Fodrin in the human polymorphonuclear leucocyte: redistribution induced

by the chemotactic peptide

TOYOSHI FUJIMOTO and KAZUO OGAWA

Department of Anatomy, Faculty of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan

Summary

Fodrin, a membrane skeletal protein, was found toaccumulate in the posterior portion of human neutro-phils polarized morphologically after stimulation bythe chemotactic peptide, JV-formylmethionyl-leucyl-phenylalanine (FMLP). In most (>90 %) unstimulatedneutrophils, the distribution of fodrin was found tobe uniform by immunofluorescence microscopy.When FMLP (10"8M) was applied at 25°C, fodrinbecame polarized in about 40% of cells by lmin,about 70% by 2min, and about 80% by lOmin. Thecells with polarized distribution decreased there-after to about 60 % of the cells at 20 min and about20% at 60 min. Using the under-agarose system, itwas confirmed that the concentration of fodrin oc-curred in the region opposite to the direction ofchemoattraction in moving cells.

By immunoelectron microscopy, most of the label-ing for fodrin was observed in the filamentous cellcortex and not associated with the plasma membrane

itself. In cells polarized morphologically by FMLP,the fodrin labeling became concentrated in the pos-terior portion of the cell; the labeling was found mostdensely in the granule-rich cytoplasm, while thefilamentous tail region was not labeled intensely. Thelamellipodium in the head region was also labeledonly sparsely.

The results indicate that in human neutrophilsfodrin exists as a cytoskeletal protein rather than asa membrane protein and that the protein accumu-lates in the endoplasm of the posterior portion inmigrating cells. The rearrangement is likely to modu-late the organization of the actin-rich cell cortex forcell locomotion.

Key words: neutrophils, chemotactic peptide, fodrin,polarization, cell locomotion.

Introduction

Locomotion of polymorphonuclear leucocytes (neutrophils)is elicited by various chemotactic and chemokinetic fac-tors. It is obvious that the cytoskeleton is engaged activelyin the generation of movement, but the mechanism bywhich the signals received on the cell surface are trans-duced to the physical rearrangement of the cytoskeletalarchitecture is only beginning to unfold (for reviews, seeOmann et al. 1987; Stossel, 1988). Since actin is undoubt-edly the most important component in generating loco-motion, actin-associated proteins that can affect its organ-ization have been one focus of investigation. For example,rapid cytoskeletal association of a--actinin was reported tooccur in the neutrophil after chemotactic peptide stimu-lation (Niggli and Jenni, 1989).

In the present study, we examined the behavior offodrin, one of the actin-associated proteins, in relation tothe neutrophil locomotion. The protein is an analogue oferythrocyte spectrin and is known to be a major calmodu-lin-binding protein (Kakiuchi et al. 1981). It is also asubstrate of protein kinases and calpain I (for reviews, seeBennett, 1985; Marchesi, 1985; Goodman et al. 1988).Although the importance of these modifications in vivo isnot fully understood, the properties suggest the possibilitythat fodrin may be modified in some manner upon signalreception and thereby modulate the cytoskeletal organiz-

Journal of Cell Science 96, 477-484 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

ation. In fact, translocation of fodrin from the cell mem-brane to other cytoplasmic sites upon receiving variousstimuli has been found in lymphocytes (Black et al. 1988;Lee et al. 1988), adrenal medullary cells (Fujimoto andOgawa, 19896) and cultured keratinocytes (Yoneda et al.1990a,6).

We studied the question of how the chemotactic stimu-lus affects distribution of fodrin in the neutrophil. Theprotein was found to be concentrated in the posterior endof polarized cells by immunofluorescence microscopy.Moreover, by immunoelectron microscopy fodrin waslocalized not in the cell membrane but in the corticalfilamentous layer. These findings suggest that fodrin inthe neutrophil is a cytoskeletal protein rather than aperipheral membrane protein as in most other cells. Theimplications of the results are discussed, especially inrelation to the mechanism of cell locomotion.

Materials and methods

AntibodiesAnti-rat brain fodrin antibody was prepared and characterized asdescribed before (Fujimoto and Ogawa, 1989a). Anti-spectrinantibody was raised in rabbits against human erythrocyte spec-trin (Bennett. 1983) and purified similarly. To examine thereactivity of the antibodies, immunoblotting was carried out.

477

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresisof solubilized neutrophils prepared using diisopropylfluorophos-phate (Amrein and Stossel, 1980) and erythrocyte ghosts was runon 7.5 % gel by the method of Laemmli (1970) and proteins wereelectro-transferred to Durapore GVHP filter (Millipore Corp.,Bedford. MA) (Towbin et al. 1979). The blots were labeled with theanti-fodrin antibody and the anti-spectrin antibody, respectively,both at l^gml"1 followed by peroxidase-conjugated goat anti-rabbit antibody (Cappel, West Chester, PA). Positive bands werevisualized by the diaminobenzidine reaction.

Human neutrophilsHuman neutrophils were isolated from heparinized venous bloodof healthy donors by dextran sedimentation and Ficoll-Hypaquedensity centrifugation (Boyum, 1968). For some experiments,hypotonic shock was applied to remove residual erythrocytes.Rinses were done with 5 mil Hepes-buffered Hanks' balanced saltsolution (pH7.4) (HBSS) containing 0.1% bovine serum albumin(BSA) (Seikagaku Kogyo, Tokyo, Japan). Cells were allowed toadhere to glass coverslips pretreated briefly with HBSS at 25°C.In some experiments, neutrophils were rinsed repeatedly withHBSS without BSA and seeded onto clean glass coverslipswithout pretreatment. In both cases, loosely attached cells werewashed off the coverslips by pipetting HBSS mildly onto thesurface.

Stimulation with chemotactic peptide andimmunofluorescence microscopyiV-formyl-L-methionyl-leucyl-phenylalanine (FMLP; SigmaChemical Co, St Louis, MO) was dissolved in dimethyl sulfoxideand stored frozen at -20°C in aliquots until use. The peptide wasdiluted to 10~ 8 M in HBSS containing BSA and given to neutro-phils on coverslips. The final concentration of dimethyl sulfoxidewas less than 0.001 % and did not affect the results at all. Controlcells as well as cells incubated with the peptide for 0.5, 1, 2, 3, 5,10, 20, 30 and 60min at 25 °C were fixed for immunofluorescencemicroscopy. Some cells were treated with 10 or 100//gml~concanavalin A (ConA; Sigma) instead of FMLP for 10-20 min at25 or 37 °C and fixed.

Cells were fixed with a mixture of 3 % paraformaldehyde and0.1% glutaraldehyde in 0 . 1 M sodium phosphate buffer (pH7.4)for 10 min at room temperature and immunolabeled using 0.5%Triton X-100 as a permeabilizing reagent (Fujimoto and Ogawa,1988); the primary and the secondary antibodies used were theaffinity-purified rabbit anti-rat brain fodrin antibody (lOfigml"1)and fluorescein isothiocyanate-conjugated goat anti-rabbit IgGantibody (20//gml~1) (Cappel), respectively. Some specimenswere doubly labeled for fodrin and F-actin by adding rhodamine-phalloidin (1:80 dilution) (Molecular Probes, Junction City, OR)to the primary antibody solution. Fluorescence and phase-con-trast micrographs were taken with Kodak Tri-X-Pan film in anOlympus Vanox photomicroscope (Tokyo, Japan) or with a ZeissAxiophot microscope (FRG) with epifluorescence illumination.

The percentage of cells in which fodrin accumulated in a smallregion was counted directly under the microscope. This seemedessential because the height of cells was variable. Cells withuniform fluorescence in one focal plane often showed polarizedfodrin distribution in other planes. Cells showing clearly concen-trated labeling for fodrin in any focal plane were counted as'polarized'.

Under-agarose systemAgarose plates were prepared upon glass coverslips in a tissueculture dish and three round wells were cut in line as described(Nelson et al. 1976). Neutrophils were placed in the central wellwith 10 ;<1 of K T 7 M FMLP in HBSS and control HBSS, bothcontaining BSA, on opposite sides. The plates were incubated at37 °C in a humidified incubator under 5% CO2/95% air for 2h.Fixation and immunolabeling for fodrin were done in the samemanner as above.

Immunoelectron microscopyNeutrophils adhered to glass coverslips in the presence of BSA

and incubated with FMLP for various periods of time were fixedwith periodate-lysine-paraformaldehyde (PLP) solution for3-5 h at room temperature. Erythrocytes and lymphocytes alsotaken from venous blood were fixed in suspension and labeled forspectrin and fodrin, respectively, in the same manner as describedbelow. After rinses with phosphate-buffered saline (PBS) contain-ing 10 mM glycine (PBS/glycine), they were treated with 0.1%saponin in PBS/glycine for 30 min at 37 °C, rinsed with PBS/glycine with 0.01 % saponin (PGS), pretreated with a mixture of0.8% bovine serum albumin, 0.1 % gelatin and 1 % normal goatserum in PGS for 30 min at room temperature (RT). Cells wereincubated with the affinity-purified anti-fodrin antibody(10-30 g ml"1) in the pretreatment protein solution for 60-90 minat 37 °C. As a negative control, nonspecific rabbit IgG at the sameconcentration was substituted for the specific antibody. Afterwashing, the specimens were treated with gold (1 nm)-conjugatedgoat anti-rabbit IgG antibody (1:40 dilution) (Janssen BiotechN.V., Olen, Belgium) in the same protein solution for 10-15 h atRT. Cells were rinsed with PBS and then with distilled water andsilver enhancement was performed for 5-10 min at RT usingIntenSE M kit (Janssen) according to the manufacturer's instruc-tions. After rinses with distilled water, cells were dehydrated andembedded in Epoxy resin in flat molds. Coverslips were detachedfrom the polymerized resin block by plunging into liquid nitrogenand hot water alternately, and sections were cut horizontally tothe substratum. Ultrathin sections counterstained with uranylacetate and lead citrate were observed with a JEOL 1200 EX or aJEOL 2000 EX electron microscope operated at 80 kV.

Distribution of silver-enhanced gold particles within a definedzone of a cell were quantitated according to Hartwig et al. (1989).Particles associated with the cell membrane or distributed in themost cortical 100 nm of cytoplasm were counted and their ratio tototal particles per cell was calculated.

Results

ImmunoblottingBy immunoblotting, the affinity-purified anti-fodrin anti-body was found to recognize the cv-chain of fodrin; reactionwith /3-chain was negligible (Fujimoto and Ogawa, 1989a).When applied to human neutrophil homogenates, theantibody reacted positively with a band of 240xl03Mr

(Fig. IB). The anti-spectrin antibody reacted specificallywith both a- and y3-chains of spectrin of the humanerythrocyte ghost (Fig. 1A).

Immunofluorescence microscopy for fodrinA majority of untreated neutrophils that adhered tocoverslips appeared round and roughly symmetrical; fod-rin was distributed evenly in most of the cells withoutlocal concentration (Fig. 2A). Only in about 6 % of the cellswas a polarized distribution of fodrin observed. WhenFMLP was added, most neutrophils gradually becameelongated; a round head and a tapered tail were unam-biguously recognized. Three minutes after the FMLPaddition at 25 °C, cells with polarized distribution of fodrinwere in the majority (Fig. 2B). The accumulation of fodrinwas most apparent around 10 min after the treatment(Fig. 2D), and thereafter became less distinctive(Fig. 2E,F). The proportion of cells with local concen-tration of fodrin reached a plateau as early as 3 min andstarted to decrease after 10 min (Fig. 3). By double label-ing of fodrin and F-actin, the accumulation of fodrin wasshown to occur only at the opposite end to the actin-richlamellipodia, that is, in the posterior portion of the cell(Fig. 2B,C).

Neutrophils plated on coverslips and treated with 10 or100 ̂ gml"1 of ConA for 10 min at either 25 or 37 °C did notchange shape and showed no local concentration of fodrin

478 T. Fujimoto and K. Ogawa

-240

A B

Fig. 1. Immunoblotting. Lane A, human erythrocyte ghost; andlane B, human leucocyte homogenate were run by SDS-gelelectrophoresis, transferred to a filter membrane and reactedwith the anti-spectrin and the anti-fodrin antibodies,respectively. The arrow indicates a band of 240 x 103 MT.

(Fig. 2G). Cells rinsed with HBSS without BSA and placedonto coverslips in the absence of BSA remained symmetri-cal even after the addition of FMLP; fodrin in those cellsdid not accumulate locally (photograph not shown).

Under-agarose systemAfter 2h of incubation at 37 °C, cells observed underagarose between the central well and the FMLP well tookon a polarized shape. Most of the cells showed accumu-lation of fodrin in the posterior portion facing the centralwell (Fig. 4A). In contrast, cells remaining in the centralwell were mostly unpolarized and the fodrin labeling wasseen without conspicuous concentration (Fig. 4B).

Immunoelectron microscopyImmunolabeling for fodrin was observed as silver-enhanced gold particles ranging from 5 to 40 nm or larger,depending upon the duration of the development. Incontrol cells without FMLP treatment, the particles wereseen scattered in the peripheral cytoplasm (Fig. 5A). Mostof them were localized in the cell cortex, which appearedfilamentous in nature, and were not associated with thecell membrane. Specimens incubated with nonspecificrabbit IgG for the primary antibody showed only a lowbackground (Fig. 5B).

Human erythrocytes treated by the same protocol lostmost hemoglobin, due to poor crosslinking by PLP fixa-tive. The labeling of spectrin was found almost exclusively

Fig. 2. Immunofluorescence microscopy of the neutrophil labeled singly for fodrin (A,D-G) or doubly labeled for fodrin (B) andF-actin (C). A. Control; B,C, 3min; D, lOmin; E, 20min; F, 60 min after addition of 1CT8M FMLP at 25 °C. Concentration of fodrin inthe posterior region is apparent in B and D, but less obvious in E and F. In the doubly labeled cells, polarization of fodrin andF-actin in opposite directions is shown. G. 10min after addition of lO^gml"1 ConA at 25°C. Fodrin is distributed without localaccumulation. X480.

Fodrin polarization in neutrophils 479

on the inner surface of the cell membrane (Fig. 5C). Theparticles were either adhered to the cell membrane orlocalized in close proximity to it. In lymphocytes, althoughsome silver-enhanced gold particles appeared in the cyto-

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Fig. 3. Percentage of cells showing polarized distribution offodrin measured under the immunofluorescence microscope. Theresult given is the average of four experiments performedindependently.

Fig. 4. Immunofluorescence microscopy of the neutrophilincubated in the gradient of FMLP ( 0 - 1 0 ~ 7 M ) using the under-agarose system for 2h at 37 "C. A. Most of the cells migratingtoward the FMLP well (arrow shows the direction) aremorphologically polarized and fodrin is accumulated m theposterior portion of the cell. B. Cells remaining in the centralwell are symmetrical and the labeling for fodrin does not showlocal concentration. A few bright spots are autofluorescencederived from eosinophils. X380.

Table 1. The ratio of immunolabeling either associatedwith the cell membrane or distributed in the most cortical100 nm of the cytoplasm (including membrane-associated)

in erythrocytes, lymphocytes and untreated neutrophilsLabeling adhering to

the cell membraneLabeling either adheringto or within 100 nm from

the cell membrane (%)

ErythrocytesLymphocytesNeutrophils

90.9±3.2051.9±8.4611.8±4.97

99.6±1.1086.8±6.2723.2±9.90

The values are significantly different from each other (Student's (-test;P<0.001); given 88 meants.D.

plasm, much of the labeling was also observed either on orimmediately beneath the cell membrane (Fig. 5D).

The ratio of immunolabels that are associated with thecell membrane and that are distributed within 100 nmfrom the membrane (including membrane-associated) wasquantitated (Table 1). Whereas almost all the labeling forspectrin in erythrocytes and the majority of the labelingfor fodrin in lymphocytes were localized in a narrowsubmembranous region, most of the labeling for fodrin inuntreated neutrophils was distributed in the deeper cyto-plasm.

After incubation with FMLP for 10 min, most neutro-phils revealed a polarized overall morphology with atypical disposition of internal structure (Fig. 6A-C): inthe head region, a filamentous lamellipodium existed, andmost granules were distributed from the mid to theposterior region of the cell behind the nucleus; in theposterior portion, a filamentous cortex that was thickest inthe tail was observed consistently. The particles indi-cating localization of fodrin were seen concentrated in theposterior portion of polarized cells. The most intenselabeling was not seen in the cortical filamentous layer, butin the inner granule-rich cytoplasm. The elongated tail,mostly appearing filamentous, was not labeled intenselyexcept for its core containing a few granules (arrows inFig. 6). The lamellipodium consistently showed immuno-labeling, but only sparsely (arrowheads in Fig. 6).

Discussion

The redistribution of fodrin observed in polarized neutro-phils appears to resemble that in lymphocytes duringcapping that is induced by various polyvalent Uganda(Levine and Willard, 1983; Nelson et al. 1983). But thereare several important differences between the twophenomena. First, FMLP is a monovalent ligand, and apolyvalent lectin, ConA, which induces capping in lym-phocytes, did not cause either morphological polarizationor fodrin concentration in neutrophils attached to thesubstratum. Depolymerization of microtubules seemsnecessary for ConA to induce a cap in neutrophils (Alber-tini et al. 1977; Sheterline and Hopkins, 1981), althoughthere has been a contradictory report (Kuehn and Epps,1980). In any case, it is important to note that accumu-lation of fodrin cannot be induced by simple crosslinking ofsurface molecules.

Second, whereas actin as well as many actin-bindingproteins coexist with fodrin beneath the lymphocyte cap,in the FMLP-treated neutrophil F-actin is distributedmost densely in the lamellipodium of the head region andnot in the tail (Oliver et al. 1978; Wallace et al. 1984;Hasten, 1987). Myosin and actin-binding protein have also

480 T. Fujimoto and K. Ogawa

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5A

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C ,

Fig. 5. Immunoelectron microscopy using 1 nm gold conjugate combined with silver enhancement. A. Neutrophil labeled for fodrin;without FMLP stimulation. Most of the particles are in the filamentous layer; those in the very periphery are fewer in number,x l l 000. B. Negative control. The primary antibody was substituted with nonspecific rabbit IgG. Only a few particles are seen in thecell. X2700. C. Erythrocyte labeled for spectrin. Virtually all labels are adhered to the inner surface of the cell membrane, x 10 200.D. Lymphocyte labeled for fodrin. The majority of labels are associated with or in a close proximity of the cell membrane, x 10 400.

Fodrin polarization in neutrophils 481

6A

B \

Fig. 6. Immunoelectron microscopy of neutrophils stimulated with FMLP for 10 min. Labeling for fodrin. The head is on the left andthe tail is on the right. The labels are concentrated in the granule-rich cytoplasm of the posterior portion. The tail with thickenedfilamentous cortex is not intensely labeled except for its core region (arrows). The lamellipodium in the anterior portion is labeledonly sparsely (arrowheads). x6700.

482 T. Fujimoto and K. Ogawa

been reported to be concentrated in the head of movingneutrophils (Valerius et al. 1981). In addition, morphologi-cal polarization and fodrin accumulation in neutrophilsoccur faster than they do in capping lymphocytes (Levineand Willard, 1983). Considering these differences as wellas the cytoplasmic distribution of fodrin in the neutrophil(compared with its mostly membranous location in thelymphocyte) as discussed below, the phenomenon dis-cussed here in the neutrophil is likely to be different fromthat in the capping lymphocyte.

The present method of immunoelectron microscopyusing a combination of 1 nm gold-conjugated antibody andsilver enhancement gave a distinct localization of theantigen. As a control experiment, we fixed and labeledhuman erythrocytes for spectrin by the same technique.Since spectrin in the erythrocyte is present exclusively onthe cytoplasmic surface of the cell membrane, the resultshould provide an example of the labeling distributionexpected for membrane proteins; almost all the spectrinlabels were associated with the cell membrane. In con-trast, only a small percentage of the immtfnolabels forfodrin were seen adhered to the cell membrane of theneutrophil. Most of them were observed in the filamentouslayer beneath the cell membrane, which probably corre-sponds to the actin-rich cell cortex described before (Brayet al. 1986; Bray and White, 1988). It is not likely that thedifference in distribution seen in the erythrocyte and theneutrophil is simply due to the presence of the cytoplasmin the latter, because a majority of the fodrin labels in thelymphocyte were either adhered to the membrane orlocalized just beneath it. Hence, most of the fodrin mol-ecules in the neutrophil probably exist as a cytoskeletalprotein rather than as a membrane protein. In view of thesimilarity of locomotion of neutrophils and lymphocytes,the difference in the distribution of fodrin is surprising.The functional significance of the protein in the two celltypes may be different. The results of immunoelectronmicroscopy also suggest that fodrin was moved to the innercytoplasm after chemotactic peptide stimulation, althoughwhether the protein was associated with any particularstructure could not be determined.

The physiological implication of the fodrin translocationin the neutrophil can be discussed only speculatively atpresent. But it is most likely a phenomenon related to celllocomotion because immobile neutrophils on non-BSA-coated glass (Keller et al. 1979; Smith et al. 1979) did notshow either morphological polarization or fodrin accumu-lation. The uneven distribution of fodrin may be importantin establishing the functional polarization necessary foreffective locomotion (Zigmond, 1989). For example, thetranslocation of fodrin to the endoplasm may make thefilamentous cortex rigid and thus inhibit pseudopodialprojections in the posterior portion; consequently pro-trusions occur only in the anterior portion. It is alsopossible that, as suggested in the exocytosis of chromaffincells (Perrin and Aunis, 1985), the sparsity of fodrin mightinduce vesicles to fuse preferentially with the cell mem-brane in the head; vesicles carrying various receptors(Walter et al. 1980; Weinbaum et al. 1980) or recyclingmembrane lipids, as supposed in the membrane-flowhypothesis (Bretscher, 1984; Singer and Kupfer, 1986),may be involved. The role of functional polarization hasbeen ascribed to conventional myosin in Dictyosteliumdiscoideum (Spudich, 1989), but myosin in the neutrophilwas found concentrated in the anterior portion (Valerius etal. 1981) in contrast to the posterior localization in Dictyo-stelium (Yumura and Fukui, 1985). It will be interesting to

study the question of whether a fodrin-like protein existsin Dictyostelium and where it is located before and afterchemotactic stimulation.

It has been shown that N-formyl chemotactic receptorsshift to high affinity after ligand binding and are tran-siently associated with the cytoskeleton (Jesaitis et al.1984; Painter et al. 1987). Recently, the occupied receptorswere shown to be segregated into actin- and fodrin-richcell membrane domains (Jesaitis et al. 1988). The presentresult suggests that the receptors are concentrated in theposterior portion of the cell; the localization of fodrin in theinner cytoplasm rather than in the cell membrane or itsunderlying cortex implies that they may be in someintracellular compartment and not exposed on the cellsurface. Accumulated fodrin may be related to endocytosisof the ligand-receptor complex (Jesaitis et al. 1984).

The molecular mechanism that causes the polarizeddistribution of fodrin in the neutrophil is not known. Thereare a few known examples in which fodrin is translocatedfrom the cell membrane to another cytoplasmic compart-ment: in lymphocytes, fodrin exists as cytoplasmic aggre-gates (Black et al. 1988; Lee et al. 1988); in rat chromaffincells, some retrieving vesicles during massive secretioncontain fodrin (Fujimoto and Ogawa, 1989); in culturedkeratinocytes, at least some of the fodrin molecules areattached to actin filaments (Yoneda et al. 1990a,£>). In theabove cases, however, the direct cause of fodrin translo-cation has not been determined. In the neutrophil stimu-lated with the chemotactic peptide, increase of cytoplasmicCa2+ and activation of protein kinase C and calpain I areknown to occur (Snyderman and Uhing, 1988; Melloni andPontremoli, 1989) and, interestingly, all three have beenshown to modify fodrin either in vitro, in other cells or inboth (Marchesi, 1985; Goodman et al. 1988; Melloni andPontremoli, 1989). Molecular details of how fodrin ischemically changed or not changed in moving cells andhow the translocation is caused obviously merit furtherstudy, both for delineation of the role of fodrin and forunderstanding of the cell locomotory mechanism.

The authors are grateful to Ms Kosei Okano for technicalassistance. This work was supported by a Grant-in-Aid from theMinistry of Education, Science and Culture of the JapaneseGovernment (#01770011) and by a grant from The WaksmanFoundation of Japan, Inc.

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(Received 2 March 1990 - Accepted 18 April 1990)

484 T. Fujimoto and K. Ogawa