incubated with reaction
buffer (20 mM Tris [pH 7.5], 137 mM NaCl, 1% Nonidet P-40, 10%
glycerol) containing 100 µM caspase 3 substrate
acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) (Calbiochem, Cambridge, Mass.) or
Z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC)
(Enzyme Systems Products, Livermore, Calif.). The reaction mixture was
incubated at 37°C for 2 h, and fluorescence excitation of AMC or
AFC at 380 or 400 nm, respectively, was measured at 460 or 505 nm,
respectively, with a CytoFluor 2350 cytofluorometer (Perseptive
Biosystems, Burlington, Ontario, Canada). Using this approach, caspase
3 activity was evident by 5 h postinfection. The increase in
caspase 3 activity from 7 to 10 h postinfection, when the maximum
level of activation was reached, was maintained through to 12 h
postinfection (Fig. 2). This protease
assay demonstrated that caspase 3 was in an active form in infected
cells and that it was capable of proteolytically processing other
caspases and substrates.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Caspase Activation and Specific Cleavage of
Substrates after Coxsackievirus B3-Induced Cytopathic Effect in
HeLa Cells
St. Paul's
Hospital,1 and
QLT PhotoTherapeutics
Inc.,2 Vancouver, British Columbia, Canada, and
Department of Pathology and Microbiology, University of
Nebraska Medical Center, Omaha, Nebraska3
ABSTRACT
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-fodrin (12, 66), focal adhesion kinase
(71), and mdm2 (18), among many others. Such cleavage events result in important alterations to normal homeostatic cellular processes and corresponding cell morphological-structural changes.
)-TNF receptor signalling complex, the Bcl-2
family of regulators, or the caspase family of executioners (49,
60). The mechanisms of death of CVB3-infected cells remain to be
determined; however, there is limited morphological evidence regarding
the induction of apoptosis in picornavirus-infected cells. Evidence
obtained with Theiler's murine encephalitis virus (32, 65)
and poliovirus (63) has indicated that picornavirus-infected
cells undergo apoptosis, based on morphological criteria including
nuclear condensation and DNA fragmentation.
20°C for further biochemical analyses.
View larger version (70K):
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FIG. 1.
Release of progeny CVB3 virus, host cell production of
CVB3 viral protein, viral protease cleavage of host eIF4G, and cell
morphology changes following infection with CVB3. (A) Culture medium
was collected and assayed for infectious virus by the agar overlay
plaque assay method. There was an increase in the amount of infectious
virus (in PFU per milliliter) released over the 12-h experiment (B).
Cellular lysate was collected from CVB3-infected HeLa cells, and
immunoblot analysis with a CVB3 polyclonal antibody that recognizes
major viral proteins was performed. (C) Cytosolic extract was then
analyzed for the presence of the 220-kDa eIF4G component of the
translation initiation complex. (D) Contrast microscopy of HeLa cells
at 1, 6, 7, and 12 h postinfection was performed. Note the
extensive cytopathic changes that occurred between 6 and 7 h
postinfection.
View larger version (25K):
[in a new window]
FIG. 2.
Caspase 3 activation and cleavage of the 32-kDa proform
following CVB3 infection of HeLa cells. (A) Ten micrograms of cell
lysate was incubated in 150 µl of reaction buffer containing the
caspase 3-specific substrate Ac-DEVD-AMC. After incubation at 37°C
for 1 h, fluorescence levels were determined with an excitation
wavelength of 380 nm and an emission wavelength of 460 nm. Note the
increase in fluorescence, representing caspase activity, beginning
after 7 h postinfection and increasing to maximum levels by
10 h postinfection. (B) HeLa cell lysates were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to
nitrocellulose. Immunoblotting for the presence of the 32-kDa proform
of caspase 3 demonstrates that this protein is processed between 7 and
12 h postinfection.
Caspase 3 cleaves specific substrates at aspartic acid residues (42). PARP, a nuclear protein involved in DNA repair (13, 69), has been shown to be a substrate for activated caspase 3 as well as other caspases (35). In apoptotic cells, PARP is cleaved from a 116-kDa protein, yielding fragments of 85 and 25 kDa, determined with antibodies for the amino and carboxyl termini of the protein, respectively. In CVB3-infected HeLa cells, PARP degradation, with the appearance of an 85-kDa fragment, was detectable by 9 h postinfection, with further reduction of levels of the 116-kDa peptide by 10 and 12 h postinfection, as determined by immunoblot analysis with mouse monoclonal anti-PARP (1:2,000; Biomol) (Fig. 3).
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DFF is a cytosolic protein which can be cleaved by caspase 3 (37). Once cleaved, this protein releases an endonuclease that migrates to the nucleus, where it can cleave DNA at internucleosomal sites, resulting in DNA fragmentation (16). It has been demonstrated previously that internucleosomal DNA degradation is a cellular feature of picornavirus infection (32). As determined by using rabbit polyclonal anti-DFF (generously provided by Xiaodong Wang, University of Texas Southwestern Medical School, Dallas), DFF is cleaved from a 45-kDa protein, producing a 30-kDa fragment beginning at 9 h following infection, with continued processing and loss of the 45-kDa protein between 10 and 12 h postinfection (Fig. 3).
To determine whether caspases directly produce the characteristic CPE which occurs following picornavirus infection or are activated subsequent to the morphological alterations, cells were treated with the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk). A stock solution (100 mM in dimethyl sulfoxide) of ZVAD.fmk (Bachem) was diluted in complete MEM to concentrations ranging from 0 to 200 µM, and the dilutions were incubated with cells for 30 min prior to infection or light treatment. After CVB3 infection, the cells were washed with PBS, and complete MEM containing 10% FBS and fresh ZVAD.fmk (0 to 200 µM) was then substituted. This peptide has been shown to inhibit the induction of morphological alterations by multiple apoptotic stimuli (46, 54). ZVAD.fmk at concentrations of 50, 100, and 200 µM blocked both caspase activity and cleavage of PARP and DFF in BPD-MA- and light-treated HeLa cells (positive control for apoptosis) (Fig. 4). In CVB3-infected HeLa cells, the cleavage of PARP and DFF was partially prevented by the inhibitor at concentrations of 50 and 100 µM (Fig. 4). At the highest concentration of the inhibitor (200 µM), there was no evidence of PARP or DFF cleavage fragments in CVB3-treated cells at 10 h following infection. Caspase cleavage of structural proteins such as actin, gelsolin, lamin B, and focal adhesion kinase is responsible for the morphological alterations observed following the induction of apoptosis (42, 45). At 2 h following photoactivation of BPD-MA, HeLa cells were condensed, had extensive membrane blebbing, and were releasing from the monolayer (Fig. 5). At increasing concentrations of ZVAD.fmk, this apoptotic phenotype was not apparent, with the cells maintaining a morphology similar to that of the control cells (Fig. 5). CVB3-infected HeLa cells were condensed and were releasing from the monolayer but exhibited no membrane blebbing at 10 h following infection (Fig. 5). Blockade of caspase activity by ZVAD.fmk at concentrations up to 200 µM did not alter the cytopathic phenotype even though cleavage of substrates (DFF and PARP) was inhibited.
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A classic feature of viruses of the family Picornaviridae is the cellular CPE following infection. Since the discovery of an extraneural cell culture technique for the multiplication of poliovirus (17), degenerative changes in cell morphology have been noted. First described by Robbins et al. (48) in 1950, these cytopathic changes include nuclear shrinkage, condensation of chromatin, cell rounding, and release from the monolayer, with eventual progression to acidophilic cytoplasm, nuclear pyknosis, and fragmentation of the nuclear chromatin (karyorrhexis) (47).
The recent understanding of cell death mechanisms sets the stage for examination of host cell death proteins and their possible role in the CPE of CVB3 infection. Many viruses inhibit or activate cell death, strategies that convey distinctive aspects of cell injury, inflammatory responses, or viral persistence. As noted above, previous picornavirus studies have revealed the morphological features of apoptotic cell death, including cell shrinkage, DNA fragmentation, and nuclear condensation (21, 32, 47).
Caspase 3, a cysteine protease with homology to the Caenorhabditis elegans protein Ced-3 (77), is considered one of the key proteins involved in the execution stage of cell death. Apoptosis induced by multiple stimuli, including Fas, TNF, etoposide, staurosporine, photodynamic therapy, ionizing radiation, and growth factor withdrawal, involves pro-caspase 3 processing and subsequent activation (15, 23, 30, 33, 51). Beginning at 7 to 8 h postinfection with CVB3, pro-caspase 3 is depleted, and caspase activation assays have demonstrated that this protein is cleaved into its active form. Several proteins can activate caspase 3, including caspase 8 (via signalling through TNF or Fas receptors) (55), granzyme B from cytotoxic lymphocytes (62), and caspase 9 (via release of mitochondrial cytochrome c and assembly of apoptotic protease activation factors) (36). Once activated, caspase 3 can degrade specific substrates, which in turn results in structural alterations and loss of homeostatic regulation of cellular processes. Numerous proteins have been shown to be cleaved by activated caspases. Consistent with the activation of caspase 3, both PARP and DFF are cleaved following CVB3 infection. Caspase activation and DNA fragmentation are directly linked through the cleavage of DFF (37). DFF is a human cytosolic factor consisting of two subunits of 45 and 40 kDa, the larger of which is degraded into smaller polypeptides by caspase 3. In recent studies by Enari et al. (16) using murine lymphoma cells, an endonuclease, caspase-activated DNase (CAD), was isolated. The murine equivalent of DFF protein was isolated and termed inhibitor of CAD (50). Caspase 3 cleavage of inhibitor of CAD (DFF) allows CAD nuclear translocation and DNA degradation. DFF is cleaved beginning at 9 h postinfection, resulting in a 30-kDa fragment (Fig. 3) which can be further processed to an 11-kDa fragment (37). PARP is located in the nucleus and is involved in DNA repair. Cleavage of PARP begins at 9 h following infection, suggesting that once caspases are activated in the cytosol, they are able to access nucleus-localized substrates.
Of note, caspase inhibition with the general caspase inhibitor ZVAD.fmk did not prevent the CPE induced by CVB3 following infection. At between 6 and 7 h postinfection, the CPE became apparent by contrast microscopy in our CVB3 infection model. The time between infection and appearance of the CPE, as observed by contrast microscopy, was consistent at ZVAD.fmk concentrations of from 50 to 200 µM. In addition to not affecting the time to CPE, ZVAD.fmk treatment resulted in cells with a morphological appearance similar to that of untreated, infected cells (Fig. 5). We used treatment with BPD-MA and light as an alternative method of inducing apoptosis in HeLa cells (22, 23). Inhibition of caspase activation with the inhibitor ZVAD.fmk prevented the apoptotic phenotype (Fig. 5). From these results, we conclude that caspase activity and cleavage of substrates do not account for the characteristic CPE associated with picornavirus infection but instead are activated subsequent to the morphological changes.
The point of intersection of the viral replicative cycle and activation of the host cell death pathway remains to be determined. Picornavirus infection soon results in inhibition of cellular RNA and protein synthesis (19, 74). Early studies of relationships between picornavirus-induced metabolic alterations and virus-induced CPE indicated that the inhibition of protein and RNA synthesis was not directly related to cell morphological changes (4). Protein and RNA synthesis inhibitors delayed cell death, but the cells displayed fewer morphological changes than did picornavirus-infected cells (5). Inhibition of protein and RNA synthesis with any one of multiple agents, such as actinomycin D, puromycin, and diphtheria toxin, results in apoptosis (39). Early studies done in poliovirus infection systems showed that puromycin, an inhibitor of the translation of viral as well as host proteins, delays the onset of cytopathic changes, suggesting that certain viral proteins may be directly cytotoxic (4). Increasing the MOI leads to a more rapid onset of CPE (unpublished observations), although almost all host protein translation is shut off within 3 h at a relatively low MOI (25) such as that used in this study (MOI = 5). Recently it has been shown that the 2B protein encoded by coxsackievirus and poliovirus associates with cellular membrane fractions, including the plasmalemma and endoplasmic reticulum, and disrupts ion movement, including the movement of Ca2+ to the cytosol (1, 67). Ca2+ influx occurs in apoptosis (7, 44), but it is not clear whether the influx occurs prior to or following caspase activation. By examination of the ionic requirements of caspases, it has been determined that the calcium ion concentration has little effect on caspase activity (56). An early calcium influx following coxsackievirus infection could result from the influence of the 2B protein on membrane permeability, and the large late calcium influx noted (>6 h postinfection) (67) could be a downstream effect of caspase activation.
During the early phases of infection, it would be advantageous for the virus to inhibit host cell death, thereby allowing for maximal production of viral progeny. At late stages of the viral life cycle, it would also be beneficial for the virus to induce apoptosis rather than necrosis. Such a mechanism of death is a potential means of host immune system evasion by the virus during its release to the surrounding tissue. Apoptosis is characterized by the rapid phagocytosis of affected cells without the release of proinflammatory cytokines (53).
Viruses have been shown to interact at various levels of the apoptotic pathway. Several gammaherpesviruses (including Kaposi's sarcoma-associated human herpesvirus 8) as well as the tumorigenic molluscum contagiosum virus contain FLICE-inhibitory proteins that interact with the Fas adapter protein FADD and compete to inhibit caspase 8 recruitment and subsequent activation (61). Expression of the cowpox virus serpin CrmA blocks the caspase 8-mediated activation of downstream caspases such as caspase 1 and caspase 3 (55). The IAP (for inhibitors of apoptosis) proteins constitute a family of proteins, expressed by baculoviruses, that block apoptosis induced by viral infection or by caspase 1 (78). Furthermore, several viral homologs of the Bcl-2 family of proteins have been discovered (2, 8, 20, 70). Adenovirus (9, 20, 57), African swine fever virus (41), and Epstein-Barr virus (26, 41, 59) encode proteins (E1B-19K, LMWS-HL, and BHRF1, respectively) that exhibit sequence homology to pro-survival genes of the Bcl-2 family.
The identification of viral proteins that directly induce apoptosis is not as extensively documented as that of viral proteins that inhibit cell death. The lentiviruses human immunodeficiency virus and human T-cell leukemia virus type 1 encode the transcription regulators Tat and Tax, which have been shown to increase expression of Fas ligand while decreasing the expression of Bcl-2 family members (79, 80). The human adenovirus-encoded E1A, E3, and E4 gene products cause cell death following expression in cell culture. The adenovirus death-inducing genes are expressed late in the infection cycle and ultimately overwhelm the virus-encoded death-inhibiting genes (38).
Our data demonstrate that caspase 3 activation follows, rather than precedes, CVB3-induced degenerative morphological changes in infected HeLa cells. Activated caspases process specific substrates, including PARP and DFF. However, inhibition of caspase activity does not eliminate the morphological appearance (CPE) of virus-infected cells, as determined by contrast microscopy. Caspase processing and cleavage of substrates may be important in the ultimate alteration of normal homeostatic processes in infected cells and may facilitate the final clearance of virus-infected cells. The viral proteases 2A, 3C, and 3CD may cleave specific structural proteins, resulting in morphological alterations consistent with CPE, in a fashion analogous to the action of caspases, which cleave separate substrates to achieve a distinct apoptotic phenotype.
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ACKNOWLEDGMENTS |
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We appreciate deeply the technical support of Lubos Bohunek. We thank Xiaodong Wang (University of Texas Southwestern Medical School, Dallas) for the generous gift of DFF antibody.
These studies have been supported by the Heart and Stroke Foundation of British Columbia and Yukon (B.M.M., C.M.C., D.J.G., and K.A.W.), the Medical Research Council of Canada (D.Y. and B.M.M.), and the B.C. Health Research Foundation (D.Y.)
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, Cardiovascular Research Laboratory, University of British Columbia, St. Paul's Hospital, 1081 Burrard St., Vancouver, B.C., Canada V6Z 1Y6. Phone: (604) 631-5200. Fax: (604) 631-5208. E-mail: mcmanus{at}unixg.ubc.ca.
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