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*
Department of Surgery, University of Pittsburgh School of Medicine, and Children’s Hospital of Pittsburgh, Pittsburgh, PA 15213; and
Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, Kangwon-do, Korea
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The caspases consist of a family of at least 14 cysteine proteases that specifically cleave proteins after the second aspartate residue. It is now well-established that they play a key role in apoptosis (9, 10). Caspase-1, or IL-1-converting enzyme (ICE), was initially implicated in mammalian cell apoptosis because of its similarity to the gene product of Caenorhabditis elegans, ced-3 (11, 12, 13). Although experimental evidence supports a role for caspase-1 in apoptosis (14, 15, 16, 17), most data suggest that other caspases, especially caspase-3, may play a more pivotal role in this process than caspase-1 (9). In fact, caspase-3 is believed to be one of the key executioners of apoptosis, being responsible either partially or totally for the proteolytic cleavage of many important proteins, such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP), which is activated during apoptosis. PARP cleaves NAD+ to nicotinamide and ADP-ribose and catalyzes the addition of poly(ADP-ribose) adducts to nuclear proteins, a process which requires energy. Cleavage of PARP by caspases is recognized as a hallmark of apoptosis (18, 19). Caspase-activated deoxyribonuclease (CAD), also known as caspase-activated nuclease or DFF-40, is another enzyme that can be activated by caspases. CAD is normally complexed with its inhibitor, ICAD (45 kDa), in the cytoplasm of growing, nonapoptotic cells (20, 21, 22). Activation of caspases as a result of apoptotic stimuli, in particular, activation of caspase-3, cleaves ICAD to a 34-kDa fragment (20). CAD, thus released from ICAD, migrates to the nucleus where it digests chromosomal DNA into nucleosomal units (21, 22).
We previously reported that NO induces apoptosis in thymocytes via a p53-dependent pathway.4 However, the role of caspases, if any, in NO-induced thymocyte apoptosis is still undefined. In this report, we demonstrate that NO induces thymocyte apoptosis via a caspase-1- but not caspase-3-dependent pathway. Caspase-1 derived from the cytosolic fraction of thymocytes treated with the NO donor S-nitroso-N-acetyl penicillamine (SNAP) was able to cleave ICAD and lead to DNA fragmentation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The NO donor SNAP was synthesized as described previously (24). SNAP was dissolved in DMSO (Sigma, St. Louis, MO), aliquoted, and kept frozen at -20°C. Active recombinant human caspase-1 (rh-caspase-1) and rh-caspase-3 were kindly provided by Dr. Robert Talanian (BASF Bioresearch Corporation, Worcester, MA) (25). For Western blot analysis, the following Abs were obtained: mouse mAb to PARP (Oncogene, Cambridge, MD) and goat anti-mouse HRP-conjugated secondary Ab (Pierce, Rockford, IL), rabbit polyclonal Abs to caspase-1 and caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA), and goat anti-rabbit HRP-conjugated secondary Ab (Pierce). The caspase inhibitors ZVAD-fmk, YVAD-fmk, DEVD-fmk, VEID-fmk, LEHD-fmk, IETD-fmk, ZFA-fmk, Ac-DEVD-cho, and Ac-YVAD-cho and the caspase substrates Ac-DEVD-pNA and Ac-YVAD-pNA were purchased from Alexis Biochemicals (San Diego, CA). ICAD was obtained from Dr. X. Wang (University of Texas Southwestern Medical Center, Dallas, TX).
Animals
Four-week-old BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME); 8- to 12-wk-old caspase-1 (ICE) knockout (KO) 164 BBC mice and their wild-type (WT) control KOH (ICE) 164 BBC mice were obtained from BASF Bioresearch Corporation. Male Sprague Dawley rats (Harlan Sprague-Dawley, Madison, WI) were purchased and were used at between 200 and 250 g. The animals were housed in a specific pathogen-free facility and were fed rodent chow and water ad libitum.
Thymocyte preparation
Briefly, single cell suspensions of thymocytes were prepared in RPMI 1640 (BioWhittaker, Walkersville, MD). The cells were washed three times and cultured in duplicate at a final concentration of 5 x 106 cells/ml in a final volume of 2 ml. The culture medium, which consisted of RPMI 1640 supplemented with 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM nonessential amino acids, 2 mM L-glutamine, and 5 x 10-5 M 2-ME, was supplemented with 5% FBS.
Preparation of S-100 fraction from thymocytes and hepatocytes
The procedure was conducted as previously described for hepatocytes (25). Briefly, freshly isolated thymocytes or hepatocytes (2 x 108 cells) were washed twice with ice-cold PBS and resuspended in 5 volumes of ice-cold buffer D (20 mM HEPES (pH 7.4), 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 4 mM EGTA, and protease inhibitors). After incubating for 20 min, cells were homogenized on ice by six strokes with a tight pestle in a Dounce homogenizer. The nuclei, intact cells, and cell debris were removed by centrifugation at 1,000 x g for 10 min at 4°C. The supernatant was further centrifuged at 100,000 x g for 1 h in a Beckman 70 Ti rotor (Beckman, Palo Alto, CA). The supernatant (S-100 fraction) was immediately frozen in liquid nitrogen and stored at -80°C for subsequent use in the cell-free apoptosis assay in the reconstitution system (see below).
Preparation of rat hepatocyte nuclei
The procedure for isolating rat hepatocyte nuclei has been described (25). Freshly isolated and purified rat hepatocytes (1.5 x 108 cells) were washed twice in ice-cold PBS and resuspended in 10 volumes of buffer E (15 mM PIPES (pH 7.4), 80 mM KCl, 15 mM NaCl, 5 mM EDTA, 0.15 mM spermidine, 0.15 mM spermine, 1 mM PMSF, and 25 mM sucrose). Cells were allowed to swell on ice for 20 min and then were homogenized with a Dounce homogenizer. Greater than 90% lysis was confirmed by microscopy. The homogenates were layered over 5 ml of buffer E containing 2.3 mM sucrose in a Beckman SW28 centrifuge tube and centrifuged at 90,000 x g for 90 min at 4°C. The pellets were washed with buffer E at 800 x g for 10 min. The pellets containing the nuclei were resuspended in buffer E at a concentration of 1 x 108 nuclei/ml and immediately used in an in vitro apoptosis assay.
Caspase-1 and caspase-3 activity
Caspase activity was measured as previously described (25). Briefly, the cells were washed with PBS, collected by plastic scraper, and subsequently pelleted by centrifugation at 400 x g for 10 min at 4°C. The cell pellets were washed with ice-cold PBS and resuspended in 100 mM HEPES buffer (pH 7.4) containing protease inhibitors (5 µg/ml aprotinin, 5 µg/ml pepstatin A, 10 µg/ml leupeptin, and 0.5 mM PMSF). The cell suspension was lysed by three freeze/thaw cycles, and the cytosolic fraction was obtained by centrifugation at 12,000 x g for 20 min at 4°C. Protein concentration was determined with the bicinchonic acid protein assay reagent (Pierce). Two hundred micrograms of cytosolic protein was combined with 400 µM of the synthetic substrate Ac-YVAD-pNA or Ac-DEVD-pNA in 150 µl of 100 mM HEPES (pH 7.4) containing 20% glycerol and protease inhibitors, and the reaction was conducted for 1 h at 37°C. Cytosolic caspase-1 and caspase-3 activity was assayed by measuring the increased absorbance at 405 nm. In preliminary experiments, we confirmed that thymocytes from caspase-1 KO mice had hardly any detectable caspase-1 activity, but they had normal caspase-3 activity.
Induction of apoptosis in a cell-free reconstitution system
This procedure was conducted as previously described
(25). The reaction mixture contained 40 µl of S-100
fraction (
5 mg/ml); 10 µl of nuclei solution (1 x
106 nuclei); and 400 ng of pretreated
rh-caspase-1 or rh-caspase-3, 15.6 µM Ac-YVAD-cho, or 15.6 µM
Ac-DEVD-cho in the final concentration, in a final volume of 80 µl of
buffer F (10 mM HEPES (pH 7.4), 40 mM b-glycerophosphate, 50 mM NaCl, 2
mM MgCl2, 4 mM EGTA, 2 mM ATP, 10 mM creatinine
phosphate, 50 µg/ml creatinine kinase, and 0.2 mg/ml BSA). The
mixtures were incubated at 37°C for 140 min and were occasionally
mixed. The reaction solution was then mixed with 500 µl of buffer G
(50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% SDS, and 0.2 mg/ml of
proteinase K) and incubated at 37°C for 1 h. The solution was
extracted with phenol/chloroform. DNA isolation and electrophoresis
were conducted as described previously.
DNA fragmentation assay
The protocol was adapted from McCarthy et al. (26). Briefly, after incubation, the cells were washed three times in PBS; lysed with a buffer containing 5 mM Trizma base, 1 mM EDTA, and 0.5% Triton X-100; and centrifuged to obtain supernatant and pellet fractions. Both fractions were sonicated (Sonics & Materials, Danbury, CT) for 60 s on ice and plated in 0.1 ml triplicate serial dilutions in Dynatech MicroFluor 96-well plates (Dynatech Laboratories, Alexandria, VA). An equal volume (0.1 ml) of a 0.6 µg/ml solution of the fluorescent DNA tag 4',6'-diamidino-2-phenylindole dye (Sigma), suspended in a buffer containing 10 mM Trizma base and 100 mM NaCl, was then added to the samples. Relative pellet and supernatant DNA concentrations were calculated from the emission at 465 nm, as measured on a Dynatech MicroFluor plate reader. After mathematical conversion, percent DNA fragmentation was quantitated as follows: % DNA fragmentation = [(DNA in supernatant)/(DNA in supernatant + DNA in pellet)] x 100.
Western blot
Whole cell lysates were obtained by incubating the cells in a buffer containing 50 mM Tris, 30 mM NaCl, 10 mM SDS, 100 mM Triton X-100, and protease inhibitors (6 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.5 mM PMSF (Sigma)). The samples were boiled in sample buffer for 60 s and loaded. Western blots were performed using 12% SDS-PAGE; 60 µg protein was loaded per lane. Equal loading was ensured by protein measurement using bicinchonic acid as substrate and BSA (Pierce Biochemicals, Madison, WI) as standard, and they were read at 465 nm on a V-max microplate reader. After protein transfer to nitrocellulose, the following Abs and their corresponding dilution in 1% milk-PBS-0.1% Tween 20 were used: mouse mAb to PARP, 1:500; rabbit polyclonal Ab to caspase-1, 1:500; and rabbit polyclonal Ab to caspase-3, 1:500. The membranes were washed and incubated in the appropriate HRP-conjugated secondary Ab diluted 1:5000 in 1% milk PBS-0.1% Tween 20 for 1 h at room temperature. Ab binding was detected with enhanced chemiluminescent reagent (Amersham Life Science, Arlington Heights, IL) and developed on Kodak X-Omat film for a period of 30 s to 10 min. Band density volume was measured using a Personal Densitometer SI, (Molecular Dynamics, Sunnyvale, CA) and was expressed as arbitrary units.
Irradiation
Thymocytes were exposed to a total of 5 Gy gamma radiation utilizing a cesium emitter irradiator (Gamma Cell 1000 Elite; Nordion International, Vancouver, Canada).
Statistical analysis
Data in the figures or tables depict the results from either a representative experiment or combined data from three or more experiments as indicated in the figure legends. Significance was determined by the Student t test using the StatView statistics program (Abacus Concepts, Berkeley, CA). Statistical significance was established at a p value of <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether caspases were involved in NO-induced
apoptosis, thymocytes from BALB/c mice were treated with the NO donor
SNAP and various concentrations of the pan-caspase inhibitor ZVAD-fmk;
DNA fragmentation was then determined (Table I
). SNAP treatment increased the rate of
apoptosis from 22 to 46%. ZVAD-fmk inhibited NO-induced thymocyte
apoptosis in a dose-dependent manner. Similarly, Ac-YVAD-cho, a
caspase-1 inhibitor, significantly reduced NO-induced thymocyte
apoptosis. However, ZVAD-fmk was slightly more effective than
Ac-YVAD-cho in suppressing apoptosis at similar concentrations (15.6
µM). In contrast, Ac-DEVD-cho, a caspase-3 inhibitor, had almost no
inhibitory effect on SNAP-induced thymocyte apoptosis. In fact, 1 mM
Ac-DEVD-cho was required to completely suppress SNAP-induced apoptosis
(data not shown). Similarly, DEVD-fmk, IETD-fmk, VEID-fmk, and ZFA-fmk
had no significant effect on SNAP-induced apoptosis (data not shown).
These data indicate that caspases, especially caspase-1, are involved
in NO-induced thymocyte apoptosis.
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). In contrast, Ac-YVAD-cho completely
blocked NO-induced apoptosis when added 8 h after SNAP treatment;
this protective effect gradually diminished at 12 h and was
completely lost by 16 h.
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We measured the kinetics of caspase-1 and caspase-3 activity in
thymocytes after SNAP treatment. A nearly 2-fold increase over baseline
caspase-1 activity was observed as early as 4 h after SNAP
treatment (Fig. 1). Caspase-1 activity
peaked by 8 h and remained elevated at 12 h and diminished at
20 h, although its activity was still 3-fold higher than
that of the media group (data not shown). Interestingly, no
increase over basal caspase-3 activity was observed during the same
periods after SNAP treatment (Fig. 1). In fact, caspase-3 activity at
4, 8, and 12 h was negligible. The results indicate that
caspase-1, but not capase-3, is activated in SNAP-treated
thymocytes.
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). In contrast, Western blot for
caspase-3 showed no cleavage of pro-caspase-3 in SNAP-treated
thymocytes (Fig. 3).
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The foregoing experiments suggested that activation of caspase-1
after SNAP treatment could lead to thymocyte apoptosis. Because
caspase-3 is believed to be the principal caspase that can cleave ICAD
and thus allow CAD to translocate to the nucleus and induce DNA
fragmentation, we attempted to determine whether caspase-1 alone
could also cleave ICAD. Fig. 4A demonstrates that both
caspase-1 and caspase-3 can cleave ICAD and that such cleavage can be
inhibited by their respective specific inhibitors, Ac-YVAD-cho, and
Ac-DEVD-cho.
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B). The addition of rh-caspase-3 or
rh-caspase-1 to the mixture resulted in DNA fragmentation that could be
blocked by the specific inhibitors of caspase-1, Ac-YVAD-cho, or
caspase-3, Ac-DVED-cho.
To further determine whether caspase-1 is responsible for cleavage of
ICAD in thymocytes after SNAP treatment, cells were incubated with 0.5
mM SNAP for 8 h, the time when maximal caspase-1 activity and
cleavage of pro-caspase-1 were detected. The cytosolic fraction (which
contains the caspases) was harvested and incubated with ICAD in the
presence or absence of caspase-1 or caspase-3 inhibitors. Cytosolic
fraction from SNAP-treated thymocytes was able to cleave ICAD (Fig. 4C, lane 2). ICAD cleavage was blocked by
Ac-YVAD-cho (lane 3), but not by Ac-DEVD-cho
(lane 4). In subsequent experiments, we also examined
the ability of DEVD-fmk or YVAD-fmk, as well as inhibitors of caspase-6
(VEID-fmk) or caspase-9 (LEHD-fmk), to cleave ICAD. Fig. 5 shows that cytosolic fractions from
SNAP-treated thymocytes cleaved ICAD to the P-11 fragment, similarly to
rh-caspase-1 (Fig. 4A). YVAD-fmk nearly completely inhibited
ICAD cleavage. LEHD-fmk was somewhat less effective than YVAD-fmk,
whereas DEVD-fmk and VEID-fmk had little to no effect. Neither
significant caspase-9 cleavage nor activity was detected in
SNAP-treated thymocytes compared with control (data not shown). Taken
together, these data suggest that caspase-1 was likely responsible for
DNA fragmentation in SNAP-treated thymocytes.
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Caspases can lead to PARP cleavage, a phenomenon that correlates
well with the process of apoptosis. To determine whether the activation
of caspase-1 in thymocytes results in PARP cleavage, thymocytes were
treated with SNAP for 12 h and PARP cleavage was examined by
Western blot (Fig. 6). PARP is a 117-kDa
molecule that can be cleaved to 85-kDa and 31-kDa fragments,
corresponding to the N-terminal DNA binding domain and the
C-terminal catalytic domain of the enzyme, respectively. Fig. 6
reveals some PARP cleavage in the untreated thymocytes; however, PARP
cleavage is greatest in SNAP-treated thymocytes at 8 and 12 h. To
determine whether inhibition of caspase-1 could block PARP cleavage,
thymocytes were pretreated with Ac-YVAD-cho before addition of SNAP or
media; PARP cleavage was measured at 12 h. Fig. 7 shows that Ac-YVAD-cho blunted PARP
cleavage in SNAP-treated thymocytes. In fact, addition of Ac-YVAD-cho
up to 8 h after SNAP treatment reduced PARP cleavage (Fig. 8).
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To confirm that caspase-1 is required for SNAP-induced apoptosis,
thymocytes were harvested from both caspase-1 WT and KO mice and then
treated with media, SNAP, or irradiation; DNA fragmentation was then
measured at 24 h. Thymocytes from both WT and KO mice had a
spontaneous apoptosis rate of about 20% (Fig. 9). Because irradiation induces apoptosis
in thymocytes via a caspase-3-dependent pathway, not surprisingly, 98%
of WT and 86% of caspase-1 KO thymocytes underwent apoptosis after
exposure to 5 Gy (Fig. 9). In contrast, thymocytes from caspase-1 KO
mice did not undergo apoptosis after SNAP treatment (20.5 ±
6.4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Caspase-1 activity was four times higher in SNAP-treated thymocytes at 8 and 12 h compared with untreated cells. Caspase-1 activity decreased thereafter. Both ZVAD-fmk and Ac-YVAD-cho inhibited NO-induced apoptosis when added up to 12 h after SNAP treatment. The inhibitory effect was lost after 16 h. PARP cleavage takes place after the amino acid sequence Ac-DEVD-cho. Originally this activity was attributed to caspase-3, but caspases-2,-4,-6,-7,-8, and -10, when added at high concentrations, can also cleave PARP (27). Because the rate of spontaneous apoptosis in our thymocyte cultures was 20%, it is not surprising that some (mild) PARP cleavage was found in untreated thymocytes. However, there was greater PARP cleavage after SNAP treatment, and it was blocked by Ac-YVAD-cho, even when added 8 h after SNAP treatment. Because neither caspase-3 nor caspase-9 was significantly activated during SNAP treatment, caspase-1 (or other caspases) was likely responsible for the cleavage of PARP in our cultures.
The decrease in caspase-3 activity in SNAP-treated thymocytes is consistent with our previous findings that NO inhibits caspase-3 activity in vitro by S-nitrosylation of the active cysteine site (24). Seven caspases have been shown to be inactivated by nitrosylation (28). Caspase-3 appears to be more sensitive than caspase-1 to NO-mediated inhibition of the catalytic activity (24, 28). Recently, caspase-3 zymogens were found to be S-nitrosylated at their catalytic cysteine site in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway (29). Thus, protein S-nitrosylation/denitrosylation can play a regulatory role in signal transduction pathways. Caspase-3 activity in thymocytes was dramatically decreased after SNAP treatment. It is possible that SNAP not only increased caspase-3 S-nitrosylation, which resulted in a decrease in intracellular caspase-3 activity, but that it also blocked the cleavage of the caspase-3 zymogen to its active subunits.
The mechanism by which caspase-1 is activated in thymocytes after SNAP treatment is not clear. NO has been shown to trigger disruption of the mitochondrial transmembrane potential (23). Thus, it is possible that NO induces release of cytochrome c and apoptosis protein activating factor-1, which leads to subsequent activation of downstream caspases including caspase-1.
However, significant caspase-9 activity was not detected. Caspase-1 was the first human caspase identified and was shown to induce apoptosis when overexpressed in the cell. However, few reports have described that caspase-1 is required or sufficient to induce apoptosis. Recently, Salmonella typhimurium (16) and Shigella flexneri (17) were reported to induce apoptosis in macrophages via a caspase-1-dependent pathway. It is possible that caspase-1 may not be required in most forms of apoptosis or that other caspases may substitute for caspase-1 in caspase-1 KO mice. Because caspase-1 can cleave IL-1ß to its mature form, it is believed to play a role mainly in inflammation rather than in the apoptosis process. It was reported that both caspase-1 WT and KO mice show a similar susceptibility to apoptosis induced by gamma-irradiation or dexamethasone. Furthermore, Virag et al. (8) suggested that peroxynitrite-induced apoptosis in thymocytes is dependent on caspase-3, but not caspase-1. However, they did not examine caspase-1 KO mice, nor did they measure activity of caspase-1 or the effect of inhibitors of caspase-1 in their cultures. In this study, we demonstrated that thymocytes from caspase-1 KO mice do not undergo apoptosis after SNAP treatment and that caspase-1 alone can cleave ICAD and lead to DNA fragmentation. Caspase-1 may play a key role in NO-induced thymocyte apoptosis, although the physiologic significance of this pathway in vivo is not clear.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Henri R. Ford, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213.
3 Abbreviations used in this paper: iNOS, inducible NO synthase; ICE, IL-1-converting enzyme; PARP, poly(ADP-ribose) polymerase; CAD, caspase-activated deoxyribonuclease; ICAD, inhibitor of CAD; SNAP, S-nitroso-N-acetyl penicillamine; rh-caspase, recombinant human caspase; KO, knockout; WT, wild type.
4 S. A. Gordon, W. Abou-Jaoude, R. A. Hoffman, S. A. McCarthy, Y. M. Kim, X. L. Zhang, R. L. Simmons, X. Zhou, and H. R. Ford. Nitric oxide induces oxidative injury and mediates murine thymocyte apoptosis via a p53- and Bax-dependent mechanism. Submitted for publication.
Received for publication August 27, 1999. Accepted for publication May 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, endotoxin, and interaction with allogeneic T cells: nitric oxide production is associated with dendritic cell apoptosis. J. Immunol. 157:3577.[Abstract]
/lipopolysaccharide. J. Neurochem. 71:88.[Medline]
-inducing factor (IL-18) release from macrophages by inhibiting caspase-1 (IL-1ß-converting enzyme). J. Immunol. 161:4122.This article has been cited by other articles:
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  Z. Liu, R. Dawes, S. Petrova, P. C. Beverley, and E. Z Tchilian CD45 regulates apoptosis in peripheral T lymphocytes Int. Immunol., June 1, 2006; 18(6): 959 - 966. [Abstract] [Full Text] [PDF]
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 Y. Ishihara and N. Shimamoto Involvement of Endonuclease G in Nucleosomal DNA Fragmentation under Sustained Endogenous Oxidative Stress J. Biol. Chem., March 10, 2006; 281(10): 6726 - 6733. [Abstract] [Full Text] [PDF]
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 P. N. Rocha, T. J. Plumb, L. A. Robinson, R. Spurney, D. Pisetsky, B. H. Koller, and T. M. Coffman Role of Thromboxane A2 in the Induction of Apoptosis of Immature Thymocytes by Lipopolysaccharide Clin. Vaccine Immunol., August 1, 2005; 12(8): 896 - 903. [Abstract] [Full Text] [PDF]
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 S. A. Gordon, W. Abou-Jaoude, R. A. Hoffman, S. A. McCarthy, Y.-M. Kim, X. Zhou, X.-R. Zhang, R. L. Simmons, Y. Chen, L. Schall, et al. Nitric oxide induces murine thymocyte apoptosis by oxidative injury and a p53-dependent mechanism J. Leukoc. Biol., July 1, 2001; 70(1): 87 - 95. [Abstract] [Full Text] [PDF]
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