J. Biol. Macromol., 6 (1) , 3-9 (2006)

Review
Functional Oligosaccharide and Its New Aspect as Immune Modulation
Kodo Otaka
Dept. Cellular Macromolecule Chemistry, Graduate School of Agriculture, Kyoto Prefectural University,
1-5, Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.

Received March 5, 2006; Accepted March 16, 2006
Short chain saccharides have beneficial functions for health of humans and animals, and are
known as functional oligosaccharides. They have been utilized as a food ingredient. Recently, newfunctions of oligosaccharides have been reported from the field of immune study. In this article, I
would like to review the immune enhancing ability of β-glucan oligosaccharides extended fromlaminarin and yeast cell wall based upon recent reports including our own results. It has been shownthat β-glucan oligosaccharides possess an activity to stimulate a secretion of cytokine such as TNF-α
from human monocytes or macrophage. In addition, oligosaccharides extending from yeast cell wall,
prepared by the method of autolysis, together with digestion by endo-1,3-β-glucanase, have enhanced
the modulation of immune system when orally administered as a diet ingredient. It is further
discussed about the most effective chain length or structure of β-glucan oligosaccharides for the
stimulation of monocytes. The yeast cell wall hydrolyzate that contains oligosaccharide can besupplied with easy process. Such hydrolyzate might become promising for the product of practicaluse in humans and animals.
Keywords: oligosaccharide, β-glucan, laminarin, yeast cell wall, immune modulation
Introduction
Oligosaccharides, a short chain saccharide
containing of homo- or hetero-sugars, are wellknown for their beneficial effects on human lifeand have been extensively utilized for long time[1]. Functional oligosaccharides, which have aphysiological function such as low cariogenicity[2] and bifidobacteria growth factor [3], improve
the health of humans and animals. The
development of oligosaccharide production
requires the identification of new enzymes andapplication technology of these enzymes because
these oligosaccharides are produced fromrespective polysaccharides or sugars by enzymatichydrolysis and/or transfer reaction. For instance,
maltooligosylsucrose is produced by thetransglucosylation reaction of cyclodextrin
glucanotransferase from starch and sucrose, andgalactooligosaccharides are produced by theCorresponding to: E-mail:ohtakakpu@yahoo.co.jp

hydrolysis and transgalactosylation reaction of
lactase from lactose. The prevention of an
infection from pathogenic microbes and viruses isimportant to maintain human health. β-Glucan,
which is derived from the yeast cell wall andfungi, is known to possess antimicrobial andantitumor activities through the enhancement of
the host immune function. Recently, new functionsof oligosaccharides, which have the ability tomodulate the immune system in humans, animals,
and fish, have been reported. For instance, it has
been suggested that mannanoligosaccharide (MO)tends to modulate the systemic immunity, and it
has
been
demonstrated
that
laminarinoligosaccharide, which is an extension fromlaminarin hydrolyzed with endo-1,3-β-glucanase,possesses the ability to induce production ofcytokines such as TNF-α from human monocytes.
Identifying the new functions of oligosaccharides
is important; furthermore, it would be of interest to
determine whether oligosaccharides or solubilizedsaccharides from the yeast cell wall can stimulate

4Functional oligosaccharide

the immune system when they are applied as asupplement like diet ingredient. In this review, thephysiological functions of oligosaccharides arereviewed, including enzymes involved in theoligosaccharide production process, and especiallythe immune modulation ability of oligosaccharides
such as MO, laminarin oligosaccharides, andoligosaccharides that are an extension from theyeast cell wall. The elucidation of thecharacteristics and immune modulation ability ofthe oligosaccharides from laminarin and watersoluble saccharides and oligosaccharides fromyeast, Hansenula anomala, is the main focus of
this review.

incubated with the mixture of starch and sucrose,various maltooligosylsucroses are produced [7].
Palatinose, 6-O-α-D-Glucosyl-D-fructofranose(isomalturose), which has a function as a noncariogenic sweetener through the suppression of
the production of water-insoluble glucans, ismainly utilized in confectionery as a sugar
substitute [8-10]. α-Glucosyltransferase derived
from bacteria catalyzes the transformation of
sucrose to palatinose. When a sucrose solution ispassed through the immobilized cell of Erwinia
rapontici, which exhibits an α-glucosyltransferase
activity, sucrose is transformed to palatinose andrelated oligosaccharides [11].

Low-cariogenic oligosaccharides
The prevention of dental caries is a serious
problem in our daily life. Oral microbials havebeen shown to cause dental caries. The bacterium
Streptococci, including Streptococcus mutans,produces a water-insoluble, highly sticky dextranlike glucan from sucrose. The reaction is catalyzed
by glucosyltransferase. The glucan covers thedental surface, forming a caries-conductive plaque.
The presence of a saccharide such as glucose,fructose or sucrose during plaque formation leads
to bacterial acid production in the plaque,subsequently initiating the occurrence of a dental
caries [4,5]. Dextranase, which hydrolyzes
polysaccharides produced by oral Streptococii,
may be useful in the prevention of dental plaque[6], and has been used extensively as an ingredientof toothpaste. Similarly, oligosaccharides,
includingmaltooligosylsucrose
known
asCoupling sugar, prevent dental caries. Those aretermed cariogenic oligosaccharides, and the effectof Coupling sugar has been extensively studied.
The result indicates that Coupling sugar is not only
unavailable for the synthesis of insoluble glucan
but also inhibits the synthesis of insoluble glucan
from sucrose by St. mutans. Furthermore, its acidproduction is low compared to that from sucrose[2]. Coupling sugar is produced by a transfer
reaction
catalyzed
bythe
enzyme,cyclomaltodextrin glucanotransferase, which has
the ability to convert starch to cyclodextrin andalso catalyzes the transglucosylation reaction
whereby glycosyl moieties are transferred fromstarch to an accepter. When the enzyme is

Bifidobacteria growth factor
Gibson and Roberfroid introduced the conceptof “prebiotics” which alter the microbial
populations in the intestines and, consequently,
improve the health of the host [12]. By definition,
prebiotics mean nondigestible food ingredientsthat beneficially affect the host by selectively
stimulating the growth and/or activity of one
bacterium or a limited number of bacteria in thecolon;
thus
improvinghost
health.
Fractooligosaccharides (FO) are nondigestable, butthey are selectively utilized by bifidobacteria in
humanintestinal
canal[13,14].
βFructofranosidase, derived from Aspergillus niger,
reacts with sucrose and produces some FO such as
1-kestose (GF2), nystose (GF3) and 1F-βfructofranosylnystose
(GF4)
[15].
The
administration of FO has been reported to beeffective in suppressing the production ofintestinal putrefactive substances in both humans
and animals. In the case of hyperlipemia patients,
the administration of FO greatly improved thelevel of lipids in serum such as cholesterol andtriglyceride as well as the blood pressure [16].
Galactooligosaccharides have a function as
bifidobacteria growth factor [17]. β-Galactosidase(lactase), derived from Aspergillus oryzae,catalyzes the transgalactosylation reaction as wellas the hydrolysis of lactose to produce
disaccharide (galactosyl glucose and galactosyl
galactose),
trisaccharide,andhigheroligosaccharide [18]. 6’-Galactosyllactose occurs
naturally in human milk and has been utilized as
milk replacement inclusion for infants. Further

Kodo Otaka

development of the enzymes and bioengineering to
produce these beneficial oligosaccharides in anindustrial scale are expected to contribute to themaintenance of human health.
Modulation of the immune system withsaccharides and oligosaccharides
Mannanoligosaccharides (MOs)
Supplementation of mannans, which have beenreported to modulate the immune system, increases
the IgA concentration in the cecal contents of rats[19], bile IgA and systemic IgG in turkeys [20],and neutrophile activity in dogs [21] and fish [22].
Secretory IgA is important in mucosal immunity
because it inhibits the attachment and penetrationof bacteria in the rumen, increases mucus secretionand prevents inflammatory reactions that would
cause damage to the epithelial tissues [23]. On theother hand, the roll of MOs in the modulation of
the immune system is not completely understood.
It has been reported that MOs tend to enhancemicrobial populations and modulate the systemic
immunity. Dogs supplemented with MOs had
lower fecal total aerobes and tended to have agreater Lactobacillus population. Furthermore,they were likely to have an enhanced immune
system with increased IgA and lymphocyte
concentration. The trends of increased serum IgA
and lymphocyte concentration are likely due to theincreased proliferation of B lymphocytes andsecretory IgA occurring in the intestine [24].
β-Glucan oligosaccharides
β-Glucans are mainly distributed among fungi,
brown seaweed, lichen and barley as cell wallcomponents of fungi or plant storage substances.
Numerous reports have indicated that β-glucans
have an activity to stimulate immunity anddecrease infectious complications in humans[25,26] and animals [27], and are also known toactivate macrophages, neutrophiles, and NK cellsby binding to the β-glucan receptor on the cells[28,29]. Immunologically active glucans have nonbranched β-1,3-linked backbone or β-1,3-linked
backbone with β-1,6-branches. Furthermore, theimmunological activities of β-glucans are
considered to depend on the structure of glucans,including the molecular mass, degree of branching,length of branch, and higher order structure [30-532]. The first step in macrophage activation by β1,3-glucan is thought to be the binding of thepolymer to specific macrophage receptors and isfollowed by the secretion of cytokines such as
TNF-α. A relatively wide range of β-glucans
seems to be recognized by specific receptors, suchas CR3 (CD11b/CD18) and dectin-1 [33-35].
However, most of the β-glucans analyzed so far
have a relative higher molecular mass, and only
limited information is available on the β-glucan
with low molecular mass in terms of thecorrelation between its immunomodulatory
function and structure.
Engstad and Robertson reported that the
Atlantic salmon macrophage possesses a receptor
that recognizes even very short β-1,3-linked
glycosyl-chains originated from yeast cell walls[36]. The Atlantic salmon receptor recognizes
small oligomers from formolyzed β-glucan
particles and linear β-1,3-linked oligomers with adegree of polymerization (DP) ≥ 3. The recognizedability of laminarin and laminaripentaose isabolished by degrading the nonreducing terminalend by means of a sodium periodate treatment. In
contrast, in the case of laminarin, this ability isregained by a complete Smith degradation. In
addition, it has been shown that salmon
macrophage does not recognize peroxidateoxidized glucan, however, this ability is regained
when the glucans are hydrolyzed to recover thenonreducing terminal end. These findings will bevery important in future studies. Recently, the β1,3-glucan oligomer (DP ≥ 4) from laminarin
derived from Laminaria digitata hydrolyzed withendo-1,3-β-glucanase from Bacillus clausii NM-1
is examined with regard to its effect on humanperipheral blood monocytes. A conditioned
medium prepared by incubating monocytes withthe β-1,3-glucan oligomer showed strong
inhibitory activity against the proliferation ofhuman leukemia U937 cells. On the other hand, aconditioned medium prepared with originallaminarin has little effect on the growth of U937
cells. The cytotoxicity of the monocyteconditioned medium prepared with the β-1,3glucan oligomer was significantly reduced by ananti-TNF-α antibody, suggesting that the
enzymaticallydepolymelized
β-1,3-glucan
oligomer induces TNF-α production in humanFunctional oligosaccharide

6

Viable cells (×104 )monocytes. These results suggest that the β-1,3glucan oligomer prepared with 1,3-β-glucanase
contains an essential structural unit to stimulate
macrophage to release TNF-α [37].
We examined the human monocyte-stimulating
ability of laminarin from Eisenia bycyclis (LB),
lichenan from Cetraria islandica, and theiroligomers depolymerized with purified endo-1,3β-glucanase from Arthrobacter sp. [38]. The
monocyte-conditioned medium pre-cultured with afraction of β-glucan oligomer (DP ≥ 8) fromlaminarin, LB-1, showed inhibitory activity against
the proliferation of human myeloid leukemia U937
cells (Fig. 1). On the other hand, the monocyteconditioned medium pre-cultured with other βglucan oligomers and original laminarin andlichenan showed little or no activity. These results
suggested that enzymatic hydrolysis is an essential
step for the monocyte-stimulation activity oflaminarin. Moreover, the average DP of LB-1 (DP≥ 8) was estimated to be 13, and the ratio of β-1,3to β-1,6-linkage was estimated to be 1.3 by NMR
analysis. Since the average DP and the ratio of β1,3- to β-1,6-linkage of original laminarin are
estimated to be 36 and 1.5 respectively, thestructure of the active β-glucan oligomer will havea higher ratio of β-1,6-linkage than that of theoriginal laminarin. These results indicate that the
140
120
100
80
60
40
20
0
Con.

LB

LB-1

LB-2

LB-3

Figure 1. Effects of the monocyte-conditioned
medium pre-cultured in the presence or absenceof β-glucans on the proliferation of U937 cells.
LB-1, -2, -3 are depolymerized oligomers of
LB, and their DP values are ≥ 8, 6-7, and 4-5,
respectively.
β-glucan oligomer with higher contents of β-1,6linkage stimulates monocytes to inhibit the
proliferation of U937 cells [35]. It is interesting tonote that a recently identified β-glucan receptor,

dectin-1, can recognize not only β-1,3 but also β1,6-linked glucans. Although the correlation of βglucan oligomer with dectin-1 is unknown, thebinding of β-glucan to one of the β-glucan receptors may induce cytokine release. It is not clear
why the hydrolysis of laminarin is essential for the
monocyte-stimulation activity; however, a significant correlation can be expected from the case of
Atlantic salmon macrophage mentioned above.
Preparation of soluble saccharide from yeast cellwall
The cell wall of the yeast Saccharomyces
cerevisiae contains β-glucan, chitin, andmannoprotein, and the β-glucan consists of alkaliinsoluble and alkali-soluble components. Yeast βglucan has been rendered soluble by sulfornylation[39], sulfation [40], phosphorylation [41], andalkali extraction methods for clinical use [42]. We
have developed the autolysis and enzymatichydrolysis method to obtain soluble saccharides
from newly isolated yeast Hansenula anomala
SB1020. Autophagic death occurs when vacuolarhydrolytic protease causes the dissolution of the
protoplasm,
andautolysis
occurswhen
carbohydrases cause the lysis of the envelope.
Glucanase, chitinase, and mannanase participate inyeast autolysis as the lytic enzymes of cell wall[43]. In the progress of autolysis with the heat
treatment, a decrease in the alkali-insoluble
fraction and an increase in the alkali-soluble
fraction were observed, but a small amount ofsoluble saccharides was released. It is interestingthat autolysis in a mixture of two strains, H.
anomala and R. glutisis, was stimulated withsynergistic effect, suggesting that R. glutisis has
strongly reactive carbohydrases and stimulates thehydrolysis of the yeast cell wall. An autolysis
product of yeast, H. anomala (YHA), stimulates
the enzyme reaction of Rhizopus sp., whichcontains endo-1,3-glucanase and protease, butlittle stimulation was observed without autolysis.
These results indicated that enzymatic hydrolysis
should be performed after the autolysis, as asignificant amount of soluble saccharides can thus
be obtained. It was confirmed by NMR and HPLC
analyses that the saccharides, enzymatic hydrolysis
product of YHA (YHAE), are composed of mono-,
oligo-, and polysaccharides, including β-1,3- andKodo Otaka

β-1,6-glucosidic bonds, and α-1,2 and α-1,6linkage [44].
Functions of soluble saccharides from the yeast
cell wall
The supplementation of feed including YHA
markedly improved the feed efficiency of thejuvenile aqua culture ayu, Plecoglossus altivelis[45]. It was deduced that the increase in the feed
efficiency resulted not from the nutritional value ofthe ingredients such as vitamins and amino acids
but from an addition of a growth-promoting factor
or as a result of the enhancement of immunity like
protective function against infection of pathogenic
microbes. In this regard, the elevation of immunity
by the supply of feed or diet including H. anomala
without autolysis (HA) or YHAE in fish and rats
has been reported. The survival rate of the red sea
bream, Pagrus major, against virulent bacteria,
Vibrio angeulum, injected intraperitoneally,
improved by supplying a feed containing HA in
advance. Furthermore, the enhancement ofperitoneal macrophages of ddY female rats fed adiet including YHAE was detected in the ability of

Fluorescent intensity

50
40
30
20
10
0
Con-1

Rho

Han

Mix-1

Posi.

Figure 2. Effects of yeast supplementation on
phagocytosis activity of peritoneal macrophage(ddY female rats). Con.; control, Rho;
supplementation of Rhodotorula glutinis, Han;
supplementation of Hansenula anomala, Mix-1;
supplementation of mixture of Rhodotorula
glutinis and Hansenula anomala, Posi.;
treatment with LPS. Values are means ± SD, n= 3.
phagocytosis as well as cathepsin activity and NO
production (Fig. 2) [46]. The absorption of thesesaccharides through the intestines is critical for theelevation of immunity, but the results suggest thatoral administration of a yeast autolysis product7

would modulate the immune system of animals
and fish. In addition, YHAE was subjected toimmune stimulation examination in vitro. YHAE
was fractionated through a Sephadex G15 column
and each fraction of oligosaccharide and higher
molecular mass was submitted to testing. The
monocyte-conditioned medium pre-cultured with afraction of oligosaccharides (Main DP = 5~6),
showed a strong inhibitory activity against the
proliferation of human myeloid leukemia U937
cells (unpublished results).
All of these results suggest that YHA and
YHAE, which include mannan, β-glucan, and its
oligomer, are promising products to elevate theimmune efficiency and are expected to maintainintestinal health as “prebiotics” for humans andanimals. Because YHAE can be easily prepared, itshould be useful for commercial applications suchas health food products.
Acknowledgements
The author thanks to Dr. Takashi Maoka and
Ms. Kumi Hidaka of Research Institute for
Production Development, for helpful advice andencouragements.
References
1) Handbook of Amylases and Related Enzymes(ed. by The Amylase Research Society ofJapan), Pergamon Press (1988)
2) Araya, S. (ed.) Proceedings of the 5th
Conference on Dental Caries and Coupling
Sugar (in Japanese), National Institute of
Health, Tokyo (1980)
3) Gibson, G. R., Beatty, E. R., Wang, X.,
Cummings, J. H.; Gastroenterology 108, 975982 (1995)
4) Hamada, S., Koga. T.; Kagaku to Seibutsu (inJapanese) 19, 695 (1981)
5) Haward, J. S.; C&EN Special Report,
Washington Feb. 25 (1980)
6) Murayama, Y., Wada, H., Hayashi, H., Uchida,T., Yokomizo, E.; J. Dent. Res. 52, 658-667(1973)
7) Okada, S., Kitahata, S.; Nippon Shokuhin
Kogyo Gakkai-shi (in Japanese) 22, 420-424(1975)
8) Ohta, K., Takazoe, I.; Bull. Tokyo Dent. Coll.
24, 1-11 (1983)

8

Functional oligosaccharide

9) Ohshima, T., Izumitani, A., Sobue, S.,
Okahashi, N., Hamada, S.; Infect. Immunol. 39,
43-49 (1983)
10) Ohshima, T., Izumitani, A., Sobue, S.,
Hamada, S.; Japan J. Med. Sci. Biol. 36, 219223 (1983)
11) Cheetham, P. S., Garrett, C., Clark, J.;
Biotechnol. Bioeng. 27, 471-481 (1985)
12) Gibson, G., Roberfroid, M. B.; J. Nutr. 125,
1401-1412 (1995)
13) Hidaka, H., Hara, T., Eida, T., Okada, J.,
Shimada, K., Mitsuoka, T.; in Chonai Flora to
Shokumotsu Inshi (Mitsuoka T. ed.), pp. 39-67,
Japan Scientific Societies Press, Tokyo (1983)
14) Oku, T., Tokunaga, T., Hosoya, N.; J. Nutr.
114, 1574-1581 (1984)15) Hidaka, H., Hirayama, M.; Kagaku to Seibutsu(in Japanese) 23, 600-605 (1985)16) Hidaka, H., Eida, T., Takizawa, T., Tokunaga,T., Tashiro, Y.; Bifidobacteria Microflora 5,
37-50 (1986)
17) Toba, T.; J. Dairy Food Sci. 34, A169-A182(1985)
18) Tanaka, R., Takayama, H., Morotomi, M.,
Kuroshima, T., Ueyama, S., Matsumoto,K.,
Kuroda, A., Mutai, A.; Bifidobact. Microflora
2, 17-24 (1983)
19) Kudoh, K., Shimizu, J., Ishiyama, A., Wada,
M., Takita, T., Kanke, Y., Innami, S.; J. Nutr.
Sci. Vitaminol. 45, 173-181 (1999)
20) Savage, T. F., Cotter, P. F., Zakrzewska, E. I.;
Poult. Sci. 75, 143 (1996)21) O’Carra, R.; M. S. thesis, National Universityof Irland, Galway, Ireland. (1997)22) Yoshida, T., Kruger, R., Inglis, V.; J. Fish Dis.
18, 195-198 (1995)
23) Russell, M. W., Reinholdt, J., Kilian, M.; Eur.
J. Immunol. 19, 2243-2249 (1989)
24) Swanson, K. S., Grieshop, C. M., Flickinger,
E. A., Bauer, L. L., Healy, H. -P., Dawson, K.
A., Merchen, N. R., Fahey, G. C., Jr.; J. Nutr.
132, 980-989 (2002)
25) Browder, W., Williams, D., Pretus, H.,
Olivero, G., Enrichens, F., Mao, P., Franchello,A.; Ann. Surg. 211, 605-613 (1990)
26) Babineau, T. J., Hackford, A., Kenler, A.,
Bistrian, B., Forse, R. A., Fairchild, P. G.,
Heard, S., Keroack, M., Caushaj, P., Benotti,P.; Arch. Surgery 129, 1204-1210 (1994)27) Williums, D. L., Mueller, A., Browder, W.;
Clin. Immunother. 5, 392-399 (1996)28) Mueller, A., Rapis, J., Rice, P. J., Kalbfleisch,J. H., Stout, R. D., Ensley, H. E., Browder, W.,
Williams, D. L.; Glycobiology 10, 339-346(2000)
29) Wasser, S. P.; Appl. Microbiol. Biotechnol. 60,
258-274 (2002)30) Miyazaki, T., Oikawa, N., Yadomae, T.,
Yamada, H., Yamada, Y., Hsu, H., Ito, H.;
Carbohydr. Res. 69, 165-170 (1979)
31) Yadomae, T., Ohno, N.; Recent Res. Devel.
Chem. Pharm. Sci. 1, 23-33 (1996)32) Kataoka, K., Muta, T., Yamazaki, S.,
Takeshige, K.; J. Biol. Chem. 277, 3682536831 (2002)
33) Thomton, B. P., Vetvicka, V., Pitman, M.,
Goldman, R. C., Ross, G. D.; J. Immunol. 156,
1235-1246 (1996)
34) Yan, J., Vetvicka, V., Xia, Y., Coxon, A.,
Carroll, M. C., Mayadas, T. N., Ross, G. D.; J.
Immunol. 163, 3045-3052 (1999)
35) Taylor, P. R., Brown, G. D., Reid, D. M.,
Willment, J. A., Martinez-Pomares, L.,
Gordon, S., Wong, S. Y.; J. Immunol. 169,
3876-3882 (2002)
36) Engstad, R. E., Robertsen, B.; Dev. Comp.
Immunol. 18, 397-408 (1994)37) Miyanishi, N., Iwamoto, Y., Watanabe, E.,
Oda, T.; J. Biosci. Bioeng. 95, 192-195 (2003)
38) Pang, Z., Otaka, K., Suzuki, Y., Goto, K.,
Ohnishi, M.; J. Biol. Macromol. 4, 57-66(2004)
39) Pang, Z., Otaka, K., Maoka, T., Hidaka, K.,
Ishijima, S., Oda, M., Ohnishi, M.; Biosci.
Biotechnol. Biochem. 69, 553-558 (2005)
40) Demleitner, S., Kraus, J., Franz, G.;
Carbohydr. Res. 226, 247-252 (1992)
41) Williams, D. L., Pretus, H. A., McNamee, R.
B., Jones, E. L., Ensley, H. E., Browder, I. W.;
Carbohydr. Res. 235, 247-257 (1992)
42) Williams, D. L., Mcnamee, R. B., Jones, E. L.,
Pretus, H. A., Ensley, H. E., Browder, I. W.,
Di Luzio, N. R.; Carbohydr. Res. 219, 203213 (1991)
43) Lee, J. -N., Lee, D. -Y., Ji, I. -H., Kim, G. -E.,
Kim, H. N., Sohn, J., Kim, S., Kim, C. -W.;
Biosci. Biotechnol. Biochem. 65, 837-841(2001)

Kodo Otaka

44) Walker, G. M.; Yeast Physiology andBiotechnol. pp. 22, John Willey & Sons,
Chichester (1998)45) Otaka, K., Hidaka, K., Nakajima, A., Maoka,
T.; J. Biol. Macromol. 2, 64-74 (2002)

946) Otaka, K., Maoka, T., Hidaka, K., Nakajima,A.; Japan Kokai Tokkyo Koho; 2004-99580
Communicated by Oda Masayuki