Vol. 81, No. 4, 1978 April 28,1978

BlOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1286-1293

BLOOD GROUP i AND I ACTIVITIES OF "IACTO-N-norHEXAOSYLCERAMIDE" AND ITS ANALOGUES: THE STRUCTURAL REQUIREMENTSFOR i-SPECIFICITIES* Heiner

Niemannf Kiyohiro

Biochemical Oncology, 1124 Columbia,

Watanabe and Sen-itiroh

Hakomori

Fred Hutchinson Cancer Research Seattle, WA 98104, U.S.A.

Center,

and Robert A. Childs and Ten Feizi Clinical Research Centre Watford Road, Harrow Middlesex, England Received

March

13,1978

Summary-A pure straight chain cersmide hexasaccharide ("lacto-N-norhexaosylceramide" Gal8l~~GlcNAc~1-t3Gal81-+~GlcNAc81~3Gal~1-t~Glc-tCersmide) showed strong i-activity determined by hemagglutination inhibition and by radioimmunoassay with five out of six anti-i antisera. Two repeating Gal&+)+GlcNAc residues and GlcNAc81+3Gal residues could be essential for the full expression of this activity; eleven closely related analogues including those derived by chemical modification had lower or no detectable activity. The same structure reacted also with some anti-1 antisera. The strong i-activity and the moderate I-activity were both abolished by elimination of the terminal Gal or by removal of the N-acetyl groups of the two GlcNAc residues. Structural

elucidation

of the molecular disease" tumors;

basis

for the Ii-determinants

anti-1

structure

of the entire

During the course

ation

with

some glycosphingolipids inhibition

(2).

Ii-antigen

with

human

Ii-antigens

of blood group ABH determinants

of the isolation

ABH activities

agglutinin

changes associated

appear in some human tumors

as the precursors

for the understanding

known as "cold

of antigenic

serum (Ma) has been shown to recognize

chemical

that

is important

of the autoimmune disorder

and for the understanding

(l),

been considered

lipids

of Ii-determinants

have

(3) and one

GalS1+4GlcNAcSl%Gal;

however

the

complex has not yet been elucidated. and characterization

from human erythrocyte

membranes (4) we have noticed

show blood group I and i activity

(5) and radioimmunoassay

(6),

of glycosphingo-

by hemagglutin-

and have found strong

i and

I activities in certain bovine erythrocyte ganglioside fractions particularly after * This investigation was supported by the National Cancer Institute Grant CAl9224. Iatrobeads silica gel used in this study was donated by Dr. T. Yamakawa, Tokyo University under the U.S.-Japan Cooperative Science Program supported by FJ-5084. %upported by Deutsche Forschungsgemeinschaft. solvent composition is expressed Abbreviations: C-M-W: chloroform-methanol-water, by volume ratio, TLC: thin-layer chromatography, DEAE: diethylaminoethyl. 0006-291x/78/0814-1286$01,00/0. Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form resewed.

1286

Vol. 81, No. 4, 1978

One such active

desialylation. found

BIOCHEMICAL

to be identical

of a ganglioside The structural studied ceramide

series

rwrhexaosylceramide"

described

requirement

1

fraction

for

of the

including

minus

of the

Ii-activities

by chemical

(7)

(5)

and by Chien

analogues

from the highly

and has been

and the

i and I activities

of eleven

derived

purified

fucose"

by Wiegandt

expression

those

RESEARCH COMMUNICATIONS

was extensively

to "H2-glycolipid

recently

by comparison

AND BIOPHYSICAL

i-active

"asialo-core" et al

(8).

has been of lactoglycosyl"lacto-N-

modification.

MATERIALS

AND METHODS

Isokztion, Preparation and Purification of GZycosphingoZipids: H2-glycolipid (5 ) , paragloboside (ll), sialosylparagloboside (ll), lacto-N-triosylceramide (12), a-gslactosylparagloboside (13), t%galactosylparagloboside (14) have been prepared according to the methods described. The i/I active gangliosides of bovine erythrocyte stroma were prepared by Folch's partition of the total lipid extract followed by successive chromatographies on DEAF,-Sephadex according to a modified method of Momoi et al (15) and Iatrobeads silica gel (16) (see Fig. 1 Desialylation of gangliosides was carried out by hydrolysis in and Legends). 1% aqueous acetic acid at 100° C for one hour or by incubating with Rbrio ChoZerae H2-glycolipid was neuraminidase (Behring Diagnostics, Somerville, N.J. 08876). degraded by fucosidase of ChaZonia lampas (l'i'), purified by affinity chromatoLacto-N-norpentaosylceramide (see Table I) was prepared by treating graphy (18). desialylated ganglioside fraction E by Jack bean meal @-galactosidase (19). Both fraction E ganglioside and the desialylated compound (lacto-N-norhexaosylcersmide) were further purified after acetylation and TLC in dichloroethaneacetone (55:45) or in butylacetate-acetone-water (25:lO:l). The products were homogeneous on TLC in these solvents and in benzene-methanol (16:3). Structural Deteninution of the Active GangZioside E and its DesiaZyZated Derivative flacto-N-norhexaosyIceramide): Analysis of the carbohydrate composition, sequence of carbohydrate by step-wise enzymatic degradation, methylation analysis, and total mass spectrometry of permethylated compounds have been carried out according to the procedures already described (4,5,11,14,20). Structural Modification of La&o-N-norhexaosyZceramide: The terminal !3-galactosyl residue of lacto-N-norhexaosylceramide was modified to C6-aldehydoGa3. by galactose oxidase in tetrahydrofuran according to the method described by Suzuki (21), and by the limited periodate oxidation according to the condition described by Hakomori et al (22). The terminal Gal was eliminated by Jack bean 8-galactosidase (23) and the resulting compound was lacto-N-norpentaosylcersmide (Table I). The N-acetyl group of N-acetylglucosaminosyl residues of lacto-N-norhexaosylceramide was removed by hydrazinolysis (in hydrazine hydrate, 100° C 5 hrs), under the condition no fatty acyl amide linkage was eliminated (unpublished data), In these chemical modifications,the rate of conversion was checked by TLC. Detemnination of Ii-Activities: Radioimmunoassays of Ii-activities were carried out using five anti-1 and six anti-i sera according to the procedure previously described (6,23). Ii-activity was also determined by hemagglutination inhibition 1. Nomenclature adopted according to IUPAC-IUB Commission on Biochemical Nomenclature (9). Lactoglycosyl series denotes glycolipids containing GlcNAcg1+3Gal Bl+kGlcl+lCer as the common structure. Lacto-N-norhexaosylcersmide signifies, in this paper, a glycolipid having a straight chain hexasaccharide containing Gal and GlcNAc. It differs from "lacto-N-hexaose" or 'lacto-N-neohexaose" which has a branched structure (10). These branched oligosaccharides should be called lacto-N-iso-hexaose or lacto-N-iso-neohexaose.

1287

yol. 81, No. 4, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IA k.3.

DEAE

Sephadex

A25-Column

. . ..I. --w--m-

-

ABC

D

5Gil

EF

I

1500

low Eluate

in

_.___

-

H

G

2ooo

2500

ml

1B

-..m. --we-.... E

-.... w......

. ..I..._

I 100

200

200 Eluate

Fig.

400 in

a00

I 800

ml

1.

Isolation of i/I active fraction E from bovine erythrocyte gangliosides by chromatography on DEAF-Sephadex A25 and Iatrobeads silica gel. Upper panel (IA) shows the separation pattern of gangliosides on DEAE-Sephadex A25: 600 mg of glycolipid mixture (Folch's upper phase) were dissolved in 20 ml of C-M-W (30:15:2) and applied on 290 ml bed volume of DEAE-Sephadex A25 (acetate After eluting neutral glycolipids with the same solform) in the same solvent. vent , gangliosides were separately eluted by a linear gradient of NH4OAc (0.030.27 M) in the same solvent. Of each fraction (15 ml) aliquots were analyzed by TLC in C-M-W (60:35:8). The fractions containing the same kind of gsngliosides were combined, concentrated, dialyzed, and lyophilized; thus fractions A to I were separated. Fraction E was i/I active. There were other Ii-active fractions with longer carbohydrate chains which will be described elsewhere. Lower panel (lB) shows the separation pattern of fraction E on Iatrobead tography. 20 mg of E was dissolved in 1.5 ml C-M (2:1), applied on an column (120 X 1.5 cm, Iatron Lab., Inc., Tokyo), and eluted by a linear of C-M-W (53:45:2) to C-M-W (16:80:4). Each 5 ml fraction was analyzed in C-M-W (60:35:8). The active fraction E yielded 9 mg (4.5% of total side).

chromaIatrobead gradient by TLC ganglio-

Dench, anti-i Hog (donated with 3 hemagglutination doses of anti-1 Step, anti-i by Mrs. M.C. Crookston, Toronto General Hospital), anti-1 Ma (donated by Dr. E. WA), anti-i McC (donated by Giblett, Puget Sound Central Blood Bank, Seattle, Dr. V. Ginsburg, National Institute of Arthritis and Metabolic Disease, National Institutes of Health). 1288

BIOCHEMICAL

Vol. 81, No. 4, 1978

TABLE I.

Structure

of i/I

active

AND 8lOPHYSlCAL

lacto-N-norhexaosylceramide

RESEARCH COMMUNICATIONS

and its

analogues.

H -glycolipid PFucosyllacto-N-norhexaosylceramide ) Fraction "E" (Sialosyllacto-N-norhexaosylceramide) Lacto-N-norhexaosylceramide (LNHC) Lacto-N-norpentaosylceramide (LNHC minus Gal)

La&o-N-neotetraosylceramide (paragloboside) La&o-N-triosylceramide Sialosylparagloboside a-galactosylparagloboside &galactosylparagloboside

RESULTSANDDISCUSSION An Ii-active successive

(E) was isolated

chromatography

"Iatrobeads" This

fraction

silicic

glycolipid

on DEAE-Sephadex

acid

(Fig.

lB),

was homogeneous

solvent

systems

ation.

The structure

(see Material

analysis

and Methods)

Glc and neuraminic and 2 moles atic

of h-linked

hydrolysis

showed

the

adjacent

acid,

alternating

acid

and 1 mole

the

E completely

terminal

by mass spectrometry.

carbohydrate

agreed

with

1289

and

derivative. in three

after

deacetyl-

findings: one mole

3 moles Glc,

each of

of 3-linked

c) sequential

no indication

d) solid the

probe

above

of two

mass spectrometry

structural

assignment.

as N-glycolylneursxninic

E was identified

Gal

enzym-

and $-N-acetylhexosaminidase,

was identified

The desialylated

by

lA),

acetylated

state

of GlcNAc,

of k-linked

residues,

free

following

of Gal and GlcNAc but

GlcNAc

(Fig.

derivative

yielded

&galactosidase,

sequence

Gal or two adjacent

analysis

stroma

as NGNeur2+3Galf31+4GlcNAc

on the

2 moles

of Gal,

elution

acetylated

and in the

based

b) methylation GlcNAc

gradient

E was determined

by neursminidase,

of the permethylated In addition

3 moles

erythrocyte

by TLC of the

on TLC as the

of fraction

showed

with

followed

~1+3Galf31+~GlcNAcg1+3Galg1+~Glc~1+lCer a) sugar

from bovine

as Galf31+4GlcNAc

Vol. 81, No. 4, 1978

TABLE II.

I and i activities five

I and

BIOCHEMICAL

AND BIOPHYSICAL

of "lacto-N-norhexaosylceramide"

six

and its

eleven

RESEARCH COMMUNICATIONS

malogues

determined

with

i antisera. Anti-I

step

Phi

>lOO 12

-----

t

MEL

era

Da

>lOO -__

-----

-----

Anti-i

1

Tho

McDon

McC

Den

Ha3

Gall

-----

-----

-----

>lOO 0.1

>lOO 0.7

-----

(35%) ---

>lOO 3

H2-glycolipid I$ minus fucose

Ii* H

Fraction

PI** (4;;) H

>lOO ---

>lOO >lOO

>lOO ---

>lOO ---

>lOO ---

La&o-N-wrhexaosylcermide(IXBC)

R H

('6%) ---

>lOO >lOO

>lOO ---

50 ---

---

---

La&a-N-norpentaosy1cermide

R H

>lOO >lOO

>lOO ---

,100 >lOO

>lOO ---

,100 ---

,100 ---

,100 ---

,100 ,100

,100 ,100

--,100

,100 ---

Sialosylparaglobaside

H

>lOO

---

>lOO

---

---

---

---

,100

,100

,100

---

a-Galactosylparegloboside

R H

>lOO >lOO

>lOO ---

>lOO >lOO

>lOO ---

>lOO ---

,100 ---

,100 _--

,100 ,100

,100 ,100

--,100

,100 ---

E-Galactosylparagloboside

R H

>lOO >lOO

>lOO ---

>lOO ,100

>lOO ---

>lOO ---

,100 ---

>lOO ---

,100 >lOO

,100 7100

-->lOO

,100 ---

Paragloboside

R H

>lOO >lOO

>lOO ---

>lOO 100

>lOO ---

>lOO ---

SlOO ---

>lOO ---

>lOO 100

>lOO 100

--100

>lOO ---

La&o-N-triosylcermide

R Ii

>lOO ---

>lOO ---

>lOO ---

>lOO ---

>lOO ---

-----

>lOO ---

>lOO >lOO

-->lOO

_->lOO

>lOO ---

R H

20 40

>lOO ---

>lOO >lOO

>lOO ---

(W) ---

23 ---

---

(40%) 23

--2.5

>lOO ---

(25%) 2.5

---

1.2

>lOO ---

Periodate oxidized

E

LNHC

2.3 10

1

2.0

2.3

R

H

2

>lOO ---

>lOO >lOO

>lOO ---

(25%) _--

(30%) ---

IL ---

de-N-acetylated LNHC

R H

>lOO >lOO

>lOO ---

>lOO >lOO

>lOO ---

>lOO ---

ZlOO ---

>lOO ---

0.8 2.5

0.6

6

Galactose oxidase treated-LNHC

>lOO 12

15.0

2.5

30.0 5.0 >lOO >lOO

>lOO >lOO

--3

>lOO ---

0.6

>lOO ---

---

-->lJO

>lOO ---

*H:

activity by hemagglutination inhibition assays, expressed as the minimum mount (ug) per 0.1 ml of sample that inhibited 3 hemagglutination units of antibody. by radioimuhoassays, expressed aa the mncentration pg/ml required t.0 give 50% inhibition of binding of 1251-labeled Ii-active antigen to the anti-1 or i antibodies. With materials giving less than inhibition the figures in parentheses indicate the amount of inhibition given at the highest concentration tested, 100 pgiml. >lOO: no inhibition at 100 ,,g/ml. :---not tested.

l *R: activity

~1~3Gal~1~~GlcNAc~1~3Gal~1~~Glc~1-tlCeramide b) methylation

analysis,

spectrometry

c) enzymatic

of the permethylated

as "lacto-N-norhexaosylceramide"~ lacto-N-norhexaosylceramide these

glycolipids, Ii-activities

inhibition

degradation

on a) sugar

analysis,

and d) solid

probe

The structure

glycolipid.

is

Lacto-N-norpentaosylcersmide by treating

paragloboside, with

based

several

or by radioimmunoassay

with

and other antisera, are

analogues determined

shown

1290

in Table

are

mass

hereby

designated

was derived

S-galactosidase,

from

The structures listed

50%

in Table

of I.

by hemagglutination II.

Strong

i-activity

was

Vol. 81, No. 4, 1978

found

BIOCHEMICAL

in la&o-N-norhexaosylceramide

H2-glycolipid

showed

indicating

the

structure

must

antibodies. anti-1

identity

inhibition. either

the

was found

this

pentaosylcersmide,

by the

like

to paragloboside ations

cold did

(6).

inhibition.

Since

terminal

and penultimate

the

Both abolished N-acetyl

chain

or lack

by 1) removal

no activity

II).

Tsai

et a2 (24)

lacto-N-

was found

specificity showed

reported

(25),

chemical

is

could

GalSl+kGlcNAc with

not

residues;

thus

sera

the

they

same

are

be due to the

structure, anti-1

Step,

but

demonstrated

and no other

&Gal the

1291

residue

activities

were

glycolipids

similarly

and 2) removal

of the

were

reduced,

similarly

in

as the

analysis,

of lacto-N-norhexaosylceramide of the terminal

share

due to contimanants,

homogeneous

prepar-

in hemagglutination

The I-activity is

directed

of anti-i

to assume that

activity

caused

and three

no inhibition

is reasonable

strong

that

antibodies

was observed

repeating

in fucosyl-

hemagglutination

of paragloboside

glycolipid

Ma by

was found

McC, however,

activity

showed

of two GlcNAc

anti-1

in H2-glycolipid,

while

it

of the

by rigorous

with

Some activity

preparation

of this

by hemagglutination

no activity

anti-i

activity

i and I activities

group

Step

Ma and Gra by radioimmunoassay.

be detected

Phi and Da by radioimmuno-

and lacto-N-norhexaosylceramide

structure,

lacto-N-norhexaosylceramide

could

anti-1

anti-1

of patient

paragloboside

length

anti-1

carbohydrate

with

inhibited

A minimum

Lacto-N-norhexaosylceramide not with

activity

paragloboside

The minimum

chain

high

showed

S-galactosylparagloboside,

show typical

purified

by radioimmunoassays

cross-reactive,

agglutinin

not

of extensively

shorter

also

with

(see Table

(lacto-N-neotetraosylceramide)

anti-i

anti-i

demonstrated

sialosyllacto-N-norhexaosylceramide

paragloboside

This various

and la&o-N-triosylcersmide.

lacto-N-norhexaosylcersmide

a-Lfucosidase,

with

showed

were

with

tested.

of i-activities

with

glycolipid

sera

lacto-N-norhexaosylceramide.

a-galactosylparagloboside,

sialosylparagloboside,

treatment

glycolipid

or weak activity

No Ii-activities

method.

with

expression

same pure

activity

However,

after

product

for

and moderate

A moderate

i-activity

of the

be essential

5 of the 6 anti-i

with

a strong

However,

Step

assay.

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 81, No. 4, 1978

BlOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NHAc

Fig.

Possible structural requirement for i and I specificities. Two formulas for the iI-active hexasaccharide are shown; the upper is the classical Haworth's structure, the lower is a possible tertiary structure according to Rees (26). Note that the GlcNAcg1+3Gal disaccharide unit forms a plane (approximately zero rotation) and that two planes are rotated around the f31+4glycosidic linkage. The terminal trisaccharide sequence of GalBl+~GlcNAc g1+3Gal is a necessary but not sufficient requirement for i-activity. The repeat of the penultimate GlcNAcg1-+3Gal disaccharide unit seems to be an additional requirement for the expression of i-specificity. R could be either lipid or protein. I specificity may require this same structure as well as an additional unknown substituent X, which could be a side chain attached to the hexasaccharide.

but

2.

not

ring

abolished

by 3) periodate

and 4) oxidation Thus,

it

by chemical

of Cb-hydroxyl

was impossible methods

used.

It

polysaccharides

Irrespective

planes

twisted

being

according that

the

invariable

to the

structural

is most

these that

I and i activities

the

studies

species,

they

@l-t& linkage

pyranosyl

Gal,

I-activity

The alternating

diffraction

glycosidic

are

g-Gal

1 eq + 3 eq) as found

assignment

groups It

the

modify

reaction,

shown by X-ray

around

terminal

of the terminal

possible

of the pyranose

two acetamido distance.

a cross

(1 eq -f 4 eq,

has been

structures.

group

is therefore is

@l-t3 pyranosides

of the

to differentially

lacto-N-norhexaosylceramide gl-&,

oxidation

in

sequence connective

are built

(26).

probable

1292

on two disaccharide that

this

compact

of tissue

to form helical up of disaccharidm A model

of lacto-N-norhexaosylcersanide

located

of

constructed indicates

planes two-plane

at an (semi-

BlOCklEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 81, No. 4, 1978

helical)

structure

determinants. this

spatial

relationship

is

the

essential

The structures arrangement is

illustrated

requirement

recognized

by anti-1

but may require

additional

in Figure

for

the majority Step,

Phi

unknown

of i-antigenic

and Da may include structures.

This

2.

REFERENCES Grune and J.V. (1962) The Haemolytic Anaemias 2nd ed. Part II. Stratton, New York; Feizi, T,, Taylor-Robinson, D., Shields, M.D., and Carter, R.A. (1969) Nature 222, 1253-1256. C., Westwood, J.H. (1975) The Lancet ;?, 391-393. Feizi, T., Tuberville, Feizi, T., Kabat, E.A., Anderson, B,, Marsh, W.L., and Vicari, G. (1971) J. Immunol. IO6, 1578-1592. Hakomori, S., Watanabe, K., and Laine, R.A. (1977) Pure & Appl. Chem. 49, 1215-1227. Watanabe, K., Laine, R.A., and Hakomori, S. (1975) Biochemistry I.&, 2725-2733. Feizi, T., Childs, R., Hakomori, S., and Powell, M. Biochem. J. in press. Wiegandt, H. (1974) ~urop. J. Biochem. 45, 367-369. (1977) Fed. Proc. g, 731. Chien, J-L., Li, S-C., Laine, R.A., and Li, Y-T. IUPAC-IUB Commission on Biochemical Nomenclature. The Nomenclature of Lipids. (1977) Lipids 12, 455-463. (1972) J. Biol. Chem. 2& 1525-1529; (1972) Kobata, A., and Ginsburg, V. Arch. Biochem. Biophys. 150, 273-281. Siddiqui, B., and Hakomori, S. (1973) Biochim. Biophys. Acta 330, 147-155. Watanabe, K., and Hakomori, S. (1976) J. Exp. Med. 144, 644-653; Ando, S., Kon, K., Isobe, M., Nagai, Y., and Yamakawa, T. (19m J. Biochem. '& 625-632. E-to, T., Ichikawa, T., Nishimura, K., Ando, S., and Yemakawa, T. O-968) J. Biochem. 64, 205-213; Stellner, K., Saito, H., and Hakomori, S. (1973). Arch. Biochem. Biophys. 155, 464-472. Stellner, K., and Hskomori, S. (1974) J. Biol. Chem. &, 1022-1025. Momoi, T., Ando, S., and Nagai, Y. (1976) Biochim. Biophys. Acta 441, 488-497. (1976) Biochim. Biophys. Acta 424, 98-105. Ando, S., Isobe, M., and Nagai, Y. Iijima, Y., Muramatsu, T., and Egami, F. (1971) Arch. Biochem. Biophys. I&

1. Dacie, 2.

3. 4. 2: 7. 8. 9. LO. 11. 12.

13. 14. 15. 16. 17.

150-154. 18. Jain, R.S., 19. 20. 21. 22. 23. 24. 25. 26.

Binder, R.L., Levy-Benshimol, A,, Buck, C.A., and Warren, L. (1977) J. Chromat. D, 283-290. Li, Y-T., and Li, S-C. (1972) Methods in Enzymol. 8, 702-713. Laine, R.A., Stellner, K., and Hakomori, S. (1974) Methods of Membrane Biol. 2, 205-247. Ed. E. Korn, Plenum Publishing, New York. Suzuki, Y., and Suzuki, K. (1972) J. Lipid Res. l3, 687-689. Hakomori, S., Siddiqui, B., Li, Y-T., Li, S-C., and Hellerqvist, C.G. (1971) J. Biol. Chem. 246, 2271-2277. Wood, E., Lecomte, J., Childs, R., and Feizi; T. (1977) Proc. of the 4th International Symposium on Glycoconjugates, Woodshole, Massachusetts. in press. Tsai, C-M., Zopf, D.A., Wistar, R., and Ginsburg, V. (1976) J. Iimnunol. 117, 717-721. Schwarting, G.A., and Marcus, D.M. (1977) J. Immunol. 118, 1415-1419. Rees, D.A. (1975) MTP International Review of Science, 2, l-42, ed. Whelan, W.J., Butterworths (London) University Park Press (Baltimore); Atkins, E.D.T., Isaac, D.H., Nieduszynski, I.A., Phelps, C.E., and Sheehan, J.K. (1974) Polymer 2, 263-271.

1293

Blood group i and I activities of "lacto-N-norhexaosylceramide" and its analogues: the structural requirements for i-specificities.

Vol. 81, No. 4, 1978 April 28,1978 BlOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1286-1293 BLOOD GROUP i AND I ACTIVITIES OF "IACTO-N-no...
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