Chicken Breast Muscle Connectin: Passive Tension and I-Band Region Primary Structure

doi:10.1016/j.jmb.2007.04.054

J. Mol. Biol. (2007) 370, 213–219

Chicken Breast Muscle Connectin: Passive Tension and I-Band Region Primary Structure Hiroshi Noguchi 1 , Shigeru Takemori 2 , Junpei Kajiwara 1 Masako Kimura 2 , Koscak Maruyama 1 and Sumiko Kimura 1 ⁎ 1

Department of Biology, Faculty of Science, Chiba University, Chiba 263-8522, Japan 2

Department of Physiology, The Jikei University School of Medicine, Tokyo 105-8461, Japan

We performed cDNA cloning of chicken breast muscle connectin. Together with previous results, our analysis elucidated a 24.2 kb sequence encoding the amino terminus of the protein. This corresponded to the I-band region of the skeletal muscle sarcomere, which is involved in extension and contraction between the Z-line and the A–I junction. There were fewer middle immunoglobulin domains and amino acid residues in the PEVK segment of chicken breast muscle connectin than in human skeletal muscle connectin, but more than in human cardiac muscle connectin. We measured passive tension generation by stretching mechanically skinned myofibril bundles. This revealed that appreciable tension development in chicken breast muscle began at longer sarcomere spacings than in rabbit cardiac muscle, but at shorter spacings than in rabbit psoas and soleus muscles. We suggest that the chicken breast muscle sarcomere remains in a relatively extended state even in unstrained sarcomeres. This would explain why chicken breast muscle does not extend under force to the same degree as rabbit psoas and soleus muscles. © 2007 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: connectin; PEVK; striated muscle; passive tension; titin

Introduction Connectin, also known as titin, is a giant fibrous elastic protein of vertebrate striated muscle with a molecular mass exceeding 3 MDa. A single molecule, ∼1 μm in length, connects the Z-line and the M-line of the striated muscle sarcomere.1–4 Because of its elasticity, connectin plays a role in maintaining the thick filament at the centre of the sarcomere during contraction and relaxation of the muscle. Observations with fluorescent antibodies and immunoelectron microscopy using antibodies against connectin have demonstrated that this function occurs only in the I-band region, and not in the entire sarcomere.5–9 Human cardiac muscle connectin cDNA is 82 kb in size and encodes a protein of 26,926 amino acid

Abbreviations used: Ig, immunoglobulin; Fn, fibronectin. E-mail address of the corresponding author: [email protected]

residues.10 Several isoforms of connectin are found in different tissues, and are produced by selective splicing of the I-band region.11 More than 70% of the I-band region is comprised of a PEVK segment (proline, glutamate, valine, lysine) and immunoglobulin (Ig)-like domains arranged in tandem. Recent investigations into myofibril elasticity changes using recombinant human connectin showed that, on stretching of the muscle sarcomere, the tandem Ig segments were the first to extend linearly, followed by extension of the PEVK segment.12 Comparative studies between phylogenetically distant animals are often useful in elucidating the molecular design of protein structure. In this study, we focused on the comparison of human connectin with chicken breast muscle connectin at the physiologically-important extensible I-band region. The primary structure of chicken breast muscle connectin from the Z-line to one half of the I-band has been reported,13,14 but the sequence from the PEVK segment to the A–I junction was undetermined. Here, we determined the primary structure of the entire I-band region of chicken breast muscle connectin to enable comparisons to be made with human skeletal and cardiac muscle connectin. We

0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.

214

Figure 1. Comparison of the molecular mass of chicken breast muscle connectin, and rabbit cardiac, psoas and soleus muscle connectins. (a) SDS-PAGE of 2.3%–4.0% gradient gel. Lane 1, chicken breast muscle; lane 2, rabbit cardiac muscle; lane 3, rabbit psoas muscle; lane 4, rabbit soleus muscle. α-Con, α-connectin; Neb, nebulin; MHC, myosin heavy chain. (b) Magnification of (a).

measured passive tension in situ using mechanically skinned myofibril bundles.

Results Comparison of α-connectin in chicken breast, rabbit skeletal and cardiac muscles SDS-PAGE analysis revealed that chicken breast muscle α-connectin has a molecular mass less than that of rabbit skeletal muscle α-connectin but similar to that of cardiac muscle connectin (Figure 1), suggesting that it could be a natural recombinant suitable for structure–function correlation analysis. Cloning of the extensible region of chicken breast muscle connectin A 600 bp probe from the 3′ end of clone CnA1 was used to screen the chicken breast muscle cDNA library, from which clone Ch32-1 was obtained (Figure 2). Then 3′ walking was used to obtain clones Ch62, Ch31, Ch12, and b11. Clones Ch32-1, Ch62, and Ch31 overlap clone Cn3.13 Clone Ch35 was obtained by immunoscreening with the monoclonal antibodies 1G1 and 3A1 (with epitopes near the A–I junction15) against chicken breast muscle

Avian Breast Muscle Connectin

connectin. Clones Ch21 and Ch32-2 were obtained by 5′ walking using Clone Ch35. These techniques enabled 9014 bp connectin cDNA corresponding to 3004 amino acid residues to be newly sequenced (DDBJ/EMBL/GenBank accession no. AB264053) from the 3′ terminus of clone Cn3. Sequencing of the I-band region of chicken breast muscle connectin revealed a 24,222 bp sequence corresponding to 8074 residues, with a predicted molecular mass of 904,782 Da (DDBJ/EMBL/GenBank accession no. AB299444) (Figure 2). The MIR region, an A–I junction site in human connectin (I-band 45–46 in the human connectin sequence),16 was obtained from clone Ch35, thus completing the entire primary structure of the I-band region of chicken breast muscle connectin. Primary-structure analysis of chicken breast muscle I-band connectin Similar to human connectin, the primary structure of chicken breast muscle I-band connectin contains immunoglobulin (Ig) domains, fibronectin type 3 (Fn3) domains and a PEVK segment (Figure 3). The number of proximal (15) and distal (22) Ig domains and Fn3 domains are preserved between the two species, while the number of middle Ig domains (13) and amino acid residues in the PEVK segment (429) in chicken breast muscle connectin are intermediate between those of human connectin in cardiac (one domain and 163 amino acid residues) and soleus (53 domains and 2174 amino acid residues) muscles. The four amino acids P, E, V and K comprise 70.4% of the sequence in chicken breast muscle connectin, and 74.3% in human soleus muscle connectin. The chicken breast muscle connectin contained N2A sequence but not N2B. That is, N2A and N2B sequences are not expressed concomitantly in chicken breast muscle as in human skeletal muscle. Homology of chicken and human skeletal muscle connectins Chicken and human skeletal (psoas and soleus) muscle connectins demonstrate high levels of identity: 80% in the proximal, 73% in the middle, and 75% in the distal Ig segments; 77% in the N2A region, and 80% in the A–I junction (Figure 4). Examination of the sequences of clones Ch62, Ch31, Ch12 and b11 suggested the presence of

Figure 2. cDNA clones used to determine primary and domain structures of chicken breast muscle connectin I-band region.

215

Avian Breast Muscle Connectin

Figure 3. Comparison of I-band region domain structures of chicken breast muscle connectin, and human cardiac, psoas and soleus muscle connectins. Numerals represent the number of proximal, middle and distal Ig domains.

alternative chicken breast muscle connectin isoforms. Although the PEVK segment of clone Ch31 is identical with clone Cn3, clone Ch62 has a 26 amino acid residue deletion between the 27th alanine and the 52nd glutamic acid of the PEVK segment. Clone Ch12 contains a 73 amino acid residue insertion between the 169th valine and the 170th proline of the PEVK segment (DDBJ/EMBL/ GenBank accession no. AB264055), while clone b11 lacks the third Ig domain of the N2A region (DDBJ/ EMBL/GenBank accession no. AB264056). Isoforms with modifications of the PEVK and middle Ig segments have been reported in human connectin,11 although no isoform with changes in the N2A region has been described.

Repeat sequence in the PEVK segment The PEVK segment of human connectin is comprised of glutamic acid-rich poly(E) segments and repeats of 28 amino acid residues, starting with PPAK (proline, proline, alanine, lysine).17 The PEVK segment of chicken breast muscle connectin contains 17 PPAK repeated motifs (Figure 5), whose consensus sequence matches that of the human PPAK repeats at 24 of the 28 residues. We found that the central five of the 17 PPAK motifs were highly preserved in chicken breast muscle connectin (Figure 5(b); PPAK motif), while the remainder was less well preserved (PPAK-like motif). This contrasts with the well-preserved sequences in the

Figure 4. Homology of chicken breast muscle and human soleus muscle connectins.

216

Avian Breast Muscle Connectin

Figure 5. Repeated motifs in the PEVK segment of chicken breast muscle connectin. (a) Domain structure and homology of the PEVK segment in chicken breast muscle and human soleus muscle connectins. Yellow, repeated motifs similar to human PPAK motif; black, poly (E) segment. (b) Amino acid and consensus sequences of chicken and human connectin PEVK segments.

60 PPAK motifs of human soleus muscle.17 A poly (E) segment sharing 56% homology with human soleus muscle connectin is present between the first and second PPAK-like motifs of chicken breast muscle connectin. Glutamic acid was found to comprise 38% of the poly(E) segment sequence. The Paircoil program18 did not show any coiled-coil potential in the PEVK segment of chicken breast muscle connectin.

though chicken breast muscle was again intermediate between rabbit cardiac and psoas muscles, the differences in the unstrained sarcomere spacings were surprisingly smaller than those in threshold sarcomere spacings at which appreciable tension develops and those in the length of the I-band connectin sequences. Connectins in unstrained myofibrils would be in relatively extended states in both cardiac and chicken breast muscles, and in more folded states in rabbit psoas and soleus muscles.

Passive tension of mechanically skinned myofibril bundles

Discussion Since the length of the PEVK segment and the number of Ig domains within connectin are considered to determine myofibril elasticity, it would be expected that chicken breast myofibrils are significantly less elastic than rabbit skeletal (psoas and soleus) muscles and more elastic than rabbit cardiac muscle. Therefore, we compared passive tension development of mechanically skinned myofibril bundles of chicken breast muscle with those of rabbit cardiac, psoas and soleus muscles (Figure 6). Under stretching, appreciable tension began to develop in skinned chicken breast muscle at the threshold sarcomere spacing of 2.25 μm. By contrast, skinned rabbit cardiac, psoas and soleus muscles began to develop tension at 2.0 μm, 2.5 μm and 2.8 μm, respectively. This is consistent with our finding that the length of PEVK and the number of Ig domains in chicken breast muscle connectin are intermediate between those in rabbit cardiac and psoas muscle connectins. As another index of myofibril elasticity, we focused on the unstrained myofibril bundles of chicken breast muscle, which showed sarcomere spacing of 1.9 μm. The sarcomere spacings of unstrained rabbit cardiac, psoas and soleus muscles were 1.85 μm, 2.0 μm and 2.1 μm, respectively. Al-

Together with previous results, our analysis elucidated the composition of an approximately 24.2 kb sequence from the N-terminus to the A–I junction of chicken breast muscle connectin. As can be seen in Figure 4, despite a phylogenetic difference between humans and chickens, the primary structure of I-band connectin is well conserved between chicken breast muscle and human skeletal muscle (73–82%), with the exception of the PEVK segment. Chicken breast muscle connectin is smaller than human skeletal muscle connectin and similar in molecular mass to human cardiac muscle connectin (Figure 1). Focusing entirely on the I-band region, chicken breast muscle connectin is intermediate between human skeletal muscle connectin and human cardiac muscle connectin with respect to the number of amino acid residues, the number of Ig domains in the middle Ig segment, and the number of amino acid residues in the PEVK segment. Human connectin contains several isoforms, each with a different number of middle Ig domains and PEVK residues.11 However, this is the first time that an isoform lacking the third Ig domain (corresponding to I82 in human skeletal muscle connectin) in the N2A region has been discovered in chicken breast

217

Avian Breast Muscle Connectin

Figure 6. (a) Passive tension generation in skinned myofibril bundles of chicken breast (red), rabbit cardiac (blue), rabbit psoas (black) and rabbit soleus (green) muscles. The number of measurements is given. Bars represent standard errors. The threshold sarcomere spacing for appreciable passive tension development is shown by an arrowhead of the corresponding color. (b) A representation of the sarcomere at the threshold sarcomere spacing. I-band connectin is drawn under the assumption that the Ig domain corresponds to a length of 100 amino acid residues in the PEVK segment. The scale for sarcomere spacing is shown together with arrowheads indicating the threshold sarcomere spacing. M, M-line; Z, Z-line. (c) A representation of the sarcomere structure in the unstrained state.

muscle connectin. Sorimachi et al. found that the I82 domain in the N2A region of human skeletal muscle interacts with calpain protease p94,19 so our isola-

tion of a chicken breast muscle connectin clone lacking the corresponding domain is extremely interesting in terms of understanding the action of calpain protease p94. The number of residues within the chicken connectin PEVK segment, which is important for connectin elasticity, would be in the range of 403 to 429. This was much fewer than the 2174 residues in human soleus muscle connectin, and suggested that chicken breast muscle does not stretch as readily under force. The PEVK segment of chicken breast muscle connectin also contained a motif similar to the human PPAK motif and sharing 56% homology with the human poly(E) segment. The PPAK motif of human connectin has a polyproline type II helix (PP II helix) secondary structure,17,20 which interacts with the SH3 domain of nebulin.21 However, the sequence of this motif was highly preserved in only five of the 17 PPAK motifs in chicken breast muscle connectin, and it remains to be determined whether the PEVK segment of chicken breast muscle has the same structure and function as those of human connectin. The unstrained sarcomere spacings of skinned myofibril bundles of chicken breast and rabbit soleus muscle were shown to be similar, ranging from 1.9 μm to 2.1 μm. However, skinned myofibril bundles of chicken breast muscle generated tension even when the sarcomere spacing was short, suggesting that chicken breast muscle connectin was maintained in a slightly stretched state within the sarcomere in resting muscle. By contrast, rabbit soleus muscle connectin was maintained in a slack state, so tension was not generated when the sarcomere was short. On the basis of these considerations, we present our hypothetical sarcomere model of connectin states in chicken breast and rabbit soleus muscles in Figure 6(c). The model is consistent with the elasticity model proposed by the study of artificially recombinant human connectin.12 That is, the tandem Ig segments are the first to extend linearly, followed by extension of the PEVK segment. Our tension measurements of skinned myofibril bundles with natural recombinant connectins suggested that the initial extension of the tandem Ig segments develop no appreciable passive tension, and could be in an already stretched state in some muscles such as chicken breast muscle.

Materials and Methods SDS-PAGE Freshly excised striated muscles were boiled for 3 min in SDS sample solution (100 mM Tris–HCl (pH 8.0), 10% (w/v) SDS, 40 mM DTT, 5 mM EDTA, 0.5 mM leupeptin), and clarified by centrifugation at 15,000g for 10 min. The supernatant proteins were separated by SDS-PAGE using 2.3%–4.0% (w/v) gradient polyacrylamide gels (acrylamide:methylenebisacrylamide, 30:1.5 (w/w)).22

218 DNA cloning and sequencing Total RNA was extracted from adult chicken breast muscle using TRIzol reagent (Gibco BRL). Poly(A) RNA was purified using a Dynabeads Oligo (dT)25 mRNA purification kit (Dynal) according to the manufacturer's instructions. cDNA was synthesized from poly(A) RNA primed with oligo(dT) or random hexameric primer using Superscript™ II RNase H– reverse transcriptase (Gibco BRL). The cDNA fragments were ligated to λZAP II expression-vector arms using a λZAP II predigested vector kit (Stratagene), and in vitro packaging into phage particles was carried out using Gigapack III gold packaging extract (Stratagene). Clone CnA1 (Figure 2) was used as a cDNA probe to isolate the 3′ terminus-directed clones by standard hybridization screening of the cDNA library. The products of the cDNA library were also screened with monoclonal antibodies 1G1 and 3A1. Clone Ch35 was used as a cDNA probe to isolate 5′ terminus-directed clones by standard hybridization screening of the cDNA library. Sequencing of both strands was carried out with an ALFexpress DNA autosequencer (Amersham Pharmacia), using a Thermo Sequenase fluorescent-labeled primer cycle-sequencing kit (7-deaza-dGTP, and T3 and T7 primers). Sequence analysis and homology searches were performed with Genetyx Win version 4.0 and a GenomeNet www server sequence similarity search (BLAST). The location of coiled-coil regions in amino acid sequences was predicted by the Paircoil program.18 Passive-tension measurement

Avian Breast Muscle Connectin Using the sharp tips of a pair of glass needles, the cell membrane of the fragment was peeled off and their ends were penetrated. Before experimental use, the fiber was treated with 0.5% (v/v) Triton X-100 for 5 min. The specimen was stretched by manipulation of the glass needle, and its sarcomere spacing was determined microscopically. The force was determined from the calibrated bending of the glass needle. Soleus muscle Mechanically skinned myofibril bundles were prepared also from rabbit soleus muscle to measure its resting tension. Soleus muscle minced with a blender was centrifuged gently to obtain an optimum-sized fragment (typical dimensions: 23 μm × 29 μm × 110 μm). Tension measurement was performed using the technique described for cardiac myofibril bundles.

Acknowledgements We thank A. Hanashima and Y. Sugo for their technical assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S.K. This work is dedicated to the memory of Drs Reiji Natori (1/12/1912–11/20/2006) and Koscak Maruyama (6/16/1930–11/19/2003).

Psoas and breast muscle The passive tension of mechanically skinned myofibril bundles isolated from rabbit psoas and chicken breast muscle was measured as described.23 In brief, the cell membrane of an isolated fiber was mechanically peeled off under a stereo microscope to prepare a skinned myofibril bundle. The bundle was treated with 0.5% (v/v) Triton X-100 for 15 min before experimental use. Typically, the bundle was ∼30 μm in diameter and ∼5 mm in length. The force response against stepwise lengthening of the skinned myofibril bundles was measured with the 406 and UL-2 force transducers (Cambridge Technology and Minebea, respectively) in a relaxing solution (20 mM PIPES (pH 7.0), 75.1 mM KMs, 6.0 mM Mg(Ms)2, 4.2 mM Na2ATP, 1.0 mM leupeptin, 10 mM EGTA, where Ms stands for methanesulfonic group -CH3SO3) at 5 °C. Sarcomere spacings were measured by He-Ne laser diffraction. Cardiac muscle The passive tension of mechanically skinned myofibril bundles obtained from rabbit left ventricular muscle was measured using a method similar to that described by Fabiato and Fabiato.24 Briefly, the whole heart dissected from a rabbit was perfused with Hepes-Tyrode solution at 38 °C (5 mM Hepes (pH 7.4), 137 mM NaCl, 5.0 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, 11 mM glucose), by the Langendorff technique. The perfusing solution was then gradually replaced by the relaxing solution in the presence of 20 mM diacetyl-monoxime (BDM). The left ventricle was minced with a blender (Ultra Turrax TP18/10S1, Janke & Kunkel GmbH & Co. KG), and the minced tissue was filtered and centrifuged gently to obtain cell-sized tissue fragments (typical dimensions: 6 μm × 10 μm × 100 μm).

References 1. Maruyama, K. (1976). Connectin, an elastic protein from myofibrils. J. Biochem. 80, 405–407. 2. Maruyama, K., Matsubara, S., Natori, R., Nonomura, Y., Kimura, S., Ohashi, K. et al. (1977). Connectin, an elastic protein of muscle: Characterization and function. J. Biochem. 82, 317–337. 3. Maruyama, K., Kimura, S., Yoshidomi, H., Sawada, H. & Kikuchi, M. (1984). Molecular size and shape of β-connectin, an elastic protein of striated muscle. J. Biochem. 95, 1423–1433. 4. Tskhovrebova, L. & Trinick, J. (2003). Titin: properties and family relationships. Nature Rev. Mol. Cell Biol. 4, 679–689. 5. Itoh, Y., Suzuki, T., Kimura, S., Ohashi, K., Higuchi, H., Sawada, H. et al. (1988). Extensible and lessextensible domains of connectin filaments in stretched vertebrate skeletal muscle sarcomeres as detected by immunofluorescence and immunoelectron microscopy using monoclonal antibodies. J. Biochem. 104, 504–508. 6. Fürst, D. O., Osborn, M., Nave, R. & Weber, K. (1988). The organization of titin filaments in the halfsarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten nonrepetitive epitopes starting at the Z line extends close to the M line. J. Cell Biol. 106, 1563–1572. 7. Nave, R., Fürst, D. O. & Weber, K. (1989). Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain? J. Cell Biol. 109, 2177–2187.

Avian Breast Muscle Connectin 8. Maruyama, K. (1994). Connectin, an elastic protein of striated muscle. Biophys. Chem. 50, 73–85. 9. Linke, W. A., Ivemeyer, M., Olivieri, N., Kolmerer, B., Ruegg, J. C. & Labeit, S. (1996). Towards a molecular understanding of the elasticity of titin. J. Mol. Biol. 261, 62–71. 10. Labeit, S. & Kolmerer, B. (1995). Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science, 270, 293–296. 11. Freiburg, A., Trombitas, K., Hell, W., Cazorla, O., Fougerousse, F., Centner, T. et al. (2000). Series of exonskipping events in the elastic spring region of titin as the structural basis for myofibrillar elastic diversity. Circ. Res. 86, 1114–1121. 12. Granzier, H. L. & Labeit, S. (2004). The giant protein titin: a major player in myocardial mechanics, signaling, and disease. Circ. Res. 94, 284–295. 13. Maruyama, K., Endo, T., Kume, H., Kawamura, Y., Kanzawa, N., Nakauchi, Y. et al. (1993). A novel domain sequence of connectin localized at the I band of skeletal muscle sarcomeres: homology to neurofilament subunits. Biochem. Biophys. Res. Commun. 194, 1288–1291. 14. Yajima, H., Ohtsuka, H., Kawamura, Y., Kume, H., Murayama, T., Abe, H. et al. (1996). A 11.5-kb 5′terminal cDNA sequence of chicken breast muscle connectin/titin reveals its Z line binding region. Biochem. Biophys. Res. Commun. 223, 160–164. 15. Maruyama, K. (1997). Connectin/titin, giant elastic protein of muscle. FASEB J. 11, 341–345. 16. Trombitas, K., Freiburg, A., Centner, T., Labeit, S. & Granzier, H. (1999). Molecular dissection of N2B cardiac titin's extensibility. Biophys J. 77, 3189–3196.

219 17. Greaser, M. (2001). Identification of new repeating motifs in titin. Proteins: Struct. Funct. Genet. 43, 145–149. 18. Berger, B., Wilson, D. B., Wolf, E., Tonchev, T., Milla, M. & Kim, P. S. (1995). Predicting coiled coils by use of pairwise residue correlations. Proc. Natl Acad. Sci. USA, 92, 8259–8263. 19. Sorimachi, H., Kinbara, K., Kimura, S., Takahashi, M., Ishiura, S., Sasagawa, N. et al. (1995). Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific sequence. J. Biol. Chem. 270, 31158–31162. 20. Gutierrez-Cruz, G., Van Heerden, A. H. & Wang, K. (2001). Modular motif, structural folds and affinity profiles of PEVK segment of human fetal skeletal muscle titin. J. Biol. Chem. 276, 7442–7449. 21. Ma, K. & Wang, K. (2002). Interaction of nebulin SH3 domain with titin PEVK and myopalladin: implications for the signaling and assembly role of titin and nebulin. FEBS Letters, 532, 273–278. 22. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. 23. Fukuzawa, A., Shimamura, J., Takemori, S., Kanzawa, N., Yamaguchi, M., Sun, P. et al. (2001). Invertebrate connectin spans as much as 3.5 μm in the giant sarcomeres of crayfish claw muscle. EMBO J. 20, 4826–4835. 24. Fabiato, A. & Fabiato, F. (1975). Contractions induced by a calcium-triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells. J. Physiol. 249, 469–495.

Edited by J. Karn (Received 30 December 2006; received in revised form 13 April 2007; accepted 19 April 2007) Available online 4 May 2007