From the Unité des Venins, Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France and the § Laboratoire
de Neurobiologie Cellulaire et Moléculaire, CNRS URA 1857, 46 rue
d'Ulm, 75005 Paris, France
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ABSTRACT |
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The venom of the snake Bungarus fasciatus contains a hydrophilic, monomeric species of acetylcholinesterase (AChE), characterized by a C-terminal region that does not resemble the alternative T- or H-peptides. Here, we show that the snake contains a single gene for AChE, possessing a novel alternative exon (S) that encodes the C-terminal region of the venom enzyme, located downstream of the T exon. Alternative splicing generates S mRNA in the venom gland and S and T mRNAs in muscle and liver. We found no evidence for the presence of an H exon between the last common "catalytic" exon and the T exon, where H exons are located in Torpedo and in mammals. Moreover, COS cells that were transfected with AChE expression vectors containing the T exon with or without the preceding genomic region produced exclusively AChET subunits. In the snake tissues, we could not detect any glycophosphatidylinositol-anchored AChE form that would have derived from H subunits. In the liver, the cholinesterase activity comprises both AChE and butyrylcholinesterase components; butyrylcholinesterase corresponds essentially to nonamphiphilic tetramers and AChE to nonamphiphilic monomers (G1na). In muscle, AChE is largely predominant: it consists of globular forms (G1a and G4a) and trace amounts of asymmetric forms (A8 and A12), which derive from AChET subunits. Thus, the Bungarus AChE gene possesses alternatively spliced T and S exons but no H exon; the absence of an H exon may be a common feature of AChE genes in reptiles and birds.
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INTRODUCTION |
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Acetylcholinesterase (AChE)1 (EC 3.1.1.7) is an essential component of cholinergic synapses, in the nervous tissues and muscles of vertebrates (1). This enzyme is also found in nonsynaptic contexts, where its function is unclear. In the blood of mammals, AChE exists in the form of soluble tetramers (G4na), probably originating from the liver, and of membrane-bound dimers (G2a), anchored by a glycophosphatidylinositol (GPI) to the surface of erythrocytes and lymphocytes (2); these enzymes could serve as a safeguard against any diffusion of acetylcholine from synapses into the circulation. The venoms of various Elapidae from the genera Bungarus, Hemachatus, Naja, and Ophiophagus represent a particularly rich source of nonsynaptic AChE (3, 4).
The presence of AChE in snake venoms is mysterious because it is nontoxic by itself and does not enhance the toxicity of other venom components. This enzyme has been characterized as a true AChE, possessing the characteristic catalytic activity of AChEs from cholinergic tissues of other species: it hydrolyses acetylcholine faster than propionylcholine or butyrylcholine and it is inhibited by eserine (5). Moreover, the primary sequences of Naja and Bungarus venom AChEs present a strong homology to those of other AChEs, as shown by analysis of partial peptidic sequences (5, 6) and by analysis of the complete sequence of Bungarus AChE deduced from cDNA clones (7).
The cloning of AChE from Bungarus venom revealed, however,
that this homology is limited to the catalytic domain and that the
C-terminal sequence is entirely different from both C-terminal H- and
T-peptides, which are encoded by alternatively spliced exons in the
single AChE gene and characterize AChEH and
AChET subunits of other vertebrates (review in Ref. 1).
These C-terminal peptides determine the mode of posttranslational
processing and quaternary associations of AChE catalytic subunits.
Thus, AChEH subunits are modified by cleavage and addition
of a GPI anchor, as well as by the formation of an intersubunit
disulfide bond, generating GPI-anchored dimers (8). The
AChET subunits produce monomers and a variety of
disulfide-linked oligomeric forms, including homo-oligomers (dimers or
tetramers) and hetero-oligomers, which incorporate structural collagen
subunits (collagen-tailed forms) or hydrophobic subunits
(hydrophobic-tailed tetramers). These hetero-oligomeric forms are
tethered to extracellular matrices at neuromuscular synapses (9) or
attached to cellular membranes, particularly in the brain (10). The
T-peptide may adopt an amphiphilic helical structure, thus
explaining the observation that monomers and dimers of
AChET subunits can interact with detergent micelles and
membranes phospholipids (1). In contrast with all molecular forms that
are normally produced by AChEH and AChET
subunits, the venom AChE consists of soluble, hydrophilic monomers.
This is clearly related to the fact that the venom AChE possesses a specific C-terminal peptide, which we called SARA after its last four
residues: this peptide is highly hydrophilic and does not contain any
cysteine residue that could establish intersubunit disulfide bonds. It
defines AChES subunits, which produce only soluble monomers
when expressed in COS cells (7).
The presence of the SARA sequence, replacing the H or T sequences, which are encoded by alternative exons in Torpedo and mammalian AChE genes, raises the problem of the relationship between the venom enzyme and the AChE molecules that occur in cholinergic synapses of the snake. Several hypotheses may be considered to explain the production of this unusual type of AChE in venom glands. First, although previously studied vertebrates possess a single AChE gene (11-14), the snake might possess two distinct AChE genes, expressed in cholinergic tissues and the venom glands, respectively. Such a duplication would be similar to the duplication of cholinesterase genes, which generate the twin enzymes AChE and butyrylcholinesterase (BChE) (EC 3.1.1.8) in vertebrates (15). Second, the venom enzyme may derive from the same gene as AChE in other tissues. In this case, the SARA sequence could be encoded by a novel type of alternative exon or by "readthrough" transcripts. Readthrough transcripts, in which the genomic sequence following the common catalytic exons is maintained, have been characterized in Torpedo electric organs (16), in mouse MEL cells (13) and embryonic diaphragm (17), and in rat embryonic liver (18). Readthrough transcripts are expected to produce nonamphiphilic, monomeric AChE, but the corresponding proteins have never been characterized in vivo.
In the present report, we show that Bungarus possesses a single AChE gene containing a novel alternative exon, S, localized downstream of the T exon. We identified the alternative splicing of the AChE transcripts in the venom glands, the liver, and the muscles, and we characterized the resulting molecular forms in vivo, as well as in COS cells expressing various constructs.
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MATERIALS AND METHODS |
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RNA Purification-- Bungarus fasciatus snakes were kindly provided by Prof. Xiong Yu-Liang and Dr. Zhang Yun (Kunming Institute of Zoology, Academia Sinica, Kunming, Yunnan, China). They were sacrificed in China, and the tissues (venom gland, muscle and liver) were immediately frozen and transported in dry ice. Total RNA was extracted using RNAsol (Bioprobe), according to the method of Chomczynski and Sacchi (19).
Reverse Transcription and PCR Experiments-- For reverse transcription-PCR experiments, 1 µg of total RNA was reverse transcribed using 200 units of Superscript-Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) with 10 pmol of oligo-dT, 2 pmol of specific primer, or 25 pmol of hexanucleotides, as described in the legend to Fig. 4. PCR was performed essentially as described previously (7) with Taq polymerase from Promega in a PTC150 thermocycler (M. J. Research).
RNase Protection Assay-- For RNase protection assays, we introduced exon 4 and the T or S exon, under control of T7 promoter to produce antisense probes. 10 µg of these constructs (described in Fig. 5) were digested overnight with 20 units of EcoRI in a final volume of 30 µl. The DNA was extracted with phenol and chloroform and then precipitated by ethanol in the presence of ammonium acetate. DNA was resuspended in 20 µl of RNase-free water and quantified using a GeneQuant spectrophotometer (Amersham Pharmacia Biotech). For probe synthesis, 3 µg of DNA were incubated with 100 µCi of [32P]dUTP (800 Ci/mmol; Amersham Pharmacia Biotech) and 150 units of T7 RNA polymerase (Promega) during 1 h at 37 °C and then digested using 2 units of RNase-free DNase (Ambion). The probe was purified by denaturing polyacrylamide gel electrophoresis and eluted for 2 h at 37 °C in a solution containing 0.5 M ammonium acetate, 0.2% SDS, and 1 mM EDTA. Total RNA (10 µg) was then co-precipitated overnight with 100,000 cpm of probe (200,000 cpm in the case of venom gland RNA, in which the AChE mRNA is more abundant) with 0.5 M ammonium acetate. The pellet was then resuspended in Hybspeed buffer (Ambion). Further incubations and RNase digestions were performed following precisely the instructions of the manufacturer. Samples of the reaction mixtures were then loaded on a sequencing denaturing polyacrylamide gel. After electrophoresis, the gel was dried and exposed to a Fuji imaging plate, which was read after 1 h. Each band was then quantified using the TINA program. The signal was corrected according to the amount in U bases in the protected fragment.
Tissue Extraction-- Tissues and transfected cells were extracted in a low salt detergent buffer (50 mM Tris-HCl, pH 7.0, 5 mM MgCl2, 1% Triton X-100), and in some cases the pellet was re-extracted in a high salt buffer (same as above, with 1 M NaCl).
Sedimentation and Electrophoretic Analyses--
AChE and BChE
were analyzed by sedimentation in 5-20% sucrose gradients containing
10 mM Tris-HCl, pH 7.0, and 5 mM
MgCl2, either without detergent or in the presence of 0.2%
Triton X-100 or 1% Brij-96. AChE and BChE activities were assayed in
the presence of specific inhibitors as indicated below. For analysis of
asymmetric forms, the gradients contained 0.4 M NaCl and
1% Triton X-100. E. coli alkaline phosphatase (6.1 S) and
E. coli -galactosidase (16 S) were included as internal
sedimentation standards. After centrifugation at 36,000 rpm for 18 h at 7 °C in a Beckman SW41 rotor, 45 fractions were collected from
the bottom of the tubes and assayed for the different enzymatic
activities. Electrophoresis in nondenaturing polyacrylamide gels was
performed as described previously (24, 25). The gels contained 0.25%
Triton X-100 with or without 0.05% deoxycholate, and they were
electrophoresed for approximately 2-3 h under 15 V/cm, with cooling at
15 °C. AChE activity was revealed by the histochemical method of
Karnovsky and Roots (26).
Collagenase and PI-PLC Treatment-- Collagenase form III (27) was purchased from Advance Biofactures Co. (Lynnbrook, NJ). A high salt extract containing AChE was incubated with 40 units of collagenase in a buffer containing 50 mM Tris-HCl, pH 8, and 5 mM CaCl2, for 1 h at 26 °C. Treatment with PI-PLC was performed as described previously (28).
Assays of AChE and BChE Activity--
AChE and BChE were assayed
by the colorimetric method of Ellman et al. (29).
Acetylthiocholine was used as a substrate for both enzymes. AChE was
assayed in the presence of the specific anti-BChE inhibitor iso-OMPA
(105 M), and BChE was assayed in the presence
of the specific anti-AChE inhibitor BW284C51
(1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide)
(10
5 M).
Genomic Structure and Plasmidic Constructs-- Genomic DNA was extracted and isolated by a salting-out protocol (30): liver was crushed in liquid nitrogen and transferred in 10 volumes of extraction buffer (10 mM Tris-HCl, pH 8.0, 0.1 M EDTA, 20 µg/ml pancreatic RNase, 0.5% SDS, and 0.5 M NaCl) and incubated at 50 °C for 30 min. Proteinase K was added at a final concentration of 100 µg/ml, and incubation was performed overnight at 50 °C. Saturated NaCl (>6 M) was then added (1/4 volume) and agitated. After centrifugation (15 min at 5000 rpm), 2 volumes of cold ethanol were added to the supernatant, which was then kept on ice for 10 min. After centrifugation (15 min at 5000 rpm), the DNA pellet was redissolved in Tris-EDTA buffer.
PCR was performed with primer oligonucleotides corresponding to sequences of exons 3, 4, T, and S. To search for the presence of a putative exon H between exons 4 and T, we made several constructs by inserting various 3' sequences, using a unique BglII site, located in exon 4, as shown in Fig. 7.Transfection of COS Cells--
COS cells were transfected by the
DEAE-dextran method, as reported previously (31), using 5 µg of DNA
encoding the catalytic subunit AChET with or without DNA
encoding the QN/HC binding protein (31, 32), as
specified. The cells were maintained at 37 °C and extracted 2-4
days after transfection. The culture medium (7 ml/10-cm dish containing
about 5 × 106 cells) was collected after variable
periods of time, as indicated, for analysis of released AChE activity.
The extracts and culture media were stored at 80 °C.
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RESULTS |
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Molecular Forms of Cholinesterases in Bungarus Liver and Muscles-- Extracts from snake liver were found to hydrolyze butyrylthiocholine, as well as acetylthiocholine, indicating the presence of both AChE and BChE. As shown in Fig. 1, an AChE component sedimented at 4.5 S (about 80% of the total cholinesterase activity), and a BChE component sedimented at 10.9 S (about 20% of the total activity); only the latter component hydrolyzed butyrylthiocholine (not shown). The sedimentation patterns were not modified by incubation with PI-PLC (not shown) and were identical in the presence of Triton X-100 (Fig. 1), in the presence of Brij-96, or without detergent (not shown), indicating that both components were nonamphiphilic, corresponding to a monomeric form of AChE (G1na) and a tetrameric form of BChE (G4na). The residual activity observed around 11 S in the presence of iso-OMPA, a specific inhibitor of BChE, and the fact that BW284C51, a specific inhibitor of AChE, reduced the cholinesterase activity of the same fractions suggests the presence of a small contribution of tetrameric AChE (G4na). The absence of amphiphilic forms and the fact that PI-PLC had no effect on these profiles indicated that the snake liver did not produce any GPI-anchored form of AChE.
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AChE Transcripts Are Generated by a Single Gene in Bungarus Muscles, Liver and Venom Glands-- A Southern blot of digested Bungarus genomic DNA was hybridized with a probe corresponding to nucleotides 1610-1727 of the AChE cDNA from venom glands, within the coding region of the catalytic domain. We obtained a single labeled band after digestion by EcoRI and BamHI, suggesting that Bungarus possesses a single gene for AChE (not shown).
Experiments involving 3' rapid amplification of cDNA ends were unsuccessful to identify the 3' region of the coding sequence of AChE cDNA in muscle. To obtain the complete coding sequence, we amplified a cDNA fragment encoding the C-terminal region of muscle AChE, by reverse transcription-PCR. Reverse transcription of mRNA was performed with random hexanucleotides associated with Ri and Ro sequences for PCR priming (20) (Fig. 4A). PCR was then performed with Ri and Ro reverse primers and a forward primer corresponding to a fragment from the AChE cDNA previously cloned from venom gland (7). We thus amplified a fragment of about 200 bp, which was subcloned and sequenced (Fig. 4B). According to this sequence, the end of the catalytic domain is identical to that of the venom cDNA clone, but it is associated with a different C-terminal region. In agreement with our analysis of AChE forms in muscles, this region corresponds to a T-peptide, as shown by its alignment with sequences from Caenorhabditis, Torpedo, avian, and mammalian AChEs (Fig. 4C). The strict conservation of eight aromatic residues and of a cysteine residue at position
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Genomic Structure of the 3' Region of the Bungarus AChE Gene-- We explored the structure of the Bungarus AChE gene by PCR amplification of genomic DNA, using primers corresponding to various exonic sequences. The results are shown in Fig. 6. The sequence encoding the catalytic domain is interrupted by an intron (about 1.3 kb) at the level of amino acid 475 (according to the Torpedo numbering), as in other vertebrates. The last common exon (encoding 35 residues) is followed by a genomic region of 1741 base pairs, preceding the T exon. The region encoding the C-terminal part of the venom AChE (SARA, or S) is located about 300 nucleotides downstream of the stop codon of the T exon. Therefore, the SARA region does not derive from a readthrough sequence but from a novel type of exon, which we call S (Fig. 7). It is interesting to note that the 300-nucleotide-long sequence located between the sequence encoding the T-peptide and exon S contains GC-rich domains.
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Expression of Bungarus AChE in Transfected COS Cells: Existence of an H Exon?-- We transfected COS cells with expression vectors containing either the cDNA sequence encoding the AChET subunit or a partial genomic construct (AChEgT), which included the 1741-bp intron preceding exon T, where a putative H exon would be expected to be localized (see Fig. 7). The total AChE activity and the proportion that was secreted in the culture medium were the same in both cases: 30-45% of the activity was recovered in the medium, 2 days after transfection. As illustrated in the case of the AChET construct (Fig. 8, A and B), sedimentation analysis showed that the cells and the culture medium contained G1a, G2a and G4na forms, as well as heavy polydisperse aggregates. These aggregates, which have not been observed in other AChE species, accounted for as much as 80% of the total activity in cell extracts. The culture medium contained the same type of molecules, including aggregates, with a higher proportion of G2a than in the cells. We obtained the same results with the AChEgT construct (not shown). We could not detect any PI-PLC-sensitive AChE, showing that only AChET subunits were produced. This demonstrates the absence of any functional exon H, that would generate AChEH subunits.
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DISCUSSION |
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Bungarus Possesses a Single AChE Gene, with a Novel Type of Alternative Exon-- Analyses of genomic DNA and of AChE cDNA from muscle showed that Bungarus, like other vertebrates, possesses a single AChE gene, and that AChES and AChET subunits are produced by alternative splicing. The S-peptide is not encoded by a readthrough sequence but by a bona fide alternative exon, called S, which is located 3' of the T exon.
The peptidic sequence encoded by the T exon is highly homologous to the C-terminal T-peptides of other AChEs and BChEs. Among the conserved residues, it is interesting to note the presence of a cysteine residue near the C terminus, involved in intersubunit disulfide bridges, as well as of several aromatic residues, probably involved in the hydrophobic character of an amphiphilicExpression in Transfected Cells--
It has been shown that the
C-terminal peptides of AChE subunits determine the fate of the enzyme
in a tissue-specific manner, and in particular its metabolic stability:
thus, the rat RBL cells express rat AChEH subunits and
expose them at their surface much more efficiently than
AChET subunits, despite the fact that the two proteins seem
to be synthesized at equivalent levels (39). We found that AChE
activities were systematically higher in transfected COS cells
expressing Bungarus AChE than in cells expressing rat AChE.
This was true for AChET subunits and was even more marked in the absence of the T-peptide (AChE or
AChES). In all cases, we observed a similar influence of
the amount of vector DNA used for transfection, with saturation at
approximately the same dose (5 µg/dish), so that the difference could
not be ascribed to transcription; translation is also very unlikely to
differ between constructions such as AChET and
AChE
, which differ only by the presence or absence of
the C-terminal 40-amino acids T-peptide. Comparisons of AChE activities
obtained with the different constructions clearly showed that the
presence of a C-terminal T-peptide reduced the yield of active enzyme
and that the catalytic domains of Bungarus and rat AChEs
present intrinsic differences. The snake enzyme may be able to fold
more efficiently into its active conformation, as suggested by its
capacity to renature after exposure to guanidinium hydrochloride.2
Expression of Cholinesterases in Bungarus Liver and Muscle-- The liver and muscles of B. fasciatus contain both AChE and BChE activities, demonstrating that Bungarus possesses two distinct cholinesterase genes, like other vertebrates.
Reverse transcription-PCR and RNase protection assays showed that AChE transcripts in the venom gland are predominantly of type S, with less than 5% type T, in agreement with the production of soluble monomers derived from AChES subunits. Liver and muscle contain both types of transcripts: about two-thirds type S and one-third type T. The liver contains mostly soluble AChE monomers, which may correspond to AChES subunits, as in the venom, and a minor proportion of tetrameric AChE, which probably consists of AChET subunits. The proportions of these two molecular forms do not correspond to those of the S and T transcripts, either because AChET subunits are partially converted into soluble monomers, by removal of their amphiphilic C-terminal region, or because AChES subunits are more efficiently produced than AChET subunits, as observed in transfected COS cells. In Bungarus muscle, the situation is opposite because the major AChE forms (G1a, G4a, and collagen-tailed molecules) derive from AChET subunits, despite the fact that this tissue also contains more S than T transcripts. The production of AChES subunits in muscle may be underestimated because of their rapid secretion, as observed in transfected COS cells. In any case, the contrast between the AChE transcripts and molecular forms in liver and muscle clearly illustrates the fact that the expression of a protein cannot be simply deduced from the level of its mRNA but also critically depends on cellular specificity of posttranslational processing.Collagen-tailed forms of AChE in Bungarus Muscles-- The small proportion of collagen-tailed AChE forms obtained in extracts from Bungarus muscle raises the question of their functional role: muscles differ widely in the proportions of collagen-tailed and globular AChE forms, depending on the species and on their physiological slow or rapid type (1); an extreme case is that of Torpedo muscle, which only contains GPI-anchored G2a AChE. On the other hand, our results may reflect the nonextractability of collagen-tailed molecules rather than their low abundance. In quail muscle, collagen-tailed AChE exists in extractable and nonextractable states (41); such molecules may be linked by disulfide bonds to other components of the extracellular matrix through the C-terminal cysteine-rich region of the collagen tail (42).
Evolutionary Significance of the S Exon-- The presence of a novel alternatively spliced exon, S, in Bungarus, raises interesting evolutionary questions. The S exons may have originated independently of the production of AChE in venoms. In fact, S transcripts are also expressed in the liver and muscles. In addition, AChE does not appear to contribute to the toxicity of the venom (7). It will therefore be interesting to examine whether the presence of an S exon is correlated with expression of AChE in the venom, in particular in Dendroaspis snakes (mambas), which do not contain AChE in their venom (4), and whether S exons also exist in other reptiles. The evolutionary significance of the absence of H exons and the presence of S exons in Elapidae snakes clearly deserves more detailed studies.
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ACKNOWLEDGEMENTS |
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We thank Dr. Yun Zhang for fruitful discussions, Dr. Eric Krejci for help in RPA experiments, and Anne Le Goff and Rizwana Nawaz for expert technical assistance.
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FOOTNOTES |
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* This research was supported by grants from the Centre National de la Recherche Scientifique, the Direction des Recherches et Etudes Techniques, the Association Française contre les Myopathies, and the Human Capital and Mobility program of the European Community.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.
¶ Recipient of a fellowship from the Institut National de la Recherche Agronomique.
To whom correspondence should be addressed. Tel.:
33-1-45688685; Fax: 33-1-40613057; E-mail: cbon{at}pasteur.fr.
1
The abbreviations used are: AChE,
acetylcholinesterase; A8 and A12, asymmetric
forms composed of two or three AChE tetramers, associated with a triple
helical collagen tail; AChEH, AChES, and
AChET, AChE subunits of type H, S and T, generated from
transcripts terminating with the H, S, and T exons; AChEgT,
construction containing the intron that precedes exon T;
AChE, truncated AChE subunit limited to the catalytic
domain; BChE, butyrylcholinesterase; G1a,
G2a, and G4a,
amphiphilic globular monomer, dimer, and tetramer, respectively; G1na and G4na,
nonamphiphilic globular monomer and tetramer; GPI,
glycophosphatidylinositol; iso-OMPA, tetraisopropyl pyrophos-phoramide;
PI-PLC, phosphatidylinositol phospholipase C; PCR, polymerase chain
reaction; bp, base pair(s).
2 D. I. Kreimer, I. Silman, C. Bon, and L. Weiner, unpublished work.
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REFERENCES |
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