Uncoordinated Expression of Myosin Heavy Chains and Myosin-Binding Protein C Isoforms in Human Extraocular Muscles

ARTICLES Uncoordinated Expression of Myosin Heavy Chains and Myosin-Binding Protein C Isoforms in Human Extraocular Muscles

Daniel Kjellgren,1 Per Stål,2 Lars Larsson,3 Dieter Fu¨rst,4 and Fatima Pedrosa-Domello¨f1,2

PURPOSE. To examine the distribution of myosin-binding pro- special features. The EOMs contain fibers with a wide array of tein C (MyBP-C) in human extraocular muscles (EOMs) and to contractile properties, varying from extremely fast to fibers correlate the myosin heavy chain (MyHC) and the MyBP-C capable of sustained, tonic contractions. Previously, we sought composition of the fibers. to elucidate the molecular basis of this unique allotype of 3–5 METHODS. Samples from 17 EOMs, 3 levator palpebrae (LP), and human EOMs at the fiber level, by determining the patterns 6 limb muscles were analyzed with SDS-PAGE and immunoblot of expression of myosin heavy chain (MyHC; the major deter- 6,7 or processed for immunocytochemistry with monoclonal anti- minant of heterogeneity of contraction force and velocity ) 8 bodies (mAbs) against MyBP-C-fast, MyBP-C-slow, MyHCIIa, My- and of SERCA-1 and -2 (determinants of the relaxation rate ). HCI, MyHCsto, MyHC␣-cardiac, and MyHCemb. The fibers in the human EOMs have very complex MyHC composition patterns, with most fibers containing more than RESULTS. In the limb muscle samples, fast fibers were labeled one MyHC isoform. Moreover, differences in the relative with anti-MyBP-C-fast and anti-MyBP-C-slow, whereas the slow amounts of a given MyHC isoform are typically observed fibers were immunostained with anti-MyBP-C-slow only, in 3 accordance with previous studies. In 11 EOM samples MyBP- among fibers sharing a particular combination of isoforms. C-fast was not detected, and weak staining with anti-MyBP-C- Despite this heterogeneity, the fibers in the human EOMs can be divided grossly into three major fiber groups, based on their fast was seen only in a few fibers in the proximal part of 2 3 muscles. The mAb against MyBP-C-slow labeled all fibers, but content of MyHCI, MyHCIIa, and MyHCeom. fibers containing MyHCI were generally more strongly stained. Myosin-binding protein C (MyBP-C) is, next to myosin, the second most abundant thick-filament protein in striated mus- In the levator palpebrae, immunostaining with anti-MyBP-C-fast 9 was present in some fibers labeled with anti-MyHCIIa and/or cles. It is located in the A band, in a restricted part of the cross-bridge–bearing region.10 MyBP-C is a Ϸ130 kDa protein anti-MyHCeom. MyBP-C-fast and -intermediate were not de- 11,12 tected biochemically in the EOMs. and both its C terminus and N terminus bind to myosin. MyBP-C is presumed to have a regulatory, although not essen- CONCLUSIONS. The lack of MyBP-C-fast and intermediate is an tial, role in sarcomere assembly13,14 and to play a physiological additional feature of the human EOM allotype. The true EOMs role in regulating contraction by modulating unloaded short- have a unique myofibrillar protein isoform composition reflect- ening velocity.15 The importance of MyBP-C for muscle func- ing their special structural and functional properties. The leva- tion is indicated by the fact that mutations in the gene for tor palpebrae muscle phenotype is intermediate between that cardiac MyBP-C (MyBP-C-card) cause familial hypertrophic car- of the EOMs and the limb muscles. (Invest Ophthalmol Vis Sci. diomyopathy.16–18 2006;47:4188–4193) DOI:10.1167/iovs.05-1496 There are three major isoforms of MyBP-C in human muscle: fast skeletal (MyBP-C-fast), slow skeletal (MyBP-C-slow), and he functional properties of muscle fibers vary considerably MyBP-C-card.19 MyBP-C-fast, detected both with in situ hybrid- between different muscles.1 The extraocular muscles T ization and immunocytochemistry, is present in fast fibers, (EOMs) are among the most complex muscles in the body and whereas MyBP-C-slow is present in both slow and fast fibers, in have been considered a separate muscle allotype,2 due to their human skeletal muscle.20 The cardiac isoform is restricted to the heart and has never been detected in conjunction with any other MyBP-C isoforms in cardiac or skeletal muscle.20 The From the 1Department of Clinical Sciences, Ophthalmology, Umeå three human MyBP-C genes have been mapped and se- University, Umeå, Sweden; the 2Department of Integrative Medical quenced.21–23 Recent analysis of single fibers by SDS-PAGE, Biology, Section of Anatomy, Umeå University, Umeå, Sweden; the revealed the coordinated expression of MyBP-C-slow in fibers 3Department of Clinical Neurophysiology, Academic Hospital, Uppsala 4 containing MyHCI (slow); MyBP-C-fast in fibers with MyHCIIx University, Uppsala, Sweden; and the Institute for Biochemistry and (fast); and an additional isoform, MyBP-C-intermediate, in fibers Biology, Department of Molecular Cell Biology, University of Bonn, 24 Bonn, Germany. containing MyHCIIa (fast) in human limb muscle. Coordi- Supported by grants from Stiftelsen KMA, Synfra¨mjandet, the nated isoform changes, indicating that MyBP-C expression is Swedish Research Council and the Medical Faculty of Umeå University. linked to MyHC expression, have been reported during skeletal Submitted for publication November 23, 2005; revised May 31, muscle hypertrophy in the rat.25 However, in the human mas- 2006; accepted August 15, 2006. seter, a masticatory muscle with rather unique properties,26–28 Disclosure: D. Kjellgren,None; P. Stål, None; L. Larsson, None; the very complex MyHC composition of its fibers was not D. Fu¨rst, None; F. Pedrosa-Domello¨f, None paralleled by an intricate MyBP-C pattern.24 The publication costs of this article were defrayed in part by page Data on the MyBP-C composition of human EOMs and its charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. correlation to the MyHC composition at the protein and cellu- Corresponding author: Fatima Pedrosa-Domello¨f, Department of lar level are lacking. In the present study, we investigated the Integrative Medical Biology, Section of Anatomy, Umeå University, distribution of the fast and slow isoforms of MyBP-C in relation S-901 87 Umeå, Sweden; [email protected]. to the MyHC profile of the fibers and found further evidence of

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the uniqueness of the molecular portfolios of the fibers in the ble 1). The tissue sections were processed as previously described,32,37 human EOMs. by using the indirect peroxidase complex (Dako, Copenhagen, Den- mark) technique to visualize bound antibody. Processed sections were photographed under a microscope MATERIAL AND METHODS equipped with a charge-coupled device (CCD) camera (Nikon, Tokyo, The muscles were collected according to the ethical recommendations Japan). The overall staining pattern of each section was examined, and of the Swedish Transplantation Law, with the approval of the Medical representative areas of each muscle sample, including the orbital and Ethics Committee, Umeå University, and in compliance with the Dec- global layers, were studied in detail. laration of Helsinki for research involving human tissue. Seventeen EOM samples were obtained at autopsy from six men and one woman (ages, 17, 26, 27, 34, 34, 81 and 86 years) who had had no known RESULTS neuromuscular disease. The samples were mounted on cardboard, rapidly frozen in propane chilled with liquid nitrogen, and stored at SDS-PAGE and Immunoblots Ϫ80°C until used. Both MyBP-C-slow and -fast were identified in the limb muscle SDS-PAGE and Immunoblots samples by SDS-PAGE (Fig. 1A) and in immunoblots (Fig. 1B). In the EOMs, only MyBP-C-slow could be detected in the gels Whole-muscle extracts were prepared from one rectus superior, one and immunoblots, whereas MyBP-C-fast and -intermediate were rectus lateralis, two obliquus superior, one levator palpebrae (LP), and absent (Fig. 1). one brachioradialis muscle, as previously described.29 MyBP-C isoforms were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 4% (wt/vol) stacking and 8% running Immunocytochemistry 24 gels. Gel buffer solutions included 10% glycerol. Proteins were iden- Validation of Antibody Specificity. The mAbs against tified on the basis of their molecular mass, immunoreactivity, and order MyBP-C-fast and slow did not label sections of human myocar- of migration in comparison with purified rabbit MyBP-C-fast and refer- dium, indicating that they do not cross-react with the MyBP-C- ence samples prepared from one vastus lateralis muscle with mostly card isoform. type 1 (slow-twitch) fibers and one vastus lateralis muscle with mostly Limb Muscles. Anti-MyBP-C-fast immunostained all fast fi- type 2 (fast-twitch) fibers, as well as cultured human skeletal muscle bers (containing MyHCIIa and/or MyHCIIx) strongly, whereas 30,31 cells rich in MyBP-C-fast. The separating gels (160 ϫ 180 ϫ 0.75 it did not label any fibers containing solely MyHCI (Fig. 2). mm) were silver stained and subsequently scanned. For immunoblot Anti-MyBP-C-slow stained all fibers strongly (Fig. 2), in accor- analysis, the separated proteins were transferred onto nitrocellulose dance with previous data.20 sheets and incubated with antibodies against MyBP-C-slow and -fast Extraocular Muscles. The antibodies against the different (Table 1). MyHC isoforms stained the EOMs heterogeneously (Fig. 3, 4, 5). The MyHC composition of the individual fibers in the Immunocytochemistry orbital and global layers was complex, because of the presence The samples for immunocytochemistry were taken from the rectus of multiple isoforms in each fiber and differences in the relative superior (n ϭ 5), rectus inferior (n ϭ 2), rectus medialis (n ϭ 2), rectus amounts of any given MyHC among the fibers. Three major lateralis (n ϭ 2), and obliquus superior (n ϭ 2) muscles. Eight samples groups of fibers were distinguished in both the orbital and were taken from the middle portion of the muscle, two from the distal global layers of the EOMs, according to their immunohisto- part (close to the bulb), and three from the proximal part (close to chemical staining patterns, as previously described3: (1) fast anulus tendineus) of the muscle. For comparison, samples were also fibers that contain MyHCIIa and in addition may contain MyH- taken from human myocardium, LP (n ϭ 2), biceps brachii, first dorsal Cemb and/or MyHCeom; (2) slow fibers that contain MyHCI interosseus, and vastus lateralis muscles. and may also contain MyHC-slow tonic, MyHC␣-cardiac, MyH- Serial cross sections, 5 ␮m thick, were processed for immunocy- Cemb, and/or MyHCeom; and (3) MyHCeompos/MyHCIIaneg- tochemistry, with a panel of previously very well characterized mono- fibers that lack MyHCI and MyHCIIa, but contain MyHCeom clonal antibodies (mAb), recognizing MyBP-C and MyHC isoforms (Ta- and may in addition contain MyHCemb.

TABLE 1. Antibodies Used for Immunocytochemistry

Antibody Speciﬁcity Short Name Gene* Reference

BB146 MyBP-C slow Anti-MyBP-C slow MYBPC1 alt MYBPCS 20 BB88 MyBP-C fast Anti-MyBP-C fast MYBPC2 alt MYBPCF 20 A4.74† MyHCIIa Anti-MyHCIIa MYH2 32, 33 A4.951† MyHCI Anti-MyHCI MYH7 32, 34 F88‡ MyHC␣-cardiac Anti-MyHC␣-cardiac MYH6 36 N2.261† MyHCI Anti-MyHCI ϩ IIa ϩ eom MYH7 32, 34 MyHCIIa MYH2 MyHCeom MYH13 MyHC␣-cardiac MYH6 ALD19§ MyHC slow tonic Anti-MyHCsto ? 37, 38 2B6࿣ MyHCembryonic Anti-MyHCemb MYH3 32, 37, 39

* Ofﬁcial gene nomenclature, according to OMIM (http://www.ncbi.nlm.nih.gov/omim/). † Obtained from The Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Dept of Biological Sciences, Iowa City, IA. ‡ Gift from Jean J. Le´ger, Institut National de la Sante´ et de la Recherche Me´dicale, Unite´ 249, Montpellier, France. § Gift from Donald A. Fischman, Cornell University, Ithaca, New York. ࿣ Gift from Alan Kelly, University of Chicago, Chicago, IL.

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FIGURE 1. (A) SDS-PAGE of whole muscle extracts from a limb muscle, the brachioradialis (BR), and a rectus medialis (RM). Note that the MyBP-C-slow band is present in both samples whereas the MyBP-C-fast band is present in the limb muscle sample only. M, molecular mass standard, showing 170 and 130 kDa. (B) Immunoblots treated with the antibodies against MyBP-C-slow (lanes 1–4) and -fast (lanes 5–8). No immunoreactivity was detected with anti-MyBP-C in the EOM samples FIGURE 3. Photomicrographs of sections from a rectus inferior muscle (lanes 5 and 7). M, molecular mass standard; RM, rectus medialis; BR, immunostained with (A) anti-MyBP-C-slow, (B) anti-MyBP-C-fast, (C) brachioradialis; OS, obliquus superior; C, cultured human muscle cells anti-MyHCI, (D) anti-MyHCIϩIIaϩeom, (E) anti-MyHCsto, (F) anti-My- rich in MyBP-C-fast. HCIIa, and (G) anti-MyHC␣-cardiac. Note that all fibers were immunolabeled by anti-MyBP-C-slow, whereas no part of the section showed immunoreactivity with anti-MyBP-C-fast. Also note that there is an area Anti-MyBP-C-fast did not label any of the fibers in 11 of the in the global layer showing a somewhat lighter immunoreaction with EOM samples examined (Figs. 3B, 4B, 5B). However, in the anti-MyBP-C-slow and that this area is also weakly immunostained with proximal part of a rectus superior and of a rectus inferior anti-MyHCIIa. muscle taken from the oldest subjects (81 and 86 years, respec- tively), this mAb labeled a few fibers weakly (Fig. 6). These stained fibers were mostly, but not exclusively, located in the contained only MyHCI, MyHCIϩMyHCsto, or MyHCIϩ periphery of the muscles. Many of them were also reactive to MyHCstoϩMyHC␣-cardiac (Figs. 4, 5). The fast fibers (MyH- anti-MyHCIIa, but there was no clear correlation between the CIIa) were labeled moderately to strongly with anti-MyBP-C- staining pattern of anti-MyHCIIa and anti-MyBP-C-fast. slow, whereas the MyHCeompos/MyHCIIaneg-fibers were la- Anti-MyBP-C-slow labeled all fibers (Figs. 3A, 4A, 5A). The beled lightly, moderately, or strongly by this mAb. At low slow fibers were strongly stained, irrespective of whether they magnification, the orbital layer appeared more heavily stained than the global layer, and the proximal and distal portions of the muscles appeared more stained than the middle portions, because the MyHCeompos/MyHCIIaneg-fibers were more abundant in the global layer and in the midbelly region. No correlation was found between the staining pattern of anti-MyBP-C- slow and the presence or absence of MyHCemb, MyHCsto, or MyHC␣-cardiac in the fibers (Fig. 5). Levator Palpebrae. Anti-MyBP-C-fast labeled some of the fast fibers and the MyHCeompos/MyHCIIaneg fibers in the LP heterogeneously. The fibers containing MyHCI were not stained with anti-MyBP-C-fast. Anti-MyBP-C-slow labeled all fibers strongly, with less heterogeneity than in the EOMs (Fig. 7).

DISCUSSION FIGURE 2. Photomicrographs of sections from a biceps brachii muscle The most important result of the present study is the striking immunostained with (A) anti-MyBP-C-fast, (B) anti-MyBP-C-slow, (C) anti-MyHCIIa, and (D) anti-MyHCI. Fibers containing MyHCIIa (arrow- difference in the MyBP-C composition between human EOM head), MyHCIIx (open arrows), and MyHCI (solid arrows) are indi- fibers and limb muscle, a finding that lends further support to cated. Note that anti-MyBP-C-fast immunolabeled the fibers containing the uniqueness of the EOMs as a separate muscle allotype. In MyHCIIa and MyHCIIx, whereas all fibers were labeled with anti-MyBP- addition, the present results further confirm the phenotype of C-slow. the LP muscle as intermediate between that of the true EOMs

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FIGURE 5. Photomicrographs of sections from the global layer of a rectus medialis muscle. Immunostaining as in Figure 4. Fibers containing MyHCIIa (arrowhead), MyHCI (solid arrow) or lacking both of these MyHC isoforms (open arrow) are indicated. Note that the fibers containing MyHCI have a stronger immunoreaction with anti-MyBP-C- FIGURE 4. Photomicrographs of sections from the orbital layer of a slow than the other fibers. Also note that anti-MyHC␣-cardiac and rectus medialis muscle immunostained with (A) anti-MyBP-C-slow, (B) anti-MyHCemb stained only a few fibers, without correlation to the anti-MyBP-C-fast, (C) anti-MyHCI, (D) anti-MyHCIϩIIaϩeom, (E) anti- staining patterns of the other mAbs. MyHCsto, (F) anti-MyHCIIa, (G) anti-MyHC␣-cardiac, and (H) anti- MyHCemb. Arrows: fibers containing MyHCI; arrowheads: fibers containing MyHCIIa. Note that anti-MyBP-C-fast did not label any fibers in feature that further strengthens the identity of the EOMs as a this muscle and that anti-MyBP-C-slow immunostained all fibers separate allotype, distinct from limb muscle. In the masseter strongly. There was no correlation between the labeling with anti- MyHCemb and any of the other mAbs. muscle, a member of the masticatory allotype, only one MyBP-C isoform, with a molecular mass similar to slow MyBP-C, was identified in single fibers with SDS-PAGE, irre- and the limb muscles, as previously indicated on the basis of spective of their MyHC composition.24 Whether this MyBP-C the MyHC composition.3 isoform detected in the masseter is identical with MyBP-C-slow or an additional isoform remains to be investigated.24 The MyHC versus MyBP-C Composition Human limb muscle fibers contain MyHCI, MyHCIIa, or MyH- CIIx and occasional hybrid fibers contain two of these isoforms (I ϩIIa or IIaϩIIx).40 Coordinated expression patterns for MyHC and MyBP-C isoforms have been reported for rat limb muscle in hypertrophy25 and for single human limb muscle fibers studied with SDS-PAGE,24 meaning that MyBP-C-fast dominates in fibers expressing MyHCIIx, MyBP-C-intermediate dominates in fibers containing MyHCIIa, and MyPB-C-slow cor- relates with a content of MyHC I.24 Our results confirm previous data in human limb muscle.20,24 Moreover, we showed in the present study that there is no such coordination between MyHC and MyBP-C isoforms in the human EOMs. We were unable to detect MyBP-C-fast or intermediate in most of the fibers of the EOMs, although these muscles have a predomi- nance of fast fibers when examined on the basis of their FIGURE 6. Photomicrograph of a section from the proximal part of a myosin heavy chain reactivity (71% of the fibers contain MyH- rectus inferior immunostained with anti-MyBP-C-fast The orbital layer pos neg 3 CIIa and 13% are MyHCeom /MyHCIIa fibers ) and they (OL) and the global layer (GL) are indicated. Scattered immunoreactiv- are among the fastest muscles in the body. This is an additional ity, preferably in some fibers of the orbital layer is evident.

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Our understanding of the function of MyBP-C is still rather limited, despite the fact that mutations on the cardiac isoform are responsible for familial hypertrophic cardiomyopa- thies.16–18 Recently, it has been shown that the S2 binding domain of MyBP-C is a modulator of contractility and that it works in a fashion that is at least partly independent of a “tether” with the MyHC molecule.45 The lack of MyBP-C theoretically may result in higher contraction rates, since experimental extraction of MyBP-C leads to an increase in contraction rate and increases the Ca2ϩ sensitivity of the force–velocity curve.46 These changes were shown to be reversible by the readdition of MyBP-C. The lack of MyBP-C at the single muscle fiber level may have an impact on regulation of muscle contraction at low levels of activation in vivo. This notion is supported by in vitro experiments demonstrating an increase in the low-velocity phase of shortening at submaximal Ca2ϩ activation levels15 and increase the Ca2ϩ sensitivity of force46 after chemical extraction of endogenous MyBP-C from skinned skeletal muscle fibers. The effects of removal of MyBP-C on regulation of contraction have been interpreted to be secondary to removal of a structural constraint of MyBP-C on the myosin S-1 domain leading to increased cross-bridge binding. Thus, lack of MyBP-C is ex- pected to reduce an internal load on the myosin head, resulting in increased shortening velocity. Single-fiber experiments are needed to elucidate the role of MyBP-C on the contractile properties of the different fiber types in the human EOMs. However, the very small size of the EOM fibers poses a tech- FIGURE 7. Photomicrographs of sections from an LP muscle immunostained with (A) anti-MyBP-C-slow, (B) anti-MyBP-C-fast, (C) anti-My- nical challenge in performing such experiments. HCI, and (D) anti-MyHCIIa, labeled as in Figure 5. Note that some of the fibers containing MyHCIIa are immunostained to a variable degree Levator Palpebrae with anti-MyBP-C-fast. We have previously shown that the human LP muscle shares common features with the true EOMs (e.g., loosely arranged MyHC composition of the fibers in the human EOMs and in the fibers and the presence of MyHC␣-cardiac, MyHCemb, and masseter is very complex. Most, if not all the fibers in the EOMs MyHCeom), but has a phenotype that is intermediate between contain more than one MyHC isoform, and in both EOMs and the EOMs and the limb muscles (e.g., intermediate fiber size, 3 masseter fibers, up to five different MyHC isoforms have been lack of organization into layers, and lack of MyHCsto). In the identified,3,24,26 including developmental and ␣-cardiac MyHC present study, immunocytochemistry indicates the presence of isoforms.24,26,41 In addition to the MyHC isoforms detected in MyBP-C-slow in all fibers and MyBP-C-fast in some fast fibers. our study, transcripts of MyHCIIb are also present in human Taken together, these findings further confirm the phenotype EOMs (Andersen J, Pedrosa-Domello¨f F, personal communica- of the LP as intermediate between that of the EOMs and the tion, 2000) and the presence of MyHCIIx cannot be excluded.3 limb muscles.

MyBP-C Composition Acknowledgments In the present study, SDS-PAGE, immunoblots, and immunocytochemistry all revealed that the human EOMs lacked MyBP-C- The authors thank Margaretha Enerstedt for excellent technical assis- fast. Furthermore, microarray data confirm at the RNA level tance. that the human EOMs differ significantly from limb muscles in the level of expression of MyBP-C-fast, which is downregulated References 42 more than 100-fold in EOMs. All these findings indicate that 1. Bottinelli R. Functional heterogeneity of mammalian single muscle MyBP-C-fast is absent or present in only trace amounts in the fibres: do myosin isoforms tell the whole story? Pflugers Arch. human EOMs. MyBP-C intermediate was not detected biochem- 2001;443:6–17. ically in the EOMs but the lack of a specific antibody does not 2. Hoh JF, Hughes S, Hoy JF. Myogenic and neurogenic regulation of allow us to explore whether it may be present in amounts myosin gene expression in cat jaw-closing muscles regenerating in below the level of detection with SDS-PAGE. fast and slow limb muscle beds (published corrections appear in In contrast, at the RNA level, MyBP-C-slow appears to be J Muscle Res Cell Motil. 1988;9:567 and 1992;13:126). J Muscle Res essentially identical in human EOMs and limb muscle.42 The Cell Motil. 1988;9:59–72. MyBP-C-cardiac isoform has been detected only in heart mus- 3. Kjellgren D, Thornell LE, Andersen J, Pedrosa-Domellof F. Myosin cle,20,43 and expression profiling of the human EOMs versus heavy chain isoforms in human extraocular muscles. Invest Oph- limb did not reveal the cardiac isoform to be part of the EOM thalmol Vis Sci. 2003;44:1419–1425. allotype.42 Multiple isoforms of MyBP-C have been identified in 4. Kjellgren D, Ryan M, Ohlendieck K, Thornell LE, Pedrosa-Domellof 44 F. Sarco(endo)plasmic reticulum Ca2ϩATPases (SERCA1 and -2) in chicken, but until now there is no evidence of the existence human extraocular muscles. Invest Ophthalmol Vis Sci. 2003;44: of isoforms other than MyBP-C-slow, -fast, -intermediate, and 5057–5062. -cardiac in human muscle. Further studies with single-fiber 5. Pedrosa-Domellof F, Holmgren Y, Lucas CA, Hoh JF, Thornell LE. SDS-PAGE in conjunction with genetic tools are needed to Human extraocular muscles: unique pattern of myosin heavy chain address the question of whether novel MyBP-C isoforms may expression during myotube formation. Invest Ophthalmol Vis exist in the human EOMs. Sci2000;41:1608-.

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