Mitochondrial DNA Restriction Fragment Length Polymorphism (RFLP) and 18S Small Subunit Ribosomal DNA PCR RFLP Analyses of Acanthamoeba Isolated from Contact Lens Storage Cases of Residents in Southwestern Korea

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Mitochondrial DNA Restriction Fragment Length Polymorphism

(RFLP) and 18S Small-Subunit Ribosomal DNA PCR-RFLP

Analyses of

Acanthamoeba

Isolated from Contact Lens

Storage Cases of Residents in Southwestern Korea

Hyun-Hee Kong,

1

_{Ji-Yeol Shin,}

1

_{Hak-Sun Yu,}

1

_{Jin Kim,}

2

_{Tae-Won Hahn,}

3

Young-Ho Hahn,

4

_{and Dong-Il Chung}

1

_*

Department of Parasitology, Kyungpook National University School of Medicine, Taegu,1_{Department of Parasitology,}

Seonam University College of Medicine, Namwon,2_{Department of Ophthalmology, Catholic University}

Medical College, Seoul,3_{and Department of Ophthalmology, Kosin University}

College of Medicine, Pusan,4_Korea

Received 6 September 2001/Returned for modification 8 October 2001/Accepted 2 December 2001

We applied ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) and mitochondrial DNA (mtDNA) RFLP analyses to 43Acanthamoebaenvironmental isolates (KA/LH1 to KA/LH43) from contact lens storage cases in southwestern Korea. These isolates were compared to American Type Culture Collection strains and clinical isolates (KA/E1 to KA/E12) from patients with keratitis. Seven riboprint patterns were seen. To identify the species of the isolates, a phylogenetic tree was constructed based on the comparison of riboprint patterns with reference strains. Four types accounted for 39 of the isolates belonging to the A.

castellaniicomplex. The most predominant (48.8%) type wasA. castellaniiKA/LH2 type, which had identical

riboprint and mtDNA RFLP patterns to those ofA. castellaniiCastellani, KA/E3 and KA/E8. The riboprint and mtDNA RFLP patterns of the KA/LH7 (20.9%) type were identical to those ofA. castellaniiMa, a corneal isolate from the United States. The riboprint and mtDNA RFLP patterns of the KA/LH1 (18.6%) type were the same as those ofA. lugdunensisL3a, KA/E2, and KA/E12. The prevalent pattern for each type ofAcanthamoebain southwestern Korea was very different from that from southeastern Korea and Seoul, Korea. It is noteworthy that 38 (88.4%) out of 43 isolates from contact lens storage cases of the residents in southwestern Korea revealed mtDNA RFLP and riboprint patterns identical to those found for clinical isolates in our area. This indicates that most isolates from contact lens storage cases in the surveyed area are potential keratopathogens. More attention should be paid to the disinfection of contact lens storage cases to prevent possible amoebic keratitis.

Acanthamoebaspp., causative agents of the sight-threaten-ing amoebic keratitis and the life-threatensight-threaten-ing granulomatous amoebic encephalitis, have ubiquitous distribution in human environments (35, 45). Amoebic keratitis was a rare corneal infection for 10 years after the first case report by Jones et al. (23). However, the number of reported cases has increased, apparently as a consequence of an association of the disease with contact lens wearing (36, 57). Amoebic keratitis has been continuously reported in Korea as well (unpublished data). Ecological studies (22, 30) showed that the contamination rate of contact lens paraphernalia byAcanthamoebain Korea was much higher than rates reported in industrialized countries (24, 29, 56). In particular, the rate in southwestern Korea was twice that found in other provinces in Korea. The pathogenic potential ofAcanthamoebaisolates from lens storage cases in Korea has yet to be determined. Because no simple and reli-able in vivo or in vitro test exists, investigators have been using strain typing methods in order to indirectly assess the potential pathogenicity of the isolates (19, 49, 60).

Although the identification of Acanthamoebaat the genus level can be easily accomplished on the basis of the distinctive morphology of the trophozoites and cysts, there have been disputes over methods of species identification for Acan-thamoeba.Pussard and Pons (42) classifiedAcanthamoebainto three groups according to the cyst size and morphological features. Group I consists ofAcanthamoebaspp. with relatively large cysts, distinctly stellate endocysts, and smooth spherical ectocysts. Group II and group III Acanthamoeba spp. have smaller cysts (diameters less than 18 ␮m). Group II species have polygonal to stellate endocysts with irregular or wrinkled ectocysts, while the cysts of group III species have rounded or slightly angular endocysts with thinner and smooth or slightly wrinkled ectocysts. However, because of variable cyst morphol-ogy by culture conditions (53), species identification by mor-phology alone can hardly be possible (54).

Therefore, investigators have used several kinds of nonmor-phological methods for the taxonomy ofAcanthamoebaspp. Studies of isoenzyme patterns (11, 12, 14, 15, 55) suggested the other groupings ofAcanthamoebastrains that are not consis-tent with previous species assignments based on morphological criteria. Kong et al. (28) and Chung et al. (6) reported in-traspecific heterogeneity of the zymograms for several kinds of isoenzymes. Costas and Griffiths (13) considered that the vari-* Corresponding author. Mailing address: Department of

Parasitol-ogy, Kyungpook National University School of Medicine, 101 Dongin-dong, Joong-ku, Taegu 700-422, Korea. Phone: 82-53-420-6956. Fax: 82-53-422-9330. E-mail: [email protected].

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ation among someAcanthamoeba species overlaps so exten-sively that the amoebae should be regarded as species com-plexes. Mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) analysis has also been applied for taxonomy analysis ofAcanthamoeba(2, 3, 58, 59). How-ever, considering the profound interstrain diversity of the mtDNA RFLP and alloenzyme profiles, Kong et al. (28) and Chung et al. (6) suggested that both analyses should be used for the strain identification, differentiation, and characteriza-tion rather than species identificacharacteriza-tion.

The sequence of the small-subunit (ssu) rRNA gene is very useful as molecular data for phylogeny and taxonomy (5, 21, 33, 52). Stothard et al. (52) studied 18S (ssu) rRNA gene phylogeny ofAcanthamoebaand classified 53 isolates into 12 sequence types. However, when the number of isolates to be studied is large, sequencing of the 2,300-bp rRNA gene is too labor-intensive, time-consuming, and expensive for identifica-tion or characterizaidentifica-tion of Acanthamoeba isolates. Thus, a simpler and less expensive method is needed. Riboprinting, the examination of restriction enzyme site polymorphism of ssu rRNA-coding DNA (rDNA) amplified by PCR, is a simple, inexpensive, and timely method and has recently been used to establish the taxonomic relationships amongEntamoebaspp. (9, 10), among Naegleria spp. (8, 16), and among Acan-thamoebaspp. (5, 27). Chung et al. (5) were the first to apply riboprinting to the subgenus classification ofAcanthamoeba. Their results coincided well with those of rRNA sequencing performed by Stothard et al. (52).Acanthamoebataxonomists generally agreed with the classification that Chung et al. (5) had suggested (T. J. Byers, personal communication). By ei-ther rDNA sequencing or riboprinting, most clinical isolates of

morphological group II were shown to belong to a clade (T4 by Stothard et al.,A. castellaniisubgroup by Chung et al.) con-tainingA. castellaniiCastellani. Stothard et al. (52) suggested that various species in T4 all might be reclassified asA. castel-laniibecause the sequence type includes the type strain for that species.

In the present study, we analyzed 43Acanthamoebaisolates from contact lens storage cases of the residents of southwest-ern Korea using riboprinting and mtDNA RFLP analyses and evaluated the potential keratopathogenicities of these isolates.

MATERIALS AND METHODS

Acanthamoebaisolation and axenic culture.In order to isolateAcanthamoeba

from contaminated contact lens storage cases, the cases were opened under aseptic conditions. The centrifuged sediment from the solution in a case was aseptically smeared on a nonnutrient agar plate covered with heat-treated Esch-erichia coli(60). A sterile cotton ball was then rubbed over the internal surface of the lens case and placed on a different agar plate covered with heat-treatedE. coli. The plates were incubated at 25°C for 1 week and examined daily for the presence and growth ofAcanthamoebaunder an inverted microscope.

TheAcanthamoebacysts encysted on the plate were grouped by their size and morphological features according to the criteria of Pussard and Pons (42).

A piece of agar plate (0.5 by 1 cm) covered with the cysts of a clone was treated with 0.1 N HCl for 24 h for axenization and washed with glass-distilled water three times. The piece of agar plate with many cysts was placed in Proteose Peptone-yeast extract-glucose medium (60) at 25°C.

Reference strains.Twenty-threeAcanthamoebastrains (Table 1) were ob-tained from the American Type Culture Collection (ATCC) (Manassas, Va.). Twelve clinical isolates (KA/E1 to KA/E12) from Korean keratitis patients were also used as reference strains.

Riboprinting: PCR-RFLP of the ssu rDNA.Chromosomal DNA of each clone was extracted as described by Kong and Chung (27). Amoebae harvested at the end of the logarithmic growth phase were washed with cold phosphate-buffered saline three times and boiled in 0.1 ml of 0.1 N NaOH for 3 min. The supernatant

Chang 30898 ⫹ Freshwater United States A. castellanii 4

BH-2 30730 ⫹ Ocean sediment United States A. hatchetti 48

RB-F-1 50388 ND Ocean sediment United States A. stevensoni 47

S-7 30731 ⫹ Beach bottom United States A. griffini 46

Ray & Hayes 30137 ND Soil United States A. astronyxis 43

OC-15C 30867 ND River United States A. tubiashi 32

A-1 30171 ⫹ Tissue culture United States A. culbertsoni 51

OC-3A 30866 ⫹ GAEb _{United States} _{A. healyi} ₃₇

GE-3a 50252 ⫺ Swimming pool France A. pustulosa 42

Reich 30870 ⫺ Soil Israel A. palestinensis 44

a_{ND, not determined.}

b_{GAE, granulomatous amoebic encephalitis.}

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was collected after centrifugation at 800⫻gfor 2 min at room temperature and mixed with 0.2 ml of glass-distilled water. An equal volume of phenol was added to the solution, and this mixture was vortexed for 1 min. The mixture was centrifuged at 15,000 rpm for 5 min at 4°C twice. The resulting aqueous phase was centrifuged at 12,000 rpm for 5 min again with 300␮l of phenol-chloroform (1:1) solution. The nuclear DNA was precipitated by adding 600␮l of cold absolute ethanol and 30␮l of 3 M sodium acetate (pH 5.2), incubating this mixture at⫺70°C for 15 min, and then centrifuging it at 15,000 rpm for 20 min at 4°C. The sediment was washed with 70% ethanol, vacuum dried, and then dissolved in 30 to 50␮l of glass-distilled water. The DNA was stored at⫺20°C until used.

The sequence of primers for PCR ssu rDNA (P3, 5⬘-CCGAATTCGTCGAC AACTGTTGATCCTGCCAGT-3⬘; P4, 5⬘-GGATCCAAGCTTGATCCTTC TGCAGGTTCACCTAC-3⬘) are designed to hybridize at the highly conserved sequences of the extreme 5⬘(P3) and 3⬘(P4) termini of eukaryotic ssu rDNA (1, 5). PCR consisted of 1 min at 94°C, 1 min at 58°C, and 2 min at 72°C in a thermal cycler (PE480; Perkin-Elmer Cetus). After 30 cycles, 10 min of extension time was given. After amplification, the PCR products of 43Acanthamoebaisolates were checked by electrophoresis in a 1.5% agarose gel at 4 V/cm for 1.5 h. The amplified ssu rDNA was examined by digestion with 10 restriction endonucleases (MspI,HaeIII,HhaI,HinfI,DdeI,TaqI,Tru9I,MboI,RsaI, andSau96I; Posco-chem, Seongnam, Korea) that have recognition sequences of four nucleotides for 2 h. The digested DNA was electrophoresed in a 2.5% agarose gel (3 parts agarose, 1 part Nusieve). When the enzyme digested the DNA into small and similarly sized fragments, 15% polyacrylamide gel with Tris-borate-EDTA buffer

was used to obtain clearer and more-accurate data of fragments. The ethidium bromide-stained gel was examined and photographed under a UV transillumi-nator.

Sequence divergence estimates were calculated by the Nei and Li equation (40) from the fragment comigration data set which was obtained by comparison of the riboprints of 43 isolates and all reference strains (5). A phylogenetic tree was constructed by the unweighted pair group method with arithmetic average using a computer program (PHYLIP, version 3.5) (7, 18).

mtDNA extraction and RFLP.mtDNA of Acanthamoebaisolates was ex-tracted by the method described by Yagita and Endo (59). Briefly,Acanthamoeba

trophozoites washed with phosphate-buffered saline (pH 7.4) were suspended in 100␮l of chilled TEG buffer (25 mM Tris-HCl, 10 mM EDTA, 50 mM glucose; pH 8.0) and incubated on ice for 5 min. Amoebae were lysed by adding 200␮l of chilled fresh 1% sodium dodecyl sulfate solution in 0.2 N NaOH and then incubated subsequently on ice for 5 min. Chilled 3 M potassium acetate buffer was mixed with the suspension and incubated on ice for 15 min. Degraded cellular proteins and genomic DNA were extracted with equal volumes of phe-nol-chloroform by centrifugation at 12,000 rpm at 4°C for 5 min. mtDNA in supernatant was precipitated by adding 1.0 ml of absolute ethanol and 40␮l of 3 M sodium acetate solution and by incubating at⫺70°C for 15 min. After centrifugation at 15,000 rpm for 20 min at 4°C, the precipitated DNA was washed with 70% chilled ethanol. The DNA sediment was vacuum dried and dissolved in 15 to 25␮l of TE buffer (5 mM Tris-HCl, pH 8.0; 1 mM EDTA) and stored at

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⫺20°C until used. mtDNAs of 43Acanthamoebaisolates were digested with restriction enzymes that have recognition sequences of six nucleotides at 37°C for FIG. 1. Agarose gel electrophoretic pattern of PCR products from seven different types of Acanthamoebaand their polyacrylamide gel electrophoretic restriction fragment patterns. Lanes M,HindIII-digested␭phage DNA or 100-bp ladder (DNA size standards).

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2 h (sometimes overnight) in a 20-␮l reaction volume with the buffers specified for each restriction enzyme (EcoRI,BglII,XbaI,SalI,ScaI,ClaI,HpaI, and

PvuII; Poscochem). Digested DNA was electrophoresed in a 0.7% agarose gel and stained with ethidium bromide. The RFLP of mtDNA of 43 strains was observed and photographed under a UV transilluminator. TheHindIII-digested

␭phage was used as a size marker.

RESULTS

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Morphology of Acanthamoeba isolates. Among 43 isolates analyzed in this study, 42 isolates belonged to the morpholog-ical group II of Pussard and Pons (42). In their system, the cyst FIG. 2. Phylogenetic tree of seven different types and 23 reference strains ofAcanthamoebabased on the rDNA PCR-RFLP analyses obtained by using the unweighted pair group method with arithmetic average and a computer program (PHYLIP, version 3.5).

FIG. 3. Agarose gel electrophoretic restriction fragment patterns byEcoRI of mtDNA of 43 environmentalAcanthamoebaisolates. Lanes M,

HindIII-digested␭phage DNA (DNA size standard).

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has a polygonal endocyst and an irregularly wrinkled thick ectocyst, with the cyst diameter ranging from 11.8 to 16.5␮m and the number of arms ranging from 3.8 to 6.2. One other isolate (KA/LH35) belonged to group III, which features round endocysts closely attached to wrinkled ectocysts.

Analysis of ssu rDNA PCR-RFLP.Figure 1 presents PCR-amplified full-length rDNA and RFLP patterns obtained by using three kinds of restriction enzymes. The size of the PCR products was approximately 2,300 bp in all isolates, meaning no intron was present in the ssu rDNAs of 43 environmental isolates. Considering all patterns by eight kinds of restriction enzymes (some data not shown), a total of seven different rDNA RFLP types emerged. Using the phylogenetic tree of the isolates with reference strains based on the estimated se-quence divergence, 41 isolates were identified asA. castellanii

complex, and 1 was identified asA. polyphaga(Fig. 2), but the remaining isolate has yet to be studied. Among the 41 isolates belonging toA. castellaniicomplex, 8 isolates (KA/LH1 type) showed the same rDNA PCR-RFLP pattern asA. lugdunensis

L3a. Twenty-one isolates (KA/LH2 type) showed patterns of rDNA PCR-RFLP very similar to those ofA. castellanii Cas-tellani, the type strain of the type species. Nine isolates (KA/ LH7 type) were identical toA. castellaniiMa in their rDNA PCR-RFLP pattern. The pattern of one isolate (KA/LH32 type) was identical to that ofA. castellaniiCastellani. An iso-late (KA/LH35 type) with morphological characteristics of group III showed a unique rDNA PCR-RFLP pattern. Two isolates (KA/LH39 type), although they belonged toA. castel-laniicomplex, showed a rDNA PCR-RFLP pattern somewhat different from that of the Castellani strain. One isolate (KA/ LH8 type) clustered with theA. rhysodesclade.

mtDNA restriction phenotypes.Figure 3 shows agarose gel electrophoretic patterns ofEcoRI digests of mtDNA from 43

Acanthamoebaisolates. They were divided into seven different types according to their patterns. Although KA/LH2 and KA/ LH7 types appeared to have almost the same patterns by

EcoRI digestion, as seen in Fig. 3, the fragment sizes were slightly different. Moreover, they showed very different RFLP

patterns with other restriction enzymes, such asBglII (Fig. 4A). The most predominant type was KA/LH2, and 21 isolates showed the same mtDNA RFLP pattern. Nine isolates were of the KA/LH7 type, and eight isolates were of the KA/LH1 type. Two isolates, KA/LH39 and KA/LH40, had the same mtDNA RFLP pattern as each other. The mtDNA RFLP pattern of KA/LH32 was very similar to that of KA/LH2 but slightly different. The other two isolates, KA/LH8 and KA/LH35, had unique mtDNA RFLP patterns. A total of seven different restriction phenotypes byBglII enzymes emerged for 43 iso-lates, as shown in Fig. 4A. The other three kinds of restriction enzymes we tested showed the same patterns (data not shown).

DISCUSSION

In the present study, 43Acanthamoebaisolates, originating from contact lens storage cases of residents in southwestern Korea, were morphologically and genetically analyzed based on riboprinting and mtDNA RFLP analysis. Except for one isolate (KA/LH35), all belonged to the morphological group II (42). These isolates may be morphologically assigned to theA. castellaniiorA. polyphagacomplexes. This indicates that Acan-thamoebaof morphological group II may be predominant in contact lens storage cases in Korea. Based on riboprints, 41 isolates were genetically very closely related to A. castellanii

Castellani, and 1 was closely related to A. polyphaga. The remaining 1 isolate showed a unique riboprint. This coincided well with the results of morphological observation and indi-cated that amoebae closely related to A. castellanii may be predominant in contact lens storage cases in Korea.

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Although rDNA sequencing or riboprinting is promising for subgenus classification ofAcanthamoeba(5, 19, 52), it is not simple to identify isolates at the species level, especially strains in group II, to which most clinical and environmental isolates belong. Stothard et al. (52) suggested that various species in T4 might be reclassified asA. castellaniibecause the sequence type includes the type strain for that species. However, the molec-ular characteristics of strains in T4 were quite different among FIG. 4. Agarose gel electrophoretic restriction fragment patterns of mtDNA byBglII restriction enzyme. Seven different types of 43 environ-mentalAcanthamoebaisolates (A), three types of environmental isolates and associated reference strains ofAcanthamoeba(B), and two types of environmental isolates and associated clinical isolates ofAcanthamoebafrom keratitis patients in Korea (C) were compared. Lanes: M,Hin dIII-digested␭phage DNA (DNA size standard); L3a,A. lugdunensisL3a; Castellani,A. castellaniiCastellani; Ma,A. castellaniiMa.

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strains, and many species which belonged to this clade have already been assigned. We would rather assign this subgroup (T4 sequence type) as a species complex (60), as Costas and Griffiths suggested (13).

Based on the riboprints and mtDNA RFLP analysis in the present study, the A. castellanii KA/LH2 type was the most predominant (48.8%) type ofAcanthamoebafrom contact lens storage cases in southwestern Korea (Table 2). It is noteworthy that the riboprint and mtDNA RFLP patterns of the KA/LH2 type were identical to those ofA. castellaniiCastellani, KA/E3, and KA/E8 (Fig. 4B and C)—the type strain and two clinical isolates from infected corneas of Korean patients, respectively. The predominance of the Castellani type ofAcanthamoebain

southwestern Korea is unique compared to the previous survey results in southeastern and Seoul, Korea. The predominance of the Castellani type was 3.6% in the southeastern area (31) and 7.7% in Seoul (60).

Acanthamoeba castellanii KA/LH7 (20.9%) and KA/LH1 (18.6%) types were the second and third most prevalent types of Acanthamoeba in the surveyed area. The riboprint and mtDNA RFLP patterns of the KA/LH7 type were identical to those ofA. castellaniiMa (Fig. 4B), a clinical isolate from the United States (34). The Ma type has never been isolated in southeastern Korea (31) but was relatively prevalent (20.5%) in Seoul (60). The riboprint and mtDNA RFLP patterns of the KA/LH1 type were the same as those of A. castellanii L3a,

KA/LH14 KA/LH1 KA/LH 1 A. castellaniicomplex KA/E2, KA/E12

KA/LH15 KA/LH7 KA/LH 7 A. castellaniicomplex Ma

KA/LH16 KA/LH2 KA/LH 2 A. castellaniicomplex KA/E3, KA/E8

KA/LH17 KA/LH 2 KA/LH 2 A. castellaniicomplex KA/E3, KA/E8

KA/LH20 KA/LH 7 KA/LH 7 A. castellaniicomplex Ma

KA/LH32 KA/LH 32 KA/LH 32 A. castellanii

KA/LH35 KA/LH 35 KA/LH 35

KA/LH39 KA/LH 39 KA/LH 39 A. castellaniicomplex

KA/LH40 KA/LH 39 KA/LH 39 A. castellaniicomplex

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KA/E2, and KA/E12 (Fig. 4B and C). The KA/LH1 type was reported to be the most prevalent type ofAcanthamoeba in southeastern Korea (64.3%) and in Seoul (51.3%). Two amoe-bic keratitis patients from Seoul and Pusan were recently re-ported to be infected with this type (unpublished data).

The prevalent pattern for each type of Acanthamoeba in southwestern Korea was very different from those in the south-eastern area. However, it was interesting that the pattern in Seoul was intermediate between those in the southeastern and southwestern areas, because the citizens in Seoul are com-posed of people from various provinces, including provinces in the southeastern and southwestern areas. The prevalence pat-tern can vary from nation to nation. Kilvington et al. (25) reported that isolates with the mtDNA RFLP type ofA. cas-tellaniiJones (formerlyA. polyphaga orA. lugdunensis) were demonstrated the most frequently among clinical isolates of

Acanthamoebain Europe. According to Gautom et al. (20), isolates with the mtDNA restriction phenotype of theA. cas-tellaniiCastellani type were demonstrated the most frequently from clinical and environmental sources in the United States. In Japan, isolates with mtDNA RFLP patterns of theA. cas-tellaniiMa type were demonstrated the most frequently from contact lens paraphernalia and infected corneas (T. Endo, personal communication).

The riboprint pattern of the KA/LH32 type was the same as that of the Castellani, KA/E3, and KA/E8 types, but the KA/ LH32 type could not be regarded as a clone of the Castellani, KA/E3, or KA/E8 types but was regarded as a closely related clone, because the mtDNA RFLP pattern of the KA/LH32 type was slightly different from those of the reference strains. This finding can be explained by the fact that mtDNA evolves faster than nuclear DNA. Therefore, the mitochondrial ge-nome can be used for phylogenetic study of closely related

Acanthamoeba isolates. The mtDNA RFLP pattern of KA/ LH32 was almost identical to that of JAC/E1, an isolate from a Japanese keratitis patient (58).

The most important fact in the present study is that 38 (88.4%) out of 43 isolates from contact lens storage cases of the residents of southwestern Korea revealed mtDNA RFLP and riboprint patterns identical to those of clinical isolates of

Acanthamoeba. This indicates that most isolates in this study are potential keratopathogens. Four of the remaining five iso-lates were new types.

In contrast to the high contamination rate of contact lens paraphernalia by potentially keratopathogenicAcanthamoeba

spp. in Korea, the incidence ofAcanthamoebakeratitis so far is low. It is suggested that the combination of wearing a contact lens contaminated with keratopathogenicAcanthamoebaand minor injury of the corneal epithelium may be the predisposing factor inAcanthamoebakeratitis among contact lens wearers. At any rate, more attention should be paid to the disinfection of contact lens storage cases to prevent possible amoebic ker-atitis.

ACKNOWLEDGMENT

This work was supported by a grant (HMP-97-2-0029 for the 1997, Good Health R&D Project) from the Ministry of Health and Welfare, Republic of Korea.

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